STRUCTURE BASED DISCOVERY OF A NEW CLASS OF ENZYME INHIBITORS. Thomas J. Stout⁺, M. Paola Costi^{[[daggerdbl]]}, Daniela Barlocco^{[[daggerdbl]]}, Marcella Rinaldi^{[[daggerdbl]]}, Brian Shoichet^*, K.M. Perry^*, I.D. Kuntz^*, Robert M. Stroud⁺ Departments of Biochemistry⁺ and Pharmaceutical Chemistry^*, University of California, San Francisco, San Francisco, CA 94143-0448 USA Dipartimento di Scienze Farmaceutiche Universitá Degli Studi di Modena 41100 Modena - Via Campi, 183 Italy^{[[daggerdbl]]}

Thymidylate Synthase (TS) is an important target for the development of anti-cancer therapeutics. TS forms a biosynthetic bottleneck for the production of the DNA nucleotide thymidine (T) by catalyzing the reductive methylation of dUMP to dTMP. As such, many inhibitors of TS have been developed as mechanism based substrate or co-factor analogues, but currently less than five have been approved for use as anti-cancer therapeutics. We have used structure-based modeling methods and single-crystal X-ray crystallography to discover an entirely new series of TS inhibitors unrelated to substrate or co-factor (1). First generation leads have subsequently been sequentially improved through cycles of design, synthesis and crystallography to yield TS inhibitors of improved potency and specificity.

These phthalid-based compounds display a new mode of general enzyme inhibition through non-specific occlusion. The crystal structures show multiple modes of ligand binding which vary significantly with small modifications to the ligands. In addition, kinetic analysis shows concentration dependent inhibition, implicating a non-specific mode of binding. Indeed, the hydrophobic non-specific interactions found within the crystal structures appear to predominate over potential hydrogen bonds. The combined occupied volumes of five different enzyme-inhibitor complexes map out a new ligand-accessible space which can now be used for future structure-based design of inhibitors with additional binding specificity.

1. Shoichet, B. K., Stroud, R. M., Santi, D. V., Kuntz, I. D., & Perry, K. M. (1993) Science 259, 1445-50.