The primary goal of this research is to attain a molecular level understanding of the biophysical mechanisms regulating folate- dependent enzymes, in particular dihydrofolate reductase (DHFR). A secondary goal is to use this understanding to facilitate the development of folate-dependent enzyme inhibitors as more effective anticancer drugs. For elucidating correlations between molecular structure and bioactivity, a database of over 100 antifolate X-ray crystal structures will be used to search for patterns of intra- and inter-molecular interactions and to test a working model relating high antifolate potency and low conformational flexibility. These patterns will be compared with those observed for the enzyme-inhibitor complexes to determine their prevalence in various environments. Computational chemistry techniques, including empirical force-field and molecular orbital calculations along with molecular computer graphics, will be employed to assess the energetic and entropic ramifications of these binding patterns and the role of solvent and electrostatic effects. Our specific focus will be on selected members from four antifolate classes, representatives of which are available from the antifolate structural database. Regarding these specified substrates and inhibitors of DHFR, our research plan is (1) to characterize their conformational profile in terms of conformational energy and entropy, preferred conformers, rotational barriers, and overall thermodynamic and dynamic flexibility; (2) to explore the influence of substituents, protonation and solvent on these conformational features; (3) to delineate correlations between molecular structure and conformation on the one hand and antifolate activity on the other, and (4) To initiate a theoretical probe of the specific binding interactions involved in formation of the DHFR-inhibitor complex. It is well known but less well understood that small changes in molecular structure often give rise to large variations in antifolate activity. Hence the proposed studies are aimed at a systematic and detailed theoretical analysis of the role of structure and conformation in dictating bioactivity, thus permitting a more rational basis for anticancer drug design.
