Exploring Iso-mukaadial acetates and other small compounds as inhibitors of recombinant Plasmodium falciparum lactate dehydrogenase.

Abstract

Malaria is a major killer disease in Sub-Saharan Africa, this disease is caused by a protozoan parasite of genus Plasmodium. It is a pressing health issue the public is facing, and the effectiveness of every treatment developed thus far is being jeopardized by the emergence of parasite drug resistance. This then creates a demand for new antiprotozoal medication, necessitating novel approaches that will assure the long- term discovery of the lead compounds. The investigation of compounds such as Iso-mukaadial acetate (IMA), Betulinic acid (BA), Ursolic acid (UA) and Oleanolic acid (OEA) which are isolated from plants shows to possess antimalarial activity. These compounds either originate from various plants or leaves, IMA which is isolated from a pepper bark tree, BA from bark of a plant species (white birch), UA from leaves of (lavender, rosemary), and OEA found in leaves and Olea europaea fruit. This study aims to investigate the inhibitory properties of these compounds against Plasmodium falciparum lactate dehydrogenase (PFLDH) an enzyme found in the parasite glycolytic pathway that converts pyruvate to lactate and in so doing, provides the energy needed for the survival of the malarial parasite. These methodologies were followed to conduct this study; Recombinant PfLDH was expressed and then purified for further analysis including colony PCR, expression, purification, interaction studies including Fourier transform infrared (FTIR) analysis and Ultraviolet-visible spectroscopy (UV-Vis), antimicrobial activity along with in silico analysis. The following results were obtained: Colony PCR confirmed the presence of a 951bp insert in the PKK223 plasmid. Metal affinity chromatography successfully purified PfLDH protein sized 34.9kDa which was confirmed by ExPasy ProtParam server. The following results were obtained from isolated compounds (BA and IMA) that were screened for IC50 to demonstrate overall activity against the asexual P. falciparum. BA and IMA had IC50 values of 1.27 and 1.03μg/ml against asexual P. falciparum, respectively. When compounds were incubated with protein, FTIR analysis showed a clear shift in the curve, which is indicative of an interaction between IMA and BA with PfLDH. UV-Vis showed that structural conformational change was induced, resulting in an interaction of the compounds with the aromatic side chains of PfLDH. The in silico analysis showed where these interactions occurred, highlighting the ligand atoms responsible for the interaction. Based on these findings, it is possible that these investigated compounds could be effective PfLDH inhibitors as they have binding affinities which are like the standard drug, chloroquine (QA).