Mechanisms of inhibition of Plasmodium falciparum growth by a novel 4-cyano-3-methylisoquinoline compound

We attempted to develop compounds that target Plasmodium falciparum protein kinase A (PfPKA), an essential parasite kinase important for replication of the malaria-causing asexual blood stage. We developed a novel series of compounds based on a 4-cyano-3-methylisoquinoline compound known to inhibit rat PKA. Our lead compounds achieved modest parasite growth inhibition; however, further investigation revealed that these compounds did not inhibit PfPKA at the concentrations required to inhibit parasite growth. To identify the biological target we selected for parasites resistant to our lead compound MB14. Genomes sequencing revealed all parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with their targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.