Metabolic studies and drug discovery in apicomplexan parasites

• Protozoan parasites belonging to the phylum Apicomplexa are causative agents for a number of important human and veterinary infectious diseases including malaria, toxoplasmosis, babesiosis, cryptosporidiosis and theileriosis to name a few. There is a critical need for new drugs to combat these parasites as the emergence of drug resistance and toxicity issues have severely hampered the use of existing few drugs. In the post-genomic era, availability of genome sequence data along with genome wide functional datasets, such as gene expression and genetic variation, for many of these parasites have allowed us to better understand their biology and their disease mechanisms. Comparative genomics facilitates phylogenetic and evolutionary studies on parasites and this approach has been very useful for identifying unique genes or pathways as potential drug targets. My laboratory is focused on studying various aspects of parasite metabolism, especially those that are unique to the parasite or distinct from host, in order to better understand their biology as well as to identify novel anti-parasitic small molecules. The approaches employed by our group will include a combination of genomic, biochemical, genetic and metabolomic techinques.



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Carbon and energy metabolism:

• Many features of central carbon and energy metabolism are distinct in apicomplexan parasites when compared to humans and these have been identified by way of comparative genomics. For instance, the glycolytic pathway in these parasites is decoupled from the citric acid cycle, as the pyruvate dehydrogenase complex is absent from parasite mitochondria. These parasites are also known to be more reliant on glycolysis for ATP production rather than mitochondrial oxidative phosphorylation. From studies using T. gondii, we have demonstrated that glucose metabolism via glycolysis is not essential for the survival of these parasites, but genetic mutants incapable of metabolizing glucose (due to hexokinase gene knock out) are compromised in their ability to differentiate and persist as tissue cysts in vivo. 13C tracer metabolomics and ATP synthesis assays have revealed that T. gondii metabolism is versatile enough to use alternate nutrients as carbon source and alternate pathways to meet cellular ATP requirements. As a follow up to these findings my laboratory will be carrying out studies to dissect the modulation and regulation of carbon and energy metabolism in Apicomplexan parasites during the course of their life cycle.



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Biology of plastid and mitochondrial organelles:

• Apicomplexan parasites are unicellular eukaryotes and posses some unique set of subcellular organelles to support their parasitic lifestyle. A key organelle is the apicoplast (a plastid organelle of secondary endosymbiotic origin), which houses metabolic functions for the biosynthesis of fatty acids (via Type II pathway), isoprenoids (via non-mevalonate [DOXP-] pathway), lipoic acid and heme (only part of the pathway is associated with the plastid). All of these pathways have distinct features in comparison to human counterparts and are therefore suitable for exploitation as drug targets. Of particular interest to us is the synthesis of heme. Phylogenetic studies suggest that the pathway for heme biosynthesis is an evolutionary mosaic of both animal and plant type pathways, and is associated with both the mitochondria and apicoplast. Detailed biochemical characterization of phorphobilinogen synthase (PBGS) has been carried out from both P. falciparum and T. gondii, and the crystal structure of T. gondii PBGS has provides insights on unique subunit interactions which will be exploited for discovering novel and selective inhibitors for parasite PBGS enzyme. In addition, interesting aspects of organelle biology, such as the basis of the plastid associated delayed death phenotype and the role of the divergent mitochondrial ATP synthase in parasite energy metabolism, will be pursued at a mechanistic level.



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Comparative genomics and drug discovery:

• Part of my laboratory activities will be focused on in silico approaches for identifying novel drug targets and probable small molecule inhibitors of these targets. Our efforts on this front will include comparative genomics and structure based drug discovery approaches. As part of an ongoing collaborative project, an open access online resource (www.tdrtargets.org) has been developed for interrogating and mining the genomes of several different pathogens that are responsible for neglected tropical diseases. The TDR Targets database resource integrates genome sequence data and several different genome wide functional datasets, such as gene/protein expression, functional annotation data (enzymes and pathways), structure, phylogenetic distribution, gene essentiality, genetic/chemical phenotypes, and druggability. Users can query the TDR Targets database for one or more of these criteria, in a genome wide fashion and across multiple genomes to identify and prioritize drug targets. Most importantly, genes can be ranked by the user, using scores assigned to weight each criteria, and query results can be combined using Boolean functionalities such as AND, NOT and OR to provide consensus list of genes. Our current interest is to integrate bioactivity data of small molecules inhibitors in TDR Targets, in order to establish readily searchable links between genes and potential inhibitors. Thus, our proposal is to make TDRtargets.org a complete chemo-genomics platform catering primarily to tropical infectious diseases.

 

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