Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

Abstract

To identify potential inhibitors of egress and invasion in the asexual blood stage of Plasmodium falciparum, we screened the Medicines for Malaria Venture (MMV) Pathogen Box. This compound library comprises of 400 drugs against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, Nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has greater implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.ImportancePlasmodium falciparum causes the most severe form of malaria and with emerging resistance to frontline treatments, there is the need to identify new drug targets in the parasite. One of the most critical processes during the asexual blood stage in the parasite’s lifecycle is the egress from old red blood cells (RBCs) and subsequent invasion of new RBCs. Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. Identifying novel compounds that inhibit these essential processes would further their development into possible antimalarials that would be highly effective at killing asexual RBC stage parasites when used in combination with drugs that target the intraerythrocytic growth phase. These compounds potentially may also be used as novel tools to study the complex biology of parasites to gain further insight into the mechanisms behind egress and invasion.

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

“…In vitro high-throughput screening efforts, such as those conducted with the Medicine for Malaria Venture's Pathogen Box, serve as a starting point for the identification and development of new antimalarial drugs through characterization of anti-Plasmodium compound attributes [37,38]. As with any in vitro system, the translatability to in vivo efficacy presents a critical hurdle.…”

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor

“…However, to seek less peptidic small molecules with similar activity, we searched the literature for descriptions of compounds displaying similar egress‐inhibitory characteristics. This alerted us to a recent targeted screen of the Medicines for Malaria Venture (MMV) Pathogen Box compound set which singled out the purine nitrile MMV676881 (Fig 6A) as an egress inhibitor that blocked RBCM rupture, but not PVM rupture or RBCM poration (Dans et al, 2020). This phenotype, which we confirmed here (Fig 6B), is remarkably similar to that of ΔSERA6 and ΔMSA180 parasites, suggesting that MMV676881 might act through inhibition of SERA6 maturation.…”

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria parasites

“…Previous studies have investigated egress and invasion as a promising target for antimalarial drug discovery, suggesting that HAD5 may likewise be of interest for antimalarial drug discovery(46, 48–51); however, phosphomannomutases are found widely throughout nature, including two genes in the human genome, Hs PMM1 and Hs PMM2(70, 71). We therefore evaluated the potential for selective inhibition of P. falciparum HAD5 over human PMM1 and PMM2 (Fig.…”

“…GPI anchors, which contain mannose (16,18,31), are required for egress of the malaria parasite from the infected host cell, as well as reinvasion of merozoites (46,(48)(49)(50)(51)(52)(53)(54)(55). Because of its essential role in mannose metabolism, we hypothesized that HAD5 may be required for efficient egress and invasion.…”