Plasmodium falciparum is the parasite responsible for malaria, a disease that continues to devastate all tropical regions of the world. A quarter of a million cases of malaria are reported annually with close to a million of these cases resulting in death. Efforts to control this disease are complicated by growing drug resistance in the parasite, leading to an imminent need for new drug targets. One avenue to the identification of novel targets is to discover and characterize essential enzymes in the parasite by inhibiting them with small molecules. The serine hydrolases are a large family of enzymes with highly reactive and covalently modifiable active sites making them attractive for inhibition with small molecules. Furthermore serine hydrolases are well represented in most organisms including humans where they comprise 1% of all proteins. In P. falciparum two essential serine hydrolases have already been identified, subtilisin-like serine protease 1 (PfSUB1) and 2 (PfSUB2). In order to find additional essential serine hydrolases in the parasite, a library of highly effective serine hydrolase inhibitors in mammals was screened in P. falciparum. This library of triazole urea compounds revealed several compounds that slow or block the growth of P. falciparum. The most efficacious compound, AA691, appears to induce a host cell invasion defect. Furthermore it has been verified that AA691 binds to at least one of the same targets as fluorophosphonate (FP), a generic serine hydrolase inhibitor, in competition assays suggesting this defect is due to inhibition of serine hydrolases. In this project the serine hydrolases inhibited by AA691 will be isolated using a probe based on the structure of AA691 that covalently modifies its targets. The targets will then be identified using tandem orthogonal proteolysis-activity-based protein profilin (TOP-ABPP) and mass spectrometry. The role(s) of these targets in host cell invasion will be characterized in culture through the creation of direct and regulatable conditional knockouts in P. falciparum. The invasion defect will be further verified in vivo with P. berghei mouse infections. Finally the effects of AA691 on the liver stage will be assessed in hepatocyte culture and in vivo. The rising prevalence of P. vivax infection, which remains dormant in the liver leading to relapse after blood stage treatment, is driving a push for therapeutics against liver stage parasites. There is an urgent need for novel malaria therapeutics and the results of this research could lead directly to the development of these drugs.
Plasmodium falciparum is the parasite responsible for malaria, a disease that continues to devastate all tropical regions of the world with a quarter of million cases reported annually and close to a million of these cases resulting in death. Efforts t control this disease are complicated by growing drug resistance in the parasite, thus leading to an imminent need for novel drug targets. Through this project, ultrapotent triazole urea inhibitors will be used to identify essential P. falciparum serine hydrolases involved in host cell invasion and the inhibitor and its target enzyme(s) will present potential new avenues for the development of malaria therapeutics.
