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Neuropeptide Inactivation By Peptidases
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Gerard O’Cuinn, Brendan O’Connor, Laura Gilmartin, Maria Smyth
Subsequently a second pyroglutamate aminopeptidase was located in synaptosomal membranes of guinea pig brain13 and of rat brain.14 Purification and characterization of solubilized preparations of this pyroglutamate aminopeptidase II activity from guinea pig brain15,16 and from rabbit brain17 revealed that the enzyme's specificity was restricted to TRH (Glp-His-Pro NH2) or to only very closely related peptides. There appeared to be an absolute requirement for the N terminal Glp-His sequence as replacement of the Glp residue by Glu or replacement of the His residue by other amino acids abolished activity17. These authors also noted that hydrogen bonding of the imidazole nitrogen of the His residue appears not to be involved in the substrate’s interaction with the active site of the enzyme as benzylation of the imidazole ring did not affect activity. The C-terminal prolineamide residue of TRH can however be replaced by proline, by 3'4'dehydroproline, by tryptophan, by tryptophan naphthylamide or by alanine naphthylamide and activity is conserved but KM or Ki values were found to be increased15,17. Activity was found to be restricted to tripeptides, tripeptideamides or tetrapeptides16 as it was shown that pyroglutamate aminopeptidase II removed the N-terminal Glp residue from Glp-His-Pro, Glp-His-ProNH2 and Glp-His-Pro-Gly but that no hydrolysis occurred with Glp-His, Glp-His-Pro-GlyNH2 or Glp-His-Pro-Gly-Lys. When each of these peptides was investigated as possible inhibitors in the hydrolysis of [3H-Pro]-TRH by pyroglutamate aminopeptidase II competitive inhibition was observed in each case and the lowest Ki value obtained (Ki = 50mM) was observed with Glp-His-ProNH2 (TRH). A similar pattern is visible in the LH-RH sequence where Glp-His-Trp (LH-RH1–3) was hydrolyzed but Glp-His-Trp-Ser-Tyr (LH-RH1–5) was not16.
Human mass balance, pharmacokinetics and metabolism of rovatirelin and identification of its metabolic enzymes in vitro
Published in Xenobiotica, 2019
Kaoru Kobayshi, Yoshikazu Abe, Asuka Kawai, Takao Furihata, Hiroshi Harada, Takuro Endo, Hiroo Takeda
From the results of in vitro studies using human liver S9 and microsomal fractions, TAMP was not expected to be directly formed from rovatirelin by the enzymes present in these fractions, such as ALDH, aminopeptidase and esterase in the liver. Furthermore, rovatirelin was resistant to pyroglutamate aminopeptidase in the rat brain homogenate and rat plasma (Kobayashi et al., 2019). These findings indicated that TAMP is not formed from rovatirelin by hydrolysis as the first metabolic reaction. In HLM, formation of TAMP and the unknown metabolite was simultaneously observed depending on the presence of NADPH, and the formation of both compounds was inhibited by a typical CYP3A4/5 inhibitor (ketoconazole). An ALDH inhibitor (disulphiram) and its active metabolite (diethyldithiocarbamate) also inhibited the formation of TAMP in HLM, but not that of the unknown metabolite. In addition, TAMP was formed by recombinant human CYP3A4 and 3A5, but not by recombinant human CYP2E1, and only recombinant human CYP3A4 and 3A5 significantly formed the unknown metabolite. These results indicated that formation of TAMP and the unknown metabolite depends on CYP3A4/5 activity. Furthermore, it is reasonable to consider that TAMP may be generated depending on the formation of the unknown metabolite and that CYP3A4/5 activity could play an important role in the formation of the unknown metabolite from rovatirelin in addition to the possible direct formation of TAMP. Additionally, ALDH inhibitors affected the formation of TAMP from the unknown metabolite, suggesting that ALDH activity contributes to the formation of TAMP from the unknown metabolite. As ALDH is known to have NAD-independent esterase activity and is widely distributed in liver cell fractions (Marchitti et al., 2008; Sidhu & Blair, 1975; Yoshida et al., 1998), there is a possibility of hydrolysing the amide bond that the unknown metabolite may possess.