Nilotinib based pharmacophore models for BCRABL

Tyrosine kinase inhibitors have revolutionized the treatment of several malignancies, converting lethal diseases in a manageable aspect. Imitanib, a small molecule ABL kinase inhibitor is a highly effective therapy for early phase chronic myeloid leukemia (CML), which has constitutively active ABL kinase activity owing to the over expression of the BCR-ABL fusion protein. But some patients develop imatinib resistance, particularly in the advanced phases of CML.The discovery of resistance mechanisms of imitanib; urge forward the development of second generation drugs. Nilotinib, a second generation drug is more potent inhibitor of BCR-ABL than imatinib. But nilotinib also develops dermatologic events and headache in patients. Large information about BCR-ABL structure and its inhibitors are now available. Based on the pharmacophore modeling approaches, it is possible to decipher the molecular determinants to inhibit BCR-ABL. We conducted a structure based and ligand based study to identify potent natural compounds as BCR-ABL inhibitor. First kinase inhibitors were docked with the receptor (BCR-ABL) and nilotinib was selected as a pharmacophore due its high binding efficiency. Eleven compounds were selected out of 1457 substances which have mutual pharmacopohre features with nilotinib. These eleven compounds were validated and used for docking study to find the drug like molecules. The best molecules from the final set of screening candidates can be evaluated in cell lines and may represent a novel class of BCR-ABL inhibitors.

Abbreviations

CML - Chronic myeloid leukemia, PDGFR - Platelet derived growth factor receptor, TKI - Tyrosine kinase inhibitors.     

Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors

BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.     

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.     

Anticipating clinical resistance to target-directed agents : the BCR-ABL paradigm

The deregulated tyrosine kinase activity of BCR-ABL is necessary and sufficient to induce chronic myelogenous leukemia (CML). This observation has paved the way for the development of small-molecule inhibitors specifically targeting the kinase activity of the BCR-ABL protein. Indeed, the amazing success of imatinib has revolutionized the whole area of targeted cancer therapeutics. However, enthusiasm for the striking efficacy of imatinib has been tempered by the development of clinical resistance. In essentially all cases, resistance results from kinase domain mutations and/or overexpression of the BCR-ABL gene. To overcome resistance, several novel BCR-ABL inhibitors have been developed and are in clinical trials, though it is inevitable that resistance to second-generation inhibitors will occur as well. Nonetheless, kinases represent an attractive target for therapeutic intervention in several diseases and, at present, some 50 different kinase inhibitors are in clinical trials. We anticipate that resistance to these compounds will follow mechanisms similar to those observed with imatinib. Resistance mutations cause their effect either by direct steric hindrance to drug binding or by allosterically modulating kinase dynamics. This review highlights the principal mechanisms underlying point mutations from these two different classes to confer drug resistance.     

Identification of a new series of potent diphenol HSP90 inhibitors by fragment merging and structure-based optimization

Heat shock protein 90 (HSP90) is a molecular chaperone to fold and maintain the proper conformation of many signaling proteins, especially some oncogenic proteins and mutated unstable proteins. Inhibition of HSP90 was recognized as an effective approach to simultaneously suppress several aberrant signaling pathways, and therefore it was considered as a novel target for cancer therapy. Here, by integrating several techniques including the fragment-based drug discovery method, fragment merging, computer aided inhibitor optimization, and structure-based drug design, we were able to identify a series of HSP90 inhibitors. Among them, inhibitors 13, 32, 36 and 40 can inhibit HSP90 with IC₅₀ about 20–40 nM, which is at least 200-fold more potent than initial fragments in the protein binding assay. These new HSP90 inhibitors not only explore interactions with an under-studied subpocket, also offer new chemotypes for the development of novel HSP90 inhibitors as anticancer drugs.     

Synthesis and evaluation of 2′-dihalo ribonucleotide prodrugs with activity against hepatitis C virus

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Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2′-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2′-deoxy-2′-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver. AIBEE prodrug 18c was compared with sofosbuvir (1) by co-dosing both compounds by oral administration in dog (5 mg/kg each) and measuring liver concentrations of the active triphosphate metabolite at both 4 and 24 h post dosing. In this study, 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir (1) at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.     

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.     

How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study

Abstract

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph?+?ALL). Ph?+?ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph?+?ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph?+?ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.     

Identification of novel small-molecule inhibitors of α-methylacyl-CoA racemase (AMACR; P504S) and structure-activity relationships

α-Methylacyl-CoA racemase (AMACR; P504S; EC 5.1.99.4) catalyses epimerization of 2-methylacyl-CoAs and is important for the degradation of branched-chain fatty acids and the pharmacological activation of ibuprofen and related drugs. It is also a novel drug target for prostate and other cancers. However, development of AMACR as a drug target has been hampered by the difficulties in assaying enzyme activity. Consequently, reported inhibitors have been rationally designed acyl-CoA esters, which are delivered as their carboxylate prodrugs. The novel colorimetric assay for AMACR based on the elimination of 2,4-dinitrophenolate was developed for high-throughput screening and 20,387 ‘drug-like compounds’ were screened, with a throughput of 768 compounds assayed per day. Pyrazoloquinolines and pyrazolopyrimidines were identified as novel scaffolds and investigated as AMACR inhibitors. The most potent inhibitors have IC₅₀ values of ~2 µM. The pyrazoloquinoline inhibitor 10a displayed uncompetitive inhibition, whilst 10j displayed mixed competitive inhibition. The pyrazolopyrimidine inhibitor 11k displayed uncompetitive inhibition. This is the first report of the identification of specific drug-like small-molecule AMACR inhibitors by high-throughput screening. Pyrazoloquinolines and pyrazolopyrimidines may also be useful as inhibitors of other CoA-utilizing enzymes.     

Molecular and cellular bases of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCR-ABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.     

Synthesis and evaluation of 7-azaindole derivatives bearing benzocycloalkanone motifs as protein kinase inhibitors

Protein kinases are important drug targets, especially in the area of oncology. This paper reports the synthesis and biological evaluation of new 7-azaindole derivatives bearing benzocycloalkanone motifs as potential protein kinase inhibitors. Four compounds 8g, 8h, 8i, and 8l were discovered to inhibit cyclin-dependent kinase 9 (CDK9/CyclinT) and/or Haspin kinase in the micromolar to nanomolar range. 8l was identified as the most potent Haspin inhibitor (IC₅₀ = 14 nM), while 8g and 8h acted as dual inhibitors of CDK9/CyclinT and Haspin. These novel compounds constitute a promising starting point for the discovery of dual protein kinase inhibitors that have potential to be developed as anticancer agents, since both CDK9/CyclinT and Haspin are considered to be drug targets in oncology.     

Inhibition of bromodomain-containing protein 9 for the prevention of epigenetically-defined drug resistance

Bromodomain-containing protein 9 (BRD9), an epigenetic “reader” of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.     

Identification of novel series of pyrazole and indole-urea based DFG-out PYK2 inhibitors

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide–urea and a pyrazole–urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.     

Discovery of a novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors

A novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors were derived from a fragment-based approach using information from X-ray crystallographic analysis of NS5B-inhibitor complexes and iterative rounds of parallel synthesis. Structure-based drug design strategies led to the discovery of potent sub-micromolar inhibitors 11a–c and 12a–c from a weak-binding fragment-like structure 1 as a starting point.     

Development of a new class of proteasome inhibitors with an epoxyketone warhead: Rational hybridization of non-peptidic belactosin derivatives and peptide epoxyketones

Proteasome inhibitors are currently a focus of increased attention as anticancer drug candidates. We recently performed systematic structure–activity relationship studies of the peptidic natural product belactosin A and identified non-peptidic derivative 2 as a highly potent proteasome inhibitor. However, the cell growth inhibitory effect of 2 is only moderate, probably due to the biologically unstable β-lactone warhead. Peptide epoxyketones are an important class of proteasome inhibitors exhibit high potency in cellular systems based on the efficient α,β-epoxyketone warhead. Importantly, belactosin derivatives bind primarily to the primed binding site, while peptide epoxyketones bind only to the non-primed binding site of proteasome, suggesting that hybridization of them might lead to the development of a new class of proteasome inhibitors. Thus, we successfully identified a novel chemotype of proteasome inhibitors 3 and 4 by rational structure-based design, which are expected to bind to both the primed and non-primed binding sites of proteasome.     

Discovery and optimization of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1

The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ～10 nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.     

Discovery of novel histone lysine methyltransferase G9a/GLP (EHMT2/1) inhibitors: Design,synthesis, and structure-activity relationships of 2,4-diamino-6-methylpyrimidines

The discovery and optimization of a novel series of G9a/GLP (EHMT2/1) inhibitors are described. Starting from known G9a/GLP inhibitor 5, efforts to explore the structure-activity relationship and optimize drug properties led to a novel compound 13, the side chain of which was converted to tetrahydroazepine. Compound 13 showed increased G9a/GLP inhibitory activity compared with compound 5. In addition, compound 13 exhibited improved human ether-a-go-go related gene (hERG) inhibitory activity over compound 5 and also improved pharmacokinetic profile in mice (oral bioavailability: 17 to 40%). Finally, the co-crystal structure of G9a in complex with compound 13 provides the basis for the further development of tetrahydroazepine-based G9a/GLP inhibitors.     

Discovery of chiral dihydropyridopyrimidinones as potent,selective and orally bioavailable inhibitors of AKT

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK₂. A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model.