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 t wo 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 T ype II isoform have great potential as cancer chemotherapeutic agents. For this study, an expression system was developed which yields 35-50 mg of sol uble, purified recombinant Type I and II protein from 1 L of bacteria. In a ddition, three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized an d shown to act as specific inhibitors of human recombinant Type II IMPDH. T he agents are competitive inhibitors with respect to the endogenous substra te LMP and K-i values range from 5 to 44 muM but were inactive as inhibitor s 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 assay s revealed that the compounds inhibited Molt-4, leukemia growth with ED50 v alues of 3.2-7.6 muM. Computational docking studies predict that the compou nds bind to IMPDH in the IMP-binding site, although interactions with resid ues differ from those previously determined to interact with bound IMP. Whi le 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 selectivi ty for the human Type II isoform. These compounds represent the first class of selective IMPDH Type II inhibitors which may serve as lead compounds fo r the development of isoform-selective cancer chemotherapy.