Structure activity refinement of phenylsulfonyl piperazines as antimalarials that block erythrocytic invasion

https://doi.org/10.1016/j.ejmech.2021.113253Get rights and content

Highlights

  • Key structural motifs necessary for asexual parasite potency were identified.

  • Analogues exhibit potent P. knowlesi and P. falciparum multi-drug resistant potency.

  • Phenylsulfonyl piperazine class has a unique erythrocyte invasion phenotype.

  • Optimized analogues are useful tools for studying Plasmodium erythrocyte invasion.

Abstract

The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.

Introduction

Malaria remains a substantial global health burden causing significant morbidity and mortality each year [1]. The World Health Organization (WHO) estimates that in 2018, 228 million cases of malaria occurred causing 405,000 deaths, with sub-Saharan Africa bearing the major proportion of the global burden [2]. Malaria is a disease caused by protozoan parasites belonging to the genus Plasmodium that are transmitted by female Anopheles mosquitoes [3]. Of the five species of Plasmodium that infect humans, P. falciparum is the deadliest, accounting for 99.7% of malaria cases in sub-Saharan Africa [2].

Artemisinin-based combination therapies (ACT) have significantly reduced malaria burden and deaths worldwide. However, P. falciparum resistance to artemisinins and their partner drugs have emerged in regions of southeastern Asia [[4], [5], [6]] and resistance mutations have recently been observed in parasites in Rwanda [7]. Beginning with the isolation of quinine in the 1820’s, an array of antimalarial drugs have been developed, however they target only a small handful of biochemical pathways within the parasite [8]. Gratifyingly, there has been a concerted effort in recent years by many organisations to develop novel scaffolds with unique mechanisms of action [9]. Despite this endeavor and coupled with the spread of resistance to ACT, there is an ongoing need for therapies that have novel and complementary mechanisms of action that ideally have efficacy against different stages of the parasite life cycle [8,10].

Malaria in humans begins with the bite of an infected Anopheles mosquito that transfers sporozoite forms of Plasmodium parasites to the bloodstream. The sporozoites then migrate to the liver and infect hepatocytes. Within hepatocytes, parasites grow and divide over 7–10 days into merozoites which are then released into the bloodstream [11]. These merozoites then immediately invade red blood cells (RBCs) and grow and replicate for 48 h to form a schizont stage parasite containing about 20 merozoites. The merozoites then egress from their schizonts and host blood cells and invade new RBCs in a rapid process that takes just a few minutes [12]. The egress and invasion of merozoites are dependent on a number of cell signaling events, proteolytic cascades, receptor-ligand interactions and an actomyosin invasion motor. As these events occur in the bloodstream, they are optimally exposed to inhibition with small molecule drugs [13,14]. Drugs which could strongly reduce invasion efficiency would result in a dramatic reduction in parasite amplification and disease progression. Furthermore, delaying merozoite egress and RBC invasion would expose the merozoites to enhanced destruction by innate and humoral immune responses [15,16]. Drugs known to target these processes include kinase, subtilisin-like protease 1, plasmepsin 9 and actin inhibitors, and is comprehensively reviewed by Burns et al. [13].

To detect compounds that interfere with either erythrocytic egress or invasion, Dans et al. recently reported the development of an assay employing transgenic P. falciparum parasites which express the bioluminescent nanoluciferase (Nluc) reporter [14,17]. This assay platform was utilized to screen the Medicines for Malaria Venture (MMV) Pathogen Box, a collection of 400 compounds with activity against a variety of neglected tropical disease pathogens including 125 compounds with antimalarial activities. The screen of the Pathogen Box identified a small set of compounds that inhibited either erythrocyte invasion or egress and were validated by further synchronized parasite phenotype assays and live cell microscopy. One of the invasion-specific hit compounds discovered was MMV020291 (1) (Fig. 1). Microscopy studies demonstrated hit compound 1 blocked merozoite RBC internalization but had minimal effects on other stages of the asexual lifecycle. MMV020291 has a unique phenylsulfonyl piperazine scaffold, that is structurally divergent from other previously described phenylsulfonyl piperazine antimalarial series [18,19], thus providing a promising starting point for the development of an invasion-specific antimalarial.

In this study we define the structure−activity relationship (SAR) and optimize the antimalarial activity of the phenylsulfonyl piperazine class (1). During this process the antimalarial activity of analogues was iteratively determined utilizing the previously reported lactate dehydrogenase (LDH) asexual P. falciparum 3D7 assay [20]. Human HepG2 cell cytotoxicity and physicochemical parameters were monitored in parallel [21]. Analogues with suitable physicochemical properties and asexual stage activity were further evaluated against P. falciparum multi-drug resistant lines, P. knowlesi parasites and NF54 gametocytes to ultimately determine their target candidate profile (TCP) suitability [22].

Section snippets

Results and discussion

The initial focus for optimizing the hit compound 1 was to establish the structure-activity relationship (SAR) and improve antimalarial activity based upon the LDH viability-based assay. This assay involves culturing asexual P. falciparum 3D7 parasites in the presence of compound for 72 h. Therefore, this assay allows the completion of one full 48 h asexual cycle and will reveal compounds that reduce parasite viability by blocking erythrocytic invasion. Subsequently selected compounds were then

Conclusions

Merozoite invasion of the RBC is an essential step in the asexual development of Plasmodium parasites. We previously exploited a novel phenotypic assay capable of detecting RBC invasion to screen the MMV Pathogen Box and identified that MMV020291 (compound 1) blocks invasion [14]. In this study, we explored the structure activity relationship of compound 1 and determined several key structural motifs were necessary for P. falciparum asexual parasite activity. Most notably, the t-butyl group,

Experimental section

P. falciparum Asexual Stage Parasite Assay. Parasite viability assays were performed as previously described [48,49].

HepG2 Cell Growth Inhibition Assay. The HepG2 cellular assay was undertaken according to previously described protocol [21].

P. falciparum Multidrug Resistant and P. knowlesi Asexual Stage Parasite Viability Assays. Mefloquine resistant W2mef, chloroquine resistant DD2 and 7G8, artemisinin resistant Cam3.I2539T Cambodian isolate [50], and P. knowlesi YH1, were cultured according

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was funded by the National Health and Medical Research Council (NHMRC) of Australia (Development Grant 1135421 to B.E.S., P.R.G. and V.M.A.; Project Grant 1143974 to D.W.W. and B.E.S.), the Australian Cancer Research Foundation, Australian Government Research Training Program Scholarship (to M.G.D.), the Victorian State Government OIS and Australian Government NHMRC IRIISS. B.E.S. is a Corin Centenary Fellow and D.W.W. is a Hospital Research Foundation Fellow. We thank and

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