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

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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 structure-

activity 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.

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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

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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].

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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.

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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  86 

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Therapeutic panels metabolic, cancer, cardiovascular, digestive/renal, inflammation,  neurological, psychiatric, reproductive, respiratory, sensory 

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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.

KINOMEscanTM panels of cell-free biochemical assay services and PathHunter®

cell-based, functional kinase assays can be performed, ideally, in parallel (Fig.

2).

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[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.

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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 

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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-

345541, a non-ATP-competitive inhibitor, (right panel) against IKK-beta.

KINOMEscanTM (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 (ATP-

competitive 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.

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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 seven-

transmembrane receptors. Trends Pharm Sci, 2004; 25:413.

2. Manning G, Whyte D, Martinez R, Hunter T, and Sudarsanam S. The protein kinase

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complement of the human genome. Science, 2002; 258(5600):1912-34.

3. Whalen EJ, Rajagopal S, and Lefkowitz RJ, Therapeutic potential of β-arrestin and

G protein-biased agonists. Trends Mol Med, 2011; 17(3):126-39.

4. Jones D, Sastry S, and Kuchibhatla S, Dawn of a new era for kinase drug discovery,

Int Drug Discov, 2011 (February/March): 34-35.

5. Sastry S and Kuchibhatla S, New momentum for kinase drug discovery. Innov

Pharmaceut Technol, 2011 (June): 42-44.

6. 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):28-39.

8. 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):6-11.

10. 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 Ca2+ flux assays. Int Drug Disc, 2011; Feb/Mar issue: 28.

11 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. Email:

equinn@discoverx.com

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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. Email: djones@discoverx.com

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