Plasmepsins on the antimalarial hit list

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Science  27 Oct 2017:
Vol. 358, Issue 6362, pp. 445-446
DOI: 10.1126/science.aaq0002

The spread of multidrug-resistant “super malaria” throughout Southeast Asia (1, 2) offers another stern warning that new antimalarial drugs are needed urgently. Our present-day arsenal of antimalarials have poorly understood mechanisms of action, and no single drug targets all of the life-cycle stages of the malaria-causing parasite. Killing all life-cycle stages would stop parasite transmission between people and potentially prevent relapsing forms of malaria. On pages 522 and 518 of this issue, Pino et al. (3) and Nasamu et al. (4), respectively, identify different aspartyl protease inhibitor scaffolds as potent antimalarials that target multiple stages of the parasite life cycle. Importantly, they also identify the targets of these drugs and their essential functions in the malaria life cycle, exposing the underlying mechanisms of action. This provides two new enzymes to target with a combined therapy that could treat malaria.

Malaria is caused when a parasite-infected mosquito takes a blood meal and delivers sporozoites from its salivary glands into the human host. Sporozoites are transported through the blood to the liver, where they infect hepatocytes (liver cells) and develop into merozoites that egress from hepatocytes and invade erythrocytes (red blood cells) (see the figure). Blood stage infection involves repeated cycles of merozoite invasion, replication, and egress, causing the clinical symptoms of malaria. Egress and reinvasion are thus vital for the parasite to survive. A proteolytic cascade enables merozoite egress from the erythrocyte, initiated by exocytosis (secretion) of the serine protease subtilisin 1 (SUB1) (5). SUB1 is matured by two cleavage events, the first being autocatalytic and the second through an unknown mechanism. Mature SUB1 cleaves serine-rich antigen 5 (SERA5; a negative regulator of merozoite egress) (6) and additional effectors, including merozoite surface protein 1 (MSP1), which help break open the erythrocyte (7, 8), and the parasite then invades a new cell. SUB1 is thus a prime drug target to prevent merozoite egress (5, 9). However, Pino et al. and Nasamu et al. show that treatment of merozoite-infected erythrocytes with aspartyl protease inhibitors blocks the maturation of SUB1, SERA5, and MSP1, preventing egress (3, 4). This implies that an aspartyl protease functions upstream of SUB1.

Plasmepsins IX and X are essential across the malaria life cycle

The malaria life cycle involves infection of the liver (asymptomatic), the blood (causes malaria), and the mosquito (spreads parasites between humans).


The malaria parasite uses more than 100 proteases throughout its life cycle, 10 of which—the plasmepsins—belong to the aspartyl protease family. Conditional knockdown of plasmepsin X (PMX) expression in merozoites phenocopies the blockade observed with the aspartyl protease inhibitors; SUB1, SERA5, and MSP1 are not matured, and merozoite egress is prevented (4). Additionally, recombinant PMX was shown to cleave SUB1 in an aspartyl protease inhibitor–dependent manner (3, 4). Altogether, this demonstrates that PMX regulates egress by licensing SUB1 to cleave SERA5, MSP1, and other proteins, and this can be potently inhibited by using aspartyl protease inhibitors, preventing egress. This prevents parasites from continuing their life cycle and obtaining nutrients, thus killing them.

These studies further demonstrate that in addition to preventing egress, aspartyl protease inhibitors also block merozoite invasion of erythrocytes. Merozoite invasion involves the production of a tight junction at the parasite–host cell interface, which acts as a traction point for the parasite to move into the cell. The tight junction is formed and maintained by interactions between apical membrane antigen 1 (AMA1) on the parasite surface and rhoptry neck protein 2 (RON2) on the erythrocyte membrane (10, 11). Pino et. al. show that aspartyl protease inhibitors prevent AMA1 processing in merozoites and block recombinant PMX from cleaving AMA1 (3). Conditional knockdown of PMX expression in merozoites also reduces erythrocyte invasion in vitro and causes parasites to become hypersensitive to aspartyl protease inhibitors, confirming the target (4). Therefore, PMX is important both for egress and invasion. Interestingly, conditional knockdown of another member of the aspartyl protease family, plasmepsin IX (PMIX), does not affect egress but potently blocks invasion (3, 4). PMIX cleaves rhoptry-associated protein 1 (RAP1) and apical sushi protein (ASP), which are associated with merozoite invasion (3, 4). Collectively, this reveals that PMIX and PMX have essential enzymatic functions during merozoite infection.

Pino et al. also show that their aspartyl protease inhibitor is lethal across the malarial life cycle. It inhibits SUB1-dependent egress by gametocytes, the sexual parasite forms transmitted to mosquitos, and prevents processing of the cell traversal protein for ookinetes and sporozoites (CelTOS) within the ookinete, the parasite form that infects mosquitos (3). The inhibitor also interferes with egress from hepatocytes during the liver stage, leading to reduced blood stage infections in vivo (3). SUB1 is required for this step as well (12, 13). Therefore, notwithstanding the important roles of other plasmepsins in these life-cycle stages, PMX is the most probable target, and its inhibition could provide a way to treat malaria but also eliminate liver- and mosquito-stage parasites, reducing the incidence of disease.

The discovery of PMIX and PMX as essential enzymes for invasion and egress could lead to further exciting discoveries in the malaria field. For example, what are all of the substrates at each life-cycle stage, and how do they function? Do sporozoites require these proteins to invade mosquito salivary glands or human hepatocytes? Could other substrates be new drug targets? The identification of PMIX and PMX as druggable enzymes has the potential to mean even more for malaria patients of the future. Aspartyl protease inhibitors have been optimized to treat several human conditions, including hypertension and HIV infections, so that the expertise and large compound libraries available should accelerate antimalarial lead identification. But, careful consideration must be given to understanding potential mechanisms of resistance. For example, mutations in HIV-1 aspartyl protease gave rise to resistant virus strains. Furthermore, amplification of the PMII and PMIII genes was recently identified as a new surrogate marker of piperaquine-resistant super malaria (14, 15). It will be imperative to understand whether malaria parasites adapt to aspartyl protease inhibitors by amplifying or mutating their PMIX or PMX genes and whether targeting multiple plasmepsins can help overcome this. The discovery of PMIX and PMX as master regulators of egress and invasion opens the door to new biology and drug discovery in the ongoing fight against malaria.


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