Designing novel inhibitors against histone acetyltransferase (HAT: GCN5) of Plasmodium falciparum

Abstract

During active proliferation phase of intra-erythrocytic cycle, the genome of P. falciparum is regulated epigenetically and evolutionary conserved parasite-specific histone proteins are extensively acetylated. The reversible process of lysine acetylation, causing transcriptional activation and its deacetylation, causing transcriptional repression is regulated by balanced activities of HATs and HDACs. They are also known to regulate antigenic variations and gametocytic conversion in P. falciparum. These histone modifying enzymes have been identified as potential targets for development of anitmalarials in literature. PfGCN5, a HAT family member of P. falciparum is predominantly involved in H3K9 acetylation. In this study, through comparative structure and sequence analysis, we elucidate differences in the catalytic pocket of PfGCN5 which can be exploited to design selective inhibitors. Through virtual screening of known antimalarials from ChEMBL bioassay database, we mapped 10 compounds with better affinity towards PfGCN5. Further, we identified 10 more novel compounds which showed remarkably better affinity towards the Plasmodium target from analogues of mapped inhibitors from ZINC database of commercially available compounds. Comparative molecular dynamics simulation study of one of the compounds (C14) complex with PfGCN5 and HsGCN5 suggested the possible reason for its selectivity. In vitro parasite growth assay in the presence of C14 showed IC50 value at lower nanomolar range (∼ 225 nM). However, no effect in mammalian fibroblast cells was observed for C14 (up to 20 μM). Further, reduced level of HAT activity of recombinant GCN5 and H3K9Ac was observed in the parasites treated with C14. Overall, this study reports 20 potential inhibitors of PfGCN5 and experimental validation of one molecule (C14) with antimalarial activity at low nanomolar range.

Introduction

Despite substantial decrease observed in global burden, malaria is still responsible for 438 thousand deaths and 214 million infections globally [1]. The emergence of resistance against artemisinin derivatives, the front-line antimalarials has further accentuated the need to develop new antimalarials with novel modes of action [2]. Actively proliferating Plasmodium parasites exhibit frequent transition between morphological states and antigenic variation to survive, leading to infection and evasion of host immune responses [3]. These antigenic variations promote pathogenesis through epigenetically controlled transcriptional variabililty [4]. One such example of epigenetically controlled antigenic variation is expression of hypervariable var gene family which encodes approximately 60 variants of P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, the main variant surface antigen expressed on infected erythrocytes prevents its clearance from circulatory system. At a time, only one or few var genes are expressed and these alterations in expression pattern help parasite to cause chronic infection [3], [5]. Epigenetic control of gene regulation is mainly accomplished by chromatin remodeling and post-translational modifications (PTMs) [6], [7]. PTMs affect protein properties such as localization, binding, folding, enzymatic activity or stability, and hence are essential for cellular functions and cell survival. Reported PTMs in malaria parasite include ubiquitination, sumoylation, nitrosylation, glutathionylation, phosphorylation, acetylation, methylation and lipidation [7]. PTMs of histones can alter chromatin structure by modulating the interactions of proteins with DNA and subsequently recruit effector proteins [3]. The factors affecting Histone modifications are also suggested to have links between mode of action of artemisinin and its resistance [8]. Recently, across Intraerythrocytic development cycle (IDC) 232 distinct histone modifications have been reported in Plasmodium which has unique signature that correlates with parasitic virulence [9]. Out of 12 epigenomic map of histone modifications viz H4K5Ac, H4K8Ac, H4K12Ac, H4K16Ac, H3K9Ac, H3K14Ac, H3K56Ac, H4K20me1, H4K20me3, H3K4me3, H3K79me3 and H4R3me2 generated using ChIP-on-chip, eight modifications are suggested to be associated with the transcription levels of up to 76% of P. falciparum genes [10]. In P. falciparum, majority of histone modification marks include lysine acetylation and methylation and are linked to active and repressed state of genes [3].

Dynamic level of histone acetylation is regulated by balanced activity of histone acetyltransferases (HATs) and Histone deacetylases (HDACs) [11]. Evolutionarily conserved from yeast to humans, the function of catalytic subunit of HATs depends on the interacting partners of HAT complexes [12]. Categorization based on their catalytic domain has grouped HATs into five major classes: GCN5 N-acetyltransferases (GNATs); CREB binding protein (p300/CBP HATs); MYSTs (MOZ, Ybf2/Sas3, Sas2 and TIP60 related HATs); general transcription factor HATs and the nuclear hormone-related HATs [13]. HATs have been shown to have potential role in pathology of cancer, asthma, Chronic Obstructive Pulmonary disorder (COPD), viral infections and neurological disorders through acetylation of both histone and non-histone proteins [14], [15]. Importance of histone modifications and its correlation with parasitic virulence suggests that enzymes involved in incorporating these modifications could be targeted to develop new antimalarials.

Plasmodium falciparum GCN5 N acetyltransferase 5 (PfGCN5), a well characterized member of GNAT family has most homologous region within HAT domain and bromodomain in Plasmodium species [16]. GCN5 catalyzes the transfer of acetyl group from acetyl-CoA to ε-amino group of lysine residue of histone by formation of a ternary complex of histones, acetyl-CoA and enzyme [17]. Both native and recombinant forms of PfGCN5 HAT have substrate specificity for H3 and preferentially acetylates at K9 and K14 [18], [16]. In vivo experiments have shown that it forms complex with PfADA2 implicating its active role in chromatin remodeling and regulation of transcription in P. falciparum [19]. Reversible and noncompetitive inhibition of PfGCN5 by Anacardic acid treatment induced hypoacetylation at H3K9 and H3K14, causing down-regulation of 207 genes in late trophozoite state [20]. Both curcumin and anacardic acid inhibited the growth of chloroquine resistant and susceptible strains of P. falciparum either by generating reactive oxygen species or by down-regulating the HAT activity of PfGCN5 which introduced disturbances in the regulation of transcription in the parasite [20], [21]. Embelin, a HAT inhibitor showed down-regulation of var gene expression with hypoacetylation at H3K9 around var gene promoters suggesting interplay among histone acetylation status, as well as subnuclear compartmentalization of different genes and their activation in P. falciparum [22]. Competitive methylation and acetylation marks at H3K9 in var 5′ flanking region is reported to epigenetically regulate mono-allelic expression pattern of var genes during parasite proliferation through activation or repression [23]. Several other HAT inhibitors have been developed but they suffer from undesired properties like anti-oxidant activity, instability, low potency or lack selectivity between HAT subtypes and other enzymes [15]. Many HAT inhibitors have been identified by applying structure based drug designing approach and virtual screening [15]. In Virtual screening, a virtual chemical database is screened against the known/predicted potential binding site of the crystal structure or homology model of target protein. Using this methodology compound C646 has been identified which is currently the most potent HAT inhibitor [15], [24]. PfGCN5 bromodomain has been crystallized (PDB ID 4QNS) but the structure of its HAT domain is not known.

In this study, through homology modeling, we generated the structure of PfGCN5 HAT domain and performed comparative sequence and structural analysis to find the differences in its active site which can be exploited to identify novel inhibitors. 20 inhibitors were identified against PfGCN5 through molecular docking. Further, MD simulation was performed to check the stability of modeled structure and calculate the binding free energy of C14 against PfGCN5 HAT domain. The potential of C14 was evaluated through in vitro experiments and was found to be effective against Plasmodium growth and H3K9 acetylation.