Production and characterization of recombinant PbPM4 and inhibition analysis of combinatorial chemistry derived compounds against PbPM4

Abstract

Plasmepsins, a series of homologous aspartic proteinases, have been considered novel anti-malarial drug targets for battling the increasing drug-resistance in malaria parasites. Plasmepsin 4 (PbPM4) of the rodent malarial parasite Plasmodium beighei shares high sequence identity with its orthologs of the human malarial species. Therefore, PbPM4 is considered to be able to mimic the target of anti-malarial compounds in a parasite-infected mouse model. Here, a semi-proPbPM4 construct was over-expressed in E. coli and refolded in vitro. PbPM4 showed full catalytic activity at pH 5.5 in the presence of the N-terminal truncated prosegment. In contrast, specific self-processing of the prosegment between L117p and L118p at acidic conditions resulted in time-dependent loss of catalytic activity. In addition, the mature PbPM4 showed comparable kcat and kcat/Km values on chromogenic substrates with plasmepsins of the human malarial parasites, indicating that it shared similar kinetic features with its human orthologs. The inhibition constants were determined for a series of peptidomimetic compounds developed by combinatorial chemistry against mature PbPM4. These inhibitors exhibited nanomolar binding affinities, which were comparable with those of plasmepsins of human parasites. This evidence further suggested PbPM4 can serve as a potential drug target in a rodent model. Supported by NIH grant AI39211.