Investigation of epigenetic inhibitors as anti-plasmodial drug leads

Abstract

Malaria, caused by Plasmodium parasite species, remains one of the most devastating infectious diseases and is a major public health problem in numerous developing countries. In 2016, an estimated 216 million malaria cases and ~445,000 deaths were reported worldwide with the heaviest burden in sub-Saharan Africa. There is currently no widely effective licensed vaccine for malaria, although recently the RTS,S vaccine has shown partial efficacy in African children (~30%). As this vaccine provides only partial protection against malaria, other measures, in particular antimalarial drugs, will continue to be a vital part of the malaria elimination program. Unfortunately, parasite resistance to currently used malaria drugs is an ongoing problem. Even the gold standard treatment for malaria, artemisinin-based combination therapies (ACTs), is now susceptible to parasite resistance in the Greater Mekong Subregion. Possible failure of ACTs worldwide would be devastating, and is driving the design of new antimalarial drugs with novel modes of action. Although there are many novel anti-malarial candidates in various stages of the drug discovery pipeline, only spiroindolones have reached late phase clinical trials. To address the resistance issue, new antimalarial agents with different modes of action to current drugs are urgently needed. Previous studies have indicated that proteins involved in epigenetic regulatory mechanisms may be potential new drug targets in malaria parasites, as these mechanisms appear to be essential to parasite survival. While some anti-plasmodial inhibitors of epigenetic regulatory proteins have been described, in particular histone deacetylase (HDAC) inhibitors, the potential of other epigenetic inhibitors for malaria has been less well investigated and none have progressed to clinical trial. Thus, the aim of this project was to identify inhibitors that putatively target P. falciparum epigenetic regulatory proteins and to evaluate the in vivo efficacy of selected compounds in a murine malaria model. A total of 125 compounds (55 putative HDAC inhibitors, 7 putative DNA methyltransferase (DNMT) inhibitors, 21 putative arginine methyltransferase (PRMT) inhibitors, and 42 bromodomain-containing protein inhibitors (BDPi)) were investigated in this project. In vitro growth inhibition assays with P. falciparum parasites identified 25 compounds with 50% inhibitory concentration (IC50) <1 μM. Of these, 17 were putative HDAC inhibitors (Chapter 3) while eight were putative methyltransferase inhibitors (Chapter 6). None of the BDPi had an IC50 <1 μM against P. falciparum (Chapter 5). Based on hit-like and lead-like criteria outlined by Medicines for Malaria Venture (MMV), a not-for-profit public-private partnership, two lead-like compounds (putative DNMT inhibitor MC2841 and putative PRMT inhibitor BSF2P (P. falciparum IC50 <0.1 μM; Selectivity Index SI; mammalian cells IC50/PfIC50 >50) and four “hit-like” compounds (putative DNMT inhibitor MC3322, and putative PRMT inhibitors BSH, BSF10, BSF3 (P. falciparum IC50 <1 μM; SI >10) were identified (Chapter 6). Although putative HDAC inhibitor MC2590 had lead-like anti-plasmodial potency (IC50 0.01 μM), the selectivity for Plasmodium (SI 35) did not meet MMV’s lead-like criteria (Chapter 3). While 16 other putative HDAC inhibitors had IC50 <1 μM, they showed low to modest selectivity for Plasmodium and did not meet MMV’s hit-like selectivity criteria (Chapter 3). While additional studies are needed to determine the targets/mode of actions of these inhibitors, preliminary investigations with HDAC inhibitors using Western blot confirmed that the compounds cause hyperacetylation of P. falciparum histone H4 (Chapter 3). This suggests that these compounds act, either directly or indirectly, by inhibiting HDAC activity. Of note, this is the first report on the anti-plasmodial activity of BDPi, with the most potent compound SGC-CBP30 having improved anti-plasmodial activity with prolonged incubation times (PfIC50 48 h 10 μM at 48 h versus 3.16 μM at 72 h) and SI 2-7 (Chapter 5). Despite the low potency of the 42 BDPi examined, these data could serve as a starting point for rational design of more potent and selective BDPi for Plasmodium parasites that can be used to explore the potential of BDPi as new drug leads. In addition, these BDPi could potentially be used as probes to understand the roles of BDPs on the growth and survival of malaria parasites. Previous work has shown that the clinically approved anticancer HDAC inhibitors (vorinostat/suberanilohydroxamic acid (SAHA), panobinostat, romidepsin and belinostat) have potent in vitro activity against P. falciparum parasites (IC50 10-200 nM) and cause hyperacetylation of parasite histone proteins. Here, these findings were extended by examining the in vivo activity of two hydroxamic acid-based drugs, SAHA (Sigma Aldrich, USA) and panobinostat (LBH-589; Selleck Chemicals, USA), in a murine model of malaria. In a clinical setting, SAHA (vorinostat) is approved for cutaneous or peripheral T-cell lymphoma while panobinostat is approved for combination therapy of multiple myeloma. In P. berghei-infected mice with orally administered SAHA or panobinostat (25 mg/kg twice daily for four consecutive days), parasitemia was significantly reduced from day 4-7 and 4-10 post infection, respectively (P<0.05), however, no cures were achieved. SAHA was less effective than panobinostat. This could be due to the higher in vitro potency (PfIC50 10-30 nM versus 120-190 nM) and improved pharmacokinetic profile of panobinostat versus SAHA in humans (Chapter 4). Together with the fact that panobinostat did not cure P. berghei-infected mice, it should be noted that a potential limitation of repurposing panobinostat for malaria is that adverse effects (such as diarrhoea, fatigue, nausea, fever etc.) are reported at the recommended daily dose for cancer patients of 20 mg. However, these data do suggest the possibility of developing panobinostat analogues with improved Plasmodium-specific potency, selectivity and safety profile. AR-42, which is a hydroxamic acid-based HDAC inhibitor currently in phase I clinical trials for cancer, was also tested in a murine model of malaria. Previous unpublished in vitro studies showed that AR-42 has potent activity against P. falciparum parasites (IC50 15-26 nM) and displays good selectivity for Plasmodium parasites versus human cells (SI 66-83). Here, twice-daily oral administration of 25 mg/kg AR-42 was found to cure 10 out of 12 mice infected with P. berghei while a 50 mg/kg single dose cured six of six the infected mice (Chapter 4). In contrast, single daily dosing of 25mg/kg AR-42 significantly attenuated parasite growth in mice from day 4 to 9 post infection (P<0.01), but did not result in cures (Chapter 4). It is speculated that the superior in vivo efficacy of AR-42 in suppressing parasites growth in P. berghei-infected mice compared to SAHA and panobinostat may be due to its longer half-life (11.1 h vs 0.75 h and 2.9 h, respectively), however this would need to be experimentally confirmed. These preliminary data do, however, suggested that AR-42 could potentially repurposed for malaria. However, further pre-clinical investigations would need to be carried out to investigate the potential of this compound as an anti-malarial drug lead. To begin to investigate this, 18 AR-42 analogues were assessed against P. falciparum 3D7 and Dd2 parasites. While seven analogues gave IC50 <1 μM (Chapter 4), the potency was significantly lower than AR-42 and all compounds had low to modest selectivity (inhibited >60% of HEK-293 cells at 10 μM; Chapter 4). The structure–activity relationship (SAR) data obtained may, however, be a useful guide for lead optimization in the future. In summary, the work in this thesis has provided new insights into the potential of different classes of known/putative epigenetic inhibitors as anti-plasmodial drug leads. In vitro studies led to the identification of one putative DNMT inhibitor (MC2841/SGI-1027) and one putative PRMT inhibitor (BSF2P) that have early-phase lead-like profiles (IC50 <0.1μM; SI >50). In addition, four hit-like compounds were also identified, one putative DNMT inhibitor (MC3322) and three putative PRMT inhibitors (BSH, BSF10 and BSF3; IC50 <1μM; SI >10). Although not potent, the first data on the anti-plasmodial activity of BDPi is also presented. Finally, while modest in vivo activity was observed in P. berghei-infected mice dosed orally with SAHA or panobinostat, AR-42 is the first HDAC inhibitor to cure the malaria infected mice when administered orally. This provides a strong starting point for further exploration of this compound.