PfHDAC1 is an essential regulator of P. falciparum asexual proliferation and host cell invasion genes with a dynamic genomic occupancy responsive to artemisinin stress.

Plasmodium falciparum, the deadly protozoan parasite responsible for malaria, has a tightly regulated gene expression profile closely linked to its intraerythrocytic development cycle. Epigenetic modifiers of the histone acetylation code have been identified as key regulators of the parasite's transcriptome but require further investigation. In this study, we map the genomic distribution of Plasmodium falciparum histone deacetylase 1 (PfHDAC1) across the erythrocytic asexual development cycle and find it has a dynamic occupancy over a wide array of developmentally relevant genes. Overexpression of PfHDAC1 results in a progressive increment in parasite load over consecutive rounds of the asexual infection cycle and is associated with enhanced gene expression of multiple families of host cell invasion factors (merozoite surface proteins, rhoptry proteins, etc.) and with increased merozoite invasion efficiency. With the use of class-specific inhibitors, we demonstrate that PfHDAC1 activity in parasites is crucial for timely intraerythrocytic development. Interestingly, overexpression of PfHDAC1 results in decreased sensitivity to frontline-drug dihydroartemisinin in parasites. Furthermore, we identify that artemisinin exposure can interfere with PfHDAC1 abundance and chromatin occupancy, resulting in enrichment over genes implicated in response/resistance to artemisinin. Finally, we identify that dihydroartemisinin exposure can interrupt the in vitro catalytic deacetylase activity and post-translational phosphorylation of PfHDAC1, aspects that are crucial for its genomic function. Collectively, our results demonstrate PfHDAC1 to be a regulator of critical functions in asexual parasite development and host invasion, which is responsive to artemisinin exposure stress and deterministic of resistance to it. IMPORTANCE: Malaria is a major public health problem, with the parasite Plasmodium falciparum causing most of the malaria-associated mortality. It is spread by the bite of infected mosquitoes and results in symptoms such as cyclic fever, chills, and headache. However, if left untreated, it can quickly progress to a more severe and life-threatening form. The World Health Organization currently recommends the use of artemisinin combination therapy, and it has worked as a gold standard for many years. Unfortunately, certain countries in southeast Asia and Africa, burdened with a high prevalence of malaria, have reported cases of drug-resistant infections. One of the major problems in controlling malaria is the emergence of artemisinin resistance. Population genomic studies have identified mutations in the Kelch13 gene as a molecular marker for artemisinin resistance. However, several reports thereafter indicated that Kelch13 is not the main mediator but rather hinted at transcriptional deregulation as a major determinant of drug resistance. Earlier, we identified PfGCN5 as a global regulator of stress-responsive genes, which are known to play a central role in artemisinin resistance generation. In this study, we have identified PfHDAC1, a histone deacetylase as a cell cycle regulator, playing an important role in artemisinin resistance generation. Taken together, our study identified key transcriptional regulators that play an important role in artemisinin resistance generation.

Cyclic Tetrapeptide HDAC Inhibitors with Improved Plasmodium falciparum Selectivity and Killing Profile.

Cyclic tetrapeptide histone deacetylase inhibitors represent a promising class of antiplasmodial agents that epigenetically disrupt a wide range of cellular processes in Plasmodium falciparum. Unfortunately, certain limitations, including reversible killing effects and host cell toxicity, prevented these inhibitors from further development and clinical use as antimalarials. In this study, we present a series of cyclic tetrapeptide analogues derived primarily from the fungus Wardomyces dimerus that inhibit P. falciparum with low nanomolar potency and high selectivity. This cyclic tetrapeptide scaffold was diversified further via semisynthesis, leading to the identification of several key structural changes that positively impacted the selectivity, potency, and in vitro killing profiles of these compounds. We confirmed their effectiveness as HDAC inhibitors through the inhibition of PfHDAC1 catalytic activity, in silico modeling, and the hyperacetylation of histone H4. Additional analysis revealed the in vitro inhibition of the most active epoxide-containing analogue was plasmodistatic, exhibiting reversible inhibitory effects upon compound withdrawal after 24 or 48 h. In contrast, one of the new diacetyloxy semisynthetic analogues, CTP-NPDG 19, displayed a rapid and irreversible action against the parasite following compound exposure for 24 h.

In silico identification of inhibitors against Plasmodium falciparum histone deacetylase 1 (PfHDAC-1).

In erythrocytes, actively multiplying Plasmodium falciparum parasites exhibit a unique signature of virulence associated histone modifications, thereby epigenetically regulating the expression of the majority of genes. Histone acetylation is one such modification, effectuated and maintained by the dynamic interplay of two functionally antagonist enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Their inhibition leads to hypo/hyperacetylation and is known to be deleterious for P. falciparum, and hence they have become attractive molecular targets to design novel antimalarials. Many compounds, including four Food and Drug Administration (FDA) approved drugs, have been developed so far to inhibit HDAC activity but are not suitable to treat malaria as they lack selectivity and cause cytotoxicity in mammalian cells. In this study, we used comparative modeling and molecular docking to establish different binding modes of nonselective and selective compounds in the PfHDAC-1 (a class I HDAC protein in P. falciparum) active site and identified the involvement of active site nonidentical residues in binding of selective compounds. Further, we have applied virtual screening with precise selection criteria and molecular dynamics simulation to identify novel potential inhibitors against PfHDAC-1. We report 20 compounds (10 from ChEMBL and 10 from analogues compound library) bearing seven scaffolds having better affinity toward PfHDAC-1. Sixteen of these compounds are known antimalarials with 14 having activity in the nanomolar range against various drug resistant and sensitive strains of P. falciparum. The cytotoxicity of these compounds against various human cell lines are reported at relatively higher concentration and hence can be used as potential PfHDAC-1 inhibitors in P. falciparum. These findings indeed show great potential for using the above molecules as prospective antimalarials.

Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs.

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50 < 500 nM) and good selectivity over the activity of human HDAC in cells (up to >50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.