Pharmacological and genetic studies on delayed death inhibitors of apicomplexan parasites

Abstract

Discovering new drugs against apicomplexan parasites, preferably acting via novel mechanisms or extending our chemical toolkit for well determined targets are much needed. We have done detailed screening effort to identify novel inhibitors of Toxoplasma gondii using three different chemical libraries- “Sigma LOPAC”, MMV “Malaria Box” and MMV “Pathogen Box”. Sigma LOPAC is a collection of 1280 bioactive small molecules from Sigma Aldrich and have known mechanism of action in mammalian system and these molecules could be used to identify some unusual and new druggable targets in apicomplexan parasites. The molecule collection from MMV ‘Malaria Box’ and ‘Pathogen Box’ includes ~800 chemically diverse small molecules in each set, with proven potency against malaria parasites as well as other infectious agents, however, through unknown mechanisms.We first compared the anti-plasmodial and anti-toxoplasma activities of these libraries and found compounds with similar as well as mutually exclusive growth inhibitory effects and determined EC50 values for selected hits showing ≥80% growth inhibition at 10 μM. By comparing the EC50 values of T. gondii and P. falciparum, we identified a set of molecules that are effective with sub-micro molar potency against both parasites. This has allowed us to select molecules for detailed phenotypic studies in T. gondii, since it is similar in many aspects of cellular biology to malaria parasites, and possess corresponding orthologous genes. Using complementary phenotypic screens on P. falciparum and T. gondii, we differentiated sub-sets of molecules as inhibitors of overall cell growth of parasite, plastid segregation, parasite invasion, and egress, thereby providing early insights on their probable modes of action. These results highlight the advantage of comparative phenotypic screens in related species, as a means to identify lead molecules with conserved mode of action. Interestingly, few molecules thatfailed to induce acute growth inhibition on T. gondii tachyzoites in a two-day proliferation assay caused delayed parasitic death upon extended exposure. At least 28 (4-Malaria Box, 23-Pathogen Box and 1-LOPAC) of these molecules appear to act by inducing apicoplast mis-segregation during daughter cell formation. We have prioritized these inhibitors for target identification and mechanistic studies. Further work on target identification and mechanism will facilitate developing anti-parasitic compounds which have novel chemical scaffolds and act via a very well-studied phenomenon of delayed death in these parasites. Collectively, our findings highlight the potential for chemical phenotype based identification of novel mechanisms and targets in related parasites, for the MMV molecules. It will help to develop a deeper understanding of novel targets that can be leveraged to develop the future generation of drugs with a wide spectrum of activity not just for malaria, but other related diseases as well. .