the current status of drug discovery against the human kinome

r e v i e w

22 AssAy and Drug Development Technologies FEBRUARY 2009 DOI: 10.1089/adt.2008.164

Richard M. Eglen1 and Terry Reisine 2

1Bio-discovery, PerkinElmer Life and Analytical Sciences, Waltham, Massachusetts.2Independent consultant.

AbstrAct Protein kinases are important targets in drug discovery programs aimed at treating many devastating diseases, including cancer, autoimmune disorders, diabetes, and neurological disorders. Most “classical” drug discovery efforts employ rational drug design meth-ods based upon structural information to identify compounds target-ing the enzyme catalytic domain. Novel information on kinase biol-ogy is opening up other approaches in the design of selective inhibitors that may provide more subtle modulation of these drug discovery targets. The identification of such modulators requires adoption of a new generation of high-throughput screening tech-niques. These approaches will allow measurement of conforma-tional changes in kinases, as well as protein–protein interactions via assessment of functional responses such as cellular transloca-tion. Therefore a range of novel techniques, together with the under-standing that numerous “orphan” kinases will provide targets for therapeutics, suggests that a new era of kinase therapies is rap-idly emerging.

introductionrotein kinases are a family of enzymes that catalyze the transfer of the g phosphate groups from ATP to serine, thre-onine, or tyrosine hydroxyl group in target protein sub-strates.1–4 This process, which is reversed by specific phos-

phatases, serves as an activation step in many signaling cascades and in turn induces a whole series of subsequent cellular responses. In the human kinome, there are over 500 genes encoding protein kinases5 with at least 30% of the human proteome being phosphorylated6 by protein kinases. These data indicate that kinases exert pervasive ef-fects on human physiology and pathophysiology. Kinases serve cen-tral roles in mediating the biological action of many neurotransmit-ters, hormones, and growth factors, as in the example of the second messenger kinases such as cyclic AMP (cAMP)-dependent protein kinases (PKAs), which mediate the cellular actions of most hormones and neurotransmitters.7

Most kinases are believed to consist of at least two domains—a catalytic domain, which serves to bind and phosphorylate target proteins, and a regulatory region (in some cases a discrete subunit) that interacts directly with ancillary proteins that allosterically modulate activity of the catalytic domain. Because of their substrate recognition sites, kinases are divided broadly into two major classes—tyrosine kinases (TKs) and Ser/Thr kinases (STKs). The for-mer catalyze phosphorylation of tyrosine residues in target pro-teins,3,6,8 and in humans, over 100 genes encode protein TKs, many

The Current Status of Drug Discovery Against the Human Kinome

P

ABBREvIATIonS: AKAP, A-kinase anchoring protein; AP-1, activator protein 1; BRET, bioluminescence resonance energy transfer; cAMP, cyclic AMP; CDK, cyclin-dependent protein

kinase; DELFIA®, dissociation-enhanced lanthanide fluoroimmunoassay; DHFR, dihyrofolate reductase; EFC, enzyme fragment complementation; EGF, epidermal growth factor;

EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FKBP, FK506 binding protein; FP, fluorescence polarization; FRET, fluorescence resonance energy

transfer; GPCR, Gprotein coupled receptor; GRK, G protein receptor kinase; GSK3, glycogen synthase kinase 3; IL, interleukin; IMAP, immobilized metal assay for phosphopeptides;

JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; NEMO, NFkB essential modu-

lator; NFkB, nuclear factor kB; PCA, protein fragment-complementation assay; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; RACK, receptor for activated

C-kinase; RTK, receptor tyrosine kinase; SPA, scintillation proximity assay; STK, Ser/Thr kinase; TK, tyrosine kinase; TNFa, tumor necrosis factor a; TR, time-resolved.

3.Eglen.ADT.7.1P.indd 22 3/30/09 7:41:40 PM

© mARY Ann lIEBERt, Inc. • VOl. 7 nO. 1 • FEBRUARY 2009 AssAy and Drug Development Technologies 23

ProTein KinAse Drug Discovery

of which are soluble, intracellular proteins, although others act as cell surface receptors, e.g., epidermal growth factor (EGF) receptor (EGFR). Activation of receptor TKs (RTKs) catalyzes phosphorylation of a range of cellular pathways, including transcription factors. Moreover, when they bind their activating ligand, TKs also catalyze autophosphorylation of their receptor domains, resulting in sus-tained receptor activation. Such constitutive activity is particularly important in the regulation of cell proliferation, with the corollary being that its disregulation is implicated in many cancer etiologies. Indeed, abnormalities in kinase activity, due to either changes in expression level or mutations in the protein sequence, have been responsible in many disease pathologies, and in the human genome, over 250 protein kinase genes map to disease loci.9 It is clear that many cancers are caused by kinase mutations, including chronic myelogenous leukemia (Abelson TK), chronic myelomonocytic leu-kemia (Tel-platelet-derived growth factor receptor kinase), papillary renal cancer (Met receptor kinase), and non-Hodgkin’s lymphoma (Alk kinase).10 Since kinases regulate cell growth, differentiation, and proliferation, it is not surprising that abnormal functioning leads to uncontrolled growth, neoplasias, or metastasis and, ulti-mately, cancer.11 This role in cancer etiology was initially suggested by the observation that viral oncogenes express constitutively active protein kinases,6,10 and it was first noted with the EGFR, a TK that has been linked with breast cancer. Indeed, her2, a proto-oncogene and member of the EGFR family, is overexpressed in breast cancer,12 providing a rationale for the therapeutic use of the monoclonal antibody trastuzumab (Herceptin®, Genentech, South San Francisco, CA) in breast cancer therapy.13 Abnormalities in TK activity also have been associated with several other diseases such as immuno-deficiency, inflammation, rheumatoid arthritis, psoriasis, diabetes, and artherosclerosis, as well as cardiovascular and neurological diseases.

As one would anticipate, the central role of kinases in disease has led to significant interest in the biopharmaceutical industry to develop kinase inhibitors as therapeutics.14–17 A major success in this area was the TK inhibitor imatinib (Gleevec®, Novartis, Basel, Switzerland), a potent inhibitor of the constitutively active BCR-ABL fusion protein. This drug is approved for the treatment of leukemia and gastrointestinal stromal tumors.10,14,15,18 This was followed by other small-molecule drugs, such as the EGFR in-hibitors erlotinib (Tarceva®, Genentech) and gefitinib (Iressa™, AstraZeneca, London, UK), both receiving approval for treatment of non–small cell lung carcinoma.10,14,15,18 A number of other pro-tein kinase inhibitors have either received Food and Drug Admin-istration approval or are in late-stage clinical development (see

Table 1 for some of the kinase inhibitor drugs approved and in late-stage development) to treat different cancers.10,17,19 Further-more, there has been intensive development of kinase inhibitors for other diseases, such as ruboxistaurin to treat diabetic retin-opathy, while another protein kinase C (PKC) inhibitor, safingol, was developed to treat atopic dermatitis. Finally, Asahi Kasei Pharma (Tokyo, Japan) has developed the kinase inhibitor fasudil to treat heart failure and has recently received approval in Japan to use this drug to treat cerebral ischemia.

Taken together, kinases are now important targets in drug development and are considered as exceedingly “druggable” from a medicinal chemical viewpoint. Since they can also be purified in relatively large amounts, crystal structures of these enzymes have been characterized, resulting in the publication of high-resolution structures of the catalytic domains for over 40 differ-ent kinases.8,19 This structural information enables the rational design of small molecules using structure-based medicinal chem-istries. Concordantly, there have been major advances in the development of HTS technologies aimed at the identification of selective, high-affinity compounds to inhibit discrete kinases.

This review aims to assess both of these rapidly advancing areas in kinase drug discovery. It first addresses our evolving understanding in kinase structure and function, and second dis-cusses emerging HTS technologies developed to undertake screen-ing at these targets.

structure–FunctionCatalytic Domain

Kinases have considerable amino acid sequence and structural variations, yet also exhibit important common structural motifs. No-tably, the structure of the catalytic domain of many kinases is highly conserved because of the fact that all kinases recognize and bind ATP at common sites.20 As a consequence, several ATP analogs, as well as other small molecule compounds targeting the ATP binding domain, nonselectively inhibit multiple kinases. Nonetheless, the catalytic domains of kinases possess several other functional motifs that differ structurally. For example, many protein kinases comprise two major domains, with the substrate and ATP bind sites buried deep within a cleft linking these two regions.20 For these kinases, an activation loop retains the substrate in close proximity to the ATP binding site in such a manner that, in the presence of ATP, the kinase catalyzes the transfer of the phosphate moiety to the substrate.

As with most enzymes, kinases exist in either an inactive or active state, because of conformational differences in location of this acti-

3.Eglen.ADT.7.1P.indd 23 3/17/09 11:37:03 AM

24 AssAy and Drug Development Technologies FEBRUARY 2009

egLen AnD reisine

vation loop, i.e., DFG loop in or out. Changes in conformation occur either by binding of substrate or by phosphorylation within the loop itself. In the inactive conformation, the activation loop resides in a compact form covering the substrate recognition site of the kinase. When the kinase is activated, the activation loop opens up, uncover-ing the catalytic domain and substrate recognition site of the kinase. Compounds that directly inhibit kinase catalytic activity generally do so either by hindering access of ATP to the enzyme or by locking

the kinase in an inactive conformation. Gleevec was one early ex-ample of a small molecule that bound to the closed inactive state of the TK, Bcr-Ablkinase (Fig. 1).10,21 Structural analysis reveals that PD173955, also an inhibitor of Bcr-Abl kinase, binds to the open conformation of the activation loop of this kinase, a phenomenon typical of many small molecule inhibitors of active kinases.21 However, unlike Gleevec, PD173955 is unselective for Bcr-Abl kinase, being also a potent inhibitor of Src kinases. This property is presumably

Table 1. Protein Kinase Inhibitor Drugs in Development and Approved

Druga Target Status Therapeutic Direction

Gleevac® ABL, KIT, PDGFR Approved GI stromal tumors, CML

Iressa® EGFR Approved Non–small cell lung cancer

Tarceva® EGFR Approved Metastatic non–small cell lung cancer

Erbitux® EGFR Approved Metastatic colorectal cancer

Vectibix® EGFR Approved Colorectal cancer

Sutent®VEGFR2, PDGFR,FLT3, KIT

Approved Kidney cancer, GI stromal tumors

Avastin® VEGFR Approved Metastatic carcinoma, non–small cell lung cancer, breast cancer

TORISEL™ mTOR kinase Approved Advanced RCC

Herceptin® Her-2 Approved Her-2-positive breast cancer

Tykerb® Her-2, other kinases Approved Advanced breast cancer

Sprycel® TK inhibitor Approved CML, acute lymphoblastic leukemia

Nexavar® Multikinase inhibitor Approved RCC

Rapamune® mTor kinase Approved Organ rejection in renal transplants

Fasudil Rho kinase Approvedb Cerebral vasospasm following surgery

Roscovitine CDK2 Phase II Different cancers

BAY-43-9006 RAF1 Phase III Melanoma, renal and solid tumors

Vatalanib VEGFR2 Phase III Colorectal and GI stromal tumors

ZD-6474 VEGFR2, EGFR Phase II Different cancers

Ruboxistaurin PKC Phase III Diabetes

BIRB-796 p38a Phase III Rheumatoid arthritis, Crohn’s disease

CML, chronic myeloid leukemia; GI, gastrointestinal; mTOR, mammalian target of rapamycin; PDGFR, platelet-derived growth factor receptor; RCC, renal cell carcinoma; VEGFR, vascular endothelial growth factor receptor.aCetuximab (Erbitux), Merck KGaA (Darmstadt) under license from ImClone Systems Inc. (New York); panitumumab (Vectibix), Amgen (Thousand Oaks, CA); sunitinib malate (Sutent), Pfizer (New York); bevacizumab (Avastin), Genentech/Roche (Indianapolis, IN); temsirolimus (TORISEL), Wyeth (Madison, NJ); lapatinib (Tykerb), GlaxoSmithKline (Research Triangle Park); dasatinib (Sprycel), Bristol-Myers Squibb; sorafenib (Nexavar), Onyx Pharmaceuticals (Emeryville, CA)/Bayer HealthCare Pharmaceuticals (Wayne, NJ); sirolimus (Rapamune), Wyeth.bApproved in Japan. Information taken from Karaman et al.,17 www.fda.gov (drugs approved), and www.clinictrials.gov.




due to the high degree of structural similarity in the open conforma-tions of many kinases. It is probable therefore that compounds bind-ing to active kinases in the open conformation are much more likely to inhibit several kinases in comparison to those compounds binding to the inactive state.

There are significant advantages to developing inhibitors of the inactive form of a kinase, not the least of which are enhanced specificity and the lack of a necessity to compete with high (mil-limolar) ATP concentrations. However, there are also several disad-vantages. In the case of Abl kinase, for example, drug resistance develops in cancer cells because of mutations in the unique struc-tural region associated with Gleevec binding.22,23 Although these mutations do not affect kinase activity in themselves, they markedly attenuate the ability of the enzyme to bind Gleevec. By contrast, such mutations do not affect the ability of PD173955 to inhibit the kinase, further suggesting that the two drugs interact with different sites on the same kinase and that the mutations influence the Gleevec binding site in the inactive conformation. When drug re-sistance is rapidly induced, several problems occur in that the com-pound is rendered therapeutically less active, ultimately resulting in remission of Gleevec therapy.

Although inhibitors of inactive ki-nase conformations hold promise in the discovery of selective drugs, sur-prisingly there is a paucity of HTS technologies that facilitate the identi-fication of such compounds. Indeed, almost all kinase screening assays are predicated upon the measurement of catalytic activity by monitoring either substrate depletion or product accumu-lation. Therefore, they preclude detec-tion of compounds binding to the inac-tive enzyme conformation. Related to this issue is the fact that most pub-lished information on kinase structure centers upon the ATP binding pocket—which has limited utility in structure-based design of compounds binding to the inactive enzyme (given that the ATP binding pocket is generally “locked” in the inactive conformation). This may be why there remain very few compounds rationally designed to inhibit the inactive kinase. Nonethe-

less, screening technologies that could measure kinase conformation changes, as they switch from inactive to active conformation, would be of marked benefit to kinase drug discovery, particularly to identify compounds abrogate such conformational shifts.

In summary, most compounds targeting kinases are directed to-wards the ATP binding pocket and interact with residues conserved across many kinases. As a consequence, such compounds are non-selective and inhibit many kinases. The prototypical example of such ATP binding site drugs is the antibiotic alkaloid staurosporine, which binds potently and surmountably to the ATP binding site of many STKs and TKs to inhibit activity.24,25

However, despite such initial concerns that compounds targeting the ATP binding domain would be nonselective (and therefore cause many unwanted side effects), several novel and therapeutically effective drugs have been identified, all of which bind in close proximity but not in the ATP recognition site and which exhibit few side effects. This arises from the fact that, while there are several structurally conserved regions in this domain, there are also numerous nonconserved residues around the vicinity of the ATP binding site to which drugs can selectively bind and still occlude ATP access to the kinase to retard enzyme activity.

FiG. 1. Schematic representation of Abl kinase (left) in the active conformation and (right) in the inactive conformation. Structural elements that shift between the two conformations include the P-loop or glycine-rich loop (red) and the activation loop (green). Tyrosine 393 in the activation loop is highlighted in magenta. In the active structure ADP is bound, and in the inactive structure Gleevec (imatinib) is bound (cyan carbons with transparent spheres in both cases). The pictures were pre-pared using Pymol and PDB entries 2G2I.pdb and 2HYY.pdb, respectively.



egLen AnD reisine

Crystallographic analyses support this notion. This analysis in-dicates that within the ATP binding pocket there is a phosphate transfer region that is highly conserved among kinases. There also exists a glycine-rich loop, hinge regions, and a hydrophobic pocket, each of which contain residues unique for each kinase (see Fig. 1). Nowadays, kinase inhibitors are not generally designed to directly interact with the conserved phosphate transfer region but instead are targeted to the hydrophobic pocket, as exemplified in crystal structure analysis of the docking of p38 selective inhibi-tors.26,27 Consequently, small molecules interacting directly with unique residues in the binding pocket confer specificity such that the three-dimensional space they occupy hinders access of ATP to the phosphate transfer site, resulting in specific inhibition of kinase activity.

Allosteric Regulatory Sites of Protein KinasesKinase activity is also regulated by sites orthogonal to the catalytic

domain. A notable example occurs with some PKC isoforms pos-sessing allosteric sites for the binding of calcium ion, diacylgycerol, and phosphatidylserine, each of which induce conformational changes causing activation of the catalytic domain without directly interacting with the ATP binding pocket.28 Furthermore, in the case of several soluble TKs, such as Her-2 described further below, regu-latory subunits (in the case of Her-2, the EGF receptor) interact with the catalytic subunit at allosteric sites in order to influence activity. This produces conformational changes in the activation loop, con-verting the catalytic domain from a closed to open state. This switch occurs in the case of positive regulators. In the case of endogenous inhibitors, allosteric sites that modulate ATP and substrate binding sites also stabilize the kinase in an inactive conformation. For inte-gral membrane TKs, growth factors additionally interact at extracel-lular sites, inducing conformational changes in the catalytic domain to increase kinase activity.

These allosteric sites clearly present novel targets for inhibitors. Since they lie outside of the catalytic domain, they frequently pos-sess unique structural motifs, allowing compounds to target them in a selective manner. Furthermore, as they modulate kinase activ-ity, occluding those sites theoretically may regulate excessive activ-ity, without necessarily affecting basal activity. Such modulation may provide distinct advantages over active-site directed inhibitors. Although few allosteric site-directed drugs have been developed to date, such sites will undoubtedly provide targets for a new genera-tion of kinase inhibitors with more selectivity than compounds hitherto developed. Examples of such allosteric regulators and the kinases they regulate are described further below.

PKA. One of the first allosteric modulators of kinase activity was identified for PKA.29 PKA comprises distinct catalytic and inhibitory regulatory subunits—the latter binding cAMP. The inactive form of the enzyme consists of two catalytic subunits associated with two regulatory subunits, which maintain the haloenzyme in an inactive state. Structural analysis reveals that residues in domains of the catalytic subunit and the activation loop interact with a phosphate binding cassette in the regulatory subunits. The interaction of these regions maintains the catalytic subunit in an inactive conformation. The binding of cAMP to the phosphate binding cassette in the reg-ulatory subunits causes unfolding of the regulatory subunits, ulti-mately releasing them from the catalytic subunit. These allosteric binding sites in the catalytic subunit consequently interact with the regulatory subunits. These sites also present targets for identifying novel drugs that increase PKA activity by attenuating the interaction of the regulatory subunit with the catalytic subunit. In effect, such compounds would de-repress or “disinhibit” the catalytic subunit.

IkB kinase. IkB kinase is a critical enzyme that regulates nu-clear factor kB (NFkB)/IkB signaling.30,31 NFkB is sequestered by IkB in the cytosol, thereby preventing NFkB translocation to the nucleus. However, serine residue-specific phosphorylation of IkB, by IkB kinase, promotes dissociation from NFkB, triggering NFkB nuclear translocation. This process then stimulates cascading changes in gene expression. Collectively, IkB kinase thus provide a critical checkpoint in NFkB signaling.30 IkB kinase mediates the effects of a large family of cytokines and chemokines on NFkB/IkB signaling. Notably, IkB kinase mediates the cellular effects of interleukin (IL)-1 and tumor necrosis factor a (TNFa), which play critical roles in inflammatory diseases such as ar-thritis and atherosclerosis as well as several autoimmune diseases including rheumatoid arthritis and Crohn’s disease.32 The regula-tion of NFkB signaling is important as NFkB stimulates expres-sion of cell adhesion molecules involved in tumor metastasis and angiogenesis and tumor growth.33 By contrast, blockade of the actions of NFkB, via suppression of IkB phosphorylation, induces tumor cell apoptosis and death.34 There is presently no way to attenuate the actions of NFkB other than preventing its disso-ciation from IkB. Consequently, inhibition of IkB kinase activity presents an attractive therapeutic target to develop antiprolif-erative agents. However, there are potential problems in develop-ing drugs that directly block the catalytic activity of IkB kinase. For example, if both basal and stimulated activities are reduced, cell apoptosis may ensue. Although this would be beneficial in developing anticancer agents, it may induce side effects when




used to treat other disorders, including inflammatory and immune diseases, where cell death is not required for seeing a beneficial therapeutic outcome.

May et al.35 showed that IkB kinase is under positive allosteric regulation by NFkB essential modulator (NEMO). Furthermore, as-sociation of IkB kinase with NEMO is obligatory for cytokine stimulation of kinase activity. Since NEMO stimulates IkB kinase activity via an allosteric site, inhibitors of this site may reduce stimulated IkB kinase activity without directly acting upon the kinase catalytic site. Indeed, small peptides corresponding to a region of the carboxyl terminus of IkB kinase reversed the inter-action with NEMO and prevented TNFa and IL-1 stimulation of IkB kinase activity. They also prevent cytokine-induced prolifera-tive responses in tissues in vitro and blocked inflammation in vivo; however, they did not block the kinase catalytic activity. Since the interaction of NEMO with IkB kinase was inhibited with only a short peptide sequence—and the contact site in IkB kinase for NEMO binding is six amino acids—it is highly probable that small molecules could be identified to block interaction of NEMO with IkB kinase. May et al.35 therefore suggested that developing such small molecule inhibitors of the NEMO/IkB kinase interaction would be effective in preventing cytokine-induced inflammatory and proliferative effects without impairing basal NFkB signaling, which is critical for normal cell survival. Antagonists of NEMO could avoid potential toxic effects of inhibitors of IkB kinase catalytic activity and be important drugs to control the NFkB signaling pathway.

Cyclin-dependent protein kinases (CDKs). CDKs are STKs that regulate the cell cycle. They have been found to be critical targets both for discovery of novel anticancer drugs but also for other diseases, including neurological disorders such as Alzheimer’s and Parkinson’s disease.36,37 Like other kinases, the catalytic subunits of CDKs comprise two domains, with the ATP and substrate bind-ing sites centered at a cleft between the protein lobes.38 In the monomeric form, the catalytic subunit is in a closed inactive con-formation. Binding of cyclin to one of the lobes of the catalytic subunit induces conformational changes in the activation loop, allowing for partial opening up of ATP and substrate binding sites and stabilization of the binding of ATP to the catalytic domain. This conformational change also allows for phosphorylation of a critical serine residue in the activation loop, stabilizing substrate binding and providing full activation of catalytic activity.39 Al-though CDKs control cell cycle progression, their expression is relatively constant throughout the cycle. By contrast, cyclin ex-

pression changes during the cell cycle as a result of transcriptional up-regulation.38,40,41 This process allows CDKs to be activated only at discrete times during the cell cycle. As expected, different CDKs are regulated by different cyclins because of the selective binding of cyclins to individual CDKs. By this means, CDKs serve as check-points during the cell cycle to facilitate conversion from one phase to another.37

CDKs are also modulated by a family of inhibitors, the Cip and INK4 proteins. These block kinase activity via allosteric sites distinct from those that bind the cyclins.41–43 INK4 protein binding alters the catalytic domain reducing affinity for ATP and also reduces affinity of the CDKs for the cyclins. INK4 proteins bind to sites near the ATP binding pocket of CDKs as well as sites in both lobes of the catalytic subunit. When bound to the monomeric CDKs, the INK4 proteins essentially convert the enzyme to an inactive form and also reduce their ability to be stimulated by cyclins.

Taken together, the dynamic interplay of cyclins and INK4 proteins through allosteric sites allows precise control of CDK activity. Since all CDKs are involved in cell cycle control and cell proliferation, abnormalities in the expression or action of these protein allosteric regulators lead to proliferative disorders including cancer. Specific alterations may involve modifications of INK4 proteins such that they are less effective in interacting with CDKs, as well as mutations in CDK such that they are less sensitive to INK4 proteins or increased expression of cyclins to overactivate CDKs.44,45

Consequently, allosteric regulation provides alternative sites for development of CDK inhibitors beyond the classical approach of developing catalytic site inhibitors. Since the cyclins bind to sites outside of the catalytic cleft, small molecule drugs targeting those sites may inhibit cyclin activation of the CDKs. Furthermore, there is a high degree of specificity with regards to which cyclin binds to which CDK, suggesting that drugs could be identified to selec-tively target CDKs. Identification of such allosteric inhibitors clearly requires the development of HTS assays that allow mea-surement of the physical association of the different cyclins with CDKs, given that simple measurement of catalytic activity would not distinguish active site inhibitors from those affecting cyclin regulation in themselves.

EGFR and other RTKs. A large family of RTKs is expressed in the human genome. Activation involves binding of ligands, such as growth factors, to a monomeric form of the receptor. This induces oligomerization and autophosphorylation, leading to kinase activa-tion. The most extensively characterized RTKs are the EGFR kinase family. These enzymes are proto-oncogenes and comprise four



egLen AnD reisine

receptors: EGFR, ErbB2, ErbB3, and ErbB4.46 EGF binding causes oligomerization of EGFR with ErbB2 (also referred to HER-2), and, while HER-2 does not bind extracellular ligand, stimulation of the kinase activity requires association with EGFR. HER-2 is particu-larly important in breast cancer, being overexpressed in more than a quarter of patients with the disease. It is also the target for treatment with the monoclonal antibody Herceptin, which blocks the forma-tion of EGFR–HER-2 complexes.47 Signaling via the EGFR complex generally involves EGF binding to EGFR monomers, which then recruit HER-2 to form a heterodimer.48 The extracellular domains of each receptor associate such that the kinase domains comes in close proximity. The kinase domains phosphorylate each other to enhance catalytic activity and then also phosphorylate substrates involved in cell proliferation and tumor genesis. In addition to induction of oligomerization of EGFR with HER-2, EGF binding induces con-formational changes in the EGFR that activates the kinase domain. Thus, in its monomeric form, the kinase activity of the EGFR is low, whereas activity is greatly enhanced following ligand binding. Zhu et al.49 have proposed that the extracellular domains of the EGFR serves as a negative constraint on TK activity since mutant forms of the EGFR lacking the extracellular domains are constitutively active and highly oncogenic.50

Protein KinAse trAnslocAtion And comPArtmentAlizAtion

Although catalytic activity is an important component of kinase function, it is not the only factor involved in the function of these enzymes. The unique functions of kinases depends not only which proteins they phosphorylate but also spatial access to their unique substrates, which in many cases are located in different cellular locations to the kinase. Consequently, many kinases translocate from the cytosol to a target in a different cellular compartment, such as the nucleus. This translocation process is an important regulatory mechanism by which kinases control cell activity. For example, b-adrenergic receptor kinase translocates from the cytosol to the cell membrane, where it phosphorylates and desensitizes G-protein coupled receptors (GPCRs).51–54 Mitogen-activated protein kinase (MAPK) translocates from the cytosol to the nucleus, where it reg-ulates gene transcription.55

Overall, translocation is an important process for multipurpose kinases that regulate several different processes in the same cell. PKCd translocates from the cytosol to the cell membrane to inhibit an inward rectifying K1 channel to cause depolarization.56 It also translocates to the mitochondria where it activates an ATP-depen-

dent K1 channel to induce apoptosis.57 PKCd also translocates to the nucleus where it regulates genes involved in cell growth. O’Flaherty et al.58 have shown that, in the same cell, PKCd simultaneously translocates to both the cell membrane and nucleus. Therefore, translocation to different cellular compartments is how PKCd in-duces widespread control over the cell and is a general mechanism by which kinases control cell activity.

The importance of kinase translocation in cellular control is em-phasized in studies by Zaccolo and Pozzan,59 who identified discrete microdomains of cAMP generation in cardiac myocytes in response to b-adrenergic receptor stimulation. This discrete formation of cAMP causes compartmental activation of PKA in specific subcel-lular locations, including the transverse tubule/junctional sarcoplas-mic reticulum membrane. These authors suggested that compart-mental localization and activation provide the basis for specificity of cellular kinase activity and that each protein kinase possesses a unique microdomain of activity due mainly to a unique transloca-tion processes.

The mechanisms involved in protein kinase translocation and compartmentalization have been most extensively studied for PKC and PKA. These two families of proteins are involved in controlling the localization of these kinases to different compartments. The receptors for activated C-kinase (RACKs) are involved in translocat-ing PKC isoforms to different cellular regions, while the A-kinase anchoring proteins (AKAPs) are involved in localizing PKA to dif-ferent sites in cells.60–62 Different members of these proteins interact with specific isoforms of PKC and PKA and not only serve as shut-tling or targeting proteins but are also able to put the kinases in close proximity to other proteins to generate networks of interact-ing proteins. Alterations in these proteins may be critical for dys-function of kinase signaling and may be part of the molecular defect involved in cardiac failure. Furthermore, since these proteins have such important roles in the activity of PKA and PKC, changes in their expression or function can significantly affect the activity of the protein kinases even if the levels of the protein kinases are normal. Importantly, the sites of interaction of the protein kinases with RACK and AKAP have been identified, raising the possibility of developing drugs that block kinase interaction with these target-ing proteins to modify selective functions of the protein kinases.

RACKsRACKs are members of the family of WD 40 proteins possessing

significant sequence similarity to the G protein subunit Gb.61 There are two main RACKs—RACK1 and RACK2. RACK1 primarily associ-ates with PKCbII, and RACK2 interact with PKC´. Activated PKC




interacts with RACKs, which transport the kinase from the cytosol to different cellular locations, such as the cell membrane and nu-cleus. Translocation is dependent on PKC activation. RACK1 associ-ates with the cytoskeleton via plectin.63 Binding of activated PKCbII to RACK1/plectin frees RACK1 from the cytoskeleton to allow for translocation of the RACK1/PKCbII complex. RACK1 directs PKCbII to different cellular locations via unique targeting sequences.

The interaction of RACK1 with PKCbII occurs at the so-called C2 region, which is distinct from the allosteric sites involved in calcium ions, diacylglycerol, and phosphatidylserine activation. Peptides corresponding to the C2 region (or the kinase binding domains) in RACK1 inhibit binding to PKCbII, thus preventing translocation of PKCbII to different cellular compartments.64 Im-portantly, dequalinium can inhibit association of RACK1 with PKCbII.65 Dequalinium is an antitumor small molecule drug that had been known to block PKC activity, but the mechanism of inhibition had not been known until its ability to block association with RACK1 was identified.65 Thus, the ability of dequalinium to block RACK1/PKC interactions is a potential mechanism by which the compound acts as an anticancer agent. Importantly, the abil-ity of dequalinium to block RACK1 association with PKCbII sug-gests that small molecule inhibitors could be developed to prevent the interaction of the kinase with its cognate transporting protein. This observation may be of therapeutic value in that PKC is in-volved in causing diseases such as cancer and cardiac failure. Thus, compounds blocking RACK1/PKC association may be devised to prevent the disease-associated actions of PKC—but not necessarily all actions of the kinase—particularly those that do not require PKCbII to associate with RACK1.

A role for RACK proteins in cancer has also been suggested be-cause in several tumors, RACK1 levels are greatly increased.66 Fur-thermore, there is a positive correlation between RACK1 levels and PKCbII translocation and activity. Since RACK1 is critical for the functions of PKC, then elevated levels of this protein can result in increased function of PKC even if the levels of the kinase itself are not altered. Interestingly, RACK1 associates with other proteins in addition to PKCbII that have a role in cancer. RACK1 binds with the proto-oncogene gene product c-Src, and the two can be co-immunoprecipitated from cancer cells.67 Binding of RACK1 inhibits c-Src activity and decreases cell proliferation induced by the TK. PKCbII stimulates association of RACK1 with c-Src, suggesting that RACK1 can mediate cross-talk between the two kinases and control of PKCbII over c-Src.

RACK1 and PKCbII have been linked to cardiac failure.68 Thus, levels of PKCbII are greatly increased in myocardium of patients

with heart failure, and selective overexpression of constitutively active PKCbII in transgenic mice produces cardiac hypertrophy and failure that can be blocked by the PKCbII inhibitor, LY-333531. Furthermore, angiotensin II-induced cardiac hypertrophy is as-sociated with increased co-localization of PKCbII with RACK1 in heart tissue.

PKCe, like PKCbII, is also involved in cardiac failure. Overexpres-sion of this isoform in mice produces cardiac hypertrophy and fail-ure.69,70 Under normal conditions, PKCe associates with RACK2, and this adaptor is responsible for the intracellular movement of PKCe to different cellular compartments. In animals overexpressing PKCe that have a cardiac hypertrophy phenotype, PKCe is able to bind to RACK1.68 As a consequence, it has been suggested PKCe can switch its function and take over the role of PKCbII in causing hypertro-phy.68 Furthermore, overexpression of PKCe increases PKCbII bind-ing to RACK1 and translocation, suggesting a synergism occurs between the two PKC isoforms with RACK1 being the nexus.

These studies collectively indicate that RACK1 is a critical element in cardiac failure and that drugs inhibiting the interaction of RACK1 with the PKC isoforms would be expected to be cardioprotective. Since PKCe normally does not interact with RACK1, yet does so in hypertrophic cardiac tissue, drugs blocking this interaction could be selective for cardiac disease tissue and serve to inhibit this synergism between PKCe and PKCbII in causing myocardial failure.

RACK1 is also involved in defects in the immune system.71 Mac-rophages, consequently, are critical elements in the ability of the immune system to target cancer cells. The cells respond to stimulation by lipopolysaccharide by increasing TNFa production. This effect, mediated by PKC, is essential for the host response. With age, mac-rophages become less responsive to lipopolysaccharide and produce less TNFa in response to challenge, and as a consequence humans are less able to respond to infection. The diminished ability of li-popolysaccharide to stimulate TNFa production is due to a defective PKC signaling pathway involving a down-regulation of RACK1 ex-pression.71 These studies suggest that RACK1 is intimately involved in controlling the immune system; loss of expression of this enzyme can produce immunodeficiencies due to reduced PKC signaling.

AKAPAKAPs are a family of proteins that anchor PKA to specific cellular

locations60,62 and bind to the inactive PKA complex. They have target-ing sequences that direct the kinase to specific locations. These target-ing sequences in AKAPs localize PKA to the cell membrane via myris-toylation and palmitoylation signals such that the kinase regulates ionic conductance channels and localize the kinase to the mitochon-



egLen AnD reisine

dria, cytoskeleton, and nucleus to modulate other cellular activities. Once anchored and when cAMP is generated at that location, the catalytic subunit is freed from the regulatory subunit to phosphorylate substrates involved in the mediation of cAMP signaling. In addition to anchoring PKA to specific subcellular locations, AKAPs also inter-act with other proteins involved in PKA signaling and by bringing those proteins together create signaling networks. Thus, AKAPs can bind both PKA and cAMP phosphodiesterase simultaneously. By bring-ing both enzymes in close proximity, the initiation and termination of PKA activity are localized and fixed. Mutations in AKAPs produce altered intracellular signaling and have been linked to disease etiolo-gies. For example, mutations in AKAP9 cause altered regulation of potassium channels involved in QT interval regulation.72 Furthermore, studies have associated variants in AKAPs with increased breast can-cer risk,73 suggesting that these proteins could have diverse cellular function in addition to kinase anchoring.

Protein KinAses As therAPeutic tArGets Due to their diverse physiological roles and the emerging under-

standing that mutations in their genes are responsible for a variety of diseases, kinases are important targets for the drug development. Some kinases, in particular, have generated considerable interest as drug targets as described below.

MAPKsMAPKs play a central role in propagating effects of growth factors,

cytokines, hormones, and neurotransmitters on gene expression and a host of other cellular responses. Abnormalities in MAPK function are linked to cancer, inflammatory diseases, and immune and central ner-vous system disorders.74–76 There are three major subfamilies: extracel-lular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 kinases.77 All have a high degree of amino acid sequence similarity but differ mainly in their activation loops sequence as well as their response to different stimuli. In general, however, ERKs respond to mitogenic and proliferative stimuli, while both JNK and p38 kinases respond mainly to environmental stressors.

ERKs. ERKs regulate activity of two critical transcription factors, activator protein 1 (AP-1) and NFkB.74 By activating c-Jun, a com-ponent of AP-1, ERKs can induce expression of cyclin D,78,79 neces-sary for activation of CDK4 and CDK6, both of which control cell division via the transition through G1 phase of the cell cycle. ERKs can also phosphorylate and activate the proto-oncogene, myc, which is important for cell cycle progression and tumor cell proliferation.74

Therefore ERK inhibitors are useful in blocking cell cycle progres-sion and serve as anticancer agents.

JNKs. There are three major members of the JNK family: JNK1, JNK2, and JNK3, which are encoded by different genes.80 These kinases are activated by TNFa and IL-1 as well as other stress and inflammatory stimuli. JNKs can phosphorylate and transactivate c-Jun and are essential for TNFa stimulation of c-Jun and AP-1 transactivation.81 They are important also in inducing apoptosis via Bcl-2 and p53.

The JNKs have a particularly critical role in regulating immune cells where they stimulate expression of TNFa, IL-2, E-selectin, and matrix metalloproteinases.82 These proteins are all involved in in-flammatory responses, suggesting a role of JNK in inflammation. Matrix metalloproteinases are essential factors in causing cartilage and bone destruction in rheumatoid arthritis. JNK, via activation of AP-1, stimulates matrix metalloproteinase expression, and SP-600125, an inhibitor of JNKs,83 decreases matrix metalloprotei-nase expression in synoviocytes from rheumatoid arthritis patients.84 It also decreases joint swelling and bone and cartilage destruction in adjuvant-induced arthritic rats.

JNK also has a role in modulating T cell function.81 The JNKs control expression of TNFa and also mediate responses induced by TNFa, indicating these kinases serve a central role in TNFa func-tions. JNKs have important roles in T cell maturation. Blockade of JNK prevents TH1-mediated immune responses but not TH2 re-sponses, indicating that JNKs are necessary for a TH1 phenotype. These studies suggest that JNK inhibitors may provide selective control of T cell function in treating immune disorders. In fact, the JNK inhibitor CC-401, produced by Celgene (Summit, NJ), is in clinical trials to test effectiveness in treating these immune disor-ders.74

JNK has also been proposed to have a role in central nervous system disorders such as Parkinson’s disease. Expression of peptide inhibitors of JNK in brain using adenovirus blocks the death of dopamine neurons and improves behavioral deficits in mouse mod-els of Parkinson’s disease.85 This suggests that JNK inhibitors may have neuroprotective effects, and small molecule JNK antagonists could be useful in treating the progression of Parkinson’s disease.

Finally, JNK may have a role in development of diabetes and obesity.86 JNK phosphorylates and inactivates insulin receptor and insulin receptor substrate. This inhibits insulin signaling and aug-ments development of insulin resistance. JNK also promotes expres-sion of inflammatory agents that decrease insulin signaling. Knock-out studies suggest that JNK1 is involved in causing obesity, and




a number of studies have suggested a link between obesity and occurrence of type 2 diabetes. These studies suggest that JNK in-hibitors may also be useful in treating metabolic disorders as well as inflammation and central nervous system diseases.

p38 MAPK. The p38 MAPK consists of a family of four splice variants, with p38a being the most well studied. p38a is primarily expressed in macrophages and monocytes, has a critical role in regulating expression of the inflammatory cytokines IL-1, IL-6, and TNFa, and has an essential role in mediating stressed induced cel-lular responses.87

At a mechanistic level, p38 can activate a number of transcrip-tion factors, including cAMP response element binding protein and activating transcription factors.74 Inhibitors of p38 block expression of IL-1 and TNFa and prevent neutrophil chemotaxis and lymphocyte proliferation induced by IL-2 and IL-7. In con-trast to ERKs, p38 blocks induction of cyclinD to arrest cell cycle progression.74,75

IL-1 and TNFa are critical cytokines involved in inflammation.88 Since p38 is involved in the production of these cytokines, drugs targeting the inhibition of p38a could be effective in treating in-flammatory disorders. In fact, p38 inhibitors have been found ef-fective in animal models of rheumatoid arthritis, including collagen- and adjuvant-induced arthritis.89–92 Furthermore, such inhibitors are effective in other animal models of inflammatory disorders such as in cardiac hypertrophy,93 suggesting that such drugs could be ef-fective in treating a number of different inflammatory disorders. In fact, the p38 inhibitor BIRB-796 is in clinical trials for treatment of rheumatoid arthritis and Crohn’s disease, while the inhibitor VX-702 is being tested for treating acute coronary syndromes.

IkB KinaseBecause IkB kinase mediates the effects of cytokines and

chemokines, which induce inflammatory diseases such as arthri-tis and atherosclerosis, on NFkB/IkB signaling,94 small molecule inhibitors of IkB kinase might have applications in treating these chronic inflammatory diseases. Selective IkB kinase inhibitors have been developed, including SPC-839 and PS-1145.95–97 These compounds are effective in blocking IkB kinase in vivo, reduce IkB kinase-mediated production of cytokines, and have been found effective in some animal models of arthritis. Bristol-Myers Squibb (Princeton, NJ) has developed an allosteric inhibitor of IkB kinase, BMS-345541, that is selective for IkB kinase b, does not compete at the ATP binding site, blocks TNFa challenge in vivo, and is effective in animal models of arthritis.98,99 This drug

may form that basis of a unique family of anti-inflammatory drugs devoid of the anti-apoptic effects of direct-acting IkB ki-nase inhibitors.

Glycogen Synthase Kinase 3 (GSK3)GSK3 plays a critical role in insulin signaling and is an impor-

tant target for development of novel diabetes drugs and drugs targeting other disorders.100–104 GSK3 phosphorylates and inhibits glycogen synthase.6 This blocks conversion of glucose to glycogen. GSK3 is normally inhibited by a variety of growth factors, includ-ing insulin. The ability of insulin to inhibit GSK3 is critical for its ability to stimulate glycogen and protein synthesis. In type 2 diabetes, cells become resistant to the effects of insulin and there-fore are unable to convert glucose to glycogen. Since GSK3 is a critical component of the insulin signaling pathway, GSK3 in-hibitors could bypass the insulin resistance to mimic actions of insulin to stimulate glycogen synthesis.

In fact, selective inhibitors of GSK3 have been found to stimulate glycogen synthase and glycogen synthesis from glucose.102 In ani-mal models of type 2 diabetes these inhibitors normalize blood glucose levels. This suggests that GSK3 inhibitors may have a role in the treatment of type 2 diabetes. Furthermore, studies have sug-gested the GSK3 inhibitors could protect pancreatic beta cells from death and be a potential therapy for type 1 diabetes.103

PKCOne of the most well-studied roles of PKC in disease is its role

in diabetes.105,106 Studies in animal models and humans with dia-betes have shown increased activation of PKC in the vasculature, including blood vessels of the retina and kidney. This enhanced activity causes damage in the blood vessels, increased permeabil-ity, endothelial cell activation, altered blood flow, leukocyte adhe-sion, and abnormal growth factor signaling. The enhanced PKC activation may be induced by elevated glucose in diabetes, which can result in increased formation of diacylglycerol, a natural stimulant of PKC. Many of the vascular problems in diabetes ap-pear to be directly linked to dysfunction of PKCb, and overactiva-tion of this isoform has been associated with retinopathy, neu-ropathy, and nephropathy.

Lilly (Indianapolis, IN) developed the selective PKCb inhibitor ruboxistaurin and has shown this drug to be effective in a num-ber of preclinical studies on diabetes.28 In human clinical trials, ruboxistaurin prevented hyperglycemia-induced impairment of endothelial-dependent vasodilatation in healthy subjects.107–109 It also decreased the development of sight-threatening macular



EGLEN AND REIsINE

edema and visual loss in diabetic patients in phase III trials, although it did not prevent the progression of retinopathy. Ru-boxistaurin is under consideration by the Food and Drug Ad-ministration for approval for the treatment of moderate to severe nonproliferative diabetic retinopathy and if approved would be the first PKC inhibitor approved for treatment of a disease.

Kinase assays in Drug DiscoveryA wide variety of technologies have been developed to measure

kinase activity and therefore to screen for novel inhibitors. Several excellent reviews have been published on these technologies in the past, from both a biochemical as well as a cell-based assay perspec-tive.110–112 These assays also provide for identification of kinase inhibi-tors not easily identified by rational design based on crystal structure analysis. Compounds that inhibit kinase activity by binding to regions in the catalytic domain other than the ATP binding sites can be iden-

tified using screening technologies allowing for detection of compounds that stabilize the kinase in an inactive state. Furthermore, screening assays can reveal allosteric regulators of protein kinases as well as those that block kinase translocation. Finally, for the RTKs, assays measuring ligand binding or dimerization of the receptors can identify compounds that inhibit activation of the kinase. Such inhibitors can confer very high degrees of specificity for particular kinases.

Cell-Free Protein Kinase Assays (Table 2)Radiometric assays. An early, and widely used, approach to mea-

sure kinase activity measured the enzymatic incorporation of 32P into peptide or protein substrates.113–115 A modification of this stan-dard basic research approach that has been utilized in screening against protein kinase employs the scintillation proximity assay (SPA) from GE Healthcare (Pollards Wood, U.K.). Here, a biotinylated 33P-labeled substrate generated by kinase action is captured on a streptavidin-coated SPA bead. Light is then emitted from the beads

Table 2. Cell-Free Protein Kinase Assays

Readout

Substrate Phosphorylation

ATP/ADP Modulation Substrate BindingRadioactive Nonradioactive

Assay FlashPlateSPA

DELFIA (TR-fluorescence)LANCE (TR-FRET)AlphaScreen (TR-FRET)LanthaScreen (TR-FRET)IMAP (TR-FRET) (FP)Far-Red PolarScreen (FP)KinEASE (FP)Z’LYTE (FRET)IQ kinase (fluorescence quench)Antibody Beacon (fluorescence quench)TruLight (fluorescence quench)

EasyLiteKinase-GloADP QuestADP-FP

AlphaScreenEFC

Advantages Simple to useDetects most protein kinasesNo antibody neededNo modification of substrate

SimpleSensitiveMost homogeneousHTSAny substrateFor some no antibody needed (AlphaScreen phosphor sensor, IMAP)Most can be used in lysate

No antibody or substrate modificationHomogeneousCost-effective

Competitive binding assayEFC assay for all kinasesNo antibody or ATP neededHTS

Disadvantages Radioactive wasteLimited HTS

Most require labeling of substrate (not AlphaScreen Surefire) and require antibody, limiting number of kinases studiedOptical interference of compoundsExpensive

Weak kinase results in low response for ATP depletion assayHigh backgroundNot a problem for ADP formation

Identifies inhibitors at ATP binding site

3.Eglen.ADT.7.1P.indd 32 3/24/09 12:47:54 PM



via 33P-labeled substrate and detected by either scintillation spec-trometry or using a charge-coupled device imager.116,117 An alterna-tive approach is the use of FlashPlate® (PerkinElmer, Waltham, MA) technology in which the radiolabeled substrate is captured on a microtiter plate surface either using streptavidin (to capture bioti-nylated substrate) or with nickel chelate (to capture His-tagged substrate). Incorporation of 33P substrate into the solid-phase sub-strate on the FlashPlate then comes in close proximity to a scintil-lant coating on the plate. The resulting signal is then measured following a washing step by scintillation spectrometry.

The main advantages of both of these technologies are that they are simple to employ and do not require the generation of specific antibodies against phosphorylated substrate, which can be costly. In addition, a wide diversity of kinases, in either crude preparations or purified recombinant form, can be studied kinetically by this approach. Consequently, the technique has been widely adopted in lead profiling activities or mechanism of action studies, in which Michaelis-Menten kinetics can be used in a wide range of assay conditions and substrate concentrations.118 Nonetheless, there are major disadvantages of employing this approach in HTS because of the use of radioactivity and the inherent high costs of waste disposal associated with the technique.

Nonradiometric assays. The development of selective antibodies that can distinguish phosphorylated from nonphosphorylated forms of the kinase substrates has accelerated the development of a range of nonradiometric assays for HTS.119,120 Presently, polyclonal and monoclonal antibodies against phosphorylated tyrosine, serine, or threonine residues in phosphoprotein or phosphopeptide or sub-strates have been used in many kinase assays. Originally developed as radioimmunoassays, the technology has been adopted to nonra-diometric protocols that do not require wash and separation steps, i.e., they are homogeneous in nature and highly amenable to the kind of automated fluid dispensing instruments used in HTS. In addition, the difficulty of obtaining high-affinity, selective antibod-ies, notably to phosphoserine/threonine residues found in STK sub-strates, has led to the adoption of Lewis metal ions, rather than antibodies, to coordinate the phosphorylated substrates. In general, the methods discussed above assess kinase activity by measurement of phosphorylated product accumulation. Other methods have also been developed that have been suggested to be more generic across kinases. These included assessment of the depletion of ATP during the kinase reaction, as well as measurement of ADP accumulation. In the sections below, each of these approaches will be discussed in turn.

A classical fluorometric assay to measure kinase activity using anti-tyrosine or anti-serine/threonine phosphoantibodies is the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA®, PerkinElmer),121 which employs a lanthanide-labeled antibody for detection. The lanthanide labels have the advantage of a long decay time, as well as a wide Stokes’ shift, collectively resulting in robust signal to noise ratio in the assay. The assay protocol comprises the use of biotinylated substrate, and following the kinase reaction the phosphorylated substrate is then transferred to streptavidin-coated microplates. The phosphorylated substrate binds to the plate, which then reacts to the europium-labeled antibody against phosphory-lated substrate. Time-resolved (TR) fluorometry of the enhanced fluorescence of the lanthanide, which is dissociated by a proprietary enhancement solution, is then carried out to increase sensitivity. This assay protocol, while sensitive, involves the use of a wash and separation step and has limited utility in HTS protocols. An alterna-tive method that can be conducted in a homogeneous fashion is the use of fluorescence resonance energy transfer (FRET)- and TR-FRET-based assays.

Kinase assays using FRET or TR-FRET techniques are widely avail-able to detect kinase activity.122–125 Here, a kinase substrate with an acceptor moiety is incubated with enzyme.126 In the LANCE® Ultra formats (PerkinElmer), the phosphorylated substrate is detected with a specific anti-phosphopeptide antibody labeled with europium chelate molecules (Eu), which serve as donors.118,126 The binding of the Eu labeled-antibody to the phosphorylated peptide substrate with an ac-ceptor moiety (ULight™ in the case of the LANCE Ultra assay) causes the donor and acceptor dyes to come in close proximity and upon excitation results in energy transfer and light emission, which is de-tected as the assay response. Analogous FRET-based assays to measure kinase activity are the Z’-LYTE™ kinase assay and LanthaScreen™ TR-FRET (Invitrogen, Carlsbad, CA) and HTRF® TR-FRET (CisBio, Bagnols-sur-Cèze, France).127,128

The homogeneous kinase AlphaScreen® technology (PerkinEl-mer) employs a bead-based proximity assay approach rather than a FRET approach to detect the phosphorylated substrate.118,129,130 Thus, biotinylated or glutathione S-transferase-tagged substrates are phosphorylated by the kinase under study, and the phospho-rylated substrate then binds to the donor beads via either strepta-vidin or glutathione. Anti-tyrosine or anti-serine/threonine anti-bodies are conjugated to the acceptor beads. Donor and acceptor beads are brought into close proximity as a result, and transfer of singlet oxygen, from the donor to acceptor bead, occurs upon laser excitation. This results in a signal generated by the acceptor bead, which is then detection in a plate reader.128,131 Since the



egLen AnD reisine

distance relies upon an oxygen channeling technique, as opposed to FRET/TR-FRET approaches, large analytes, such as phosphop-roteins, can be detected. Indeed, the distance between the two beads may be as much as 200 nm, which is very much larger than the distance than can be accommodated by a FRET pair.131 Practi-cally, this means that the AlphaScreen format can be used in kinase assays where the kinase substrate is a protein, as opposed to a peptide.

As mentioned above, in recent years the use of Lewis metals, rather than antibodies, has been suggested as a means to detect phosphorylated substrates. These metals are known to bind phos-phate groups and have been used in assays involving AlphaScreen, immobilized metal assay for phosphopeptides (IMAP) and TR-FRET (MDS Analytical Technologies, Toronto, Canada).132,133 Advantages of the approach are that it is sensitive and homogeneous and does not require phosphor-specific antibodies. However, there are limits to the use of the assay, notably in terms of the concentrations of ATP than can be used in the assay. High concentrations of ATP will also bind to the Lewis metals and thus raise assay background. Moreover, that several kinases have moderate activity and require high ATP concentrations to attain maximal velocity suggests limits on the use of the technique.

Fluorescence polarization (FP) assays have also been developed to measure kinase activity.134,135 These assays are simple to use, homogeneous, and relatively simple to automate. Commercially available FP assays include the Far-Red PolarScreen™ FP assay (Invitrogen), KinEASE™ (Millipore, Billerica, MA), and the IMAP-FP assay (MDS Analytical Technologies). However, use of FP by itself as a technique has declined in recent years, principally because of the levels of interference from compounds in the screening library that either autofluoresce or quench the fluores-cent signal. In many laboratories the use of FP techniques in kinase screening has declined in favor of TR-FRET-based ap-proaches where the assay background is inherently lower. As discussed above, several formats of TR-FRET assays are available, including those using HTRF technology (CisBio) and LANCE Ultra (PerkinElmer).

Finally, other assay technologies available to measure accumulation of phosphopeptides include the Antibody Beacon TK fluorescence quenching assay (Invitrogen), the IQ™ kinase assay (Thermo Fisher, Waltham, MA),125 the TruLight™ kinase assay (Merck Biosciences, Darmstadt, Germany), and the enzyme fragment complementation (EFC) assay using b-galactosidase (DiscoveRx, Fremont, CA).136,137 All of these assays utilize a competitive immunoassay format via an anti-phosphopeptide antibody.

ATP depletion assays. Kinase activity converts ATP into ADP via incorporation of a phosphate group into a substrate. Therefore, mea-surement of either ATP depletion or ADP accumulation serves as a measure of kinase turnover.138–140 The Kinase-Glo® luminescent kinase assay (Promega, Madison, WI) and the easyLite-Kinase™ assay (Perki-nElmer) determine the depletion of ATP in a kinase assay via firefly luciferase readout. These assays are homogeneous, do not require specific substrates or antibodies, and can be read on simple lumines-cent readers. Although the format is such that almost any kinase can be screened (since all kinases convert ATP to ADP), the technique has several drawbacks. One of these involves the use of purified kinases, since it does not distinguish ATP depletion induced by a kinase from induced by contamination levels of enzymes such as ATPases.

In parallel to the ATP depletion assays, the ADP Quest™ assay from DiscoveRx measures the formation of ADP as a consequence of kinase activity.138 The newly formed ADP reacts with the fluorescence emit-ter, and the change in light emitted is the readout. In letion assay. Similarly, Bellbrook Labs (Madison, W I) has developed TR-FRET and FP assays to measure ADP generation as a result of protein kinase activity. These assays comprise a terbium-conjugated monoclonal antibody that selectively recognizes ADP. When bound, ADP couples with the fluorscein trace, and a TR-FRET signal is detected because of the close proximity of the fluorscein and terbium. ADP generated from the action of protein kinases displaces the trace reducing the TR-FRET signal in a concentration-dependent manner.

Substrate-kinase binding assays. An alternative, less widely used, screening approach to identify selective inhibitors of kinases is to employ a competitive binding assay in order to assess either binding of substrates to a purified kinase or the binding of small molecules known to bind to the catalytic domain. Consequently, inhibitors of the kinase could be detected by their ability to compete with either the substrate or small molecule ligand from the catalytic domain.

AlphaScreen (PerkinElmer) has been used to measure binding of substrate to protein kinase.129 Here, a glutathione S-transferase-tagged substrate was bound to a donor bead, and a His-tagged target protein kinase bound to an acceptor bead. When substrate and kinase bind, the donor and acceptor beads come into close proximity to allow for a luminescent response.

A small molecule ligand binding assay using the EFC technology of DiscoveRx is based on the use of small molecules like stauro-sporine that bind to the ATP binding site of most protein kinases.141 This complementation assay consists of two components: EA, which is a truncated b-galactosidase that is inactive, and Prolabel™, a peptide that spontaneously recombines with EA to form an active




b-galactosidase that can be detected in a chemiluminescent or fluorescent assay. For the protein kinase assay, Prolabel is covalently coupled to staurosporine to generate the signaling molecule Prola-bel-staurosporine. This molecule is generated in such a way that Prolabel-staurosporine can recombine with EA to induce b-galac-tosidase activity and that Prolabel-staurosporine can bind to most any protein kinase with high affinity. When Prolabel-staurosporine binds to a protein kinase it is no longer accessible to EA, so no b-galactosidase activity is measurable. In the presence of competi-tive inhibitors of protein kinase (staurosporine, ATP, or active site inhibitors), Prolabel-staurosporine is released from the kinase and is able to recombine with EA to restore b-galactosidase activity.

Cell-Based Protein Kinase Assays (Table 3)In vivo protein phosphorylation assays. While molecular assays

have been the standard for primary screening efforts to identify protein kinase inhibitors, cell-based assays are essential in establish-ing the biological effectiveness of any kinase-targeted drug. The physiological actions of protein kinases are dependent on a number of cellular factors, including location, adaptor proteins, and a host of regulatory proteins. These factors cannot be easily simulated in cell-free assays. Thus, while it is possible to identify a compound that can inhibit the activity of the purified kinase, it may be another matter whether that same compound works in cells. Importantly, the cell-based assays can determine whether a compound identified in initial screening efforts is cell permeable and whether it is toxic to cells, and therefore likely to cause side effects.

The initial approaches to cell-based assays to measure protein kinase activity involved loading cells with [32P]ATP and measuring activation of particular kinases by their ability to incorporate 32P into cellular substrates. This approach provided information on the gamut of po-tential targets for a kinase. A nonradiometric modification of this approach is still used today as a proteomic approach to identify kinase targets. Instead of labeling ATP pools with [32P]ATP, phosphorylation is accessed by matrix-assisted laser desorption ionization-time of flight mass spectroscopy, which is able to distinguish whether particular proteins are phosphorylated. These approaches are primarily used for research purposes and are not easily employed for drug screening.

Phosphorylation of proteins in response to protein kinase activation in cells can also be measured using simple enzyme-linked immunosor-bent assays against target substrates that can distinguish phosphorylated from nonphosphorylated proteins. Using a similar idea, the AlphaScreen Surefire™ technology is a luminescent assay for phosphoproteins (TGR Biosciences, Adelaide, Australia/PerkinElmer). This homogeneous ap-proach involves an antibody against a substrate that is coupled to bio-tin so that it can be captured by a streptavidin-coated donor bead and a second antibody directed against the phosphorylated form of the substrate that is coupled to an acceptor bead by protein A. Luminescence is detected when both antibodies bind to the same phosphorylated substrate bringing donor and acceptor in close proximity.

As a second approach to measure kinase-induced phosphorylation to monitor protein kinase activity in cells, Violin et al.142 and Gal-legos et al.143 developed a cell-based assay to measure PKC-induced phosphorylation using FRET. They generated a reporter (C-kinase

Table 3. Cell-Based Protein Kinase Assays

Readout

Phosphorylation of Substrate

Kinase Translocation Receptor Dimerization Radioactive NonradioactiveAssay 32P labeling AlphaScreen Surefire

BRET, FRETEFC, PCAConfocal microscopy

BRET, PCAEFC

Advantages Detects all cell substrates

Targets selective substratesSensitiveHomogeneousAlphaScreen amenable for HTS

Measures movement of activated kinaseCan distinguish different functions of kinaseTotally cell basedAmenable for HTSCan detect allosteric inhibitors of kinases

Detects allosteric inhibitors of receptor kinases

Disadvantages Waste removalInsensitiveNot easy to quantifyNot amenable for HTS

Requires labeling of kinase and substrateCannot be used for orphan kinases

Kinase labelingRequires extensive knowledge about kinase cell biology



egLen AnD reisine

activity reporter) of PKC consisting of a peptide substrate of the kinase fused to cyan fluorescent protein and yellow fluorescent protein and an FHA2 phosphopeptide-binding module and ex-pressed the fusion protein in target cells. When the substrate is not phosphorylated, a robust FRET signal is detected, and when PKC phosphorylates C-kinase activity reporter, the FRET response is lost. They showed that the reporter responded in a predictable manner to known stimulators and inhibitors of PKC. Interestingly, Gallegos et al.143 further modified the assay so that C-kinase activity reporter could be targeted to different cellular locations such as the plasma membrane, cytosol, mitochondria, Golgi apparatus, and nucleus us-ing subcellular targeting sequences. This allowed them to assess PKC activity at different cellular compartments and to determine effect of different drugs on PKC activity in those different locations. They found drugs affected activity differently in different compart-ments. This may provide an important technology to identify drugs that can discriminate distinct functions of PKC. Similar approaches should be possible with other protein kinases in which substrates have been identified.

Cellular imaging to measure protein kinase activity: protein–protein interactions. Besides measuring protein phosphorylation, activity of kinases can be measured using other parameters. As described above, the interaction of protein kinases with other cel-lular proteins is critical for their activity and also provides the subtlety of the complex nature by which they regulate multiple cellular functions in the cell. Thus, interaction of IkB kinase with NEMO is essential for full catalytic activity, and association of cyclins with CDKs is necessary for activation of this family of ki-nases. Cell-based assays that can measure these protein–protein interactions provide a way to detect the activation of different protein kinases and also yield assay formats that can be used to identify drugs that modulate activity without necessarily directly interacting with the catalytic domain of the kinases. That is, if a drug can block the interaction of NEMO with IkB kinase, it will prevent the activation of the kinase but not the basal activity. Such allosteric regulators may provide a number of therapeutic advan-tages over more classical drugs that directly affect the catalytic domain of kinases.

An example of such protein–protein interaction assay in one focused on PKA. PKA is known to be activated when the catalytic subunit dissociates from the regulatory subunit. Measuring the dissociation of these two proteins reveals the activation of this kinase. Using bioluminescence resonance energy transfer (BRET), Prinz et al.144 and Moll et al.145 tagged the regulatory and catalytic

subunits of PKA with the donor Renilla luciferase or green fluo-rescent protein. When the subunits are associated, the catalytic activity is inhibited, and a BRET signal is detected. Stimulation by cAMP causes the subunits to dissociate to increase kinase ac-tivity. This reduces the BRET signal. This assay can be used to identify activators of PKA as well as compounds that stabilize the complex to inhibit PKA activity.

Similarly, Stefan et al.146 have developed a Renilla luciferase complementation assay in which fragments of the luciferase are attached to the regulatory and catalytic subunits of PKA. When the kinase is inactive, the fragments are in close proximity, and full luciferase activity is detected. When PKA is stimulated by cAMP, the subunits and the fragments dissociate, and no luciferase activ-ity is detected. These authors suggested that the sensitivity of this assay is far greater than BRET- or FRET-type assay formats and can be employed for drug screening against GPCRs because of the high sensitivity of the cell-based readout.

Numerous technologies employing either FRET or BRET as an assay readout have been developed to measure protein–protein in-teractions to provide other measures of protein kinase activity. For example, Hundsrucker et al.147 were able to study interactions of PKA with its AKAP anchor proteins. As described in Protein Kinase Translocation and Compartmentalization, this association is impor-tant for the activation of PKA in discrete locations in cells. Hund-srucker et al.147 used BRET technology to measure AKAP association with the regulatory subunit of PKA. BRET probes were inserted into AKAP and the regulatory subunit of PKA. When the proteins were associated, a BRET signal was detected, and when they dissociated, the signal was lost.

Employing FRET to study protein kinase dynamics, Fujioka et al.148 studied interactions of the MAPKs ERK2 and MAPK/ERK kinase (MEK) in intact cells. First, they developed an assay to study intra-molecular changes in ERK2 conformation by placing FRET probes (yellow and cyano fluorescence proteins) at the ends of ERK2. When in an open conformation, the FRET signal is minimal, but when ERK2 binds to MEK, an interaction needed for the activation of ERK2, a conformational change is induced causing an increase in FRET signal. Thus, this simple assay can be employed as a cell-based assay to identify inhibitors of the activation of ERK2 by MEK.

Technologies measuring protein–protein interactions have now been adapted for HTS of compound libraries for protein kinase drug discovery. Odyssey Thera (San Ramon, CA) has developed and em-ploys a protein fragment-complementation assay (PCA) to measure protein–protein interactions in cells in a HTS format.149 The technol-ogy employs fragments of reporters such as dihyrofolate reductase




(DHFR) attached to interacting proteins.149 The fragments themselves are inactive, but when the target proteins to which they are linked associate, the fragments combine to form an active reporter that can be measured with a luminescent readout. For example, Remy and Michnick149 generated two fragments of DHFR and linked the fragments to either the FK506 binding protein (FKBP) or the FKBP–rapamycin binding protein, which are known from a number of studies to associate in cells. When FKAP and FKBP–rapamycin bind-ing protein associate, DHFR is formed. Methotrexate is known to bind with high affinity to DHFR in cells but does not interact with the fragments of DHFR. Thus, they could detect formation of DHFR in cells by using the binding of fluorescein-methotrexate and fluo-rescent microscopy as a measure of FKAP and FKBP–rapamycin binding protein interaction. This technology has been adapted by Odyssey Thera to use other reporters such as b-lactamase, Renilla luciferase, green fluorescent protein, and yellow fluorescent protein to detect intracellular protein–protein interactions, and the green fluorescent protein and yellow fluorescent protein tags have now been employed in an HTS format that can be used for drug discov-ery. While their approach can be used to measure many different biomolecular interactions, it is most suited for detecting weak as-sociations of allosteric regulators of protein kinase activity. Fur-thermore, Remy and Michnick149 also adapted the technology to measure binding of growth factors to their cell surface receptors, and thus the approach can be used to discover allosteric inhibitors of RTKs.

In this regard, several technologies focused on measuring protein–protein interactions have now become available to measure the interaction of growth factors with their RTKs. These technologies use BRET, FRET, or DiscoveRx’s EFC technology and can be adapted for HTS to identifying small molecule inhibitors of the receptor subunit interactions or growth factor binding. BRET has been used to measure dimerization of cell surface receptors. Boute et al.150 used BRET to study insulin receptor activation. They showed that they could measure insulin dimer formation and that BRET signal increased upon activation of the receptor complex with insulin and other growth factors. They suggested that the BRET signal was due to conformational changes in the receptor complex and suggested that the assay could be employed for HTS of small molecule stim-ulators or inhibitors of the complex. Couturier and Jockers151 have used BRET to measure in intact cells dimerization of the leptin receptor, a member of the cytokine receptor family. Similarly, BRET has been used to measuring subunit interactions of the growth hormone receptor152 and IL-2 receptor.153 Using a version of Dis-coveRx’s EFC technology, Wehrman et al.154 developed an assay to

measure interaction of the EGFR and Erb2 subunits. In follow-up studies, Wehrman et al.154 reported that EFC could be employed to study the dynamic interactions of TrkA and p75, suggesting that it could be employed to study subunit interactions of a host of growth factor receptors. Since the assay is adapted to an HTS format, much like the PCA technology of Odyssey Thera, it could be used as a primary screen to identify either small molecule antagonists or growth factor receptor ligands.

Cellular imaging to measure protein kinase activity: protein kinase translocation assays. Protein kinases translocate between different cellular compartments when they are activated. This process is nec-essary because for many kinases their substrates are in different cellular locations than the sites at which they are activated. Moni-toring the movement of kinases provides a means to detect the different functions of kinases and also provides approaches to dis-cover drugs capable of blocking their selective functions.

Imaging technologies have been available for a number of years to measure movement of proteins in cells, especially those employ-ing confocal microscopy.155 Confocal microscopy has been em-ployed to study the activation and translocation of one specialized family of protein kinases, the G protein receptor kinases (GRKs). The activation of most GPCRs induces the translocation of GRKs from the cytosol to the cell membrane, where they catalyze the phosphorylation of cytoplasmic domains of the receptors. This results in the recruitment of b-arrestins to the receptor. Because the activation of most if not all GPCRs is believed to involve the recruitment and translocation of GRKs and b-arrestins to the cell membrane,156 Norak Biosciences (Research Triangle Park, NC) de-veloped both GRK and b-arrestin translocation assay as a drug discovery technology to identify agonists at any GPCR. The tech-nology employs confocal microscopy to measure movement of either GRK or b-arrestin from the cytosol to the cell membrane as a readout of GPCR activation.

Translocation of other protein kinases in cells can also be measured by confocal microscopy. While this approach is widely employed for research purposes, only until lately have technologies been developed to allow for use of confocal microscopy for drug screening. Specifi-cally, Evotec (now PerkinElmer) developed instrumentation and tech-nology referred to as Opera™ that can be used to detect movement of proteins in individual cells. Employing a family of fluorescent microscopic techniques run in parallel,157 the confocal microscopy can be used in a microplate image reader system that allows for HTS of compound libraries. In essence, they can use protein kinase trans-location as a readout for drug screening.



egLen AnD reisine

While the use of confocal microscopy provides a very sensitive and spatially precise measurement of protein kinase movement, the technology is expensive, especially the use of the Opera instrumen-tation. Thus, in addition to confocal microscopy, less costly alterna-tive approaches for HTS of protein translocation involve the use of complementation assays such as Odyssey Thera’s PCA technology and EFC technology158 can be used to detect protein kinase trans-location as a drug screening end point.

conclusionsKnowledge gained on the biology of protein kinases has provided

new insights on how to discover kinase regulators that are more specific and potentially more subtle in their action. Technologies are becoming available to target allosteric regulatory sites of ki-nases. These sites should confer a high degree of specificity of targeted drug since the structures of the allosteric sites are unique for each kinase. They may also provide ways to subtly affect some functions of the kinases but not necessarily all that may be of therapeutic value. This could also occur through the use of ap-proaches to discover drugs that selectively alter the translocation of kinases to some but not all compartments of cells.

While growth factor and cytokine receptors are important for normal physiological control of cells and are critical in certain diseases, the discovery of small molecule regulators of these protein kinases has been limited. Antibodies directed at the protein growth factor have been developed basically to sequester circulating growth factors. Furthermore, antibodies that block growth factor binding to the receptors have been developed. In general, there are many drawbacks to the use of protein therapeutics, including cost, immune reactivity, and other toxicity issues. With the development of tech-nologies to measure protein–protein interactions, it should now be possible to identify small molecules that block oligomerization of growth factor receptors, an essential step in the activation of the transmembrane receptors.

The kinome has been one of the most important targets for drug discovery and development in the pharmaceutical industry. Based on the new discoveries in the field, one would predict that this popularity should continue in the future.

AcKnowledGmentsThe authors wish to acknowledge Drs. Sandra Jacob (CPC/Protein

Structure Unit, Novartis Institutes for BioMedical Research, Basel, Switzerland) and Doriano Fabbro (Center for Proteomic Chemistry,

Novartis Pharma AG, Basel, Switzerland) for preparing Fig. 1 for this paper. Their help is very much appreciated.

disclosure stAtementNo competing financial interests exist.

ReFeRenCeS

Nishzuka Y: Intracellular signaling by hydrolysis of phospholipid and activa-1. tion of protein kinase C. Science 1992;258:607–614.

Edelman A, Blumenthal D, Krebs E: Protein serine/threonine kinases. 2. Annu Rev Biochem 1987;56:567–613.

Fantl W, Johnson, D, Williams LT: Signaling by receptor tyrosine kinases. 3. Annu Rev Biochem 1993;62:453–481.

Yarden Y, Ulrich A: Growth factor receptor tyrosine kinases. 4. Annu Rev Biochem 1988;57:443–478.

Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: The protein 5. kinase complement of the human genome. Science 2002; 298:1912–1234.

Cohen P: The role of protein phosphorylation in human health and disease. 6. The Sir Hans Krebs Medal Lecture. Eur J Biochem 2001;268:5001–5010.

Nestler E, Greengard P: Protein phosphorylation in the brain. 7. Nature 1983;305:583–588.

Uckun F, Mao C: Tyrosine kinases as new molecular targets in treatment of 8. inflammatory disorders and leukemia. Curr Pharm Des 2004;10:1083–1091.

Knuutila S, Bjorkqvist AM, Autio K, Tarkkaqnen M, Wolf M, Monni O, 9. et al.: DNA copy number amplifications in human neoplasms: review of compara-tive genomic hybridization studies. Am J Pathol 1998;152: 1107–1123.

Noble M, Endicott J, Johnson L: Protein kinase inhibitors: insights into drug 10. design from structure. Science 2004;303:1800–1805.

Dancy J, Sausville EA: Issues and progress with protein kinase inhibitors for 11. cancer treatment. Nat Rev Drug Discov 2003;2:296–313.

DiGiovanna MP, Stern DF, Edgerton SM, Whalen SG, Moore D, Thor AD: 12. Relationship of epidermal growth factor receptor expression to ErbB-2 signaling activity and prognosis in breast cancer patients. J Clin Oncol 2005;23:1152–1160.

Piccart-Gebhart M, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, 13. Smith I, et al.: Herceptin Adjuvant (HERA) Trial Study Team. N Engl J Med 2005;353:1659.

Druker B, Tamura S, Buchdunger E, Ohno S, Segal G, Fanning S, 14. et al.: Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2:561–566.

Druker B, Talpaz M, Resta D, Peng B, Buchdunger E, Ford J, 15. et al.: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031–1037.

Barker AJ, Gibson KH, Grundy W, Godfrey AA, Barlow JJ, Healy MP, 16. et al.: Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg Med Chem Lett 2001;11:1911–1914.




Karaman M, Herrgard S, Treiber D, Gallant P, Atteridge C, Campbell B, 17. et al.: A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008;26:127–132.

Druker BJ: STI571 (Gleevec) as a paradigm for cancer therapy. 18. Trends Mol Med 2002;8(Suppl):S14–S18.

Sawyer T: Novel oncogenic protein kinase inhibitors for cancer therapy. 19. Curr Med Chem Anti-Cancer Agents 2004;4:449–455.

ter Haar WP, Walters S, Pazhanisamy P, Taslimi AC, Pierce GW, Bemis W, 20. et al.: Kinase chemogenomics: targeting the human kinome for target valida-tion and drug discovery. Mini Rev Med Chem 2004;4:235–253.

Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach DR, Miller WT, 21. et al.: Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res 2002;62:4236–4243.

Corbin A, La Rosee P, Stoffregen E, Druker B, Deininger M: Several Bcr-Abl 22. kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib. Blood 2003;10:4611–4614.

Gorre M, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao P, 23. et al.: Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876–880.

Zhu X, Kim JL, Newcomb JR, Rose PE, Stover DR, 24. et al.: Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors. Structure 1999;7:651–661.

Walker E, Pacold M, Perisic O, Stephens L, Hawkins P, Wymann M, 25. et al.: Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 2000;6:909–919.

Tong L, Pav S, White D, Rogers S, Crane K, Cywin C, 26. et al.: A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. Nat Struct Biol 1997;4:311–316.

Fitzgerald C, Patel S, Becker J, Cameron P, Zaller D, Pikounis V, 27. et al.: Structural basis for p38alpha MAP kinase quinazolinone and pyridol-pyrimi-dine inhibitor specificity. Nat Struct Biol 2003;10:764–769.

Shen G: Selective protein kinase C inhibitors and their applications. 28. Curr Drug Targets Cardiovasc Haematol Disord 2003;3:301–307.

Kim C, Xuong NH, Taylor SS: Crystal structure of a complex between the cat-29. alytic and regulatory (RIalpha) subunits of PKA. Science 2005;307:690–696.

Karin M, Yamamoto Y, Wang Q: The IKK NF-kappa B system: a treasure trove 30. for drug development. Nat Rev Drug Discov 2004;3:17–26.

May M, Ghosh S: I31. kB kinases: kinsmen with different crafts. Science 1999;284:271.

Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in 32. chronic inflammatory diseases. N Engl J Med 1997;336:1066–1071.

Haefner B: NF-kappa B: arresting a major culprit in cancer. 33. Drug Discov Today 2002;7:653–663.

Karin M, Lin A: NF-kappaB at the crossroads of life and death. 34. Nat Immunol 2002;3:221–227.

May M, D’Acquisto F, Madge L, Glockner J, Pober J, Ghosh S: Selective inhibi-35. tion of NFkB activation by a peptide that blocks the interaction of NEMO with the IkB kinase complex. Science 2000;289:1550–1554.

Pallàs M, Verdaguer E, Jordà EG, Jiménez A, Canudas AM, Camins A: 36. Flavopiridol: an antitumor drug with potential application in the treatment of neurodegenerative diseases. Med Hypoth 2005;64:120–123.

Knockaert M, Greengard P, Meijer L: Pharmacological inhibitors of cyclin-37. dependent kinases. Trends Pharmacol Sci 2002;23:417–425.

Pavletich NP: Mechanisms of cyclin-dependent kinase regulation: struc-38. tures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J Mol Biol 1999;287:821–828.

Stevenson LM, Deal MS, Hagopian JC, Lew J: Activation mechanism 39. of CDK2: role of cyclin binding versus phosphorylation. Biochemistry 2002;41:8528–8534.

Hirai H, Kawanishi N, Iwasawa Y: Recent advances in the development of 40. selective small molecule inhibitors for cyclin-dependent kinases. Curr Top Med Chem 2005;5:167–179.

Jeffrey P, Tong L, Pavletich N: Structural basis of inhibition of CDK-cyclin 41. complexes by INK4 inhibitors. Genes Dev 2000;14:3115–3125.

Roy KK, Sausville E: Early development of cyclin dependent kinase modula-42. tors. Curr Pharm Des 2001;7:1669–1687.

Parry D, Bates S, Mann DJ, Peters G: Lack of cyclin D-Cdk complexes in 43. Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour sup-pressor gene product. EMBO J 1995;14:503–511.

Hall M, Peters G: Genetic alterations of cyclins, cyclin-dependent kinases, 44. and Cdk inhibitors in human cancer. Adv Cancer Res 1996; 68:67–108.

Zuo L, Weger J, Yang Q, Goldstein AM, Tucker MA, Walker GJ, 45. et al.: Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma. Nat Genet 1996;12:97–99.

Ghosh S, Liu X, Zheng Y, Uckun F: Rational design of potent and selec-46. tive EGFR tyrosine kinase inhibitors as anticancer agents. Curr Cancer Drug Targets 2001;1:129–140.

Wehrman TS, Raab WJ, Casipit CL, Doyonnas R, Pomerantz JH, Blau HM: 47. A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions. Proc Natl Acad Sci U S A 2006;103:19063–19068.

Schlessinger J, Ullrich A: Growth factor signaling by receptor tyrosine kinas-48. es. Neuron 1992;9:383–391.

Zhu H, Iaria J, Orchard S, Walker F, Burgess A: Epidermal growth fac-49. tor receptor: association of extracellular domain negatively regulates intracellular kinase activation in the absence of ligand. Growth Factors 2003;21:15–30.

Frederick L, Wang XY, Eley G, James CD: Diversity and frequency of epider-50. mal growth factor receptor mutations in human glioblastomas. Cancer Res 2000;60;1383–1387.

Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ: Beta-adrenergic recep-51. tor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci U S A 1986;83: 2797–2801.

Strasser RH, Benovic JL, Caron MG, Lefkowitz RJ: Beta-agonist- and pros-52. taglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors. Proc Natl Acad Sci U S A 1986;83;6362–6366.



egLen AnD reisine

Benovic JL, Mayor F Jr, Somers RL, Caron MG, Lefkowitz RJ: Light-dependent 53. phosphorylation of rhodopsin by beta-adrenergic receptor kinase. Nature 1986;321:869–872.

Benovic JL, DeBlasi A, Stone WC, Caron MG, Lefkowitz RJ: Beta-adrenergic 54. receptor kinase: primary structure delinates a multigene family. Science 1989;246:235–240.

Pouyssegur P, Lenormand P: Fidelity and spatio-temporal control in MAP 55. kinase (ERKs) signalling. Eur J Biochem 2003;270:3291–3299.

Perillan P, Chen M, Potts E, Simard J: Transforming growth factor-beta 1 56. regulates Kir2.3 inward rectifier K1 channels via phospholipase C and pro-tein kinase C-delta in reactive astrocytes from adult rat brain. J Biol Chem 2002;277:1974–1980.

Majumder P, Mishra N, Sun X, Bharti A, Kharbanda S, Saxena S, 57. et al.: Targeting of protein kinase C delta to mitochondria in the oxidative stress response. Cell Growth Differ 2001;9:465–470.

O’Flaherty J, Chadwell BA, Kearns MW, Sergeant S, Daniel LW: Protein kinases 58. C translocation responses to low concentrations of arachidonic acid. J Biol Chem 2001;276:24743–24750.

Zaccolo M, Pozzan T: Discrete microdomains with high concentra-59. tion of cAMP in stimulated rat neonatal cardiac myocytes. Science 2002;295:1711–1715.

Wang L, Sunahara R, Krumins A, Perkins G, Crochiere M, Mackey M, 60. et al.: Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein. Proc Natl Acad Sci U S A 2001;98: 3220–3225.

Mochly-Rosen D, Khaner D, Lopez J: Identification of intracellular recep-61. tor proteins for activated protein kinase C. Proc Natl Acad Sci U S A 1991;88:3997–4000.

Carnegie G, Scott J: A-kinase anchoring proteins and neuronal signaling 62. mechanisms. Genes Dev 2003;17:1557–1568.

Osmanagic-Myers S, Wiche G: Plectin-RACK1 (receptor for activated C kinase 63. 1) scaffolding: a novel mechanism to regulate protein kinase C activity. J Biol Chem 2004;279:18701–18710.

McCahill A, Warwicher J, Bolger G, Houslay M, Yarwood S: The RACK1 scaf-64. fold protein: a dynamic cog in cell response mechanisms. Mol Pharmacol 2002;62:1261–1273.

Rotenberg SA, Sun XG: Photoinduced inactivation of protein kinase C by 65. dequalinium identifies the RACK-1-binding domain as a recognition site. J Biol Chem 1998;273:2390–2395.

Schechtman D, Mochly-Rosen D: Adaptor proteins in protein kinase 66. C-mediated signal transduction. Oncogene 2001;20:6339–6347.

Miller L, Lee K, Mochly-Rosen D, Cartwright C: RACK1 regulates Src-mediated 67. Sam68 and p190RhoGAP signaling. Oncogene 2004;23: 5682–5686.

Pass J, Gao J, Jones W, Wead W, Wu X, Zhang J, 68. et al.: Enhanced PKC beta II translocation and PKC beta II-RACK1 interactions in PKC epsilon-induced heart failure: a role for RACK1. Am J Physiol Heart Circ Physiol 2001;281:H2500–H2510.

Pass JM, Zheng YT, Wead WB, Zhang J, Li R, Bolli R, 69. et al.: PKCepsilon activa-tion induces dichotomous cardiac phenotypes and modulates PKCepsilon-RACK interactions and RACK expression. Am J Physiol Heart Circ Physiol 2001;280:H946–H955.

Takeishi Y, Ping P, Bolli R, Kirkpatrick DL, Hoit BD, Walsh R: Transgenic over-70. expression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy. Circ Res 2000;86:1218–1223.

Corsini E, Battaini F, Lucchi L, Marinovich M, Racchi M, Govoni S, 71. et al.: A defective protein kinase C anchoring system underlying age-associated impairment in TNF-alpha production in rat macrophages. J Immunol 1999;163:3468–3473.

Chen L, Marquardt ML, Tester DJ, Sampson KJ, Ackerman MJ, Kass RS: 72. Mutation of an A-kinase-anchoring protein causes long-QT syndrome. Proc Natl Acad Sci U S A 2007;104:20990–20995.

Frank B, Wiestler M, Kropp S, Hemminki K, Spurdle AB, Sutter C, 73. et al.: Association of a common AKAP9 variant with breast cancer risk: a collabora-tive analysis. J Natl Cancer Inst 2008;100:437–442.

Boldt S, Kolch W: Targeting MAPK signalling: Prometheus’ fire or Pandora’s 74. box? Curr Pharm Des 2004;10:1885–1905.

Kumar S, Boehm J, Lee J: p38 MAP kinases: key signalling molecules 75. as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2003;2:717–726.

Sebolt-Leopold J, Herrera R: Targeting the mitogen-activated protein kinase 76. cascade to treat cancer. Nat Rev Cancer 2004;4:937–947.

Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, 77. et al.: Mitogen-activated protein (MAP) kinase pathways: regulation and physi-ological functions. Endocr Rev 2001;22:153–183.

Marshall C: How do small GTPase signal transduction pathways regulate cell 78. cycle entry? Curr Opin Cell Biol 1999;11:732–736.

McMahon M, Woods D: Regulation of the p53 pathway by Ras, the plot 79. thickens. Biochim Biophys Acta 2001;1471:M63–M71.

Gupta S, Barrett T, Whitmarsh AJ, Cavanagh J, Sluss HK, Derijard B, 80. et al.: Selective interaction of JNK protein kinase isoforms with transcription fac-tors. EMBO J 1996;15:2760–2770.

Manning A, Davis R: Targeting JNK for therapeutic benefit: from junk to 81. gold? Nat Drug Discov 2003;2:554–565.

Manning AM, Mercurio F: Transcription inhibitors in inflammation. 82. Expert Opin Investig Drugs 1997;6:555–567.

Bennett BL, Sasaki DT, Murray BW, O’Leary EC, Sakata ST, Xu W, 83. et al.: SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci U S A 2001;98:13681–13686.

Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, 84. et al.: c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 2001;108:73–81.

Xia X, Harding T, Weller M, Bieneman A, Uney J, Schulz J: Gene transfer of the 85. JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson’s disease. Proc Natl Acad Sci U S A 2001;98:10433–10438.

Hirosumi J, Tuncman G, Chang L, Gorgun C, Uysal K, Maeda K, 86. et al.: A central role for JNK in obesity and insulin resistance. Nature 2002;420:333–336.

Adams JL, Badger AM, Kumar S, Lee JC: p38 MAP kinase: molecular target for 87. the inhibition of pro-inflammatory cytokines. Prog Med Chem 2001;38:1–60.

Smolen JS, Steiner G: Therapeutic strategies for rheumatoid arthritis. 88. Nat Rev Drug Discov 2003;2:473–488.




Badger AM, Bradbeer J, Votta B, Lee J, Adams J, Griswold D: 89. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther 1996;279:1453–1461.

Regan J, Breitfelder S, Cirillo P, Gilmore T, Graham AG, Hickey E, 90. et al.: Pyrazole urea-based inhibitors of p38 MAP kinase: from lead compound to clinical candidate. J Med Chem 2002;45:2994–3008.

Badger AM, Griswold DE, Kapadia R, Blake S, Swift BA, Hoffman SJ, 91. et al.: Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis. Arthritis Rheum 2000;43:175–183.

Mclay LM, Halley F, Souness JE, McKenna J, Benning V, Birrell M, 92. et al.: The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy. Bioorg Med Chem 2001;9:537–554.

Ma XL, Kumar S, Gao F, Louden C, Lopez B, Christopher T, 93. et al.: Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation 1999;99:1685–1691.

Gutkind JS: The pathways connecting G protein coupled receptors to the 94. nucleus through divergent MAPK’s. J Biol Chem 1998;273:1839–1842.

Wen D, Nong Y, Morgan JG, Gangurde P, Bielecki A, Dasilva J, 95. et al.: A selective small molecule IkappaB Kinase beta inhibitor blocks nuclear factor kappaB-mediated inflammatory responses in human fibroblast-like synoviocytes, chondrocytes, and mast cells. J Pharmacol Exp Ther 2006;317:989–1001.

Palanki MS, Gayo-Fun L, Shevlin G, Erdman P, Sato M, Goldman M, 96. et al.: Structure-activity relationship studies of ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-4-(trifluoromethyl)pyrimidine-5-carboxylate: an inhibitor of AP-1 and NF-kappaB mediated gene expression. Bioorg Med Chem Lett 2002;12:2573–2577.

Castro AC, Dang L, Soucy F, Grenier L, Mazdiyasni H, Hottelet M, 97. et al.: Novel IKK inhibitors: beta-carbolines. Bioorg Med Chem Lett 2003; 13:2419–2422.

Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW, 98. et al.: BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent tran-scription in mice. J Biol Chem 2003;278:1450–1456.

McIntyre KW, Shuster DJ, Gillooly KM, Dambach DM, Pattoli MA, Lu P, 99. et al.: A highly selective inhibitor of I kappa B kinase, BMS-345541, blocks both joint inflammation and destruction in collagen-induced arthritis in mice. Arthritis Rheum 2003;48:2652–2659.

Bhat R, Budd S, Haeberlein L, Avila J: Glycogen synthase kinase 3: a drug tar-100. get for CNS therapies. J Neurochem 2004;89:1313–1317.

Murphy E: Inhibit GSK-3beta or there’s heartbreak dead ahead. 101. J Clin Invest 2004;113:1526–1528.

Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, 102. et al.: Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 2000;7:793–803.

Mussmann R, Geese M, Harder F, Kegel S, Andag U, Lomow A, 103. et al.: Inhibition of GSK3 promotes replication and survival of pancreatic beta cells. J Biol Chem 2007;282:12030–12037.

Lucas J, Hernandez F, Gomez-Ramos P, Moran M, Hen R, Avila J: Decreased 104. nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J 2001;20:27–39.

Rondinone CM: Diabetes: the latest developments in inhibitors, insulin sen-105. sitisers, new drug targets and novel approaches. October 18–19, 2004, The Hatton, London, UK. Expert Opin Ther Targets 2005;9:415–418.

Leal EC, Santiago AR, Ambrósio AF: Old and new drug targets in diabetic 106. retinopathy: from biochemical changes to inflammation and neurodegenera-tion. Curr Drug Targets CNS Neurol Disord 2005;4:421–434.

Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Creager MA: Inhibition of 107. protein kinase Cbeta prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circ Res 2002;90:107–111.

Meier M, King GL: Protein kinase C activation and its pharmacological inhibi-108. tion in vascular disease. Vasc Med 2000;5:173–185.

Frank RN: Potential new medical therapies for diabetic retinopathy: protein 109. kinase C inhibitors. Am J Ophthalmol 2002;133:693–698.

Whitney JA: Reference systems for kinase drug discovery; chemi-110. cal genetic approaches to cell-based assays. Assay Drug Dev Technol 2004:2:417–430.

Klumpp M, Boettcher A, Becker D, Meder G, Blank J, Leder L, 111. et al.: Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format. J Biomol Screen 2006;11:617–633.

Jia Y, Quinn CM, Kwak S, Talanian RV: Current in vitro kinase assays; the 112. quest for a universal format. Curr Drug Discov Technol 2008;5:59–69.

Ishida A, Kameshita I, Sueyoshi N, Takanobu T, Taniguchi T, Shigeri Y: Recent 113. advances in technologies for analyzing protein kinases. J Pharmacol Sci 2007;103:5–11.

Hardie DG: 114. Protein Phosphorylation: A Practical Approach. Oxford University Press, New York, 1999.

Bischoff KM, Shi L, Kennelly PJ: The detection of enzyme activity following 115. sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem 1998;260:1–17.

Beveridge M, Park YW, Hermes J, Marenghi A, Brophy G, Santos A: Detection 116. of p56lck kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format. J Biomol Screen 2000;5:205–212.

Cook N, Harris A, Hopkins A, Hughes K: Scintillation proximity assay (SPA) 117. technology to study biomolecular interactions. Curr Protoc Protein Sci 2002(May);Chapter 19:Unit 19.8.

Diks SH, Kok K, O’Toole T, Hommes DW, van Dijken P, Joore J, 118. et al.: Kinome profiling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells. J Biol Chem 2004;279:49206–49213.

Sachsenmaier C, Schachtele C: Integrated technology platform protein kinases 119. for drug development in oncology. Biotechniques 2002 Oct;Suppl:101–106.

Wesche H, Xiao SH, Young SW: High throughput screening for protein kinase 120. inhibitors. Comb Chem High Throughput Screen 2005;8:181–195.

Olive DM: Quantitative methods for the analysis of protein phosphorylation 121. in drug development. Expert Rev Proteomics 2004;1:327–341.

Han S, Zhou V, Pan S, Liu Y, Hornsby M, McMullan D, 122. et al.: Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors. Bioorg Med Chem Lett 2005;15:5467–5473.



egLen AnD reisine

Schröter T, Minond D, Weiser A, Dao C, Habel J, Spicer T, 123. et al.: Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). J Biomol Screen 2008;13:17–28.

Braunwalder AF, Yarwood DR, Sills MA, Lipson KE: Measurement of the 124. protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates. Anal Biochem 1996;238:159–164.

Morgan AG, McCauley TJ, Stanaitis ML, Mathrubutham M, Millis SZ: 125. Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectiv-ity and site-specific inhibitor determination. Assay Drug Dev Technol 2004;2:171–181.

Zhang WG, Shor B, Yu K: Identification and characterization of a constitu-126. tively T-loop phosphorylated and active recombinant S6K1: expression, puri-fication, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay. Protein Expr Purif 2006;46:414–420.

Rodems SM, Hamman BD, Lin C, Zhao J, Shah S, Heidary D, 127. et al.: A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev Technol 2002;1:9–19.

Mathis G: HTRF® technology. 128. J Biomol Screen 1999;4:309–314.

Warner G, Illy C, Pedro L, Roby P, Bosse R: AlphaScreen kinase HTS platforms. 129. Curr Med Chem 2004;11:721–730.

Von Leoprechting A, Kumpf R, Menzel S, Reulle D, Griebel R, Valler MJ, 130. et al.: Miniaturization and validation of a high-throughput serine kinase assay using the AlphaScreen platform. J Biomol Screen 2004;9:719–725.

Guenat S, Rouleau N, Bielmann C, Bedard J, Maurer F, Allaman-Pillet N, 131. et al.: Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates. J Biomol Screen 2006;11:1015–1026.

Corthals GL, Aebersold R, Goodlett DR: Identification of phosphorylation sites 132. using microimmobilized metal affinity chromatography. Methods Enzymol 2005;405:66–81.

Moser K, White FM: Phosphoproteomic analysis of rat liver by high capacity 133. IMAC and LC-MS/MS. J Proteome Res 2006;5:98–104.

Seethala R, Menzel R: A fluorescence polarization competition immunoassay 134. for tyrosine kinases. Anal Biochem 1998;255:257–262.

Sportsman JR, Gaudet EA, Boge A: Immobilized metal ion affinity-based fluo-135. rescence polarization (IMAP): advances in kinase screening. Assay Drug Dev Technol 2004;2:205–214.

Eglen RM: Enzyme fragment complementation: a flexible high throughput 136. screening assay technology. Assay Drug Dev Technol 2002;1:97–108.

Eglen R, Singh R: 137. b Galactosidase enzyme fragment complementation (EFC) as a novel technology for high throughput screening. Comb ChemHigh Throughput Screen 2003;6:381–387.

Charter NW, Kauffman L, Singh R, Eglen RM: A generic, homogenous method 138. for measuring kinase and inhibitor activity via adenosine 5’-diphosphate accumulation. J Biomol Screen 2006;11:390–399.

Singh P, Harden BJ, Lillywhite BJ, Broad PM: Identification of kinase inhibi-139. tors by an ATP depletion method. Assay Drug Dev Technol 2004; 2:161–169.

Koresawa M, Okabe T: High-throughput screening with quantitation of ATP 140. consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev Technol 2004;2:153–160.

Vainshtein I, Silveria S, Kaul P, Rouhani R, Eglen RM, Wang J: A high-141. throughput, nonisotopic, competitive binding assay for kinases using nonse-lective inhibitor probes (ED-NSIP™). J Biomol Screen 2002;7:497–504.

Violin JD, Zhang J, Tsien RY, Newton AC: A genetically encoded fluorescent 142. reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 2003;161:899–909.

Gallegos L, Kunkel M, Newton A: Targeting protein kinase C activity 143. reporter to discrete intracellular regions reveals spatiotemporal differ-ences in agonist-dependent signaling. J Biol Chem 2006;281:30947– 30956.

Prinz A, Diskar M, Herberg FW: Application of bioluminescence resonance 144. energy transfer (BRET) for biomolecular interaction studies. Chembiochem 2006;7:1007–1012.

Moll D, Prinz A, Gesellchen F, Drewianka S, Zimmermann B, Herberg FW: 145. Biomolecular interaction analysis in functional proteomics. J Neural Transm 2006;113:1015–1032.

Stefan E, Aquin S, Berger N, Landry C, Nyfeler B, Bouvier M, 146. et al.: Quantification of dynamic protein complexes using Renilla luciferase frag-ment complementation applied to protein kinase A activities in vivo. Proc Natl Acad Sci U S A 2007;104:16916–16921.

Hundsrucker C, Krause G, Beyermann M, Prinz A, Zimmermann B, 147. Diekmann O, et al.: High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides. Biochem J 2006;396:297–306.

Fujioka A, Terai K, Itoh R, Aoki K, Nakamura T, Kuroda S, 148. et al.: Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes. J Biol Chem 2006;281:8917–8926.

Remy I, Michnick S: Clonal selection and in vivo quantitation of protein 149. interactions with protein-fragment complementation assays. Proc Natl Acad Sci U S A 1999;96:5394–5399.

Boute N, Pernet K, Issad T: Monitoring the activation state of the insulin 150. receptor using bioluminescence resonance energy transfer. Mol Pharmacol 2001;60:640–645.

Couturier C, Jockers R: Activation of the leptin receptor by a ligand-151. induced conformational change of constitutive receptor dimers. J Biol Chem 2003;278:26604–26611.

Brown RJ, Adams JJ, Pelekanos RA, Wan Y, McKinstry WJ, Palethorpe K, 152. et al.: Model for growth hormone receptor activation based on subunit rotation within a receptor dimer. Nat Struct Mol Biol 2005;12:814–821.

Damjanovich S, Bene L, Matko J, Alileche A, Goldman C, Sharrow S, 153. et al.: Preassembly of interleukin 2 (IL-2) receptor subunits on rest-ing Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: a fluorescence resonance energy transfer study. Proc Natl Acad Sci U S A 1997;94:13134–13139.

Wehrman T, He X, Raab W, Dukipatti A, Blau H, Garcia K: Structural and 154. mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 2007;53:25–38.

Lang P, Yeow K, Nichols A, Scheer A: Cellular imaging in drug discovery. 155. Nat Rev Drug Discov 2006;5:343–356.




Barak L, Ferguson S, Zhang J, Caron M: A beta-arrestin/GFP biosensor for 156. detecting GPCR activation. J Biol Chem 1997;272:27497–27500.

Palo K, Brand L, Eggeling C, Jager S, Kask P, Gall K: Fluorescence intensity and 157. lifetime distribution analysis: toward higher accuracy in fluorescence fluc-tuation spectroscopy. Biophys J 2002;83:605–618.

Fung P, Peng K, Kobel P, Dotimas H, Kauffman L, Olson K, 158. et al.: A homo-geneous cell-based assay to measure nuclear translocation using beta-galactosidase enzyme fragment complementation. Assay Drug Dev Technol 2006:4:263–272.

Address reprint requests to:Richard M. Eglen, Ph.D.

Bio-discoveryPerkinElmer Life and Analytical Sciences

940 Winter StreetWaltham, MA 02451

E-mail: [email protected]


the current status of drug discovery against the human kinome

Documents