ISOZYME-SPECIFIC TRANSITION-STATE INHIBITORS FOR THE TRYPANOSOMAL NUCLEOSIDE HYDROLASES

Protozoan parasites lack de novo purine biosynthesis and require purin e salvage from the host. Nucleoside hydrolases are involved in nucleos ide salvage and are not found in mammals, making them protozoan-specif ic targets for inhibitor design. Several protozoan nucleoside hydrolas e isozymes with distinct substrate specificities have been characteriz ed. Novel substituted iminoribitols have been synthesized to resemble the transition state structure of the nonspecific inosine-uridine nucl eoside hydrolase from Crithidia fasciculata (IU-nucleoside hydrolase). These inhibitors have been characterized for this enzyme and for a pu rine-specific nucleoside hydrolase (IAC-nucleoside hydrolase) from Try panosoma brucei brucei. Inhibitors which provide nanomolar inhibition constants for IU-nucleoside hydrolase exhibit micromolar inhibition co nstants for the IAG-enzyme. For example, p-bromophenyliminoribitol inh ibits the IU- and IAG-enzymes with dissociation constants of 28 nM and 190 mu M, respectively. Substrate specificity, the action of transiti on state inhibitors and the pH-dependence of the kinetic constants est ablish that the catalytic mechanisms and transition state structures a re fundamentally different for the IU- and IAG-isozymes. The finding i s remarkable since these isozymes share significant homology at the ca talytic sites and both use inosine as a preferred substrate. The speci ficity of the transition state analogues indicates that logically-desi gned transition state inhibitors are isozyme specific, with (K-m/K-i I U-nucleoside hydrolase)/ (K-m/K-i IAG-nucleoside hydrolase) values up to 39 000. The mechanism of the differential inhibition is based on th e relative leaving group activation and ribosyl-oxocarbenium-forming a bilities of these enzymes. In addition to providing isozyme-specific i nhibitors, the novel molecules described here have diagnostic value fo r the nature of the transition states for N-ribohydrolase enzymes.