ABSTRACT
We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.
Subject(s)
Indoles/chemical synthesis , Janus Kinase 3/antagonists & inhibitors , Maleimides/chemical synthesis , Cell Line , Cell Membrane Permeability , Crystallography, X-Ray , Humans , Immunosuppressive Agents/chemical synthesis , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Indoles/chemistry , Indoles/pharmacology , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 3/chemistry , Maleimides/chemistry , Maleimides/pharmacology , Models, Molecular , Molecular Structure , Phosphorylation , Piperidines , Pyrimidines/pharmacology , Pyrroles/pharmacology , STAT5 Transcription Factor/metabolism , Structure-Activity RelationshipABSTRACT
The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2(V617F) mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2(V617F) and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2(V617F)-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2(V617F) cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats.
Subject(s)
Cell Proliferation/drug effects , Janus Kinase 2/antagonists & inhibitors , Polycythemia/prevention & control , Quinoxalines/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Apoptosis/drug effects , Cell Line , Cell Line, Tumor , Erythropoiesis/drug effects , Humans , Janus Kinase 2/chemistry , Janus Kinase 2/genetics , K562 Cells , Mice , Mice, Inbred BALB C , Mice, SCID , Models, Molecular , Molecular Structure , Mutation , Phosphorylation/drug effects , Polycythemia/metabolism , Polycythemia/pathology , Protein Structure, Tertiary , Quinoxalines/chemistry , Rats , STAT5 Transcription Factor/metabolism , Splenomegaly/metabolism , Splenomegaly/pathology , Splenomegaly/prevention & controlABSTRACT
Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.
Subject(s)
Amides/chemistry , Antiviral Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Hepacivirus/drug effects , Thiophenes/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Amides/chemical synthesis , Amides/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Protein Structure, Tertiary , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology , Viral Nonstructural Proteins/metabolismABSTRACT
We have designed and synthesized a novel series of 2,8-diaryl-quinoxalines as Janus kinase 2 inhibitors. Many of the inhibitors show low nanomolar activity against JAK2 and potently suppress proliferation of SET-2 cells in vitro. In addition, compounds from this series have favorable rat pharmacokinetic properties suitable for in vivo efficacy evaluation.
Subject(s)
Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinoxalines/chemistry , Quinoxalines/pharmacology , Administration, Oral , Animals , Cell Line , Drug Discovery , Drug Evaluation, Preclinical , Models, Molecular , Protein Kinase Inhibitors/pharmacokinetics , Quinoxalines/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
Carbohydrate-protein interactions play a key role in many biological processes. Cramoll is a lectin purified from Cratylia mollis seeds that is taxonomically related to concanavalin A (Con A). Although Cramoll and Con A have the same monosaccharide specificity, they have different glycoprotein binding profiles. We report the primary structure of Cramoll, determined by Edman degradation and mass spectrometry and its 1.77 A crystallographic structure and compare it with the three-dimensional structure of Con A in an attempt to understand how differential binding can be achieved by similar or nearly identical structures. We report here that Cramoll consists of 236 residues, with 82% identity with Con A, and that its topological architecture is essentially identical to Con A, because the Calpha positional differences are below 3.5 A. Cramoll and Con A have identical binding sites for MealphaMan, Mn2+, and Ca2+. However, we observed six substitutions in a groove adjacent to the extended binding site and two in the extended binding site that may explain the differences in binding of oligosaccharides and glycoproteins between Cramoll and Con A.
