PROGRAM & ABSTRACTS

PROGRAM

Wednesday (August 28, 2013)

10:30-10:45 Opening words: Hannu Koistinen & Erik Wallén

10:45-11:30 Chair: Perttu Permi

10:45-11:30 L-1 Peter Tompa (Vrije Universiteit Brussel, Belgium) “Structural Disorder of Proteins”

11:30-12:45 Lunch & Exhibition

12:45-14:30 Chairs: Per Saris & Janne Weisell

12:45-13:30 L-2 Margit Mahlapuu (Pergamum, Sweden) “Clinical Development of Antimicrobial Peptides in Advanced Wound Care” 13:30-14:00 L-3 Sirpa Jalkanen (University of Turku, Finland) “Cell Trafficking-Associated Peptides for Imaging Inflammation and Cancer” 14:00-14:30 L-4 Jyrki Kukkonen (University of Helsinki, Finland) “Orexins – Promises and Pitfalls”

14:30-16:00 Posters & Coffee/Tea & Exhibition

16:00-17:50 Chairs: Marc Baumann & Johanna Mattsson

16:00-16:20 OC-1 Henna Ylikangas (University of Eastern Finland) “From Cyclic Peptides to Drug-Like Compounds Stimulating Kallikrein-Related Peptidase 3 (KLK3)” 16:20-16:40 OC-2 Kristian Meinander (University of Helsinki, Finland) “Hydrocarbon Isosteres of Disulfide Bridges in Peptides that Stimulate the Proteolytic Activity of KLK3” 16:40-17:00 OC-3 Elena Martinova (Russian Medical Academy of the Post-Diploma Education) “Estimation of the Cellular Activity of Synthetic Peptide with Thrombin Activity on the Mouse Model” 17:00-17:20 OC-4 Pavel Myhailiuk (Enamine Ltd, Ukraine) “Design, Synthesis and Application of Fluorinated Amino Acids as Labels to Study the Membrane Active Peptides by Solid State 19F NMR”

17:20-17:50 L-5 David Fewer (University of Helsinki, Finland) “Bioactive Peptides from Cyanobacteria”

18:00-19:00 Finnish Peptide Society (FIPS) General Assembly, only for FIPS and Biobio Society members

19:30- FIPS Dinner (Restaurant Sipuli)

Thursday (August 29, 2013)

9:00-10:30 Chairs: Ale Närvänen & Kristina Luthman

9:00-9:45 L-6 Victor Hruby (University of Arizona, USA) “The Impact of Genomics and Proteomics in Peptide Drug Design: Multivalent Ligands That Address the Disease State” 9:45-10:30 L-7 David Fairlie (University of Queensland, Australia) “Downsizing Proteins: Cyclic Peptidomimetics Beyond the Rule of Five”

10:30-11:00 Coffee/Tea & Exhibition and shift to FinMedChem part of the symposium

Emerging Areas and New Technologies

11:00-11:45 Chair: Kristiina Wähälä

11:00-11:45 L-8 Ian Baxendale (University of Durham, UK) “Flow Chemistry: A Tool for Synthesis and Scale-up”

11:45-13:00 Lunch & Exhibition

13:00-14:30 Chair: Olli Pentikäinen & Pia Vuorela

13:00-13:45 L-9 Chris Abell (University of Cambridge, UK) “Fragment-Based Approaches for Drug Discovery and Chemical Biology” 13:45-14:30 L-10 Robert Owen (Pfizer Neusentis, UK) “Aldehyde Oxidase Metabolism: An Emerging but Surmountable Problem in Drug Discovery”

14:30-15:00 Coffee/Tea & Exhibition

15:00-16:30 Chairs: Erik Wallén & Maija Lahtela Kakkonen

15:00-15:30 L-11 Matthias D’hooghe (Ghent University, Belgium) “Application of small-ring azaheterocyclic building blocks for the synthesis of biologically relevant nitrogen compounds” 15:30-15:45 OC-5 Vania Moreira (University of Helsinki, Finland) “Design and Synthesis of New Bioactive Diterpenes“ 15:45-16:00 OC-6 Muthusamy Venkatraj (University of Antwerp, Belgium) “Synthesis and Antiviral Activity of Novel Diaryltriazines with Cyanovinyl Spacers for Prevention of HIV Infections” 16:00-16:15 OC-7 Tina Seifert (University of Gothenburg, Sweden) “Design and Synthesis of Chroman-4-one-Based SIRT2 Inhibitors” 16:15-16:30 OC-8 Piia Kokkonen (University of Eastern Finland) “SIRT6 In Vitro Deacetylation Assay Substrates“

16:30-18:00 Poster session & Refreshments & Exhibition

18:00-19:00 Annual Meeting of the Medicinal Chemistry Committee of the Finnish Pharmaceutical Society open to all interested, in the end of the meeting a presentation of EU-OpenScreen by Krister Wennerberg (University of Helsinki, Finland)

19:30- FinMedChem Dinner (Restaurant Saaristo)

Friday (August 30, 2013)

Kinase Research

9:00-10:30 Chairs: Josef Messinger & Mikko Myllymäki

9:00-9:45 L-12 (Orion Lecture) Karl-Heinz Altmann (ETH Zürich, Switzerland) “Chemical Diversity in Kinase Inhibitors” 9:45-10:30 L-13 Gerhard Müller (Mercachem, Netherlands) “Next-Generation Inhibitors for Kinases, HDACs, and HKMTs: The Discovery of Slowness”

10:30-11:00 Coffee/Tea & Exhibition

11:00-11:45 Chair: Adyary Fallarero

11:00-11:45 L-14 Jan Ehlert (ProQinase, Germany) “Development of Multi-Kinase Inhibitor 4SC-203 for the Treatment of AML and the Relevance of Cellular Phosphorylation Assays”

11:45-13:00 Lunch & Exhibition

13:00-14:30 Chairs: Leena Otsomaa & Heikki Käsnänen

13:00-13:45 L-15 Morten Grøtli (University of Gothenburg, Sweden) “Targeting the ERK Kinase Signalling Pathway and Upstream Receptor Tyrosine Kinases; Developing MEK and RET Inhibitors” 13:45-14:15 L-16 Timo Heinrich (MerckSerono, Germany) “FAK Inhibitor Hits from Kinase Platform- & Fragment-Screening” 14:15-14:30 OC-9: Jari Yli-Kauhaluoma (University of Helsinki, Finland) ”Design, Synthesis and Biological Activity of Novel PKC C1 Domain Targeted Compounds”

14:30-15:00 Coffee/Tea & Exhibition

15:00-15:45 Chairs: Tapio Nevalainen & Leo Ghemtio

15:00-15:15 OC-10: Vigneshwari Subramanian (University of Helsinki, Finland) “Visually Interpretable Models of Kinase Selectivity Related Features Derived from Field-Based Proteochemometrics” 15:15-15:30 OC-11: Anu Airaksinen (University of Helsinki, Finland) “Synthesis and Biological Evaluation of Novel 123I-Labeled Prolyl Oligopeptidase (POP) Inhibitors” 15:30-15:45 OC-12: Jayendra Patel (University of Eastern Finland) “Identification of Chiral 1,3,4-Oxadiazol-2-one Analogues as Potent and Selective FAAH Inhibitors”

15:45-16:00 Closing words: Hannu Koistinen & Erik Wallén

L-1

Structural disorder of proteins

Peter Tompa

VIB Department of Structural Biology, Brussels and Institute of Enzymology, Budapest

Our traditional view of proteins is rooted in the notion that a well-defined three-dimensional structure is the prerequisite of their function. The evidence is steadily growing, however, that for a significant fraction of the proteome the functionally relevant state is not structured1,2. This recognition has called for the extension of the protein structure-function paradigm to encompass such proteins and protein domains, now termed intrinsically unstructured, intrinsically disordered (IDPs) or natively unfolded. Here the current state of this rapidly advancing field is surveyed3. It is shown that such proteins are common in living organisms and play important roles in the regulation of key cellular processes of signaling and transcription. It will be shown that we have multiple experimental techniques at our disposal for the characterization of the structural ensemble of IDPs, and also that structural disorder provides advantages so that IDPs often surpass globular proteins in terms of functional specificity, versatility, speed and control4. Because of their important functions, however, IDPs are also often involved in disease, such as cancer and neurodegeneration. Thus, it is argued that understanding this structural phenomenon at atomic resolution not only represents a novel challenge for extending the structure-function paradigm, but also presents novel targets and raises new hope for drug discovery5.

1. Tompa, P. (2002) Intrinsically unstructured proteins. Trends Biochem. Sci. 27, 527-533. 2. Tompa, P. (2009) Structure and function of intrinsically disordered proteins, CRC Press (Taylor and Francis

Group), Boca Raton, Fl. 3. Tompa, P. (2011) Unstructural biology coming of age. Curr. Opin. Struct. Biol. 21, 419-25. 4. Kovacs, D., Kalmar, E., Torok, Z. and Tompa, P. (2008) Chaperone activity of ERD10 and ERD14, two

disordered stress-related plant proteins. Plant Physiol. 147, 381-90. 5. Metallo, S.J. (2010) Intrinsically disordered proteins are potential drug targets. Curr. Opin. Chem. Biol. 14,

481-8.

L-2

Clinical development of antimicrobial peptides in advanced wound care

Margit Mahlapuu

Pergamum AB, Stockholm, Sweden; the Sahlgrenska Academy at University of Gothenburg, Sweden

AntiMicrobial Peptides (AMPs), also called Host Defense Peptides, are an evolutionarily conserved component of the innate immune system. In recent years, these peptides have emerged as promising novel drug candidates based on their anti-infectious or immuno-modulatory properties.

Pergamum is a Swedish R&D company located on the campus of Karolinska Institutet. The company is focusing on early stage clinical development of state-of-the-art products based on AMPs for local or topical application in advanced wound care. The current development pipeline includes three candidate products in clinical development, addressing bacterial infections, preventing adhesions and scars, and promoting healing of chronic ulcers.

The Pergamum’s product candidate PXL01 is a short synthetic AMP being developed with the purpose to reduce the formation of obstructing scars and adhesions in connection to surgery or trauma. The anti-scarring effects of PXL01 are anticipated to relate to its capacity to limit the amount of inflammatory response as well as inhibiting fibrin deposition, both leading to excessive scarring. Effective scar prevention by PXL01 has been demonstrated in animal models using the rat multiple abrasion model as well as the rabbit model of digit surgery. A Phase I study in healthy volunteers demonstrated the product is safe and well tolerated. The top-line data from the Phase II clinical trial of PXL01 for the treatment of post-surgical adhesions in hand surgery are now available. In total, 138 patients undergoing flexor tendon repair surgery were treated in this prospective, double-blind, randomized, placebo controlled clinical trial conducted in Sweden, Denmark and Germany. Treatment was not associated with any safety issues or increase in the rate of tendon rupture. The efficacy results of the trial demonstrate a statistically significant improvement in functional hand recovery by PXL01, compared to placebo, supporting that PXL01 has benefits in this patient category

L-3

Cell Trafficking-Associated Peptides for Imaging Inflammation and Cancer

Sirpa Jalkanen

MediCity, University of Turku, Turku, Finland

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the trafficking-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Based on the molecular modeling of VAP-1 we first designed a lysine-containing peptide (GGGGKGGGG) fitting to the enzymatic groove of VAP-1. This peptide inhibited the enzymatic activity of VAP-1 and also the lymphocyte interaction with vascular endothelial cells in flow conditions. However, the real leukocyte counter-receptors for VAP-1 remained unknown for 15 years after the discovery of VAP-1 despite several different attempts in several laboratories. Finally, a phage display approach utilizing a peptide library displaying CX8C decapeptides was able to identify Siglec-9 as a candidate ligand on granulocytes and Siglec-10 on lymphocytes. The binding between Siglec-9/10 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9/10 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium- labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of experimental inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

L-4

Orexins – Promises and Pitfalls

Jyrki P. Kukkonen

Biochemistry and Cell Biology, Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland

Orexins are neuropeptides produced in the hypothalamus. They mediate their responses via two G-protein-coupled receptors, OX1 and OX2 receptors. Orexins regulate a wide range of central nervous system (CNS) functions including sleep–wakefulness, appetite–metabolism, stress response, reward–addiction, and pain gating, and possibly also processes outside the CNS. Orexins are currently most well known for their role in promotion of wakefulness and stabilization of the arousal states, which most dramatically comes to expression in narcolepsy, which is thought, in its human form, to be caused by a death of the orexinergic neurons. Orexin receptors are also known for their multi-faceted cellular signaling.

There has been an active industrial development of small-molecular antagonists against orexin receptors. While the first aim appeared to be appetite suppression, the current development mainly has had sleep induction (for insomnia) as the main goal. On the research side, orexin receptor antagonists have found much use for the mapping of the orexinergic systems. In contrast, there are no unequivocal reports on small-molecular orexin receptor agonists. While the effect of receptor activation (including potential side-effect) is more speculative, readily thinkable uses would be found in the treatment of narcolepsy (and related disorders), metabolic disturbances, and some cancer forms (as orexins induce cell death in come cell types, including colon carcinoma), in addition to the research use. Active orexin peptides include orexin-A (33 aa) and -B (28 aa), both processed from the same precursor, preproorexin. The peptide sequences are fully conserved for the last 10 aa:s (except for the most C-terminal aa) and there are also some other conserved aa:s. In contrast, the N-terminal parts have diverged. Truncation/addition and amino acid exchange studies have indicated that the C-terminus indeed is most important for the peptide–receptor interaction, while the N-terminus is clearly more redundant. Truncation from the N-terminus causes a successive loss of potency (likely via lost affinity), but the loss becomes too high at the remaining length of 6–16 aa:s (depending on the receptor and the study) to allow effective classical peptidomimetic approach. According to the published NMR solution structure studies, major parts of both

orexin peptides are found in two α-helices. One explanation to the lost activity upon truncation is that this conformation is destabilized, which might be mended by additional manipulations (see the poster by Leino et al.). Such modifications would also stabilize the peptide in biological context. Another targeted approach would be to try to transform receptor antagonists into agonists.

In summary, the orexin system has been a target for intensive basic research as well as commercial interest. While the antagonist ligand development has progressed well, with licensing of the first drug soon in sight, development of agonist ligands is seriously lagging behind, probably due to its greater challenges and lower immediate economical gain.

L-5

Bioactive peptides from cyanobacteria

Niina Leikoskia, Liwei Liua, Jouni Jokelaa, Matti Wahlstena, Alexandra Calteaub, Perttu Permic, Kaarina Sivonena, Cheryl A. Kerfeldd, e, Muriel Guggerf, David P. Fewera

aDepartment of Food and Environmental Sciences, Division of Microbiology and Biotechnology, PO Box

56, Viikki Biocenter, Viikinkaari 9, FIN-00014, University of Helsinki, Finland bCEA, DSV, IG, Genoscope and CNRS-UMR8030, Laboratoire d’Analyse Bioinformatiques en

Genomique et Metabolisme (LABGeM), 91057 Evry, France cProgram in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of

Helsinki, FI-00014 Helsinki, Finland dDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA

eUS Department of Energy, Joint Genome Institute, Walnut Creek, CA 94598, USA fInstitut Pasteur, Collection des Cyanobacteries, 75724 Paris Cedex 15, France

The cyanobacterial phylum encompasses oxygenic photosynthetic prokaryotes that play key roles in global carbon and nitrogen cycles. Cyanobacteria are infamous toxin producers but also a promising microbial group in the search for novel bioactive compounds. Low-molecular weight peptides containing a range of proteinogenic and non-proteinogenic amino acids are one of the major classes of bioactive compounds produced by cyanobacteria. These peptides are produced by both non-ribosomal and ribosomal biosynthetic pathways in cyanobacteria assembly lines which work independently of the ribosome to churn out complex peptides. Here we trace the distribution of non-ribosomal and ribosomal peptide biosynthetic pathways in cyanobacteria using a genome mining approach.1 The extent and distribution of the capacity for bioactive peptide synthesis in cyanobacteria has been underestimated and we retrieved 384 non-ribosomal pathways from 126 genomes.1 Our results reveal that 70% of cyanobacterial genomes encode non-ribosomal pathways with just a fraction encoding known end-products. Likewise, gene clusters involved in ribosome-dependent synthesis of diverse peptides through the post-translational modification of short precursor proteins are even more broadly distributed across the phylum.1 The most abundant corresponds to the newly discovered cyanobactin and bacteriocin families with multiple representatives in almost every genome analyzed. We used this information to carry out genome-driven discovery of new natural products. Extensive chemical analyses demonstrated that some cyanobacteria produce short linear cyanobactins with a chain length ranging from three to five amino acids.2 The linear peptides were N-prenylated and O-methylated on the N and C termini, respectively, and named aeruginosamide and viridisamide.2 These findings broaden the structural diversity of the cyanobactin family to include highly modified linear peptides with rare post-translational modifications.

1. Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, Calteau A, Cai F, Tandeau de Marsac N, Rippka R, Herdman M, Sivonen K, Coursin T, Laurent T, Goodwin L, Nolan M, Davenport KW, Han CS, Rubin EM, Eisen JA, Woyke T, Gugger M, Kerfeld CA.. Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci U S A. 2013 110, 1053-8.

2. Leikoski N, Liu L, Jokela J, Wahlsten M, Gugger M, Calteau A, Permi P, Kerfeld CA, Sivonen K, Fewer DP.. Genome mining expands the chemical diversity of the cyanobactin family to include highly modified linear peptides. Chem Biol. 2013 20, 1–11.

L-6

The Impact of Genomics and Proteomics in Peptide Drug Design: Multivalent Ligands That Address the Disease State

Victor J. Hruby

Department of Chemistry and Biochemistry, 1306 East University Boulevard, The University of Arizona, Tucson, Arizona, 85721, U.S.A.

Approaches to the design of drugs for the treatment of degenerative diseases such as cancer, prolonged and neuropathic pain, obesity, diabetes, etc. have had only limited success. It is now clear that these diseases are the result of multiple changes in the expressed genome. These findings suggest that new approaches in drug design are needed. Over a decade ago, we proposed that multivalent drugs that targeted 2 or more receptors/acceptors responsible for the disease state might provide numerous advantages in drug design. In this presentation, we will discuss two of our major approaches that have had considerable success: 1) the design, synthesis and in vitro and in vivo analysis of trivalent ligands that have mu and delta opioid receptor agonist and NK-1 receptor antagonist activities all in a single molecule. These novel compounds cross the blood brain barrier and show very potent analgesia in neuropathic pain models in animals, and none of the toxic side effects of current opioids. Nor can any tolerance or addiction potential with prolonged use be detected; 2) development of homodivalent and heterodivalent ligands for the detection and treatment of cancer.

Supported by grants from the U.S. Public Health Service, National Institutes of Health

L-7

Downsizing Proteins: Cyclic Peptidomimetics Beyond the Rule of Five

David Fairlie

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia

Most proteins interact with other proteins through large, solvent exposed, surface areas to achieve high binding affinity and exquisite selectivity. Often these lack a well defined hydrophobic cleft for targeting by small organic molecules, presenting significant challenges to medicinal chemists. The need to increase interacting surface area in small molecule ligands usually defies the rule of five found so useful for predicting oral bioavailability.

Proteins and peptides have many potential uses in medicine, but their applications have been limited by high manufacturing costs, chemical instability, low bioavailability, low patient responsiveness or poor compliance with injectables. Traditionally considered to be insufficiently drug-like, injectable recombinant proteins (especially monoclonal antibodies) and peptides have nevertheless become blockbuster therapeutics and their market share is projected to grow spectacularly. This contrasts with the stagnating number of new chemical entities reaching registration as human pharmaceuticals over the last two decades.

This talk will summarize some efforts to downsize proteins to small cyclic peptidomimetics, constrained to adopt peptide conformations that bind and activate/inhibit a range of protein targets, including bacterial, viral and human proteins. Examples will be given of compounds of MW 500–2500 that combine advantages of small organic molecules (low cost, stability, bioavailability) with some of polypeptides (target specificity, solubility). Can cyclic peptidomimetics of protein strands, turns and helices realize these objectives and what are some limitations with approaches used to date.

L-8

Flow Chemistry: A Tool for Synthesis and Scale-up

Ian R. Baxendale

Department of Chemistry, University of Durham, Durham, DH1 3LE. United Kingdom.

Synthetic chemistry is the complex art, science and craft of molecular assembly. As a discipline it has undergone many significant changes over the past centuries as our understanding of the fundamentals of bond forming and breaking including how to exert control over these processes has been refined. Throughout this time the basic approach and apparatus available to the experimentalist has remained relatively constant with the occurrence of test tubes, flasks and beakers being historically synonymous with chemistry. This is still true today in that our approach to synthesis is a batch based practice that utilises laboratory glassware such as round bottomed flasks and a defined sequence of discrete reaction steps, work-ups and purifications.

An alternative approach is being evaluated based upon the concept of flow chemical processing which makes use of small capillary reactors and packed bed columns as dynamic vessels for performing chemical transformations.1,2 This more holistic approach also attempts to integrate other aspects of the synthesis such as work-up and purification into the overall process delivering a clean product as one single operation. Consequently the ability to further telescope and assimilate these individual transformations into more elaborate sequences enables the generation of advanced product outputs.

This presentation will detail some of the achievements that our laboratory has made in the area of flow chemistry with the emphasis being placed on the application of this technique to synthetic organic chemistry. By way of exemplification a selection of small molecule building blocks, pharmaceutical libraries and natural products will be illustrated. A concurrent theme to the talk will be the incorporation of immobilised reagents and scavengers for direct in-line reaction promotion and product purification enabling multi-step chemical transformations. Further discussion on the greater scope of reaction conditions attainable through the use of higher system pressures and temperatures as well as processing capability and scale up will also be covered.

1. Baxendale, I. R.; Millia, C. J.; Brocken, L.; (Flow chemistry approaches directed at improving chemical synthesis) Green Process Synth. 2013, 2, 211–230. (DOI: 10.1515/gps-2013-0029).

2. Baxendale, I. R. (The integration of flow reactors into synthetic organic chemistry) J. Chem. Technol.

Biotechnol. 2013, 88, 519–552. (DOI: 10.1002/jctb.4012).

L-9

Fragment-based approaches for drug discovery and chemical biology

Chris Abell

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK

We have developed several approaches based on different biophysical methods to identifiy low affinity fragments binding to macromolecules. These fragments act as starting points for subsequent elaboration into more potent ligands. Our approach is illustrated by the development of inhibitors again enzymes from Mycobacterium tuberculosis.1,2,3 The talk will also describe studies targetting protein-protein interactions4,5 and also riboswitches.6

1. A W Hung, H L Silvestre, S Wen, A Ciulli, T L Blundell & C Abell. Angew. Chemie Intl. Ed. 2009, 48, 8452-8456.

2. G L Abrahams, A Kumar, S Savvi, A W Hung, S Wen, C Abell, C E Barry III, D R Sherman, H I M Boshoff, V Mizrahi. Chem. & Biol. 2012, 19, 844-854.

3. S A Hudson, K J McLean, S Surade, Y-Q Yang, D Leys, A Ciulli, A W Munro, C Abell. Angew. Chemie Intl. Ed. 2012, 51, 9311-9316.

4. From crystal-packing to molecular recognition: prediction and discovery of a binding site on the surface of Polo-Like Kinase 1. P Sledz, C J Stubbs, S Lang, Y Yang, G J McKenzie, A R Venkitaraman, M Hyvönen, C Abell Angew. Chemie Intl. Ed. 2011, 50, 4003-4006.

5. D E Scott, M T Ehebauer, T Pukala, M Marsh, T L Blundell, A R Venkitaraman, C Abell, M Hyvönen.

ChemBioChem 2013, 14, 332-342. 6. Chen, E Cressina, F J Leeper, A G Smith, & C Abell. A.C.S. Chem. Biol. 2010, 5, 355-358.

L-10

Aldehyde Oxidase Metabolism: An Emerging but Surmountable Problem in Drug Discovery

Robert M. Owen

World Wide Medicinal Chemistry, Pfizer – Neusentis, Cambridge, UK

Over the last few decades, reducing compound attrition due to PK-related clinical failures has been a major focus for the pharmaceutical industry. Since 2000, clinical failures due to poor pharmacokinetic properties have decreased substantially although some recent, and unexpected, examples of PK-related compound attrition are thought due to metabolism via the cytosolic metabolizing enzyme aldehyde oxidase (AO). This talk will decribe AO-driven metabolism and how it has manifested itself in a broad range of drug discovery programs. Beyond providing a basic background of the AO enzyme, the talk will also focus on the prevalence of AO-susceptible chemotypes across a broad range of target classes, cross species differences in AO metabolism and the lack of correlation between physicochemical properties and rates AO metabolism. In addition, short case studies will be given that exemplify how AO-mediated metabolism can be tackled in design.

L-11

Application of small-ring azaheterocyclic building blocks for the synthesis of biologically relevant nitrogen compounds

Matthias D’hooghe

SynBioC Research Group, Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

Small-ring azaheterocycles have acquired a pivotal position as building blocks in medicinal chemistry. In

particular, aziridines, azetidines and β-lactams comprise eligible synthons for the construction of a broad variety of stereodefined (heterocyclic) frameworks, often endowed with pronounced biological activities. In this presentation, the synthetic flexibility of these three- and four-membered systems toward the preparation of different types of nitrogen-containing target structures will be unravelled.

In the first part, a number of novel synthetic strategies toward a broad set of important heterocyclic scaffolds will be discussed (see figure below). In particular, different new methods for the regio- and stereoselective rearrangement of aziridines, azetidines and β-lactams into functionalized four- to seven-membered hetero(bi)cycles will be explained. All these compounds represent relevant moieties encountered as substructures in a variety of biologically active synthetic and natural products.

In the second part, the application of aziridines, azetidines and β-lactams for the preparation of specific types of bioactive (hybrid) compounds will be illustrated by means of selected examples. To that end, the synthesis and antimalarial or antiviral assessment of different classes of nitrogen compounds will be reviewed (see figure below).

L-12

Chemical Diversity in Kinase Inhibitors

Karl-Heinz Altmann

Swiss Federal Institute of Technology (ETH) Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences

Kinases have emerged as the most successful target class for new drug discovery in the 21st century, with a total of 23 low-molecular weight (LMW) kinase inhibitors currently being in clinical use for the treatment of different types of cancer, as immunosuppressants and, most recently, for the treatment of rheumatoid arthritis. Of these, as many as 12 compounds have been approved only since the beginning of 2010. Structurally, the vast majority of marketed kinase inhibitors are based on different types of heterocyclic or urea-derived core structures and they bind to the ATP binding site of their respective kinase target(s) either in its (their) active or inactive conformation(s) by forming at least one backbone hydrogen bond to the hinge region that connects the N-and C-terminal lobe of the kinase domain. While many kinase inhibitors are based on identical or similar core structures their potencies and specificities are modulated by the nature of the groups appended to a particular scaffold, which also determine whether binding occurs to the active or the inactive conformation of the kinase. This presentation will review the structures of marketed kinase inhibitors and some compounds that are in advanced stages of clinical development and it will discuss how specific structural features determine inhibitor binding modes and potencies. In addition, selected approaches will be presented that have been developed for the identification of new core structures and kinase inhibitor scaffolds, in order to expand the structural and chemical diversity of available lead structures. In a last part, the potential of natural products as leads for kinase-directed drug discovery will be investigated.

L-13

Next-Generation Inhibitors for Kinases, HDACs, and HKMTs: The discovery of Slowness

Gerhard Müller

Mercachem b.v., Dept. of Medicinal Chemistry, Nijmegen, The Netherlands

In Medicinal Chemistry, a paradigm shift has occurred over the last decade in that more emphasis is laid on the improvement of ADME-related properties and off-target effects early in the drug discovery process, thus avoiding a mere IC50-hunting campaign. Despite these advances, lead finding and optimization programs frequently struggle in achieving high cellular and in-vivo efficacy for a given compound series, despite excellent biochemical activity and good physicochemical properties of the frontrunner candidates. To improve the correlation between e.g. biochemical and cellular or in-vivo efficacy, it is advantageous to consider the lifetime of the ligand-target complex by measuring and optimizing the residence time of compound-target complexes.

In this presentation the relevance of binding kinetic attributes of inhibitors against kinases, as well as a variety of epigenetic targets, such as histone-deacetylases (HDACs) and histone-lysinemethyl-transferases (HKMTs) is emphasized. In the area of protein kinases, the prospective engineering of a binding kinetic signature into inhibitors that exhibit a slow koff by applying “deep-pocket-directed” scaffolds is exemplified (see figure 1).

Figure 1: A type II kinase inhibitor accommodated by a conformationally rearranged kinase target (left); Surface Plasmon Resonace sensogram of a novel CDK8 inhibitor exhibiting a long residence time on target (right).

It will be demonstrated that a thorough understanding of the precise pharmacophoric requirements on the target’s binding site is essential to pre-engineer the desired slow off-rate into new, thus literature-unprecedented scaffolds that qualify as privileged structures for the target family of kinases. The details of the so-called “retro-design” approach for type II kinase inhibitors (see figure 1) will be highlighted by a hit-to-lead and lead optimization campaign that yielded novel and highly efficacious CDK-8 inhibitors (1).

In addition to kinases, novel design principles to next-generation HDAC and HKMT inhibitors with pre-designed slow off-rates will be introduced that exploit the conformational flexibility associated with transient binding sites and product release channels.

(1) Müller, G., Sennhenn, P. C., Woodcock, T., Neumann, L., IDrugs, 2010 13, 457-466.

L-14

Development of multi-kinase inhibitor 4SC-203 for the treatment of AML and the relevance of cellular

phosphorylation assays

Jan E. Ehlerta, D. Fegera, H. Webera, F. Totzkea, A. Lingnaua, S. Taslerb, T. Herzb, C. Henryb, R. Baumgartnerb, R. Doblhoferb, B. Dietrichb, B. Hentschb, R. Kraussb, M.H.G.

Kubbutata, C. Schaechtelea

aProQinase GmbH, Freiburg, Germany; b4SC AG, Martinsried, Germany

4SC-203 will be introduced, a novel multi-kinase inhibitor with a unique selectivity profile which was co-developed by ProQinase GmbH and 4SC AG. During lead development, kinase inhibition of precursors of this compound was not only analysed biochemically but also in various cellular phosphorylation assays in iterative cycles. 4SC-203 turned out as potent inhibitor of FLT3, FLT3 mutants and VEGFR2 in biochemical as well as in cellular phosphorylation assays. Moreover, 4SC-203 inhibited proliferation of FLT3-transformed AML (acute myeloid leukemia) cells down to the picomolar range, and caused tumor regression in AML animal models. Favourable results from preclinical ADMET analyses cleared the way for Phase I clinical trial in healthy donors, in which 4SC-203 proved to be safe and well tolerated. A central experience in the lead development phase of 4SC-203 was, that biochemical kinase inhibitory activity of compound groups from medicinal chemistry optimization could often not be translated into cellular activity, an observation that could not be stringently attributed to lack of cellular permeability, as for some compounds the ratio of cellular to in vitro activity differed among the kinases tested. To elucidate this phenomenon more systematically, we analysed the inhibitory activity of 7 clinically tested kinase inhibitors on 16 oncologically relevant kinases in biochemical and cellular assays. Our results suggest the necessity to test compounds in cellular systems as soon as possible. We conclude that biochemical and cellular analyses represent important complementary components in lead development.

L-15

Targeting the ERK Kinase Signalling Pathway and Upstream Receptor Tyrosine Kinases; Developing MEK and RET

Inhibitors

Morten Grøtli

Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden

The highly conserved mitogen activated protein kinases (MAPKs) are essential regulators in signal transduction pathways and play crucial roles in cellular processes such as transcription, proliferation, differentiation and apoptosis. Dysregulation of MAPKs, in particular the extracellular regulated kinases (ERKs), effector kinases of the RAF/MEK/ERK pathway, is strongly associated with human cancers and is therefore attractive targets for the development of anticancer drugs.1

Extracellular signals such as hormones, growth factors and differentiation factors, as well as tumour- promoting substances, employ the RAF/MEK/ERK pathway to execute their functional effect. In general, most of these stimuli arrive at the cell surface and activate receptor tyrosine kinases (RTKs) through their binding properties. Activation of RTKs (e.g. REarranged during Transfection [RET]) leads to the activation of GTP-binding proteins (e.g. RAS) which in turn activate the core unit of the cascade composed of a MAPKKK (RAF), a MAPKK (MEK1/2), and MAPK (ERK1/2).2 An activated ERK dimer can regulate targets in the cytosol and also translocate to the nucleus where it phosphorylates a variety of transcription factors regulating gene expression.

The RET tyrosine kinase receptor has emerged as important target for the treatment of certain cancer subtypes. RET inhibitors are valuable tools for dissecting its cellular functions. However, commonly used kinase inhibitors used to study RET activity also target other kinases that lie downstream of, or interact directly with RET. We have developed an RET inhibitor with efficient inhibition in vitro and good selectivity when tested on a panel of kinases that makes it a useful tool for investigating RET kinase signaling in human cancer cell lines.3

Inhibition of MEK1/2 represents a promising strategy for the discovery of a new generation of specific anticancer agents. Structure-based design was used to study potential structural modifications on the chromone-based lead compound to obtain highly potent derivatives that target the allosteric pocket on MEK1. Subsequently, a series of analogs were synthesized and evaluated as potential MEK inhibitors.

The design, synthesis and biological evaluation of both kinase inhibitors will be discussed.

1. Dhillon, A.S. et al. Oncogene 2007, 26, 3279. 2. Malumbres, M. et al. Nat. Rev. Cancer, 2003, 3, 459. 3. Dinér, P. et al. J. Med. Chem., 2012, 55, 4872.

L-16

FAK Inhibitor Hits from Kinase Platform- & Fragment-Screening

Timo Heinrich,a Ulrich Grädler,a Felix Rohdich,a Christina Esdar,a Hartmut Greiner,a Nadia Bruggerb, Jeyaprakashnarayanan Seenisamy,c Jayashankaran Jayadevan,c

Venkatraman Sundararamanc

aMerckSerono, Merck KGaA, Darmstadt, Germany; bEMDSerono, Billerica, USA; cSyngene International Ltd., Bangalore, India

Focal Adhesion Kinase (FAK), also known as Protein Tyrosine Kinase 2 (PTK2), is a non-receptor protein tyrosine kinase. Thus, it is located in the cytosol, and is found and recruited to focal adhesion areas at the cell membrane. These focal adhesions are engaged in the contact of the cells to extracellular matrix. FAK is involved in the regulation of cell adhesion to their matrix and in cell migration.

FAK integrates signals from integrin and growth factor receptors and is reported to play a role in the regulation of cell survival, cell growth, proliferation and migration of cells.1

Activation of FAK (in part by autophosphorylation) is induced by interaction with integrin receptors and in response to growth factor stimulation. Phosphorylated FAK is recruited to adhesion foci and interacts there with various adhesion points and other signaling proteins. FAK inhibitors are expected to act in a context related fashion anti-proliferative, anti-invasive & anti-angiogenic, i.e. for the treatment of cancerous diseases. In addition, FAK inhibitors are also expected to be used in the treatment of non-hyperproliferative diseases like psoriasis, arthritis, inflammation, benign prostate hyperplasia, immunological and autoimmune diseases.

This talk describes the first optimization of screening hits from two different approaches: kinase platform ‘KP’- and fragment-screening ‘FS’. The KP initiative, based on testing the enzymatic activity of compounds, resulted in selective pyridine-based FAK inhibitors.2 The ‘FS’ campaign by surface plasmon resonance (SPR) discovered mono- and bidentate hinge interacting fragments that were used as starting points in combination with a type II inhibitor discovered by HTS for optimization by fragment merging .3 The presentation focuses on type-I ligands but also touches type-II FAK inhibitors.4

N NH

R1

R2

R3

N

arylNH

NH

ON

Naryl

RN

R1aryl

NH

R3

R2

Type-I Type-II

1. Mclean et al. Nat. Rev. Cancer 2005, 5, 505-515. 2. Heinrich et al., ISMC: Berlin, 2012, P421. 3. Heinrich et al. J. Med. Chem. 2013, 56, 1160-1170. 4. Grädler et al. Bioorg. Med. Chem. Lett. 2013 manuscript in press

http://dx.doi.org/10.1016/j.bmcl.2013.07.0

OC-1

From Cyclic Peptides to Drug-Like Compounds Stimulating Kallikrein-Related Peptidase 3 (KLK3)

Henna Ylikangas,a Johanna M Mattsson,b Hannu Koistinen,b Antti Poso,a

Maija Lahtela-Kakkonen a

aSchool of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

bDepartment of Clinical Chemistry, University of Helsinki and Helsinki University Central Hospital, Finland

Kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen (PSA), is a serine protease widely used as a biomarker of prostate cancer. It has also been suggested to play a more direct role in regulating cancer growth via its anti-angiogenic properties, which are closely related to its proteolytic activity. 1,2 Therefore, compounds that stimulate the enzymatic activity of KLK3 might be able to inhibit angiogenesis, and in turn slow down the progression of prostate cancer. Previously, cyclic KLK3-stimulating peptides that bind specifically to KLK3 and increase its enzymatic activity have been developed 3. However, the usage of peptides as drug molecules is limited due to their poor physicochemical properties and susceptibility to proteolysis. We have recently reported the 3D pharmacophore for compounds that stimulate KLK3.4 A successful pharmacophore-based virtual screening resulted in finding the first published drug-like molecule that stimulates KLK3 (5E) (Figure 1). Based on the molecular structure and molecular interaction fields of 5E, we have discovered new active analogs of 5E which also increase the enzymatic activity of KLK3. These compounds can be applied in the optimization of the 3D pharmacophore for novel KLK3 stimulating compounds, which clarify the binding of small molecules to KLK3 and provide further insights on the structural features of efficient KLK3-stimulating compounds.

Figure 1: The first small compound that stimulates the enzymatic activity of KLK3, 5E, fitted with the 3D pharmacophore.

1. Fortier AH, et al. Prostate. 2003, 56, 212-19 2. Mattsson, JM et al. Scand. J. Clin. Lab. Invest. 2009, 69, 447-51 3. Wu P, et al. Eur. J. Biochem. 2000, 267, 6212-20 4. Härkönen, HH et al. ChemMedChem. 2011, 6, 2170-78

OC-2

Hydrocarbon Isosteres of Disulfide Bridges in Peptides that Stimulate the Proteolytic Activity of KLK3

Kristian Meinander,a Janne Weisell,b Miikka Pakkala,b Can Hekim,c Roope Kallionpää,a Ale Närvänen,b Hannu Koistinen,c Ulf-Håkan Stenman,c Kristina Luthmand and

Erik A.A. Walléna

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; bSchool of Pharmacy, University of Eastern Finland, Kuopio, Finland; cDepartment of Clinical Chemistry, University of Helsinki, Finland; dMedicinal Chemistry, Department of Chemistry and Molecular Biology, University of

Gothenburg, Sweden

Stimulation of the enzymatic activity of kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) may be beneficial for patients with prostate cancer due to the antiangiogenic activity of KLK31. So far, only a few peptides have been reported to stimulate KLK3. The most potent ones are denoted C4 and B2, both of which increase the proteolytic activity of KLK3 several-fold at micromolar concentrations.2 However, no small molecule compounds with comparable activity have been reported.

The in vivo use of natural peptides is highly challenging. With this in mind we set out to construct synthetic protocols for replacing both internal and terminal disulfide bridges with more stable hydrocarbon isosteres. We have synthesized several pseudopeptide analogs based on both the C4 and B2 peptides. All of shese pseudopeptides show a significant stimulating effect on KLK3, the most potent ones shoving a more than two-fold activation at a concentration of 20 µg/ml (13-14 µM).3

Table 1. Synthesized peptides and their effect on the proteolytic activity of KLK3. All activities were measured at a peptide concentration of 20 µg/ml corresponding to 13-14 µM. 100 % corresponds to the activity of KLK3 alone.

Peptide Linker R Xaa Yaa Zaa Activity

(% ± SE)

1 A

n.a. Ile Glu His 625 ± 36

2 A

n.a. Ile Ala Ala 225 ± 15

3 A

n.a. Ile Glu His 189 ± 9

4 A

n.a. Phe Glu His 223 ± 15

5 B

NH2 n.a. n.a. n.a. 363 ± 5

6 B

NH2 n.a. n.a. n.a. 161 ± 8

7 B

NH2 n.a. n.a. n.a. 247 ± 10

8 B

H n.a. n.a. n.a. 150 ± 0.5

9 B

H n.a. n.a. n.a. 174 ± 15

1. Koistinen H., et al., Biol. Chem. 2008, 389, 633-642. 2. Wu P. et al., Eur. J. Biochem. 2000, 267, 6212-6220. 3. Meinander K. et al, Med. Chem. Commun., 2013, 4 (3), 549-553.

OC-3

Estimation of the cellular activity of synthetic peptide with thrombin activity on the mouse model

Elena A. Martinova*, Lev D. Rumsh#

*Dept. Fundamental Investigations, Russian Medical Academy of the Post-Diploma Education, Ministry of the Public Health & Social Development, Moscow, 123995, Russian Federation;

# Lab. Chemistry of the Proteolitic Enzymes, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences (IBC RAS), Moscow, 117997, Russian Federation

Aim of this work was to test the cellular effects of synthetic peptide with thrombin activity which was previously synthesized in the IBC RAS for a wound healing. To achieve this goal, we use the simple and informative mouse model which can be used for testing any peptide. Our peptide in the concentrations ranged from 1 nanoM to 10 microM has been dissolved in the 0.5 ml physiological solution (PS) and administered intraperitoneally (i.p.) to C57Bl/6 mice (male, 12 weeks old, 3 mice per dose). Control mice received PS. All experiment was been made according the Good Laboratory Practices and repeated three times. The peritoneal cells were removed after 1, 3, and 18 hours followed by a fixation in 2% paraformaldehyde in phosphate buffer (PBS). The immediate fixation allows us to keep the intact composition of cell populations and subpopulations including neutrophils which rapidly undergo an apoptosis followed by a loss of important information. Peritoneal cells were analyzed initially of the channels FSC / SSC to look the whole picture of all cell population with specific gate disposition standard for each type of cells that can be numerically calculated. It should be underlined that there is no need for cell staining on this stage of experiment due to the initial mapping of cell populations. We can see the low-lying gate of lymphocyte, over it neutrophils with macrophages located to the right. The left part of the FSC/SSC mapping includes the apoptotic cells and the debris in the low left corner and sometimes the erythrocytes over it. The upper right part of this mapping may represent the epithelial cells and the large immune cells. Our peptide has been found to change the picture of cell population dose-dependently. After 1h we found the lost of macrophages and increasing number of neutrophils. After 3h the number of phagocyting macrophages and phagocyting neutrophils was elevated but some part of neutrophils underwent an apoptosis. After 18 h the percentage of lymphocytes was dose-dependent increased as well as the percentage of phagocyting macrophages and neutrophils. On the next phase of experiment we stained the fixed cell with propidium iodide (which binds the DNA ends) in the hypotonic water buffer with sodium citrate and triton X-100 that allowed us to analyze the percentage of the apoptotic cells as well as a proliferation of each cell population on the channels FL2 or FL3. Usually we can see the very specific histograms including the central diploid peak, left apoptotic peak clear separated from the first one, and right peak of the proliferating cells densely adjacent to the diploid peak. Using the marker we can calculate the percentage of apoptosis, percentage of cells in the cell cycle that is very convenient for the initial screening of the working dose of peptide. According this one, we have received some information about our synthetic thrombin-like peptide action aimed at the DNA of neutrophils and lymphocytes. We can see the decreased mean of DNA fluorescence in one part of neutrophil’s gate 3h after peptide administration and increased number of apoptotic cells. Also we found the specific bimodal effect of peptide on the lymphocyte. Whole population of lymphocyte has been activated statistically confirmed 18 h after the peptide injection. The conclusions from this low-cost part of investigation are that thrombin-like peptide modulates dose-dependently the lymphocytes, increases the phagocyte activity of neutrophils and macrophages and induces some apoptotic signals in neutrophils. That may be enough for initial screening and an estimation of optimal doses for the subsequent experiments. The next part of work is dependent of the next goal. In out case, we stained the cells (remaining after previously investigations) with antibodies directed to the cell-cycle proteins to achieve the mechanisms of lymphocyte activation, we stained the cell with antibodies attitude to an apoptosis. Additional experiment has been provided with i.p. administration to mice the FITC-labeled fixed Staphylococcus aureus that elucidated the mechanism of the peptide regulation of phagocyte activity.

OC-4

Design, synthesis and application of fluorinated amino acids as labels to study the membrane active peptides by solid

state 19F NMR

Pavel Mykhailiuk,1,2 Anton Tkachenko,1,2 Anna Zhdanova,2 Vladimir Kubyshkin,3 Dmytro Radchenko,1,2 Sergii Afonin,3 Oleg Babii,3 Parvesh Wadhwani,3 Anne S.

Ulrich,3,4 Igor Komarov1,2

1 Enamine Ltd., Oleksandra Matrosova Street, 23, Kyiv 01103, Ukraine; 2 Department of Chemistry, Kyiv National Taras Shevchenko University, Volodymyrska Street, 64,

Kyiv 01033, Ukraine; 3 Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), P.O.B. 3640, D-

76021 Karlsruhe, Germany 4 KIT, Institute of Organic Chemistry and CFN, Fritz-Haber-Weg 6, D-76021 Karlsruhe, Germany

Solid state 19F-NMR is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. For this purpose the native peptide has to be selectively labeled with a suitable 19F-containing amino acid at several different positions. The resulting 19F-labeled peptides are then analyzed by solid state 19F-NMR. The main limitation to this approach currently lies in the poor arsenal of available 19F-labels.1

F3CCO2H

NH2NH

CO2H

F3C CO2H

NH2

F3C

HO

CO2H

NH2

F3C

CO2H

NH2

F3C

1 (Leu, Ile, Val, Ala) 2 (Pro) 3 (Ser, Thr)

4 (Phe) 5 (Phe)

We have therefore rationally designed and synthesized the conformationally restricted amino acids 1-5 bearing a CF3-reporter group, as the 19F-labels.1 We have successfully substituted the residues of hydrophobic amino acids (1),2 proline (2),3 polar amino acids (3)4 and phenylalanine (4) by 19F-labels in peptides.

By now we have applied the developed 19F-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (Gramicidin S, PGLa, Magainin 2, KIGAKI, SAP, Temporin A, BP100, etc).1b

1. a) Ulrich A. S. Prog. NMR Spectr. 46, 2005, 1-21; b) V. Kubyshkin et al. Fluorine in Pharmaceutical and Medicinal Chemistry: From Biophysical Aspects to Clinical Applications. (Eds.: V. Gouverneur, K. Müller), Imperial College Press, London, 2012, p. 91-139.

2. Mikhailiuk P. K. et al. Angew. Chem. Int. Ed. 2006, 45, 5659-5661. 3. Mykhailiuk P. K. et al. Angew. Chem. Int. Ed. 47, 2008, 5765-5767. 4. Tkachenko A. et al. Angew. Chem. Int. Ed. 2013, 52, 1486–1489.

OC-5

Design and Synthesis of New Bioactive Diterpenes

Vânia M. Moreira,a Jari Yli-Kauhaluomaa

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland

Abietic and dehydroabietic acids are naturally occurring diterpenoids of the rosin of coniferous trees. In Finland, some 23 million ha (3/4 of the land and about 11% of the forest area in Europe) is under forests, with 90% of these predominantly coniferous of pine and spruce. The chemistry and availability of the rosin acids is now well-established and diverse biological activities have been reported for the diterpenoids including antitumoral, antileishmanial, anti-inflammatory, and antifungal. Our group has extensive expertise in natural products chemistry,1 and has successfully produced bioactive molecules starting from marine sources and forest residues and waste. One aspect of our current research is the production of semi-synthetic derivatives of commercially available wood diterpenes with relevant biological activities.2 Selected chemical modifications of both abietic and dehydroabietic acids have afforded compounds with antimicrobial activity against several pathogens including S. aureus and E.coli, with some of the new compounds bearing the interesting ability to prevent the formation of and to destroy existing S. aureus biofilms in micromolar concentrations, without significant toxicity towards mammalian cells. Results of this bioactivity screening will be presented.

1. Moreira VM, Salvador JAR, Simões S, Destro F., Gavioli R. Eur. J. Med. Chem. 2013, 63: 46. Salvador JAR, Moreira VM, Gonçalves BMF, Leal AS, Jing Y. Nat. Prod. Rep., 2012, 29:1463. Moreira VM, Salvador JAR, Matos Beja A, Paixão JA Steroids, 2011, 76: 582. Alakurtti S, Heiska T, Kiriazis A, Sacerdoti-Sierra N, Jaffe CL, Yli-Kauhaluoma J. Bioorg. Med. Chem., 2010, 18, 1573. Alakurtti S, Bergstrom P, Sacerdoti-Sierra N, Jaffe CL, J. Yli-Kauhaluoma J. J. Antibiotics, 2010, 63, 123. Pohjala L, Alakurtti S, Ahola T, Yli-Kauhaluoma J, Tammela P. J. Nat. Prod., 2009, 72, 1917. Salin O, Alakurtti S, Pohjala L, Siiskonen A, Maass V, Maass M, Yli-Kauhaluoma J, Vuorela P. Biochem. Pharmacol., 2010, 80, 1141.

2. Fallarero A., Skogman M, Kujala J., Rajaratnam M., Moreira VM, Yli-Kauhaluoma J., Vuorela P. Int. J. Mol. Sci. 2013, 14, 12054.

OC-6

SYNTHESIS AND ANTIVIRAL ACTIVITY OF NOVEL DIARYLTRIAZINES WITH CYANOVINYL SPACERS FOR

PREVENTION OF HIV INFECTIONS

Muthusamy Venkatraj,a Kevin K. Ariën,b Jurgen Joossens,a Johan Michiels,b Pieter Van der Veken,a Paul J. Lewi,a Guido Vanham,b,c Jan Heeres,a Koen Augustynsa

aLaboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium; bVirology Unit, Division of Microbiology, Department of Biomedical Sciences, Institute of

Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium; cDepartment of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium

HIV/AIDS remains a major global health issue. Within the epidemic, heterosexual transmission represents a major pathway for HIV-1 infection. As a preventive strategy, topical microbicide products designed to be applied vaginally or rectally to prevent HIV-1 are being developed. A number of different classes of antiretrovirals are in development as microbicides. One specific class which has been widely studied in the field is the HIV-1 reverse transcriptase inhibitors (RTI), including nucleos(t)ide RTI (such as Tenofovir) and non-nucleoside RTI such as Dapivirine (TMC120). Tenofovir was shown to be safe and active as a microbicide (CAPRISA004 study) and Dapivirine (TMC120) is now in phase III trial to test the long-term safety and effectiveness for prevention of HIV in African women. Dapivirine is cytotoxic (CC50 = 2.88 µM) and show only submicromolar activity against Efavirenz resistant viruses. In search of antiretrovirals with improved activity and toxicity profile as microbicides, we have synthesized a library of 20 triazines with cyanovinyl spacer. All the compounds were evaluated for their in vitro activity against the laboratory strain Ba-L and a primary subtype C isolate. Cellular toxicity on TZM-bl cells was evaluated using WST-1. Subsequently, a selection of 8 compounds was further evaluated for anti-HIV activity in different primary cells, including PBMCs, dendritic cells and CD4+ T lymphocytes. In addition, the activity against NNRTI-resistant viruses (Nevirapine, Efavirenz and Dapivirine) was tested. We present data on the anti-HIV activity and toxicity of these compounds in comparison with TMC120.

N N

NHN NH

NH2

CN

CN

N

NHN NH

CN

Dapivirine (TMC120)Triazine withcyanovinyl spacer

Activity against wild type EC50 (nm) 2 1.3Activity against Resistant EC50 (nm)Nevirapine 2-11 2-3Efavirenz 673 6.8Dapivirine >1000 20-90

Cytotoxicity CC50 (nm) 2877 24540

OC-7

Design and Synthesis of Chroman-4-one-based SIRT2 Inhibitors

Tina Seifert,a Maria Fridén-Saxin,a Tiina Suuronen,b Elina Jarho,c and Kristina Luthmana,*

aDepartment of Chemistry and Molecular Biology, Medicinal Chemistry, University of Gothenburg, SE-412 96 Göteborg, Sweden. bDepartment of Neurology, Institute of Clinical Medicine, University of

Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland and School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland.

Sirtuins (silent information regulator human type protein) are an enzyme family consisting of seven isoforms (SIRT1- SIRT7) which function as NAD+-dependent lysine deacetylases.1 Histones were found to be the first substrates for these enzymes, thereby indicating their involvement in gene silencing. However, the deacetylation is not limited to histones. Other targets including tumor suppressors, transcription factors, and proteins involved in insulin signaling, metabolism and aging have also shown to be deacetylated by SIRTs.2 As the enzymes are implicated to be important in certain diseases such as cancer, neurodegeneration, and diabetes, the development of SIRT modulating agents has become highly interesting.3

Our group has a long-term interest in the chemistry and biological activities of chromone and chroman-4-one derivatives.4 We have recently shown that trisubstituted chroman-4-ones can selectively inhibit SIRT2 with IC50 values in the low micromolar range (Figure 1).5 A set of chroman-4-ones based on this lead compound has been synthesized to explore the structure-activity relationship and improve the physicochemical properties.

Figure 1. The most potent SIRT2 inhibitor with an IC50 value of 1.5 μM.

1. Imai, S.; Armstrong, C. M.; Kaeberlein, M.; Guarente, L. Nature 2000, 403, 795–800. 2. Sinclair, D.; Michan, S. Biochem. J. 2007, 404, 1–13. 3. Sebastián, C.; Satterstrom, K. F.; Haigis, M. C.; Mostoslavsky, R. J. Biol. Chem. 2012, 287, 42444–42452. 4. Friden-Saxin, M.; Pemberton, N.; Andersson, K. D.; Dyrager, C.; Friberg, A.; Grötli, M.; Luthman, K. J. Org. Chem. 2009, 74, 2755–2759. 5. Fridén-Saxin, M.; Seifert, T.; Rydén Landergren, M.; Suuronen, T.; Lahtela-KakkonenM., Jarho, E. M., Luthman, K. J. Med. Chem. 2012, 55, 7104–7113.

OC-8

SIRT6 in vitro deacetylation assay substrates

Piia Kokkonen, Minna Rahnasto-Rilla, Tarja Kokkola, Elina Jarho, Maija Lahtela-Kakkonen

Faculty of Health Sciences, School of Pharmacy, University of Eastern Finland

SIRT6 is a nuclear epigenetic enzyme, which has both deacetylation and mono-ADP-ribosylation activity. Recently it has been discovered to hydrolyse also longer chain fatty acids from lysine residues.1 The function of SIRT6 is important for aging related cellular processes, such as DNA damage repair and telomere maintenance.2 Overexpression of SIRT6 has been shown to lengthen the lifespan male mice and its dysfunction is connected to the formation and maintenance of cancer.3,4 Yet, all the physiological functions of SIRT6 have not been thoroughly studied. Therefore, chemical compounds that regulate activity of SIRT6 are required to further investigate the relevance of this enzyme.

In our previous study, a set of compounds from the in-house database of SIRT1-3 designed regulators was tested for their inhibitory activity against SIRT6.5 These compounds can inhibit the deacetylation reaction of SIRT6 in micromolar range, although generally in lesser extent than they inhibit SIRT1-3.

In the same study, we observed that the assay was not well designed for SIRT6. It uses p53-based substrate, even though p53 is not natural substrate of SIRT6. Natural deacetylation substrate of SIRT6 is histone 3 (H3), and therefore H3 based assay substrates could provide greater affinity and thus improve the quality of the screening results. The influence of incubation time, fluorescence wavelengths, and the use of DNA or BSA as a nonspecific binding partner in the assay was also studied.

The modified assay will improve the screening of SIRT6 deacetylation regulators and increase the reliability of the hits.

1. Jiang H, Khan S, Wang Y, et al. SIRT6 regulates TNF-alpha secretion through hydrolysis of long-chain fatty acyl lysine. Nature. 2013;496(7443):110-113.

2. Jia G, Su L, Singhal S, Liu X. Emerging roles of SIRT6 on telomere maintenance, DNA repair, metabolism and mammalian aging. Mol Cell Biochem. 2012;364(1-2):345-350.

3. Sebastian C, Zwaans BM, Silberman DM, et al. The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism. Cell. 2012;151(6):1185-1199.

4. Kanfi Y, Naiman S, Amir G, et al. The sirtuin SIRT6 regulates lifespan in male mice. Nature. 2012;483(7388):218-221.

5. Kokkonen P, Rahnasto-Rilla M, Kiviranta PH, et al. Peptides and pseudopeptides as SIRT6 deacetylation inhibitors. ACS Med Chem Lett. 2012;3(12):969-974.

OC-9

Design, Synthesis and Biological Activity of Novel PKC C1 Domain Targeted Compounds

Gustav Boije af Gennäs,a Virpi Talman,b Olli Aitio,a Alessia Pascale,c Anna Galkin,d Anu Surakka,e Timo Ruotsalainen,a Kari Kreander,d Päivi Tammela,d Kaarina Sivonen,e Pia Vuorela,d Marialaura Amadio,c Cecilia Osera,c Salla Sorvari,b Daniela Rossi,f Stefano

Govoni,c Simona Collina,f Elina Ekokoski,b Raimo Tuominenb and Jari Yli-Kauhaluoma,a

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; bDivision of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, Finland; cDepartment of Drug

Sciences, Section of Pharmacology, University of Pavia, Italy; dCentre for Drug Research, Faculty of Pharmacy, University of Helsinki, Finland; eDepartment of Applied Chemistry and Microbiology, Division

of Microbiology,University of Helsinki, Finland; fDepartment of Drug Sciences, Section of Section of Medicinal Chemistry and Technology, University of Pavia, Italy

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases that consists of at least ten isoenzymes.1 PKC is involved in proliferation, differentiation and apoptosis2 and is a potential therapeutic target in cancer, neurological (Alzheimer’s disease), immunological, and cardiovascular diseases.3 Indeed, Picato® (ingenol mebutate), a diterpene targeting the regulatory domain of PKC, was recently approved by EMA4 and the US FDA5 for the treatment of actinic keratosis.

PKCs have a catalytic domain that phosphorylates proteins and a regulatory domain that controls kinase activity and subcellular localization of the enzyme. The ATP-binding pocket of the catalytic domain is highly conserved and it is therefore hard to develop specific inhibitors for it. However, one of the regulatory subdomains, the C1 domain, is an attractive target since there are only six other protein families that contain a 1,2-diacylglycerol (DAG)-responsive C1-domain.6 DAG, the natural ligand of PKC, binds to the C1 domain and subsequently activates PKC and translocates it to subcellular compartments of the cell. It is possible to develop both inhibitors and activators of the C1 domain, depending on the PKC isoenzymes and the pathological state.

We used the crystal structure of the PKCδ C1b domain7 and designed and synthesized a novel class of C1 domain binding compounds. These dialkyl (5-hydroxymethyl)isophthalates are conveniently synthesized in only four steps, bind to PKCα and PKCδ with Ki values between 210 and 590 nM and the structure-activity relationship (SAR) of the compounds was studied.8 Depending on the cell type, these compounds and especially HMI-1a3 and HMI-1b11, penetrate the cell membrane, induce apoptosis in HL-60 cells,9 exhibit antiproliferative effect in both HeLa10 and SH-SY5Y cells,11 and show differentiation-inducing effect in SH-SY5Y cells. In more detail, particularly HMI-1b11, which was non-toxic in SH-SY5Y cells, supported neurite outgrowth of these cells and induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation.

1. Newton, A.C. Chem. Rev. 2001, 101, 2353-2364. 2. Griner, E.M. and Kazanietz, M.G. Nat. Rev. Cancer 2007, 7, 281-294. 3. Boije af Gennäs, G.; Talman, V.; Tuominen, R. et al. Curr. Top. Med. Chem. 2011, 11, 1370-1392. 4. European Medicines Agency (EMA). http://www.ema.europa.eu/ema/ (accessed May, 2013). 5. US Food and Drug Administration (FDA). http://www.fda.gov/ (accessed May, 2013). 6. Blumberg, P.M.; Kedei, N.; Lewin N.E. et al. Current Drug Targets 2008, 9,641-652. 7. Zhang, G.; Kazanietz, M.G.; Blumberg, P.M. et al. H. Cell 1995, 81, 917-924. 8. Boije af Gennäs, G.; Talman, V.; Aitio, O. et al. J.Med.Chem. 2009, 52, 3969-3981. 9. Galkin, A.; Surakka, A.; Boije af Gennäs, G. et al. Drug Dev. Res. 2008, 69, 185-195. 10. Talman, V.; Tuominen, R.K.; Boije af Gennäs, G. et al. PLoS One, 2011, 6, e20053. 11. Talman, V.; Amadio, M.; Osera, C. et al. Pharmacol. Res. 2013, 73, 44-54.

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Visually interpretable models of kinase selectivity related features derived from field-based proteochemometrics

Vigneshwari Subramanian#§, Peteris Prusis#, Lars-Olof Pietilä#, Henri Xhaard§, Gerd Wohlfahrt#

# Computer-Aided Drug Design, Orion Pharma, Orionintie 1, FIN-02101 Espoo, Finland.

§ Centre for Drug Research and Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, FIN-00014 Helsinki Finland.

Protein kinases are amongst the most important drug targets as they play crucial roles in various processes such as cell growth, differentiation, apoptosis and intracellular signal transmission. Several hundred diseases are related to the dysregulation of kinases1. The highly conserved nature of ATP binding sites poses a major challenge to design selective inhibitors for kinases. Therefore the structural characterization of kinase ATP binding sites is an integral part in the development of more selective inhibitors/drugs.

Comparison of molecular interaction fields of binding sites within a protein family is a valuable tool to (qualitatively) interpret the selectivity of ligands2. A more quantitative approach to address selectivity issues of receptors is proteochemometrics, a multivariate statistical method, which aims to correlate both ligand and protein description with affinity3. Unlike conventional QSAR models, proteochemometric models provide good predictability and interpretability for both activity and specificity simultaneously for ligands and for targets3. Employing molecular interaction fields to describe proteins in combination with 2D and 3D ligand descriptors in proteochemometric models provides a way for visualizing, understanding and modifying selectivity profiles of small-molecule inhibitors.

The method is demonstrated for 50 kinases with ~2600 activity values collected from the Protein Data Bank and literature. Proteochemometric models using field-based protein descriptors, ligand descriptors and experimentally measured affinity values were generated by Partial Least Squares methods (PLS). Visual interpretation of the models highlights protein field points and ligand functional groups which influence binding affinity and selectivity.

To date, proteochemometrics has been used mostly with sequence-based descriptors. Proteochemometric models based on protein-fields contain more structural details than sequence-based models, which can be visualized and used to support the design of selective inhibitors.

(1) Melnikova, I. and Golden, J. (2004) Targeting protein kinases. Nature Reviews Drug Discovery, 3, 993-994.

(2) Hoppe, C., Steinbeck, C., Wohlfahrt, G. (2006) Classification and comparison of ligand-binding sites derived from

grid-mapped knowledge-based potentials. J. Mol. Graph. Model, 24, 328-340.

(3) Prusis, P., Muceniece, R., Andersson, P., Post, C., Lundstedt, T. and Wikberg, J.E.S. (2001) PLS modeling of

chimeric MS04/MSH-peptide and MC1/MC3-receptor interactions reveals a novel method for the analysis of ligand-

receptor interactions. Biochim. Biophys. Acta, 1544, 350-357.

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SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL 123I-LABELED PROLYL OLIGOPEPTIDASE (POP) INHIBITORS

Annukka Kallinen,a Boyan Todorov,a Roope Kallionpää,b Susanne Bäck,c Mirkka Sarparanta,a Mari Raki,d Juan A. García-Horsman,c Kim A. Bergström,d,e Erik A.A.

Wallén,b Pekka T. Männistö,c Anu J. Airaksinena

aLaboratory of Radiochemistry, Department of Chemistry and bDivisions of Pharmaceutical Chemistry, cPharmacology and Toxicology and dCentre for Drug Research, Faculty of Pharmacy, University of

Helsinki, Finland; e HUS Medical Imaging Center, Helsinki University Central Hospital, Finland

Prolyl oligopeptidase (POP) is an endopeptidase, which hydrolyses L-proline peptide bond of short peptides. POP may be associated with neuromodulation and development of neurodegenerative diseases. Recent studies indicate that the levels of POP are altered in several inflammatory diseases, and the peptidase has been found to participate in the inflammatory cascade along with matrix metalloproteinases.1,2 Radiotracers, which can be used for non-invasive imaging, are needed for investigating the role of POP in normal physiology and in pathophysiological conditions in vivo. We synthesized two novel POP-specific 123I-radiolabeled 4-phenylbutanoyl-L-prolyl-pyrrolidines ([123I]1a and [123I]1b) of which 4-(4-[123I]iodophenyl)butanoyl-L-prolyl-2(S)-cyanopyrrolidine ([123I]1b, Ki = 4.2 nM) was selected. The selected inhibitor 1b has an electrophilic cyano group that is known to increase the dissociation time of POP inhibitors.3 [123I]1b was synthesized in high radiochemical yield and purity (87 ± 4%, >99%, respectively) and with a specific activity of 456 ± 98 GBq/µmol. [123I]1b was evaluated in healthy mice (C57Bl/6JRccHsd) by ex vivo biodistribution studies and SPECT imaging. Pretreatment with the known inhibitor 4-phenylbutanoyl-L-prolyl-(2S)-cyanopyrrolidine (KYP-2047) showed that binding of [123I]1b was POP specific and the labeled inhibitor allowed detection of peripheral POP enzyme with SPECT imaging. In addition, [123I]1b was evaluated in models of neuroinflammation and acute localized inflammation. A minor increase in binding of [123I]1b was observed in the inflamed region in the acute localized inflammation model. Similar increase in binding was not observed in the neuroinflammation model.

Figure 1. Structures of 123I-radiolabeled 4-phenylbutanoyl-L-prolyl-pyrrolidines, [123I]1a and [123I]1b. [123I]1b was evaluated in vivo.

1. Penttinen A, Tenorio-Langa J, Siikanen A, Morawski M, Roβner S, García-Horsman JA. CNS Neurol. Disord. Drug Targets 2011, 10, 340-348.

2. O’Reilly P, Hardison MT, Jackson PL, Xu X, Snelgrove RJ, Gaggar A, Galin FS, Blalock JE. J. Neuroimmunol. 2009, 217, 51-54.

3. Venäläinen JI, García-Horsman JA, Forsberg MM, Jalkanen A, Wallén EAA, Jarho EM, Christiaans JAM, Gynther J, Männistö PT. Biochem. Pharmacol. 2006, 71, 683-692.

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Identification of chiral 1,3,4-oxadiazol-2-one analogues as potent and selective FAAH inhibitors

Jayendra Z. Patel,a Teija Parkkari,a, b Tuomo Laitinen,a Agnieszka A. Kaczor,a,c Susanna M. Saario,b Juha R. Savinainen,b Dina Navia-Paldanius,b Mariateresa

Cipriano,d Jukka Leppänen,a Igor O. Koshevoy,e Antti Poso,a Christopher J. Fowler,d Jarmo T. Laitinen,a Tapio Nevalainena

a School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland; b School of Medicine, Institute of Biomedicine/Physiology, University of Eastern

Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland; c Department of Synthesis and Chemical Technology of Pharmaceutical Substances, Faculty of Pharmacy with Division of Medical Analytics,

Medical University of Lublin, 4a Chodzki St., PL-20093 Lublin, Poland; d Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-90187, Umeå, Sweden; e Department of Chemistry,

University of Eastern Finland, P.O. Box 111, FIN-80101 Joensuu, Finland

Fatty acid amide hydrolase (FAAH), a member of serine hydrolase family, is responsible for in vivo degradation of arachidonoyl ethanolamine (anandamide, AEA), an endogenous ligand of the cannabinoid receptors. Inhibitors of fatty acid amide hydrolase (FAAH) have clinical potential in treatment of various disorders, such as pain, inflammation, depression, and anxiety. Advantage of the FAAH inhibitors over cannabinoid receptor ligands is that they do not produce CNS related side effects. Till date, only a few FAAH inhibitors have reached clinical trials and hence there is still a need of novel FAAH inhibitors. Our research group have identified 1,3,4-oxadiazol-2-one derivatives (Fig. 1, I) as dual inhibitors of FAAH and MGL.1,2 Along with FAAH, dual inhibitor of FAAH and COX are of interest3-5 due to the nominal side effects observed compared to sole use of COX inhibitors (Fig. 1, II).

In this study, we have developed novel oxadiazolone series (Fig. 1, III) by hybridizing previously disclosed 1,3,4-oxadiazol-2-ones (Fig. 1, I) with the marketed phenylalkanoic acid derivatives of NSAIDs (Fig. 1, II) using a convenient two-step synthesis (Fig. 1, III) approach. Novel, chiral 1,3,4-oxadiazol-2-one analogues are acting as potent and selective FAAH inhibitors, and more importantly, they show clear enantioselectivity. The most promising compound of the series is found to be highly potent FAAH inhibitor with a high selectivity towards other endocannabinoid targets and in mouse brain proteomes.

1. Minkkilä, A. et. al. ChemMedChem. 2009, 4, 1253

2. Käsnänen, H. et. al. Eur. J. Pharm. Sci. 2013, 49, 423

3. Fowler, C. J. et. al. J. Enzym. Inhib. Med. Chem. 2012, 1

4. Favia, A. D. et. al. J. Med. Chem., 2012, 55, 8807

5. Jacqueline M. B. et. al. Eur. Med. Chem., 2010, 49, 3564.

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Role of Intrinsically Disordered Protein EspFU in EHEC Pathogenesis

Olli Aitio,a Maarit Hellmana, Brian Skehanb, Arunas Kazlauskasc, Didier F Vingadassalomb,Tapio Kestic, John M Leongb, Kalle Sakselac, Perttu Permia

aProgram in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland; bDepartment of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, USA; cDepartment of Virology, Haartman Institute, University of Helsinki and HUSLAB,

University of Helsinki Central Hospital, Finland

Enterohaemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes bloody diarrhea and strong stomach cramps. Some 10% of EHEC infections lead to serious complications such as haemolytic uremic syndrome which can be fatal especially among young patients. The hallmark of EHEC infection is an actin pedestal, generated beneath the host bound bacterium, which promotes intestinal colonization. To that end, EHEC translocates two effectors, Tir and EspFU, which establish a complex with the cellular protein IRTKS. Association of this complex with the host protein N-WASP ignites localized actin polymerization that is commandeered by the pathogen.

We have used biophysical tools such as NMR spectroscopy and isothermal titration calorimetry together with biochemical and cell biological techniques to study EspFU free in solution as well as in complex with IRTKS and N-WASP1,2. We show that EspFU is an intrinsically disordered protein (IDP), composed of disordered 47-residue repeats, each repeat containing a transient α-helical N-terminus and a dynamic C-terminus2. We show that bacterial and cellular ligands interact with IRTKS SH3 domain in a similar fashion. However, the bacterial protein has evolved to supersede cellular targets by utilizing a targeted single amino acid replacement that offers superior binding affinity to IRTKS. This enables hijacking of cellular ligand and formation of Tir-IRTKS-EspFU-N-WASP complex that triggers actin pedestal formation2.

1. Aitio O, Hellman M, Kazlauskas A, Vingadassalom DF, Leong JM, Saksela K, Permi P. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 21743-21748.

2. Aitio O, Hellman M, Skehan B, Kesti T, Leong JM, Saksela K, Permi P. Structure 2012, 20, 1692-1703.

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N O

R1

1 R1=

HO

O

2 R1=O

HO

O

3 R1=O

HO

O

O

R2-A-I-P-A-S-W-F-R-NH2

Structures of firefly luciferase inhibitor compounds (FLICs) Trypsin inhibitor peptide (TIP) conjugates

4 R2= Ac

5 R2= 1

6 R2= 2

7 R2= 3

R3-R-Q-I-K-I-W-F-Q-N-R-R-M-K-W-K-K-NH2

Penetratin (Pen) conjugates

8 R3= Ac

9 R3= 3

10 R3= Fluorescein

Firefly Luciferase Inhibitor-conjugated Peptide Quenches Bioluminescence: A Versatile Tool for Real Time Monitoring

Cellular Uptake of Biomolecules

Pekka K. Poutiainen,a Teemu Rönkkö,a Ari E. Hinkkanen,b Jorma J. Palvimo,c Ale Närvänen,a Petri Turhanen,a Reino Laatikainen,a Janne Weisell,a Juha T. Pulkkinena

aSchool of Pharmacy, bA. I. Virtanen Institute for Molecular Sciences and cInstitute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland

In this study, novel firefly luciferase-specific inhibitor compounds (FLICs) previously developed by us1 are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond, which is not degraded during the cellular internalization. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the trypsin inhibitor model peptide. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that Penetratin-FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 µM) and inhibited bioluminescence, without affecting the viability of the cells. On the other hand inhibitor conjugated to trypsin inhibitor peptide without internalization properties was not active, indicating that inhibitor do not have transporter properties itself. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.2

1. Poutiainen PK, Palvimo JJ, Hinkkanen AE, Valkonen A, Väisänen TK, Laatikainen R, Pulkkinen JT. J. Med. Chem. 2013, 56, 1064−1073.

2. Poutiainen PK, Rönkkö T, Hinkkanen AE, Palvimo JJ, Turhanen P, Närvänen A, Laatikainen R, Weisell J, Pulkkinen JT. Submitted to Bioconjugate Chem.

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Purification of tumor-associated trypsin inhibitor (TATI) from serum for quantification by LC-MS

Outi Itkonena,b, Suvi Ravelab, Ulf-Håkan Stenmanb and Leena Valmub,c

aHUSLAB, Helsinki University Central Hospital, bDepartment of Clinical Chemistry, University of Helsinki, cBiOva Ltd, Helsinki, Finland

AIM. Sample pre-treatment for absolute quantification of peptides in complex biological matrices by mass spectrometry (MS) is a challenge. We aimed to develop and optimize an immunopurification step for tumor-associated trypsin inhibitor (TATI) employing magnetic Sepharose beads for subsequent quantification by LC-MS.

METHODS. We used a mouse monoclonal antibody (MAb 6E8) to TATI coupled to protein A Mag Sepharose (GE Healthcare) (1 mg MAb/ 100 µL bead slurry) according to the instructions of the manufacturer. Pooled serum from healthy volunteers was spiked with human TATI (17-758 ng/mL) and used to optimize the following conditions: the time needed for capture (5, 15, 30 and 60 min) and elution (15 and 30 min), and the elution solution [0.1 mol/L glycine-HCl without and with 2 mol/L urea, 0.1 %formic acid (FA), 0.01%, 0.05% and 0.1% trifluoroacetic acid (TFA) ]. TATI concentration in the various fractions was determined by a time-resolved immunofluorometric method or by LC-MS.

RESULTS and DISCUSSION. TATI recovery was poor (<20%) when eluted (2 x 50 µL) with glycine-HCl, glycine-HCl-urea, FA and 0.01% TFA. On the other hand, the recovery with 0.1% TFA (90%) was only slightly better than that with 0.05% TFA (83%). There was a minor increase in recovery when elution time was increased from 15 min (90%) to 30 min (100%). The binding of serum TATI to the affinity beads was fast: maximal binding was observed already in 5 min.

CONCLUSIONS. We have optimized an immunopurification procedure for serum TATI employing MAb 6E8 coupled to magnetic Sepharose beads. The most efficient and practical procedure was to allow binding and elution to occur in 15 min using 0.1% TFA for elution. The optimized procedure allows quantification of serum TATI by LC-MS.

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New LC-MS based assay of serum tumour-associated trypsin inhibitor (TATI) allows simultaneous quantitation and

detection of TATI variants

Suvi Ravelaa, Leena Valmua, Ulf-Håkan Stenmana,b and Outi Itkonenb

aDepartmet of Clinical Chemistry, University of Helsinki, Helsinki, Finland, bHUSLAB, Helsinki University Central Hospital, Helsinki, Finland

Background: We aimed to develop an LC-MS based assay for quantification of serum tumour-associated trypsin inhibitor (TATI) and known TATI variants (N34S and P55S) associated with pancreatitis. TATI is a 6 kDa peptide associated with many cancers, especially mucinous ovarian carcinoma. Elevated serum TATI concentrations are also found in severe inflammations including pancreatitis1.

Methods: Serum TATI was captured using an antibody (MAb 6E8) coupled protein G Mag Sepharose beads (GE Healthcare). TATI was then quantified by an LC-MS pseudo-MRM assay using an Agilent 1200 LC (Agilent Technologies) with Polaris C18-A column (Varian Inc.) and a 4000 QTRAP mass spectrometer (AB Sciex). The newly developed LC-MS assay was validated and compared with a time-resolved immunofluorometric assay (TR-IFMA) using 90 serum samples.

Results: The analytical recovery of the assay was 87%-100% and the linear range 1-500 ng/ml. Within-assay CV was <6.4 %. The LC-MS assay correlates well with TR-IFMA. With this assay it was also possible to detect the N34S variant from a confirmed carrier and from a patient sample.

Conclusions: Our novel LC-MS assay for serum TATI shows good correlation with TR-IFMA. Most importantly, it enables the detection and quantification of TATI variants known to be associated with familial pancreatitis.

1. Stenman UH.Tumor-associated trypsin inhibitor. Clin Chem. 48(8):1206-1209, 2002

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Mesoporous silicon nanoparticles functionalized with cell penetrating peptides: a novel delivery platform for

oligonucleotide drugs

Jussi Rytkönen,a Piret Arukuusk,b Wujun Xu,c Kaido Kurrikoff,b Ülo Langel,b,d Vesa-Pekka Lehto,c Ale Närvänena

aSchool of Pharmacy, University of Eastern Finland, Finland; bInstitute of Technology, University of Tartu, Estonia; cDepartment of Applied Physics, University of Eastern Finland, Finland, dDepartment of

Neurochemistry, University of Stockholm, Sweden

RNA interference with antisense oligonucleotides has not yet lived up to its promises of a magic bullet. The largest barrier in the use of oligonucleotide drugs is the delivery of these large and negatively charged biomolecules trough cellular membranes into the cytosol and the nucleus.

As of lately, mesoporous silicon (PSi) has become of interest in drug delivery due to its biocompatibility and tunable properties. In this study, PSi-nanoparticles, with thermal stabilization and subsequent aminosilane modification1, were utilized as an oligonucleotide delivery platform. Splice correcting oligonucleotides (SCOs), a type of oligonucleotide drug, were loaded into the positively charged PSi-nanoparticles up to 14,3% (w/w). The fast loading is driven by electrostatic interactions, the loading efficiency being 100% within 5 minutes in room temperature.

The nanoparticles were able to deliver and release biologically active SCO inside cells in vitro, when formulated together with novel cell penetrating peptide (NickFect51)2. The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells3. Furthermore, the use of PSi-carrier platform in oligonucleotide delivery did not reduce the cell proliferation, contrary to commercial counterpart. Additionally, the peptide-oligonucleotide cargo was shown to be protected against proteolytic digestion inside the porous nanocarrier.

1. Xu et al. J. Phys. Chem. C, 116 (2012), 22307–22314 2. Arukuusk et al. Biochimica et Biophysica Acta 1828 (2013) 1365-1373 3. Kang et al. Biochemistry, 37 (1998), 6235-6239

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Peptides stimulating kallikrein-related peptidase 3 (KLK3) enhance its activity towards protein substrates

Johanna Mattsson,a Ale Närvänen,b Magnus Jonsson,c Johan Malm,c Ulf-Håkan Stenman,a Hannu Koistinen a

aDepartment of Clinical Chemistry, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland; bDepartment of Biosciences, University of Eastern Finland, Kuopio, Finland.

cDepartment of Laboratory Medicine, Lund University, Malmö, Sweden.

Kallikrein-related peptidase 3 (KLK3, also called prostate-specific antigen, PSA) is a serine protease expressed in prostate and secreted into seminal fluid. KLK3 is a useful biomarker for prostate cancer as it leaks from malignant prostatic tissue into surrounding extracellular fluid and serum. The slow growth of prostate cancer is potentially dependent on high KLK3-levels around the tumor, since KLK3 has been shown to exert anti-angiogenic activity.1 Therefore, it may be possible to control prostate cancer growth by modulating the proteolytic activity of KLK3. Previously, phage display was used to identify peptides that stimulate the enzymatic activity of KLK3 towards a small chromogenic peptide substrate.2,3 From those peptides, B2 and C4 were the most effective stimulators of KLK3, and they also enhanced the anti-angiogenic activity of KLK3 in an endothelial cell tube formation model.4 Here, we characterized the effect of the two peptides on the activity of KLK3 towards several protein substrates in addition to the chromogenic and a fluorometric peptide substrate.

B2 (CVFAHNYDYLVC) is a 12-amino acid long cyclic peptide with disulfide bridge between cysteines 1-12 and C4 (CVAYCIEHHCWTC) is a 13-amino acid long double cyclic peptide that has disulfide bridges between cysteines 1-13 and 5-10. The effect of these peptides on KLK3 activity towards peptide substrates was studied using colorimetric and fluorometric assays and towards known protein substrates, such as semenogelin 1 and 2, IGFBP-3 and fibronectin, using SDS-PAGE followed by silver staining and immunofluorometric assay (IFMA).

Peptides B2 and C4 stimulated the activity of KLK3 towards small peptide substrates by 4-fold and 6-8-fold, respectively. Peptides also stimulated the activity of KLK3 towards protein substrates, but their relative efficiency was different from that with peptide substrates, with B2 being more active. These results suggest that the peptides would be useful in stimulating the activity of KLK3 towards physiological substrates and, therefore, they could be useful lead molecules for development of drugs that stimulate the anti-angiogenic activity of KLK3.

1. Fortier AH, Nelson BJ, Grella DK, Holaday JW. J. Natl. Cancer Inst. 1999, 91, 1635-1640. 2. Wu P, Leinonen J, Koivunen E, Lankinen H, Stenman UH. Eur. J. Biochem. 2000, 267, 6212-6220. 3. Pakkala M, Jylhäsalmi A, Wu P, Leinonen J, Stenman UH, Santa H, Vepsäläinen J, Peräkylä M, Närvänen

A. J. Pept. Sci. 2004, 10, 439-447. 4. Mattsson JM, Närvänen A, Stenman UH, Koistinen H. Prostate. 2012, 72, 1588-1594.

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Truncated constrained orexin-A peptides

Teppo O. Leino,a Niklas G. Johansson,a Janne Weisell,b Lasse Karhu,c Henri Xhaard,c Jyrki P. Kukkonen,d Erik A.A. Walléna

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; bSchool of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Finland; cCentre for Drug Research,

University of Helsinki, Finland; dDepartment of Veterinary Biosciences, University of Helsinki, Finland

The effect of the mature orexin peptides orexin-A and -B is transmitted through the G-protein-coupled orexin receptors (OX1R and OX2R), which have shown to play a significant role in the regulation of the wakefulness.1 Orexin-A consists of 33 and orexin-B of 28 amino acids, respectively. The peptides need to contain at least 19 C-terminal amino acids for the biological effect to remain significant.2 13 of these 19 amino acids are identical in both peptides (RL**LL***GNHAAGILT*). An orexin receptor agonist could be helpful especially in narcolepsy, but also e.g. in the treatment of daytime sleepiness, metabolic disorders or some cancer forms, or to act as chemical probe in biochemical or pharmacological studies. The NMR structures of the orexin peptides in a buffered water solution are mostly in an α-helical conformation. Whether an α-helical conformation of the peptide remains upon binding to OX1R and OX2R, is currently unknown. Here, we have tested whether the α-helical conformation of the peptides could be stabilized by cross-linking side chains using alkene-metathesis reaction, which has been reported to change the characteristics of the peptides (e.g. cell permeability).3 The aim of the synthesis of constrained peptides is also to shorten the length of orexin peptides to less than 19 amino acids without loss of action, since it is problematic to use peptides as drugs due to their poor pharmacokinetic properties. In this study, 19- and 12-amino acids-long peptides, which correspond to the C-terminus of orexin-A, were synthetized. Peptides were synthesized with two unnatural amino acid residues containing terminal alkene groups in their side chain for the ring-closing metathesis reaction at a distance of i and i+4. The optimal placement of these building blocks was chosen based on molecular modeling. The alkene-metathesis reaction was successfully conducted when a commercial building block (2-amino-2-methylhept-6-enoic acid) was used, but not with L-O-allyl-serine.

1. Scammell T. E., Winrow C. J. Orexin receptors: pharmacology and therapeutic opportunities. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 243-266.

2. Ammoun S., Holmqvist T., Shariatmadari R., Oonk H. B., Detheux M., Parmentier M., Åkerman K. E., Kukkonen J.P. Distinct recognition of OX1 and OX2 receptors by orexin peptides. J. Pharmacol. Exp. Ther. 2003, 305, 507-514

3. Verdine G.L., Hilinski G.J. Stapled peptides for intracellular drug targets. Methods Enzymol. 2012, 503, 3-33.

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Polypharmacological blockage of estradiol synthesis and signaling

Sanna P. Niinivehmas, Olli T. Pentikäinen

Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, Finland

Estradiol is an important factor in the development of normal mammary glands. Unfortunately, estradiol also stimulates cell proliferation and gene expression through estrogen receptor (ER), and thus may increase the probability for breast cancer growth. Lately, two enzymes, aromatase (ARM) and 17-beta hydroxysteroid dehydrogenase type 1 (HSD-1), that are responsible for the synthesis of estradiol, have become popular targets for the drug discovery. The idea behind this approach is to decrease the estradiol levels in the body to stop the cancer growth. Our research group has earlier developed inhibitors of HSD-1 that simultaneously inhibit ER.1

In this project we utilize predictive HSD-1, ARM and ER models (and also models for several other proteins e.g. nuclear receptors) for virtual high-throughput screening (vHTS).2,3 In vHTS huge molecular libraries of millions of molecules are rapidly examined by shape and electrostatic matching in order to find novel potential drug candidates. Molecules that are predicted to be highly active are studied with more accurate in silico methods like quantitative structure-activity relationship, molecular docking and molecular dynamics simulations merged with post-processing method molecular mechanics – generalized Born surface area.

Molecules that show promise in triple role in inhibiting HSD-1 and ARM, and antagonizing ER, are tested experimentally and optimized further. Furthermore, by using de novo ligand discovery, we have identified highly promising HSD-1 inhibitor core-structure, which optimization will employ the same methodology. The novel vHTS method used in this study enables chemogenomics and polypharmacology approaches to be used in the development of novel ligands for medical purposes. Simple shape-electrostatic models allow the rapid prediction of binding probability of any given small molecule to a wide variety of proteins so that in addition to desired function also possible side effects of a potential drug can be predicted through them.

1. Messinger J et al., Mol. Cell. Endocrinol. 2006, 248, 192-198. 2. Virtanen SI, Pentikäinen OT. J. Chem. Inf. Model. 2010, 50, 1005-1011. 3. Niinivehmas SP et al., J. Chem. Inf. Model. 2011, 51, 1353-1363.

P-9

Novel structural elements for drug discovery: fluorinated amines, morpholine mimics and uncommon scaffolds

Mykhailiuk P.; Denisenko O.; Datcenko O.; Martirosov R.; Artemenko A.; Tkachuk G.; Parhomenko, G.; Gorulia O.; Andrushko O.; Kondratov I.; Tolmachev A.

Enamine Ltd, 78 Chervonotkatska Street, 02094 Kyiv, Ukraine

It is now commonly accepted that structural diversity of the known drugs is very low. Moreover, it is believed to be one of the reasons, why it is getting more and more difficult to launch the principally novel drugs in the recent years.

In the contemporary paradigm of drug discovery under the pressure of shortening turnaround time in lead optimization projects, medicinal chemists are to use only commercially available building blocks that can quickly be delivered. Under these circumstances even the most conceptually attractive compounds have low chances to find the true practical application if they are not commercialized. For example, being highly accessible conventional laboratory reagents pyrrolidine, piperidine and morpholine, left their deep footprint in drug discovery. These are the most frequently reported secondary amines in medicinal chemistry. We wanted to upgrade this toolkit of primitive cyclic amines and synthesized other family members including fluorine-containing ones allowing for a variety of forms:[1-9]

For the representative synthetic affords, please see: [1] Yarmolchuk, V. et al. Eur. J. Org. Chem. 2013, in press. [3] Artamonov, O. et al. Synthesis 2013, 45, 225. [4] Shcherbatiuk, A. et al. Tetrahedron 2013, 69, 4066. [5] Radchenko, D. Tetrahedron. Lett. 2013, 54, 1897. [6] Alekseenko, A. Synthesis 2012, 44, 1974. [7] Bezdudny, A. et al. Eur. J. Org. Chem. 2011, 9, 1782. [8] Denisenko, O. et al. Org. Lett. 2010, 12, 4372. [9] Mityuk, A. et al. Tetrahedron Lett. 2010, 51, 1790.

P-10

High-throughput screening with a miniaturized radioligand competition assay identifies new modulators of human a2-

adrenoceptors

Fallarero Ac, Pohjanoksa Kc, Wissel Ga,b, Parkkisenniemi-Kinnunen UMd, Xhaard Ha, Scheinin Md, Vuorela Pc

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; bCentre for Drug Research, Faculty of Pharmacy ,University of Helsinki, Finland; cPharmaceutical Sciences,

Department of Biosciences, Abo Akademi University, BioCity, Turku, Finland; dDepartment of Pharmacology, Drug Development and Therapeutics, University of Turku, Unit of Clinical Pharmacology,

Turku University Hospital, Turku, Finland.

G Protein-Coupled Receptors (GPCR) are one of the most targeted receptors and 40% of all drugs available in the market target one or more GPCRs. GPCRs are divided into five different families and α-2 Adrenoceptors (ARs) belong to the A family, these receptors bind adrenaline and noradrenaline.

In human, the α2-ARs have three subtypes A, B and C, all of which have a high sequence identity with about 66% of identical amino acids in the transmembrane region.

Subtype-specific ligands can be used for therapeutic applications or as chemical probes in specific in-vitro assays that can be used to understand the function of these receptors. To date there is no general explanation about subtype specificity, although for a few specific cases a reasonable explanation exists.1,

2, 3

The aim of this work was to identify active compounds for human α-2 Adrenoceptors screening a large chemical library with miniaturized assay.

Based on our studies, we identified the first non-protonable molecule which binds with high affinity to all α2-ARs.4

1. Ruuskanen et al., J Neurochem. 2005, 94, 1559-69. 2. Laurila et al., Br J Pharmacol. 2007, 151, 1293-304. 3. Laurila et al., Br J Pharmacol. 2011, 164, 1558-72. 4. Fallarero et al., Eur J Pharm Sci. 2012, 47, 190-205

P-11

Human Serum Albumin-Based Adducts with Hydrophobic Therapeutic and Diagnostic Agents

Pavel S. Chelushkin,a Maria V. Leko,a Dmitry V. Krupenya,b Roman A. Akasov,c Alexey S. Melnikov,d Yu-Jui Tseng,e Elena A. Markvicheva,c Pi-Tai Chou,e Sergey P. Tunik,b

Sergey V. Burova

a Institute of Macromolecular Compounds, Russian Academy of Sciences, Russia; b Department of Chemistry, Saint-Petersburg State University, Russia; c Shemyakin & Ovchinnikov Institute of Bioorganic

Chemistry, Russian Academy of Sciences, Russia; d Saint-Petersburg State Polytechnic University, Russia; e Department of Chemistry, National Taiwan University, Taiwan

Many promising anticancer drugs and imaging agents are highly hydrophobic. This results in their lower solubility and thus bioavailability. Furthermore, biodistribution profiles of these compounds are often far from desirable which can lead to incorrect imaging and/or severe side effects. To address these drawbacks, various types of carriers are being proposed. Human serum albumin (HSA) is one of the most promising carriers for both drugs and imaging agents. This carrier protein has several hydrophobic pockets and thus can bind various endo- and exogenous hydrophobic compounds thereby increasing their solubility. On the other hand, HSA was shown to accumulate in solid tumors owing to enhanced permeability and retention (EPR) effect. This feature leads to better biodistribution profiles as well as lower systemic toxicity of drugs or imaging agents bound to HSA.

In this work we present two types of HSA-based adducts with hydrophobic compounds potentially useful for either diagnostics (fluorescent imaging) or therapy. In both cases, highly water-soluble adducts were formed upon direct mixing of components in DMSO/water mixture followed by dialysis against pure water and lyophilization. Adduct formation was proved by UV spectroscopy.

For diagnostic purpose we have prepared HSA adducts with several organometallic complexes of the copper subgroup metals (Cu, Ag, Au) that contain homonuclear gold or heteronuclear gold-metal (Au-Cu, Au-Ag) clusters. These complexes demonstrate unprecedented photophysical characteristics compared with other transition metal luminophors: unusually intense room-temperature phosphorescence of wide color range with quantum efficiencies up to 100% and negligibly small effect of emission quenching by molecular oxygen. HSA/complex adducts appeared to be highly soluble in water. UV spectroscopy and luminescent study have shown that photophysical properties of adducts were essentially similar to those of initial complexes with unsubstantial loss in quantum yield. Confocal imaging experiments on HeLa cells have shown that HSA-based adducts were readily internalized into cell cytoplasm.

For therapeutic purpose we have synthesized two palmitoyl-modified doxorubicin (Dox) derivatives: N-palmitoyl-substituted Dox (Palm-N-Dox) and hydrazone of Dox with pamitoyl hydrazide (Palm-hydr-Dox). MTT tests on sensitive MCF7 and Dox-resistant MCF7/R cell lines demonstrated that Palm-hydr-Dox had EC50 value (4 and 25 μM for MCF7 and MCF7/R, respectively) comparable to Dox (1 and 15 μM,

respectively). HSA/Palm-hydr-Dox adduct also had low EC50 (7 and 35 μM, respectively). Despite little lower EC50 values for HSA/Palm-hydr-Dox compared to Dox, we believe that this adduct will show much lower toxicity at systemic administration because of EPR effect.

Overall, the present study shows that binding of hydrophobic molecules to HSA dramatically increases their solubility in water with no or little loss in functionality and makes them useful in both diagnostic and therapeutic applications.

The present work was performed using equipment of "Analytical center of nano- and biotechnology Saint-Petersburg Polytechnic University" core facility and founded by Russian Federation State Budget (contract № 12.37.132.2011) and RFBR (grant № 11-03-92010-NNC-а).

P-12

Triazolothiadiazines with potential anticancer activity against solid tumors

Peri Sevim Aytac1, Irem Durmaz2, Rengül Cetin-Atalay2, Birsen Tozkoparan1

1 Department of Pharmaceutical Chemistry, Hacettepe University, Ankara, Turkey

2 Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey

According to International Agency for Research on Cancer (IARC)-World Cancer Report, cancer is the second leading cause of death worldwide (12.6%) and its projected that this rate will increase twice as much in year 2030. Therefore, there are many scientific effort on the field of cancer research in terms of therapy, prevention and early diagnosis. There are many aspects of cancer drug discovery but one of the ultimate goals is the development of target specific anti-cancer therapeutic agents with high efficacy but low side effects.

Protein kinases are vital component of the cell signaling and have important functions in most intracellular events such as growth and cell cycle, cellular metabolism or DNA damage response. Recent studies have revealed that aberrant catalytic activity of many kinases, via mutation or overexpression, plays an important role in numerous pathological conditions, including cancer. Therefore kinases have become attractive targets in cancer chemotherapy and many small compounds have been developed directly inhibit the catalytic activity of the kinases. Recent studies showed that the molecules with triazolothiadiazine ring have cytotoxic activities in various cancer cells compared to normal cells via inhibition of various kinases. Based on these knowledge, in this study, synthesis and identification of cytotoxic activities of triazolo-[3,4-b]-1,3,4-thiadiazine derivatives is aimed.

Title compounds (1a-c) was obtained by the reaction of an aminomercaptotriazole derivative (1) with appropriate phenacyl bromides (or chlorides) in anhydrous ethanol. Initially, the cytotoxic activities of the compounds were analyzed using NCI60 SRB assay on liver (Huh7), breast (MCF7) and colon (HCT116) cancer cell lines in triplicate. Camptothecin was used as a positive control and DMSO was the vehicle control. IC50 values were ~18-40 µM for compound 1, ~7-19 µM for compound 1a, ~10-17 µM for compound 1b and ~4-9 µM for compound 1c. Due to promising results, additional liver carcinoma cell lines (HepG2, Hep3B and Mahlavu) were examined in the presence of selected coumpunds. The time-dependent cytotoxic activity surveillance was also performed using RT-CES platform for Huh7 cell line. All triazolothiadiazines derivatives (1a-c) had an irreversible growth inhibitory effect on Huh7 cell line in a time and dose dependent manner. Additionally luminescence based kinase assay (with Promega KinaseGlo Assay Kit), demonstrated that compound 1a had the highest kinase inhibitory effect on human liver cancer lines (Mahlavu, Hep3B) and compound 1 had the lowest. The results of the biological activities revealed that cyclization of aminomercaptotriazole skeleton with a thiadiazine ring gives rise to more active compounds and triazolothiadiazine structure could be an attractive scaffold for further modification and design of novel potent compounds.

Acknowledgement: This work was supported by The Scientific and Technological Research Council of Turkey Grant #112S211.

P-13

EVALUATION OF SELECTIVE HUMAN MONOAMINE OXIDASE INHIBITORY ACTIVITIES OF SOME

NOVEL HYDRAZONE DERIVATIVES CONTAINING 2-BENZOXAZOLINONES

Umut Salgın-Gökşen1,2, Samiye Yabanoğlu-Çiftçi3, Açelya Yalovaç3, Gülberk Uçar3, Kemal

Yelekçi4, Nesrin Gökhan-Kelekçi1* 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, 06100, Ankara, 2 Turkish Medicines and Medical Devices Agency, Analyses and Control Laboratories, 06100, Ankara,

3 Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhıye, Ankara-Turkey.

4 Bioinformatics and Genetics Department, Faculty of Engineering and Natural Science, Kadir Has University, 34083 Fatih, İstanbul-Turkey.

*Corresponding author. e-mail:onesrin@hacettepe.edu.tr, onesrin@gmail.com

Depression is one of the most prevalent psychopathologies in the Western world (1). Its therapy relies on classical antidepressant drugs such as monoamine oxidase (MAO) inhibitors and drugs that inhibit the reuptake of catecholamines (2). A common problem with the current antidepressant therapies is the several side effects produced by these drugs besides their slow onset of action. In addition to, there is a delay of about 4 weeks to alleviate the symptoms of depression (3). The classical period of the MAO inhibitors (MAOIs) started with hydrazine derivatives (4) and it was reported that hydrazines were as useful antidepressant agents in treating psychic disturbances associated with over-activity of MAO. Hydrazine/hydrazido-based antidepressants were demostrated as a group of nonselective and irreversible MAOIs, however, most have been withdrawn due to toxic reactions like hepatotoxicity. On the basis of this clinical profile, researchers focused their attention on structural modification of the hydrazine group to enhance the pharmacological activity and to limit the toxicity. The biological activity associated with these compounds was attributed to the presence of the (-CONHN=CH-) moiety. In this context, pyrazole and pyrazoline derivatives was considered as a cyclic hydrazine moiety and their MAO inhibitory activity was also shown in earlier studies (5,6) Prompted by these findings, we have combined 2-(2-benzoxazolinone-3-yl)acetylhydrazine with chalcones to obtain a series of hydrazone derivatives, which were tested against MAO inhibitor activities in order to uncover new leads in the development of novel antidepressant agents. Synthesized compounds were docked computationally to the active site of the human MAO-A and MAO-B isoenzymes. The AutoDock 4.2.1.5 docking simulation and Accelrys’ Discovery Studio visualization programs were employed to find the inhibition constants and their poses in the active sites of both MAO isozymes. The MAO inhibition activities of the inhibitors were compared with the properties of their experimental values. Effects of novel compounds on the activity of MAO were determined using recombinant human MAO-A and –B according to a method previously described (7). hMAO inhibitory activity of the compounds were compared with those of known MAOIs (selegiline and moclobemide).Tested compounds inhibited hMAO-A selectively and potently. Their inhibition was found as competitive and reversible. Compound 3 appeared as the most selective and potent MAO-A inhibitor with the Ki value for hMAO-A as 0.20x10-3 μM and SI value as 7.27x10-5 suggesting that this compound is more potent and selective comparing to moclobemide. Experimental data were in a good agreement with the data obtained from the docking studies. [1] Belmaker, R. H., Agam, G. N., Eng. J. Med. 358, 55 (2008). [2] Tran, P. V., et al, J. Clin. Psychopharmacol. 23 78, (2003). [3] Cryan, J. F., et al, Trends Pharmacol. Sci. 23, 238 (2002). [4] Sahoo, A., et al, Bioorg. Med. Chem. Lett. 20, 132-136 (2010). [5] Gökhan-Kelekçi, N., et al, Bioorg. Med. Chem. 17, 675-689 (2009). [6] Chimenti, F., et al, Med. Chem. Commun. 1, 61-72 (2010).[7] Salgın-Goksen, U. et al, J. Neural. Transm. 120, 883-891 (2013). Acknowledgement: This study was supported by Scientific and Technoloical Research Council of Turkey (Project Number:112T746) and the Hacettepe University Research Fund the Hacettepe University Research Fund (Project Number: 012D06301001).

P-14

SYNTHESIS AND ANTIPROTOZOAL ACTIVITY OF NOVEL TRIAZINE DIMERS

Muthusamy Venkatraj,a Jan Heeres,a Jurgen Joossens,a Pieter Van der Veken,a Paul Cos,b Paul J. Lewi,a Louis Maes,b Koen Augustynsa

aLaboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium; bLaboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp,

Groenenborgerlaan 171, 2020 Antwerp, Belgium

The World Health Organization estimates that neglected tropical diseases (NTDs) caused by protozoan parasites affect over one billion people and in aggregate cause almost 550 000 deaths annually. African sleeping sickness, Chagas disease, leishmaniasis and malaria are the main ones for which the currently used drugs were developed several decades ago. They lack adequate efficacy, cause side effects or are not affordable and increasingly suffer from drug-resistance. Because most of the affected patients live in developing countries and cannot afford drugs, the pharmaceutical industry has traditionally neglected these diseases. Hence, a reasonable approach to discover new lead compounds at a lesser cost is the screening of already available molecules for antiparasitic activity. As part of our research program for the development of novel antiviral agents as microbicides we synthesized and evaluated a library of triazines and triazine dimers (NNRTIs) for antiviral activity.1-3 In the literature, several groups have reported substituted triazines as antiprotozoal agents, which prompted us to screen our in-house microbicidal compounds against these tropical parasites. All the compounds were evaluated for their in vitro activity against T. b. brucei, T. b. rhodesiense, T. cruzi, L. infantum and P. falciparum, and cytotoxicity on a human cell line (MRC-5). Selected compounds were evaluated next for in vivo activity against T. b. brucei (suramin-sensitive Squib 427 strain) in Swiss mice. The determination of antiprotozoal activity (IC50) of triazine dimers in comparison with monomers allowed to draw preliminary conclusions about the structure-activity relationship (SAR) of these series of compounds.

1. Venkatraj M, Ariën KK, Heeres J, Joossens J, Messagie J, Michiels J, Van der Veken P, Vanham G, Lewi PJ, Augustyns K. Bioorg. Med. Chem. Lett. 2012, 22, 7174–7178.

2. Heeres J, Augustyns K, Van der Veken P, Joossens J, Venkatraj M, Maes L, Lewi PJ. PCT Int. Appl. 2013, WO 2013068551 A1 20130516

3. Ariën KK, Venkatraj M, Michiels J, Joossens J, Vereecken K, Van der Veken P, Abdellati S, Cuylaerts V, Crucitti T, Heyndrickx L, Heeres J, Augustyns K, Lewi PJ, Vanham G,. J. Antimicrob. Chemother. 2013, doi:10.1093/jac/dkt166

P-15

Modulation of the kinase mTOR signaling pathways by mycotoxin Fumonisin B1

Elena A. Martinova

Dept. Fundamental Investigations, Russian Medical Academy of the Post-Diploma Education, Ministry of the Public Health & Social Development, Moscow, 123995, Russian Federation

Introduction. The serine / threonine kinase mTOR (Mammalian Target of Rapamycin, M 289 kDa) is considered to be a key kinase integrating a number of extracellular signaling pathways (from nutrients, hormones, energy, etc.) that allows it to regulate the important cellular events including protein synthesis, cell proliferation, autophagy, apoptosis, etc. The enzyme activity of kinase mTOR is requires ATP and is regulated by specific and non-specific inhibitors and activators. In response to the specific signals, kinase mTOR assembles a number of proteins and forms the complexes which are known as mTORC1 (rapamycin is inhibitor) and mTORC2 (DEPDC6, Deptor, Protor are inhibitors). The mTORC1 complex includes mTOR, Raptor, mLST8, PRAS40, HSP70, Akt1S1, etc, and provides signals to the mRNA translation and protein synthesis, regulates signaling pathways of autophagy and apoptosis. The mTORC2 complex includes mTOR, Rictor, mLST8, mSIN1, Protor-1, Deptor, MAPKAP-1, etc. and coordinates the signals from insulin and PI-3-kinase/Akt, couples extracellular signals with cytoskeleton. Nonetheless, some evidences indicate that mTOR may create other protein-specific complexes, for example, on the mitochondrial outer membrane. When we investigated mTOR complex on mitochondria, we found that it was regulated by mycotoxin fumonisin B1 (C34H59NO15, M 721 kDa) which was a member of group B related mycotoxins sphingolipid origin produced by fungi Fusarium proliferatum, F. moniliforme and allied species spread widely abroad. Fumonisins of group B are the human and animal carcinogens. Because activation of mTORC1 is very important question in the regulation of normal and tumor cell proliferation, we made an effort to study an influence of fumonisin B1 on an expression of some proteins from upstream and downstream signaling pathways of mTOR. Methods. Fumonisin B1 has been injected intraperitoneally to the male mice (CBA and C57Bl/6) in the doses 0.01–10 microM per mouse. Mice were killed after 15, 30, 60, 90 minutes, 2h, 3h, and 6h after injection. All experiment was been made according to the rules of work with laboratory animals. Statistical analysis from 5 experiments was provided using ANOVA. Cells from thymus, spleen, liver, and whole brain has been received by homogenization in the complete DMEM medium with 5% of serum. Separated cells were stained with antibodies to the mTOR-related proteins followed by an analysis on the flow cytometer. Results. We found the similar action of fumonisin B1 on all types of normal mouse cells in the case of its regulation of mTOR-related proteins. Fumonisin B1 dose-dependently inhibits an expression of protein mTOR during 6h with the maximum effect at 1h. Both mTORC1 and mTORC2 complexes are regulated by upstream inhibitors TSC1 and TSC2 (proteins hamartin and tuberin). After 60 min fumonisin B1 inhibited the TSC1 and TSC2 protein expression followed by the partial restoration of their expression. Scavenger protein 14-3-3 provides, particularly, a connection between mTORC1 and mTORC2 complexes that may be regulated by fumonisin B1 which inhibits 14-3-3 protein expression during 6h without restoration of its activity. Expression of other proteins connected with mTOR (Raptor, Rictor, etc.) has been found to be regulated by fumonisins B1 dose-dependently. Downstream mTOR signaling protein 4E-BP1 (eukaryotic initiation factor 4E binding protein-1) is regulator of translation activation. Fumonisin B1 has the bimodal effect of the 4E-BP1 expression during 6h and acts dose-dependently. Other downstream mTOR messenger is the ribosomal kinase p70 S6K1 which initiates the protein synthesis is also regulated by fumonisin B1 bimodal and dose-dependently. General negative influence of fumonisin B1 on the mTORC1 signaling proteins leads to the decreased cell proliferation and activation of an apoptosis. References: 1) Martinova, E.A. & Ivanchenko, O.B. Mycotoxin fumonisin B1 and regulation of microibiota. – Moscow, Publish House of the Federal State Institution “All-Russian Scientific Research Institute of Civil-Defense and Emergencies”, EMERCOM of Russia, - 2006. – 350 P., ill. – ISBN 5–93970–021–7. 2) Martinova E.A. mTOR kinase and its role in the cell stress response Biochemistry (Moscow), Series A: Membrane and Cell Biology (ISSN 1990-7478), 2012, Vol.6, N1, P.9-15.

P-16

Phosphate and ribose structural isosteric replacement in the Protein Data Bank

Yuezhou Zhanga, Alexandre Borrela,c, Gustav Boije af Gennäsb, Jari Yli-Kauhaluomab, Henri Xhaarda

a Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Finland

b Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland

c INSERM, UMRS 973, MTi, Paris, France; Univ Paris Diderot, Sorbonne Paris Cité, UMRS 973, MTi,

Paris, France.

In this work, we have studied the structural isosteric replacements of phosphate and ribose found in protein-ligand complexes available in the Protein Data Bank database (August of 2012 release). We developed a computational protocol that was used to construct 157 datasets. Each of these datasets is composed of several superimposed ligands, including POP, AMP or ADP to use as references, derived from superimposed molecular complexes. Structural replacements of ribose and phosphate groups were then extracted and studied: we identified15 common structural isosteres of phosphate and 43 structural isosteres of ribose. In addition to classical isosteres of phosphate, we found unexpected types of replacements that do not conserve charge or polarity, for example phosphate and ribose replaced by aliphatic groups, phenyl, or carbamoyl groups. The structural mechanism involved in structural isosteres appears varied: New interactions may be created, water molecules are important, in some case ion plays a role, and of course large and small conformational changes do occur at the binding sites. This study has implications both in the field of medicinal chemistry, i.e. it expands our knowledge of structural isosteres, and in the field of chemoinformatics, since our results have implications with respect to the definitions of chemical similarity.

P-17

Direct Targets Identification of a Bioactive Compound Using Chemical Biology

Sylvain Blanc1, Paul Bradley1, Marie-Edith Gourdel2, Michaël Cholay2, Pierre Gontard2, Gisèle Guimèse2, Mike Mckenzie1, George Nasi1, Jean-Christophe Rain2 and Barbara

Rugiero2

1 Charnwood Molecular Ltd, the Heritage Building, 7 Beaumont Court, Prince William Road, Loughborough, LE11 5GA, UK

2 Hybrigenics Services SAS, 3-5 impasse Reille, 75014 Paris, France.

Identifying protein partners of a small bioactive molecule is of great interest in many aspects of life sciences and specifically in the drug discovery and development process cycle. It is a support to (i) decipher the mechanism of action after for example a “High Content” screening, (ii) study “off-target” effects, (iii) adjust therapeutic indications and clinical regimens of a drug and (iv) consider drug repositioning.

ULTImate YChemH is a chemical biology tool for direct targets identification. This method is based on the well-established yeast two-hybrid (Y2H) technology. The small molecule of interest is used as a bait to screen highly complex protein domain libraries. The small molecule - protein interactions are detected thanks to the reconstitution of an active transcription factor from DNA Binding Domain (DBD) and Activation Domain (AD) moieties. When an interaction takes place, the bait derivative bridges the gap between the DBD and the AD. This enables the expression of the reporter gene and subsequent yeast growth on a selective medium. Positive clones are then analyzed by sequencing to identify the protein partners.

One example with a hit coming from a “black box” screen highlights how this technology can be easily applied for target identification.

P-18

Salbutamol increases MKP-1 expression and inhibits the production of inflammatory mediators TNF and NO in

activated macrophages.

Keränen Tiina, Hömmö Tuija, Hämäläinen Mari, Moilanen Eeva, Korhonen Riku

The Immunopharmacology Research Group, University of Tampere school of Medicine and Tampere University Hospital, Tampere, Finland

Introduction: Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a nuclear phosphatase that dephosphorylates (i.e. inactivates) p38 MAP kinase, and inhibits cytokine production and inflammatory response. Salbutamol is a bronchodilating drug that activates β2-receptors and elevates cAMP levels. We have recently shown that a PDE4 inhibitor rolipram up-regulated MKP-1 expression by a mechanism dependent on cAMP-PKA-CREB pathway, and using MKP-1 knockout mice we found that MKP-1 mediated anti-inflammatory effects of rolipram in vivo. Therefore we investigated the effect of salbutamol on the expression of MKP-1 and the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO).

Methods: The effects of salbutamol on MKP-1 expression, p38 MAP kinase phosphorylation and the production of TNF and NO were investigated in primary mouse peritoneal macrophages (PM) and J774 mouse macrophages. MKP-1 expression was determined by quantitative RT-PCR and Western blot, and p38 MAP kinase and CREB phosphorylation by Western blot. TNF and NO accumulated into the culture medium were measured by ELISA and Griess reaction, respectively.

Results: Salbutamol (1-1000 nM) alone and in combination with LPS (10 ng/mL) increased intracellular cAMP and, interestingly also MKP-1 expression in J774 macrophages in a dose-dependent manner. cAMP analog 8-Br-cAMP (100 µM) also enhanced LPS-induced MKP-1 expression. Increase in MKP-1 expression by salbutamol was reversed by a PKA inhibitor. Salbutamol (100 nM) inhibited LPS-induced production of TNF and NO. Salbutamol reduced LPS-induced phosphorylation of p38 MAP kinase. Accordingly, p38 MAP kinase inhibitor BIRB 796 (1 µM) inhibited TNF and NO production in J774 macrophages and TNF production in PMs.

Conclusions: Salbutamol increased the expression of MKP-1 and concomitantly inhibited the phosphorylation of p38 MAP kinase and the production of TNF and NO in mouse J774 macrophages and PMs. The results suggest that the anti-inflammatory effects of salbutamol on TNF and NO production are mediated, at least partly, by increased MKP-1 expression, which limits p38 MAP kinase pathway and, subsequently, inflammatory gene expression.

P-19

Novel triterpene derivatives as antineoplastic agents

Raisa Haavikko,a Matthias Nees,b Johannes Virtanen,b Ville Härmä,b Kirsi-Marja Oksman-Caldentey,c and Jari Yli-Kauhaluomaa

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; b

Biotechnologies for Health and Well-being, Bio and Process Technology, VTT technical Research Centre of Finland, Turku, Finland; c Cell Factory, Bio and Process Technology, VTT technical Research Centre

of Finland, Espoo, Finland

Betulin is a plentiful naturally occurring lupane-type pentacyclic triterpene. It can be easily extracted from outer bark of white birch trees, which are widespread in the northen latitudes of the world.1 Betulinic acid and other betulin derivatives show antiviral, anti-HIV, anti-inflammatory, anti-malarial, and cytotoxic effects.2 Now we have synthesized a new set of heterocyclic betulin derivatives and tested their activity against cancer cell lines PC-3 (AR negative, spontaneously invasive), LNCaP (AR positive), LAPC-4 (AR positive, androgen-dependent), and EP156T (benign basal-type epithelial). Results were promising and will be presented more detailed in the poster including structure-activity relationship analysis.

1. C. Eckerman, R. Ekman, Comparison of solvents for extraction and crystallisation of betulinol from bich bark waste. Pap. Puu 1985, 67, 100.

2. Alakurtti, S.; Mäkelä, T.; Koskimies, S.; Yli-Kauhaluoma, Pharmacological properties of the ubiquitous natural product betulin. J. Eur. J. Pharm. Sci. 2006, 29, 1-13.

P-20

PDE 4 inhibitor rolipram inhibits TNF production and carrageenan-induced paw inflammation by elevating MKP-1

through cAMP-PKA-CREB pathway

Tuija Hömmöa, Mirka Laavolaa, Tiina Keränena, Mari Hämäläinena, Eeva Moilanena ja Riku Korhonena

aThe Immunopharmacology Research Group, University of Tampere School of Medicine and Tampere University Hospital, Tampere, Finland

Mitogen-activated protein kinase phosphatase -1 (MKP-1) expression is regulated by cAMP-PKA-CREB pathway, and MKP-1 suppresses p38 MAPK activity and serves as an endogenous factor limiting inflammatory response. Phosphodiesterase (PDE) 4 coordinates cAMP signalling by rapidly hydrolyzing cAMP. PDE4 inhibitors prevent cAMP degradation leading to the elevation of cAMP levels. In this study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and inflammatory gene expression, and whether MKP-1 would be involved in the anti-inflammatory effects of rolipram.

Rolipram increased intracellular cAMP levels, as expected. Rolipram enhanced also CREB phosphorylation and MKP-1 expression, and those effects were reversed by a PKA inhibitor. Rolipram inhibited the phosphorylation of p38 MAPK and it was returned by the PKA inhibitor. Rolipram inhibited TNF production in PM from WT mice, while rolipram was clearly less effective to inhibit TNF production in PM from MKP-1 (–/–) mice. Carrageenan-induced paw oedema was markedly attenuated by rolipram in WT mice, while rolipram did not have any effect on paw oedema in MKP-1(–/–) mice.

In conclusion, PDE4 inhibitor rolipram increased MKP-1 by a mechanism dependent on cAMP-PKA-CREB pathway. The inhibition of TNF production and acute inflammation in vivo by rolipram was mediated by MKP-1. The results suggest that MKP-1 is an important factor in mediating anti-inflammatory effects of PDE4 inhibitors.

1. Korhonen R, Hömmö T, Keränen T, Laavola M, Hämäläinen M, Vuolteenaho K, Lehtimäki L, Kankaanranta H, Moilanen E.: Attenuation of TNF production and experimentally-induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase-1. Brit J Pharmacol (in press), 2013

P-21

Synthesis of Clathrodin Analogues for Antibacterial and Antiviral Screening

Paula S. Kiurua, Teppo O. Leinoa, Sofia Montalvãob,c, Päivi Tammelab, Jari Yli-Kauhaluomaa

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, bCentre for Drug Research, Faculty of Pharmacy, University of Helsinki, cDivision of Pharmaceutical Biology, Faculty of

Pharmacy, University of Helsinki ,P.O. Box 56, FIN-00014 University of Helsinki, Finland

Clathrodin is a marine alkaloid isolated from the marine sponge Agelas clathrodes. The methanol extracts of this Caribbean sea sponge have shown weak antibacterial and cytotoxic activity.1 Several 2-aminobenzothiazole and benzimidazole analogues based on the clathrodin structure have been synthesized. For the 2-aminobenzothiazole analogues a convenient four-step synthesis route was developed. The benzimidazole analogues were obtained by a three-step synthesis. The compounds were submitted to antibacterial and antiviral screening. One 2-aminobenzothiazole derivative showed moderate antiviral (Hepatitis C virus (HCV) replicon model) activity.

1. Morales, J. J.; Rodriguez, A. D. J. Nat. Prod. 1991, 54, 629-631.

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Hydrocarbon Isosteres of Disulfide Bridges in Peptides that Stimulate the Proteolytic Activity of KLK3

Kristian Meinander,a Janne Weisell,b Miikka Pakkala,b Can Hekim,c Roope Kallionpää,a Ale Närvänen,b Hannu Koistinen,c Ulf-Håkan Stenman,c Kristina Luthmand and Erik A.A.

Walléna

aDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland; bSchool of Pharmacy, University of Eastern Finland, Kuopio, Finland; cDepartment of Clinical Chemistry, University of Helsinki, Finland; dMedicinal Chemistry, Department of Chemistry and Molecular Biology, University of

Gothenburg, Sweden

Poster presentation of OC-2, same abstract

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An improved in vitro method for studying bisphosphonate binding to hydroxyapatite by 31P NMR spectroscopy

Elina Puljula, Aino-Liisa Alanne, Petri Turhanen, Janne Weisell, Jouko Vepsäläinen

School of Pharmacy, Biocenter Kuopio, University of Eastern Finland, PL 1627, 70210 Kuopio, Finland

Bisphosphonates (BPs) are used in the treatment of osteoporosis and other skeletal disorders and also for bone imaging.1 BPs chelate especially divalent cations and can therefore bind to calcium containing hydroxyapatite (HAP), which is the main mineral found in human bone.2 Their ability to bind to bone is the basis of their use in medicine, which is why bone affinity is usually a crucial factor when producing new medically interesting BPs. Thus, we were interested in developing a fast and easy method for assessing BP binding to HAP.

Several methods have already been used to determine BPs affinity to HAP and bone powder. For example, solid state NMR3, constant composition kinetic studies of crystal growth4 and fast performance liquid chromatography with HAP columns5 have been used. We chose NMR spectroscopy because quantitative data can be obtained with minimal sample preparation steps. 31P-nucleus was preferred to avoid dominating solvent peaks in the spectra and to enable the use of this method with all BP structures. A similar method by using 1H NMR has been previously described by Jahnke.6

We analyzed the HAP binding of nine different BPs, including several clinically used BPs. The concentrations of BPs were determined from solutions before and after they were incubated with HAP by 31P NMR by using an external standard. We also measured BP concentrations from the dissolved HAP to compare them with the difference between the before- and after-concentrations. While the method presented here seems quite obvious for determining HAP binding for phosphorus containing BPs we encountered various unexpected difficulties during this project. This led us to find several factors affecting the measurement procedure and the reliability of this kind of method.

1. R. G. G. Russell, Bone, 2011, 49, 2-19. 2. H. Uludag, Curr Pharm Design, 2002, 8, 1929-44. [Arial, 9 pt, left, numbered list] 3. G. Grossmann, A. Grossmann, G. Ohms, E. Breuer, R. Chen, G. Golomb, H. Cohen, G. Hägele, R. Classen.

Magn Reson Chem, 2000, 38, 11-6. 4. G.H. Nancollas, R. Tang, R.J. Phipps, Z. Henneman, S. Gulde, W. Wu, A. Mangood, R.G.G. Russell, F.H.

Ebetino. Bone, 2006, 38, 617-27. 5. M.A. Lawson, Z. Xia, B. L. Barnett, J.T. Triffitt, R. J. Phipps, J. E. Dunford, R. M. Locklin, F. H. Ebetino, R.

G. G. Russell. J Biomed Mater Res B Appl Biomater, 2010, 92, 149–55. 6. W. Jahnke, C. Henry. ChemMedChem, 2010, 5, 770-6.

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Surface structure of the drug delivery liposome and its interactions with elements of blood stream – a molecular

dynamics view

Aniket Magarkara, Michal Stepniewskia, Esra Karakasa, Tomasz Rogb, Marjo Yliperttulaa , Arto Urttia , Alex Bunkera

aCentre for Drug Reseaerch, Faculty of Pharmacy, University of Helsinki, Finland; bTechnical University of Tampere

The criteria for effectiveness of drug delivery liposomes (DDLs) are structural stability, site specific targeting, and lifetime in the bloodstream. Often to increase the lifetime in bloodstream DDL is coated with poly ethylene glycol (PEG). Although it helps to improve the lifetime, there exists plenty of room for improvement in bloodstream lifetime efficacy. The search for an alternative to PEG is a very active field of research, but to apply rational design to this, a knowledge of the mechanism through which PEG functions in a superior fashion to other superficially similar polymers must be determined, and currently our understanding of this is incomplete. We have used molecular dynamics simulation of a set of PEGylated membranes in varying conditions to gain insight into this. We have also performed MD simulation with the Cholesterol as a formulation component of DDL at its effect on stability of PEGylated DDL. Lastly we looked at the factors in the targeted delivery of the novel targeting moiety identified with phage display experiments. The moiety couldn’t increase the efficacy of DDL when tested invitro and invivo. By MD we could identify the factors responsible for this by investigating surface structure of DDL.

7. M. Stepniewski et al., Langmuir 2011, 27, 7788–7798 8. K. Knop et al., Angew. Chem. Int. Ed. 2010, 49, 6288– 6308 9. M. Stepniewski et al., J. Phys. Chem. B 2010, 114, 11784 – 11792. 10. J. W. Holland et al., Biochemistry 1996, 35, 2618 -2624

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First bisphosphonate hydrogelators: potential composers of biocompatible gels

Aino-Liisa Alannea, Manu Lahtinenb, Miika Löfmanb, Petri Turhanena, Erkki Kolehmainenb, Jouko Vepsäläinena, Elina Sievänenb

aSchool of Pharmacy, Biocenter Kuopio, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland; bDepartment of Chemistry, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland

Recently, investigation of hydrogels has gained ever increasing attention mostly because of their biomedical and pharmaceutical properties, and novel hydrogelators are constantly studied to find functional applications. The traditional hydrogels consist of synthetic and natural polymers, but recently low-molecular-weight gelators (LMWG) have intrigued because of their special properties related to self-assembly.1 Hydrogels have been used extensively in the development of drug delivery systems, and synthesis of new hydrogelators with better biocompatibility is essential for successful applications.

Bisphosphonates (BPs) are well-known compounds mostly used in clinic as drugs for bone-related diseases, such as osteoporosis and Paget’s disease.2 In this study, a novel class of BP-hydrogelators together with a BP-organogelator was found, three of which have a long alkyl chain (with the length of 11 to 13 carbons) attached to the central carbon (1-3) and one being an acetyl derivative of etidronate (4) (Fig. 1). Compounds 1-3 formed hydrogels and compound 4 a gel in a mixture of two organic solvents. The gelating properties of the compounds were studied, and several techniques were used to analyze the structure of the formed gels. These included solid state 13C and 31P CPMAS and solution state NMR spectroscopy (variable temperature and concentration-dependent 1H NMR), IR spectroscopy, PXRD, thermoanalysis, as well as SEM. Since BPs are generally known to be non-toxic and well-tolerated compounds, these novel BP-gels could be used, for example, as matrices that bind drugs to administrate, deliver, and release them.

Figure 1. Structures of bisphosphonate gelators 1–4.

1. Estroff LA, Hamilton AD. Chem. Rev. 2004, 104, 1201-1218. 2. Russel RGG. Bone 2011, 49, 2-19.

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Radiosynthesis Development for a 11C-Radiolabeled Ketoprofen–Lysine Prodrug

Mirkka Sarparanta,a Venla Kuhmonena, Dave Luména, Krista Laineb, Jarkko Rautiob, and Anu J. Airaksinena

aLaboratory of Radiochemistry, Department of Chemistry, University of Helsinki, Finland; bDepartment of Pharmacy, University of Eastern Finland, Kuopio, Finland

Prodrug strategies are intensely investigated as a means for drug transport across the blood-brain barrier (BBB) to the central nervous system (CNS). Herein, the radiosynthesis development for a carbon-11-radiolabeled ketoprofen–lysine prodrug1 capable passing into the CNS via uptake by the large neutral amino acid transporter 1 (LAT1) is presented. Radiosynthesis of [11C]ketoprofen was set up from [11C]CH3I produced from [11C]CO2 using the “wet method” on a Scintomics Hotbox Three radiosynthesis module. Briefly, [11C]CO2 was trapped to 0.1 M LiAlH4 in THF at -10°C, treated with aqueous hydriodic acid (<55%) at +112°C. The labeling precursor was subsequently distilled to a vial containing a benzoylphenyl acetic acid methyl ester precursor (4 µmol) and base (KOH, LDA, or LiHMDS, 10 µmol) in either THF or 1:2 (v/v) DMSO:DMF 2. [11C]ketoprofen was purified with solid-phase extraction and analyzed with HPLC (Waters µBondapak C18 column, 70:30 0.01M H3PO4–CH3CN, 2.5 ml min-1). The 11C-radiolabeled ketoprofen–lysine prodrug can be synthesized analogously from the corresponding 9-borabicyclononane –protected precursor. Initial radiolabeling syntheses for [11C]ketoprofen yielded only modest decay-corrected radiochemical yields (RCY) from 7–19%. Further optimization of the reaction conditions was thus clearly needed in order to improve the RCY, and the optimization together with the radiolabeling synthesis of the ketoprofen–lysine prodrug are presented. The synthesized [11C]ketoprofen–lysine prodrug is envisioned to have utility positron emission tomography (PET) imaging of LAT1-mediated drug delivery and neuroinflammation.

1. Gynther M, et al. (2010) Int. J. Pharm., 399: 121–128 2. Thominiaux C, et al. (2007) J. Labelled Compounds Radiopharm., 50: 229–236.

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Insights on the structure-activity relationships of compounds binding to Large Amino Acid Transporter 1

(LAT1)

Henna Ylikangas, Lauri Peura, Kalle Malmioja, Mikko Gynther, Kristiina M. Huttunen, Jukka Leppänen, Krista Laine, Maija Lahtela-Kakkonen, Antti Poso, Jarkko Rautio

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland

LAT1 is a transmembrane protein located at the blood-brain barrier (BBB) where it functions mostly as an influx transporter and carries important nutrients from blood to brain. Its endogenous substrates include neutral amino acids with hydrophobic side chain (e.g. L-tyrosine, L-phenylalanine, and L-leucine). LAT1 is also involved in brain delivery of drug molecules mimicking amino acids (e.g. levodopa, gabapentin,and baclofen). The broad substrate specificity and its relatively high expression at the BBB make LAT1 a promising carrier of poorly penetrable drugs into the brain in central nervous system disorders such as Alzheimer’s and Parkinson’s diseases and brain tumours. Using prodrug technology in which a substrate of LAT1 is conjugated with an active drug molecule in a bioreversible manner it is possible to transport relatively higher amounts of the drug into the brain1,2 and reduce side effects in the peripheral tissues.

In order to design new substrates for LAT1 it is very important to identify the molecular features and their three-dimensional (3D) arrangement in space required for efficient binding. Here, the 3D quantitative structure-activity relationships (QSAR) of amino acid mimicking prodrugs, endogenous substrates of LAT1 and other amino acid derivatives were analyzed using pharmacophore modeling and comparative molecular field analysis (CoMFA). LAT1-affinity measured with the in situ rat brain perfusion technique was used to determine the biological activity of the compounds for the models.

Based on 3D pharmacophore model the most important molecular features which can benefit efficient LAT1-binding have been identified: 1) hydrogen bond donor and negative charge ~3Å distance from each other, 2) aromatic region next to the donor atom and negative charge, 3) hydrogen bond acceptor following the aromatic region at ~3.8Å.3 Moreover, CoMFA was able to suggest chemical modifications that can improve the biological activity of LAT1-binding compounds. The data obtained from CoMFAwas employed succesfully in optimizing the structures of five new LAT1-binding substrates.4 Such compounds can be applied in further design of novel prodrugs for improved LAT1-mediated permeation of the BBB.

1. Gynther M, Laine K, Ropponen J, Leppänen J, Mannila A, Nevalainen T, Savolainen J, Järvinen T, Rautio J. J. Med. Chem. 2008, 51, 932-936

2. Peura L, Malmioja K, Laine K, Leppänen J, Gynther M, Isotalo A, Rautio J. Mol. Pharm.2011, 8, 1857-1866 3. Ylikangas H, Peura L, Malmioja K, Leppänen J, Laine K, Lahtela-Kakkonen M, Poso A, Rautio J. Eur. J.

Pharm. Sci. 2012, 48, 523-531. 4. Malmioja K, Ylikangas H, Peura L, Gynther M, Huttunen KM, Leppänen J, Laine K, Lahtela-Kakkonen M,

Poso A, Rautio J. 2013, unpublished results