Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity

Abstract

Summary The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

A multipass membrane protein interacts with the cGMP-dependent protein kinase to regulate critical calcium signals in malaria parasites

“…was detected in a very recent elegant study profiling a potent small compound inhibitor of PKG. However, conditional knock down of ICM1 in that work resulted only in a minor growth defect, perhaps due to the different conditional system used (Vanaerschot et al, 2020).…”

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites

“…One benefit of using Assay 1 for the initial liver stage screen is highlighted by data with the PKG inhibitor MMV030084 that blocks parasite invasion of host cells. This compound yielded an IC 50 of 199 nM when used to pre-treat hepatocytes 4 h prior to adding sporozoites, versus an IC 50 > 10 μM when added 2 h post sporozoite addition 50 .…”

An Update on Development of Small-Molecule Plasmodial Kinase Inhibitors

“…Pf PKG In terms of inhibitor development, Pf PKG is one of the plasmodial kinases that has been studied extensively thus far. It is regarded as a very attractive antimalarial drug target as its inhibition offers simultaneous prophylactic, curative and transmission-blocking potential [72]. As Pf PKG is an essential enzyme for multiple life cycle stages, there is a relatively low risk of the parasite developing high-grade resistance to Pf PKG-selective inhibitors [72].…”

“…It is regarded as a very attractive antimalarial drug target as its inhibition offers simultaneous prophylactic, curative and transmission-blocking potential [72]. As Pf PKG is an essential enzyme for multiple life cycle stages, there is a relatively low risk of the parasite developing high-grade resistance to Pf PKG-selective inhibitors [72]. The PKG enzyme is also highly conserved in all human malaria species, with an overall sequence identity of 90-92% and identical catalytic site and gatekeeper residues [73].…”

“…Vanaerschot and co-workers [72] identified Pf PKG as the primary target of the Medicines for Malaria Venture compound MMV030084 (29, Figure 12). MMV030084 had an IC 50 value of 0.4 nM against recombinant Pf PKG, and docking studies using the Pf PKG crystal structure (PDB ID: 5DYK) showed a strong interaction between MMV030084 and the ATP-binding site.…”