RESUMO
Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogues, show modest inhibition of TbrPDEB1 and B2 and quickly kill the bloodstream form of the subspecies T. brucei brucei . We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Tripanossomicidas/química , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Domínio Catalítico , Humanos , Modelos Moleculares , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
We have investigated the roles of four active-site carboxylates in the formation of a prepolymerase ternary complex of Escherichia coli DNA polymerase I (Klenow fragment), containing the template-primer and dNTP. The analysis of nine mutant enzymes with conserved and nonconserved substitutions of Asp(705), Glu(710), Asp(882), and Glu(883) clearly shows that both catalytically essential aspartates, Asp(705) and Asp(882), are required for the formation of a stable ternary complex. Of the two glutamates, only Glu(710) is required for ternary complex formation, while Glu(883) does not participate in this process. This investigation also reveals two interesting properties of the Klenow fragment with regard to enzyme-template-primer binary and enzyme-template-primer-dNTP ternary complex formation. These are (a) the significant resistance of enzyme-template-primer-dNTP ternary complexes to the addition of high salt or template-primer challenge and (b) the ability of the Klenow fragment to form ternary complexes in the presence of noncatalytic divalent cations such as Ca(2+), Co(2+), Ni(2+), and Zn(2+).