7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

Day 1 - Wednesday, May 30, 2012

7:00 Registration & Continental Breakfast 7:55 Welcome & Opening Remarks

KEYNOTE PRESENTATION

8:00 Dynamic Re-Wiring of Signaling Networks - A

Rational Approach to Optimizing Combination Therapy for Cancer

Michael B. Yaffe, M.D., Ph.D., Professor, Biology and Biological Engineering, Massachusetts Institute of Technology Protein kinases and phosphoserine/threonine-binding domains such as 14-3-3 proteins, FHA domains, Polo-box domains, and BRCT domains function together within signaling networks to control growth factor responses, cell cycle progression, and the response to DNA damage. How signals from these pathways are integrated and processed to control cell phenotypic responses is unclear. To address this, we have been developing systems biology-based models of DNA damage signaling where kinase activities, protein phosphorylation, and phosphoprotein-binding events for multiple signaling pathways are quantitatively measured at densely sampled points in time, along with cellular responses such as cell cycle arrest, autophagy, and apoptosis. The resulting large dataset of signals and responses are then related to each other mathematically using partial least squares regression, principal components analysis, and time-interval stepwise regression. We have used this approach to examine the response of breast cancer and osteosarcoma cells to DNA damaging chemotherapy and gamma radiation in the presence or absence of small molecule inhibitors of growth factor signaling pathways. The resulting models, built from thousands of signaling measurements and hundreds of cellular response assays, reveals surprisingly paradoxical context-dependent roles for several well-established protein kinase signaling pathways in controlling cell cycle arrest, apoptosis and senescence after DNA damage. Furthermore, the analysis has identified novel treatment approaches that can therapeutically re-wire signaling pathways within tumor cells to dramatically improve tumor cell killing by conventional clinical agents.

8:45 Kinase Inhibitor Selectivity Jeffrey Peterson, Associate Professor, Fox Chase Cancer Center

We recently reported a large-scale analysis of kinase inhibitor selectivity (Anastassiadis et al., Nat Biotech, 2011). 178 compounds were tested against 300 recombinant human protein kinases in substrate phosphorylation assays. The compounds included FDA-approved drugs, compounds in the clinical pipeline, and research tool compounds. Many off-target interactions were observed with seemingly unrelated kinases, revealing how large-scale profiling can identify multi-targeted inhibitors of specific, diverse kinases. I will make the case for large-scale inhibitor profiling as a new approach to screening for drug candidates and will highlight novel and surprising inhibitor-kinase interactions revealed by this study. This represents a new paradigm in drug discovery in which chemical entities are screened in parallel against large kinase panels (a compound-centric approach) up front, rather than screening compounds against individual kinases (a target-centric approach). This approach reveals the full spectrum of kinase targets early in the process, thus reducing late stage compound attrition due to unknown off-target activities. Furthermore, this approach will greatly facilitate the identification of "multi-targeted" inhibitors which are currently of great interest. 9:10 Discovery of a Series of Potent, Selective and Orally Available Bruton’s Tyrosine Kinase (BTK) Inhibitors by Rational Drug Design Yan Lou, Principal scientist, Discovery Chemistry, Hoffmann-La Roche Structure based drug design was used to guide the optimization of a series of selective BTK inhibitors with poor drug-like properties. Highlights included the discovery of a benzyl alcohol group with over tenfold increase of activity due to cooperative hydrogen bonding with Lys_Asp salt bridge and a fluorine substitution with over tenfold increase of activity. Concurrent optimization to address other issues such as high lipophilicity, primary aniline metabolite, and low solubility led to RN486 with whole blood IC50 of 17 nM for CD69 inhibition. RN486, as a reversible, potent and selective BTK inhibitor, showed significant in vivo efficacy in various animal models for RA. The discovery and subsequent profiling of RN486 further strengthened the biological rationale of BTK inhibition as a potential novel approach to treat and benefit RA patients. 9:35 Dustin Maly, Assistant Professor, Chemistry, University of Washington 10:00 Networking & Refreshment Break 10:30 Compound Centric Kinome-wide Profiling for Kinase Inhibitor Discovery Xianming Deng, Principal Investigator, School of Life Sciences, Xiamen University Potent and selective kinase inhibitors are highly desirable, both as tool compounds for dissecting complex signaling pathways and as potential therapeutics for treating kinase-related diseases. Hence there is an urgent need to develop methodologies that will allow for the rapid identification of

Session I: Trends in Drug Design - Structure/ Selectivity/ Pathway/ Epigenetics

Moderator: Jonathan Higgins, Assistant Professor, Medicine, Brigham and Women's Hospital

7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

kinase inhibitors of interest. Compound centric kinome-wide profiling enables rapid discovery of the full range of kinase-targets for a particular scaffold. Here we describe the development of potent and selective ERK5(BMK1), LRRK2 inhibitors using 'compound-centric' profiling of designed focus kinase-directed library deriving from a pyrimido-diazepine scaffold. Benefits: •Compound centric kinome-wide profiling approach presented • Focus kinase-directed library design presented • Potent and selective kinase inhibitors of ERK5(BMK1) and LRRK2 presented 10:55 Incorporation of Binding Kinetics into Lead Optimization Peter Brandt, Ph.D., Principal Scientist, Beactica AB Binding kinetics has emerged as a key parameter for understanding clinical efficacy and. pharmacodynamics

1,2,3,4

In addition, binding kinetics, as manifested by the residence time, can be of significance to target selectivity and off-target effects. However, there has been a lack of enthusiasm among medicinal chemists to add kinetic considerations to their already long list of parameters to optimize. One of the reasons is the lack of literature describing approaches for kinetics optimizations. Another reason is the lack of public datasets for method development. In fact, there are only a few datasets available describing kinetic data suitable for QSKR analysis. One of them, describing a series of compounds binding to p38 MAP kinase, was published by Regan et al. in 2003 (Boehringer Ingelheim Pharmaceuticals, Ridgefield).5 Using this dataset, we are able to show that kinetic data is as amenable to QSKR modelling as affinity data are to QSAR modelling. Thus, as most of us agree that kinetics can play a role in the effect of a drug, it seems like the incorporation of binding kinetics into lead optimization should be as doable as ordinary multiobjective optimization of affinity and preclinical properties. Highlights of the presentation: • Importance of binding kinetics • Quantitative Structure-kinetics modeling 1. Holdgate, G. A.; Gill, A. L. Drug Discovery Today 2011,

16, 910. 2. Lu, H.; Tonge, P. J. Current Opinion in Chemical

Biology 2010, 14, 467. 3. Swinney, D. C. Current Opinion Drug Discovery and

Development 2009, 12, 31. 4. Tummino, P. J., Copeland, R. A. Biochemistry 2008,

47, 5481. 5. Regan, J. et al. Bioorganic & Medicinal Chemistry

Letters 2003, 13, 3101. 11:20 Knowledge Based Target Evaluation and Compound Design for Spleen Tyrosine Kinase (Syk) Li Xing, Ph.D., Senior Principal Scientist, Pfizer

SYK (spleen tyrosine kinase) is a non-receptor tyrosine kinase that forms part of the immune complexes via binding of its SH2 domain to the diphos-ITAM in the cytoplasmic region. Syk acts upstream at the initiation of multifactorial signaling pathways thus could be more advantageous to other drugs that act on a single downstream event. A SYK inhibitor from Rigel (R788) has demonstrated positive proof of concept (POC) in phase II rheumatoid arthritis (RA) patients. However, several adverse effects were also associated with R788 in clinical studies, possibly attributed to its non-selective inhibition of a large number of kinases. To assess the doability of a specific SYK inhibitor we have taken a knowledge-based approach. Through bioinformatics analysis of human kinome combined with chemoinformatics mining of kinase selectivity data, a selectivity hypothesis was generated and tested. 11:45 The Resurgence of Covalent Drugs Juswinder Singh, Ph.D., Founder & Chief Scientific Officer, Celgene Avilomics Research 12:10 Lunch On Your Own

FEATURED PRESENTATION

1:40 Development and Characterization of Covalent Kinase Inhibitors Nathanael S. Gray, Ph.D. , Principal Investigator, Biological Chemistry and Molecular Pharmacology, Dana Farber Cancer Institute , Harvard Medical School There is an ever increasing demand for new protein kinase inhibitors both as potential therapeutics and as tools for dissecting complex signaling pathways. The vast majority of currently developed kinase inhibitors target the ATP-binding site and bind in a reversible fashion. An alternative mode of kinase inhibition involves developing compounds that form permanent covalent bonds with nucleophilic residues such as cysteines or lysines. A survey of the kinome reveals that there are over 150 kinases that possess a potentially accessible cysteine in or around the ATP-binding site. Despite this abundance of potential targets only a very limited of selective covalent kinase inhibitors are currently known. I will describe two distinct and general methods for efficiently developing novel covalent inhibitors with examples provided for T790M EGFR, FGFR and JNK. Specific benefits- • Broad survey of kinase potential targetable by covalent inhibitors • Description of general approaches for covalent kinase modification • Specific examples of covalent inhibitors of T790M EGFR, FGFR and JNK • Examples for how covalent inhibitors can be characterized

7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

Session II: Oncology Kinases & Inhibitor Candidates

Moderator: Juswinder Singh, Ph.D., Founder & Chief Scientific Officer, Celgene Avilomics Research

2:05 Using High Content Imaging of Cultured 3D Tumour Micro-tissues to Profile Kinase Inhibitors Leo Price, Faculty of Science, Leiden Amsterdam Center for Drug Research Because cells cultured in a three-dimensional extracellular matrix environment acquire many of the functional characteristics of human tissues, the effects of compounds are more likely to reflect their effects in humans. Furthermore, the complex phenotype of 3D cell cultures can be captured by high content imaging and exploited to provide rich information on the effect of a treatment on the biological system. However, 3D cultures have yet to be incorporated in routine screening of small molecules and biologics. Here, we discuss some of the pro’s and con’s of using 3D cell cultures in the screening and characterization of novel kinase inhibitors and other bioactive molecules. While posing challenges in imaging and image analysis, the high-content image data that can be obtained can provide rich information on the effects of compounds on the biological system. Multi-parametric analysis methods can be applied to the highly complex image data to enable phenotypic profiling of compounds and their subsequent classification and comparison with reference molecules. Some of these features are illustrated with examples from our own 3D screening platform, which we have used in the profiling of small molecule inhibitors and therapeutic antibodies. 2:30 Discovery of Selective CK1g Antagonists that Inhibit Canonical Wnt-Signaling Erin DiMauro, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen CK1g phosphorylates the cytoplasmic domain of the Wnt co-receptor LRP6 to create multiple Axin binding sites, which are required for the transduction of the Wnt signal to the nucleus. An ATP-competitive, small molecule kinase inhibitor of CK1g is hypothesized to function as “pan-Wnt pathway inhibitor” for the treatment of cancers that have evidence of Wnt pathway activation. A major hurdle in developing an effective CK1g inhibitor for use as an antagonist of the Wnt pathway is gaining selectivity for the gamma isoform over the closely related family members (alpha, delta and epsilon) which have been shown to have multiple and sometimes opposing effects on Wnt signaling. Beginning with hits from two different chemotypes, we used a structure-based approach to design a hybrid pyrrolopyrimidine pyridine series. Optimized leads affording good potency, selectivity and oral exposure in rodents were used to establish a powerful PK/PD relationship in a mouse tumor model of proximal signaling. Lead compound 10

(CK1g IC50 = 4 nM, >150x selective over other Wnt-pathway kinases) demonstrated dose-dependent inhibition of LRP6 phosphorylation with an unbound in vivo IC50 of 20 nM. The successful development of highly selective, orally bioavailable CK1g inhibitors affords strong pharmacological tools for exploring the importance of CK1g-mediated signaling in tumor growth, and a potentially new therapeutic strategy to treat human cancers. 2:55 Identification of Novel, Potent and Selective Polo-like Kinase 1 (PLK1) Inhibitors for Treatment of Cancer Shaoqing Chen, Ph.D., Senior Scientist, Discovery Chemistry, Hoffmann-La Roche The Polo-like kinases (PLKs), members of the serine/threonine family of kinases, play a variety of roles in G2/M phase transition and have been identified as key regulators of cell mitosis. Inhibition of PLK1 kinase activity by a small molecule represents a novel approach to target mitosis and may be clearly differentiated from other mitosis-targeting agents on the market. PLK1 is a promising therapeutic target for cancer and several PLK1 inhibitors have shown promising results in phase I or II clinical trials. In this talk we report the discovery of a novel class of pyrimidodiazepinones as potent and selective ATP-competitive PLK1 inhibitors. The lead compound exhibits potent antiproliferative activity in a wide range of cancer cell lines, and shows strong antitumor activity in xenograft mouse models. 3:20 Networking & Refreshment Break 3:50 Discovery of a Glycogen Synthase Kinase-3 (GSK-3) Inhibitor for Oncology Thomas Engler, Senior Research Advisor, Discovery Chemistry Research and Technologies, Eli Lilly Glycogen synthase kinase-3 (GSK-3) is a constitutively active protein kinase involved in diverse signaling pathways. Initially described as a regulator of glycogen synthesis and metabolism, GSK-3 plays a role in a variety of cellular functions including maintenance of stem cell function, regulation of cell cycle and apoptosis. There are two highly homologous isoforms (GSK-3a/-3b) which share substrate specificity and functional properties. GSK-3b is a key component of the b-catenin destruction complex and phosphorylation of b-catenin by GSK-3b targets the substrate protein for ubiquitylation and proteosomal degradation. Inhibition of GSK-3b results in stabilization of b-catenin in the cytoplasm and nucleus. GSK-3 expression and/or activity have also been reported to be elevated in multiple tumor types, and inhibition of GSK-3 expression in colorectal tumor cell lines via specific siRNAs induced apoptosis and attenuated proliferation. LY2090314 is a novel, potent and selective inhibitor of both GSK-3a/b. LY2090314 did not exhibit useful antitumor activity in its own right against colorectal, ovarian or non-small cell lung tumor lines. However, when combined with different chemotherapeutic agents, LY potentiated their pro-

7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

apoptotic activity. In vivo, administration of a single dose of LY2090314 to xenograft-bearing mice led to a time- and dose-dependent accumulation of b-catenin in tumor tissues. In vivo xenograft growth studies revealed that administration of LY2090314 in combination with cisplatin every 7 or 14 days was sufficient to improve the efficacy as compared to cisplatin alone given at the same intervals. These findings provide evidence for the potential therapeutic utility of LY2090314 for cancer. 4:15 The Discovery and Optimisation of a Novel Class of Potent, Selective and Orally Bioavailable Anaplastic Lymphoma Kinase (ALK) Inhibitors with Potential Utility for the Treatment of Cancer Richard Lewis, Ph.D., Principal Scientist, Amgen Anaplastic lymphoma kinase (ALK) is an attractive therapeutic target in Oncology. Cytogenetic translocations and inversions are known to lead to the generation of novel fusion-ALK proteins which have constitutive kinase activity, and contribute to the oncogenic process. The best validated of these fusion proteins include nucleophosmin –ALK (NPM-ALK) which has been characterized in a subset of anaplastic large-cell lymphomas, and a fusion of echinoderm microtubule-associated protein-like 4 with ALK (EML4-ALK) which is a driving oncogene in a subset of non-small cell lung carcinomas (NSCLC). A potent and selective inhibitor of the ALK kinase domain would have potential therapeutic utility in a range of cancers. Crizotinib afforded the first clinical validation of EML4-ALK as an oncology target, and is now FDA approved as a first-line treatment for this patient population. Reports of developing resistance to crizotinib have provided added impetus to seek significantly more potent molecules with potential to treat refractory patients. The optimization of a novel screening hit to generate potent, kinase selective, and orally bioavailable candidates suitable for clinical development as second generation ALK inhibitors will be described. Benefits: Disclosure of a new class of ALK inhibitor Discussion of building in kinase selectivity An in-vitro strategy for building in-vivo potency Addressing mechanisms of resistance 4:40 ADP Glo: An Ideal Assay for Screening and Profiling Kinase Inhibitors in Drug Discovery Said Goueli, Research Fellow, Research & Development, Promega Because of the high sensitivity, universality, and robustness of ADP Glo in monitoring the activity of any enzyme reaction that producers ADP, it offers an ideal platform for screening multiple kinase with wide range of substrates. Thus, it has been successfully used in monitoring the activity of protein kinases with peptides as well as protein substrates and with lipid kinases with varying lipid structures and with small molecule kinases such as sugar and alcohol kinases. Furthermore, the assay does not

require any modification of enzyme substrates by either fluorescent labels or conjugation to a fluorescent moiety. Because of the high signal to background (S/B) values even at low % ATP to ADP conversion, the assay allows significant savings of enzyme usage in kinase assays. The fact that ADP-Glo™ assay offers so many positive attributes makes it an ideal assay not only for primary and secondary screening but also for profiling of lead compounds. We will present data demonstrating the assay principle and examples of use of the assay in both screening and profiling of compounds using known kinases. Benefits: 1. Providing the principle of an assay that is universal for all classes of kinases (Protein, lipid, sugar, etc) 2. Demonstrate the robustness and sensitivity of the assay in monitoring enzyme activity 3. Demonstrate the capability of this technology in screening and compound profiling 5:05 [Oral Presentation from Exemplary Submitted Abstracts] To be considered for an oral presentation, please submit an abstract here by April 30. 5:35 Networking Reception

Day 2 - Thursday, May 31, 2012

7:30 Continental Breakfast

Joint Plenary Keynote Session

Moderator: Shidong Jia, Scientist, Oncology Biomarker Development Group, Genentech

KEYNOTE PRESENTATION

8:00 Steven Burrill, Chief Executive Officer, Burrill & Company Pharmaceutical R&D spending is falling, the demand for innovation is increasing, and the traditional business model for the industry is failing. What does innovation look like today in the new austerity and what will it take to be successful? What’s required is creativity in raising money, making deals, and forging new business models. Burrill brings his 45 years of successful dealmaking, company building, and investing to this discussion.

KEYNOTE PRESENTATION 8:45 The Personal Genome Project - Open Access to Genome Sequences + Trait data. George Church, Ph.D. Professor, Genetics; Director, Center for Computational Genetics Harvard Medical School

7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

The PGP enables open observation and critique of a large cohort "test-driving" comprehensive participatory personalized medicine. Since 2004, we have helped push the cost of reading and writing DNA (and biological systems) down by a million-fold (5-fold faster exponential than Moore's law) and enabled fully open-access human Genome+Environment=Trait (GET) data, stem cells, and clinical community curation/interpretation tools (Evidence.PersonalGenomes.org). This involves inherited genomes plus day-to-day genomic variation -- cancers, microbes, allergens, vaccines, & subcellular-resolution epigenomics. We are also sequencing centenarians and long-lived mammals. Benefits include human genome engineering technologies for personalized diagnostics as well as stem cell, synthetic organ, microbiome and immunome transplantation therapies.

KEYNOTE PRESENTATION 9:30 RNAi and Immortality: Recognition of Self/non-Self Nucleic Acids Craig C Mello, Ph.D., Nobel Laureate, Blais Professor in Molecular Medicine, University of Massachusetts Medical School, Howard Hughes Investigator, HHMI Organisms exhibit a fascinating array of gene-silencing pathways, which have evolved, in part, to confront invasive nucleic acids such as transposons and viruses. Not surprisingly, these pathways are highly active in the germline and can be elicited upon the introduction of transgenes. A key question raised by the existence of these pathways is how do they distinguish self- from non-self nucleic acids? Evidence exists for a number of cues that might facilitate the recognition of foreign sequences including, copy-number sensing, sensing of unpaired DNA, or the sensing of aberrant RNA (e.g. dsRNA). Here we report on a remarkable silencing pathway that can permanently silence even single-copy transgenes. We show that the initiation of silencing depends on the piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors. Our findings support a model in which PRG-1 scans for foreign sequences while two other Argonaute pathways serve as epigenetic memories of "self" and "non-self" RNAs. These findings suggest that organisms utilize RNAi-related mechanisms to keep inventory of all genes expressed in the germ-line, and to recognize and silence foreign genes. 10:15 Networking & Refreshment Break

Session III: Other Kinases & Inhibitor Candidates

Moderator: Alexei Degterev, Assistant Professor, Biochemistry, Tufts University

10:45 Exploring New Kinase Targets Through Structural Genomics at SGC Alex Bullock, Ph.D., Principal Investigator, SGC, University of Oxford Academic and industry research has a traditionally narrow focus on well-known clinically validated kinase targets. As a result, 50% of all kinases are largely uncharacterized and more than 100 have completely unknown function. Many of these targets are emerging as possible candidate intervention points from unbiased kinome-wide synthetic lethality, RNAi and inhibitor screens. The open access public-private partnership of the SGC has released over 60 novel human kinase structures and built a network of academic and industry collaborators to stimulate this early stage target discovery. We hope that such efforts will revitalize the kinase drug discovery field, initiate new drug discovery projects and lead to new biological insights into the large number of unexplored kinases. This presentation will address the unexplored kinome and the opportunities it may bring. Topics covered will include: • An introduction to the unexplored kinome • A review of the freely-available SGC resource on human kinase structures and inhibitor complexes • Highlights of the first structures and chemical probes for new CDK-related families in neurobiology and TKL proteins in BMP signaling • The promise of parallel approaches in epigenetics 11:10 The G2019S Mutant of LRRK2 Increases Kinase Activity by Stabilizing the Active Conformation Min Liu, Ph.D., Assistant Director, the Laboratory of Drug Discovery, Brigham and Women's Hospital Leucine-rich repeat kinase2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson’s disease (PD). The most common mutant G2019S, like some of other LRRK2 mutations, has increased kinase activity, which correlates with increased neuronal cytotoxicity. We demonstrate that two classic DFG-out inhibitors imatinib and sorafenib inhibit both wild-type LRRK2 and the mutant G2019S but with different mechanisms of inhibition: these two classically ATP noncompetitive inhibitors in fact inhibit the mutant G2019S by a surprising ATP competitive mechanism, while the wild type protein follows the standard ATP noncompetitive kinetics. Our modeling and kinetic studies suggest that G2019S may result in an increased kinase activity by stabilizing the active/DYG-in conformation. In addition, we also provide kinetic evidence based on b-Raf, a structurally related kinsae to LRRK2, that inhibitors behave dramatically different toward the peptide and physiological protein substrates. In summary, all these results will have significant influence on drug discovery, specifically on designing selective inhibitors for LRRK2.

7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

11:35 Interplay of Akt, JNK and RIP1 Kinases in Necroptotic Cell Death Signaling Alexei Degterev, Assistant Professor ,Biochemistry Tufts University Necroptosis is a novel form of programmed cell death that is distinct from apoptosis and is necrotic in morphology. Necroptotic cell death has been implicated in the pathogenesis of many diseases such as myocardial infarction, brain ischemia, and glutamate-induced excitotoxicity. This form of cell death is known to be regulated by Receptor Interacting Protein Kinase 1 (RIP1), however a complete understanding of the molecular underpinnings of necroptosis remains unclear. Here, using the mouse fibrosarcoma L929 cell death model, we investigate cellular signaling in response to necroptotic death induced by TNF? or a pan-caspase inhibitor zVAD.fmk. We found that growth factor signaling initiated by bFGF plays a key role in the activation RIP1-dependent necroptosis. We further show that the growth factor-dependent interplay of the Akt and JNK signaling pathways promote necroptosis and synthesis of TNF?, tying together necrotic cell death and pro-inflammatory signaling. Overall, our data reveal a new signaling network downstream from RIP1 kinase that is involved in necroptosis. - The emerging mechanisms of regulated necrotic cell death, necroptosis, will be reviewed - Current evidence for the pathologic role of necroptosis will be summarized - New role of Akt as a regulator of pro-inflammatory signaling during necroptosis will be presented - The current mechanism of Akt involvement in necroptosis will be described - The effect of pharmacological inhibition of Akt signaling on necroptosis will be discussed

Session IV: Overcoming Challenges in Kinase Drug Development

Moderator: Thomas Chan, Chief Scientific Officer, Discovery and Early Development, ArQule

12:00 Mechanism of Highly Specific, Bi-substrate- Competitive Src Inhibitors from DNA-Templated Macrocycles Markus Seeliger, Assistant Professor, Pharmacological Sciences, Stony Brook University Medical School Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated gatekeeper mutant. We solved two co-crystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity

in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bi-substrate competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties, and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance. 12:25 Lunch Provided by GTC 1:30 Matthew Soellner, Ph.D., Assistant Professor, Medicinal Chemistry, University of Michigan My research program seeks to identify selective probes of kinases involved in human disease. While there are many known probes for kinases, these are often promiscuous. My group aims to develop methodology and create novel-binding compounds that have unprecedented selectivity for their target. Selective probes enable chemical genetics students where, for example, specific signaling pathways in cancer and their correlation to cell proliferation can be interrogated. Here, we describe our efforts to develop kinase inhibitors highly selective for their target, using c-Src as a model. 1:55 Rapid Deployment of Structure-Based Drug Discovery For Novel Kinase Inhibitors Thomas Chan, Chief Scientific Officer, Discovery and Early Development, ArQule Protein kinases are enzymes that regulate many cellular functions in normal and malignant cells. As an enzyme class, they have become favorite targets for new drug development. Indeed, discovery and clinical development of inhibitors of kinase functions have been a productive endeavor. Currently, there are more than a dozen kinase inhibitors approved for commercial sale in the US, the majority of them are ATP mimetics. However, inhibitors that compete for binding to the highly conserved ATP recognition region of kinases tend to have poor selectivity (they are usually referred to as multi-kinase inhibitors). Often the inhibition of off-target kinases can translate to substantial toxic side effects which have stopped development and precluded the combination of kinase inhibitors with other cancer chemotherapeutics. The recent identification of a novel, non-ATP competitive mode of inhibition that caused hydrophobic collapses (1) in many kinases points to new approaches in structure-based drug design (2). Such efforts may yield more selective and less toxic kinase inhibitors that have the potential to treat diseases outside the field of oncology. Experience to date suggests that potent and selective non-ATP competitive inhibitors can be designed to target many human protein kinases. Drug discovery campaigns that take advantage of new modes of kinase inhibition through in silico models have resulted in shorter timeline from hit-generation though to IND-candidate identification.

7th Protein Kinases in Drug Discovery – May 30-31, 2012 – Boston, MA

2:20 Dual Inhibition of p38 Kinase Activation and Activity Provides Efficacy in the Treatment of Rheumatoid Arthritis Gary L. Schieven, Ph.D., Research Fellow, Immunology Drug Discovery, Bristol-Myers Squibb Although p38 kinase has been an intense focus of drug discovery, p38 inhibitors have shown little or only transient efficacy in rheumatoid arthritis (RA) clinical trials. This tachyphylaxis suggests development of resistance in patients. We report that BMS-582949 is a dual action p38 kinase inhibitor, blocking p38 activation in cells in addition to p38 kinase activity. By contrast, the three compounds reported to give tachyphylaxis did not block p38 activation. Cells treated with diverse p38 inhibitors showed changes in gene expression expected to drive p38 activation more strongly, suggesting a resistance mechanism that activation blockade could avoid. In a 28 day clinical study a daily dose of 300 mg BMS-582949 gave similar inhibition of Hsp27 phosphorylation and LPS-induced TNF alpha production in the blood of subjects after the first and last doses, demonstrating durable inhibition of p38 signaling that was not overcome by any resistance mechanism. Treatment with BMS-582949 in combination with methotrexate for 12 weeks in a RA clinical trial was well tolerated and demonstrated rapid and durable efficacy that correlated with trough exposures and baseline CRP. These results suggest that dual action p38 inhibitors may overcome resistance mechanisms causing tachyphylaxis, and merit further investigation for the treatment of RA. • Durability of benefit in patients has been an issue for p38 inhibitors • Understanding how to overcome this could be important for many kinase targets • Resistance may be due to driving p38 activation more strongly • Durable benefit of BMS-582949 may be due to blocking p38 activation 2:45 High-throughput Analysis of the Biochemical Properties of Irreversible Covalent Kinase Inhibitors Jennifer Piesvaux, Research Biochemist, Merck A majority of kinase inhibitors known today are ATP-competitive. These compounds are typically identified using high throughput assays containing relatively low concentrations of ATP. Although these compounds regularly demonstrate good biochemical potency, they often lack kinase specificity and are less effective in cells due to higher cytoplasmic ATP concentrations. Because of this, there is increased interest in targeting kinases using irreversible covalent inhibition, where physiological concentrations of ATP inhibit small-molecule binding to a lesser extent. However, because irreversible inhibitors act in a time-dependent manner, conventional IC50 measurements are often misleading and do not provide accurate structure-activity relationship (SAR) data. Methods for studying irreversible inhibitors require measurement of inhibition at several time points for a number of inhibitor

concentrations, thus providing information about both the non-covalent affinity (KI) of a compound and the rate at which it inactivates its target (kinact). While these measurements provide an in-depth understanding of irreversible inhibition and fully enable SAR, historically these methods have proven to be technically challenging and time consuming, both in the lab and during data analysis. Here we describe a novel high-throughput platform for the biochemical characterization of irreversible covalent inhibitors. Using automation and a unique XLFit data analysis template, we were able to effectively increase experimental throughput about 10-fold over manual methods while decreasing analysis time 6-fold. In addition, implementation of robotics and routine analysis procedures has effectively improved data quality and assay robustness, providing enhanced SAR. 3:10 Conference Concludes