Advanced Lead Optimization Efforts Through Innovative and Comprehensive GPCR and Kinase Panels

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1 Advanced Lead Optimization Efforts Through Innovative and Comprehensive GPCR and Kinase Panels Elizabeth R. Quinn, Ph.D. and Daniel Jones [subhead] Use of industry leading GPCR and kinase panels to gain key insights into late stage compounds INTRODUCTION G-protein coupled receptors (GPCRs) and protein kinases represent two of the largest and most diverse druggable target classes in modern day drug discovery [1,2]. With greater than 40% of all marketed therapeutics targeting GPCRs (the majority of which are inhibitors) and 11 FDA-approved small molecule kinase inhibitors plus 500 more in active development, both target classes have proven popular and effective therapeutic targets for treatment of malignancies, inflammation, central nervous system diseases, diabetes, osteoporosis, and other diseases [3-5 and references therein]. Rapidly shifting and complex FDA regulations are constantly mandating stricter requirements on compound safety and efficacy, thereby escalating the cost and time to market an effective drug. As a result of this high cost and disappointingly sluggish pace of approval to market a new drug, now more than ever, the drug discovery community is under pressure to identify novel strategies to gain 1

2 comprehensive understanding of compound mechanism of action while aiming to improve the effectiveness of marketed drugs by minimizing potential adverse side effects - all in the shortest amount of time possible. This necessitates the need for novel technologies that can fuel the development of safe and highly efficacious GPCR and protein kinase drugs. TURNING HITS INTO LEADS Identifying and selecting a lead compound continues to be a pivotal stage of any drug discovery programme. During the process of lead selection, chemical hits are evaluated for drug-like properties including potency, stability and various other attributes that help determine whether or not the chemical entity can become a viable drug candidate [6]. Results from lead optimization assays need to drive chemists and biologists to make informed decisions on which compound to synthesize and which compound to move into more expensive in vivo animal models. Therefore, this stage requires the generation of rapid, reliable and biologically relevant results from cost-effective assays with robust predictive value [1]. During this stage, it is critically important to determine the compound specificity, selectivity and activity profiles of each new chemical candidate in order to understand and predict their full therapeutic potential. Historically, hit identification for GPCRs and kinases has relied on cell-free assays such as radioligand binding and biochemical activity based assays, respectively. Although these assays provide potency and selectivity data and are required for analysis of structureactivity relationship (SAR), these assays query only limited parameters and do not report on factors such as cellular permeability, physiological implications of compound potency, and other less well understood aspects of the cellular milieu. These important factors necessitate the need for a multi-pronged approach combining cell-free and cell-based assays to provide maximum insight on compound function, potency, and selectivity. 2

3 Although highly specific and fairly simple to run, radioligand binding assays have limited utility in GPCR drug discovery because they cannot distinguish between compound modes of action (MOA), such as full or partial agonism, antagonism or inverse agonism. Nor can they easily detect allosteric compounds that bind to sites independent of the radioligand binding site. As a result of these limitations, over the past 10 years, whole cell assays that provide a functional measure of receptor activation through G-protein signaling (GTPγS binding), second messenger signaling (camp, IP, Calcium flux) or β-arrestin recruitment have begun to replace radioligand binding assays [9 and references therein]. Most recently, the availability of a quantitative GPCR receptor internalization platform has afforded greater in depth compound pharmacology measurements and has broadened the understanding of the therapeutic implications of receptor removal and/or stabilization following compound binding and receptor activation. In addition to providing functional readouts, these next generation GPCR assays are quickly becoming a requirement in drug development programs as they provide not only a convenient, cost effective, highly quantifiable and easy to automate platform, but the availability of the same GPCR targets in multiple assay formats allows for a much more thorough and complete understanding of compound efficacy and therefore serves as a better predictor of compound activity in whole animals. Similarly, kinase inhibitor screening programs saw a resurgence with the 2001 approval of Novartis Gleevec (imatinib), for the treatment of chronic myelogenous leukemia. Despite early doubts initially surrounding the tractability of protein kinases as drug targets owing to their highly conserved catalytic domains which is where most small molecule inhibitors exert their influence, these fears fortunately proved untrue. Not only was Gleevec, a therapeutic and commercial success, it also demonstrated that active-site directed kinase inhibitors could be an effective therapeutic approach and that absolute selectivity is not an absolute necessity for commercial success or efficacy.. Although off-target kinase activity 3

4 can lead to in vivo toxicity, not all off-target activity is undesirable; indeed, some off-target activity may also extend a drug s therapeutic utility (e.g., the tyrosine kinase inhibitor, imatinib mesylate, is used to treat chronic myeloid leukemia and gastrointestinal stromal tumors). However, compound selectivity continues to present challenges to optimizing potent, highly selective, lead compounds targeted towards kinases and has led to increased interest in identifying lead compounds possessing different binding modes and exhibit affinity preferences for different kinase conformations (eg: compounds that preferentially bind an inactive versus active kinase conformation), which is thought to be a strategy to identify inhibitors with an attractive intellectual property position, greater selectivity and cellular potency. Therefore, rapid and cost-effective kinome-wide in vitro profiling using assays capable of not only detecting compounds with different binding modes but additionally able to assess and discriminate between these compounds with robust predictive value are necessary for rigorous assessment of on- and off-target potency of leads during optimisation. DISCOVERX GPCR PROFILING MULTIPATHWAY INFORMATION. GREATER INSIGHTS. DiscoveRx has been committed to the development of novel assay platforms that enable the creation of smarter and safer drugs. It is well understood that successful binding of the compound to an active site leads to activation of GPCRs results in not only signalling by both G-proteins and β- arrestin but also receptor desensitization and internalization (Figure 1). The complex relationship between G-protein and β-arrestin signaling as well as the possible effects of removing receptors from the cell surfaces determines the overall efficacy and potential side effects of GPCR-targeted drugs [1,3]. 4

5 DiscoveRx now offers the largest collection (>400) of functional cell-based assays designed to detect GPCR signaling for both known and orphan GPCRs based on second messenger signaling, arrestin binding, and receptor internalization (Table 1). Assays are designed using the same simple, homogeneous, chemiluminescent, read-out based on β-galactosidase based enzyme fragment complementation (EFC) to confer universality and flexibility for identification of lead candidates in agonist, antagonist, inverse agonist, or allosteric modes. Using the δ opioid receptor (hdor) as a model receptor system, we have previously shown that a combination of second messenger, Arrestin recruitment, and GPCR internalization formats can be used to obtain unique receptor activation and internalization profiles for a distinct set of δ opioid receptor agonists that is consistent with effects seen using in vivo mouse models of pain ([11], Figure 2). With its comprehensive, functional whole cell GPCR portfolio, for the first time, compound activity can be measured using multiple signaling readouts for the same GPCR thus enabling high content compound analysis and providing a deeper understanding of the effects of a compound on overall GPCR activation. This allows researchers to interrogate all GPCR signaling pathways and determine potency of the lead candidates for therapeutic target(s) of interest and cross-reactivity of lead candidates. Importantly, this approach can lead to the identification of functionally selective compounds while providing valuable information on lead therapeutic candidates in a high biological context. 5

6 Figure 1. DiscoveRx offers a Comprehensive Menu for Studying Receptor Biology and Complex Compound Pharmacology. More than 400 PathHunter non-force coupled cell lines detect GPCR signaling through second messenger activation, β-arrestin binding, and receptor internalisation Table I. Revolutionary GPCR Profiling Panel for Lead Optimization. Feature Solution Total receptors 200 Receptors in more than 1 mode 135 Receptors in all 3 modes 40 Orphan receptors Therapeutic panels metabolic, cancer, cardiovascular, digestive/renal, inflammation, neurological, psychiatric, reproductive, respiratory, sensory 6

7 Figure 2. Comparison of Arrestin recruitment, camp and GPCR Internalization assays at the Opioid Receptor δ (OPRD1) receptor DISCOVERX KINOME-WIDE PROFILING NOVEL SOLUTIONS FOR KINASE INHIBITOR OPTIMISATION DiscoveRx offers the broadest panel of kinase assays and services for investigation of functional activity, characterisation, profiling, and pathway information during evaluation of lead therapeutic candidates in kinase pathways. KINOMEscan TM panels of cell-free biochemical assay services and PathHunter cell-based, functional kinase assays can be performed, ideally, in parallel (Fig. 2). 7

8 [caption] Figure 2. Kinase assays currently available from DiscoveRx. Red circles denote kinase assays available in the cell-free, KINOMEscan panel; blue circles denote PathHunter cell-based assays. Mutant and lipid kinases are not represented in the figure. 8

9 Table 2. Broad Kinase Profiling Portfolio for Lead Optimization. Target KINOMEscan PathHunter MET MET C MET DDR1 DDR1 DDR1 DDR2 DDR2 DDR2 EPHB4 EPHB4 EphB4 EGFR EGFR ErbB1 ERBB2 ERBB2 ErbB2/ErbB3 ERBB4 ERBB4 ErbB4 FGFR4 FGFR4 FGFR4 FLT3 FLT3 Flt3 IGF1R IGF1R IGFR1 INSR INSR INSR PDGFRB PDGFRB PDGFRb TRKA TRKA TrkA TRKB TRKB TrkB TRKC TRKC TrkC P75 JAK1 JAK1(JH1domain catalytic) JAK1 JAK2 JAK2(JH1domain catalytic) JAK2 9

10 Binding constant (Kd) determinations for dasatinib, a known Type I inhibitor (left panel), and imatinib, a known Type II inhibitor (middle panel) against ABL1 preparations differentially phosphorylated on the A-loop, and BMS , a non-atp-competitive inhibitor, (right panel) against IKK-beta. KINOMEscan TM (acquired by DiscoveRx from Ambit Biociences in November 2010) represents the largest commercially available panel of biochemical assays of kinases to determine MOA; true thermodynamic binding affinities (Kd); and association/dissociation kinetics/reversibility of type I, type II, and allosteric (ATPcompetitive and non-atp competitive) inhibitors. Kinome-wide services offered include more than 450 kinase assays (including clinically relevant, mutant variants of kinases and catalytically inactive pseudokinases), with a rapid turnaround time. During the iterative process of lead optimisation, the customised, quantitative assay services can provide accurate, precise, and reproducible measurements of the impact of chemical modification on the biochemical potency and selectivity of the candidates. Also, DiscoveRx offers a universal platform of PathHunter cell-based assays for high-value, druggable kinases with established roles in cell signaling and disease pathophysiology. These phosphorylation and activity-based Enzyme Fragment Complementation (EFC) assays allow researchers to explore effect of compounds on ligand binding, phosphorylation, non-atp competitive inhibition, interaction with downstream adaptor proteins to various types of receptor tyrosine kinases (homodimer, heterodimer, and pre-dimerised), and subsequent inhibition of dimerisation, protein translocation, secretion, and degradation. Moreover, the flexibility of PathHunter assays permits study of various types of receptor tyrosine kinase inhibitors, including allosteric modulators, small molecules, and monoclonal antibodies. 10

11 As permeability of small molecule inhibitors of kinases into cells can affect measurement of compound activity, inclusion of cell-based, functional assays in the lead evaluation process can provide information on potential physiological efficacy of lead candidates. The simple, rapid assays, which are performed with physiologically concentrations of substrate and ATP, utilise sensitive chemiluminescent detection. INFORMATION THAT DRIVES DECISION MAKING DiscoveRx offers a comprehensive portfolio of technology, tools, and services to accelerate discovery and develop an efficient, integrative strategy for evaluation of lead candidates for GPCR and kinase pathways. Biologically rich information from these innovative technologies streamline and accelerate lead optimisation efforts and facilitate elucidation of structure-activity relationships, thereby providing timely, cost-effective lead identification and optimisation. DiscoveRx has always focused on creating technologies that enable the highest levels of innovation and development to enrich GPCR and kinase drug discovery. And by focusing on this goal, the company has become a well-known and trusted supplier of high quality products and services for the drug discovery community. With the largest and most comprehensive menu of both GPCR and kinase assays for screening and profiling, the company has now embarked on an era of drug development that will enable drug discovery researchers to develop smarter and more effective drugs. DiscoveRx s GPCR and Kinase profiling technologies enable a broader understanding of compound mechanism of action and targetbiology thereby accelerating early discovery, screening through lead optimization and into the preclinical stages. References 1. Lefkowitz RJ, Historical review: A brief history and personal retrospective of seventransmembrane receptors. Trends Pharm Sci, 2004; 25: Manning G, Whyte D, Martinez R, Hunter T, and Sudarsanam S. The protein kinase 11

12 complement of the human genome. Science, 2002; 258(5600): Whalen EJ, Rajagopal S, and Lefkowitz RJ, Therapeutic potential of β-arrestin and G protein-biased agonists. Trends Mol Med, 2011; 17(3): Jones D, Sastry S, and Kuchibhatla S, Dawn of a new era for kinase drug discovery, Int Drug Discov, 2011 (February/March): Sastry S and Kuchibhatla S, New momentum for kinase drug discovery. Innov Pharmaceut Technol, 2011 (June): Liszewski K, Drug discovery: successful lead optimization strategies: line begins to blur with lead discovery. Gen Eng & Biotech News, 2006; 26(14):1. 7. Zhang J, Yang PL, and Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer, 2009; 9(1): Kerr E, Refining GPCR discovery approaches: emerging platforms afford timelines more comparable to those for soluble targets. Gen Eng & Biotech News, 2011; 31(1):1. 9. Zhang L and Banks M, Screening strategy for lead optimization. Adv Drug Discov, 2006; 1(1): Burford, N. et al. Hit identification practices for positive allosteric modulators of G protein-coupled receptors: The need for multiple-mode screening approaches in dynamic Ca 2+ flux assays. Int Drug Disc, 2011; Feb/Mar issue: Quinn, E and Wehrman, T. Discovery of novel G-Protein or arrestin-biased ligands using a suite of GPCR signaling platforms. Int Drug Discov, 2011 (August/September): 2-3. Elizabeth Quinn, PhD, is Associate Marketing Director for GPCR portfolio at DiscoveRx Corporation (Fremont, CA, US). Having more than a decade of marketing, new product development, and research experience in life sciences and drug discovery industries, Dr. Quinn manages an extensive, diverse line of functional, cell-based GPCR assays. equinn@discoverx.com 12

13 Daniel Jones, MBA, is Director of Marketing at KINOMEscan (San Diego, CA, US), a division of DiscoveRx Corporation. With over 14 years of expertise in marketing, sales, and research in life sciences, drug discovery, and reagent markets, Mr. Jones has responsibility for marketing of KINOMEscan s high-throughput kinase screening service. djones@discoverx.com 13