Epigenetic inhibitors target multiple stages of Plasmodium falciparum parasites

Abstract

The epigenome of the malaria parasite, Plasmodium falciparum, is associated with regulation of various essential processes in the parasite including control of proliferation during asexual development as well as control of sexual differentiation. The unusual nature of the epigenome has prompted investigations into the potential to target epigenetic modulators with novel chemotypes. Here, we explored the diversity within a library of 95 compounds, active against various epigenetic modifiers in cancerous cells, for activity against multiple stages of P. falciparum development. We show that P. falciparum is differentially susceptible to epigenetic perturbation during both asexual and sexual development, with early stage gametocytes particularly sensitive to epi-drugs targeting both histone and nonhistone epigenetic modifiers. Moreover, 5 compounds targeting histone acetylation and methylation show potent multistage activity against asexual parasites, early and late stage gametocytes, with transmission-blocking potential. Overall, these results warrant further examination of the potential antimalarial properties of these hit compounds. The almost inevitable development of drug resistance in malaria parasites enforces continued discovery of novel classes of antimalarial compounds 1. To contribute to global malaria elimination strategies, such compounds would need to target multiple life cycle stages of the parasite 2. This includes targeting the rapidly dividing (~48 h) asexual parasites to reduce parasite burden, as well as targeting mature, terminally differentiated sexual gametocytes to block onward human-to-mosquito transmission of the parasite, or exo-erythrocytic liver stage development to block mosquito-to-human transmission. Importantly, to prolong or prevent resistance development, new chemical matter should target novel biological activities in the parasite 3. In oncology research, epigenetic therapeutics ('epi-drugs') evidently hold great promise as targets for anticancer therapies 4 , with several drugs approved for clinical use, including Azacitidine, Decitabine, Vorinostat and Romidepsin 5. The antitumor activity is ascribed to epigenetic deregulation as a result of inhibition of epigenetic modulators, including histone modifying enzymes and DNA methyltransferases. This results in particular changes in histone post-translational modifications (PTMs), disruption of transcriptional processes, chromatin structure maintenance and DNA repair 6,7. Plasmodium falciparum relies heavily on epigenetic mechanisms to drive both asexual proliferation and sexual differentiation (reviewed in 8-11). The parasite's genome encodes a unique complement of histone modifying enzymes including histone deacetylases (HDACs), histone acetyltransferases (HATs), histone methyltransferases (HMTs, including lysine HKMT), protein arginine methyltransferases (PRMTs), and histone demethylases (HDMs) 12 in addition to other non-histone epigenetic modifiers. As a result, inhibitors of histone modifying enzymes have been investigated as no...

H3K36 Methylation Reprograms Gene Expression to Drive Early Gametocyte Development in Plasmodium Falciparum

“…Here, we con rmed the dynamic yet stage-speci c nature of H3K36me2&3 (11) and delineate their roles in the transcriptional reprogramming occurring post-commitment in the gametocyte sexual stage of P. falciparum blood-stage development (34). Our chemical interrogation of P. falciparum HDMs supports these conclusions, highlights the importance of the dynamics of histone methylation for transcriptional control and links aberrant H3K36me2&3 patterns with the increased potency of Jumonji HDM inhibitors towards gametocytes reported before (38,65,66).…”

“…To investigate the functional relevance of these enzymes for H3K36me2&3, we chemically interrogated HDM activity and determined the effects of this inhibition on H3K36 methylation levels on days associated with stage II gametocyte development. Jumonji HDM inhibitors with activity against gametocytes (38,65,66). JIB-04 (pan-selective) and ML324 (targeting KDM4, PfJMJ3) resulted in the hypermethylation of both H3K36me2&3 relative to the untreated controls (36-to 89-fold, Fig.…”

A novel multistage antiplasmodial inhibitor targeting Plasmodium falciparum histone deacetylase 1

“…There are five putative HDACs in P. falciparum; PfHDAC1 (PF3D7_0925700) is the only class I HDAC representative in P. falciparum, while PfHDA1 (PF3D7_1472200) and PfHDA2 (PF3D7_1008000) belong to class II HDACs, and PfSir2a (PF3D7_1328800) and PfSir2b (PF3D7_1451400) are phylogenetically close to class III HDACs 47 . Several HDAC inhibitors with in vitro and in vivo antiplasmodial activity have been reported previously [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]37,38,[57][58][59][60][61] . For instance, WR301801 was discovered via chemical modification based on the structure of SAHA and can inhibit the growth of plasmodial at a low-nanomolar range in vitro, and the selectivity index is about 1000.…”

“…Histone modifications, such as histone lysine acetylation, deacetylation, and methylation, are associated with the regulation of key processes in the life cycle of malaria parasites [15][16][17][18] . Previous research reported that three out of five P. falciparum HDAC (PfHDAC) genes in the parasite genome are essential for the blood stage of the parasite 18,19 , and numerous studies have identified potent antiplasmodial HDAC inhibitors [20][21][22][23][24][25][26][27][28][29][30] , including dual-/ multistage-targeting compounds [31][32][33][34][35] , suggesting the possibility of targeting PfHDACs in drug development. In addition, indirect effects on histone acetylation, deacetylation activity, and transcriptomic changes have been demonstrated 36,37 .…”

An ELISA method to assess HDAC inhibitor-induced alterations to P. falciparum histone lysine acetylation

“…( Andrews et al, 2009 ; Andrews et al, 2012b ; Andrews et al, 2012c ; Fioravanti et al, 2020 )). HDAC inhibitors with a hydroxamic acid zinc binding group have generally demonstrated the highest potency against P. falciparum in vitro, with varying levels of selectivity for the parasite versus human cells ( Andrews et al, 2009 ; Andrews et al, 2012c ; Giannini et al, 2015 ; Coetzee et al, 2020 ; Fioravanti et al, 2020 ). However, the lack of recombinant P. falciparum HDAC enzymes ( Pf HDAC1 is commercially available but has low purity ( Ontoria et al, 2016 )) and crystal structures (none available) remains a major limitation to the rational design of new compounds with improved potency and parasite-specific selectivity.…”