Small-molecule inhibitors targeting prokaryotic ClpQ protease in Plasmodium display anti-malarial efficacies against blood and liver stages

Abstract

The ATP-dependent ClpQY system is a prokaryotic proteasome-like machinery in the mitochondrion of malaria parasite, Plasmodium. This protease system is identified as a validated target for malaria intervention. Here we have identified drug-like small chemical compounds, pyrimidotriazine derivatives, as specific inhibitors of P. falciparum ClpQ (PfClpQ) having effective parasiticidal efficacies on asexual and liver stages. Screening of the Malaria Box compound library, using a robust in vitro activity assay, identified a hit compound targeting the PfClpQ protease with IC50 at ~750nM. Further, Structure-Activity Relationship (SAR) analysis, using a small library of derivatives of the hit compound, identified three potential lead compounds, ICGEB-L1, ICGEB-L2 and ICGEB-L3, which inhibited enzyme activity with IC50 in 100-200nM ranges. The three lead compounds effectively inhibited in vitro asexual stage parasite growth in similar concentration ranges with ~100-fold selectivity over mammalian cell line; in addition, these compounds effectively inhibited in vitro growth and maturation of hepatocytic stage parasites with EC50 in low nano-molar ranges. These compounds were found to effectively block the development of trophozoite stages to schizont stage, concurrently arresting development and segregation of parasite mitochondria, in asexual as well as hepatocytic stages of the parasite. These morphological and developmental effects of the drugs on parasites mimic the data from genetic ablation of PfClpQ. Importantly, all three compounds showed significant in vivo anti-parasitic efficacies on blood stages of P. berghei mouse malaria model. Overall these results suggest that these PfClpQ targeting compounds are promising candidates to develop new anti-malarials.