Transition state (TS) analysis from isotope effects and computational chemistry provides frontier technology for understanding the chemistry of bond change at the instant of enzymatic TS formation. Transition state analogues can be designed from the molecular electrostatic potential surfaces of TSs and have provided unique design parameters for some of the most powerful enzymatic inhibitors known. First and second generation TS analogues for human purine nucleoside phosphorylase (PNP) have advanced from first principles of TS design into human clinical trials for cancer and autoimmune diseases. Third generation PNP inhibitors will be compared to 1st and 2nd generation analogues for binding, structure, thermodynamics and biological lifetimes on PNP in cells. Binding isotope effects are an emerging technology for understanding the geometric and electronic constraints experienced by molecules as they become immobilized at their binding sites on macromolecules, including enzymes and receptors. Binding isotope effects will explore the atomic constraints of substrates and tight- binding TS analogues at the binding sites of human PNP. A surprising diversity of TS structure exists in the same enzyme isolated from different species, establishing the possibility of species-specific TS analogue design. Transition state structures of bacterial 5'- methylthioadenosine nucleosidases (MTANs) will be solved and matched to specific analogues for affinity and structures of reactant and TS-complexes. Biological efficacy of MTAN inhibitors will be analyzed in bacterial quorum sensing pathways. In theory, all enzymatic TSs should be accessible to isotope effect analysis but some provide technical challenges because the chemical step is obscured by non-chemical steps. Human thymidine phosphorylase is a prototype for kinetically difficult TS analyses. TS analysis methods will be established to expose the chemical step by rapid reaction kinetics and altered reaction conditions. Atomic understanding of enzymatic TS chemistry has been developed primarily in enzymes involved in N-ribosyltransferases and deaminases. Expanding the frontier of TS analysis to hydrolysis at carbonyl carbons will be accomplished in the well-known system of HIV-protease and in the important but poorly understood target of human 2'-O-acetyl-ADP-ribosyl esterase. Goals of this research are to push the frontier of enzymatic TS theory to enhance understanding of catalysis and drug design for human targets.
Transition state theory provides an approach to design better drugs for human disease. Expanded methods of drug design will be applied to targets of human disease. Purine nucleoside phosphorylase is a target for leukemia and for autoimmune diseases including psoriasis and tissue transplant rejection;methylthioadenosine phosphorylase is a target for antibiotic-resistant bacteria;thymidine phosphorylase is a target for solid tumors;HIV protease is a target for AIDS infections;and acetyl-ADP-ribosyl hydrolase is a target for diseases of ageing. New methods will be established for the broader application of this theory and the results may lead to new drugs to treat cancer, autoimmunity, bacterial infections and diseases of ageing.
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