Selective inhibition of human molt-4 leukemia type II inosine 5′-monophosphate dehydrogenase by the 1,5-diazabicyclo[3.1.0]hexane-2,4-diones

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo purine biosynthesis. IMPDH activity results from expression of two isoforms. Type I is constitutively expressed and predominates in normal resting cells, while Type II is selectively up-regulated in neoplastic and replicating cells. Inhibitors of IMPDH activity selectively targeting the Type II isoform have great potential as cancer chemotherapeutic agents. For this study, an expression system was developed which yields 35-50 mg of soluble, purified recombinant Type I and II protein from 1 L of bacteria. In addition, three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized and shown to act as specific inhibitors of human recombinant Type II IMPDH. The agents are competitive inhibitors with respect to the endogenous substrate LMP and K-i values range from 5 to 44 muM but were inactive as inhibitors of the Type I isoform at concentrations ranging from 0.5 to 500 muM. IC50 values for recombinant Type II inhibition were determined and compared to IC50 values obtained from Molt-4 cell extracts of IMPDH. Cytotoxicity assays revealed that the compounds inhibited Molt-4, leukemia growth with ED50 values of 3.2-7.6 muM. Computational docking studies predict that the compounds bind to IMPDH in the IMP-binding site, although interactions with residues differ from those previously determined to interact with bound IMP. While all residues predicted to interact directly with the bound compounds are conserved in the Type I and Type II isoforms, sequence divergence within a helix adjacent to the active site may contribute to the observed selectivity for the human Type II isoform. These compounds represent the first class of selective IMPDH Type II inhibitors which may serve as lead compounds for the development of isoform-selective cancer chemotherapy.