RESUMO
Peptidic α-ketoamides have been developed as inhibitors of the malarial protease PfSUB1. The design of inhibitors was based on the best known endogenous PfSUB1 substrate sequence, leading to compounds with low micromolar to submicromolar inhibitory activity. SAR studies were performed indicating the requirement of an aspartate mimicking the P1' substituent and optimal P1-P4 length of the non-prime part. The importance of each of the P1-P4 amino acid side chains was investigated, revealing crucial interactions and size limitations.
Assuntos
Amidas/farmacologia , Peptídeos/química , Proteínas de Protozoários/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Subtilisinas/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas de Protozoários/metabolismo , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/química , Relação Estrutura-Atividade , Subtilisinas/metabolismoRESUMO
Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.