Structure-guided Inhibitor Design of Mycobacterium Tuberculosis Drug Targets from Central Carbon Metabolism

A key determinant of the pathogenicity of M. tuberculosis (Mtb) is its ability to conserve energy and resources by altering its intermediate metabolism response to host-derived hostile conditions. Growing evidence supports that the central carbon metabolism (CCM) of M.tuberculosis including glycolysis, gluconeogenesis, the pentose phosphate pathway, and the TCA cycle, is regulated to allow for simultaneous utilization of carbohydrates and fatty acids-derived carbon sources. The genes involved in CCM represent attractive anti TB drug-targets. Although the role of Mtb phosphoenolpyruvate carboxykinase (PEPCK) in gluconeogenesis pathway and pyruvate kinase (PykA) enzymes in glycolysis pathway are relevant for the M. tuberculosis pathogenesis, targeting these enzymes for the drug development is not straight forward because of the human orthologs existence. Here, X-ray crystallographic, biochemical, and inhibitory studies of Mtb PEPCK and PykA highlight distinct features of the pathogenic’s enzyme that differ from those of the host orthologs’ and provide opportunities to develop selective and potent inhibitors. Structural data of Mtb PEPCK has revealed that the conformation changes of the flexible loops in response to substrates binding differ from the one reported for host versions of the enzyme. GTP-competitive inhibitors of human cytosolic PEPCK bind to Mtb PEPCK in similar fashion, but the discovery of two unique small pockets in Mtb has created an opportunity for the design of a selective Mtb PEPCK inhibitor. By using structure-guided medicinal chemistry for GTP-competitive inhibitor series with PEPCK, we are able to improve the inhibitory effect of these inhibitors against the enzyme, creating inhibitors with a nano-molar range IC50 and develop 10 fold selective inhibitors against Mtb PEPCK over human PEPCK. Structural study of Mtb PykA also demonstrated that adenosine mono-phosphate (AMP) is an allosteric effector, and we identified the binding mode and interaction of AMP in the allosteric binding site for the first time. Screening diverse compound libraries gave us insight into the potential scaffolds for the development of more potent pathogen-specific inhibitors. The structural and inhibitor studies of Mtb PEPCK and PykA have allowed a better understanding of their role in gluconeogenesis and glycolysis, and provide a framework for the development of selective inhibitors