Alternative nitric oxide-producing substrates for no synthases

Nitric oxide (NO) is a key inter- and intracellular molecule involved in the maintenance of vascular tone, neuronal signaling, and host response to infection. The biosynthesis of NO in mammals involves a two-step oxidation of L-arginine (L-Arg) to citrulline and NO catalyzed by a particular class of heme–thiolate proteins, called NO-synthases (NOSs). The NOSs successively catalyze the Nω-hydroxylation of the guanidine group of L-Arg with formation of Nω-hydroxy-L-arginine (NOHA) and the oxidative cleavage of the C N(OH) bond of NOHA with formation of citrulline and NO. During the last decade, a great number of compounds bearing a C NH or C NOH function have been synthesized and studied as possible NO-producing substrates of recombinant NOSs. This includes derivatives of L-Arg and NOHA, N-alkyl (or aryl) guanidines, N,N′- or N,N-disubstituted guanidines, N-alkyl (or aryl) N′-hydroxyguanidines, N- (or O-) disubstituted N′-hydroxyguanidines, as well as amidoximes, ketoximes, and aldoximes. However, only those involving the NHC(NH2) NH (or NOH) moiety have led to a significant formation of NO. All the N-monosubstituted N′-hydroxyguanidines that are well recognized by the NOS active site lead to NO with catalytic efficiences (kcat/Km) up to 50% of that of NOHA. This is the case of many N-aryl and N-alkyl N′-hydroxyguanidines, provided that the aryl or alkyl substituent is small enough to be accommodated by a NOS hydrophobic site located in close proximity of the NOS “guanidine binding site.” As far as N-substituted guanidines are concerned, few compounds bearing a small alkyl group have been found to act as NO-producing substrates. The kcat value found for the best compound may reach 55% of the kcat of L-Arg oxidation. However, the best catalytic efficiency (kcat/Km) that was obtained with N-(4,4,4-trifluorobutyl) guanidine is only 100-fold lower than that of L-Arg. In a general manner, NOS II is a better catalyst that NOS I and III for the oxidation of exogenous guanidines and N-hydroxyguanidines to NO. This is particularly true for guanidines as the ones acting as substrates for NOS II have been found to be almost inactive for NOS I and NOS III. Thus, a good NO-producing guanidine substrate for the two latter isozymes remains to be found.