Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4

Abstract

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na + homeostasis and affect parasite pH, exhibited a fast-tomoderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets

“…The PfFNT inhibitor MMV007839 gave rise to an acidification (Figure C, purple trace), consistent with the lactate and H + produced from glycolysis being unable to exit the parasite effectively. An increase in pH cyt was observed in the presence of the PfATP4 inhibitor cipargamin (Figure C, blue trace), consistent with previous findings . PfATP4 is an “acid-loader”, , and the increase in pH cyt suggests that in this condition, when glucose was restored, the V-type H + ATPase inhibited, and PfATP4-mediated H + entry eliminated, more H + ions were leaving the parasite or being consumed in the parasite than were entering or being produced.…”

“…An increase in pH cyt was observed in the presence of the PfATP4 inhibitor cipargamin ( Figure 1 C, blue trace), consistent with previous findings. 12 PfATP4 is an “acid-loader”, 9 , 52 and the increase in pH cyt suggests that in this condition, when glucose was restored, the V-type H + ATPase inhibited, and PfATP4-mediated H + entry eliminated, more H + ions were leaving the parasite or being consumed in the parasite than were entering or being produced. PfATP4 inhibitors have been shown previously to give rise to a cytosolic alkalinization in standard pH experiments in which parasites are continuously maintained in glucose-containing media, 9 although this can be difficult to detect with a plate reader.…”

“…The “spiroindolones” were the first new class of antimalarial reported to act through inhibition of PfATP4 and have been found to give rise to a range of readily detectable physiological perturbations including an increase in the parasite’s cytosolic Na + concentration ([Na + ] cyt ), which is accompanied by osmotic swelling of the parasite and parasitized erythrocyte, and an increase in the parasite’s cytosolic pH (pH cyt ) . Since then, a large number of chemically diverse antiplasmodial compounds have been found to give rise to the physiological hallmarks of PfATP4 inhibition, − including 7% of compounds in the Medicines for Malaria Venture’s (MMV’s) Malaria Box, and 9% of the compounds in the MMV’s Pathogen Box that were annotated as having antiplasmodial activity . The most clinically advanced PfATP4 inhibitor, cipargamin (a spiroindolone), has progressed well through multiple Phase 1 and 2 clinical trials. , …”