Inhibition of the histone deacetylase family as a drug target in the human malaria parasite, plasmodium falciparum

Abstract

The asexual life cycle of Plasmodium falciparum parasites takes only 48 hours, allowing for rapid replication. The continuous infection, rupturing and re-infection of erythrocytes results in the pathogenicity of this disease. Schizogony (nuclear division) in P. falciparum parasites occurs via alternation between the S and M phases of the cell cycle where DNA synthesis occurs in the mature trophozoite and schizont stages, followed by mitosis to form daughter merozoites. Merozoites then give rise to ring stages after they have infected erythrocytes and the ring stages continue their development to trophozoites. This cyclic development, known as the intraerythrocytic developmental cycle, has a unique transcriptional regulation, which is closely linked to cell cycle regulation. However, the intricacies that these mechanisms are controlled by are still unidentified. One of the means in which the P. falciparum parasite’s complex life cycle is controlled is by means of epigenetics. Epigenetics refers to the heritable changes on a phenotypic level, which are independent of changes on a genetic level. One group of enzymes that participates in the parasite’s epigenetic control is the Plasmodium histone deacetylases. Inhibition of histone deacetylases (HDACs) results in hyperacetylation, which causes aberrant gene transcription and eventually results in parasite death. Comparative analyses of three histone isolation methods and analysis of P. falciparum parasite histones and their post-translational modifications (PTMs) by mass spectrometry techniques identified both epigenetically relevant and novel PTMs in P. falciparum parasite histones and led to the discovery of an adapted histone isolation method for investigation of histone PTM landscapes. When this modified method was used for the investigation of histones that were isolated from P. falciparum parasites treated with HDAC inhibitors compared to untreated parasites, differences were seen in the PTM landscape. Subsequent in silico screening strategies were used to identify ten compounds from the Medicines for Malaria Venture (MMV) Malaria Box, which target the active site of the zinc-requiring PfHDAC1. From these, eight compounds showed inhibition of proliferation of cultured P. falciparum parasites. Ensuing, the adaptation of an HDAC assay to investigate histone deacetylase inhibition was used to validate these compounds as possible PfHDAC1 inhibitors, with at least two of the compounds showing significant inhibition of PfHDAC1 activity, comparable to that of the known HDAC inhibitor, suberoylanilide hydroxamic acid, SAHA. The use of in silico screening of a large library of compounds, such as the MMV Malaria Box, successfully narrows down candidates for possible anti-malarials with drug-like properties by identification of their cellular targets. This work is method-based and facilitates the investigation of the epigenetic landscape of histones, and the identification of novel HDAC inhibitors.