DIHYDROOROTATE DEHYDROGENASE IS A HIGH-AFFINITY BINDING-PROTEIN FOR A77-1726 AND MEDIATOR OF A RANGE OF BIOLOGICAL EFFECTS OF THE IMMUNOMODULATORY COMPOUND

A protein with high affinity (K-d 12 nM) for the immunomodulatory comp ound A77 1726 has been isolated from mouse spleen and identified as th e mitochondrial enzyme dihydroorotate dehydrogenase (EC 1.3.3.1), The purified protein had a pi 9.6-9.8 and a subunit M(r) of 43,000, Peptid es derived from the mouse protein displayed high microsequence similar ity to human and rat dihydroorotate dehydrogenase with, respectively, 35 and 39 out of 43 identified amino acids identical. Dihydroorotate d ehydrogenase catalyzes the fourth step in de novo pyrimidine biosynthe sis. The in vitro antiproliferative effects of A77 1726 are mediated b y enzyme inhibition and can be overcome by addition of exogenous uridi ne, The rank order of potency of A77 1726 and its analogues in binding or enzyme inhibition was similar to that for inhibition of the mouse delayed type hypersensitivity response. It is proposed that inhibition of dihydroorotate dehydrogenase is an in vivo mechanism of action of the A77 1726 class of compounds. This was confirmed using uridine to c ounteract inhibition of the murine acute graft versus host response.