Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.     

Allosteric interactions between the myristate- and ATP-site of the Abl kinase

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.     

Synthesis and docking study of 2-phenylaminopyrimidine Abl tyrosine kinase inhibitors

Six analogs of imatinib, an Abl kinase inhibitor clinically used as a first-line therapeutic agent for chronic myeloid leukaemia (CML), have been synthesized and characterized. And their potency as Abl kinase inhibitors have been screened by a robust virtual screening method developed based on the crystal structure (PDB code 2hyy) of Abl-imatinib complex using Surflex-Docking. The docking results are consistent with the inhibitory potency of the compounds characterized by MS method. And the H-bonds between imatinib analogs and Thr315 and Met318 residues in Abl kinase are shown to be crucial for achieving accurate poses and high binding affinities for the ATP-competitive kinase inhibitors.     

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.     

Growth arrest of BCR-ABL positive cells with a sequence-specific polyamide-chlorambucil conjugate

Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model.     

Extended kinase profile and properties of the protein kinase inhibitor nilotinib

As a drug used to treat imatinib-resistant and -intolerant, chronic and advanced phase chronic myelogenous leukaemia, nilotinib is well characterised as a potent inhibitor of the Abl tyrosine kinase activity of wild-type and imatinib-resistant mutant forms of BCR-Abl. Here we review the profile of nilotinib as a protein kinase inhibitor. Although an ATP-competitive inhibitor of Abl, nilotinib binds to a catalytically inactive conformation (DFG-out) of the activation loop. As a consequence of this, nilotinib exhibits time-dependent inhibition of Abl kinase in enzymatic assays, which can be extrapolated to other targets to explain differences between biochemical activity and cellular assays. Although these differences confound assessment of kinase selectivity, as assessed using a combination of protein binding and transphosphorylation assays, together with cellular autophosporylation and proliferation assays, well established kinase targets of nilotinib in rank order of inhibitory potency are DDR-1 > DDR-2 > BCR-Abl (Abl) > PDGFRα/β > KIT > CSF-1R. In addition nilotinib has now been found to bind to both MAPK11 (p38β) and MAPK12 (p38α), as well as with very high affinity to ZAK kinase. Although neither enzymatic nor cellular data are yet available to substantiate the drug as an inhibitor of ZAK phosphorylation, modeling predicts that it binds in an ATP-competitive fashion.     

A small molecule-kinase interaction map for clinical kinase inhibitors

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.     

A systematic analysis of the resistance and sensitivity of HER2YVMA receptor tyrosine kinase mutant to tyrosine kinase inhibitors in HER2-positive lung cancer

Human epidermal growth factor receptor 2 (HER2) has become a well-established target for the treatment of HER2-positive lung cancer. However, a frequently observed in-frame mutation that inserts amino acid quadruplex Tyr776-Val777-Met778-Ala779 at G776 (G776^YVMA) in HER2 kinase domain can cause drug resistance and sensitivity, largely limiting the application of reversible tyrosine kinase inhibitors in lung cancer therapy. A systematic investigation of the intermolecular interactions between the HER2^YVMA mutant and clinical small-molecule inhibitors would help to establish a complete picture of drug response to HER2 G776^YVMA insertion in lung cancer, and to design new tyrosine kinase inhibitors with high potency and selectivity to target the lung cancer-related HER2^YVMA mutant. Here, we combined homology modeling, ligand grafting, structure minimization, molecular simulation and binding affinity analysis to profile a number of tyrosine kinase inhibitors against the G776^YVMA insertion in HER2. It is found that the insertion is far away from HER2 active pocket and thus cannot contact inhibitor ligand directly. However, the insertion is expected to induce marked allosteric effect on some regions around the pocket, including A-loop and hinges connecting between the N- and C-lobes of HER2 kinase domain, which may exert indirect influence to inhibitor binding. Most investigated inhibitors exhibit weak binding strength to both wild-type and mutant HER2, which can be attributed to steric hindrance that impairs ligand compatibility with HER2 active pocket. However, the cognate inhibitor lapatinib and the non-cognate inhibitor bosutinib were predicted to have low affinity for wild-type HER2 but high affinity for HER2^YVMA mutant, which was confirmed by subsequent kinase assay experiments; the inhibitory potencies of bosutinib against wild-type and mutant HER2 were determined to be IC₅₀?>?1000 and =27?nM, respectively, suggesting that the bosutinib might be exploited as a selective inhibitor for mutant over wild-type HER2. Structural examination revealed that formation of additional non-bonded interactions such as hydrogen bonds and hydrophobic contacts with HER2 A-loop region due to G776^YVMA insertion is the primary factor to improve bosutinib affinity upon the mutation.     

Conformational flexibility,binding energy,role of salt bridge and alanine-mutagenesis for c-Abl kinase complex

Abl kinase plays a decisive role in the mechanism of the most fatal human pathogen chronic mylogenous leukemia (CML). Here, we have carried out a comprehensive study about the conformational flexibility, role of salt bridge and the protein- ligand interaction for this kinase with its well-known inhibitor, Imatinib. We have performed molecular dynamics simulations for conformational behavior, investigated the salt bridges and calculated the binding free energy of Imatinib with MM-PB/SA method for Abl kinase complex. We also explored the role of salt-bridge in the kinase complex and its effect on binding activity of inhibitors. Furthermore, to investigate the importance of those residues which form salt bridges, we mutated them by Alanine with the help of Alanine scanning program. We noticed significant variations in total free energy of Imatinib in all possible mutations. The binding free energy of ligand for kinase receptor was analyzed by molecular mechanics Poission Boltzmann surface area (MM-PB/SA) method. These results suggest that conserved glutamic acid and lysine are necessary for stability of complex.     

Allosteric enhancement of the BCR-Abl1 kinase inhibition activity of nilotinib by cobinding of asciminib

Inhibitors that bind competitively to the ATP binding pocket in the kinase domain of the oncogenic fusion protein BCR–Abl1 are used successfully in targeted therapy of chronic myeloid leukemia (CML). Such inhibitors provided the first proof of concept that kinase inhibition can succeed in a clinical setting. However, emergence of drug resistance and dose-dependent toxicities limit the effectiveness of these drugs. Therefore, treatment with a combination of drugs without overlapping resistance mechanisms appears to be an appropriate strategy. In the present work, we explore the effectiveness of combination therapies of the recently developed allosteric inhibitor asciminib with the ATP-competitive inhibitors nilotinib and dasatinib in inhibiting the BCR–Abl1 kinase activity in CML cell lines. Through these experiments, we demonstrate that asciminib significantly enhances the inhibition activity of nilotinib, but not of dasatinib. Exploring molecular mechanisms for such allosteric enhancement via systematic computational investigation incorporating molecular dynamics, metadynamics simulations, and density functional theory calculations, we found two distinct contributions. First, binding of asciminib triggers conformational changes in the inactive state of the protein, thereby making the activation process less favorable by ∼4 kcal/mol. Second, the binding of asciminib decreases the binding free energies of nilotinib by ∼3 and ∼7 kcal/mol for the wildtype and T315I-mutated protein, respectively, suggesting the possibility of reducing nilotinib dosage and lowering risk of developing resistance in the treatment of CML.     

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.     

Advances in the structural biology, design and clinical development of Bcr-Abl kinase inhibitors for the treatment of chronic myeloid leukaemia

The constitutively activated Abl tyrosine kinase domain of the chimeric Bcr-Abl oncoprotein is responsible for the transformation of haematopoietic stem cells and the symptoms of chronic myeloid leukaemia (CML). Imatinib targets the tyrosine kinase activity of Bcr-Abl and is a first-line therapy for this malignancy. Although highly effective in chronic phase CML, patients who have progressed to the advanced phase of the disease frequently fail to respond to imatinib or develop resistance to therapy and relapse. This is often due to the emergence of clones expressing mutant forms of Bcr-Abl, which exhibit a decreased sensitivity towards inhibition by imatinib. Considerable progress has recently been made in understanding the structural biology of Abl and the molecular basis for resistance, facilitating the discovery and development of second generation drugs designed to combat mutant forms of Bcr-Abl. The first of these compounds to enter clinical development were BMS-354825 (BristolMyersSquibb) and AMN107 (Novartis Pharma) and, from Phase I results, both of these promise a breakthrough in the treatment of imatinib-resistant CML. Recent advances with these and other promising classes of new CML drugs are reviewed.     

A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, because patients are treated indefinitely with this drug to maintain remission, resistance is increasingly becoming an issue. Although there are many ways to detect kinase activity, most lack the ability to “multiplex” the analysis (i.e., to detect more than one substrate simultaneously). Here we report a novel biosensor for detecting Abl kinase activity and sensitivity to inhibitor in live intact cells overexpressing a CML model Abl kinase construct. This straightforward methodology could eventually provide a new tool for detecting kinase activity and inhibitor drug response in cancer cells that overexpress oncogenic kinases.     

Structure–activity relationships of 6-(2,6-dichlorophenyl)-8-methyl-2-(phenylamino)pyrido[2,3-d]pyrimidin-7-ones: Toward selective Abl inhibitors

We report the design, synthesis, and structure–activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well-characterized cancer cell lines. Our study reveals that substitutions in the 3- and 4-positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell-based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.     

Study of the role of "gatekeeper" mutations V654A and T670I of c-kit kinase in the interaction with inhibitors by means mixed molecular dynamics/docking approach

The over-expression of c-kit proto-oncogene has been reported in hematopoietic cells, small cell lung cancer, and gastrointestinal stromal tumors. The clinical importance of c-kit expression in tumors focused the research towards inhibitors of this tyrosine kinase. Imatinib (Gleevec®) was the first compound used in therapy, but mutations on c-kit led to reduced effectiveness or ineffectiveness of this treatment. Other compounds are likely to be effective against mutants, such as Sunitinib (Sutent®), but the need for new and most effective inhibitors against mutants is still critical. We report mixed Molecular Dynamics/Docking study with the aim to unveil the molecular mechanism involved in the resistance of Imatinib, Sunitinib, and other known compounds against the “gatekeeper” mutants V654A e T670I. We tried to evidence strong and weak features of actual inhibitors in order to identify the guidelines to design new and most potent inhibitors against c-kit mutants.     

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction  Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H₂O₂, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods  Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl⁺ cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl⁺ CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl⁺ cells. Conclusion  NAC enhances imatinib-induced apoptosis of Bcr-Abl⁺ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.     

Blockade of the ERK or PI3K-Akt signaling pathway enhances the cytotoxicity of histone deacetylase inhibitors in tumor cells resistant to gefitinib or imatinib

Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.     

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.     

Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis

Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.