A Symposium on Drug Discovery for the Brain

Virginia Drug Discovery Consortium

VirginiaBrainRx:

May 23 - 24, 2016 Omni Richmond Hotel

Richmond, Virginia

Poster Session Abstracts

Author Poster Number Abeyawardhane Dinendra L. T24 Akbarali Hamid M9 Allen Irving C. T19 Alwassil Osama I. T13 Argade Malaika D. T13 Asmar Anthony T27 Audette Michel T3 Aylor Kevin W. M2 Bagdas Deniz T15, T18 Balakathiresan Nagaraja T6 Banks Matthew L. M16, M19, M20, M21 Barrett Eugene J. M2 Bartsch Victoria T8 Bassaganya-Riera Josep T17 Baumann M. H. T2 Betz Heinrich M27 Bevan David M15, T5, T20 Bhomia Manish T6 Bickston Stephen T17 Blatt Jason E. T3 Bledsoe Douglas M27 Blough Bruce E. M20 Boroda Salome M25 Boulton Adam T10 Bowers Devin M28 Brown Anne M15, T20 Bruno Robert D. T26 Bullock Timothy T1 Burk Joshua T4 Bushweller John H. T10 Butman John T3 Cao Shugeng M14 Carbo Adria T17 Carlier Paul M22 Cetas Justin T3 Chen Keqiang M6 Cheng Kejun M21 Chojnacki Jeremy E. M17 Christian Phil M7 Coogle Constance M28 Cook James M16 Costa Blaise M27 Costa Anthony T3 Damaj Imad T15, T18 Daniel Dianne M5 Davalos Rafael T19 Davis Richey M24 De Felice L. J. T2 Dervisis Nikolaos T19 Dewey William M9, T7, T11, T12 Dukat Malgorzata T13 Ehrich Marion M18, T17

Electron Kebebew M14 Eltit J. M. T2 Ewend Matthew T3 Fadel Jim R. T4 Fauq Abdul M22 Fitzpatrick Mairen E. T6 Foster Johan M1 Franck Christopher T. T21 Fuss Babette M3 Gandour Richard T17 Gaskins Kelly M14 Gaultier Alban M10, T1 Gemta Leslie T1 Glennon R. A. T2 Godfrey Earl W. M5 Gonek Maciei T12 Gourdie Robert M1 Gray Sarah M2 Green Michael R. T22 Guan Ting T21 Guo Sujuan T22 Hallum Olga Y. T6 Hancock Sandy M18 Handing Kasia T10 Haq Rabia T3 Harris Robert M7 Harris Thurl E. M25 Heitger Denver R. M26 Heller Richard T27 Heller Loree T27 Helm Rich T5 Hontecillas Raquel T17 Hsu Ku-Lung T16 Hutsell Blake M21 Iyer Kavita T13 Jackson Asti T15, T18 Jacob Joanna T7 Jali Abdulmajeed T25 Jo Ami M24 Johnson Amy M20 Johnson Edward M. M5 Jurmain J.L. T14 Kayandan Sanem M24 Kelly Deborah F. T21 Kingston David M11, M14 Knudsen Giselle M13 Lamouille Samy M1 Laube Bodo M27 Lazo John S. T10 Lee Yueh T3 Li Liwu M6 Li Dai J. M7 Li Guanguan M16

Liang Yanping T21 Libutti Steven K. M14 Lile Joshua A. M20 Lippold Kumiko T11 Lister James A. M3 Liu Kai M17 Lucas Heather M26 Lucas Heather R. T24 Machida Mayumi T6 Mackey Zachary M13 Madry Christian M27 Maheshwari Radha K. T6 Malone Robert T9 Malone Jill Glasspool T9 Martin del Campo J. S. M11 McDevitt Jason M23, T23 McQueeny Kelley T10 Mesic Ivana M27 Meyers Gregory A. M7 Miles M. F. T14 Minor Wladek T10 Mollica Peter A. T26 Monceaux Christopher M22 Murphy Susan T22 Negus S. Stevens M16, M19, M20, M21, T2 Niccum Maria M25 Nicholson Katherine L. M20 Nilubol Naris M14 O’Hanlon Katherine A. M7 Obeng Samuel M12, T25 Ogle Roy C. T26 Paciotti Guilio F. M14 Pakhomov Andrei M4 Pan Xiaolei T13 Pang Priscilla T3 Partilla J. S. T2 Petrella Ross M4 Philipson Casandra T17 Philipson Noah T17 Pilitsis Julie T3 Poe Michael M. M16 Pridham Kevin T22 Purow Benjamin W. M1 Purow Benjamin W. M25 Qin Qizhi T19 Raje Vidisha M25 Rashid Tanweer T3 Ray Keith T5 Reid John A. T26 Reynolds Thomas M7 Rice Kenner C. M21 Richelson Elliott M22 Riffle Judy M24

Roberts Rose M1 Rosen Abagail M10, T1 Rossmeisl John M1 Ruchala I. T2 Russell Melissa M7 Saathoff John M17 Sachs Patrick T26 Sakloth F. T2 Salamoun Joseph M. T10 Sanford Larry D. T6 Schoenbach Karl H. M4 Schwienteck Kathryn M16, T2 Seki Scott M10, T1 Selley Dana M9 Selley Dana E. T25 Sharlow Elizabeth T10 Sheng Zhi M1, T21, T22 Slattum Patricia M28 Sobrado Pablo M11 Solis Jr. E. T2 Stacey Michael T27 Stevenson Max T1 Sultana Sharmin T3 Sutton Amy M. T6 Sweet Douglas T13 Tamarkin Lawrence M14 Tamer Ceyhun M27 Valenciano Ana Lisa M13 Varghese Robin T21 Velander Paul T5 Virbasius Ching-Man T22 Vogelaar N. M11 Wellman Laurie L. T6 Wheeler Natalie M3 Williams Brook T6 Williams Dwight M9 Wipf Peter T10 Wolz Gabriella M7 Wu Ling T5 Xiao Shu M4 Xu Guoyan M9 Xu Bin T5 Yang Hu M9 Young Richard M8 Yuan Ruoxi M6 Yuan Yunyun M9 Yuan ZiQiang M14 Zhai Min T6 Zhang Yan M9, M12, T25 Zhang Shijun M17, T5 Zhao Jielu M14 Zolotarskaya Olga M9 Zylka Mark J. T8

VirginiaBrainRx Poster Session M 1

TRANSLATION OF A NOVEL GLIOBLASTOMA DRUG TO CLINICAL TRIALS

Robert G. Gourdie1,2,3, Johan Foster4, Rose Roberts4, Benjamin W. Purow5, John Rossmeisl6, Samy Lamouille1,7 and Zhi Sheng1,3

1Virginia Tech Carilion Research Institute, Roanoke, VA, 24016; 2Department of Biomedical Engineering and Mechanics, 3Virginia Tech Center for Drug Discovery; and 4Macromolecules Innovation Institute, Department of Materials Science and Engineering, Virginia Tech, Blacksburg, VA, 24061, 5Department of Neurology, University of Virginia School of Medicine, Charlottesville, VA, 22908, 6Department of Small Animal Clinical Sciences, VA-MD College of Veterinary Medicine and Hospital, Blacksburg, VA, 24061,7FirstString Research Inc., 300 W Coleman Blvd # 203, Mt Pleasant, SC 29464 gourdier@vtc.vt.edu Glioblastoma (GBM) is the most common and deadly malignant brain tumor in adults. The standard-of-care treatment for GBM usually involves surgery to remove the tumor, followed by radiation and chemotherapy with temozolomide (TMZ). Unfortunately, this treatment is largely ineffective as tumors recur, with patients surviving an average of around a year. One of the main reasons for this failure is that patients develop resistance to TMZ chemotherapy. Our Virginia Tech team recently reported that the channel protein connexin43 (Cx43) is a key determinant of TMZ chemotherapy resistance in patients (Cancer Research, 76:139, 2016). Moreover, we showed that treatment with the Cx43 inhibitory peptide αCT1 recovered sensitivity of human GBM cells and glioma stem cells to TMZ in vitro and in GBM animal models. To advance αCT1 to human clinical testing for GBM we are pursuing two inter-related goals: Goal 1) Formulation of a controlled-release CT1: FirstString Research is presently testing αCT1 in Phase III clinical trials for chronic skin wounds (J Inv Dermatology (Nature), 135:289-98, 2015), where it is being applied in a topical gel formulation. A different strategy is required in dog and human clinical trials, wherein αCT1 will be delivered directly into tumors by convection-enhanced-delivery (CED). To achieve this, we have encapsulated αCT1 in poly(lactic-co-glycolic acid) nanoparticles (an FDA-approved vehicle), which provide for its controlled-release following CED injection. We have optimized nanoparticle size to <200 nm and have characterized release kinetics over 3 weeks at 37 °C and nanoparticle uptake and dissolution in cultured cells over similar time courses. Goal 2) To do a clinical trial in dogs with GBM tumors: Veterinary clinical tests are an invaluable tool in the study of human disease and its treatment. High-grade glioma/GBM occurs naturally at a similar frequency in dogs as it does in humans. Around 30 dogs are diagnosed with glioma each year in the Neurosurgery clinic at the Virginia Maryland School of Veterinary Medicine and Hospital. In collaboration with FirstString Research, our Virginia Tech team has obtained regulatory approval for clinical testing of oral TMZ in combination with CED of αCT1 into dog brain tumors - the FACT-CG clinical trial. The first of the canine patients entered the protocol in April 2016 and it is expected that 6 to 8 dogs will have received the combined treatment by Q4 2016. If safety outcomes from FACT-CG are favorable, we anticipate seeking regulatory approval from the FDA to advance to clinical trials in humans with GBM in Q1 2017. Acknowledgement: This research is supported by the Virginia Biosciences Health Research Corporation (VBHRC- the Catalyst), FirstString Research Inc, the Virginia Center for Innovative Technology (CIT) and Virginia Tech

VirginiaBrainRx Poster Session M 2

INSULIN UPTAKE BY THE BRAIN ENDOTHELIAL CELL IS RECEPTOR-DEPENDENT AND BLUNTED BY HIGH-FAT DIET FEEDING

Sarah M. Gray1, Kevin W. Aylor2, and Eugene J. Barrett1,2* 1Department of Pharmacology, University of Virginia, Charlottesville, VA 22908 2Division of Endocrinology & Metabolism, University of Virginia, Charlottesville, VA 22908 sg2ae@virginia.edu Insulin access to the brain may be critical for appetite regulation, metabolism, and cognition1-4. Despite its importance, little is known of how insulin crosses the blood-brain barrier (BBB) to reach brain interstitial fluid and act on target neurons. We hypothesized that the BBB endothelial cell (BEC) has an insulin receptor-mediated vesicular transport system and that high fat diet (HFD)-induced insulin resistance could interfere with this process. To test whether insulin reaches brain tissue in vivo via the CSF circulation or by crossing the BBB, rats received 0.7 pmol 125I-insulin (125I-ins). Afterward, the vasculature was flushed for 5 min and brain tissue collected. 125I-ins appeared in cerebellum, cortex, and basal nuclei before reaching CSF. An insulin receptor selective antagonist (S-961) blocked brain 125I-ins clearance (p<0.01) as did 4 weeks of HFD (p<0.05, vs chow fed rats). We also isolated brain ECs from HFD and chow fed (ND) rats and measured 125I-ins uptake (200 pM). Again, HFD decreased EC 125I-ins uptake (p<0.01). A comparison of insulin receptor mRNA and protein expression showed no difference between ND and HFD BECs. To test whether canonical insulin signaling pathways were blunted in HFD rats, we stimulated isolated BECs with 10 nM insulin or vehicle control and western blotted for phosphorylated and total proteins. To our surprise, 10 nM insulin increased p-Akt(Ser473) and p-eNOS(Ser1177) similarly in BECs from HFD and ND rats. Insulin did not increase ERK(Thr202/Tyr204), Src(Tyr416), or caveolin-1(Tyr14) phosphorylation in either group. To further examine the pathway of insulin uptake by rat brain microvascular ECs (RBMVECs, Cell Applications), we measured the time course of insulin uptake and sensitivity to agents interfering with insulin signaling or lipid raft formation. 125I-ins uptake by RBMVECs reached a plateau after 15 min and was completely blocked by 10 nM S-961 (p<0.001), but not by an IGF-I receptor neutralizing antibody (Ab-3). Pharmacological inhibitors of either insulin signaling (wortmannin, genistein, and PD89059) or Src kinase (PP1) decreased 125I-ins uptake. Depleting cholesterol from lipid rafts with 10 mM methyl-β-cyclodextran or 5 ug/ml filipin significantly decreased EC insulin uptake (p<0.05, each). We conclude that: a) insulin enters brain interstitial fluid principally by crossing the BBB; b) HFD feeding compromises brain insulin transport; c) insulin’s transport is mediated by its receptor and lipid rafts; and d) canonical insulin signaling does not mediate insulin transport in brain endothelial cells. These findings underscore the need to unravel the precise mechanisms regulating insulin uptake in BECs. References 1. Hallschmid M, Higgs S, Thienel M, Ott V, Lehnert H. Postprandial administration of intranasal insulin intensifies satiety and reduces intake of palatable snacks in women. Diabetes. 2012;61(4):782-789. doi: 10.2337/db11-1390; 10.2337/db11-1390. 2. Benedict C, Kern W, Schultes B, Born J, Hallschmid M. Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin. J Clin Endocrinol Metab. 2008;93(4):1339-1344. doi: 10.1210/jc.2007-2606; 10.1210/jc.2007-2606. 3. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature. 2006;443(7109):289-295. doi: 10.1038/nature05026. 4. Schrijvers EM, Witteman JC, Sijbrands EJ, Hofman A, Koudstaal PJ, Breteler MM. Insulin metabolism and the risk of alzheimer disease: The rotterdam study. Neurology. 2010;75(22):1982-1987. doi: 10.1212/WNL.0b013e3181ffe4f6; 10.1212/WNL.0b013e3181ffe4f6. Acknowledgement This work was supported by NIH (NK 057878 to EJB, T32 HL007284 and F31 DK104521 to SMG) and AHA (14PRE20100048 to SMG).

VirginiaBrainRx Poster Session M 3

THE ATX-LPA AXIS MODUALTES HISTONE ACETYLATION AND GENE EXPRESSION DURING OLIGODENDROCYTE DIFFERENTIATION

Natalie A. Wheeler1, James A. Lister2 and Babette Fuss1* 1Department of Anatomy and Neurobiology, 2Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, 23298. allenna@vcu.edu A specialized neural cell type, named the oligodendrocyte (OLG), generates the lipid rich myelin membrane sheath that enables fast and efficient signal propagation in the central nervous system (CNS) and provides metabolic support to axons, i.e. nerve fibers. Under demyelinating conditions as they are seen in one of the major disabling neurologic conditions in human, Multiple Sclerosis (MS), progenitor cells with the potential to differentiate into fully functional OLGs are present but often fail to give rise to myelinating cells for reasons that are currently not fully understood. Identifying therapeutic targets with the potential to enhance OLG differentiation is thus seen as a promising approach for diseases associated with a loss and/or dysfunction of the myelin sheath as seen MS for which there are currently no curative treatments available. Previous studies from our laboratory identified the extracellular protein Autotaxin (ATX) as a critical factor regulating OLG differentiation1-3. More specifically, ATX, via its MORFO (modulator of OLG remodeling and focal adhesion organization) domain mediates primarily the morphological changes that are associated with the later stages of OLG differentiation. Here, we show that ATX’s second functionally active domain, its enzymatic lysoPLD (lysophospholipase D) active site, which is known to generate the extracellular lipid signaling molecule lysophosphatidic acid (LPA), drives gene expression changes crucial for early stage OLG differentiation. Intriguingly, these transcriptional changes were found to be mediated via epigenetic mechanisms that have recently been recognized as fundamental contributing players in OLG differentiation4,5. Taken together, our findings point toward the ATX-LPA axis as an attractive candidate to be targeted in a novel therapeutic approach for diseases associated with a loss and/or dysfunction of the myelin sheath as seen in MS. References 1. Yuelling, L. W., Waggener, C. T., Afshari, F. S., Lister, J. A. and Fuss, B. Autotaxin/ENPP2 regulates

oligodendrocyte differentiation in vivo in the developing zebrafish hindbrain. Glia 60, 1605-1618 (2012).

2. Dennis, J., White, M. A., Forrest, A. D., Yuelling, L. M., Nogaroli, L., Afshari, F. S., Fox, M. A. and Fuss, B. Phosphodiesterase-Ialpha/autotaxin's MORFO domain regulates oligodendroglial process network formation and focal adhesion organization. Mol Cell Neurosci 37, 412-424 (2008).

3. Yuelling L.M. and Fuss.B. Autotaxin (ATX): a multi-functional protein possessing enzymatic lysoPLD activity and matricellular properties. Biochim Biophys Acta 1781: 525-530 (2008)

4. Emery, B. and Lu, Q.R. Transcriptional and epigenetic regulation if oligodendrocyte development and myelination in the central nervous system Cold Spring Harb Perspect Biol 7:a020461 (2015)

5. Hernandez, M and Casaccia, P. Interplay between transcriptional control and chromatin regulation in the oligodendrocyte lineage. Glia 63:1357-1375 (2015).

Acknowledgement This work was supported by grants from the NIH/NINDS (B.F. and VCU’s Department of Anatomy and Neurobiology Microscopy Facility) and the NIH/NCI (VCU’s Massey Cancer Center Flow Cytometry Core)

VirginiaBrainRx Poster Session M 4

A DIELECTRIC ROD ANTENNA FOR STIMULATING NEUROLOGICAL TISSUE

Ross A. Petrella1,2, Karl H. Schoenbach1, Andrei Pakhomov1, and Shu Xiao1,2,3* 1Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, 23529 2Biomedical Research Institute, Old Dominion University, Norfolk, VA, 23529 3 Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA, 23529 rpetr005@odu.edu Neurological tissue has been noninvasively stimulated by repetitive Transcranial Magnetic Stimulation (rTMS); however, the resolution of this technique is limited. Subnanosecond pulsed electric fields in the range of 20 kV/cm can significantly improve on these results. This was shown in a simulation of a dielectric rod antenna. The antenna comprises of three sections (conical wave launcher, cylindrical wave guide, conical emitting section). Together these work to confine the energy to the center of the rod and then emit the energy at the tip of a cone. By making direction contact with the skin, the antenna is better coupled to the skin and reflection is reduced. In a simulation on a human voxel model, the electric field present in the brain tissue at a depth of 2 cm was 11.5 V/m. To create the critical electric field for biological effects at this location, the input voltage needs to be 175 kV. The spot size at this depth is approximately 1 cm2. To confirm the accuracy of the simulation environment the conical launching section was constructed and tested in free space. These results of this set foundation for high voltage in situ experiments of the complete antenna system. It should be noted that the antenna could be repurposed for stimulation of surface tissue such as skin. Acknowledgement This project was sponsored by NIH, R21(1R21EB016912-01A1

VirginiaBrainRx Poster Session M 5

SYNTHETIC POLYNUCLEOTIDE-BINDING PUR PEPTIDE AND USES THEREOF

Dianne C. Daniel1*, Earl W. Godfrey2, Edward M. Johnson1* 1Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, Virginia, 23507 2Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk, Virginia, 23507 danieldc@evms.edu Amyotrophic lateral sclerosis (ALS) is a highly debilitating neurodegenerative disease. Although ALS has a variety of causes, the most common familial form of the disease involves an expanded hexanucleotide repeat on chromosome 9, at C9ORF72. This abnormal repeat is present in DNA and in transcribed RNA. Pur-alpha is a sequence-specific single-stranded DNA- and RNA-binding protein whose nucleotide-binding element strongly resembles the C9ORF72 hexanucleotide (1). Pur-alpha deficiency has been linked to disorders in brain development and Pur family members are being brought into focus as important players in ALS (2). Recently published data from our labs reveal an important role for expression of Pur-alpha in the ALS disease variant caused by mutated FUS (3). Our unpublished data additionally reveal a role for expression of Pur-alpha in reversing cellular pathology in the C9ORF72 expanded repeat variant of the disease. The TZIP peptide is a Pur protein segment that can agonistically displace bound Pur in an affected cell, thereby preventing cellular damage due to Pur sequestration. Overexpression of Pur-alpha proteins alleviates cellular abnormalities caused by the expanded G-rich repeats. The TZIP peptide can modify the binding of Pur proteins alpha and beta to this expanded repeat. Pur beta binds the hexanucleotide element, but its role in ALS is unknown. TZIP functions cooperatively at less than 1μM with Pur-alpha to enhance binding to the GGGGCC repeat. If Pur proteins are indeed sequestered away, TZIP can be used to bind to the G-rich repeats instead of Pur family members, freeing up Pur proteins to proceed with their normal function. We have obtained preliminary results indicating that TZIP can reduce aspects of abnormal autophagy and neuronal damage in cultured cells from ALS patients. Our future direction is to seek collaborations to test these peptides on a variety of endpoints in ALS patient iPSC-derived neurons and to test peptide toxicity in mice.

References 1. Bergemann AD, Ma ZW, Johnson EM. Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Mol Cell Biol. 1992 Dec;12(12):5673-82. PMID: 1448097; PMCID: PMC360507. 2. Orr HT. Toxic RNA as a driver of disease in a common form of ALS and dementia. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7533-4. doi: 10.1073/pnas.1305239110. PMID: 23630297; PMCID: PMC3651504. 3. Daigle JG, Krishnamurthy K, Ramesh N, Casci I, Monaghan J, McAvoy K, Godfrey EW, Daniel DC, Johnson EM, et al. (2016) Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity. Acta Neuropathol. 2016 Apr;131(4):605-20. PMID: 26728149; PMCID: PMC4791193. Acknowledgement The peptides disclosed in this poster were developed with funding from NIH & NCI (EMJ). A patent application has been filed by Eastern Virginia Medical School.

Fig. 1. Mobility Shift Assays: Pura and TZIP mixed together demonstrated the ability of TZIP to alter the binding activity of Pura to the ALS repeat sequence. Increasing the concentrations of TZIP causes the shifted band to migrate more slowly. Densitometry revealed a Kd value of 1.6x10-8 for Pura to ALS repeat and 5.5x10-10 for TZIP to ALS repeat.

VirginiaBrainRx Poster Session M 6

DISRUPTION OF NOVEL INNATE IMMUNITY MODULATOR TOLLIP LEADS TO NEURONAL CELL DEATH

Keqiang Chen1, Ruoxi Yuan2 and Liwu Li*1 1Department of Biological Sciences, Biomedical Engineering and Sciences, Medicine, Virginia Tech, Blacksburg, VA, 24061. lwli@vt.edu Inflammation is closely intertwined with human chronic diseases such as neurodegenerative disease 1. However, clear molecular mechanisms that underlie such correlation are not well understood. Toll-interacting-protein (Tollip) is a key homeostatic regulator of innate immunity and inflammation. Our previous studies suggest that Tollip is critically involved in the modulation of micro-autophagy, a key process in clearing cellular debris and maintaining homeostasis 2. Tollip deficient cells have deficiency in the completion of autophagy and may lead to cellular stress 3. To test the functional relevance of our mechanistic findings, we examined the neuronal cell death in Tollip deficient mice. We used the ApoE deficient mouse model due to its correlation of brain cholesterol transport and Alzheimer’s disease. Compared to ApoE knockout mice, the brain tissues of ApoE/Tollip double knockout mice had significantly higher levels of neuronal cell death throughout the brain regions that we sampled, through immuno-histochemical staining analyses. We also observed elevated levels of beta-amyloid, and alpha-synuclein in the ApoE/Tollip double knockout mouse brain. Mechanistically, we observed significant disruption of autophagy completion as measured by elevated levels of p62. Taken together, our data suggest that disruption of autophagy completion due to Tollip deficiency may undermine the proper neuronal cell homeostasis and lead to Alzheimer-like neuro-inflammation. Future strategies that target at restoring Tollip function may hold potential promise in treating related chronic inflammation. References

1. Morris, M.; Gilliam, E. A.; Li, L. Front Immunol 2015, 5, 689. 2. Baker, B.; et al. J Biol Chem 2015, 290, 6670-6678. 3. Yuan, R.; et al. J Pathol 2016, 238, 571-583.

Acknowledgement We appreciate the funding supports from the National Institute of Health.

VirginiaBrainRx Poster Session M 7

COMPARISON OF A MULTI-GENE PHARMACOGENETIC COMBINATORIAL ANALYSIS USING A 27 GENE NGS PANEL WITH SINGLE GENE GENETIC ANALYSIS FOR PREDICTION OF PSYCHIATRIC DRUG RESPONSE. Thomas R. Reynolds* 1, Gregory A. Meyers1, Katherine A. O’Hanlon1, Dai J Li1, Gabriella Wolz1, Robert B. Harris*1, Melissa Russell1, Phil Christian1. 1Next Molecular Analytics, 11601 Ironbridge Road, Suite 101, Chester, Virginia 23831. * Presenters. treynolds@nextmolecular.com rharris@nextmolecular.com Psychiatric disorders cover a wide range of clinical symptoms which are diagnosed and treated with a wide variety of techniques including counseling and other forms of behavioral treatment as well as with one or more psychotropic medications that are aimed at alleviating symptoms of the disorder. Although there are a wide variety of effective medications, treatment of psychiatric disease has been a challenge with as high as 50% of patients experiencing inadequate response to first-line drug therapy (1). Hence, optimal response can require numerous trials of and combinations of medications to achieve optimal response. Genetic testing including Pharmacogenetics (PGX) has recently gained acceptance in psychiatric practices in a variety of ways including help in stratifying patients’ risks of developing a particular disorder, aiding in diagnosis, targeting medication therapy, and optimally dosing medication. Effective use in psychiatric practices of PGX testing may lead to an improved ability to target medications based on the patient’s genetic profile and ability to metabolize and tolerate often potent psychiatric drugs. Proper use of PGX testing in psychiatric practice should lead to an improvement in the efficiency and efficacy of treatment. This approach is particularly important for the treatment of patients with a variety of anti-depressant and other psychiatric drugs because 80% of all drugs are metabolized through one of the CYP450 (cytochrome 450) enzymatic pathways. Patients with variations in these genes often show poor response to a given drug or have serious side effects. Additionally many psychoactive drugs are impacted by brain serotonin transporter and receptor gene variants. Moreover, recent research shows a large range of genetic variation in these genes in the patient population. For example, with exception of CYP2C9, the wild type genotype is not the most common in CYP450 genes. These findings call into question the concept of a 'normal' patient. These studies highlight the need for, and applicability of, PGX testing as an important tool for physicians for drug selection and management (2). A large number of PGX panels have come to the market in the past five years and have been used by physicians to help select treatment options. Most of these panels rely on a “single gene per drug analysis” to predict drug response and have been received with mixed success by physicians, researchers and the insurance industry/CMS. A combinatorial approach to pharmacogenetics has shown significant improvement in the reliability of PGX testing and is based on the analysis of variations in multiple genes which combine to effect an individual’s response to medications. The concept has been pioneered by groups like PharmGKB and the Clinical Pharmacogenetics Implementation Consortium (CPIC). CPIC use a combinatorial approach for many of the psychiatric drugs in common use (3). NEXT has implemented a combinatorial gene analysis approach for assessment of a patient’s response to psychotropic drugs. The NEXT (27 gene) Medicine Management Panel, like other panels such as Assurex, GeneSight (CPGxTM) panel which use a combinatorial genetic algorithm, should perform better in predicting antidepressant treatment outcomes for patients with depression compared with analysis which only use individual genes analyzed separately. In fact, clinical trials performed at the Mayo Clinic using the GeneSight test suggested just this outcome (4). In this poster, we now show results obtained from a number of individuals using the 27 gene Next Generation Sequencing (NGS) panel. The analysis shows a side–by-side comparison of the combinatorial genetic approach to a single gene approach. Differences in these analytical paradigms which have potential to impact drug selection and treatment options are highlighted and compared. References

1. Sinyor M, Schaffer A, Levitt A: Can J Psychiatry. 2010, 55: 126-135. 2. Reynolds, et. al. Int. J. of Computational Biology and Drug Design, 2016 Vol.9, No.1/2, pp.54 - 86 3. https://www.pharmgkb.org/page/cpic 4. Altar, CA, et al. (2015) Pharmacogenomics J advance online publication, February 17, 2015;

doi:10.1038/tpj.2014.85.

VirginiaBrainRx Poster Session M 8

RY-1002: A SPECIFIC AND POTENT LIGAND AT SIGMA RECEPTORS

Richard Young Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219 USA ryoung@vcu.edu Sigma 1 (σ1) and sigma 2 (σ2) receptors have been implicated in a variety of medical conditions: amyotrophic lateral sclerosis (ALS), anxiety, attention/learning/memory dysfunctions (Alzheimer’s disease, ADHD), cancer, depression, HIV, pain, Parkinson’s disease, schizophrenia, stroke and ischemia, traumatic brain injury (TBI) and substance use disorder (SUD)1,2. Although a number of drugs have been promoted as “specific" sigma receptor ligands (for either σ1 and/or σ2), their greatest drawback is the lack of specific affinity for just sigma receptors. Most often, these drugs (a) also interact (potently) with serotonin (5-HT), dopamine (D), norepinephrine (NE) and/or other neurotransmitter receptor sites or (b) have been tested only at sigma sites3. RY-1002 is a prototype drug that was evaluated in 44 receptor binding assays, which indicated specific and potent affinities at σ1 and σ2 receptors [σ1 (Ki = 12 nM) and σ2 (Ki = 28 nM)]. RY-1002 displayed no appreciable affinity at α-adrenergic 1A, 1B, 1D,

2A, 2B, 2C, β-adrenergic 1, 2, 3, benzodiazepine BDZ, peripheral BDZ, dopamine D 1, 2, 3, 4, 5, GABAA, histamine 1, 2, 3, muscarinic 1, 2, 3, 4, 5, opiate DOR, KOR, MOR, serotonin 5-HT 1A, 1B, 1D,

1E, 2A, 2B, 2C, 3, 5A, 6, 7 receptors or transporters DAT, NET, SERT. In comparison and contrast, the reference drugs rimcazole and (+)-3-PPP [(+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine)] were not as specific or as potent at sigma receptors. RY-1002 is structurally distinct from previously reported sigma (or marketed neuropsychiatric) drugs. To date, however, its exact functional interactions at sigma receptors and its potential efficacy in sigma-related therapeutic-like indications have not been determined. Nonetheless, RY-1002 could represent “first-of-its-kind” pharmacotherapy that relies solely on sigma receptor interactions to treat medical conditions. References 1. Guo, l.; Zhen, X. Curr Med Chem. 2015, 22, 989-1003. 2. Su, T-P.; Su, T-C.; Nakamura, Y.; Tsai, S-Y. Trends Pharmacol Sci. 2016, 37, 262-278. 3. Banister, S. D.; Kassiou, M. Curr Pharm Des. 2012, 18, 884-901.

Acknowledgement Patent pending: U.S. and foreign rights available. This technology is available for licensing to industry for further development and commercialization. Inquiries are welcome for chemical and/or pharmacological collaborations. Ki data were generously provided by the National Institute of Mental Health/Psychoactive Drug Screening Program (NIMH/PDSP).

RY-1002 Rimcazole (+)-3-PPP Ki (nM) Ki (nM) Ki (nM)

Sigma1 12 784 27 Sigma2 28 56 58 DAT >10,000 165 593 NET >10,000 >10,000 182 SERT >10,000 356 >10,000 5-HT2B >10,000 511 >10,000 5-HT2C >10,000 >10,000 375 5-HT7 >10,000 >10,000 754 KOR >10,000 >10,000 195 D4 >10,000 >10,000 151

VirginiaBrainRx Poster Session M 9

SYNTHESIS, CHARACTERIZATION, AND IN VITRO/IN VIVO BIOLOGICAL STUDIES OF PERIPHERY SELECTIVE NANOCONJUGATED NAP IN THE TREATMENT OF OPIOID INDUCED CONSTIPATION

Guoyan G. Xu,1 Olga Yu. Zolotarskaya,2 Dwight A. Williams,1,3, Yunyun Yuan,1 Dana E. Selley,3 William L. Dewey,3 Hamid I. Akbarali, 3 Hu Yang,2,4,5 Yan Zhang,1,5* 1. Department of Medicinal Chemistry, Virginia Commonwealth University, 800 E Leigh Street, Richmond, Virginia 23298 2. Department of Chemical and Life Science Engineering, Virginia Commonwealth University, 401 West Main Street, Richmond, Virginia 23284 3. Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North 12th Street, Richmond, Virginia 23298 4. Department of Pharmaceutics, Virginia Commonwealth University, 410 N 12th Street, Richmond, Virginia 23298 5. Massey Cancer Center, Virginia Commonwealth University, 401 College Street, Richmond, Virginia 23298 gxu@vcu.edu Opioid-induced constipation (OIC) is the most common adverse effect associated with the use of opioids, which are however the mainstay for cancer and non-cancer pain management.1 Periphery selective opioid antagonists may alleviate the symptoms of OIC without compromising the analgesic effects of opioids.2 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-pyridyl)acetamido]morphinan (NAP) was recently developed as a peripherally selective mu opioid receptor ligand carrying subnanomolar affinity to the mu opioid receptor and over 100 folds of selectivity over both the delta and kappa opioid receptors, with reasonable oral availability and half-life, and potential to treat OIC.3,4 Nanoparticle-based drug delivery systems are widely considered due to their technological advantages such as good stability, high carrier capacity, and low therapeutic side effects etc.5 Herein we report the nanoparticle supported NAP as a potential treatment for OIC. The in vitro and in vivo results showed the NAP nanoparticles retained potency and selectivity to the mu opioid receptor with improved periphery selectivity over the original lead compound NAP. References 1. Manchikanti, L.; Singh, A. Therapeutic opioids: a ten-year perspective on the complexities and complications of the escalating use, abuse, and nonmedical use of opioids. Pain Physician 2008, 11, S63-88. 2. Yuan, C. S. Clinical status of methylnaltrexone, a new agent to prevent and manage opioid-induced side effects. J Support Oncol 2004, 2, 111-7. 3. Li, G.; Aschenbach, L. C.; Chen, J.; Cassidy, M. P.; Stevens, D. L.; Gabra, B. H., et al. Design, synthesis, and biological evaluation of 6alpha- and 6beta-N-heterocyclic substituted naltrexamine derivatives as mu opioid receptor selective antagonists. J Med Chem 2009, 52, 1416-27. 4. Mitra, P.; Venitz, J.; Yuan, Y.; Zhang, Y.; Gerk, P. M. Preclinical disposition (in vitro) of novel mu-opioid receptor selective antagonists. Drug Metab Dispos 2011, 39, 1589-96. 5. Zolotarskaya, Q. Y.; Yuan, Q.; Wynne, K. J.; Yang, H. Synthesis and Characterization of Clickable Cytocompatible Poly(ethylene glycol)-Grafted Polyoxetane Brush Polymers. Macromolecules 2013, 46, 63-71. Acknowledgement The authors thank NIH/NIDA, National Science Foundation, and Virginia Commonwealth University for financial support.

VirginiaBrainRx Poster Session M 10

SIGMA-1 RECEPTOR IS A NOVEL INHIBITOR OF ENDOPLASMIC RETICULUM DRIVEN INFLAMMATION

Abagail Rosen1, Scott Seki1, Alban Gaultier2 1Department of Pharmacology, University of Virginia, Charlottesville, VA, 22903 2Department of Neuroscience, University of Virginia, Charlottesville, VA, 22903 amr3hu@virginia.edu The endoplasmic reticulum (ER) is classically defined as the site of secreted protein synthesis and

trafficking. Recently, the ER functions have been extended to a plethora of new biological roles, including

inflammation. Activation of the ER stress sensor IRE1 is essential for the normal inflammatory response

to stimuli such as LPS1. However, the mechanisms by which IRE1 regulates inflammation remain unclear.

We have identified a novel role of the ER protein Sigma-1 receptor (S1R) as a critical inhibitor of LPS-

induced cytokine production. Mice lacking S1R succumb quickly to hypercytokinemia after administration

of a sub-lethal dose of LPS. Mechanistically, S1R controls IRE1 endonuclease activity required for

cytokine expression and regulates the biosynthesis of IRE1, without an impact on cytosolic inflammatory

signaling pathways. Our data reveal the contribution of S1R to the restraint of the inflammatory response,

and place S1R as a promising therapeutic target to treat inflammatory disorders. References

1. Martinon F, Chen X, Lee AH, Glimcher LH. TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages. Nat Immunol. 2010;11(5):411-418.

Acknowledgement UVA Pharmacological Sciences Training Grant

VirginiaBrainRx Poster Session M 11

VIRGINIA TECH CENTER FOR DRUG DISCOVERY SCREENING LABORATORY (VTCDDSL)

P. Sobrado1,2*, J. S. Martin del Campo2, N. Vogelaar3 and D.G.I. Kingston 2,3 1Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061 (USA) 2Virginia Tech Center for Drug Discovery, Blacksburg, VA 24061 (USA) 3 Department of Chemistry, Virginia Tech, Blacksburg, VA, 24061 (USA) psobrado@vt.edu A high-throughput screening laboratory has been established at Virginia Tech and is available for use by the scientific community for biochemical and cell-based assays. Instrumentation for the preparation of microtiter plates includes various liquid dispensers and a robot capable of high-accuracy plate-to-plate transfers and plate replication. Detection of assay results can be done using UV-Vis absorbance, fluorescence, time-resolved fluorescence, fluorescence polarization, glow luminescence, or by UPLC. The facility has over 35,000 compounds available for screening use. Results of a high-throughput screening methodology to identify inhibitors of SidA from Aspergillus fumigatus will be presented. SidA is a highly-specific ornithine hydroxylase in the siderophore biosynthetic pathway and is essential for A. fumigatus virulence.1 References 1Hissen, A., et al. The Aspergillus fumigatus Siderophore Biosynthetic Gene sidA, Encoding L-Ornithine N5-Oxygenase, Is Required for Virulence. Infect Immun 73 (2005) 5493-5503.

Acknowledgement The Fralin Life Science Institute at Virginia Tech, The Virginia Tech College of Science, The Virginia Tech College of Agriculture and Life Sciences and NSF- MCB 1021384

VirginiaBrainRx Poster Session M 12

ALLOSTERIC INHIBITORS OF GLYCOGEN SYNTHASE KINASE (GSK-3β) AS POTENTIAL ALZHEIMER’S DISEASE THERAPEUTICS Samuel Obeng1, Yan Zhang1* Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23298 obengs@vcu.edu Alzheimer’s disease (AD) is an age-dependent neurodegenerative disorder that results in the progressive loss of cognition, personal function and independence. At the molecular level, it is characterized by extracellular deposition of β-amyloid (Aβ) plaques and accumulation of intracellular neurofibrillary tangles (NFTs).1 Accumulating evidence has been shown that elevated glycogen synthase kinase 3β (GSK-3β) activity seems to be linked to increased cleavage of amyloid precursor protein (APP) to form Aβ peptides which will oligomerize to form Aβ plaques. Meanwhile GSK-3β is also known to phosphorylate tau protein and hyperphosphorylation of tau may lead to the formation of NFTs.2 Palinurin, a marine natural sesquiterpene has recently been identified as an allosteric inhibitor of GSK-3β, and its binding mode to GSK-3β has been proposed through molecular dynamics (MD) simulation studies.3 Allosteric inhibitors may carry fewer side-effects compared to ATP and substrate competitive inhibitors because allosteric binding sites are typically less conserved. We hereby report molecular modeling studies on palinurin and its analogs as potential GSK-3β allosteric inhibitors in order to design more potent and selective GSK-3β inhibitors as potential treatment for AD. Palinurin, ircinin-1, ircinin-2, and Asc 1-3 (Figure 1) were first docked into the allosteric site of GSK-3β (1PYX) close to amino acid residue of Phe116. The docking results indicated that palinurin, ircinin-1, and ircinin-2 recognized two binding pockets (one close to Lys86 and the other Thr43) linked by a hydrophobic groove. Meanwhile Asc 1-3 seemed to only bind to one of them (the one near Lys86) with their alkyl chain fitting into the hydrophobic groove, which may explain their observed relatively lower inhibitory activity on the target protein. In addition, as the alkyl chain of the ascorbic acid derivatives (i.e. Asc 1-3) was elongated, the inhibitory activity was also improved probably due to increased interaction with hydrophobic residues in the hydrophobic groove. It was observed that in the pocket near Lys86, all the compounds formed hydrogen bonds with Tyr56, Ser118 and Lys86. It was further observed that the furan ring of ircinin-1 formed additional interactions with the backbone nitrogen atom of Tyr117, which may be the reason for its highest inhibitory activity (IC50=0.8) to GSK-3β among all the compounds. It is our belief that all above information collected will help design next generation allosteric inhibitors for GSK-3β as potential AD therapeutics. References 1. Imbimbo, B. P.; Lombard, J.; Pomara, N. Pathophysiology of Alzheimer's disease. Neuroimag. Clin. N. Am. 2005, 15, 727-753. 2. Hooper, C.; Killick, R.; Lovestone, S. The GSK3 hypothesis of Alzheimer’s disease. J. Neurochem. 2008, 104, 1433-1439. 3. Bidon-Chanal, A.; Fuertes, A.; Alonso, D.; Pérez, D. I.; Martínez, A.; Luque, F. J.; Medina, M. Evidence for a new binding mode to GSK-3: Allosteric regulation by the marine compound palinurin. Eur. J. Med. Chem. 2013, 60, 479-489.

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VirginiaBrainRx Poster Session M 13

EXAMINING UNIQUE PROPERTIES OF TBERK8 THAT CAN BE CHEMICALLY EXPLOITED FOR DRUG DISCOVERY

Zachary B. Mackey1*, Ana Lisa Valenciano1 and Giselle M. Knudsen2 1Virginia Tech, VA USA, Fralin Life Science Institute, and Virginia Tech Center for Drug Discovery (VTCDD) Blacksburg, VA (USA) 2University of California San Francisco, CA USA mackeyzb@vt.edu Trypanosoma brucei subspecies are brain pathogens that cause sleeping sickness or Human African Trypanosomiasis (HAT), a meningoencephalitic disease that is 100% fatal if left untreated. T. brucei is categorized as one of the 17 pathogenic organism that cause neglected tropical diseases (NTDs). Like the other NTDs, treatments for HAT are inadequate and have toxic side effects, owing to the need for improved therapeutics against T. brucei. One approach to developing new therapies is to identify novel targets that can be chemically exploited. Tb927.10.5140 is a mitogen-activated protein kinase (MAPK) that is essential for normal proliferation in bloodstream form T. brucei and homologous to the human extracellular-signal regulated kinase 8 (ERK8). We characterized TbERK8 and found that it was inefficient at pulling down the proliferating cell nuclear antigen of T. brucei (TbPCNA). We demonstrated that TbERK8 efficiently phosphorylated TbPCNA and preferred TbPCNA to myelin basic protein, the generic kinase substrate. TbERK8 phosphorylates TbPCNA at Thr202, Ser211 and Ser216 in vitro, but only Thr202 and S211 of TbPCNA are phosphorylated in vivo. These results from our characterization of TbERK8 indicate that the inefficient interaction between TbERK8 and TbPCNA correlated to a relationship between an enzyme and its substrate. In contrast, HsERK8 forms a tight complex with HsPCNA that does not result in HsPCNA phosphorylation. We screened a kinase inhibitor library to test the hypothesis that the active site of TbERK8 can be chemically differentiated and identified several compounds that selectively inhibited HsERK8 over TbERK8. We also identified AZ960, which preferred TbERK8 over HsERK8. This study reveals fundamental differences in the biochemistry of these two ERK8 homologs that have the potential to be exploited for T. brucei-specific inhibitors. References Valenciano, A. L., Ramsey, A. C., and Mackey, Z. B. (2015) Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression. Cell Cycle 14, 674-688

Acknowledgement VTCDD Small Grant VTCDD #119286

AZ960

VirginiaBrainRx Poster Session M 14

SYNTHESIS AND PRECLINICAL EVALUATION OF CYT-21625, AN EXCITING GOLD NANOPARTICLE DRUG DELIVERY SYSTEM FOR PACLITAXEL

David G. I. Kingston*,1, Shugeng Cao1, Jielu Zhao1, Giulio F. Paciotti,2 Lawrence Tamarkin,2 Naris Nilubol3, Kelly Gaskins,3 Kebebew Electron,3 Steven K. Libutti,4 ZiQiang Yuan4

1 Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA, 24061. 2 CytImmune Sciences Inc., 9640 Medical Center Drive, Rockville, MD 20850. 3 Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. 4 Department of Surgery, Albert Einstein College of Medicine, Bronx, NY 10467. dkingston@vt.edu Paclitaxel (Taxol®) (1) is one of the most widely used cytotoxic agents, and it is an effective first-line

treatment for breast, ovarian, lung, and colon cancer1-3. However, it has significant toxicity when systemically administered, including alopecia, nausea and vomiting, myelosuppression,4 and peripheral neuropathy.5 The major problem with paclitaxel is thus a drug delivery problem; if it could be delivered specifically to the tumor, its side effects would be minimized and it would

potentially become even more effective. The novel nanomedicine CYT-21625 has been developed to address the problem of effective paclitaxel delivery. It consists of a gold nanoparticle core, decorated with PEG-thiol for biocompatibility, paclitaxel linked through a self-immolating linker, and recombinant human tumor necrosis factor (rhTNF). The rhTNF has antitumor effects, including tumor targeting, reduction of interstitial fluid pressure,6 and apoptosis of cancer cells,7 but it is highly toxic, and cannot be used systemically. CYT-21625 allows the benefits of thTNF to be obtained without any resulting toxicity because of its linkage to gold nanoparticles. The in vivo antitumor activity of CYT-21625 has been evaluated in anaplastic thyroid cancer (ATC) and pancreatic neuroendocrine tumors. Compared to vehicle control and soluble paclitaxel, mice with metastatic human ATC xenografts (8505C cell line) treated with CYT-21625 for 3 weeks had significantly less tumor burden demonstrated by luciferase activity. References 1. Kingston, D. G. I. In Anticancer Agents from Natural Products; Cragg, G. M., Kingston, D. G. I.,

Newman, D. J., Eds.; CRC Press: Boca Raton, FL, 2012, pp. 123. 2. Saloustros, E.; Mavroudis, D.; Georgoulias, V. Expert Opin. Pharmacother. 2008, 9, 2603. 3. Yared, J. A.; Tkaczuk, K. H. R. Drug Des., Dev. Ther. 2012, 6, 371. 4. Chiorazzi, A. Horiz. Cancer Res. 2011, 45, 1. 5. Rivera, E.; Cianfrocca, M. Cancer Chemother. Pharmacol. 2015, 75, 659. 6. Kristensen, C. A.; Nozue, M.; Boucher, Y.; Jain, R. K. Br. J. Cancer 1996, 74, 533. 7. Goodsell, D. S. The Oncologist 2006, 11, 83. Acknowledgement Partial support of this work by NIH under SBIR award R43 CA119399 is gratefully acknowledged.

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VirginiaBrainRx Poster Session M 15

ATOMISTIC MOLECULAR DYNAMICS SIMULATIONS OF AMYLOID Β-PEPTIDE (1-42): TETRAMER FORMATION, REARRANGEMENT, AND MEMBRANE INTERACTIONS

Anne M. Brown1 and David R. Bevan*,1,2 1Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061 (USA) 2Virginia Tech Center for Drug Design, Virginia Tech, Blacksburg, VA, 24061 (USA) ambrown7@vt.edu The aggregation cascade and peptide-membrane interactions of the amyloid β-peptide (Aβ) are implicated as toxic events in the development and progression of Alzheimer’s disease. Aβ42 forms oligomers and ultimately plaques; these oligomeric species have been hypothesized as the main toxic species contributing to neuronal cell death. To better understand oligomerization events and subsequent oligomer-membrane interactions of Aβ42, united-atom molecular dynamics (MD) simulations were performed to characterize both inter-peptide interactions and perturbation of model membranes by the peptides. MD simulations were utilized to first show formation of a tetramer unit by four separate Aβ42 peptides. Aβ42 tetramers adopted an oblate ellipsoid shape and showed a significant increase in β-strand formation in the final tetramer unit relative to the monomers, indicative of on-pathway events for fibril formation The Aβ42 tetramer unit formed in the initial simulations was used in subsequent MD simulations in the presence of a pure POPC or cholesterol-rich raft model membrane. Tetramer-membrane simulations resulted in elongation of the tetramer in the presence of both model membranes, with tetramer-raft interactions giving rise to rearrangement of key hydrophobic regions in the tetramer and the formation of a more rod-like structure indicative of a fibril-seeding aggregate. Membrane perturbation by the tetramer was manifested in the form of more ordered, rigid membranes, with the pure POPC being affected to a greater extent than the raft membrane. These results provide critical atomistic insight into the aggregation pathway of Aβ42 and indicate putative routes for therapeutic design. References

Acknowledgement The authors thank Advanced Research Computing at Virginia Tech for usage of the BlueRidge supercomputer.

VirginiaBrainRx Poster Session M 16

ABUSE-RELATED EFFECTS OF GABAA RECEPTOR POSITIVE ALLOSTERIC MODULATORS IN AN ASSAY OF INTRACRANIAL SELF-STIMULATION IN RATS. Kathryn L. Schwienteck1, Guanguan Li2, Michael M. Poe2, James M. Cook2, Matthew L. Banks1, and S. Stevens Negus1,*

1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298 2Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, 53211

schwienteckl@vcu.edu GABAA receptor positive allosteric modulators (GABAA PAMs), such as the benzodiazepine diazepam, are used clinically to treat disorders that include insomnia and anxiety; however, therapeutic use of these compounds is limited in part by abuse liability.1 In an effort to improve therapeutic efficacy and safety, novel GABAA PAMs have been developed that vary in efficacy at, and selectively for, GABAA receptor subtypes that contain α1, α2 or α3 subunits.2 Intracranial self-stimulation (ICSS) is one preclinical procedure that has been used to evaluate abuse potential of drugs,3 and this study compared effects on ICSS produced by diazepam (high-efficacy and relatively non-selective), zolpidem (high-efficacy and selective for GABAA receptors containing an α1 subunit), and the compounds JY-XHe-053, XHe-II-053 and HZ-166 (intermediate-efficacy with putative selectivity for GABAA receptors that contain α2/α3 subunits).4 Adult, male Sprague-Dawley rats (n=17), implanted with a bipolar electrode in the medial forebrain bundle, were trained to respond under a fixed-ratio 1 schedule for brain stimulation delivered at 10 different frequencies (56-158 Hz in 0.05 log increments). Under baseline conditions, brain stimulation maintained a frequency-dependent increase in response rates. Diazepam (0.1-10 mg/kg) and zolpidem (0.032-3.2 mg/kg) produced a transient and abuse-related facilitation of ICSS at low doses but primarily depressed ICSS at higher doses. JY-XHe-053 (3.2-32 mg/kg) and HZ-166 (3.2-32 mg/kg) produced significant but weaker and less reliable ICSS facilitation, and XHe-II-053 (3.2-32 mg/kg) had no effect on ICSS. These results are consistent with other clinical and preclinical evidence for abuse potential of diazepam and zolpidem and also suggest that high efficacy and/or selectivity at α1 GABAA receptor subtypes contributes to abuse-related effects of GABAA PAMs in this ICSS procedure. References 1. Woods, J. H., Katz, J. L., & Winger, G. (1987). Abuse liability of benzodiazepines. Pharmacological reviews, 39(4), 251-413. 2. Tan, K. R., Rudolph, U., & Lüscher, C. (2011). Hooked on benzodiazepines: GABAA receptor subtypes and addiction. Trends in neurosciences, 34(4), 188-197. 3. Negus, S.S., & Miller, L. L. (2014). Intracranial self-stimulation to evaluate abuse potential of drugs. Pharmacological reviews, 66(3), 869-917. 4. Fischer, B. D., Licata, S. C., Edwankar, R. V., Wang, Z. J., Huang, S., He, X., Yu, J., Zhou, H., Johnson, E.M., Cook, J.M., Furtmüller, R., Ramerstorfer, J., Sieghart, W., Roth, B. L., Majumder, S., and Rowlett, J.K. (2010). Anxiolytic-like effects of 8-acetylene imidazobenzodiazepines in a rhesus monkey conflict procedure. Neuropharmacology, 59(7), 612-618. Acknowledgements Study funded by NIH grants R01-NS070715, R01-MH096463, and VCU School of Medicine.

VirginiaBrainRx Poster Session M 17

CURCUMIN/MELATONIN HYBRIDS AS NEUROPROTECTIVE AGENTS FOR ALZHEIMER’S DISEASE

John M. Saathoff1, Jeremy E. Chojnacki1, Kai Liu1 and Shijun Zhang1,* 1Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, 23298 szhang2@vcu.edu Alzheimer's disease (AD) is a progressive neurodegenerative disorder that effects ~5.2 million Americans. Current FDA approved medications provide mainly symptomatic relief and there are no agents available to delay or cure this disease. Multiple factors such as amyloid-β (Aβ) aggregates, dyshomeostasis of biometals, oxidative stress, and neuroinflammation have been indicated roles in development of AD. Even though significant advances have been made in understanding the mechanisms leading to AD, the exact etiology of AD still remains elusive. Given AD’s multifactorial nature, a multifunctional strategy of small molecule design would help to identify novel chemical templates. Curcumin (1) displays activity against Aβ plaque formation, inflammatory cytokines, and oxidative stress; while melatonin (2) also reduces Aβ plaque formation and neuroinflammation. Recently our lab has developed hybrid molecules (Figure 1) of curcumin (1) and melatonin (2) that exhibit potent neuroprotections in various AD models. Initial biological characterization of 3 from in vitro assays established that the hybrid strategy is a viable approach in providing novel chemotypes with novel pharmacology. Further modifications identified 4 as a lead compound with potent neuroprotections in MC65 cells (EC50 = 27.60 ± 9.60 nM). Lead compound 4 was displayed antioxidative activity in MC65 cells (IC50 = 68.41 nM). Preliminary mechanistic studies suggested that antioxidative effects might be the major mechanism leading to their neuroprotection, and it is likely that the manifested antioxidative effects of 4 are through interference of the interactions of AβOs with the mitochondria in MC65 cells. Furthermore, 4 has been shown to penetrate the BBB efficiently after oral administration in intact mice, thus confirming that it is orally bioavailable and therapeutically relevant concentrations are attainable in CNS. These results strongly encourage further studies and optimization of 4 in development of more potent analogs. These findings also support the hybridization strategy as a novel design approach to provide effective disease-modifying agents for AD. Herein, we report the optimization of the discovered hybrid lead compound 4 through structure-activity relationship (SAR) studies. Sturcutral modifications at the phenyl ring, olefin double bond, and the 5-postion of the indole ring, led to 15 hybrid molecules. Biological charatcerization demonstarted that the 4-OH of the lead structure is crutical for activity. On the other hand hybrids lacking the olefin still displayed activity, showing that the olefin is not required. Finally, SAR studies also confirmed thatodifcation at the 5-postion of indole ring is tolerated. References 1. Bush, A. I. J. Alzheimers Dis., 2008, 15, 223-240. 2. Hardy, J.; Selkoe, D. J. Science, 2002, 297, 353-356. 3. Kirkitadze, M. D.; Bitan, G.; and Teplow, D. B. J. Neurosci. Res., 2002, 69, 567-577. 4. Selkoe, D. J. Behav. Brain Res., 2008, 192, 106-113. 5. Walsh, D. M.; Selkoe, D. J. J. Neurochem., 2007, 101, 1172-1184. 6. Zhu, X.; Su, B.; Wang, X.; Smith, M. A.; Perry, G. Cell. Mol. Life Sci., 2007, 64, 2202-2210. 7. Chojnacki, J. E.; Liu, K; Yan, X.; Toldo, S.; Selden, T.; Estrada, M.; Rodríguez-Franco, M. I.; Halquist, M. S.; Ye, D.; Zhang S. ACS Chem. Neurosci., 2014, 5 (8), 690-699.

Acknowledgement We thank Professor George M. Martin of the University of Washington for providing the MC65. Similarly, we thank Dr. Hilda Meth (JMS), Commonwealth of Virginia (SZ), and the National Institute of Health/National Institute of Aging (R01AG041161, SZ) for funding.

VirginiaBrainRx Poster Session M 18

DRUG DEVELOPMENT USING THE COLLEGE OF VETERINARY MEDICINE AT VIRGINIA TECH: INCORPORATING QUALITY INTO EARLY PRECLINICAL SAFETY ASSESSMENT

Sandy Hancock* and Marion Ehrich* Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24061 (USA) hancocksk@vt.edu; marion@vt.edu Translational medicine has the goal of bringing research innovation from the laboratory bench to the patient bedside. In the United States, the drug developmental pathway is regulated by the Food and Drug Administration (FDA), which ensures that new therapeutic agents are safe and effective. Due to the nature of research in the discovery phase of drug development, there are no regulatory requirements governing nonclinical product efficacy, proof of concept or early animal testing for toxicity. However, it can be an advantage for academic researchers in drug discovery to consider the early adoption of quality

laboratory standards to assure data confidence and build a strong foundation to support the decision to move a compound forward and invest in further development. This becomes important because preclinical studies required to support FDA drug approval must comply with the Good Laboratory Practice (GLP) regulations to ensure the quality, reliability and accuracy of the laboratory study data submitted to the agency.

Resources and expertise providing valuable support for early preclinical efficacy and safety assessment available at the Virginia-Maryland College of Veterinary Medicine for Virginia Tech’s Center for Drug Discovery include rodent behavioral assessments, pharmacology/toxicology, and pathology. A quality system infrastructure modeled after the GLP regulations ensures the study data are accurate, reliable and reproducible. Early preclinical safety research at the College is led by a board-certified toxicologist and a board-certified veterinary pathologist, each bringing more than 25 years of experience in conducting regulated research in an academic environment. The team also includes a board-certified clinical pathologist, an experienced analytical chemist, and an on-site statistician. Expertise in pharmacokinetics, animal dosing, behavioral observations, sample collection and histopathology provides a strong program for supporting early preclinical work. Unique to the College is an established GLP Program that, for more than 20 years, has supported faculty who partner with industry for work requiring compliance with the GLP regulations. Elements of the GLP quality system, including robust documentation practices, are incorporated into the team’s preclinical study conduct. Merging scientific research expertise with quality laboratory practices early in drug discovery adds value to the study data and can provide potential industry partners with increased confidence to transition a product from an academic laboratory to the commercial developmental pipeline.

VirginiaBrainRx Poster Session M 19

PRECLINICAL ASSESSMENT OF THE 5HT2A INVERSE AGONIST PIMAVANSERIN AND 5HT2C AGONIST LORCASERIN AS CANDIDATE THERAPEUTICS FOR COCAINE USE DISORDER

S. Stevens Negus*1 and Matthew L. Banks1

1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond VA 23227 sidney.negus@vcuhealth.org Cocaine use disorder remains a significant clinical challenge for which there are no medications currently approved by the Food and Drug Administration. Abuse-related effects of cocaine are dependent on activation of mesolimbic dopamine (DA) neurons, and activity of these neurons can be modulated by serotonin (5HT) acting at 5HT2A receptors (to stimulate DA neuron activity) or 5HT2C receptors (to inhibit DA neuron activity). These findings have been interpreted to suggest that treatment with 5HTA inverse agonists or 5HT2C agonists could decrease DA neuron activity, attenuate abuse-related effects of cocaine, and serve as potential therapeutics for cocaine use disorder.1,2 To test this hypothesis, this study evaluated the effects of chronic 7-day maintenance on the 5HT2A inverse agonist pimavanserin (0.32-10 mg/kg/day), the 5HT2C agonist lorcaserin (0.1-0.32 mg/kg/day), and a combination of pimavanserin (0.32 mg/kg/day) + lorcaserin (0.1 mg/kg/day) in a preclinical assay of cocaine self-administration that has been used to assess other candidate medications and non-pharmacological treatments.3,4 Adult male rhesus monkeys (N=4) fitted with chronic IV catheters engaged in daily experimental sessions that permitted choice between increasing cocaine doses (0-0.1 mg/kg/injection) and a single banana-flavored food pellet. Under baseline conditions, cocaine maintains a dose-dependent increase in cocaine choice in this procedure, and previous studies have shown that clinically effective treatments decrease cocaine choice, whereas ineffective treatments do not alter or increase cocaine choice.4 Pimavanserin, lorcaserin, and the pimavanserin + lorcaserin combination all failed to decrease cocaine choice. Rather, both pimavanserin and lorcaserin produced dose-dependent increases in cocaine choice, and the combination of inactive pimavanserin and lorcaserin doses had no effect on cocaine choice. The failure of pimavanserin and lorcaserin to reduce cocaine choice could not be attributed to their anorectic effects, because other clinically effective anorectic medications that function as DA releasers (e.g. amphetamine and phendimetrazine) are effective in reducing cocaine choice.5,6 Rather, pimavanserin and lorcaserin effects on cocaine choice by rhesus monkeys are similar to effects of other medications (e.g. DA receptor antagonists, kappa opioid receptor agonists) that reduce DA signaling and that are clinically ineffective to treat cocaine use disorder.3,7 Results of the present study do not support the utility of 5HT2A inverse agonists or 5HTC agonists as candidate medications to treat cocaine use disorder. References 1. Bubar, M.J.; Cunningham, K.A. Prog. Brain. Res. 2008, 172, 319-346. 2. Howell, L.L.; Cunningham, K.A. Pharmacol. Rev. 2014, 67, 176-197. 3. Negus, S.S. Neuropsychopharmacol. 2003, 28, 919-931. 4. Banks, M.L.; Hutsell, B.A.; Schwienteck, K.L.; Negus, S.S. Curr Treat Option Psych. 2015, 2, 136-150. 5. Negus, S.S.; Henningfield, J. Neuropsychopharmacol. 2015, 40, 1815-1825. 6. Banks, M.L.; Blough, B.E.; Negus, S.S. Drug Alcohol Depend 2013, 131, 204-213. 7. Negus, S.S. Psychopharmacol. 2004 176, 204-213.

Acknowledgement Supported by R01 DA026946. Cocaine, pimavanserin and lorcaserin were provided by the NIDA Drug Supply System via Research Triangle Institute, Research Triangle Park, NC

VirginiaBrainRx Poster Session M 20

PRECLINICAL ASSESSMENT OF LISDEXAMFETAMINE MAINTENANCE AS A CANDIDATE THERAPEUTIC FOR COCAINE USE DISORDER

Amy R. Johnson*1, Matthew L. Banks1, Bruce E. Blough2, Joshua A. Lile3, Katherine L. Nicholson1, S. Stevens Negus1

1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond VA 23227 2Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC 27709 3Departments of Behavioral Science, Psychiatry, and Psychology, U. of Kentucky, Lexington KY 40536 johnsonar24@mymail.vcu.edu In its context as a candidate treatment for cocaine use disorder, amphetamine is considered to be an “agonist” medication analogous to methadone for the treatment of opioid abuse, and amphetamine maintenance has been found to decrease cocaine use in rats, nonhuman primates, human laboratory studies, and clinical trials.1 Lisdexamfetamine is a prodrug for amphetamine that has been approved for treatment of attention-deficit hyperactivity disorder.2 A recent clinical trial of lisdexamfetamine observed a trend to decrease cocaine use at the highest dose that could be tested (approximately 1 mg/kg/day), and the study authors suggested that higher doses might warrant testing.3 The goal of the present study was to evaluate effects of chronic 7-day treatment with lisdexamfetamine treatment (0.32-3.2 mg/kg/day) on cocaine self-administration in nonhuman primates. Four adult male rhesus monkeys were trained to choose between IV cocaine injections (0, 0.043, 0.14, or 0.43 mg/kg/injection) and food pellets (10 1g banana-flavored food pellets) using a procedure designed to match a parallel cocaine-vs.-money choice procedure that has been established for human laboratory studies.4,5 During daily 5h sessions, a single cocaine dose and a single food-reinforcer magnitude were available in 10 30-min trials. During the initial “sample” trial, the available cocaine and food reinforcer were delivered non-contingently. During each of the subsequent nine “choice” trials, responding could produce either the cocaine or food reinforcer. Choice of either cocaine or food increased the response requirement for the chosen reinforcer on the next trial under a concurrent independent progressive-ratio schedule. Under baseline conditions, cocaine maintained a dose-dependent increases in cocaine choice. Effects of lisdexamfetamine were examined on choice between 0.14 mg/kg/injection cocaine and 10 pellets, which maintained an average of approximately 6 cocaine choices and 3 food choices. Lisdexamfetamine decreased cocaine choice in all monkeys and produced variable effects on food choice. In one monkey, the decrease in cocaine choice was accompanied by a complete reallocation of behavior to food choice, which is a desirable treatment outcome. Partial reallocation of behavior to food choice was observed in two monkeys, and a decrease in food choice was observed in the fourth monkey. Overall, lisdexamfetamine dose-dependently decreased cocaine choice without producing a significant change in food choice. These results support further consideration of lisdexamfetamine as a candidate medication for cocaine use disorder and further suggest that doses greater than 1.0 mg/kg/day may be required to decrease cocaine use. References 1. Negus, S.S.; Henningfield, J. Neuropsychopharmacol. 2015, 40, 1815-1825. 2. Blick S.K.A.; Keating, G.M. 2007 Paeditr. Drugs 9, 129-135. 3. Mooney, M.E.; Herin, D. V.; Specker, S.; Babb, D.; Levin, F.R.; Grabowski, J. 2015, Drug Alcohol Depend. 153, 94-103. 4. Johnson, A.R.; Banks, M.L.; Blough, B.E.: Lile, J.A.; Nicholson, K.L.; Negus, S.S. In press, Drug Alcohol Depend 5. Lile, J.A.; Stoops, W.W.; Rush, C.R.; Negus, S.S.; Glaser, P.E.A.; Hatton, K.W.; Hays, L.R. In press, Drug Alcohol Depend Acknowledgement Supported by R01 DA033364.

VirginiaBrainRx Poster Session M 21

PRECLINICAL ASSESSMENT OF THE KAPPA OPIOID RECEPTOR ANTAGONIST NORBINALTORPHIMINE AS CANDIDATE THERAPEUTIC FOR COCAINE USE DISORDER

Blake A. Hutsell*1, Kejun Cheng2, Kenner C. Rice2, S. Stevens Negus1 and Matthew L. Banks1

1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond VA 23227 2Chemical Biology Branch, NIDA & NIAAA, Bethesda, MD 20892 blake.hutsell@vcuhealth.org The dynorphin/kappa opioid receptor (KOR) system has been implicated as one potential neurobiological modulator of the abuse-related effects of cocaine and as a potential target for medications development.1 KOR antagonism can be achieved with selective antagonists such as norbinaltorphimine (nor-BNI) or, less selectively, by combining the mu agonist/kappa antagonist opioid buprenorphine with naltrexone to block buprenorphine’s mu agonist effects (Bup/NTX). Both nor-BNI and Bup/NTX blocked the escalated cocaine intake in rats exposed to a long-access conditions of cocaine self-administration.2,3 However, nor-BNI has failed to block cocaine self-administration in rhesus monkeys,4,5 and Bup/NTX failed to decrease primary measures of cocaine use in a recent clinical trial.6 The present study7 determined effects of norBNI on cocaine self-administration in adult male rhesus monkeys responding under a novel procedure that featured two daily components: (1) a 2 h “choice” component (9-11 am) when monkeys could choose between food pellets and cocaine injections (0-0.1 mg/kg/inj, IV), and (2) a 20 h “extended-access” component (noon-8 am) when cocaine (0.1 mg/kg/inj) was available under a fixed-ratio schedule to promote high daily cocaine intakes. Rhesus monkeys (n=4) were given 14 days of exposure to the choice + extended-access procedure, then treated with nor-BNI (3.2 or 10.0 mg/kg, IM), and cocaine choice and extended-access cocaine intake were evaluated for an additional 14 days. Consistent with previous studies, cocaine maintained both a dose-dependent increase in cocaine choice during choice components and a high level of cocaine intake during extended-access components. nor-BNI did not significantly alter cocaine choice or extended-access cocaine intake. In two additional monkeys, nor-BNI also had no effect on cocaine choice or extended-access cocaine intake when it was administered at the beginning of exposure to the extended-access components. These results are consistent with previous studies showing no effect of nor-BNI on cocaine self-administration4 or cocaine-vs.-food choice5 in rhesus monkeys and with the failure of Bup/NTX to decrease primary measures of cocaine use in humans.6 Overall, these results do not support a major role for the dynorphin/KOR system in modulating cocaine self-administration, nor do they support the clinical utility of selective KOR antagonists, or surrogate approaches such as Bup/NTX, as a pharmacotherapeutic strategy for cocaine addiction. References 1. Koob, G.F. 2014 Eur. J. Pharmacol. 753, 73-87. 2. Wee, S.; Orio, L.; Ghirmai, S.; Cashman, J.R.; Koob, G.F. 2009, Psychopharmacology 205, 565-575. 3. Wee, S.; Vendruscolo, L.F.; Misra, K.K.; Schlosburg, J.E.; Koob, G.F. 2012, Sci. Transl. Med. 4, 146ra110 4. Negus, S.S.; Mello, N.K.; Portoghese, P.S.; Lin, C.E. 1997, J. Pharmacol. Exp. Ther. 282, 44-55. 5. Negus, S.S. 2004, Psychopharmacology 176, 204-213. 6. Ling, W. et al. 2016, Addiction doi: 10.1111/add.13375 7. Hutsell, B.A.; Cheng, K.; Rice, K.C.; Negus, S.S.; Banks, M.L. 2016 Addict. Biol. 21, 360-373. Acknowledgement Supported by R01 DA026946 and T32 DA007027.

VirginiaBrainRx Poster Session M 22

TRIPLE REUPTAKE INHIBITOR ANTIDEPRESSANT CANDIDATES: VARIATIONS ON THE PRC200 SCAFFOLD

Christopher J. Monceaux1, Abdul Fauq2 and Elliott Richelson2, Paul R. Carlier*1 1Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA, 24061. 2Neuropsychopharmacology Laboratory, Mayo Foundation for Medical Education and Research and Mayo Clinic, Jacksonville FL, 32224. cmonceau@radford.edu, pcarlier@vt.edu Approximately 15% of the U.S. population is subject to depression; of those receiving pharmacotherapy, roughly 30-50% are resistant to treatment with serotonin-selective reuptake inhbitors (SSRIs) or serotonin/norepinephrine reuptake inhibitors (SNRIs).1 Combination therapy with SSRIs and bupropion (a norepinephrine/dopamine reuptake inhibitor) in the STAR*D trial suggested improved antidepressant response and reduced side effects relative to monotherapy.2,3 Addition of a dopamine reuptake component to antidepressant therapy may also address anhedonia, and lead to a faster onset of action.1 PRC200 potently inhibits reuptake of all three neurotransmitters4 and like DOV21,9471 is considered a triple reuptake inhibitor. PRC200 was thus explored for its antidepressant-like activity, and was found to dose-dependently decrease immobility in the forced-swim test in rats and in the tail-suspension test in mice.4 Importantly PRC200 did not affect locomotion in mice or rats, and was not self-administered by rats.4 Based on these favorable results an extensive campaign was undertaken to improve the drug-like properties of PRC200 while retaining triple reuptake inhibition. Several compounds with promising pharmacological profiles were identified5 and will be disclosed in this poster. References 1. Skolnick, P.; Basile, A. S. Drug Discovery Today: Therapeutic Strategies 2006, 3, 489-494. 2. Zisook, S.; Rush, A. J.; Haight, B. R.; Clines, D. C.; Rockett, C. B. Biological Psychiatry 2006, 59, 203-210. 3. Trivedi , M. H.; Fava , M.; Wisniewski , S. R.; Thase , M. E.; Quitkin , F.; Warden , D.; Ritz , L.; Nierenberg , A. A.; Lebowitz , B. D.; Biggs , M. M.; Luther , J. F.; Shores-Wilson , K.; Rush , A. J. N. Eng. J. Med. 2006, 354, 1243-1252. 4. Liang, Y. Q.; Shaw, A. M.; Boules, M.; Briody, S.; Robinson, J.; Oliveros, A.; Blazar, E.; Williams, K.; Zhang, Y.; Carlier, P. R.; Richelson, E. J. Pharmacol. Exp. Ther. 2008, 327, 573-583. 5. Richelson, E.; Fauq, A.; Carlier, P. R.; Monceaux, C. J. “Inhibiting Neurotransmitter Reuptake,” PCT/US2014/022684, filed March 25, 2014. Acknowledgement The compounds disclosed in this poster were developed with funding and collaboration from AstraZeneca; all rights to them reverted to Virginia Tech and the Mayo Foundation in 2012. We gratefully acknowledge partnership with Michael Wood and Thomas R. Simpson (AstraZeneca).

VirginiaBrainRx Poster Session M 23

D-CYCLOSERINE FOR TREATMENT OF DEMENTIA, INCLUDING FRONTOTEMPORAL DEMENTIA AND MILD COGNITIVE IMPAIRMENT

Jason McDevitt William and Mary Research Institute, College of William and Mary, Williamsburg, VA 23187 jpmcde@wm.edu This is a 505(b)(2) drug opportunity to treat (i) the rare/orphan disease Frontotemporal Dementia, one of the most common forms of early dementia for which there is no FDA-approved treatments; and/or (ii) the extremely common affliction Mild Cognitive Impairment, the early stage of dementia that afflicts an enormous number of people and often develops into Alzheimer’s Disease. D-cycloserine (“DCS”, an old off-patent, rarely-used drug to treat tuberculosis, approved only as a 250 mg capsule) is an NMDA receptor partial agonist that enhances neuroplasticity and facilitates certain kinds of learning. Recent research1 from the University of Alabama-Birmingham showed potential efficacy for DCS in a rat model of frontotemporal dementia. DCS has shown significant efficacy2 (comparable to donepezil) for Alzheimer’s in some smaller human clinical trials, but a 400-patient study conducted by Searle/Pfizer showed no benefits3. This result is not surprising (the author spent many years as CEO of a startup company that tried to develop the same drug for augmenting talk therapy in treatment of anxiety disorders), as tolerance to DCS starts to develop within one day. Therefore, long-term daily administration of DCS is unlikely to be effective. Moreover, timing of administration can be important. We have pending patents covering novel DCS dosing methods that do not induce tolerance, and also provide additional advantages. For conditions such as FTD or MCI, with expected long-term administration of a drug for many years (e.g., the rest of one’s life), it is critical to develop a drug treatment protocol that is safe, does not shut off crucial biological pathways, does not induce tolerance, and does not create other problems. While DCS has significant CNS side effects when dosed at 1,000 mg (four 250 mg pills) per day for tuberculosis, the side effects under our dosing strategy (a much lower dose given much less frequently) are almost non-existent. The drug will be extremely safe, not terribly expensive, and we expect it to halt or significantly slow disease progression. We are seeking academic and commercial collaborators to help develop the drug, and in particular would like to conduct a pilot human clinical study. References

1. Warmus et al., The Journal of Neuroscience, 3 December 2014, 34(49): 16482-16495 2. Tsai et al., Am J Psychiatry, 1999 Mar; 156(3): 467-469. 3. Fakouhi et al., J Geriatr Psychiatry Neurol, October 1995, vol. 8, no. 4, pp. 226-230

Acknowledgement Thanks to Michael Davis and Kerry Ressler for helpful discussions.

VirginiaBrainRx Poster Session M 24

FABRICATION AND CHARACTERIZATION OF POLYMERIC NANOCARRIERS FOR DRUG DELIVERY AND BIOIMAGING

4. Ami Jo1, Sanem Kayandan2,3, Judy S. Riffle2,3, and Richey M. Davis1,3 1Department of Chemical Engineering, Virginia Tech, Blacksburg, VA 2Department of Chemistry, Virginia Tech, Blacksburg, VA 3The Macromolecules and Interfaces Institute (MII), Virginia Tech, Blacksburg, VA rmdavis@vt.edu The use of nanoparticles in drug delivery can address many of the difficulties faced by traditional methods such as systemic toxicity, transport limitations, and drug solubility. By loading drugs into biocompatible polymeric nanoparticles, drugs that were previously unusable due to their solubility or toxicity can now be used. Because the properties of the polymeric carriers can be manipulated, it is possible now to improve circulatory residence time, targeting, and drug-release depending upon application needs. This work focuses on the engineering of nanoparticles in which drug delivery and bioimaging are combined to enable better quantitation of biodistribution. The bioimaging functionality is imparted by fluorescent dyes and magnetite incorporated into the polymer nanoparticles. Fluorescent dyes such as 6,13-Bis(triisopropylsilylethynyl)pentacene (TIPS pentacene) are ideal for imaging nanoparticle transport and processing in cell cultures and in model animal studies. The MRI contrast effect generated by superparamagnetic magnetite included in the nanoparticles can lead to theranostic particles capable of both on-demand therapeutic drug release and MRI imaging. The latter function is crucial for following the real-time biodistribution of drugs in a patient and for assessing local drug dosage in targeted tissues. We provide an overview of recent work done in our group in fabricating polymeric nanoparticles to deliver small molecule drugs and DNA. Acknowledgement We would like to acknowledge and thank the Institute of Critical Technology and Applied Science (ICTAS) and the Department of Chemical Engineering at Virginia Tech for their financial support.

VirginiaBrainRx Poster Session M 25

RITANSERIN IS A NOVEL INHIBITOR OF DGKα, AND THE CLASSIC DGK INHIBITORS ARE ALSO SEROTONIN RECEPTOR ANTAGONISTS IN VITRO

Salome Boroda1, Maria Niccum1, Vidisha Raje1, Benjamin W. Purow2, Thurl E. Harris1 1University of Virginia, Department of Pharmacology, 2University of Virginia, Department of Neurology, Charlottesville, VA

Email of presenting author: sb9jq@virginia.edu

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Until recently, the only described inhibitors of DGKα were R59022 and R59949. DGKα is a potential therapeutic target in glioblastoma multiforme (GBM) and attenuation of DGKα activity with R59022 induces GBM cell death in vitro and in vivo. However, the pharmacokinetics of R59022 in mice is very poor. Ritanserin is a serotonin (5-HT) receptor antagonist, which is structurally similar to R59022 and has been used in clinical trials for the treatment of schizophrenia with low incidence of adverse side effects. In the present study, we show that Ritanserin is a potent inhibitor of DGKα in vitro with a lower IC50 than R59022. Pharmacologically, these molecules decrease the Vmax for ATP and DAG and increase the affinity of DGKα for ATP. While recognizing Ritanserin as a DGK inhibitor, we also find that R59022 and R59949 function as 5-HT receptor (5-HTR) antagonists. Despite their activity against 5-HTRs, we show that both R59022 and Ritanserin are functional DGKα inhibitors in cultured cells and increase phosphorylation of downstream targets of protein kinase C (PKC). In conclusion, we describe Ritanserin as a viable compound for pharmacologically targeting DGKα while also recognizing that commercially available DGK inhibitors may have significant effects on 5-HTRs. References 1Dominguez CL, Floyd DH, Xiao A, Mullins GR, Kefas BA, Xin W, et al. Diacylglycerol kinase alpha is a critical signaling node and novel therapeutic target in glioblastoma and other cancers. Cancer.Discov. 2013;3:782-97. 2de Chaffoy de Courcelles D, Roevens P, Van Belle H, Kennis L, Somers Y, De Clerck F. The role of endogenously formed diacylglycerol in the propagation and termination of platelet activation. A biochemical and functional analysis using the novel diacylglycerol kinase inhibitor, R 59 949. J.Biol.Chem. 1989;264:3274-85 3 de Chaffoy de Courcelles DC, Roevens P, Van Belle H. R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. J.Biol.Chem. 1985;260:15762-70. 4Wiesel FA, Nordstrom AL, Farde L, Eriksson B. An open clinical and biochemical study of ritanserin in acute patients with schizophrenia. Psychopharmacology (Berl) 1994;114:31-8.

Acknowledgements We want to thank Tyler Basting for assistance with statistics and Dr. Bryan Roth the use of his National Institute of Mental Health (NIMH) psychoactive drug screening program (PDSP) at the University of North Carolina, Chapel Hill. This research was supported by the National Institutes of Health (N.I.H) R01 1R01DK101946 (to T.E.H), the N.I.H R01 CA180699 (to B.W.P), the N.I.H. R01 CA189524 (to B.W.P), the N.I.H Pharmacology graduate student training grant and the University of Virginia Wagner Graduate Fellowship.

VirginiaBrainRx Poster Session M 26

USING PEPTIDES FROM α-SYNUCLEIN TO LOCATE AND DISASSEMBLE LEWY BODIES.

Denver R. Heitger, Heather R. Lucas* Department of Chemistry, Virginia Commonwealth University, Richmond, VA, 23284 (United States) heitgerdr@vcu.edu α-Synuclein has been a major focus in the scientific community for its pivotal role in the formation of Lewy bodies, which are insoluble proteinaceous aggregates found in the brain of Parkinson’s disease (PD) patients. With PD being the second most common neurological disease, there has been strong motivation to understand the molecular mechanisms associated with PD and to specifically elucidate how α-synuclein forms these aggregates.1 Recent studies have shown that a specific sequence, 68GAVVTGVTAVA78, located in the NAC region of α-synuclein acts as the toxic core that promotes α-synuclein aggregation.2 This study and others have led the Lucas group to focus on using this aggregation-prone sequence to benefit rather than harm. Tethering an anti-aggregate or cleaving agent to these recognition peptides would allow for a more site specific interaction with the protein.3 Pre-treatment may hinder aggregation, hence mitigating Lewy body formation, while placing the tethered cleaving agent in proximity to α-synuclein fibrils may result in proteolysis, thus achieving breakdown of these toxic aggregates. Results from this work may lead to a targeted route for attenuating and/or preventing the symptoms associated with PD. References 1. Lucas, H. R.; Lee, J. C.; Emerging Role for Copper-Bound α-Synuclein in Parkinson’s Disease Etiology. In Brain Diseases and Metalloproteins; D. R. Brown, Ed.; Pan Stanford, 2013, pp 295-316. 2. Rodrigues, J. A.; Ivanova, M. I.; Cascio, D.; Reyes, F. E.; Shi, D.; Sangwan, S.; Guenther, E. L.; Johnson, L. M.; Xhang, M.; Jiang, L.; Arbing, M. A.; Nannenga, B. L.; Hattne, J.; Whitelegge, J.; Brewster, A. S.; Messerschmidt, M.; Boutet, S.; Sauter, N. K.; Gonen, T.; Eisenberg, D. S. Nature 2015, 525,486-492. 3. Chu, T.; Li, Q.; Qiu, T.; Sun, Z.; Hu, Z.; Chen, Y.; Zhao, Y.; Li Y. Mol. BioSyst. 2014, 10, 3081-3085.

Acknowledgement This work was supported by Virginia Commonwealth University, College of Humanities and Sciences and the Department of Chemistry.

VirginiaBrainRx Poster Session M 27

NOVEL ALLOSTERIC MODULATOR BINDING SITES IN GLUN1/2A AND GLUN1/2B SUBUNIT CONTAINING NMDA IONOTROPIC GLUTAMATE RECEPTORS Douglas Bledsoe1, Ceyhun Tamer2, Ivana Mesic2, Christian Madry2, Heinrich Betz3, Bodo Laube2, Blaise Costa1* 1 Virginia College of Osteopathic Medicine, Department of Biochemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24060. 2 Technical University, Darmstadt, Germany. 3 Max-Planck- Institute for Institute for Medical Research, Heidelberg, Germany. *bcosta@vcom.vt.edu Abstract: N-methyl-D-Aspartate (NMDA) subtype of glutamate receptors play a crucial role in normal brain function, pathogenesis of schizophrenia and neurodegenerative disorders. A functional hetero-tetrameric NMDAR channel is formed by two glycine binding GluN1 and two glutamate binding GluN2 subunits (of which there are four types 2A-2D). In the present study, we have made the following amino acid point mutations in the GluN1 subunit ligand binding domain (LBD) based on the crystal structure [1]: N521A, N521D (Site I); K531A, Y535A (Site II); E781A (Site III). These GluN1 mutants were co-expressed with the wild type GluN2A or 2B subunits in Xenopus oocytes and the receptor function was studied by using Two Electrode Voltage Clamp (TEVC) technique. These mutations were also incorporated into the crystal structure of GluN1/2A and GluN1/2B LBD to study the changes in intersubunit interactions. The results show that mutations at Site-I&II affects the agonist efficacy, activation and deactivation kinetics of GluN1/2A receptor, whereas these properties are mostly unaltered in mutant GluN1/2B receptors when compared to the wild type. Conversely, E781A mutation at Site-III alters the agonist efficacy in GluN1/2B receptor but not in GluN1/2A. Furthermore, the GluN1 Site-I&II interaction was found to be critical for channel activation and desensitization in GluN1/2A receptors, but not for GluN1/2B. These results are consistent with the structural analysis of GluN1/2 LBD. These distinct intersubunit interactions at LBD validate our recent identification [2] of the novel allosteric modulator [3] binding sites. References:

1. Furukawa, H., et al., Subunit arrangement and function in NMDA receptors. Nature, 2005. 438: p. 185-192.

2. Kane, L. T. and B. M. Costa, Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study. J. Mol Graph Model, 2015. 61: p. 204-213.

3. Costa, B. M., et al., A novel family of negative and positive allosteric modulators of NMDA receptors. J Pharmacol Exp Ther, 2010. 335(3): p. 614-621.

VirginiaBrainRx Poster Session M 28

VIRGINIA’S ALZHEIMER’S DISEASE AND RELATED DISORDERS COMMISSION: RESEARCH-RELATED GOALS OF THE DEMENTIA STATE PLAN

Patricia Slattum1, Devin Bowers2, Constance Coogle3

1School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia, 23298 (USA) pwslattu@vcu.edu 2Department for Aging and Rehabilitative Services, Richmond, Virginia 23229 (USA) devin.bowers@dars.virginia.gov 3Virginia Center on Aging, Virginia Commonwealth University, Richmond, Virginia 23298 (USA) The Alzheimer’s Association estimates 140,000 individuals aged 65 and older are living with Alzheimer’s Disease in Virginia and this number is projected to increase by nearly 36% by 2025 (190,000). In response to the growing number of individuals and families affected by dementia the Alzheimer’s Disease and Related Disorders Commission (www.alzpossible.org) was established in 1982 by the Virginia General Assembly and has maintained 15 active members who are appointed and meet quarterly. The Commission produced Virginia’s first Dementia State Plan in 2011 and updates the plan every four years. In addition to advising the Governor and General Assembly, the Commission works to achieve the five goals of the Dementia State Plan through the efforts of four work groups. Goals two and five support using dementia related data to improve public health outcomes and expanding resources for dementia-specific translational research and evidence-based practices. The Data and Research Work Group is dedicated to achieving these goals. Accomplishments of the work group include increasing funding for the Alzheimer’s and Related Diseases Research Award Fund by just over 60% to $125,000 annually, supporting the collection of the 2012 and 2013 cognitive decline module of the Behavioral Risk Factor Surveillance System, and the publication of a report including available federal and state-level data relevant to dementia. Moving forward, the Data and Research Work Group will provide training for Institutional Review Boards on the changes involving informed consent due to the signing of HB 337 and will seek opportunities to promote collaboration among dementia researchers in Virginia. This poster presentation will provide an overview of the history and function of the Commission and the Data and Research Work Group, highlight available funding opportunities in Virginia for dementia research, and provide an opportunity for researchers to express an interest in collaborating or networking, which will inform and influence the activities of the work group moving forward. References Alzheimer’s Association. 2016 Alzheimer’s Disease Facts and Figures. Alzheimer’s & Dementia 2016; 12(4).

VirginiaBrainRx Poster Session T 1

LINEAGE-SPECIFIC METABOLIC PROPERTIES AND VULNERABILITIES OF T CELLS IN THE DEMYELINATING CENTRAL NERVOUS SYSTEM Scott M. Seki1-3, Max Stevenson1, Abagail M. Rosen1, Lelisa Gemta4, Timothy N.J. Bullock4, Alban Gaultier1,# 1Center for Brain Immunology and Glia, Department of Neuroscience 2Graduate Program in Neuroscience 3Medical Scientist Training Program 4Department of Pathology, School of Medicine, University of Virginia, Charlottesville, VA 22908, USA sms4ad@virginia.edu Multiple Sclerosis (MS) is a disease characterized by immune-mediated destruction of central nervous system (CNS) myelin. Current MS therapies aim to block peripheral immune cells from entering the CNS.1,2 While these treatments limit new inflammatory activity in the CNS, no treatment effectively prevents longterm disease progression and disability accumulation in MS patients. One explanation for this paradox is that current therapies are ineffective at targeting smoldering immune responses already present in the CNS. To this end, we sought to understand the metabolic properties of T cells mediating ongoing inflammation in the demyelinating CNS. Using experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, a well-studied model of MS, we discovered that CD4+ and CD8+ T cells that invade the EAE CNS are highly glycolytic, and furthermore, that glycolysis is essential for inflammatory response s to myelin. Unexpectedly, we found that an inhibitor of glyceraldehyde-3 phosphate (GAPDH), 3-bromopyruvic acid (3-BrPa), robustly blocks IFN-γ but not IL-17A production in T cells isolated from the EAE CNS – further studies on purified T cell populations suggest this is due to T helper lineagespecific responses to 3-BrPa. Finally, in transfer models of EAE, transient blockade of GAPDH in a fully activated encephalitogenic T cell population significantly delays onset and severity of disease. These data are the first to demonstrate the metabolic properties of T cells in the demyelinating CNS in vivo, and provide insight into the therapeutic potential of pharmacological targeting of T cell glycolysis in MS. References

1. Tramacere, I., C. Del Giovane, G. Salanti, R. D'Amico, and G. Filippini. 2015. Immunomodulators and immunosuppressants for relapsing-remitting multiple sclerosis: a network meta-analysis. The Cochrane database of systematic reviews 9: CD011381

2. Castro-Borrero, W., D. Graves, T. C. Frohman, A. B. Flores, P. Hardeman, D. Logan, M. Orchard, B. Greenberg, and E. M. Frohman. 2012. Current and emerging therapies in multiple sclerosis: a systematic review. Therapeutic advances in neurological disorders 5: 205-220

Acknowledgement The authors are supported by NIH grants R01 NS083542 (A.G.), T32 GM008328 (S.M.S) and T32 GM007267 (S.M.S).

Figure 1: 3-Bromopyruvic Acid

VirginiaBrainRx Poster Session T 2

INTEGRATED STRATEGY FOR DRUG DISCOVERY WITH MONOAMINE TRANSPORTER LIGANDS: APPLICATION TO STRUCTURE-ACTIVITY STUDIES WITH 4-METHYLAMPHETAMINE ANALOGS

Sakloth F1,2*, Eltit JM3, Solis, Jr. E3,4, Partilla JS4, Ruchala I3, Schwienteck K2, Baumann MH4, De Felice LJ3, Glennon RA1, Negus SS2

1Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, 23298 2Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298 3Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, 23298 4Designer Drug Research Unit, Intramural Research Program, NIDA/NIH, Baltimore, MD, 21224 saklothft@vcu.edu Subtle structural changes to the amphetamine scaffold can produce large changes in (1) drug potency and selectivity at transporters for dopamine, norepinephrine and serotonin (DAT, NET and SERT, respectively), (2) drug function as a substrate or blocker at these transporters, and (3) physiological and behavioral drug effects.1 Structure-activity relationships for amphetamine derivatives have been targeted in designer drug syntheses, and because modulation of monoaminergic neurotransmission constitutes the basis for treatment of several mental conditions,1 amphetamine derivatives have also been examined as candidate treatments for a range of disorders including drug abuse. 2-4 The present study used an integrated battery of in vitro and in vivo techniques to study the impact of N-alkyl chain length on effects produced by four N-alkyl substituted 4-methyl amphetamines (N-R 4-MA): N-methyl, N-ethyl, N-propyl and N-butyl 4-MA . Three complementary in vitro assays were used to evaluate the potency and function of each compound as a substrate or blocker at DAT, NET and SERT. First, compounds were tested in monoamine-release- and uptake-blocking protocols in rat brain synaptosomes. Second, Ca2+-imaging was used to monitor drug effects on HEK293 cells that co-expressed a monoamine transporter and a voltage-gated Ca2+ channel. Drugs that produced sufficient depolarization to open Ca2+ channels and increase fluorescence of a Ca2+ dye were categorized as substrates, whereas drugs that blocked fluorescence elicited by known substrates were categorized as blockers. Lastly, two-electrode voltage clamp was used to evaluate drug effects on membrane potential of DAT- or SERT-expressing oocytes. Substrates and blockers induced currents that would lead to membrane depolarization or hyper polarization, respectively. Consistent results were obtained in all three assays. N-methyl 4-MA was a substrate with similar potencies at all three transporters. N-Ethyl 4-MA was a blocker at DAT but remained a substrate at NET and SERT with slightly reduced potency. N-Propyl 4-MA was a blocker at both DAT and NET but remained a substrate at SERT with further reduction in potency at all three transporters. N-Butyl 4-MA had low potency at all transporters. Behavioral studies evaluated abuse-related effects of each drug in an assay of intracranial self-stimulation in male Sprague Dawley rats. N-Methyl and N-ethyl 4-MA produced weak abuse-related effects, whereas N-propyl and N-butyl 4-MA did not. In summary, these studies indicated that increased chain length at the N-position resulted in: a) a loss in potency at DAT, NET and SERT, b) a shift in function from substrate to blocker, with this shift occurring at shorter chain lengths for DAT than for NET or SERT, and c) progressive loss of dopamine-mediated abuse-related effects. Additionally, these studies illustrate a research strategy that can be used in drug discovery to identify monoamine transporter ligands with novel neurochemical and behavioral effects and potential to serve as novel therapeutics.

References 1. Liechti, M. Swiss. Med. Wkly. 2015, 145, w14043. 2. Seeman, P.; Madras, B. K. Mol. Psychiatry 1998, 3, 386-396. 3. Blough, B. E.; Landavazo, A.; Partilla, J. S.; Baumann, M. H.; Decker, A. M.; Page, K. M.; Rothman, R.

B. ACS Med. Chem. Lett. 2014, 5, 623-627. 4. Rothman, R. B.; Baumann, M. H. Ann. N. Y. Acad. Sci. 2006, 1074, 245-260. Acknowledgement: Supported by R01 DA033930

VirginiaBrainRx Poster Session T 3

DEFORMABLE SURFACE AND CONTOUR MODELS FOR NEUROANATOMICAL MODELING SUITABLE FOR NEUROSURGERY PLANNING AND SIMULATION Michel A. Audette1*, Rabia Haq1, Tanweer Rashid1, Sharmin Sultana1, Matthew Ewend2, Jason E. Blatt2,

Yueh Lee2, Justin Cetas3, Priscilla Pang3, Julie Pilitsis4, Anthony Costa5, John Butman6.

1Dept. Modeling, Simulation and Visualization Engineering, Old Dominion University, Norfolk, VA, 23529. 2 Depts. Neurosurgery and Radiology, University of North Carolina, Chapel Hill, NC, 27514. 3 Dept. Neurosurgery, Oregon Health and Science University, Portland, OR, 97239. 4 Albany Medical Center, Albany, NY 12208. 5 Dept. Neurosurgery, Mount Sinai Hospital, New York, NY. 6 Dept. Radiology and Imaging Sciences, NIH Clinical Center, Bethesda, MD 20892. *email: maudette@odu.edu This poster summarizes on-going work on anatomical modeling for applications to neurosurgery simulation and planning, which involve collaborations with several neurosurgeons and neuroradiologists. All of this research involves adaptations to the Simplex model, which is deformable mesh consisting of vertices linked by edges with a prescribed connectivity, whose dynamic behavior is governed by a Newtonian physics of the mesh vertices in relation to underlying structures found in 3D images. An N-Simplex is characterized by N+1 connectivity, whereby a 2-Simplex is a surface mesh with 3-connectivity, displayed in black in figure 1a (and leading to geometric duality with a triangulated surface mesh), while a 1-Simplex is a 3D contour with 2-connected vertices, except at end points, as in figure 1b. The Newtonian expression for vertex motion features acceleration and damping terms as well as internal and external forces; the internal force imposes prior assumptions such as continuity, while external forces nudge each vertex towards salient image structures, namely gradients for the surface mesh and points of locally tubular image structure for the contour model. Also, this mesh features topological operators that enable us to precisely control the resolution of the mesh: refining or decimating the 2-Simplex surface mesh faces by adding or deleting edges, or endowing the 1-Simplex contour with smaller or longer edges as needed. The 2-Simplex is well published, as pioneered by Delingette for single surfaces and extended by Gilles for multiple surfaces; the 1-Simplex was introduced by us for identifying curvilinear structures. Our refinements of the 2-Simplex deformable surface model include the following: i) a multi-surface model that imbed shape statistics, which enables us to segment both vertebrae and intervertebral discs from MRI data at once, with no spatial overlap; ii) a surface mesh with shared vertices for representing a deep-brain atlas for surgery planning for a MRI-compatible deep-brain stimulation robotic assistant. Our 1-Simplex deformable contour model emphasizes modeling of the skull base for both planning and simulation, with a particular attention to minimally supervised identification (segmentation) of cranial nerves from T2-weighted MRI data. The latter project will lead to descriptive modeling of the skull base anatomy for simulation and planning, in conjunction with the development underway of digital atlases of the brainstem and cranial foramina.

(a) (b) (c) Figure 1. Applications for Simplex deformable models. (a) Shape-aware multi-surface 2-Simplex model (clockwise from top left): mesh topology in black (dual triangulated mesh in blue), multi-surface spine segmentation, and modes of shape variation extracted from 10-patient population of L1 vertebra. (b) 1-Simplex contour model: (clockwise from top left) mesh topology, nerve III centerline identified from two end points and validation based on linearization of expert-provided points in red. (c) Multi-surface 2-Simplex with shared faces for deep brain atlas personalization and intraoperative warping (clockwise from

VirginiaBrainRx Poster Session T 3

top left): MRI-compatible deep-brain stimulation robot, multi-surface representation of deep brain atlas, axial slice of brain MRI and overlaid deep brain atlas.

VirginiaBrainRx Poster Session T 4

OREXIN PROJECTIONS TO THE BASAL FOREBRAIN CONTRIBUTE TO ATTENTIONAL PROCESSING: IMPLICATIONS FOR ALZHEIMER’S DISEASE Joshua A. Burk*1 and Jim R. Fadel2 1Department of Psychology, College of William and Mary, Williamsburg, VA 23187 (USA) 2Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, SC 29208 (USA) jabur2@wm.edu Orexins are neuropeptides that are released from neurons located within the lateral hypothalamus and contiguous perifornical area and are thought to regulate several motivated behaviors, including hunger and sleep. More recently, orexinergic activation has been observed following exposure to several drugs of abuse, suggesting that this system may contribute to addiction-related motivated behaviors. Our work has focused on how orexins gain access to circuitry related to attentional processing, in order to guide and execute motivated behaviors toward relevant environmental stimuli. Basal forebrain corticopetal cholinergic neurons are known to be necessary for normal attentional processing and degenerate in Alzheimer’s Disease. Orexins are a major input onto basal forebrain cholinergic and noncholinergic neurons. Using a measure of sustained attention in rats that requires discrimination of short visual signals from trials when no signal is presented, we have shown that systemic or direct infusions into the basal forebrain of the orexin-1 receptor antagonist, SB-334867, disrupt attentional performance. Moreover, infusions of orexin A into the lateral ventricle enhance task performance during conditions when attentional demands are increased by distracter presentation. Our recent work has suggested that the effects of orexin A are mediated via basal forebrain cholinergic neurons as immunotoxic lesions that destroy only noncholinergic neurons do not affect performance in this sustained attention task, whereas immunotoxic lesions of basal forebrain corticopetal cholinergic neurons disrupt sustained attention task performance. Collectively, our findings indicate that orexins can regulate attentional performance via projections onto basal forebrain cholinergic neurons.

Acknowledgement Supported by R01AG050518 and R01AG030646.

VirginiaBrainRx Poster Session T 5

DISCOVERY AND MECHANISMS OF SMALL MOLECULE INHIBITORS AGAINST AMYLIN AMYLOIDOSIS IN THE PANCREAS AND THE BRAIN

Bin Xu*,1,2,3, Ling Wu1, Paul Velander1, Keith Ray1, Rich Helm1, Shijun Zhang4, David Bevan1 1Department of Biochemistry, 2Center for Drug Discovery, 3Translational Obesity Research Center, Virginia Tech, Blacksburg, VA 24061. 4Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298. binxu@vt.edu Epidemiological and clinical studies showed significant association between type 2 diabetes (T2D) and the risk for neurodegeneration (including Alzheimer’s disease). Amylin is a 37-residue peptide hormone that is co-secreted with insulin from pancreatic -cells. It is highly amyloidogenic and amylin amyloid deposition in the pancreas are hallmark features of T2D. Recent clinical studies reported that amylin plaques were deposited in the brain of diabetic patients, but not in healthy controls. We performed cell-based studies, demonstrating that amylin amyloid is highly toxic to multiple cell lines, including pancreatic -cells INS-1 and neuronal cells SH-SY5Y and Neuro2A. From a 384-well fluorescence-based screening of a collection of natural compounds used in complementary and alternative medicine, we identified multiple potent inhibitors, including rosmarinic acid (RA) (estimated to be 200 nM in apparent IC50). These lead compounds disaggregate amylin fibrils from transmission electron microscopic observations and significantly reduce amylin-induced cytotoxicity. Dissecting the functional groups of these natural compounds, we identified that the catechol groups contributed vitally to amyloid inhibition in more than a dozen catechol-containing compounds. Compounds with multiple catechol groups exhibited additive effects. From systematic analog analyses, we demonstrated, for the first time to our knowledge, that the vicinal hydroxyl groups of the catechol groups played key functions in amyloid inhibition. We provided further mass spectrometric evidence that incubating several of these catechol-containing inhibitors with amylin leads to (1) covalent adducts consistent with Schiff bases as a proposed mechanism of amyloid inhibition; (2) loss of the amino-terminal lysine on peptide as a potentially novel mechanism of inhibition. The inhibition effects by these compounds were demonstrated in computational simulation analyses, providing additional non-covalent mechanisms. Lastly, we will present our newly developing, powerful technical platform using combinatorial scaffolds libraries of synthetic small molecules and peptides in combination with high throughput screening for rapid lead compound discoveries for future applications to protein amyloid diseases in the brain, such as Alzheimer’s disease and Parkinson’s disease. References

1. Westermark P, Andersson A, and Westermark GT. (2011) Islet amyloid polypeptide, islet amyloid, and diabetes mellitus. Physiol Rev. 91: 795-826. 2. Jackson K, Barisone GA, Diaz E, Jin LW, DeCarli C, and Despa F. (2013) Amylin deposition in the brain: A second amyloid in Alzheimer disease? Ann Neurol. 74: 517-526. 3. Despa F and Decarli C. (2013) Amylin: what might be its role in Alzheimer's disease and how could this affect therapy? Expert Rev Proteomics.10: 403-405. 4. Wu L, Velander P, Brown AB, Zhang S, Bevan DR, Xu B. Rosmarinic acid, catechol-containing natural product, is a potent inhibitor of amylin amyloidosis. submitted, 2016.

Acknowledgement This work is in part supported by Virginia Tech new faculty start-up funds, the National Institute of Food and Agriculture of USDA, Alzheimer’s and Related Diseases Research Award Fund from Virginia Center on Aging (Award No. 16-1), Diabetes Action Research And Education Foundation, and Virginia Tech Center for Drug Discovery.

VirginiaBrainRx Poster Session T 6

MiRNA EXPRESSION IN BASOLATERAL AMYGDALA ASSOCIATED WITH STRESS-INDUCED CHANGES SLEEP: IMPLICATIONS FOR STRESS VULNERABILITY AND RESILIENCE AND MODELLING PTSD Brook L. Williams1, Laurie L. Wellman1, Mairen E. Fitzpatrick1, Olga Y. Hallum1, Amy M. Sutton1, Mayumi Machida1, Nagaraja S Balakathiresan2, Manish Bhomia2, Min Zhai, Radha2 K Maheshwari2 and Larry D. Sanford1* 1Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk, Virginia, 23507 (USA) 2Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814 williaBL@evms.edu Post-Traumatic Stress disorder (PTSD) is characterized by hypervigilance, flashbacks, intrusive thoughts, and sleep disturbances after experiencing or witnessing a traumatic event1. However, not all people who experience traumatic stress develop PTSD indicating that individual vulnerability or resilience can be an important determinant of stress outcome2. In animal models, intensely stressful events as well as memories of those events can be followed by changes in sleep, especially rapid eye movement sleep (REM)1. REM is implicated in the processing of emotional events suggesting that these variations may reflect differences in coping associated with individual resilience and vulnerability1. Functional studies have demonstrated that the amygdala, a region central to current concepts of PTSD, is a major mediator of the effects of stress and stressful memories on sleep3. Inactivation of the basolateral amygdala (BLA) with the GABAA receptor agonist muscimol (MUS) before shock training blocked the post training reductions in REM sleep often seen in control rats3. This suggests that BLA plays a role in sleep regulation after a stressful event, however the molecular mechanisms underlying the effects of BLA on sleep are currently unknown. MicroRNAs (miRNAs) are small (~22 nucleotide), endogenous, non-coding RNAs that posttranscriptionally regulate gene expression4. In this project, we examined REM sleep as a potential outcome measure of stress resilience and vulnerability, and we identified miRNAs in BLA that were altered in putative stress vulnerable (Vul) and resilient (Res) rats. Adult, male Wistar strain rats (n=25) were prepared for sleep recording and microinjections into BLA. The rats were then randomly assigned to a non-stressed, home cage control group (HC; n=6) or received ST (20 trials, 0.8 mA, 0.5 sec, 1 min ISI). Based on relative amounts of REM in the first two h following ST, the rats were designated as Vul (lower REM) or Res (higher REM). One subgroup of rats was sacrificed at two h after ST (ST-Vul (n=5) and ST-Res (n=5)) and another subgroup received microinjections of MUS prior to ST and was sacrificed at two h after context re-exposure (CTX-Vul (n=5) and CTX-Res (n=4)). HC rats were sacrificed at the same circadian time. Global miRNA expression profiles for the BLA were generated using TaqMan® MicroRNA Array cards. StatMiner® software was used to assess expression of miRNAs in BLA across groups. Compared to HC rats, BLA miRNA expression profiling showed 12 and 3 miRNAs were differentially expressed in the ST-Vul and ST-Res rats, respectively. Additionally, compared to HC rats, 19 and 7 BLA miRNAs were altered in the CTX-Vul and CTX-Res rats, respectively. These data demonstrate that directionally different alterations in REM following stress are associated with differential miRNA expression in BLA, a region critical for forming fear memory and mediating its effects on sleep. Further studies are needed to determine the potential role of REM in mediating emotional memories and analyses of the altered miRNAs by bioinformatics and system biology approaches will be needed to delineate their role(s) in mediating stress outcomes. However, these data demonstrate that it should be possible to incorporate stress vulnerability and resilience into animal models of PTSD. References 1. Wellman, L. Individual Differences in Animal Stress Models: Considering Resilience, Vulnerability, and the Amygdala in Mediating the Effects of Stress and Conditioned Fear on Sleep. SLEEP, 2016 (In Press). 2. McEwen, B., Recognizing resilience: Learning from the effects of stress on the brain. Neurobiology of Stress, 2015 1: p1-11 3. Wellman, L., The basolateral amygdala determines the effects of fear memory on sleep in an animal model of PTSD. Exp. Brain Res., 2014 232(5): p1555-1565 4. Sun, E. MicroRNAs: Small Molecules with big roles in neurodevelopment and diseases. Exp. Neurol., 2015 268:46-53 Acknowledgement

VirginiaBrainRx Poster Session T 6

This work was supported by funding from DMRDP (RKM) and MH64827 from the NIH (LDS).

VirginiaBrainRx Poster Session T 7

THE EFFECTS OF ETHANOL ON ACUTE AND CHRONIC PRESCRIPTION OPIOIDS IN MICE Joanna C. Jacob and William L. Dewey* Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298 jacobjc@vcu.edu Opioids such as morphine and oxycodone have long been clinically used for pain management, but their use is limited by high abuse potential. Prescription opioid abuse has been recognized as a national epidemic since 2011, with estimates of 100 individuals dying each day from opioid overdose in the United States (CDC, 2011). Chronic opioid use, whether for clinical or recreational purposes, is often coupled with the use of other substances, such as ethanol. Dangerous drug interactions are known to occur with opioids and ethanol, yet the mechanisms are not understood. Post-mortem data have shown that individuals who consumed ethanol along with opioids had much lower blood concentrations of the opioid than individuals who overdosed on opioids alone1. This led to the investigation and hypothesis that ethanol reverses tolerance to at least some opioid effects, such as analgesia and respiratory depression, ultimately leading to overdose and death. Previously, we found a significant and dose-dependent reversal of morphine-induced analgesic tolerance by ethanol, as measured by warm-water tail withdrawal in Swiss Webster mice2. This finding was consistent in subsequent experiments in mice made tolerant to oxycodone and hydrocodone via repeated subcutaneous injections, where it was found that 1 g/kg ethanol .i.p fully reversed those tolerances. We then investigated the potency of ethanol to reverse oral oxycodone tolerance, which is the primary route of administration in man. 1 g/kg ethanol i.p. did not reverse oral oxycodone, however, 2 g/kg oral ethanol produced a partial reversal to oral oxycodone in mice. We further investigated the development of cross-tolerance between oral oxycodone and morphine, where significant cross-tolerance was observed in mice chronically treated with oral oxycodone and challenged with 10mg/kg s.c. morphine. Ethanol at 2g/kg po fully reversed the observed cross-tolerance between oral oxycodone and morphine. These data suggest that oral ethanol is more effective in reversing the cross-tolerance between oral oxycodone and morphine than it is in reversing the tolerance to oral oxycodone alone. References 1. Thompson, J.G.; Vanderwerf, S.; Seningen, J.; Carr, M.; Kloss, J.; Apple, F.S. J. Anal. Toxicol. 2008, 32(8), 673-679. 2. Hull, L.C.; Gabra, B.H.; Bailey, C.P.; Henderson, G.; Dewey, W.L. J. Pharmacol. Exp. Ther. 2013, 345(3), 512-519. Acknowledgements This work was funded by RO1 DA036975 and DA007027.

VirginiaBrainRx Poster Session T 8

DIACYLGLYCEROL KINASES REGULATE PAIN AND ITCH BY ALTERING RECEPTOR SIGNALING IN PERIPHERAL NOCICEPTORS

Victoria B. Bartsch*1 and Mark J. Zylka1,2 1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 2Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 brings@email.unc.edu Chronic pain and chronic itch severely impair daily function and quality of life.1 Current treatments lack sufficient efficacy and have serious side effects.2,3 Characterizing the mechanisms by which painful and pruritogenic (itch-causing) stimuli are processed may reveal candidate molecules to target with novel pharmacological treatments. Pain- and itch-sensing neurons (nociceptors) whose cell bodies lie in the dorsal root ganglia (DRG) detect noxious stimuli in peripheral tissues and transduce those signals to the central nervous system. Inflammatory mediators released after injury both activate and sensitize nociceptors, shifting patients toward a state of chronic sensitivity that can persist after the injury heals.4 These inflammatory mediators activate several receptor classes, including Gαq-protein-coupled receptors (Gαq-GPCRs). Gαq-GPCR activation produces diacylglycerol (DAG), which activates protein kinase C (PKC). PKC phosphorylates and desensitizes GPCRs, terminating their signaling.5 Diacylglycerol kinase (DGK) phosphorylates DAG, attenuating activation of PKC. Therefore, DGK potentially regulates PKC-mediated modulation of receptor activity. RNA and protein expression implicate two DGK isozymes (eta, η, and iota, ι) as important for nociception. We have shown that DGKη and DGKι enhance Gαq-GPCR-induced calcium activity via decreased PKC activity.5 We hypothesize that these kinases regulate GPCR signaling in nociceptors and modulate pain sensitization and itch. Using DRG neurons cultured from our DGKη and DGKι knockout mice, we have found that DGKη or DGKι loss alters calcium responses to pain- and itch-causing compounds that signal through Gαq- GPCRs expressed in nociceptors. Furthermore, behavior analyses demonstrate that our knockout mice have differential responses to pruritogens. Baseline mechanical and thermal pain sensitivities are slightly diminished by DGK loss, and we expect that inflammatory and neuropathic injury will amplify these differences. In vitro signaling experiments coupled with in vivo behavioral assays reveal the role of DGKη and DGKι in nociception and itch and their potential as targets for analgesic therapies. References 1. Institutes of Medicine Committee on Advancing Pain Research, C., and Education, Relieving pain in America: a blueprint for transforming prevention, care, education, and research. 2011: The National Academies Press. 382. 2. Ong, C.K.S., P. Lirk, C.H. Tan, and R.A. Seymour, An evidence-based update on nonsteroidal anti-inflammatory drugs. Clinical Medicine and Research, 2007. 5(1): p. 19-34. 3. Benyamin, R., A.M. Trescot, S. Datta, R. Buenaventura, R. Adlaka, N. Sehgal, S.E. Glaser, and R. Vallejo, Opioid complications and side effects. Pain Physician, 2008. 11(2 Suppl): p. S105-120. 4. Julius, D. and A.I. Basbaum, Molecular mechanisms of nociception. Nature, 2001. 413(6852): p. 203-210. 5. Rittiner, J.E., V.E. Brings, and M.J. Zylka, Overexpression of diacylglycerol kinase eta enhances Gαq-coupled G protein-coupled receptor signaling. Molecular Pharmacology, 2014. 85(5): p. 800-810.

Acknowledgement Funding provided by NIH/NINDS grant 5R01NS081127-03: “Lipid kinase regulation of pain signaling and sensitization”

VirginiaBrainRx Poster Session T 9

RE-PURPOSING LICENSED DRUGS FOR PROPHYLACTIC AND THERAPEUTIC USE IN TREATMENT OF ZIKA VIRUS DISEASE

Robert W. Malone1,2*, Jill Glasspool Malone1,2 1. Atheric Pharmaceutical, LLC, Scottsville, VA, USA 24590. 2. Class of 2016, Harvard Medical School Global Clinical Scholars Research Training Program, Boston, MA 02115. robert.malone@atheric.com Purpose of the study: Antiviral agent development. Clinical care for Zika virus infection is supportive, and there are no prophylactic or therapeutic drugs, vaccines, or other biologicals licensed for use to prevent or treat Zika virus infection and disease. A Zika virus threat assessment and evaluation of medical countermeasure development options has been completed; re-purposing existing licensed drugs was identified as the most efficient strategy for rapid development of licensed medical countermeasures suitable for prevention, treatment, or containment of the pathogen1. Methods/summarized description of the project: Hypothesis-driven high throughput re-purposed drug screening. An iterative multi-step drug selection and screening algorithm was established; 1) drug targets involving inhibition of virus-host cell interactions were identified, 2) compounds with significant clinical pharmacokinetic and safety data (preferably including in pregnancy) which inhibit the pathways were selected, 3) selected pharmaceuticals were tested for inhibition of Zika virus infection and replication using multiple cell types and Zika viral isolates, 4) pharmaceuticals with single-digit micromolar to nanomolar IC50 and CC50/IC50 ratios consistent with anti-viral specificity were selected for subsequent development as prophylactic and therapeutic anti-Zika drug candidates. Results: Re-purposed licensed drugs with anti-Zika activity which are safe for use in pregnancy. Four general compound have been identified and corresponding compounds have been screened. Multiple re-purposed drugs have been identified which meet selection criteria for subsequent development. We are now finishing screening combinations of approved drugs to increase IC50 and CC50/IC50 ratios (individually safe in pregnancy) with animal studies designed and commencing. Conclusion: Hypothesis-driven high throughput re-purposed drug selection can expedite identification of emerging infectious disease medical countermeasure candidates. A summary of the pathways targeted, drugs identified, with the top lead candidates being selected, and viral inhibition results obtained will be presented. Discussion: Zika infection of the recipient host requires viral envelope protein binding and particle uptake into susceptible cells, is mediated by multiple specific receptors, and triggers transcriptional activation of Toll-like receptor 3 (TLR3), RIG-I, MDA5, interferon stimulated genes including OAS2, ISG15, and MX1, and beta interferon2. Zika virus subsequently exploits autophagy to facilitate uptake and replication3, and pharmacologic manipulation of Zika infected cells with 3-Methyladenine (3-MA), an inhibitor of autophagosome formation, strongly reduces viral copy numbers in infected fibroblasts2. Based on prior studies, autophagy of Zika virus has been postulated as playing a key role in the pathogenesis of Zika-associated primary microcephaly4. Pharmacological mechanisms of currently licensed drugs include inhibition of autophagy and broad-spectrum cathepsin B-mediated inhibition of viruses which require endosomal acidification5. References 1. Malone RW, Homan J, Callahan MV, Glasspool-Malone J, et al. Zika Virus: Medical Countermeasure Development Challenges. PLoS Negl Trop Dis. 2016 Mar 2;10(3):e0004530. PMID: 26934531 2. Robert W Malone, Zachary Klase, Svetlana Khakhina, Michael V Callahan, Jill Glasspool-Malone, et al. Zika Fetal Neuropathogenesis: Etiology of a Viral Syndrome PLoS Negl Trop Dis. 2016 (submitted) bioRxiv doi: http://dx.doi.org/10.1101/050674 3. Carneiro LA, Travassos LH. Autophagy and viral diseases transmitted by Aedes aegypti and Aedes albopictus. Microbes Infect. 2016. doi: 10.1016/j.micinf.2015.12.006 PMID: 26774331. 4. Tetro JA. Zika and microcephaly: Causation, correlation, or coincidence? Microbes Infect. 2016. doi: 10.1016/j.micinf.2015.12.010 PMID: 26774330. 5. Zilbermintz L, Leonardi W, Jeong SY, Sjodt M, et al. Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting. Sci Rep. 2015 Aug 27;5:13476. doi: 10.1038/srep13476. PMID: 26310922

VirginiaBrainRx Poster Session T 10

DISCOVERING POTENT PTP4A3 PHOSPHATASE SMALL MOLECULE INHIBITORS FOR COLORECTAL CANCER

Kelley E. McQueeney*1, Joseph M. Salamoun2, Kasia Handing3, Adam Boulton3, Elizabeth R. Sharlow1, John H. Bushweller3, Wladek Minor3, Peter Wipf2 and John S. Lazo1. 1Department of Pharmacology, University of Virginia, Charlottesville, VA 22908; 2Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260; 3Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908 kem5ee@virginia.edu Although there has been significant progress in understanding colorectal cancer pathogenesis, the 5-year patient survival rate in the United States of ~60% underlines the need for better treatment options when surgical resection and current therapies are not sufficient. In recent years, protein tyrosine phosphatase 4A3 (PTP4A3) has garnered significant interest and extensive validation as an anticancer target1,2. The Oncomine database documents PTP4A3 is in the top 1% of the most highly expressed colorectal cancer genes with >650 tumor samples. Moreover, it has been noted that increased PTP4A3 expression correlates with increased tumor invasiveness and poor patient prognosis3,4. We synthesized the thienopyridone JMS-631-050, which is the most potent published PTP4A3 small molecule inhibitor. Using 6,8-difluoro-4-methylumbelliferyl phosphate as an artificial substrate, we found JMS-631-050 non-competitively inhibited recombinant PTP4A3 in vitro phosphatase activity with an IC50 value of 138 nM. We have generated the first NMR spectra of PTP4A3 in complex with JMS-631-050 to more thoroughly characterize the nature of their interaction. Preliminary analysis of NMR data suggests that JMS-631-050 binds to a region of the phosphatase outside of the active site, which we hypothesize decreases protein flexibility and proximity of the general acid to the catalytic cysteine. This binding site is also consistent with molecular docking simulation studies. Recently, we discovered a new, more potent thienopyridone analog, JMS-631-053, which inhibited recombinant PTP4A3 in vitro phosphatase activity with an IC50 value of 8 nM and showed limited or no inhibition against other phosphatases at concentrations < 1 M. Both JMS-631-050 and JMS-631-053, which computationally have drug-like properties, reduced cellular migration of mouse colorectal tumor cells and prevented the formation of cohesive spheroids in an extracellular matrix phenocopying results observed with PTP4A3 null colorectal cancer cells. JMS-631-050 and JMS-631-053 should be valuable reagents for further validation of PTP4A3 as a cancer target as well as potential leads for drug discovery. References 1. Saha S, Bardelli A, Buckhaults P, Velculescu VE, Rago C, St Croix B, Romans KE, Choti MA, Lengauer C, Kinzler KW, Vogelstein B. A phosphatase associated with metastasis of colorectal cancer. Science. 2001;294(5545):1343-6. 2. Ooki A, Yamashita K, Kikuchi S, Sakuramoto S, Katada N, Waraya M, Kawamata H, Nishimiya H, Nakamura K, Watanabe M. Therapeutic potential of PRL-3 targeting and clinical significance of PRL-3 genomic amplification in gastric cancer. BMC Cancer. 2011;11(122). 3. Kato H, Semba S, Miskad UA, Seo Y, Kasuga M, Yokozaki H. High expression of PRL-3 promotes cancer cell motility and liver metastasis in human colorectal cancer: a predictive molecular marker of metachronous liver and lung metastases. Clin Cancer Res. 2004;10(21):7318-28. 4. Kim NW, Chu CW, Ahn TS, Kim CJ, Jung DJ, Son MW, Bae SH, Lee MS, Kim CH, Baek MJ. Correlation between liver metastases and the level of PRL-3 mRNA expression in patients with primary colorectal cancer. J Korean Soc Coloproct. 2011;27:231-6. Acknowledgement NIH R21CA191944, the Farrow Fellowship, and NCI Cancer Center Support Grant P30 CA44579.

VirginiaBrainRx Poster Session T 11

CHARACTERIZATION OF THE EFFECT OF LORCASERIN, A 5-HT2C AGONIST, ON THE ACUTE PHARMACOLOGICAL EFFECTS OF OXYCODONE Kumiko Lippold*1 and William Dewey1 1Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA, 23298. lippoldkm@vcu.edu Prescription opioids are important therapeutic agents for the treatment of pain that with chronic use, produce tolerance, dependence, and/or addictions. With prescription opioid abuse emerging as a major epidemic, there is a need to develop new approaches for minimizing the risk of opioid abuse and dependence and as a result of this, reducing unnecessary overdose-related deaths. The underlying neurobiological mechanisms of these conditions suggest a modulatory role of the serotonergic system in the pharmacological effects of opiates. Lorcaserin is a selective agonist, at the 5-HT2C serotonergic receptor, that was approved by the FDA for the treatment of obesity but recently has been investigated as a potential treatment for drug abuse and dependence. Several preclinical studies have demonstrated the efficacy of lorcaserin to reduce the abuse-related effects of oxycodone, as well as opioid dependence behaviors in animals dependent on either morphine or heroin1-3. The studies described herein evaluated the role of lorcaserin in the acute pharmacological effects of oxycodone by using tests of antinociception and motor activity. We observed that lorcaserin altered the acute antinociceptive effects of oxycodone, in a divergent manner, in models of spinally-mediated and supraspinally-mediated thermal nociception. Pretreatment with lorcaserin yielded a trend towards potentiation in the tail immersion test (a spinally-mediated reflex) and in the hot plate assay (a test of supraspinally-mediated pain), there was an attenuation of the antinociceptive effects of oxycodone. Additionally, lorcaserin dose-dependently attenuated oxycodone-induced locomotor activity in mice and exploratory activity in naïve mice. In a similar fashion, lorcaserin also suppressed stretching activity in mice that received an intraperitoneal injection of acetic acid. These data suggest a contrasting role of the 5-HT2C receptor in effects elicited by oxycodone in the brain and spinal cord. References 1. Wu X, Pang G, Zhang Y-M, Li G, Xu S, Dong L et al. Neurosci Lett 2015; 607: 23–28. 2. Zhang G, Wu X, Zhang YM, Liu H, Jiang Q, Pang G et al. Neuropharmacology 2015; 101: 246–

254. 3. Fox RG, Stutz SJ, Comer S, Haney M, Anastasio NC, Moeller FG et al. Drug Alcohol Depend

2015; 156: e73. Acknowledgement We gratefully acknowledge the expertise of David Stevens and NIDA funding DA 036975.

VirginiaBrainRx Poster Session T 12

REVERSAL OF PRESCRIPTION OPIOID TOLERANCE Maciej M. Gonek* 1 and William L. Dewey1

1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond VA, 23298. gonekmm@vcu.edu The United States is in the midst of an unprecedented opioid overdose epidemic with over 120 people dying every day. Most individuals who become tolerant and dependent on opioids, including the prescription opioids oxycodone and hydrocodone, co-abuse other substances. The most widely co-abused drug is ethanol but other depressants such as diazepam and other benzodiazepines are also co-abused. Several studies suggest that benzodiazepines may play a role in as much as 80% of unintentional overdose deaths involving opioids.1-5 Post mortem examination of individuals who have died from the chronic administration of opioids and ethanol had lower blood levels of opioids than individuals who overdosed on opioids alone.5 Similar results have not been reported in individuals co-abusing diazepam. Following published results on morphine tolerance reversal7, we found that tolerance developed to oxycodone and hydrocodone in the mouse tail immersion assay. The administration of 0.5 mg/kg and 2 mg/kg diazepam, doses that have no observable behavioral effect on their own, fully reversed antinociceptive tolerance to oxycodone and hydrocodone, respectively. There was no potentiation of acute opioid effects. Conversely, opioid tolerance in the stretching assay, a measurement of visceral pain, was not reversed by diazepam. Further research into the differences of opioid tolerance mechanisms can lead to new insights for the development of drugs that decrease the risk of opioid overdosing and the development of drugs to relieve severe pain with limited tolerance. References 1. Kintz P. Deaths involving buprenorphine: a compendium of French cases. Forensic Sci Int. 2001; 121(12):65–69. [PubMed: 11516889] 2. Ernst E, Bartu A, Popescu A, Ileutt KF, Hansson R, Plumley N. Methadone-related deaths in Western Australia 1993–99. Aust N Z J Public Health. 2002; 26(4):364–370. [PubMed: 12233959] 3. Wolf BC, Lavezzi WA, Sullivan LM, Flannagan LM. Methadone-related deaths in Palm Beach County. J Forensic Sci. 2004; 49(2):375–378. [PubMed: 15027564] 4. Oliver P, Keen J. Concomitant drugs of misuse and drug using behaviours associated with fatal opiate-related poisonings in Sheffield, UK, 1997–2000. Addiction. 2003; 98(2):191–197. [PubMed: 12534424] 5. Jones JD, Mogali S, Comer SD. Polydrug abuse: a review of opioid and benzodiazepine combination use. Drug Alcohol Depend. 2012; 125(1–2):8–18. [PubMed: 22857878] 6. Darke S and Hall W (2003) Heroin overdose: research and evidence-based intervention. J Urban Health 80:189–200. 7. Hull LC, Gabra BH, Bailey CP, Henderson G, Dewey WL. Reversal of morphine analgesic tolerance by ethanol in the mouse. J Pharmacol Exp Ther [Internet]. 2013;345(3):512–9. Acknowledgement We gratefully acknowledge the expertise of David Stevens and NIDA funding DA 036975 and DA007027.

VirginiaBrainRx Poster Session T 13

2-AMINODIHYDROQUINAZOLINES: A MECHANISTICALLY NOVEL CLASS OF ANTIDEPRESSANTS

Kavita A. Iyer1, Xiaolei Pan2, Malaika D. Argade1, Osama I. Alwassil1, Douglas H. Sweet2 and

Małgorzata Dukat1,* 1Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, 23219 (USA). 2Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, 23219 (USA). iyerka@vcu.edu

The WHO estimates ~350 million people suffer from depression,1 and many are refractory to current drug treatment. Available antidepressants block the uptake-1 mechanism to increase synaptic concentration of the biogenic amines serotonin (5-HT) and/or norepinephrine (NE).2 Uptake-1, a high-affinity/low-capacity transporter, includes the serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters.3 An alternative mechanism, uptake-2, a low-affinity/high-capacity transporter, identified as the organic cation transporter 3 (OCT3), has been implicated to play a role in depression.4,5 The Dukat laboratory found that 2-amino-6-chloro-3,4-dihydroquinazoline (A6CDQ) produces antidepressant-like action (ED50 = 0.23 mg/kg)6 in the mouse tail suspension test (TST) and inhibits hOCT3 (IC50 = 3.91 ± 2.35 μM). We formulated initial structure-activity-relationships for the 2-aminodihydroquinazolines at hOCT3 and generated the first homology model of hOCT3. The positional isomers, 5-Cl (A5CDQ), 7-Cl (A7CDQ), 8-Cl (A8CDQ), and the des-Cl (ADQ) analog were synthesized in order to determine if the presence and/or position of the Cl group is important for hOCT3 inhibition. ADQ (IC50 = 12.16 ± 3.35 μM) was found to be >10-fold less potent than A5CDQ (IC50 = 0.88 ± 0.19 μM), suggesting the position of the Cl group was not critical since the positional isomers (A5CDQ, A6CDQ and A7CDQ)

were found to possess comparable inhibitory potencies at hOCT3. Our molecular modeling studies suggested that the 6-Cl group of A6CDQ is involved in hydrophobic interactions at hOCT3; therefore, the lipophilic character of the substituent might be important. A CH3 group has similar lipophilic character as a Cl group but opposite electronic character. The 6-CH3 (A6MDQ, IC50 = 2.00 ± 0.40 μM) analog possesses comparable inhibitory potency as A6CDQ and supports our modeling studies. To further explore the effect of bulk at the 6 position, the other 6-halo-substituted (i.e., 6-F, 6-Br, 6-I) analogs were synthesized. A5CDQ, a positional isomer of A6CDQ, as well as A6MDQ and A6FDQ displayed antidepressant-like action in the mouse TST. 2-Aminodihydroquinazolines represent a promising class of potential antidepressants with a novel mechanism of action. References 1. World Health Organization (WHO). http://www.who.int/mental_health/management/depression/en/

(accessed April 26, 2016). 2. Williams, D. A. Antidepressants. In Foye’s Principles of Medicinal Chemistry; 7th ed.; Lemke; T. L.;

Williams, D. A.; Roche, V. F.; Zito S. W., Eds.; Lippincott Williams & Wilkins: Baltimore, 2013; pp 570 -631.

3. Eisenhofer, G. Pharmacol. Ther. 2001, 91, 35-62. 4. Daws, L. C. Pharmacol. Ther. 2009, 121, 89-99. 5. Wu, X.; Kekuda, R.; Huang, W.; Fei, Y.-J.; Leibach, F. H.; Chen, J.; Conway, S. J.; Ganapathy, V. J.

Biol. Chem. 1998, 273, 32776-32786. 6. Dukat, M.; Alix, K.; Worsham, J.; Khatri, S.; Schulte, M. K. Bioorg. Med. Chem. Lett. 2013, 23, 5945-

5948.

Acknowledgement Presidential Research Quest (PeRQ) Fund, Lowenthal Award 2013-2015 (K.A.I.)

R = 5-Cl (A5CDQ) = 6-Cl (A6CDQ) = 7-Cl (A7CDQ) = 5-Cl (A5CDQ) = 8-Cl (A8CDQ) = 6-CH3 (A6MDQ) = 6-F (A6FDQ)

VirginiaBrainRx Poster Session T 14

DOWNREGULATION OF MYELIN GENE EXPRESSION IN THE ENTORHINAL CORTEX OF FINGOLIMOD TREATED MICE

J.L. Jurmain1, M.F. Miles1 1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, 23298 (United States) jurmainjl@vcu.edu In 2014, approximately 17.0 million people in the U.S. reported being dependent on or abusing alcohol within the past year.1 Studies in human and mouse models of alcoholism suggest a potential role for myelin in the development of alcohol use disorders (AUDs) and their associated pathologies. Myelin genes are downregulated in the frontal cortex of alcohol dependent patients and in mouse models.2 Studies in C57BL/6J and DBA/2J mice show correlations between basal myelin gene expression and behavioral responses to acute ethanol exposure.3 Fingolimod (FTY720), a sphingosine-1-phosphate (S1P) analogue, is an FDA approved drug for the treatment of relapsing multiple sclerosis, a demyelinating disorder. Several literature reports on FTY720 suggest a direct role for S1P receptors in demyelination and remyelination. A recent study of immune-deficient mice revealed a group of genes in hippocampus whose expression changed with FTY720 treatment.4 This gene set significantly overlapped with a gene set from our laboratory5 whose expression was altered by chronic intermittent ethanol exposure in mice, and several myelin genes were present in both gene sets. We hypothesized that this overlap suggested a common mechanism between the effects of FTY720 and chronic ethanol treatment on myelin gene expression, and this mechanism may be useful in elucidating a treatment for the prevention or management of myelin-related pathologies associated with ethanol consumption. In a pilot study to determine the effects of oral FTY720 treatment on healthy, ethanol-naïve mice, using qRT-PCR we observed no significant changes in myelin gene expression in medial prefrontal cortex, caudate putamen, or nucleus accumbens with FTY720 treatment. Strikingly, however, two of the myelin genes investigated (Mbp and Plp) were significantly downregulated in the entorhinal cortex of FTY720 treated mice. Downregulation of these genes by FTY720 treatment was also strongly correlated with the downregulation of NFκB and Tlr4 expression, two genes involved in inflammatory signaling pathways. This downregulation of NFκB and Tlr4 is consistent with the proposed mechanism of action for FTY720 in treating multiple sclerosis, but further studies are necessary to determine the mechanism by which FTY720 downregulates myelin gene expression in the entorhinal cortex and the consequences of this downregulation for myelin protein levels and myelin structure. Rather than functioning as a treatment or preventative for demyelination associated with ethanol use, these results suggest that FTY720 may aid in revealing a molecular mechanism by which that demyelination occurs. References 1. SAMHSA. Behavioral Health Trends in the United States. (Center for Behavioral Health Statistics and

Quality, 2015). 2. Lewohl, J. M. et al. Gene Expression in Human Alcoholism: Microarray Analysis of Frontal Cortex.

Alcoholism: Clinical and Experimental Research 24, 1873–1882 (2000). 3. Kerns, R. T. et al. Ethanol-responsive brain region expression networks: implications for behavioral

responses to acute ethanol in DBA/2J versus C57BL/6J mice. J. Neurosci. 25, 2255–2266 (2005). 4. Hait, N. C. et al. Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear

extinction memory. Nature Neuroscience 17, 971–980 (2014). 5. Wolen, A. R. et al. Genetic Dissection of Acute Ethanol Responsive Gene Networks in Prefrontal

Cortex: Functional and Mechanistic Implications. PLoS ONE 7, (2012). Acknowledgement Funded by NIAAA grants U01AA016667, P20AA017828, P50AA022537 and R01AA020634 to M.F. Miles

VirginiaBrainRx Poster Session T 15

INVESTIGATING THE ROLE OF THE ALPHA4BETA2 NICOTINIC RECEPTOR POSITIVE ALLOSTERIC MODULATOR DESFORMYLFLUSTRABROMINE IN NICOTINE DEPENDENCE Asti B. Jackson 1, Deniz Bagdas1, M. Imad Damaj 1 * 1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, 23298 Jacksonab2@vcu.edu Tobacco dependence remains one of the leading causes of preventable death1. The current pharmacological interventions available have modest efficacy; therefore, there is a dire need for a better understanding of the mechanisms and substrates involved in nicotine dependence to design and develop more effective smoking cessation aids2. Nicotine, the primary addictive component in tobacco products, exerts its rewarding effects primarily through nicotinic acetylcholine receptors (nAChR)3 located in the mesolimbic pathway. The heteromeric α4β2* nAChR plays an important role in mediating nicotine reward and reinforcement4. This receptor may be a potential target as a pharmacological intervention of smoking behavior. The utility of positive allosteric modulators (PAMs) for the α4β2 receptor as smoking cessation aids has recently gained attention with the finding that desformylflustrabromine (dFBr) dose-dependently attenuates nicotine i.v. self-administration in rats5. The α4β2 PAM dFBr not only reduced nicotine reinforcement but did not substitute for nicotine suggesting it may not have abuse liability on its own5 . Despite this report, dFBr in nicotine dependence is relatively understudied. Therefore, this current study focuses on the effect of dFBr in a mouse model of nicotine withdrawal. The α4β2 PAM dFBr (0.1 and 1 mg/kg, s.c.) in a dose-related manner reversed nicotine withdrawal signs (somatic signs anxiety-like behaviors and hyperalgesia) in nicotine-dependent mice. These preliminary findings suggest that dFBr may be a potential pharmacological tool used to attenuate some nicotine withdrawal signs. Further studies will be conducted to elucidate the effects of this compound in nicotine dependence. References 1. Benowitz, N. Clinical Pharmacology of Nicotine: Implications for Understanding, Preventing, and Treating Tobacco Addiction. Clin. Pharmacol. Ther. 83, 531–541 (2008). 2. Casella, G., Caponnetto, P. & Polosa, R. Therapeutic advances in the treatment of nicotine addiction: present and future. Ther. Adv. Chronic Dis. 1, 95–106 (2010). 3. Changeux, J. P. et al. Brain nicotinic receptors: structure and regulation, role in learning and reinforcement. Brain Res. Brain Res. Rev. 26, 198–216 (1998). 4. Picciotto, M. R. et al. Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391, 173–7 (1998). 5. Liu, X. Positive allosteric modulation of α4β2 nicotinic acetylcholine receptors as a new approach to smoking reduction: evidence from a rat model of nicotine self-administration. Psychopharmacology (Berl). 230, 203–13 (2013).

Acknowledgement: This research was supported by the National Institute on Drug Abuse R01DA032933

VirginiaBrainRx Poster Session T 16

DISCOVERY OF BIOACTIVE LIPID NETWORKS BY CHEMICAL PROTEOMICS AND METABOLOMICS

1Ku-Lung Hsu*1 1Department of Chemistry & Pharmacology, University of Virginia, Charlottesville, 22904 kenhsu@virginia.edu The human genome contains a vast number of enzymes that lack functional annotation. As a result, our basic understanding of enzymatic pathways that underlie human physiology and pathology has so far remained largely incomplete. Consequently, molecular profiling technologies have emerged as an unbiased means to globally examine the biochemical makeup of cells and tissues and identify genes and proteins associated with pathophysiological states. In contrast, “systems biology” methods for the functional characterization of enzymatic pathways in native biological systems are still lacking. To address this problem, I will describe our efforts aimed at integrating activity-based protein profiling (ABPP) with functional proteomic/metabolomics methods to map novel bioactive lipid networks involved in inflammation. I will describe the discovery and development of the first selective and in vivo-active small-molecule inhibitors for the putative 2-arachidonoylglycerol (2-AG) biosynthetic enzyme, diacylglycerol lipase-beta (DAGLB), along with paired negative-control and tailored activity-based probes for the functional analysis of DAGLB in living systems1. We utilize our newly developed chemical probes to discover and show that DAGLB inactivation lowers 2-AG production in mouse peritoneal macrophages, as well as arachidonic acid and eicosanoids in a manner that is distinct and complementary to cytosolic phospholipase-A2. A corresponding reduction in lipopolysaccharide (LPS)-induced TNF-alpha release was observed, indicating that DAGLB serves as a key metabolic hub within a lipid signaling network that regulates proinflammatory responses in macrophages2. Consistent with our biological findings in peritoneal macrophages, we will present recent findings that identify DAGLB as a promising anti-inflammatory target for treatment of inflammatory and neuropathic pain3. References 1. Hsu, K.L., Tsuboi, K., Whitby, L.R., Speers, A.E., Pugh, H., Inloes, J. & Cravatt, B.F. Development and optimization of piperidyl-1,2,3-triazole ureas as selective chemical probes of endocannabinoid biosynthesis. J Med Chem 56, 8257-8269 (2013). pmcid:PMC3984011 2. Hsu, K.L., Tsuboi, K., Adibekian, A., Pugh, H., Masuda, K. & Cravatt, B.F. DAGLbeta inhibition perturbs a lipid network involved in macrophage inflammatory responses. Nat Chem Biol 8, 999-1007 (2012). pmcid:PMC3513945 3. Wilkerson, J.L., Ghosh, S., Bagdas, D., Mason, B.L., Crowe, M.S., Hsu, K.L., Wise, L.E., Kinsey, S.G., Damaj, M.I., Cravatt, B.F. & Lichtman, A.H. Diacylglycerol lipase beta inhibition reverses nociceptive behaviour in mouse models of inflammatory and neuropathic pain. Br J Pharmacol 173, 1678-1692 (2016). pmcid:PMC4842918 Acknowledgement UVa startup Funds NIH R00 DA035864 NIH Training Grant T32 GM007055

VirginiaBrainRx Poster Session T 17

BT-11: A FIRST-IN-CLASS THERAPEUTIC FOR CROHN’S DISEASE Raquel Hontecillas*,1, Adria Carbo1, Casandra Philipson1, Richard D. Gandour1, Noah Philipson1, Marion Ehrich2, Stephen Bickston3 and Josep Bassaganya-Riera1

1: Biotherapeutics Inc., 1800 Kraft Dr. Suite 200, Blacksburg, Virginia 24060 (USA) 2: Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA 24061 (USA)

3: VCU Center for Digestive Health, 1250 E Marshall St. Richmond, VA 23219 (USA) rmagarzo@biotherapeuticsinc.com Crohn’s Disease (CD) afflicts over 1.4 million Americans and over 4 million people worldwide. Current therapies have limited efficacy and significant side effects. Thus, there is an unmet clinical need for safer and more effective CD therapeutics. We validated the novel lanthionine synthetase C-like 2 (LANCL2) pathway as a new therapeutic target for CD [1, 2] (Fig. 1A, 1B). We created N,N-bis(benzimidazolylpicolinoyl)piperazine (BT-11), which is orally active, binds to LANCL2 locally in the lower gastrointestinal (GI) tract, results in 90% reduction in GI inflammation in 3 validated mouse models of IBD, and exhibits a benign safety profile [3]. To compare the efficacy of current and IND treatments with the LANCL2 technology, we treated mice with DSS colitis with BT-11 (8mg/Kg/d, p.o.), 5’ASA (50mg/Kg/d, p.o.), anti-TNFα (200μg/mouse, i.p.; days 3-7) ,and GED-0301 (250μg/mouse/d, p.o.) for 7 days.. Oral treatment with BT-11 induced colonic LANCL2 expression and lowered clinical activity more effectively than current and IND drugs for CD (Fig. 1C). Histopathology analyses indicated that leukocytic infiltration was significantly lower in colons of mice treated with BT-11 than in mice treated with GED0301, anti-TNFα, 5’ASA, or untreated mice (Fig. 1D). The loss of LANCL2 abrogated the protective actions of BT-11 in mice with DSS colitis; this confirms the selectivity of BT-11 for LANCL2 in vivo. Further, treating human PBMC with increasing concentrations of BT-11 ex vivo induced IL-10 production and expression of FOXP3 in CD4+ T cells. Further, BT-11 treatment suppressed the production of inflammatory markers (i.e. TNFα, IFNγ, and MCP1) by PBMCs from CD patients. Together with the animal pharmacology and toxicology results previously published, these data demonstrate that oral treatment with BT-11 is more efficacious than current and IND drugs for CD in mice with colitis. These findings are supported by translational data demonstrating the ability of BT-11 to suppress inflammatory mediators in PBMCs from CD patients. Thus, BT-11, a first-in-class oral therapeutic for CD that targets the novel LANCL2 MoA in the GI tract to address the unmet clinical need for safer and more effective therapeutics in a growing $5 billion market. References and Acknowledgements 1. Carbo A.,et al. Lanthionine Synthetase C-like Receptor 2 (LANCL2): A Novel Therapeutic Target

for Inflammatory Bowel Disease. Gastroenterology, 2015. 148(4): p. S686-S687. 2. Carbo A., et al. BT-11: A Novel Lanthionine Synthetase C-like 2-Based Therapeutic for IBD.

Gastroenterology. Digestive Disease Week 2016, San Diego., 2016. 3. Bissel P, et al. Exploratory Studies on Safety of BT-11: A proposed orally active therapeutic for

Crohn's disease. Int J Tox - In Press, 2015. Supported by the Virginia Biosciences Health Research Corporation (VBHRC), and NIH SBIR/STTR funds (1R43DK097940 and 1R41DK099027).

Figure 1. BT-11 is a first-in-class therapeutic for CD.

VirginiaBrainRx Poster Session T 18

ALLOSTERIC MODULATION OF α4β2 NICOTINIC ACETYLCHOLINE RECEPTORS BY DESFORMYLFLUSTRABROMINE POTENTIATES THE ANTIALLODYNIC EFFECTS OF NICOTINE IN A MOUSE NEUROPATHIC PAIN MODEL

Deniz Bagdas1,2, Asti Jackson1, M. Imad Damaj1,* 1 Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613 USA. 2 Experimental Animals Breeding and Research Center, Faculty of Medicine, Uludag University, Bursa 16059, Turkey. deniz.bagdas@vcuhealth.org Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. The alpha4beta2 (α4β2) subtype of nAChRs plays an important role in the mediation of pain and several nicotine-evoked behavioral responses.1 The antinociceptive properties of nicotine are well-known in animal and human studies. However, nicotine was poorly tolerated at the efficacious doses, particularly with respect to the side effects.2 In addition, agonists and partial agonists of α4β2 nAChRs show efficacy in acute and chronic animal pain models. There is a growing body of evidence pointing to allosteric modulation of α4β2 nAChRs as an alternative treatment strategy in experimental pain.1,3

Desformylflustrabromine (dFBr), is a positive allosteric modulator (PAM) at α4β2 nAChRs, amplifies effects of nicotine at α4β2 nAChRs without interacting with the agonist binding sites or possessing intrinsic activity at the receptor per se.4 We hypothesized that dFBr will enhance nicotine-induced antinociception in mice. Therefore, the present study investigated whether dFBr to attenuate mouse chronic constriction injury (CCI)-induced neuropathic pain in mice by increasing endogenous cholinergic tone or potentiating the nicotine-evoked antiallodynic response. We found that subcutaneous administration of dFBr (1 to 9 mg/kg) failed to reduce pain behaviors in CCI mice. However, the combination of dFBr with subcutaneous nicotine (0.5 mg/kg) significantly reversed neuropathic pain behavior dose- and time-dependently without motor impairment. We also provided the proof for α4β2 nAChRs mediation in the antinociceptive effect of combination by using competitive α4β2 antagonist dihydro-β-erythroidine. In addition, dFBr failed to potentiate the antiallodynic effect of morphine, which shows that the effect of dFBr is unique to α4β2 nAChRs. The present results suggest that selective positive modulation of α4β2 nAChR by PAM may represent a new analgesic approach in chronic neuropathic pain. References 1. Rode, F.; Munro, G.; Holst, D.; Nielsen, E.Ø.; Troelsen, K.B.; Timmermann, D.B.; Rønn, L.C.; Grunnet, M. Brain Res. 2012, 1458:67-75. 2. Matthews, A.M.; Fu, R.; Dana, T.; Chou, R. Cochrane Database Syst Rev. 2016, 1:CD009634. 3. Zhu, C.Z.; Chin, C.L.; Rustay, N.R.; Zhong, C.; Mikusa, J.; Chandran, P.; Salyers, A.; Gomez, E.; Simler, G.; Lewis, L.G.; Gauvin, D.; Baker, S.; Pai, M.; Tovcimak, A.; Brown, J.; Komater, V.; Fox, G.B.; Decker; M.W.; Jacobson, P.B.; Gopalakrishnan, M.; Lee, C.H.; Honore, P. Biochem Pharmacol. 2011, 82:967-976. 4. Weltzin, M.M.; Schulte, M.K. J Pharmacol Exp Ther. 2010, 334:917-926. Acknowledgement This research was supported by the National Institute on Drug Abuse Grant R01DA032933.

VirginiaBrainRx Poster Session T 19

EVALUATION OF AKT SIGNALING PATHWAY AND THERAPUTIC PONTENTIAL OF AKT-TARGETED THERAPY IN CANINE HISTIOCYTIC SARCOMA Qizhi Qin1, Irving C. Allen1, Rafael V. Davalos2, Nikolaos G Dervisis1,* 1Virginia Maryland Regional College of Veterinary Medicine, Blacksburg, VA, 24061 2School of Biomedical Engineering and Sciences Virginia Tech – Wake Forest University, Blacksburg, VA, 24061 qqizhi87@vt.edu Histiocytic sarcoma (HS) is an extremely rare malignant neoplasm derived from dendritic cells and histiocytes.1 Canine histiocytic sarcoma (cHS) is an aggressive tumor of the same cellular lineage and with uniformly-fatal outcome.2 Neoplastic histiocytic infiltrates are most often found in the spleen, liver, lung, bone marrow and lymph nodes, while atypical locations include ocular and central nervous system sites.3 Aberrations in the Akt signaling pathway have been implicated in the pathogenesis of this disease, both in humans and dogs.4, 5 The aim of this study is to investigate the importance of Akt signaling pathway in the canine model of the disease and the therapeutic potential of Akt-targeted therapy. The Akt pathway appeared to be constitutively active in the DH82 cHS cells when compared to the control normal dendritic cells. Serum starvation resulted in significant decrease in cell viability, while with no reduction on Akt phosphorylation status or Akt activity. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation and Akt activity, cell viability, and increased sensitization to proteasome inhibition depended apoptosis in a synergistic manner. Akt signaling appears to be active in 9/10 clinical cHS tumor samples, while PTEN mRNA and protein was detected in all tumor samples. These data indicates the Akt pathway appears to be active in the majority of canine tumor samples and may represent a potential therapeutic target. References 1. Ansari J, Naqash AR, Munker R, El-Osta H, Master S, Cotelingam JD, Griffiths E, Greer AH, Yin H,

Peddi P, Shackelford RE: Histiocytic Sarcoma as a Secondary Malignancy: Pathobiology, Diagnosis, and Treatment. Eur J Haematol 2016.

2. Affolter VK, Moore PF: Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs. Veterinary pathology 2002;39:74-83.

3. Chandra AM, Ginn PE: Primary malignant histiocytosis of the brain in a dog. Journal of comparative pathology 1999;121:77-82.

4. Moore PF: A review of histiocytic diseases of dogs and cats. Vet Pathol 2014, 51:167-184. 5. Carrasco DR, Fenton T, Sukhdeo K, Protopopova M, Enos M, You MJ, Di Vizio D, Nogueira C,

Stommel J, Pinkus GS, Fletcher C, Hornick JL, Cavenee WK, Furnari FB, Depinho RA: The PTEN and INK4A/ARF tumor suppressors maintain myelolymphoid homeostasis and cooperate to constrain histiocytic sarcoma development in humans. Cancer Cell. 2006 May;9(5):379-90.

Acknowledgement We would like to thank Morris Animal Foundation (D08CA-308) for partially funding this project, Melissa Makris, and Jen Averill for their help and support.

VirginiaBrainRx Poster Session T 20

UTILIZATION OF COMPUTATIONAL TECHNIQUES TO UNDERSTAND PROTEIN STRUCTURE-FUNCTION RELATIONSHIPS AND GUIDE DRUG DESIGN David R. Bevan *,1,2, Anne M. Brown1, and The Bevan Lab 1Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061 (USA) 2Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA, 24061 (USA) drbevan@vt.edu The research in the Bevan lab focuses on the application of computational molecular modeling of macromolecules to relate structure and dynamics of biomolecular systems to function. Two techniques that we utilize the most are molecular dynamics (MD) simulation and molecular docking. MD simulations provide insight into dynamic properties of molecular phenomena including protein structure-function relationships, protein folding/misfolding, protein-membrane interactions, and ligand-receptor interaction. Molecular docking and virtual screening offer a time- and cost-effective method to identify potential agonists and antagonists for receptors and inhibitors for enzymes. We will present a summary of ongoing projects in the lab, many of which are done in collaboration with other labs, in which our computational methods are used to complement wet-lab approaches. Two projects involve examination of amyloidogenic peptides, specifically amyloid -peptide (A ) a s s ocia te d islet amyloid polypeptide (IAPP, also called amylin) associated with type 2 diabetes and possibly Alzheimer’s disease. As part of these projects, we also are attempting to identify inhibitors of A a nd IAPP aggregation. In another project we apply molecular docking to examine the binding of inhibitors to sphingosine kinases, which are implicated in the development of cancer. A project focused on peroxisome proliferator activated receptors, which are associated with inflammation, diabetes, and obesity, among other disorders, is designed to identify isoform-specific agonists and antagonists of these important nuclear receptors. We also apply MD simulations in attempting to identify novel inhibitors of HIV. In this case, we are examining a series of compounds that inhibit viral entry by binding to gp120 on the surface of HIV. In all of these studies, our goal is to apply computational methods to provide increased understanding of protein structure, dynamics, and function as a way to rationally design potential therapeutics.

Acknowledgement The authors thank Advanced Research Computing at Virginia Tech for time on the BlueRidge supercomputer and all collaborators involved in the aforementioned projects.

VirginiaBrainRx Poster Session T 21

NOVEL PROGNOSTIC MARKERS FOR GLIOBLASTOMA Robin T. Varghese1, 3, Yanping Liang1, Ting Guan2, Christopher T. Franck1, 2, Deborah F. Kelly1, 4, 6, 7, Zhi Sheng1, 4, 5, 6* 1Virginia Tech Carilion Research Institute, Roanoke, VA, 24016 (United States of America (USA)) 2Department of Statistics, Laboratory for Interdisciplinary Statistical Analysis, Blacksburg, VA, 24061 (USA) 3Genetic Bioinformatics and Computational Biology Program, Virginia Tech, Blacksburg, VA 24061 (USA) 4Department of Internal Medicine, 1Virginia Tech Carilion School of Medicine, Roanoke, VA 24016 (USA) 5Department of Biological Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061 (USA) 6Faculty of Health Science, Virginia Tech, Blacksburg, VA 24061 (USA) 7Department of Biological Sciences, College of Sciences at Virginia Tech, Blacksburg, VA 24061 (USA) robinvarghese@vt.edu and zhisheng@vtc.vt.edu Glioblastoma is the most common and lethal malignant brain tumor. A major cause for therapeutic failure in glioblastomas derives from the lack of powerful prognostic markers. To search for new prognostic markers, we hypothesized that kinase genes that are critical for the survival of glioblastoma cells present prognostic power. We first identified 20 survival kinase genes (SKGs) in glioblastoma using a high throughput screen. The enrichment of these SKGs in glioblastoma cells or tissues was verified using the data obtained from several publically available databases (BioGPS, Oncomine, and The Human Protein Atlas). To investigate whether these SKGs are prognostic markers for glioblastoma, we analyzed patient survival data retrieved from The Cancer Genome Atlas patient database. We found that the expression of CDCP1, CDKL5, CSNK1 , IRAK3, LAT overall survival of glioblastoma with or without the treatment of temozolomide a front line chemotherapeutics for this disease. Furthermore, we found that patients with increased levels of NEK9 and PIK3CB mRNA expression had a higher probability of tumor recurrence and that expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary glioblastoma tumors was associated with recurrence-related prognosis. These genes have not been shown in glioblastoma prognosis and progression. Hence, our results reveal novel prognostic markers for this deadly disease. Given their essential role in cell survival, these SKGs are also important for future therapeutic intervention. References 1. Ostrom, Q.T., et al., CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro Oncol, 2015. 2. Cancer Genome Atlas Research, N., Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature, 2008. 3. Omberg, L., et al., Enabling transparent and collaborative computational analysis of 12 tumor types within The Cancer Genome Atlas. Nat Genet, 2013. 4. Guo, D., et al., RNA interference therapy for glioblastoma. Expert Opin Biol Ther, 2010. Acknowledgement The authors would like to thank the Genetics Bioinformatics and Computational Biology program and the Virginia Tech graduate school for advice, guidance, and support. This work was supported by the startup fund from VTCRI and the research grant from the Elsa U. Pardee Foundation to Z.S.

VirginiaBrainRx Poster Session T 22

IDENTIFICATION OF AUTOPHAGY/AUTOPHAGY FLUX-REGULATING GENES THAT MODULATE IMATINIB SENSITIVITY IN CHRONIC MYELOID LEUKEMIA Sujuan Guo1, Kevin J Pridham1, 2, Susan Murphy1, Ching-Man Virbasius3, Michael R Green3, and Zhi Sheng1, 4-6*

1. Virginia Tech Carilion Research Institute, Roanoke, VA 24016, United States.

2. Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA 24061, United States.

3. Howard Hughes Medical Institute and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, United States. .

4. Department of Biological Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, United States.

5. Department of Internal Medicine, Virginia Tech Carilion School of Medicine, Roanoke VA 24016, United States.

6. Faculty of Health Science, Virginia Tech, Blacksburg, VA 24061, United States

sjguo@vtc.vt.edu; kjprid89@vt.edu; zhisheng@vtc.vt.edu Cancer cells employ autophagy, a self-eating program that digests and recycles cellular materials, to sustain cell survival and elicit drug resistance. In chronic myeloid leukemia (CML) driven by an oncoprotein BCR-ABL, autophagy has been shown to protect tumor cells from cell death induced by imatinib (IM), a BCR-ABL inhibitor used as the front-line treatment for CML. Thus, autophagy/autophagy flux (the dynamic process of autophagy) is one of the prime targets for circumventing IM resistance. However, applying autophagy flux blocker chloroquine or inducing autophagy-associated cell death has not yet been successful in the clinic to treat cancer. New autophagy-related therapeutic targets are needed. Based upon a large-scale RNA interference (RNAi) screen in human K562 CML cells, we identified 82 ARGs, among which 71 ARGs are novel autophagy regulators. Through assessing autophagy flux, ARGs are grouped into autophagy flux-promoting genes (AFPGs) and autophagy initiation-suppressing genes (AISGs). Importantly, inhibition of AFPGs sensitizes IM-resistant K562R cells and tumors to this drug, through impeding autophagy flux and potentiating IM-induced apoptosis. Blockade of AISGs, however, induces autophagy and autophagy-independent apoptosis and/or necrosis. Collectively, our study has revealed new and important regulators in autophagy/autophagy flux, which will help us further understand how this dynamic process is precisely regulated in cancer and provide new therapeutic targets for cancer. Acknowledgement This work was supported by the Startup Funds provided to Z.S. by the Virginia Tech Carilion Research Institute.

VirginiaBrainRx Poster Session T 23

BLOCKING THE NOCEBO EFFECT FOR TREATMENT OF FEAR OF MEDICAL PROCEDURES (PARTICULARLY CHILDBIRTH)

Jason McDevitt William and Mary Research Institute, College of William and Mary, Williamsburg, VA 23187 jpmcde@wm.edu When you’re anxious about pain, the pain is amplified. This is not psychological mumbo-jumbo, but rather a neurobiological phenomenon manifested by tangible physiological changes1. The nocebo effect is real and significant, and the drug proglumide blocks this effect2, an effect which is not targeted by any existing pharmaceuticals. Proglumide is an old and safe anti-ulcer drug used around the world, although never approved in the United States. It is fairly ineffective and not widely used anymore, although proglumide is still manufactured and the API could be obtained inexpensively. Proglumide acts as a cholecystokinin antagonist, blocking both the CCKA and CCKB subtypes. Cholecystokinin (CCK) is widely distributed in the central nervous system, and its levels increase to counteract endogenous and exogenous opioids. Proglumide potentiates opioid administration, and by blocking CCK, proglumide enhances the effect of the body’s natural endorphins. Accordingly, proglumide augments the body’s natural response to pain, and crucially for the targeted indications, proglumide also blocks the nocebo effect3. We have developed a patented method for treatment of fear of medical procedures, including surgery, dental phobia, and childbirth, by administering proglumide prior to commencement of the medical procedure. Proglumide is not an analgesic, but instead reduces the significant component of pain that is attributable to anxiety about pain. In particular, administering proglumide after onset of labor can reduce anxiety-related pain and thus can reduce the need for analgesic medications for many women. These analgesic medications (e.g., opioids, epidurals), while reasonably safe, correlate with increased financial costs and a much higher incidence of complications (e.g., C-sections, instrument-assisted deliveries). Moreover, those analgesic drugs adversely impact a woman’s ability to participate in childbirth to her fullest capacity. Significantly, women in developing countries around the world frequently do not have access to quality medical care or strong analgesics during childbirth. A drug that is inexpensive, orally available, safe, does not require cold storage, has no analgesic benefits in and of itself, and does not alter physical or mental capabilities would potentially be very beneficial to these women, not only reducing pain but improving outcomes.

Understandably due to liability concerns, no pharmaceutical company is likely to develop this drug for childbirth in the US. Yet we believe it could be tremendously helpful. We are seeking academic or commercial collaborators (in the U.S. or abroad) who are intrigued by the ability to harness the nocebo effect to improve childbirth for women around the world. References

1. Scudellari, M., “Worried Sick”, The Scientist Magazine, Article 36126, December 9, 2015. 2. Benedetti et al., The Journal of Neuroscience, November 15, 2006, 26(46):12014 –12022. 3. Benedetti et al., Pain, (1997). 71:135-40.

VirginiaBrainRx Poster Session T 24

EFFECT OF BIOMETALS ON α-SYNUCLEIN CONFORMATION AND REACTIVITY

Dinendra L. Abeyawardhane, and Heather R. Lucas* Department of Chemistry, Virginia Commonwealth University, Richmond, VA, 23284 abeyawardhadl@vcu.edu Parkinson’s disease (PD) is an age-related progressive disease, and it is the second most prevalent neurodegenerative disorder in the world. The pre-synaptic protein α-synuclein (αSyn) has a clear linkage to the pathology of PD.1,2 Lewy bodies, the cytopathological hallmarks of PD, are found to be rich in aggregates of misfolded αSyn protein. There is also evidence to suggest that aggregation of monomeric αSyn is triggered by metal ions, specifically copper. CuII ions are known to bind strongly to the free N-terminal amine of αSyn,3 but the physiologically relevant form of αSyn is likely to be N-terminally acetylated (NAc-αSyn);4 thus, alternative coordination sites must be considered. Moreover, CuII/I ions have been proposed to promote cell membrane perforation by αSyn,3 hence metal-bound protein/membrane interactions are under investigation. In an attempt to elucidate the pathological mechanism and to gain a greater understanding of the interaction of redox-active metal ions with both αSyn and NAc-αSyn, investigations were carried out with CuI, CuII, FeII, and FeIII ions – the most prevalent redox active metals in the brain. The structural consequences of metal binding to αSyn and NAc-αSyn were analyzed through circular dichroism spectroscopy (CD), and Thioflavin T assays have suggested that β-sheet formation differs under aerobic and anaerobic conditions.3,5-6 Post-translational modifications, such as di-tyrosine crosslinking6 induced by the presence of reactive oxygen/nitrogen species will also be addressed. References 1. McDowall, J. S.; Brown, D. R. Metallomics. 2016, 8, 385-397. 2. Lucas, H. R.; Lee, J. C.; Emerging Role for Copper-Bound α-Synuclein in Parkinson’s Disease

Etiology. In Brain Diseases and Metalloproteins; D. R. Brown, Ed.; Pan Stanford, 2013, pp 295-316. 3. Lucas, H. R.; Lee, J. C. J. Inorg. Biochem. 2010, 104, 245-9. 4. Dikiy, I.; Eliezer, D. J. Biol. Chem. 2014, 289, 3652-3665. 5. Lucas, H. R.; Lee, J. C. Metallomics. 2011, 3, 280-283. 6. Lucas, H. R.; DeBeer, S.; Hong, M.; Lee, J. C. J. Am. Chem. Soc. 2010, 132, 6636-6637.

Acknowledgement This work was supported by Virginia Commonwealth University, College of Humanities and Sciences and the Department of Chemistry.

VirginiaBrainRx Poster Session T 25

SELECTIVE MU-OPIOID RECEPTOR (MOR) ANTAGONISTS AS POTENTIAL OPIOID ADDICTION TREATMENTS Samuel Obeng1, Abdulmajeed Jali2, Dana E. Selley2 and Yan Zhang1 1Department of Medicinal Chemistry and 2Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298 obengs@vcu.edu, jaliam@vcu.edu Substance abuse costs the United States of America over $600 billion annually. Effective drug addiction treatment has been shown to reduce associated health and social cost by far more than the cost of treatment itself. For example, the cost of 1 full year of methadone maintenance therapy is approximately

$4,700 per patient whereas 1 full year of imprisonment costs approximately $24,000 per person. The opioid receptor agonist methadone and the partial agonist buprenorphine are the major agents used in opioid addiction treatment, but 40-60% of these patients relapse. Recently, opioid receptor antagonists such as naltrexone have been shown to block relapse and curb drug craving in opiate addicts, but some severe side effects of this antagonist have been reported.1 Our laboratory recently reported the development of novel non-peptidyl MOR ligands, NAQ and NAP, which were experimentally characterized through in vitro and in vivo studies as MOR selective antagonists. Molecular modeling studies indicated that the selectivity for MOR is due to pi-pi stacking with W3182 (Figure 1). Therefore, to improve binding to

W318, new morphinan analogues were designed and synthesized (Figure 2). The binding affinities of several compounds in this series were determined by competition for [3H]naloxone binding to membranes from Chinese hamster ovary (CHO) cells stably expressing the mouse MOR. Results showed that all compounds tested so far bound with high affinity (≤ 5 nM), and in fact several bound with sub-nM affinity (in the range of 100-500 pM). These results indicate that our molecular design strategy was successful in producing very high affinity MOR ligands. Studies are underway to determine selectivity of these ligands for MOR relative to other opioid receptor types as well as efficacy to induce MOR-mediated G-protein activation and/or act as antagonists of MOR agonist-induced G-protein activation. Based on our design strategy, we predict that multiple compounds in this series will show high MOR selectivity and low pharmacodynamic efficacy, thus providing novel MOR-selective antagonists. References 1. Minozzi, S.; Amato, L.; Vecchi, S.; Davoli, M.; Kirchmayer, U.; Verster, A. Oral naltrexone maintenance treatment for opioid dependence. Cochrane Database Syst Rev. 2011, 4 2. Zaidi, S. A.; Arnatt, C. K.; He, H.; Selley, D. E.; Mosier, P. D.; Kellogg, G. E.; Zhang, Y. Binding mode characterization of 6α- and 6β-N-heterocyclic substituted naltrexamine derivatives via docking in opioid receptor crystal structures and site-directed mutagenesis studies: Application of the ‘message–address’ concept in development of mu opioid receptor selective antagonists. Bioorg. Med. Chem. 2013, 21, 6405-6413.

OH

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Figure 1: NAQ Binding mode in MOR2

VirginiaBrainRx Poster Session T 26

HUNTINGTON’S DISEASE INDUCED-PLURIPOTENT STEM CELLS SHOW ENHANCED DNA REPAIR GENE EXPRESSION AND CONTRACTION-BIASED TRINUCLEOTIDE REPEAT INSTABILITY Peter A. Mollicaa , John A. Reidb, Roy C. Oglea, Robert D. Brunoa* and Patrick C. Sachsa*

aDepartment of Medical Diagnostics and Translational Sciences, Old Dominion University, Norfolk, Virginia, 23529. bDepartment of Engineering and Technology, Old Dominion University, Norfolk, Virginia, 23529. pmollica@odu.edu Huntington’s disease (HD) is a neurodegenerative disease that exhibits genetic anticipation and somatic cell mosaicism due to instability of a trinucleotide repeat (TNR) region within the Huntingtin (HTT) gene1, 2. While the comprehensive mechanism by which TNR instability is allowed to occur in HD is unknown, several studies have revealed that DNA repair pathways can be effectors of genomic TNR instability3, 4. Previously we identified four DNA repair genes (APEX1, BRCA1, RPA1, and RPA3) to be down-regulated in multiple HD-affected fibroblast cells, and identified differential methylation patterning in the promoter of one of the identified genes5. Based on our previous report, we hypothesized that gene expression of our selected DNA repair genes is recovered during pluripotency as a result of global epigenetic changes that occur during cellular reprogramming6. To investigate whether this DNA repair deficiency is present during developmental pluripotency, we reprogrammed HD-fibroblasts into induced-pluripotent stem cells (iPSC) and saw that gene expression had recovered, relative to unaffected iPSC lines. Further examination into TNR regions of HD-iPSC lines revealed contraction-biased instability over prolonged passaging. Interestingly, we witnessed unaffected allele contractions as well in multiple HD-iPSC lines during pluripotency. We further examined DNA repair gene expression and TNR instability in HD-iPSC differentiated into mesenchymal stem cells and found gene expression of APEX1 remained unchanged, while the pathogenic TNR region maintained stability through 10 population doublings. Our findings identify possible targets for pharmacological intervention that could curb TNR instability in HD. References [1] Ranen NG, Stine OC, Abbott MH, Sherr M, Codori AM, Franz ML, Chao NI, Chung AS, Pleasant N, Callahan C, et al.: Anticipation and instability of IT-15 (CAG)n repeats in parent-offspring pairs with Huntington disease. American journal of human genetics 1995, 57:593-602. [2] Langbehn DR, Hayden MR, Paulsen JS, Group P-HIotHS: CAG-repeat length and the age of onset in Huntington disease (HD): a review and validation study of statistical approaches. American journal of medical genetics Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2010, 153B:397-408. [3] Wheeler VC, Persichetti F, McNeil SM, Mysore JS, Mysore SS, MacDonald ME, Myers RH, Gusella JF, Wexler NS, Group US-VCR: Factors associated with HD CAG repeat instability in Huntington disease. Journal of medical genetics 2007, 44:695-701. [4] Manley K, Shirley TL, Flaherty L, Messer A: Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice. Nature genetics 1999, 23:471-3. [5] Mollica PA, Reid JA, Ogle R, Sachs PC, Bruno RD: DNA methylation leads to DNA repair gene down-regulation and trinucleotide repeat expansion in patient derived Huntington's diseases cells. The American journal of pathology (In Press). [6] Sullivan GJ, Bai Y, Fletcher J, Wilmut I: Induced pluripotent stem cells: epigenetic memories and practical implications. Molecular human reproduction 2010, 16:880-5. Acknowledgement This work was funded by institutional start-up support from the College of Health Sciences at Old Dominion University.

VirginiaBrainRx Poster Session T 27

FUNDAMENTAL AND APPLIED BIOELECTRICS Richard Heller1*, Loree Heller1, Anthony Asmar1 and Michael Stacey1

Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508. rheller@odu.edu Bioelectrics has emerged as a new and exciting research field dealing with the effects of pulse electric fields on biological cells and tissue. In this field, the initial emphasis was on electroporation of the plasma membrane with pulses of a duration in excess of microseconds. Advances in pulsed power technology provided an opportunity to explore and utilize the effects of submicrosecond electric pulses. The effects of such short pulses reach the cell interior, opening up new applications and allowing the use of novel pulse delivery devices such as wideband antennas. In addition, plasmas (ionized gases) can be generated by high voltage electrical discharges in gases and the chemical species produced may positively or negatively affect cells. Bioelectrics research is at the intersection of the biological and physical sciences, and many medical advances have been made with these technologies. Examples of promising medical breakthroughs from this research include killing cancer cells and eliminating tumors, enhanced drug delivery, and accelerated wound healing. Plasma (ionized gas), either by itself or in combination with nanosecond electric pulses, disinfects the exposed areas of wounds. The Frank Reidy Research Center for Bioelectrics (CBE) at Old Dominion University is a global leader in advancing the field of bioelectrics. Advancements in this area rely heavily on the intersection of the physical and biological sciences. The CBE has made these significant contributions because it is able to bring together multiple disciplines to focus on the interactions between pulsed electric fields (picoseconds to millisecond) and biological cells and tissues. The 3rd international scientific workshop on Fundamental & Applied Bioelectrics will be held 25th-29th July at the Frank Reidy Research Center for Bioelectrics at Old Dominion University in Norfolk, VA, USA. Workshop topics include pulse power physics, atmospheric pressure and temperature plasmas, modeling of tissues and cells in electric fields, cell sensitization, drug and gene delivery, and the development of devices and electrodes. Applications of these technologies will be discussed. Examples of promising medical breakthroughs from this research include killing cancer cells producing tumor ablation, enhanced delivery of chemotherapeutic agents and/or plasmid DNA, affecting neuromuscular cell function, and accelerating wound healing. In addition to these medical applications, researchers have also made significant advancements in environmental applications such as chemical remediation. This workshop is open to all researchers (students, post-docs, and investigators) interested in this field. There are two options. In the full version, participants attend all events including lectures, socials, meet the expert lunches, and wet laboratory sessions. We are also offering daily ‘lectures only with lunch’ option so that researchers may listen to specific speakers only. Each day is organized with lectures in the morning followed by hands-on laboratory exercises in the afternoon. For more information, visit www.bioelectricsworkshop.com