Barry M. Goldstein - US grants

The conformation of a drug molecule is initimately related to its function. This research will identify potential constraints on drug conformation in the experimental antitumor agents tiazofurin and selenazofurin. Tiazofurin (TF) and selenazofurin (SF) are new C-glycosyl nucleosides currently undergoing clinical trials. In vivo, TF and SF are incorporated into analogues of the cofactor NAD. These NAD analogues act as inhibitors of inosine monophosphate dehydrogenase, the putative cause of cytotoxicity. Crystal structures of TF and SF show unusual close contacts between the heteroatom in the base (S or Se) and the furanose ring oxygen, suggesting that the conformations of these agents and/or their NAD analogues may be restricted in solution. This would have important implications for drug binding and activity. Computational results suggest that the heteroatom-oxygen interaction is in part electrostatic. Thus, the correlation between heterocycle charge and glycosyl bond conformation will be examined in a series of new base-substituted analogues of tiazo- and selenazofurin. Crystallographic, computational and NMR techniques will be employed. A crystallographic data base survey of compounds showing close intramolecular contracts will be performed in order to further identify the origins of the heteroatom-oxygen interaction. NMR experiments employing a variety of proton and heteronuclear techniques will examine the conformation of the parent compounds and the NAD analogues in solution. Enzyme inhibition and modeling studies will investigate binding of the NAD analogues to other dehydrogenases and correlate binding ability with changes in glycosyl bond conformation.

9710129 Jones This proposal details studies on the use of cytochrome P450 enzymes in the pursuit of green chemistry. Studies on the nature of P450-ligand interactions will be exploited as a biocatalyst to design an innovative synthesis for the industrially important carboxylic acid, 2-ethylhexanoate. The biocatalysis pathway will lead to pollution prevention since it will obviate the need for toxic and carcinogenic heavy metals in this synthesis. This synthesis was chosen as a model for how the P450 enzyme family can be exploited as a biocatalyst in general. These same studies will be used to develop predictive methodology that can be exploited in the design of safer chemicals. The predictive methodology will allow for rapid decision making about the potential of bioactivation in the development of new chemicals. Thus, less toxic chemicals can be designed into chemicals for new applications and potentially hazardous chemical identified rapidly. The cytochromes P450 are a ubiquitous family of enzymes that catalyze the reduction of molecular oxygen to a very reactive monooxygen species. This enzyme system is responsible for the metabolism of both endogenous and exogenous chemicals. The cytochrome P450 (CYP) enzyme family has been called the most versatile biological catalyst known. This trait is a double edged sword that makes the CYP enzyme family a diverse detoxifier of chemicals, as well as, the major enzyme system responsible for the production of toxic metabolites. Thus, while the enzymeis important in biosynthetic reactions, it is also important in bioactivation of benign chemicals to carcinogens and toxins. The general objective of this proposal is to understand the structural factors that govern cytochrome P450-ligand interactions. Active site mutants of P450cam will be constructed and kinetically characterized. Structural information from these mutants, gained from Xray crystallography and molecular dynamics studies, will define factors important in substrate binding to P450cam. This information can be used in the design of P450 mutants to perform benign synthesis, in the design of safer chemicals and in the design of P450 enzymes for bioremediation. While the development of bacteria for bioremediation is not within the scope of this grant, the results obtained during this project will be directly applicable to this endeavor. Furthermore, general knowledge about the mechanism of ligand- enzyme interactions, that can be applied to enzyme systems other than P450, will be obtained. The following are the specific objectives of this proposal: (1.1) Produce P450cam active-site mutants F87W, Y96W, L244A, and T185F. (1.2) Chemically and kinetically characterize regio- and stereospecificity of oxidations to the industrially important product 2-ethylhexanoic acid by the wild-type and mutant enzymes. (1.3) Study enzyme-ligand interactions at the active site using molecular dynamics. (1.4) Test the dynamics models by examination of crystal structures of the enzyme and enzyme-ligand complexes. ***

structural biology; proteins; biomedical resource; biological products;

DESCRIPTION (provided by applicant): Inosine monophosphate dehydrogenase (IMPDH, E.C. I .1.1.205) is an enzyme whose function is closely linked with the control of cell proliferation and differentiation. It has long been recognized as an important target in the design of both anti-tumor, immunosuppressive and antiviral drugs. This project will use X-ray crystallography to provide the first structures of chemotherapeutically active dinucleotide analogues bound to human IMPDH. This information will be correlated with biochemical and biological data. The goal is the identification of specific structural features required for binding. The long-range goal is the design of new agents with improved activity and clinical efficacy. IMPDH catalyzes the committed step in the de novo synthesis of the guanine nucleotides. Inhibition of IMPDH compromises the ability of G proteins to act as transducers of intracellular signals. Inhibition results in reductions in nucleic acid synthesis, oncogene expression, and, ultimately, cell proliferation and differentiation. Two isoforms of IMPDH have been identified, labeled type I and type II. Type I is constitutively expressed in normal cells. Expression and activity of type II is dramatically up-regulated in neoplastic and other rapidly dividing cells. This isoform has been a primary target in drug design. Tiazofurin is an antitumor agent that functions by inhibiting IMPDH. In Phase II clinical trials, tiazofurin has produced complete hematologic remissions in patients with end-stage acute leukemias. Tiazofurin is a prodrug. In vivo, it is converted to an analogue of the cofactor nicotinamide adenine dinucleotide (NAD). This NAD analogue, called TAD (thiazole-4-carboxamide adenine dinucleotide) is the major inhibitor of IMPDH. The selenium analogue of TAD, called SAD, binds IMPDH with equal efficacy. The principal investigator has obtained a structure of a complex between the human type II isoform of IMPDH and the dinucleotide inhibitor SAD. These data demonstrate the location and conformation of the bound inhibitor, and indicate specific interactions which can be exploited in the development of compounds with improved affinity and specificity. The principal investigator will examine type I and II IMPDH binding by agents designed to 1) test the hypothesis that intramolecular constraints enhance binding and 2) enhance specificity for the type II isoform and overcome clinical problems associated with drug resistance and rapid metabolism.