High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

In situ kinase profiling reveals functionally relevant properties of native kinases

Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in?vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.     

MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule-protein interactions in mammalian cells

Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.     

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.     

Recent Advances in Pain Management: Relevant Protein Kinases and Their Inhibitors

The purpose of this review is to underline the protein kinases that have been established, either in fundamental approach or clinical trials, as potential biological targets in pain management. Protein kinases are presented according to their group in the human kinome: TK (Trk, RET, EGFR, JAK, VEGFR, SFK, BCR–Abl), CMGC (p38 MAPK, MEK, ERK, JNK, ASK1, CDK, CLK2, DYRK1A, GSK3, CK2), AGC (PKA, PKB, PKC, PKMζ, PKG, ROCK), CAMK, CK1 and atypical/other protein kinases (IKK, mTOR). Examples of small molecule inhibitors of these biological targets, demonstrating an analgesic effect, are described. Altogether, this review demonstrates the fundamental role that protein kinase inhibitors could play in the development of new pain treatments.     

Multiplexed tyrosine kinase activity detection in cancer cells using a hydrogel immobilized substrate

Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752.

Current chemical biology tools for studying protein phosphorylation and dephosphorylation

Amongst different posttranslational events involved in cellular-signaling pathways, phosphorylation and dephosphorylation of proteins are the most prevalent. Aberrant regulations in the cellular phosphoproteome network are implicated in most major human diseases. Consequently, kinases and phosphatases are two of the most important groups of drug targets in medicinal research today. A major challenge in the understanding of protein phosphorylation and dephosphorylation is the sheer complexity of the phosphoproteome network and the lack of tools capable of studying protein phosphorylation and dephosphorylation as they occur in cells. We highlight herein various chemical biology tools that have emerged in the last decade for such studies. First, we discuss the use of small-molecule mimics of phosphoamino acids and their use in elucidating the function of protein phosphorylation and dephosphorylation. We also introduce recent advances in the field of activity-based protein profiling (ABPP) for proteome-wide detection of protein phosphorylation and dephosphorylation. We next discuss the key concepts in the design of peptide- and protein-based biosensors capable of real-time reporting of phosphorylation/dephosphorylation events. Finally, we highlight the application of peptide and small-molecule microarrays (SMMs), and their applications in high-throughput screening and discovery of new compounds related to phosphorylation/dephosphorylation.     

Substrate Activity Screening with Kinases: Discovery of Small‐Molecule Substrate‐Competitive c‐Src Inhibitors

Substrate‐competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Substrate activity screening was applied to tyrosine kinases. By using this methodology, the first small‐molecule substrates for any protein kinase were discovered, as well as the first substrate‐competitive inhibitors of c‐Src with activity in both biochemical and cellular assays. Characterization of the lead inhibitor demonstrates that substrate‐competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP‐competitive inhibitors.     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Encodable activators of SRC family kinases

There is considerable current interest in the design of encodable molecules that regulate intracellular protein circuitry and/or activity, ideally with a high level of specificity. Src homology 3 (SH3) domains are ubiquitous components of multidomain signaling proteins, including many kinases, and are attractive drug targets because of the important role their interactions play in diseases as diverse as cancer, osteoporosis, and inflammation. Here we describe a set of miniature proteins that recognize distinct SH3 domains from Src family kinases with high affinity. Three of these molecules discriminate effectively between the SH3 domains of Src and Fyn, which are expressed ubiquitously, and two of these three activate Hck kinase with potencies that rival HIV Nef, one of the most potent kinase activators known. These results suggest that miniature proteins represent a viable, encodable strategy for selective activation of Src family kinases in a variety of cell types.     

Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors

Some protein kinases are known to acquire resistance to selective small molecule inhibitors upon mutation of a conserved threonine at the ATP binding site to a larger residue. Here, we performed a comprehensive mutational analysis of this structural element and determined the cellular sensitivities of several disease-relevant tyrosine kinases against various inhibitors. Mutant kinases possessing a larger side chain at the critical site showed resistance to most compounds tested, such as ZD1839, PP1, AG1296, STI571, and a pyrido[2,3-d]pyrimidine inhibitor. In contrast, indolinones affected both wild-type and mutant kinases with similar potencies. Resistant mutants were established for pharmacological analysis of betaPDGF receptor-mediated signaling and allowed the generation of a drug-inducible system of cellular Src kinase activity. Our data establish a conserved structural determinant of protein kinase sensitivity relevant for both signal transduction research and drug development.     

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.     

Creating chemical diversity to target protein kinases

Protein kinases play crucial roles in regulating virtually every cellular process and are currently attracting tremendous interest as drug targets from the pharmaceutical industry. The major challenges facing the development of the potential kinase inhibitor drugs are: selectivity, physical properties (solubility, molecular weight), and pharmacological properties (bioavailability, half life, toxicity, etc.) This review focuses on how selective protein kinase inhibitors that target the ATP and allosteric binding sites are currently being identified and optimized.     

MALDI‐TOF Mass‐Spectrometry‐Based Versatile Method for the Characterization of Protein Kinases

We describe a MALDI‐TOF mass‐spectrometry‐based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALS G), CaMKII substrate (KRQQS FDLF), erktide (ATGPLS PGPFGRR), abltide (EAIY AAPFAKKK), srctide (AEEEIY GEFEAKKKK), neurogranin (AAAKIQAS FRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMS ITA). There are two fundamental points on which the proposed method is based to improve the mass‐spectrometric response: 1) mass tag technology by N‐derivatization through stable isotope labeling and 2) C‐terminal conjugation with tryptophanylarginine (WR). It was suggested that C‐terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5–13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac‐EAIY AAPFAKKKWR‐NH₂), that WR conjugation at the C‐terminus in combination with stable‐isotope labeling at the N‐terminus allowed the quantitative assay of recombinant c‐Abl kinase in the presence of adenosine 5′‐triphosphate (ATP; K_M,ATP=18.6 μM and V_max=642 pmol min^?1 μg^?1). The present protocol made a simple and reliable inhibition assay of recombinant c‐Abl kinase by imatinib possible (IC_{50(recombinant)}=291 nM ; STI571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c‐Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC_{50(active form)}=1049 nM and IC_{50(inactive form)}=54 nM ). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer‐cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple‐kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high‐throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope‐free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.     

Small molecule recognition of c-Src via the Imatinib-binding conformation

The cancer drug, Imatinib, is a selective Abl kinase inhibitor that does not inhibit the closely related kinase c-Src. This one drug and its ability to selectively inhibit Abl over c-Src has been a guiding principle in virtually all kinase drug discovery efforts in the last 15 years. A prominent hypothesis explaining the selectivity of Imatinib is that Abl has an intrinsic ability to adopt an inactive conformation (termed DFG-out), whereas c-Src appears to pay a high intrinsic energetic penalty for adopting this conformation, effectively excluding Imatinib from its ATP pocket. This explanation of the difference in binding affinity of Imatinib for Abl versus c-Src makes the striking prediction that it would not be possible to design an inhibitor that binds to the DFG-out conformation of c-Src with high affinity. We report the discovery of a series of such inhibitors. We use structure-activity relationships and X-ray crystallography to confirm our findings. These studies suggest that small molecules are capable of inducing the generally unfavorable DFG-out conformation in c-Src. Structural comparison between c-Src in complex with these inhibitors allows us to speculate on the differential selectivity of Imatinib for c-Src and Abl.     

Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.     

Direct binding assay for the detection of type IV allosteric inhibitors of Abl

Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site.     

Acquisition of a "Group A"-selective Src kinase inhibitor via a global targeting strategy

A "global" strategy for the acquisition of selective high affinity inhibitors for the Src kinase subfamily of tyrosine kinases is described. Members of the Src family exhibit a strong amino acid sequence homology. However, recent studies have revealed differences in the relative spatial relationships of the three distinct protein-binding domains present in these enzymes. We have constructed an inhibitor, using an amalgamation of combinatorial methods and directed design, which simultaneously associates with the active site and an ancillary protein-binding region (SH2 domain). The inhibitor exhibits high inhibitory potency and selectivity for the Group A versus Group B subset of Src kinases.