Exploring sequence-structure relationships in the tyrosine kinome space: functional classification of the binding specificity mechanisms for cancer therapeutics

MOTIVATION: Evolutionary and structural conservation patterns shared by more than 500 of identified protein kinases have led to complex sequence-structure relationships of cross-reactivity for kinase inhibitors. Understanding the molecular basis of binding specificity for protein kinases family, which is the central problem in discovery of cancer therapeutics, remains challenging as the inhibitor selectivity is not readily interpreted from chemical proteomics studies, neither it is easily discernable directly from sequence or structure information. We present an integrated view of sequence-structure-binding relationships in the tyrosine kinome space in which evolutionary analysis of the kinases binding sites is combined with computational proteomics profiling of the inhibitor-protein interactions. This approach provides a functional classification of the binding specificity mechanisms for cancer agents targeting protein tyrosine kinases. RESULTS: The proposed functional classification of the kinase binding specificities explores mechanisms in which structural plasticity of the tyrosine kinases and sequence variation of the binding-site residues are linked with conformational preferences of the inhibitors in achieving effective drug binding. The molecular basis of binding specificity for tyrosine kinases may be largely driven by conformational adaptability of the inhibitors to an ensemble of structurally different conformational states of the enzyme, rather than being determined by their phylogenetic proximity in the kinome space or differences in the interactions with the variable binding-site residues. This approach provides a fruitful functional linkage between structural bioinformatics analysis and disease by unraveling the molecular basis of kinase selectivity for the prominent kinase drugs (Imatinib, Dasatinib and Erlotinib) which is consistent with structural and proteomics experiments.     

Computational proteomics of biomolecular interactions in the sequence and structure space of the tyrosine kinome: deciphering the molecular basis of the kinase inhibitors selectivity

Understanding and predicting the molecular basis of protein kinases specificity against existing therapeutic agents remains highly challenging and deciphering this complexity presents an important problem in discovery and development of effective cancer drugs. We explore a recently introduced computational approach for in silico profiling of the tyrosine kinases binding specificity with a class of the pyrido-[2,3-d]pyrimidine kinase inhibitors. Computational proteomics analysis of the ligand-protein interactions using parallel simulated tempering with an ensemble of the tyrosine kinases crystal structures reveals an important molecular determinant of the kinase specificity. The pyrido-[2,3-d]pyrimidine inhibitors are capable of dynamically interacting with both active and inactive forms of the tyrosine kinases, accommodating structurally different kinase conformations with a similar binding affinity. Conformational tolerance of the protein tyrosine kinases binding with the pyrido[2,3-d]pyrimidine inhibitors provides the molecular basis for the broad spectrum of potent activities and agrees with the experimental inhibition profiles. The analysis of the pyrido[2,3-d]pyrimidine sensitivities against a number of clinically relevant ABL kinase mutants suggests an important role of conformational adaptability of multitargeted kinase inhibitors in developing drug resistance mechanisms. The presented computational approach may be useful in complementing proteomics technologies to characterize activity signatures of small molecules against a large number of potential kinase targets.     

Imprint of evolutionary conservation and protein structure variation on the binding function of protein tyrosine kinases

MOTIVATION: According to the models of divergent molecular evolution, the evolvability of new protein function may depend on the induction of new phenotypic traits by a small number of mutations of the binding site residues. Evolutionary relationships between protein kinases are often employed to infer inhibitor binding profiles from sequence analysis. However, protein kinases binding profiles may display inhibitor selectivity within a given kinase subfamily, while exhibiting cross-activity between kinases that are phylogenetically remote from the prime target. The emerging insights into kinase function and evolution combined with a rapidly growing number of publically available crystal structures of protein kinases complexes have motivated structural bioinformatics analysis of sequence-structure relationships in determining the binding function of protein tyrosine kinases. RESULTS: In silico profiling of Imatinib mesylate and PD-173955 kinase inhibitors with protein tyrosine kinases is conducted on kinome scale by using evolutionary analysis and fingerprinting inhibitor-protein interactions with the panel of all publically available protein tyrosine kinases crystal structures. We have found that sequence plasticity of the binding site residues alone may not be sufficient to enable protein tyrosine kinases to readily evolve novel binding activities with inhibitors. While evolutionary signal derived solely from the tyrosine kinase sequence conservation can not be readily translated into the ligand binding phenotype, the proposed structural bioinformatics analysis can discriminate a functionally relevant kinase binding signal from a simple phylogenetic relationship. The results of this work reveal that protein conformational diversity is intimately linked with sequence plasticity of the binding site residues in achieving functional adaptability of protein kinases towards specific drug binding. This study offers a plausible molecular rationale to the experimental binding profiles of the studied kinase inhibitors and provides a theoretical basis for constructing functionally relevant kinase binding trees.     

Inhibition of Aurora Kinase B Is Important for Biologic Activity of the Dual Inhibitors of BCR-ABL and Aurora Kinases R763/AS703569 and PHA-739358 in BCR-ABL Transformed Cells

ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.     

In-silico identification of inhibitors against mutated BCR-ABL protein of chronic myeloid leukemia: a virtual screening and molecular dynamics simulation study

Aberrant and proliferative expression of the oncogene BCR-ABL in the bone marrow cells had been proven as the prime cause of chronic myeloid leukemia (CML). It has been established that tyrosine kinase domain of BCR-ABL protein is a potential therapeutic target for the treatment of CML. Imatinib is considered as a first-generation drug that can inhibit the enzymatic action by inhibiting the ATP binding with BCR-ABL protein. Later on, insensitivity of CML cells towards Imatinib has been observed may be due to mutation in tyrosine kinase domain of the ABL receptor. Subsequently, some other second-generation drugs have also been reported viz. Baustinib, Nilotinib, Dasatinib, Ponatinib, Bafetinib, etc., which can able to combat against mutated domain of ABL tyrosine kinase protein. By taking into account of bioavailability and resistance developed, there is an utmost need to find some more inhibitors for the mutated ABL tyrosine kinase protein. For virtual screening, a data-set has been generated by collecting the all available drug like natural compounds from ZINC and Drug Bank databases. Comparative docking analysis was also carried out on the active site of ABL tyrosine kinase receptor with reported reference inhibitors. Molecular dynamics simulation of the best screened interacting complex was done for 50 ns to validate the stability of the system. These selected inhibitors were further validated and analyzed through pharmacokinetics properties and series of ADMET parameters by in silico methods. Considering the above said parameters proposed molecules are concluded as potential leads for drug designing pipeline against CML.     

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation.     

Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.     

Conformational flexibility and binding energy profile of c-Abl tyrosine kinase complexed with Imatinib: an insight from MD study

Structural biology of kinase and in particular of tyrosine kinase has given detailed insights into the intrinsic flexibility of the catalytic domain and has provided a rational basis for obtaining selective inhibitors. In this paper, we have studied the conformational flexibility of c-Abl tyrosine kinase complexed with Imatinib (STI), in the presence of TIP3P water in physiological conditions at neutral pH. The conformational studies suggest that the flexibility of activation loop is responsible to facilitate the nucleotide binding and release. Owing to the conformational adaptability, adenosine triphosphate (ATP) binds at a particular site in the loop region of the tyrosine kinase. The molecular mechanics Poisson–Boltzmann surface area methods are analysed, as is a free-energy pathways method, which shows the stable binding with free energy ? 6.04 kcal/mol for STI. The binding energy calculated by the Sietraj method is approximately the same as the experimental binding energy of STI with c-Abl kinase. It is suggested that the conserved glutamic acid and lysine residues are necessary for the stability and optimum activity of inhibitor. This study may be helpful in rational drug designing of new kinase inhibitors.     

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.     

Tyrosine kinases. New target of anticancer therapy

Recently, clinical studies of new drugs development to target specific forms of cancer were reported. Herceptin, a monoclonal antibody against the Her2/neu receptor tyrosine kinase, prolonged the survival of women with Her2/neu positive metastatic breast cancer. STI571, a small molecule inhibitor of the BCR/ABL, c-Kit and platelet derived growth factor receptor tyrosine kinase, produced pronounced clinical responses in patients with BCR/ABL positive chronic myeloid leukemia and c-Kit positive gastrointestial stromal tumors. In order to consider the use of the inhibitor of tyrosine kinases activity as anticancer drug, their mechanisms of the oncogenic activation and their impact on tumor transformation should be studied. The treatment with tyrosine kinase inhibitors such as STI571 or herceptin was a spectacular clinical success which stimulated research on the structure and function of both kinases and their inhibitors.     

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL,Jak2-V617F,or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.     

Overcoming kinase resistance in chronic myeloid leukemia

Imatinib is a small-molecule inhibitor of BCR-ABL tyrosine kinase activity, with proven efficacy and tolerability. Despite imatinib's activity, the development of resistance, whether BCR-ABL dependent or independent, is a concern. BCR-ABL-dependent resistance is commonly a result of mutations in the BCR-ABL gene, which can induce a structural predisposition towards the active conformation of the protein, resulting in a shift in the equilibrium of BCR-ABL from inactive, which imatinib binds, to active, which imatinib is unable to bind. BCR-ABL gene amplification may play a role in the development of imatinib resistance in patients with CML. There are a number of BCR-ABL-independent mechanisms of imatinib resistance, including the efflux protein multidrug resistance protein-1, of which imatinib is a substrate. Another mechanism may be the development of alternative pathways of disease progression, leading to less reliance on BCR-ABL; indeed, the SRC family tyrosine kinases LYN and HCK have been frequently implicated in treatment resistance and progression of CML. Clearly, imatinib resistance requires the development of other treatment options. Dasatinib, with increased binding potency (325-fold greater potency than imatinib for wild-type BCR-ABL), inhibition of both the active and inactive formation of BCR-ABL, and targeting of SRC family kinases, is the only agent approved for the treatment of patients with imatinib-resistant or -intolerant CML and Ph+ ALL. Dasatinib is highly active in all phases of these diseases, and is active in the majority of imatinib-resistant mutations, with the exception of T315I. The development of agents that effectively inhibit T315I mutations suggests that future treatment options will include combination therapy.     

Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC₅₀ of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.     

Induction of heparin-binding EGF-like growth factor and activation of EGF receptor in imatinib mesylate-treated squamous carcinoma cells

Imatinib mesylate is a tyrosine kinase inhibitor of the ABL, platelet-derived growth factor receptor (PDGFR), and c-kit kinases. Inhibition of BCR-ABL and c-kit accounts for its clinical activity in leukemia and sarcoma, respectively. In this report, we describe other cellular targets for imatinib. Treatment of head and neck squamous carcinoma cells with clinically relevant concentrations of imatinib-induced changes in cell morphology and growth similar to changes associated with epidermal growth factor receptor (EGFR) activation. Imatinib-induced changes were blocked with the EGFR antagonist cetuximab, which suggested direct involvement of EGFR in this process. Western blot analysis of cells incubated with imatinib demonstrated activation of EGFR and downstream signaling that was reduced by inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) and EGFR, but not Her2/ErbB2. An in vitro kinase assay showed that imatinib did not directly affect EGFR kinase activity, suggesting involvement of EGFR-activating molecules. Inhibitors and neutralizing antibodies against heparin-binding epidermal growth factor-like growth factor (HB-EGF), and to a lesser extent transforming growth factor-alpha, reduced imatinib-mediated mitogen activated protein kinase (MAPK) activation. Imatinib stimulated the rapid release of soluble HB-EGF and the subsequent induction of membrane-bound HB-EGF, which correlated with biphasic MAPK activation. Together, these results suggested that imatinib affects EGFR activation and signaling pathways through rapid release and increased expression of endogenous EGFR-activating ligands. Although, imatinib primarily inhibits tyrosine kinases, it also stimulates the activity of EGFR tyrosine kinase in head and neck squamous tumors. This finding demonstrates the need for careful use of this drug in cancer patients.     

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.     

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.     

Inhibiting Tyrosine Phosphorylation of Protein Kinase Cδ (PKCδ) Protects the Salivary Gland from Radiation Damage

Radiation therapy for head and neck cancer can result in extensive damage to normal adjacent tissues such as the salivary gland and oral mucosa. We have shown previously that tyrosine phosphorylation at Tyr-64 and Tyr-155 activates PKCδ in response to apoptotic stimuli by facilitating its nuclear import. Here we have identified the tyrosine kinases that mediate activation of PKCδ in apoptotic cells and have explored the use of tyrosine kinase inhibitors for suppression of irradiation-induced apoptosis. We identify the damage-inducible kinase, c-Abl, as the PKCδ Tyr-155 kinase and c-Src as the Tyr-64 kinase. Depletion of c-Abl or c-Src with shRNA decreased irradiation- and etoposide-induced apoptosis, suggesting that inhibitors of these kinases may be useful therapeutically. Pretreatment with dasatinib, a broad spectrum tyrosine kinase inhibitor, blocked phosphorylation of PKCδ at both Tyr-64 and Tyr-155. Expression of “gate-keeper” mutants of c-Abl or c-Src that are active in the presence of dasatinib restored phosphorylation of PKCδ at Tyr-155 and Tyr-64, respectively. Imatinib, a c-Abl-selective inhibitor, also specifically blocked PKCδ Tyr-155 phosphorylation. Dasatinib and imatinib both blocked binding of PKCδ to importin-α and nuclear import, demonstrating that tyrosine kinase inhibitors can inhibit nuclear accumulation of PKCδ. Likewise, pretreatment with dasatinib also suppressed etoposide and radiation induced apoptosis in vitro. In vivo, pre-treatment of mice with dasatinib blocked radiation-induced apoptosis in the salivary gland by >60%. These data suggest that tyrosine kinase inhibitors may be useful prophylactically for protection of nontumor tissues in patients undergoing radiotherapy of the head and neck.     

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl

The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamily often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.