ABSTRACT
A novel series of 3-amino-1H-thieno[3,2-c]pyrazole derivatives demonstrating high potency in inhibiting Aurora kinases was developed. Here we describe the synthesis and a preliminary structure-activity relationship, which led to the discovery of a representative compound (38), which showed low nanomolar inhibitory activity in the anti-proliferation assay and was able to block the cell cycle in HCT-116 cell line. This compound demonstrated favorable pharmacokinetic properties and good efficacy in the HL-60 xenograft tumor model.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Thiophenes/pharmacology , Animals , Antineoplastic Agents/chemistry , Aurora Kinases , Cell Cycle/drug effects , Cell Proliferation/drug effects , Computational Biology , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , HL-60 Cells , Humans , Male , Mice , Mice, SCID , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Neoplasms, Experimental/drug therapy , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Stereoisomerism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistry , Transplantation, HeterologousABSTRACT
Mutations in the kinase domain of Bcr-Abl are the most common cause of resistance to therapy with imatinib in patients with chronic myelogenous leukemia (CML). Second-generation Bcr-Abl inhibitors are able to overcome most imatinib-resistant mutants, with the exception of the frequent T315I substitution, which is emerging as a major cause of resistance to these drugs in CML patients. Structural studies could be used to support the drug design process for the development of inhibitors able to target the T315I substitution, but until now no crystal structure of the T315I Abl mutant has been solved. We show here the first crystal structure of the kinase domain of Abl T315I in complex with PHA-739358, an Aurora kinase inhibitor currently in clinical development for solid and hematologic malignancies. This compound inhibits in vitro the kinase activity of wild-type Abl and of several mutants, including T315I. The cocrystal structure of T315I Abl kinase domain provides the structural basis for this activity: the inhibitor associates with an active conformation of the kinase domain in the ATP-binding pocket and lacks the steric hindrance imposed by the substitution of threonine by isoleucine.
Subject(s)
Benzamides/chemistry , Benzamides/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-abl/chemistry , Proto-Oncogene Proteins c-abl/genetics , Pyrazoles/chemistry , Pyrazoles/metabolism , Aurora Kinases , Crystallography, X-Ray , Drug Resistance, Neoplasm/genetics , Humans , Imatinib Mesylate , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Piperazines/pharmacology , Protein Binding , Proto-Oncogene Proteins c-abl/metabolism , Pyrimidines/pharmacologyABSTRACT
The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an approximately 72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this "parasynaptic" location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.
Subject(s)
Carrier Proteins/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Phosphotransferases/physiology , Proto-Oncogene Proteins/physiology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/enzymology , Adaptor Proteins, Signal Transducing , CD3 Complex/immunology , CD3 Complex/metabolism , CSK Tyrosine-Protein Kinase , Carrier Proteins/analysis , Carrier Proteins/genetics , DNA Helicases , Humans , Jurkat Cells , Ligands , Lymphocyte Activation/immunology , Lymphocyte Activation/physiology , Membrane Microdomains/metabolism , Membrane Microdomains/physiology , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation , Phosphotransferases/analysis , Phosphotransferases/metabolism , Poly-ADP-Ribose Binding Proteins , Protein-Tyrosine Kinases , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/metabolism , RNA Helicases , RNA Recognition Motif Proteins , RNA, Small Interfering/genetics , Receptors, Antigen, T-Cell/physiology , Signal Transduction/genetics , Signal Transduction/physiology , T-Lymphocytes/chemistry , T-Lymphocytes/immunology , Tyrosine/metabolism , src Homology Domains/physiology , src-Family KinasesABSTRACT
PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition-related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status-dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser(10) is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers.
Subject(s)
Benzamides/pharmacology , Neoplasms/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Animals , Aurora Kinase B , Aurora Kinases , Benzamides/pharmacokinetics , Benzamides/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Immunohistochemistry , Male , Mice , Mice, Nude , Neoplasms/enzymology , Phosphorylation , Pyrazoles/pharmacokinetics , Pyrazoles/therapeutic use , Rats , Rats, Sprague-DawleyABSTRACT
PURPOSE: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy. EXPERIMENTAL DESIGN: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer. RESULTS: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632. CONCLUSIONS: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Mammary Neoplasms, Experimental/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrroles/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Aurora Kinase B , Aurora Kinases , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , HL-60 Cells , HeLa Cells , Humans , Inhibitory Concentration 50 , Mice , Mice, Transgenic , Molecular Structure , Phenotype , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/therapeutic use , Pyrroles/therapeutic use , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives toward the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of Aurora kinases that also shows low nanomolar potency against additional anticancer kinase targets. Based on its high antiproliferative activity on different cancer cell lines, favorable chemico-physical and pharmacokinetic properties, and high efficacy in in vivo tumor models, compound 9d was ultimately selected for further development.
Subject(s)
Antineoplastic Agents/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrroles/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Aurora Kinases , Cell Line, Tumor , Drug Screening Assays, Antitumor , Male , Mice , Models, Molecular , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrroles/pharmacokinetics , Pyrroles/pharmacology , Solubility , Structure-Activity RelationshipABSTRACT
Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed to target the ATP pocket of protein kinases. The most potent compound reported in this study had an IC(50) of 0.027 microM in the enzymatic assay for Aur-A inhibition and IC(50)s between 0.05 microM and 0.5 microM for the inhibition of proliferation of different tumor cell lines.
Subject(s)
Antineoplastic Agents/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Piperazines/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrroles/chemical synthesis , Adenosine Triphosphate/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Aurora Kinases , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Combinatorial Chemistry Techniques , Drug Screening Assays, Antitumor , Humans , Models, Molecular , Piperazines/chemistry , Piperazines/pharmacology , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Pyrroles/chemistry , Pyrroles/pharmacology , Structure-Activity RelationshipABSTRACT
In recent years telomerase has been identified as a new promising target in oncology and consequently new telomerase inhibitors have been intensely explored as anticancer agents. Focused screening of several polyhydroxylated flavonoids has allowed us to identify 7,8,3',4'-tetrahydroxyflavone 1 as a new telomerase inhibitor with an interesting in vitro activity in a Flash-Plate assay (IC50 = 0.2 microM) that has been confirmed in the classical TRAP assay. Starting from this compound, we developed a medicinal chemistry program to optimize our lead, and in particular to replace one of the two catechols with potential bioisosteres. From this study, new structural analogues characterized by submicromolar potencies have been obtained. Their synthesis and biological activity are described.