Multiple inhibitor analysis of the brequinar and leflunomide binding siteson human dihydroorotate dehydrogenase

Brequinar and the active metabolite of leflunomide, A77 1726, have been cle arly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but confl icting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduc tion of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarbo xylic acid (PQC), the core moiety of brequinar also shows competitive inhib ition versus ubiquinone. Multiple inhibition experiments indicate that PQC land thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitiv e inhibitor versus DHO). In addition, we failed to observe brequinar bindin g to E.orotate by isothermal titration calorimetry (ITC). These results ind icate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site p ing-pong mechanism reported for DHODH. This kinetic data suggests that rece nt crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for t hese inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].