RESUMO
New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high-throughput screen of a focussed kinase set of ~3400 compounds to identify potent and parasite-selective inhibitors of an enzymatic Leishmania CRK3-cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3-CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low-nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Leishmania/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Leishmania/enzimologia , Leishmaniose/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Relação Estrutura-Atividade , Ureia/química , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 x EC(50) for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.