Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

Abstract

The peptide hormone ghrelin plays a key role in regulating hunger and energy balance within the body. Ghrelin signaling presents a promising and unexploited target for development of small molecule therapeutics for treatment of obesity, diabetes, and other health conditions. Inhibition of ghrelin O-acyltransferase (GOAT), which catalyzes an essential octanoylation step in ghrelin maturation, offers a potential avenue for controlling ghrelin signaling. Through screening a small molecule library, we have identified a class of synthetic triterpenoids that efficiently inhibit ghrelin acylation by the human isoform of GOAT (hGOAT). These compounds function as covalent reversible inhibitors of hGOAT, providing the first evidence of the involvement of a nucleophilic cysteine residue in substrate acylation by a MBOAT family acyltransferase. Surprisingly, the mouse form of GOAT does not exhibit susceptibility to cysteine-modifying electrophiles, revealing an important distinction in the activity and behavior between these closely related GOAT isoforms. This study establishes these compounds as potent small molecule inhibitors of ghrelin acylation and provides a foundation for the development of novel hGOAT inhibitors as therapeutics targeting diabetes and obesity.

Figure 1. Ghrelin O-acyltransferase (GOAT) as a target for blocking ghrelin signaling

a) Ghrelin octanoylation catalyzed by GOAT. b) Structures of reported GOAT inhibitors. Ahx denotes aminohexanoate; Tat denotes a Tat peptide sequence (-YGRKKRRQRRR).

Figure 2. Screening of Diversity IV library compounds reveals a novel small molecule hGOAT inhibitor

a) Fluorescence-based hGOAT activity assay used for compound screening. B) Protocol for screening assay to identify hGOAT inhibitors; b) Structure of CDDO-Im (1), the initial hit from the Diversity IV library; c) Inhibition of hGOAT octanoylation activity by CDDO-Im (1). Reactions were performed and analyzed to determine percent activity as described in the inhibitor assay protocol included in the Experimental section. Error bars reflect the standard deviation from a minimum of three independent measurements.

Figure 3. Multiple CDDO derivatives effectively inhibit hGOAT

Structures and IC₅₀ values for CDDO derivatives with substitutions at the carboxyl group at position 28: R = imidazole (CDDO-Im, 1); R = methyl ester (CDDO-Me, 2); R = ethylamide (CDDO-EA, 3); R = trifluoroethylamide (CDDO-TFEA, 4); R = carboxylic acid (CDDO, 5).

Figure 4. Structure-activity analysis reveals multiple pharmacophores contribute to synthetic triterpenoid inhibition of hGOAT

Compounds tested as inhibitors of hGOAT octanoylation activity, organized by overall hydrocarbon skeleton family (triterpenoid, steroid, or cyclohexane parent structure) and colored to reflect potential pharmacophores (α,β-unsaturated ketone, green; steroid scaffold, purple; CDDO derivative functional groups in rings C-E, orange) Measured IC₅₀ values are provided for each compounds, with lower limits established based on compound solubility and lack of inhibition observed at the highest experimentally accessible concentration. Errors reflect standard deviations from a minimum of three determinations. Reactions were performed and analyzed to determine percent activity and IC₅₀ values as described in the inhibitor assay protocol included in the Experimental Section. Synthetic protocols for compounds 9-18 are provided in the Online Materials and Methods and Supplementary Information sections.

Figure 5. hGOAT inhibition profile supports the involvement of a catalytically essential cysteine residue

a) Topological model of hGOAT, with cysteine residues highlighted in yellow. The conserved functionally essential residues N307 and H338 are denoted in red. This model was constructed by comparison to the experimentally developed topology model for mouse GOAT using the Protter online server.^, b) Inhibition of hGOAT octanoylation activity by N-ethylmaleimide (NEM, structure shown in inset). c) Time dependence of hGOAT inhibition by CDDO-EA (3). hGOAT activity was measured as a function of preincubation time in the presence of 30 μM CDDO-EA. Inset: IC₅₀ values for CDDO-EA (3) inhibition of hGOAT activity as a function of inhibitor preincubation time. d) Inhibitor dilution assays reveal irreversible hGOAT inhibition by NEM and reversible inhibition by CDDO-EA (3) and α-cyanoenone steroid 9. Dap-C8 denotes the GS(octanamide-Dap)FL product-mimetic GOAT inhibitor used as a control for reversible inhibition.^, Errors bars reflect the standard deviation from a minimum of three determinations.

Figure 6. hGOAT and mGOAT exhibit dramatically different susceptibility to inhibition by cysteine modifying agents

a) Inhibition of hGOAT and mGOAT octanoylation activity by CDDO-EA 3; hGOAT, filled circles; mGOAT, open circles. b) Inhibition of hGOAT and mGOAT octanoylation activity by N-ethylmaleimide (NEM). hGOAT, black bar; mGOAT, white bar. c) Inhibition of hGOAT (left) and mGOAT (right) octanoylation activity by α-cyanoenone 9 and ketone 12. α-cyanoenone (compound 9, black bar); ketone (compound 12, white bar), treatment with ketone 12. d) Clustal Omega alignment of hGOAT and mGOAT sequences. Cysteine residues are indicated in bold, with cysteines conserved in both isoforms highlighted in gray and cysteine residues unique to hGOAT highlighted in yellow. Reactions were performed and analyzed to determine percent activity and IC₅₀ values as described in the Experimental section. Error bars reflect the standard deviation from a minimum of three independent measurements.

Figure 7. A potential mechanism for hGOAT-catalyzed ghrelin octanoylation employing a cysteine acyl-enzyme intermediate

Following formation of an octanoyl-enzyme intermediate, transfer of the octanoyl group to the serine acylation site near the N-terminus of ghrelin can be catalyzed through involvement of a general base such as the conserved and functionally essential H338 histidine residue within hGOAT.^,