Antimalarial activity of novel 4-cyano-3-methylisoquinoline inhibitors against Plasmodium falciparum: design, synthesis and biological evaluation.

Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum parasites. Following each cycle of intracellular development and replication, parasites activate a cellular program to egress from their current host cell and invade a new one. The orchestration of this process critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number of central kinases. Parasite kinases are emerging as novel antimalarial targets as they have diverged sufficiently from their mammalian counterparts to allow selectable therapeutic action. Parasite protein kinase A (PfPKA) is highly expressed late in the cell cycle of the parasite blood stage and has been shown to phosphorylate a critical invasion protein, Apical Membrane Antigen 1. This enzyme could therefore be a valuable drug target so we have repurposed a substituted 4-cyano-3-methylisoquinoline that has been shown to inhibit rat PKA with the goal of targeting PfPKA. We synthesised a novel series of compounds and, although many potently inhibit the growth of chloroquine sensitive and resistant strains of P. falciparum, they were found to have minimal activity against PfPKA, indicating that they likely have another target important to parasite cytokinesis and invasion.

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4.

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant 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 the compound 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.