BAD TO SAD: ALCOHOL DEHYDROGENASE AS A "CRYSTALLOGRAPHIC ASSAY" FOR NAD ANALOGS Barry M. Goldstein, Thomas D. Colby, Krzysztof Pankiewicz and Kyoichi Watanabe, Dept. of Biophysics, University of Rochester Medical Center, Rochester, NY 14642, and OncorPharm Corp., Gaithersburg, MD 20877.

We have used liver alcohol dehydrogenase (LADH) to determine the ability of analogs of the cofactor nicotinamide adenine dinucleotide (NAD) to structurally mimic normal cofactor binding. LADH undergoes a cofactor-induced conformational transformation which can be used to discriminate between different classes of analogs.

NAD binding to LADH induces a conformational transition from an open to closed form of the enzyme. This transition requires the formation of specific hydrogen bonds by the nicotinamide carboxamide group, and is very sensitive to perturbations at the nicotinamide end of the ligand. An analog that can adopt the conformation required by the cofactor site will induce the transition to the closed form. An analog subject to constraints incompatible with the binding site cannot stabilize the closed form. The complex then remains in the open conformation.

NAD-dependent dehydrogenases are involved in numerous metabolic processes, and have become attractive targets for drug design. A number of laboratories have been involved in the design and synthesis of neutral isosteric NAD analogs. These compounds differ in the heterocycle used to replace the nicotinamide group, as well as in the particular functional groups modified to improve both specificity and transport properties.

LADH complexes with several classes of C-glycosyl analogs have been examined. The thiazole and selenazole dinucleotides have constrained rotation about their C-glycosyl bonds. These complexes crystallize in the open conformation. (Li et al., Biochemistry, 1994, v33, 23). The pyridine dinucleotides are unconstrained, closely mimic NAD binding, and stabilize the closed conformation (Li et al., Biochemistry, 1994, v33, 11734).

We have recently examined complexes with the new dinucleotide inhibitor benzamide adenine dinucleotide (BAD), the antitumor agent selenazole-4-carboxamide adenine dinucleotide (SAD), and analogs of these compounds with additional modifications. SAD is constrained, binding in the open conformation. BAD is unconstrained, and is accommodated by the cofactor site in the closed conformation. The enzyme may also distinguish between more subtle changes in the ligand, such as the replacement of the phosphate ester oxygen with a methylene bridge.