A Quantitative Assay of Recombinant Malarial Lactate Dehydrogenase as a Platform for Screening Inhibitors from Crude Herbal Extracts

The potential of malaria parasite (Plasmodium falciparum) generating resistance to currently used antimalarial drugs is a major scourge for malaria patients. The multiple targets on which antimalarial drugs act hinder the occurrence of such resistance. As an essential metabolic enzyme involved in energy production in the parasite, the lactate dehydrogenase of P. falciparum (PfLDH) provides an alternative target for parasite killing. In the present study, the PfLDH gene was amplified from the FCC1/HN isolate of PfLDH and functionally expressed in Escherichia coli, which had been subsequently identified by Western blotting with the recombinant PfLDH protein-raised rabbit polyclonal antiserum, and further confirmed by the hybridization with lytic suspension of cultured parasites. The recombinant PfLDH protein was purified for uniform measurement of enzymatic activity and immobilized on 96-well plates for detection of PfLDH-targeted inhibitors. Hematin (HT) and chloroquine (CQ), two well-known inhibitors of PfLDH, were chosen for evaluating the feasibility of the in vitro colorimetric enzyme assay. The results showed that the enzymatic activity of PfLDH sharply dropped in a concentration-dependent pattern, as HT was involved, but only slightly dropped in the presence of CQ. Furthermore, this enzyme assay was applied to screening inhibitory component (s) of PfLDH from the crude extracts of medicinal plants, Pueraria lobata, Amomum villosum, Dichroa febrifuga, and Polygonum cuspidatum, in which the suppression of malaria growth was previously observed for P. lobata and D. febrifuga. Consequently, the correlation of enzyme inhibition with growth suppression was observed for the crude extracts of P. lobata although further investigations should be pursued for elucidation of the exact compound exhibiting inhibitory effect to PfLDH. The present recombinant PfLDH-based enzymatic activity determination provides a simplified, reproductive and economic platform for screening inhibitors of PfLDH, which contributes to the discovery of inhibitory compounds from either pure chemicals or crude extracts of medicinal plants, animals, and fungi towards development of novel antimalarial drugs.