RESUMO
Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Domínio Catalítico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.