Floyd Brownewell, Ph.D. Albany Molecular Research, Inc. 21 Corporate Circle PO Box Albany, NY

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1 Technical Reports Volume 8, umber 17 Trip Report: IBC s 3 rd International Conference, Protein Kinases and Phosphatases: Advances in Target Selection, Drug Design and Clinical Research San Diego, California March 23-26, 2003 loyd Brownewell, Ph.D. Albany Molecular Research, Inc. 21 Corporate Circle P Box Albany, Y Abstract. The conference covered a broad range of topics within the fields of kinases and phosphatases. The lectures were grouped by clinical updates, lead discovery, and case studies. A frequent theme was the so called compound gap. Many technologies described were aimed at improving the production of biologically relevant new chemical entities, while controlling costs. The industry continues to focus on kinase ATP site binders, illustrating many examples of what are described as successful HTS campaigns. The field is still plagued by modest selectivity within kinase families. A growth in use of crystallography and in silico techniques is widely believed to be valuable in bridging the selectivity issue. Phosphatases represented a much smaller contingent at the conference, and were frequently described as a fledgling field, relative to kinases, but with rapidly growing interest and relevance to drug discovery. Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 1/9

2 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 Chemogenomics Approach to Small Molecule Inhibitors of Protein Kinases R. S. Kauffman (Vertex Pharmaceuticals). The lecture attempted to refute the kinase drug target perspective that this class of proteins is too closely related to achieve specificity and that the lack of specificity leads to toxicity. A program to develop a selective inhibitor of p38 was used to illustrate this case. Inhibition of p38 interrupts the cascade involving IL-1β and Tα providing therapeutic relevance in inflammatory diseases. The concept of Scaffold Morphing was introduced: S H 2 S H 2 The following benefits were identified: information reuse, multiple new chemical entities, development success, enhanced intellectual property. Eventually VX-745 was developed: Cl Cl S This compound was screened against eight other kinases. The least selective was p38-β which was ~20 fold less potent compared to the desired p38-α. VX-745 was fold less potent against the other seven kinases screened. This compound has been through phase II RA studies. The Development of PTP-1B Inhibitors for the Treatment of Diabetes and besity B. P. Kennedy(Merck rosst). It is widely recognized that type II diabetes is an acquired form of the disease and closely linked to obesity. PTP-1B (protein tyrosine phosphatase 1B) has been implicated in the insulin receptor signal cascade, verified through mouse knockout studies. A molecule as small as a tripeptide has been shown to be a ~10 nm inhibitor of PTP-1B. A nonpeptide inhibitor was desired. A diphosphate compound was developed with good inhibition properties, but exhibited poor bioavailability. ne phosphotyrosine moiety was removed, improving bioavailability, but with a significant loss of inhibition. P 3 3 P P 3 Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 2/9

3 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 Mouse model studies show that mice on a high fat diet exhibit better weight control when a PTP inhibitor is administered. Removal of the PTP inhibitor shows a marked rise in weight. Reintroduction of the PTP inhibitor again normalizes weight gain. The model is claimed to now be well established and consistent. The next goal is to move to a human clinical trial. Protein Tyrosine Phosphatases in Signal Transduction and Cell Physiology T. Mustelin (The Burnham Institute). This lecture provided a general overview of phosphatases and their role in cells. There are 438 serine/threonine kinase genes known. There are 90 tyrosine kinase genes known. There are ~30 serine/threonin phosphatase genes known. There are ~95 tyrosine phosphatase genes known. Based on these numbers, one is led to believe tyrosine phosphatases are very substrate specific, compared to serine/threonine phosphatases. Tyrosine phosphorylation generally occurs at low stoichiometry. Tyrosine phosphatase turnover is very fast in vivo. Tyrosine phosphatase activity is 1000x higher than kinase activity. Given these facts, inhibition of tyrosine phosphatases results in massive tyrosine phosphorylation. As a result, dramatic effects can be seen from manipulation of tyrosine phosphatases. To date, this is a relatively unstudied area of signal transduction. These proteins are generally specific to a cell type (i.e. no crossover between cell types). requently the -terminus has a catalytic domain for protein protein interactions. Protein Tyrosine Phosphatase Superfamily Classic Dual Specific Atypical (~50% of PTPs) RPTP Intracell Low MW CDC25 Anti Cancer Activities of MKP-1 and Cdc25 Phosphatase Inhibitors M. Pfahl (Incyte San Diego, Inc.). MKP-1 has been implicated in the following cancers: prostate, 58% of ovarian, 80% non small cell lung cancers (SCLC), 20% small cell lung cancers, breast cancer. MKP-1 plays a role in the JK signaling pathway. Activated JK triggers the caspase cascade leading to apoptosis. MKP-1 dephosporylates JK rendering it inactive, avoiding the caspase cascade, apoptosis, and ultimately cell death. The result can be proliferation of cell growth. MX7091 was designed to inhibit MKP-1: H S H Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 3/9

4 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 MX7091 was found to be more efficacious than paclitaxel in reducing solid pancreatic tumor growth in nude mice. MX7091 exhibits significant delay in the growth of aggressive SCLC tumors in nude mice. MX7091 exhibits synergistic effect in inducing apoptosis when combined with cisplatin. Similar results were found for JK activation and SCLC tumors in nude mice. Cdc25 is over expressed in 40-50% of common human cancers. Cdc25 associated cancers are often given a poor prognosis. A compound (not identified) was found to be specific versus MKP-1. These compounds block S phase entry, inhibit DA synthesis, and induce apoptosis. Potent anti tumor activity was found for a variety of cancer cell lines as a result of reduced Cdk2 activity through Cdc25 inhibition. The analogues were determined to be effective in vivo. This program is progressing rapidly to Phase I trials. The Chemical Biology Paradigm and its Application to the Kinase Target amily P. Cox (Aventis Pharmaceuticals). Aventis is/has adopted a paradigm shift to chemical biology for the discovery of new lead compounds. The shift has largely been initiated by the compound gap (i.e. more money invested in R&D is not translating to greater numbers of drugs on the market). The Aventis approach involves the concept of chemical and biological space and understanding each individually and how they overlap. This new direction requires a shift in mindset and to some extent skills. ocus is on cooperation across sites and therapeutic areas to capitalize on all skills and expertise available to attack a problem. Aventis targets kinase ATP sites because they consider the substrate binding cleft to be too shallow and difficult to find small molecule binders. Allosteric sites are not generally targeted because they are not defined (or do not exist at all) in most kinases. The process begins with a focused screen of the Aventis kinase subset, a four step process typically resulting in ~12 K compounds. ext, this relatively small library is screened against preprofiled libraries. This screen involves a kinase panel, ADME, CYP, etc. ften compounds are assayed against a crystallized collection of kinases. Soaking experiments for x ray structures are also often performed. Twenty five co structures were solved in Structure Based ragment Screening of Protein Kinases H. Jhoti (Astex Technology, Ltd.). The foundation of this company is screening using x ray crystallography. A typical drug discovery progression includes target identification and validation, screening, hits to lead, and lead optimization, in that order. Crystallography is traditionally applied during the lead optimization phase. Astex is applying this technique during screening and hits to lead phases. urther, compound fragments are screened once a scaffold is identified because combing two incompatible fragments may preclude binding even though one or both may fit an area of the target exclusive of the other. Since this system produces a crystal structure, binding affinity is not a concern. If a fragment binds the desired protein well enough to produce a crystal structure, the fit is considered viable. Poor fits will generally average out during data acquisition. As an Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 4/9

5 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 example, a p38 lead generation program was started. A fragment was identified as a mm hit. ewer than 50 molecules were synthesized to increase potency into nm range within 22 weeks. verall, a 1 mm fragment hit to <10 nm highly tractable lead series was accomplished within 12 mo from <300 compounds by four chemistry TE s. Lead generated shows good cellular activity and good drug like properties: MW <500, clogp <4.5, no Lipinski violations, generally good ADMET profile. Rat PK/PD studies have been initiated. ovel Kinase Inhibitors from Affinity Based Screens of Unactivated Kinases S. P. Adams (eogenesis Pharmaceuticals). The eogenesis technology is described as ultra high capacity affinity screening. The target protein is mixed with mass encoded libraries as a free solution. Bound ligands are rapidly separated by automated micro scale size exclusion chromatography. The ligands are dissociated from the target protein, analyzed by mass spec, and deconvoluted by ALIS spectral search engine. Identified compounds are subjected to a rigorous synthetic proof to verify structure. Using this system, a drop out rate of <25% is claimed. ALIS is capable of screening up to 500,000 compounds/day. The system requires 50 nmoles of protein (~2 mg of a 40 kd protein). General performance characteristics include: compatible with most types of proteins, high affinity ligands identified for 81% of targets (<10 µm), averages 18 ligands and 4 chemotypes/target, 60% of all targets screened deliver biologically active ligands. ALIS can screen both active and inactive protein forms. Keynote Presentation Lead Discovery Process: Lessons Learned and Trends to Meet uture eeds K. A. Stoeckli (Aventis Pharma AG). The focus of this keynote address was on how to overcome the investment versus productivity gap. In general, today s screening processes tend to be more customized, output oriented, and flexible. Screening tends to be quality oriented, greater use of in silico methods, earlier ADMET concern, use of informatics, more science, trend toward integration of mature technologies, and partial industrialization of aligned processes. Still, production of new medicinal entities is not rising proportionally with R&D investment. In order to shift the balance, a shift in thinking is necessary. Paradigm I: Industrialize where appropriate. Standardization Automation and miniaturization Parallelization Scale and critical mass Streamlining processes Aligning interfaces Information processing and management Connectivity This should grow out of effectiveness, efficiency, and quality Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 5/9

6 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 Consistent and coherent high quality data generation Integrated knowledge base Knowledge mining Paradigm II: Use family approach and be knowledge based where possible. Chemical space: compound structure Biological space: biological structure Maximize overlap between spaces Work to understand each of these spaces and increase overlap of the two spaces wherever possible. ptimized match of structure spaces will lead to faster and better problem solving through knowledge accumulation. The shift to chemical biology is a strategy to match chemical and biological structural space. Be aware of the vastness of chemical space: estimated possible drug molecules, ~10 10 compounds described in chemical abstracts, ~10 4 are drug like. Biological space can hardly be quantified. However, there are ~500 in vivo targets. Companies have million compounds for HTS, trending toward continued increase. Increase in screening capacity does not begin to tackle the basic issue. Vast chemical libraries with poor biological space overlap lead to poor results complementary discovery strategies are needed. Many compounds coming out of HTS campaigns are not specific, have poor ADMET profiles, and are not biologically relevant. The target family approach would use different types of inhibitors for different kinase subfamilies, for example. Two targets can be approached: ATP site inhibitors, non ATP competitive inhibitors. In the case of ATP competitive inhibitors, the knowledge base should be utilized, as there is a wealth of information available. or non competitive inhibitors a diversity approach must be taken, since much less is known. As a result, the knowledge base for non competitive inhibitors is expanded. The use of focused screening, random screening, or chemical genomics will be dictated by the amount of information available for the target of interest. Involving in vitro ADME profiling early on can provide critical decision support. Some parts of drug discovery can be industrialized. The issue is balance. Reports show introduction of parallel and automated approached has resulted in increased productivity for protein production at Aventis. Efficient compound management is also very important to productivity. Logistics of software and hardware are critical. Automation and miniaturization has had a large impact on productivity and cost. The shift from 96 well format to 1536 well has decreased screen time from 9 wks to 1 wk and a reagent cost of $1.3 million to $50 thousand. High quality assays maintain quality of science in the midst of streamlining. Mechanistic understanding is the key to effective assay development. Main Lessons: Screening Shift from throughput oriented goals to output oriented quality Sometimes less is more and more meaningful Greater use of in silico methods Automation and Miniaturization Critical to productivity, but underestimated in terms of implementation Suppliers promise too much Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 6/9

7 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 Customer hopes too high Compound logistics much more important than thought Assay Development Requires scientific rigor combined with good sense of pragmatism Process Alignment/Technology Integration Interfaces to chemistry, eadmet sciences and informatics are key Technology innovation is important, but don t lose sight of current capabilities Profiling has become increasingly important in decision support Specificity Profiling of Kinase Inhibitors Based on Binding Affinity P. P. Zarrinkar (Ambit Biosciences). This lecture described the concept of reverse screening. The technology exploits drugs or other biologically active compounds with unknown mechanisms of action as probes for discovering valuable targets. The screening utilizes global and directed protein libraries. Compounds are on beads. Proteins are expressed on T7 phage. They are mixed, washed, separated, bacteria infected, and recycled to enrich the pool. The phage can be analyzed by PCR and DA sequencing to identify gene products. nce hits are identified, a more normal approach is adopted. In other words, the compounds are in the absence of beads and linkers to determine binding affinity. The system was tested with a known p38 inhibitor. Compound came through the process as a 40 nm inhibitor, compared to a published K D 37 nm. Many compounds have been reported to be specific to a particular kinase family. In some cases specific even within that family. Ambit has used this technology to test some of these compounds and found that many are considerably less specific than believed. Many were found to bind phylogenetically diverse kinases. BAY , an Activator of Soluble Guanylate Cyclase: Lessons Learned from a Successful HTS Campaign J. Huser (Bayer Healthcare). Target was chosen due to identification as a clinical drug candidate, a drugable target, and potential for a compatible cell based assay (1536 well plates). Cyclic GMP formation monitored via Ca 2+ influx. Pros identified for assay include physiological relevance, visualization of all possible drug target interactions, discrimination between agonist/antagonist interactions, sensitivity, PK and toxicology information. Cons identified for assay include complex development/optimization, noise of activity data, frequently no direct readout of target activity, high rate of unspecific/trivial hits. Example compounds: Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 7/9

8 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 H 2 H 2 Testing was fully automated for hit identification and retesting to narrow to true hits. Cluster analysis used to control rate of false negatives. Pharmacological characterization showed potent vasodilation of circulatory system, activity preserved in nitrate tolerant preparations, and potent inhibition of platelet aggregation. Tested >1 million compounds. ound 1536 well technology compatible with liquid handling technologies. The conclusion is that complex cell based assays can be achieved without compromising data quality. Highlights of Three Successful HTS Projects: Substance P, CCR1, and actor Xa R. M. Snider (Berlex Biosciences). Substance P is a regulator in pain and inflammation (K 1 receptor). Substance P is relevant to arthritis, migraine, asthma, emesis, depression, etc. The following compound is described as the first nonpeptide substance P receptor antagonist. H 3 C H K i 0.33 nm at K 1 receptor K i > 10 µm at K 2 and K 3 receptors K i >>1 µm at D 1, D 2, 5-HT 1, 5-HT 2, 5-HT 3, many opiate receptors, benzodiazepine Enantiomer K i > 1 µm Science 1991, 251, 435.; J. Med. Chem. 1992, 35, 4768.; J. Med. Chem. 1992, 35, Inflammation: Regulatory Peptides 1993, 46(1-2), 440.; Annu. Rep. Med. Chem. 1993, 28, 99. Emesis: Eur. J. Pharmacol. 1993, 249, R3.; TIBS 1999, 20, 485.; Eur. J. Pharmacol. 1996, 305, 181.; J. at. Cancer Inst. 1997, 89, 817. Depression: Science 1998, 281, CCR1 is implicated in multiple sclerosis (MS), transplant rejection, renal fibrosis, and Alzheimer s disease (AD). ound efficacious in rat models for MS. CCR1 antagonist combines synergistically with cyclosporine to extend survival of heart transplant patients. CCR1 is expressed in AD brain. CCR1 not expressed in normal aged brain, therefore may serve as a diagnostic for onset of AD. Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 8/9

9 Albany Molecular Research, Inc. Technical Reports Volume 8, umber 17 Cl H CH 3 H 2 K i 1nM; % = 70%; T 1/2 = 3 h Phase II human trials 2Q 2003 Xa plays a central role in the blood coagulation cascade. Therapeutic applications include acute coronary syndrome, prevention of thromboembolic stroke, prevention/treatment of acute deep vein thrombosis. S Cl H CH 3 H Above compound determined to be a competitive antagonist of human Xa, K app 17 nm. Selective against seven trypsin like proteases, of which Xa is a member. The worst example was prothrombinase, K app 454 nm. All others had K app >10000 nm. Compound is described as an unoptimised screening hit. Copyright 2003 Albany Molecular Research, Inc. All Rights Reserved Page 9/9