Activity of a Trisubstituted Pyrrole in Inhibiting Sporozoite Invasion and Blocking Malaria Infection

Abstract

Malaria infection is initiated by Plasmodium sporozoites infecting the liver. Preventing sporozoite infection would block the obligatory first step of the infection and perhaps reduce disease severity. In addition, such an approach would decrease Plasmodium vivax hypnozoite formation and therefore disease relapses. Here we describe the activity of a trisubstituted pyrrole, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine, in inhibiting motility, invasion, and consequently infection by P. berghei sporozoites. In tissue culture, the compound was effective within the first 3 h of sporozoite addition to HepG2 cells. In vivo, intraperitoneal administration of the compound significantly inhibited liver-stage parasitemia in P. yoelii sporozoite-infected mice and prevented the appearance of blood-stage parasites. P. berghei sporozoites lacking the parasite cGMP-dependent protein kinase, the primary target of the compound in erythrocyte-stage parasites, remained infectious to HepG2 cells and sensitive to the drug. These results suggest that the drug has an additional target(s) in sporozoites. We propose that drugs that inhibit sporozoite infection offer a feasible approach to malaria prophylaxis.The malaria parasite, Plasmodium spp., infects 500 million people a year, killing over 1 million globally. Plasmodium parasites are introduced into humans via mosquito bites, in the form of sporozoites that infect the liver to form liver-stage parasites (LS). LS are eventually released into the bloodstream, where they infect erythrocytes and initiate the symptomatic phase of malaria. Although the liver stage of infection by Plasmodium falciparum and P. vivax, the two major human parasites, is asymptomatic, P. vivax infections often result in the formation of dormant LS called "hypnozoites." When reactivated, hypnozoites cause disease relapses up to 1 year after the initial infection. Therefore, achieving global eradication of malaria will require prevention or treatment of P. vivax hypnozoites in addition to treatment of erythrocyte stages of both species.The spread of drug-resistant parasites, limited vector control measures, and lack of an effective vaccine make novel drug discovery vital. Drugs that prevent sporozoite infection of the liver would block an obligatory step in the parasite's life cycle. They would also prevent the formation of hypnozoites by P. vivax and the pathology caused by the erythrocyte stages of all human Plasmodium species. Although the liver stages are asymptomatic, reducing the parasite burden in the liver is likely to significantly diminish blood-stage infection and reduce disease severity in a subpopulation of patients (1). Furthermore, the small number of parasites and the limited number of replications during the liver phase of malaria infection compared to those in the erythrocytic phase reduces the potential for the emergence of drug-resistant parasites (13). However, of the currently licensed drugs, only primaquine has been formally demonstrated to act against sporozoit...

Chemical probes of a trisubstituted pyrrole to identify its protein target(s) in Plasmodium sporozoites

“…Sporozoites that lack the PKG gene retain their ability to infect hepatocytes, 5 and infection of hepatocytes by these sporozoites is still inhibited by TSP. 3 These data demonstrate that sporozoites express an additional unidentified protein target(s) of the TSP that is essential for sporozoite invasion of the liver. These proteins represent potentially new targets for the development of novel therapeutics to prevent malarial infection.…”

“…1) has activity against both erythrocytic 2 and sporozoite 3 stages of the parasite, in vitro and in vivo. TSP’s inhibition of the parasite’s erythrocytic stages results primarily from inhibition of a cGMP-dependent protein kinase (PKG) in the parasite.…”

“…2). 3 Briefly, P. berghei sporozoites were added to the human hepatoma cell line, HepG2, in the presence of different compounds. Sporozoite infectivity was determined by quantifying the number of intracellular liver stages that developed 40 h post-infection.…”

4(1H)-Quinolones with Liver Stage Activity against Plasmodium berghei

“…These procedures require that animals be sacrificed for tissue collection or imaging and do not allow for real-time monitoring of parasite development following drug treatment. The indirect and most common method of studying liver stage activity requires assessment of the prepatent period (the time elapsed until parasites appear in the peripheral blood following sporozoite infection); however, this technique does not distinguish between liver and blood stage activity of compounds (23,24).…”

“…These procedures require that animals be sacrificed for tissue collection or imaging and do not allow for real-time monitoring of parasite development following drug treatment. The indirect and most common method of studying liver stage activity requires assessment of the prepatent period (the time elapsed until parasites appear in the peripheral blood following sporozoite infection); however, this technique does not distinguish between liver and blood stage activity of compounds (23,24).Recent studies have shown that bioluminescent imaging (BLI), a noninvasive technique that works by capturing the light emitted from the reaction of luciferase and its substrate, can be used to study the development of malaria parasites, as well as the effects of commercially available antimalarials in vivo (25)(26)(27). This technique is beneficial because it allows for real-time monitoring of a single individual over time.…”