Inhibition of stimulated Jurkat cell adenosine 3 ',5 '-cyclic monophosphate synthesis by the immunomodulatory compound HR325

HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'(trifluoromethyl)-phe nyl]-propenamide) is an immunomodulatory compound through pyrimidine biosyn thesis inhibition with antiproliferative properties which was derived from the isoxazol compound A77 1726 [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide]. During studies of the effects on early si gnal transduction events of this type of compound, it was found that HR325 dose-dependently inhibited adenosine 3',5'-cyclic monophosphate (cAMP) synt hesis by Jurkat cells stimulated with prostaglandin E-2 (PGE(2)), cholera t oxin (CTX), or forskolin (FKN). The potency of inhibition by HR325 of FKN-s timulated cells (IC50 30.4 muM) was approximately 3-fold higher than that o f the other agonists (11.6 and 11.7 muM) and was independent of time of pre incubation for both PGE(2) and FKN. Interestingly, A77 1726, an analogue of HR325, displayed a markedly different profile of stimulus-dependent potenc ies. The inhibition of cAMP synthesis by HR325 when stimulated by both PGE( 2) and FKN was unaffected by glucose supplementation, in contrast to HR325- inhibited ATP levels, which were restored under such conditions. Further st udies revealed that HR325 reduced intracellular ATP levels by uncoupling ox idative phosphorylation, albeit with a 1000-fold lower potency than the ant ihelmintic drug niclosamide. In addition, glucose supplementation experimen ts showed that, in contrast to HR325, the niclosamide-mediated reduction of ATP levels was wholly responsible for its inhibition of PGE(2)- and FKN-st imulated cAMP synthesis. (C) 2001 Elsevier Science Inc. All rights reserved .