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

Abstract

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.

Spatial organization of protein export in malaria parasite blood stages

“…Here, we have utilized a trappable nanoluciferase (Nluc) reporter construct containing the murine dihydrofolate reductase (mDHFR) enzyme which is refractory to unfolding in the presence of WR99210 (WR) to further probe PTEX function . Nluc reporter proteins are highly quantifiable and have been used previously to examine the export of PEXEL proteins, resistance to sorbitol lysis, merozoite egress and invasion and other aspects of the parasite's life cycle . Here, we generated multiple exported constructs containing both Nluc and mDHFR, but with different additional epitope and affinity tags and followed their fates.…”

“…20 Nluc reporter proteins are highly quantifiable and have been used previously to examine the export of PEXEL proteins, resistance to sorbitol lysis, merozoite egress and invasion and other aspects of the parasite's life cycle. [21][22][23][24][25][26] Here, we generated multiple exported constructs containing both Nluc and mDHFR, but with different additional epitope and affinity tags and followed their fates. We show that different constructs do indeed behave differently upon trapping at the PVM with some constructs partially resuming export after WR removal, while others remain completely blocked.…”

Disrupting the Allosteric Interaction between the Plasmodium falciparum cAMP-dependent Kinase and Its Regulatory Subunit

“…1 (AMA1), which is a highly validated target of PfPKA (8,23,24), was used as our kinase substrate. The AMA1-tail is phosphorylated by PfPKA at amino acid Ser-610, and this event is required for merozoite invasion of erythrocytes by P. falciparum (8).…”

“…Following the reaction, phosphorylation of Ser-610 was detected with a highly specific antibody raised to a synthetic Ser(P)-610 phosphopeptide ( Fig. 2A) (23). In the absence of supplementary apo-Pf-PKA-R or cAMP, phosphorylation of AMA1 was quite low, indicating minimal levels of PfPKA-C activity in the parasite lysates (Fig.…”

“…H89 dihydrochloride hydrate was from Sigma. After culturing, lactate dehydrogenase activity was measured using a modified form of the Malstat assay (23,37).…”