The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase

Background: The p38 mitogen-activated protein (MAP) kinase regulates signal transduction in response to environmental stress. Pyridinylimidazole compounds are specific inhibitors of p38 MAP kinase that block the production of the cytokines interleukin-1 β and tumor necrosis factor α, and they are effective in animal models of arthritis, bone resorption and endotoxin shock. These compounds have been useful probes for studying the physiological functions of the p38-mediated MAP kinase pathway.Results: We report the crystal structure of a novel pyridinylimidazole compound complexed with p38 MAP kinase, and we demonstrate that this compound binds to the same site on the kinase as does ATP. Mutagenesis showed that a single residue difference between p38 MAP kinase and other MAP kinases is sufficient to confer selectivity among pyridinylimidazole compounds.Conclusions: Our results reveal how pyridinylimidazole compounds are potent and selective inhibitors of p38 MAP kinase but not other MAP kinases. It should now be possible to design other specific inhibitors of activated p38 MAP kinase using the structure of the nonphosphorylated enzyme.     

Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors

The generation of synthetic compounds with exclusive target specificity is an extraordinary challenge of molecular recognition and demands novel design strategies, in particular for large and homologous protein families such as protein kinases with more than 500 members. Simple organic molecules often do not reach the necessary sophistication to fulfill this task. Here, we present six carefully tailored, stable metal-containing compounds in which unique and defined molecular geometries with natural-product-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six reported metal compounds displays high selectivity for an individual protein kinase, namely GSK3α, PAK1, PIM1, DAPK1, MLCK, and FLT4. Although being conventional ATP-competitive inhibitors, the combination of the unusual globular shape and rigidity characteristics, of these compounds facilitates the design of highly selective protein kinase inhibitors. Unique structural features of the octahedral coordination geometry allow novel interactions with the glycine-rich loop, which contribute significantly to binding potencies and selectivities. The sensitive correlation between metal coordination sphere and inhibition properties suggests that in this design, the metal is located at a "hot spot" within the ATP binding pocket, not too close to the hinge region where globular space is unavailable, and at the same time not too far out toward the solvent where the octahedral coordination sphere would not have a significant impact on potency and selectivity. This study thus demonstrates that inert (stable) octahedral metal complexes are sophisticated structural scaffolds for the design of highly selective chemical probes.     

Features of selective kinase inhibitors

Small-molecule inhibitors of protein and lipid kinases have emerged as indispensable tools for studying signal transduction. Despite the widespread use of these reagents, there is little consensus about the biochemical criteria that define their potency and selectivity in cells. We discuss some of the features that determine the cellular activity of kinase inhibitors and propose a framework for interpreting inhibitor selectivity.     

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.     

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.     

Extraction and validation of substructure profiles for enriching compound libraries

Compounds known to be potent against a specific protein target may potentially contain a signature profile of common substructures that is highly correlated to their potency. These substructure profiles may be useful in enriching compound libraries or for prioritizing compounds against a specific protein target. With this objective in mind, a set of compounds with known potency against six selected kinases (2 each from 3 kinase families) was used to generate binary molecular fingerprints. Each fingerprint key represents a substructure that is found within a compound and the frequency with which the fingerprint occurs was then tabulated. Thereafter, a frequent pattern mining technique was applied with the aim of uncovering substructures that are not only well represented among known potent inhibitors but are also unrepresented among known inactive compounds and vice versa. Substructure profiles that are representative of potent inhibitors against each of the 3 kinase families were thus extracted. Based on our validation results, these substructure profiles demonstrated significant enrichment for highly potent compounds against their respective kinase targets. The advantages of using our approach over conventional methods in analyzing such datasets and its application in the mining of substructures for enriching compound libraries are presented.     

Understanding and modulating cyclin-dependent kinase inhibitor specificity: molecular modeling and biochemical evaluation of pyrazolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors

Summary Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.     

Covalent‐Allosteric Kinase Inhibitors

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation‐dependent signaling of protein kinases. Herein the structure‐based design, synthesis, and evaluation of pleckstrin homology (PH) domain‐dependent covalent‐allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.     

Cytoprotective Role of Mitogen-activated Protein Kinase Phosphatase-1 in Light-damaged Human Retinal Pigment Epithelial Cells

The role of the mitogen-activated protein (MAP) kinase phosphatases (MKPs) in light-damaged cells is unclear. Therefore we investigated the involvement of MKP-1 in the regulation of apoptosis and cell survival mediated by MAP kinase pathways in light-damaged human retinal pigment epithelial cells (ARPE-19). Light dose-dependent changes in the expression of MKP-1 and in the phosphorylation status of the MAP kinases, c-Jun-N-terminal kinase (JNK) and p38 were demonstrated. Low light doses up to 2 J cm⁻² led to an upregulation of MKP-1 which resulted in the prevention of cell death by inactivating JNK kinase. However, higher light doses (≥3 J cm⁻²) significantly reduced MKP-1 protein expression and subsequently led to an increased JNK kinase activity followed by a significant increase in cell death. JNK kinase inactivation by the JNK inhibitor SP600125 significantly reduced light-induced cell death, suggesting that the cytoprotective properties of MKP-1 are mediated mainly by the JNK MAP kinase pathway. Physiological concentrations of ascorbic acid or taurine were seen to prevent apoptosis and cell death in light-damaged ARPE-19 cells by reducing oxidative stress within cells, thus maintaining MKP-1 at high levels, leading to an inactivation of the JNK kinase pathway which resulted in an increased cell viability.     

Optimized Target Residence Time: Type I Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R‐Spine

Skepinone‐L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP‐competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I binders for p38α MAP kinase. Type I inhibitors interfere with the R‐spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X‐ray crystallography.     

Identification of polyoxometalates as nanomolar noncompetitive inhibitors of protein kinase CK2

Protein kinase CK2 is a multifunctional kinase of medical importance that is dysregulated in many cancers. In this study, polyoxometalates were identified as original CK2 inhibitors. [P2Mo18O62](6-) has the most potent activity. It inhibits the kinase in the nanomolar range by targeting key structural elements located outside the ATP- and peptide substrate-binding sites. Several polyoxometalate derivatives exhibit strong inhibitory efficiency, with IC50 values < or = 10 nM. Furthermore, these inorganic compounds show a striking specificity for CK2 when tested in a panel of 29 kinases. Therefore, polyoxometalates are effective CK2 inhibitors in terms of both efficiency and selectivity and represent nonclassical kinase inhibitors that interact with CK2 in a unique way. This binding mode may provide an exploitable mechanism for developing potent drugs with desirable properties, such as enhanced selectivity relative to ATP-mimetic inhibitors.     

Systematic identification of the druggable interactions between human protein kinases and naturally occurring compounds in endometriosis

Diverse kinase signaling pathways have been involved in the pathogenesis of endometriosis (EM), which can be modulated either by directly targeting the hub kinases or by indirectly regulating marginal members in the pathways. Here, a systematic kinase–inhibitor interaction profile was created for 8 naturally occurring compounds against 20 human protein kinases. The compounds are all non-sterid that have been reported as pharmacologically active molecular entities potential for EM therapeutics, while the kinases were curated via gene ontology terms enriched from the gene co-citation network with EM. The resulting profile was analyzed at structural, energetic and dynamic levels to identify druggable kinase–compound interactions. The compounds Gossypol, Curcumin and EGCG showed a similar interaction profile across these kinases; they can bind tightly to the top-listed kinases in gene ontology, while the compounds Marrubiin, Apigenin and DIM were predicted to exhibit generally weak affinity for the 20 curated kinases. The JNK kinase, a MAPK family member, was identified as a putative candidate of druggable target for EM therapeutics; the inhibitory activity of eight naturally occurring compounds as well as a sophisticated kinase inhibitor SP600125 against the JNK was tested using enzymatic activity analysis. As might be expected, the Gossypol and EGCG were determined to have high inhibitory activity at namomolar level (IC₅₀ = 55 and 94 nM, respectively), which are comparable with or better than the positive control SP600125 (IC₅₀ = 76 nM), while other tested compounds exhibited weak inhibition (IC₅₀ > 100 nM) or bad potency (IC₅₀ = n.d.) against the kinase.     

Systematic Profiling and Evaluation of Structure-based Kinase–Inhibitor Interactome in Cervical Cancer by Integrating In Silico Analyses and In Vitro Assays at Molecular and Cellular Levels

Various protein kinases are implicated in the pathogenesis of human cervical cancer and many kinase inhibitors have been used to regulate the activity of protein kinases involved in the disease signaling networks. In the present study, a systematic kinase–inhibitor interactome is created for various small-molecule inhibitors across diverse cervical cancer-related kinases by using ontology enrichment, molecular docking, dynamics simulation and energetics analysis. The interactome profile is examined in detail with heatmap analysis and heuristic clustering to derive promising inhibitors that are highly potential to target the kinome of human cervical cancer in a multi-target manner. A number of hit and unhit inhibitors are selected and their cell-suppressing effects are tested against human cervical carcinoma HeLa, from which several inhibitor compounds with high cytotoxicity are successfully identified. A further kinase assay confirms that these inhibitors can generally target their noncognate kinases HER3 and BRaf in cervical cancer with a high or moderate activity; the activity profile are comparable with or even better than that of cognate kinases inhibitors, with IC₅₀ values ranging between 4.8 and 340.6 nM for HER3 and between 37.2 and 638.2 nM for BRaf. This work would help to identify those unexpected kinase–inhibitor interactions in human cervical cancer and to develop new and efficient therapeutic strategy combating the disease.     

Structure-based design of Aurora A & B inhibitors

The Aurora family of serine/threonine kinases are mitotic regulators involved in centrosome duplication, formation of the bipolar mitotic spindle and the alignment of the chromosomes along the spindle. These proteins are frequently overexpressed in tumor cells as compared to normal cells and are therefore potential therapeutic oncology targets. An Aurora A high throughput screen revealed a promising sub-micromolar indazole-benzimidazole lead. Modification of the benzimidazole portion of the lead to a C2 linker with a phenyl ring was proposed to achieve novelty. Docking revealed that a conjugated linker was optimal and the resulting compounds were equipotent with the lead. Further structure-guided optimization of substituents on the 5 & 6 position of the indazole led to single digit nanomolar potency. The homology between the Aurora A & Aurora B kinase domains is 71% but their binding sites only differ at residues 212 & 217 (Aurora A numbering). However interactions with only the latter residue may be used for obtaining selectivity. An analysis of published Aurora A and Aurora B X-ray structures reveals subtle differences in the shape of the binding sites. This was exploited by introduction of appropriately sized substituents in the 4 & 6 position of the indazole leading to Aurora B selective inhibitors. Finally we calculate the conformational energy penalty of the putative bioactive conformation of our inhibitors and show that this property correlates well with the Aurora A binding affinity.     

Investigating the role of c-Jun N-terminal kinases in the proliferation of Werner syndrome fibroblasts using diaminopyridine inhibitors

ABSTRACT: Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.     

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)₆ fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.     

Design and synthesis of coumarin substituted oxathiadiazolone derivatives having anti-inflammatory activity possibly through p38 MAP kinase inhibition

Compounds containing oxathiadiazolone nucleus bearing substituted coumarin ring were designed and synthesized while retaining the pharmacophores required for binding with p38 MAP kinase. A four-step synthetic scheme was employed for the synthesis of 7-methoxy-4-(3t?-substituted-2t?-oxo-1t?,2t?,3t?,5t?-oxathiadiazol-4t?-yl)-coumarin. The reactions were monitored by TLC and structures of the intermediates and the target compounds were ascertained by IR, NMR, Mass spectral data. The compounds were found to possess anti-inflammatory activity comparable to indomethacin. Superimposition studies of the target compounds with the lead p38 kinase inhibitors suggested that anti-inflammatory activity of the target compounds may be due to p38 MAP kinase inhibition. It was also suggested that methoxy group on coumarin nucleus may improve the binding profile with p38 MAP kinase.     

Molecular dynamics simulation and free energy calculation studies of the binding mechanism of allosteric inhibitors with p38α MAP kinase

p38 MAP kinase is a promising target for anti-inflammatory treatment. The classical kinase inhibitors imatinib and sorafenib as well as BI-1 and BIRB-796 were reported to bind in the DFG-out form of human p38α, known as type II or allosteric kinase inhibitors. Although DFG-out conformation has attracted great interest in the design of type II kinase inhibitors, the structural requirements for binding and mechanism of stabilization of DFG-out conformation remain unclear. As allosteric inhibition is important to the selectivity of kinase inhibitor, herein the binding modes of imatinib, sorafenib, BI-1 and BIRB-796 to p38α were investigated by molecular dynamics simulation. Binding free energies were calculated by molecular mechanics/Poisson-Boltzmann surface area method. The predicted binding affinities can give a good explanation of the activity difference of the studied inhibitors. Furthermore, binding free energies decomposition analysis and further structural analysis indicate that the dominating effect of van der Waals interaction drives the binding process, and key residues, such as Lys53, Gly71, Leu75, Ile84, Thr106, Met109, Leu167, Asp168, and Phe169, play important roles by forming hydrogen bond, salt bridge, and hydrophobic interactions with the DFG-out conformation of p38α. Finally, we also conducted a detailed analysis of BI-1, imatinib, and sorafenib binding to p38α in comparison with BIRB-796 exploited for gaining potency as well as selectivity of p38 inhibitors. These results are expected to be useful for future rational design of novel type II p38 inhibitors.