A fluorescent peptide substrate facilitates investigation of ghrelin recognition and acylation by ghrelin O-acyltransferase

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

“…A number of in vitro assays have been published for GOAT using microsomes prepared from insect cells[7, 8, 20–24] or human cells[10]. These assays have established N-terminal sequence requirements for recognition of ghrelin by GOAT and identified a number of peptide-based inhibitors as well as initial small-molecule scaffolds.…”

A new class of ghrelin O-acyltransferase inhibitors incorporating triazole-linked lipid mimetic groups

“…The most significant challenge in generating novel hGOAT inhibitors is the paucity of structural and mechanistic information available regarding hGOAT, which is beginning to be addressed in recent biochemical studies. 22,28,29 A predominant and constant feature among the small pool of reported hGOAT inhibitors is incorporation of an amide-linked lipid group to mimic the octanoyl moiety of acylated ghrelin. 20,[25][26][27] Structure-activity studies have highlighted the importance of the lipid group in product-mimetic inhibitors for potency in inhibiting hGOAT.…”

“…22 The panel of triazole inhibitors was assayed for inhibitory activity against hGOAT using a fluorescently labeled hexapeptide hGOAT substrate, GSSFLC AcDan , which has been shown to act as a hGOAT substrate. 22,29 This substrate incorporates the N-terminal sequence elements shown to be essential for ghrelin recognition by hGOAT, 20,22,48 and has exhibited comparable behavior in GOAT inhibitor studies as compared to other assays using proghrelin or other ghrelin-derived peptides. 20,26,27 In initial screening of unpurified inhibitors at 0.1 and 1 lM concentrations, the inhibitor bearing the phenylpropyl triazole group (8d) exhibited the largest degree of inhibition (Fig.…”

“…The SAR for the triazole peptide inhibitors in this work exhibits similar behavior to that previously observed for acylated inhibitors of hGOAT and supports the importance of optimizing the size and Octanoylation activity of hGOAT in the presence of increasing concentrations of 8d was monitored using a fluorescently labeled peptide substrate, GSSFLC AcDan , which exhibits increased fluorescence and longer HPLC retention time upon octanoylation. 22,29 Briefly, 10-50 lg of membrane protein from Sf9 cells expressing hGOAT was incubated with 500 lM octanoyl CoA, 1.5 lM GSSFLC AcDan substrate, and either varying concentrations of peptide 8d or carrier control in 50 mM HEPES buffer (pH 7). Reactions were performed and analyzed to determine percent activity as described in the inhibitor assay protocol included in the Supplementary material.…”

“…(phenylethyl triazole) 3.8 ± 0.9 8d (phenylpropyl triazole) 0.7 ± 0.2 8e (phenylbutyl triazole) >>1 a The IC 50 value for each compound was determined by evaluation of inhibition of hGOAT octanoylation activity in an established in vitro assay. 29 Reported IC 50 values are the average of three independent determinations, with the standard deviation reported as the error.…”