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:
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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: [email protected]
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