| Bioactive agents whose mechanisms of action are unknown can nevertheless be used to probe biological mechanism. I performed molecular design studies of such agonist and antagonist probes of the bacterial receptor mediating the transition of Agrobacterium tumefaciens to virulence and oncogenesis. Initial designs were based on the native phytochemical agonist acetosyringone, and on the Lynn mechanistic model for receptor action (Proc. Natl. Acad. Sci. U.S.A. 1992, 88:7854 and therein). I improved preparations of known covalent antagonists (process syntheses), and designed and synthesized noncovalent antagonist candidates (discovery syntheses). While none of the candidates were active, the first non-saccharide modulator of agonism, a halonicotinate, was discovered. Guided by the acetosyringone-based Lynn reagent, [125I]-2-bromo-5 ′-iodo-acetovanillone, I purified the primary receptor candidates p21 and p10. In parallel, I recharacterized the radiochemistry, and found that the reagent was neither specific nor quantitative for proteins whose primary function is phenol binding. Based on these and the results of protein microchemistry and subsequent molecular genetics (Y. Wang, Ph.D. Dissertation, The University of Chicago, 1999), I conclude that p21 likely does not play a direct role in Agrobacterium virulence. I sought reasons for our misdesigns and turned to further synthetic and computational studies. I analyzed agonist structure-activity relationships (SAR) data, calculating the first quantitative descriptors of agonist structures and properties, the first formal estimates of their potencies and efficacies, and the first quantitative models of the agonist-receptor interaction (2D-Hansch and 3D-CoMFA, p < 0.05, r ≥ 0.825). These studies suggest that the commonly studied agonist, acetosyringone, is neither physicochemically representative nor optimally potent, that agonists act by more than one physiologic/pharmacologic mechanism, and that agonist basal activity and potency are defined by distinct, complex interplays of substituent effects. These models suggest new regions of agonist structure space for exploration and new combinations of agonist substructures for further synthesis and testing. Hence, there now appears to be a straightforward approach to the design of highly potent reversible agonists and antagonists (important practical goals in the chemical biology of a bacterial receptor), as well as to a significant, testable binding site model for this key receptor-mediated process underlying pathogenesis in Agrobacterium. |
