A fluorescence lifetime based binding assay to characterize kinase inhibitors

The authors present a fluorescence lifetime-based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)-binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.     

Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002

Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.     

The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides

All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unknown. Crystallographic data from our laboratory revealed that the N-terminal kinase domain of p90 ribosomal S6 kinase, isoform 2, binds three different flavonol rhamnosides in a highly unusual manner, distinct from other kinase inhibitor interactions. The kinase domain undergoes a reorganization of several structural elements in response to the binding of the inhibitors. Specifically, the main β-sheet of the N-lobe undergoes a twisting rotation by ~ 56° around an axis passing through the N- and C-lobes, leading to the restructuring of the canonical ATP-binding pocket into pockets sterically adapted to the inhibitor shape. The flavonol rhamnosides appear to adopt compact, but strained conformations with the rhamnose moiety swept under the B-ring of flavonol, unlike the structure of the free counterparts in solution. These data suggest that the flavonol glycoside scaffold could be used as a template for new inhibitors selective for the RSK family. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Real-time fluorogenic kinase assay using protein as substrate

A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C βII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K_m^ATP), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.     

Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase

Pim-1 kinase is a member of a distinct class of serine/threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a non-conserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate) complex to 2.1-angstroms resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

A fluorescence lifetime-based assay for abelson kinase

We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.     

Ligand-based and e-pharmacophore modeling, 3D-QSAR and hierarchical virtual screening to identify dual inhibitors of spleen tyrosine kinase (Syk) and janus kinase 3 (JAK3)

The clinical efficacy of multiple kinase inhibitors has caught the interest of Pharmaceutical and Biotech researchers to develop potential drugs with multi-kinase inhibitory activity for complex diseases. In the present work, we attempted to identify dual inhibitors of spleen tyrosine kinase (Syk) and janus kinase 3 (JAK3), keys players in immune signaling, by developing ideal pharmacophores integrating Ligand-based pharmacophore models (LBPMs) and Structure-based pharmacophore models (SBPMs), thereby projecting the optimum pharmacophoric required for inhibition of both the kinases. The four point LBPM; ADPR.14 suggested the presence of one hydrogen bond acceptor, one hydrogen bond donor, one positive ionizable, and one ring aromatic feature for Syk inhibitory activity and AADH.54 proposed the necessity of two hydrogen bond acceptor, one hydrogen bond donor, and one hydrophobic feature for JAK3 inhibitory activity. To our interest, SBPMs identified additional ring aromatic features required for inhibition of both the kinases. For Syk inhibitory activity, the hydrogen bond acceptor feature indicated by LBPM was devoid of forming hydrogen bonding interaction with the hinge region amino acid residue (Ala451). Thus merging the information revealed by both LBPMs and SBPMs, ideal pharmacophore models i.e. ADPRR.14 (Syk) and AADHR.54 (JAK3) were generated. These models after rigorous statistical validation were used for screening of Asinex database. The systematic virtual screening protocol, including pharmacophore and docking-based screening, ADME property, and MM-GBSA energy calculations, retrieved final 10 hits as dual inhibitors of Syk and JAK3. Final 10 hits thus obtained can aid in the development of potential therapeutic agents for autoimmune disorders. Also the top two hits were evaluated against both the enzymes.     

Modulation of the Na–;H antiport by insulin: Interplay between protein kinase C,tyrosine kinase,and protein phosphatases

The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2′,7′ bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insulin stimulates the Na? H antiport. The dose-response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26± 0.05 and 0.18 ± 0.022 pH units for 1 nM and 1 μM insulin respectively). The use of phorbol, 12-myristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na? H antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 μM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 μM. Okadaic acid 1 μM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time-course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), confirming the specificity of these effects. © 1994 wiley-Liss, Inc.     

Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.     

Strong and weak hydrogen bonds in protein-ligand complexes of kinases: a comparative study

Strong and weak hydrogen bonds between protein and ligand are analyzed in a group of 233 X-ray crystal structures of the kinase family. These kinases are from both eukaryotic and prokaryotic organisms. The dataset comprises of 44 sub-families, out of which 35 are of human origin and the rest belong to other organisms. Interaction analysis was carried out in the active sites, defined here as a sphere of 10 A radius around the ligand. A majority of the interactions are observed between the main chain of the protein and the ligand atoms. As a donor, the ligand frequently interacts with amino acid residues like Leu, Glu and His. As an acceptor, the ligand interacts often with Gly, and Leu. Strong hydrogen bonds N-H...O, O-H...O, N-H...N and weak bonds C-H...O, C-H...N are common between the protein and ligand. The hydrogen bond donor capacity of Gly in N-H...O and C-H...O interactions is noteworthy. Similarly, the acceptor capacity of main chain Glu is ubiquitous in several kinase sub-families. Hydrogen bonds between protein and ligand form characteristic hydrogen bond patterns (supramolecular synthons). These synthon patterns are unique to each sub-family. The synthon locations are conserved across sub-families due to a higher percentage of conserved sequences in the active sites. The nature of active site water molecules was studied through a novel classification scheme, based on the extent of exposure of water molecules. Water which is least exposed usually participates in hydrogen bond formation with the ligand. These findings will help structural biologists, crystallographers and medicinal chemists to design better kinase inhibitors.     

Staurosporine scaffold–based rational discovery of the wild‐type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog‐sensitive kinase technology

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first‐line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small‐molecule kinase inhibitors in EGFR‐targeted chemotherapy. Previously, the reversible pan‐kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR^T790M) over wild‐type kinase (EGFR^WT), suggesting that the staurosporine scaffold is potentially to develop the wild‐type sparing reversible inhibitors of EGFR^T790M. Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold–based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico–in vitro analog‐sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild‐type sparing inhibitors UCN‐01, UCN‐02, AFN941, and SB‐218078 with high or moderate selectivity of 30‐, 45‐, 5‐, and 8‐fold for EGFR^T790M over EGFR^WT, respectively, which are comparable with or even better than that of the parent compound staurosporine (24‐fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN‐02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase‐inhibitor binding. A hydroxyl group of UCN‐02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN‐02 than staurosporine for mutant over wild‐type kinase.     

Characterization of kinase inhibitors using different phosphorylation states of colony stimulating factor-1 receptor tyrosine kinase

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.     

Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein.

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.     

A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase

Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR–FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.     

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.     

Structure-based and shape-complemented pharmacophore modeling for the discovery of novel checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a member of the serine/threonine kinase family, is an attractive therapeutic target for anticancer combination therapy. A structure-based modeling approach complemented with shape components was pursued to develop a reliable pharmacophore model for ATP-competitive Chk1 inhibitors. Common chemical features of the pharmacophore model were derived by clustering multiple structure-based pharmacophore features from different Chk1-ligand complexes in comparable binding modes. The final model consisted of one hydrogen bond acceptor (HBA), one hydrogen bond donor (HBD), two hydrophobic (HY) features, several excluded volumes and shape constraints. In the validation study, this feature-shape query yielded an enrichment factor of 9.196 and performed fairly well at distinguishing active from inactive compounds, suggesting that the pharmacophore model can serve as a reliable tool for virtual screening to facilitate the discovery of novel Chk1 inhibitors. Besides, these pharmacophore features were assumed to be essential for Chk1 inhibitors, which might be useful for the identification of potential Chk1 inhibitors.     

Synthesis and SAR of inhibitors of protein kinase CK2: novel tricyclic quinoline analogs

Protein kinase CK2 is a potential drug target for many diseases including cancer and inflammation disorders. The crystal structure of clinical candidate CX-4945 1 with CK2 revealed an indirect interaction with the protein through hydrogen bonding between the NH of the 3-chlorophenyl amine and a water molecule. Herein, we investigate the relevance of this hydrogen bond by preparing several novel tricyclic derivatives lacking a NH moiety at the same position. This SAR study allowed the discovery of highly potent CK2 inhibitors.