HIGH-AFFINITY OF ERGOPEPTIDES FOR CYTOCHROMES P450 3A - IMPORTANCE OFTHEIR PEPTIDE MOIETY FOR P450 RECOGNITION AND HYDROXYLATION OF BROMOCRIPTINE

The interaction between rat and human liver cytochromes P450 with a se ries of lysergic acid derivatives and ergopeptide alkaloids was studie d by difference visible spectroscopy. Ergopeptides, like bromocriptine , ergocryptine and dihydroergotamine, strongly interacted with rat liv er microsomes with the appearance of a difference spectrum which is ch aracteristic of their binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P4 50s 3A in the microsomes and was at its maximum in dexamethasone-treat ed rat microsomes. All the ergopeptides studied exhibited a high affin ity for rat P450s 3A (K-s around 1 mu M), although lysergic acid deriv atives not bearing the tripeptide moiety failed to give significant in teractions with these P450s. A cyclic azatripeptide exhibiting a struc ture very similar to that of the tripeptide moiety of ergopeptides als o interacted with P450s 3A with appearance of an intense type I differ ence spectrum. Very similar results were observed with two allelic for ms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeas t. In both cases all the ergopeptides studied showed high affinities f or the P450s (K-s 0.6-2.2 mu M) and an intense shift from the low-spin to the high-spin state upon substrate binding (60-100% spin shift). L ysergic acid derivatives not bearing the tripeptide group of ergopepti des also completely failed to interact with P450s 3A4. Liver microsome s from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of brom ocriptine, which occurs at the level of its tripeptide moiety. Human l iver microsomes as well as P450 NF25 and P450 hPCN1 also exhibited a h igh activity for bromocriptine hydroxylation at this level. These resu lts show that ergopeptides exhibit a particularly high affinity for P4 50s of the 3A subfamily. The tripeptide moiety of ergopeptides is esse ntial for their recognition by P450s 3A and binds at a site close to P 450 heme, producing type-I difference spectra. Accordingly, at least o ne of the studied ergopeptides, bromocriptine, is hydroxylated by P450 s 3A at the proline ring of the cyclopeptide moiety. As cyclosporine i s known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.