The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin’s biological interactions and avenues for controlling ghrelin signaling

Abstract

Ghrelin is a small peptide hormone that requires a unique post-translational modification, serine octanoylation, to bind and activate the GHS-R1a receptor. Initially demonstrated to stimulate hunger and appetite, ghrelin-dependent signaling is implicated in a variety of neurological and physiological processes influencing diseases such as diabetes, obesity, and Prader-Willi syndrome. In addition to its cognate receptor, recent studies have revealed ghrelin interacts with a range of binding partners within the bloodstream. Defining the scope of ghrelin’s interactions within the body, understanding how these interactions work in concert to modulate ghrelin signaling, and developing molecular tools for controlling ghrelin signaling are essential for exploiting ghrelin for therapeutic effect. In this review, we discuss recent findings regarding the biological effects of ghrelin signaling, outline binding partners that control ghrelin trafficking and stability in circulation, and summarize the current landscape of inhibitors targeting ghrelin octanoylation.

Keywords:

• Ghrelin
• ghrelin O-acyltransferase
• protein acylation
• ghrelin O-acyltransferase inhibitors

Discovery and characterization of ghrelin

Ghrelin is a 28-amino acid peptide hormone discovered in 1999 by Kojima and co-workers in their search to find the endogenous ligand for a growth hormone secretagogue receptor (GHS-R1a) (Kojima et al., 1999). Ghrelin was originally found to be expressed by endocrine X/A cells (P/D cells in humans) in the gastric mucosa of the stomach and small intestine (Date et al., 2000; Mizutani et al., 2009; Rindi et al., 2002; Stengel & Tache, 2012). In subsequent studies, both ghrelin and GHS-R1a expression have been detected in a range of tissues including the pancreatic alpha cells, the pituitary gland and hypothalamus using a range of techniques including in situ hybridization, immunohistochemistry monitored by optical microscopy, immunostaining coupled with electron microscopy, and RT-PCR (Date et al., 2000, 2002; Howard et al., 1996; Kojima et al., 1999; Korbonits et al., 2001). A broad study of human tissues identified the presence of ghrelin and GHS-R1a mRNA in multiple tissues, indicating that both ghrelin and the ghrelin receptor are expressed throughout the body (Gnanapavan et al., 2002). This widespread expression of ghrelin and its cognate receptor could provide the molecular foundation for the multiple physiological effects attributed to ghrelin signaling.

In the relatively short time since its discovery, ghrelin and associated signaling pathways have been linked to a wide range of physiological processes. These include growth hormone secretion (Kojima et al., 1999; Peino et al., 2000; Takaya et al., 2000), appetite stimulation and adiposity (Kamegai et al., 2001; Nakazato et al., 2001; Shintani et al., 2001; Tschop et al., 2000; Wren et al., 2001), insulin secretion and glucose homeostasis (Egido et al., 2002; Gagnon et al., 2015; Heppner et al., 2012; Reimer et al., 2003; Tong et al., 2010; Yada et al., 2014), and organismal response to starvation (Goldstein et al., 2011; Li et al., 2012). In recent work, ghrelin has been associated with a growing number of neurological processes such as memory, stress response, learning, sleep, mood levels and behavior (Andrews et al., 2009; Broglio et al., 2002, 2004; Gahete et al., 2011; Lutter et al., 2008). Recent studies have also implicated ghrelin as a major factor in neonatal hypothalamus development which influences lifelong metabolic regulation (Steculorum et al., 2015). We direct the reader to a recent review of ghrelin’s role in physiology and signaling for a comprehensive discussion of these topics (Muller et al., 2015).

Ghrelin expression and processing

Like other peptide hormones, ghrelin is expressed as a larger polypeptide that undergoes a series of processing steps prior to secretion from the cell (Figure 1) (Chen et al., 2009; Romero et al., 2010; Takahashi et al., 2009; Zhu et al., 2006). Ghrelin is expressed as a 117-amino acid precursor (preproghrelin), which contains an N-terminal signal peptide targeting preproghrelin for secretion. Cotranslational recognition of the signal peptide leads to preproghrelin trafficking to the endoplasmic reticulum (ER) where the signal peptide is cleaved to yield the 94-amino acid ghrelin precursor proghrelin (Zhu et al., 2006). In a processing step unique to ghrelin and essential for ghrelin’s biological activity through the GHS-R1a receptor, proghrelin is then acylated with octanoic acid on a specific serine side chain hydroxyl (Ser-3) near its N-terminus. Ghrelin octanoylation is catalyzed by ghrelin O-acyltransferase (GOAT), one of three members of the membrane bound O-acyltransferase (MBOAT) superfamily of enzymes that modify protein substrates (Buglino & Resh, 2012; Doubravska et al., 2011; Hofmann, 2000; Konitsiotis et al., 2015; Matevossian & Resh, 2015; Pepinsky et al., 1998; Takada et al., 2006; Willert et al., 2003). Acylated proghrelin is packaged into secretory vesicles and cleaved by a prohormone convertase (PC 1/3, PC2, or furin) to yield the mature 28-amino acid acylated ghrelin (‘ghrelin’), which can then be secreted into the bloodstream (Zhou et al., 1999; Zhu et al., 2006). In a recent study, Seim and co-workers identified an exon-deleted splice variant of ghrelin in multiple vertebrate species that yields a 13-amino acid ‘minighrelin’ upon expression and maturation (Seim et al., 2016). Minighrelin exhibits similar biological activity to full length ghrelin at the cell and organism level, supporting the potential for ghrelin splice variants to play significant roles in ghrelin signaling.

Figure 1. Ghrelin maturation and processing. Following several proteolytic steps and octanoylation by GOAT, ghrelin is secreted into the bloodstream where it can undergo deacylation via esterases in circulation. The dotted arrow (left) reflects the potential for acylation of desacyl ghrelin in bone marrow adipocytes as recently reported (Hopkins et al., 2017).

While this processing pathway for ghrelin maturation and acylation has been well established, a recent study suggests a potential new level of regulation within ghrelin signaling (Hopkins et al., 2017). Hopkins and co-workers demonstrated that both unacylated and acylated ghrelin promote bone marrow adipogenesis in mice in the presence of GOAT, with these effects absent in GOAT knockout mice. Their study also identifies new cellular localization of GOAT including both large and small lipid-trafficking vesicles as well as the plasma membrane (Hopkins et al., 2017). This is the first evidence of GOAT localization beyond the ER (Taylor et al., 2013). The observed GOAT localization, fatty acid availability in bone marrow tissue, and the observed effects of desacyl ghrelin treatment on adipogenesis in bone marrow support a model wherein unacylated ghrelin in circulation can be reacylated at the downstream cellular site of ghrelin signaling (Hopkins et al., 2017).

Ghrelin interactions within the body

GHS-R1a receptor

Ghrelin’s discovery occurred during the search for the endogenous ligand for the GHS-R1a receptor following its cloning and expression, with this receptor initially linked to growth hormone secretion (Howard et al., 1996). Studies of this orphan receptor initially utilized synthetic peptide growth hormone secretagogues to investigate receptor signaling and function (Callaghan & Furness, 2014; Howard et al., 1996). Ghrelin was identified as the endogenous ligand for the GHS-R1a receptor in 1999 (Kojima et al., 1999), at which time the functional necessity of ghrelin octanoylation for receptor binding and activation was realized (Figure 2). Structure-activity analysis of ghrelin mimetic peptides modified with variable length acyl groups established that the GHS-R1a receptor requires the first five amino acids of ghrelin for recognition and activation equivalent to that observed with full-length ghrelin (Bednarek et al., 2000). The first four amino acids of ghrelin bound weakly to the receptor but yielded activation nearly equivalent to ghrelin, with complete receptor activation also requiring a large hydrophobic group at the Ser-3 equivalent position (Bednarek et al., 2000). Receptor activation can be achieved using ligands bearing medium to long acyl chains (up to 16 carbons) at the equivalent position to Ser-3, with reduced efficiency observed for acyl chains with less than 7 carbons (Bednarek et al., 2000). In addition to its signaling role, GHS-R1a mediates ghrelin uptake and degradation via endosomal processing followed by receptor recycling to the plasma membrane (Camina et al., 2004). In light of the potential of ghrelin signaling as a therapeutic target, there have been sustained efforts to develop agonists, antagonists, and inverse agonists targeting the GHS-R1a receptor. Several of these molecules have now progressed to clinical investigations, as discussed in several recent reviews of the topic (Avau et al., 2013; Cameron et al., 2014; Chollet et al., 2009; McGovern et al., 2016).

Figure 2. Ghrelin interacts with multiple partners in circulation. Ghrelin and desacyl ghrelin can interact with multiple binding partners in the bloodstream (equilibrium arrows), with esterases converting ghrelin to desacyl ghrelin by serine ester hydrolysis (single arrows). In interaction with lipoproteins (VLDL, HDL, and LDL), desacyl ghrelin shows a preference for binding to HDL (bold).

Lipoproteins in circulation

Both ghrelin and desacyl ghrelin lacking the serine octanoyl ester bind to lipoproteins in the blood plasma, with an increased abundance of cholesterol lipoproteins in the bloodstream leading to higher levels of ghrelin supporting the potential for these particles to act as ghrelin transporters (Purnell et al., 2003). The two different forms of ghrelin in circulation exhibit distinct binding behaviors to serum lipoproteins, with acylated ghrelin binding VLDL, LDL and HDL equally while desacyl ghrelin exhibits preferential binding to HDL (Figure 2) (De Vriese et al., 2007; Holmes et al., 2009). The binding of both ghrelin and desacyl ghrelin to HDL suggests these HDL-ghrelin complexes can serve as a ghrelin reservoir, effectively immobilizing circulating ghrelin in the bloodstream (Purnell et al., 2003). The interaction of ghrelin with HDL-associated esterases such as paraoxonase 1 (PON1) also accelerates its deacylation and degradation (Beaumont et al., 2003). Lipoprotein binding and associated enzymatic deacylation broadens the array of biological interactions regulating ghrelin trafficking, with the impact of these processes on ghrelin signaling remaining largely unexplored.

Ghrelin autoantibodies

Ghrelin-reactive autoantibodies are a recently discovered part of the body’s native immune system with recent evidence that these antibodies can be stored and released upon physiological stimulus and need (Fetissov et al., 2017). Ghrelin-reactive IgG was identified in human plasma by Takagi and co-workers, who proposed these autoantibodies stabilize ghrelin in the plasma by reducing serine ester hydrolysis and peptide degradation (Figure 2) (Takagi et al., 2013). In addition to protecting ghrelin from enzymatic deacylation, ghrelin autoantibodies are reported to enhance the ability of ghrelin to stimulate appetite (Francois et al., 2016a, 2016b; Takagi et al., 2013). Ghrelin-reactive IgG in obese individuals was found to exhibit higher affinity for ghrelin compared to ghrelin-reactive IgG from lean (non-obese) patient serum, which was proposed to enhance ghrelin trafficking and stability in obese patients (Francois et al., 2016a; Takagi et al., 2013). In contrast, individuals with anorexia nervosa have lower plasma levels of ghrelin autoantibodies and exhibit lower affinity ghrelin-IgG complexes which could lead to inefficient ghrelin signaling through a loss of effective ghrelin stabilization (Fetissov et al., 2017). As with the ghrelin-lipoprotein interactions described above, the impact of autoantibody binding on ghrelin trafficking and stabilization remains an active area of inquiry.

Ghrelin esterases

While in circulation, the octanoyl serine ester required for ghrelin binding and activation of the GHS-R1a receptor has a limited lifetime before removal by enzymatic hydrolysis to yield desacyl ghrelin (Figure 2) (De Vriese et al., 2004). Many protein components of human serum and tissues have been demonstrated to exhibit ghrelin deacylation ability, including platelet activating factor (PAF), paraoxanase (PON), carboxypeptidase, butyrylcholinesterase, carboxylesterases, APT1, and alpha 2-macroglobulin (Dantas et al., 2011; De Vriese et al., 2004, 2007; Eubanks et al., 2011; Satou et al., 2010). Butyrylcholinesterase, carboxylesterases and alpha 2-macroglobulin have been identified as potential ghrelin esterases in rat blood serum (De Vriese et al., 2004; Eubanks et al., 2011). Ghrelin deacylation was reduced in human and rat serum following the addition of phenylmethylsulfonyl fluoride or water-soluble derivatives thereof that serve as protease and esterase inhibitors (Delhanty et al., 2015; De Vriese et al., 2004). Subsequent studies have explored additional treatments to stabilize ghrelin acylation in serum, with a recent report using an alkyl fluorophosphonate reagent demonstrating rapid and complete protection of ghrelin from deacylation in biological samples (McGovern-Gooch et al., 2016).

Several recent studies have supported butyrylcholinesterase (BChE) as a potential ghrelin esterase within human circulation (Brimijoin et al., 2016; Chen et al., 2015, 2017; Schopfer et al., 2015). BChE composes a large proportion of total esterase activity in human serum, and recombinantly expressed BChE can hydrolyze ghrelin to desacyl ghrelin (Schopfer et al., 2015; Yao et al., 2016). BChE knock-out mice challenged with a high fat diet exhibit larger weight gain compared to control mice, with associated implications for BChE expression involvement in body energy regulation and weight control (Chen et al., 2015; Li et al., 2008; Schopfer et al., 2015). While these studies support the potential for BChE to act as a ghrelin esterase in human circulation, studies of human populations with BChE activity deficiencies such as the Vysya community suggest another esterase can either compensate for loss of BChE-catalyzed ghrelin hydrolysis or serve as the predominant ghrelin esterase (Manoharan et al., 2006, 2007). Defining the esterase-catalyzed deacylation limb of the ghrelin signaling pathway in circulation remains an important challenge in understanding the regulation of ghrelin-dependent processes at the organismal level.

Ghrelin O-acyltransferase: Catalyzing a unique protein modification required for biological signaling

As described above, ghrelin undergoes a unique posttranslational modification – serine octanoylation – during its maturation process (Figure 1). While intriguing as a matter of protein biochemistry, this modification carries immense biological and physiological significance as octanoylation of Ser-3 is essential for ghrelin to bind and activate its cognate receptor (Kojima et al., 1999; Muller et al., 2015). The enzyme that catalyzes this modification, ghrelin O-acyltransferase (GOAT), was reported in 2008 by two research groups (Gutierrez et al., 2008; Yang et al., 2008a). GOAT is an integral membrane protein found most prominently in the ER (Taylor et al., 2013), and is a member of the MBOAT (membrane-bound O-acyltransferase) enzyme superfamily (Hofmann, 2000). GOAT is a topologically complex membrane protein containing 11 predicted transmembrane helices and one reentrant loop, with the topology of the C-terminal ‘MBOAT domain’ of GOAT matching closely that determined for fellow MBOAT member Hedgehog acyltransferase (Hhat) (Konitsiotis et al., 2015; Matevossian & Resh, 2015; Taylor et al., 2013). GOAT has proven resistant to purification in active form (Barnett et al., 2010; Taylor et al., 2013), which has complicated biochemical studies of GOAT-catalyzed ghrelin octanoylation.

GOAT substrate selectivity and potential catalytic domains

The first study of ghrelin recognition by GOAT utilized mutagenesis of the proghrelin precursor and determined that several residues near the N-terminus of proghrelin are crucial for effective octanoylation (Yang et al., 2008a, 2008b). Several early investigations established the ability of ghrelin mimetic short peptides to serve as GOAT substrates and determined that the N-terminal sequence of ghrelin/proghrelin is essential for recognition by GOAT (Ohgusu et al., 2009; Yang et al., 2008b). Studies by our group and others have used these synthetic peptide substrates to characterize GOAT substrate selectivity, confirming the importance of the N-terminal sequence of ghrelin and establishing the contribution of each side chain in the first four amino acids of ghrelin for recognition by GOAT (Barnett et al., 2010; Darling et al., 2013, 2015; Taylor et al., 2015). There is some tolerance for modification at the site of acylation as the mouse and human isoforms of GOAT accept substrates containing a threonine at the third residue, and bullfrog ghrelin has been confirmed to be octanoylated at a threonine (Darling et al., 2015; Kaiya et al., 2001, 2006, 2011; Yang et al., 2008b). In addition to its natural activity modifying a hydroxyl group, GOAT can catalyze modification of a substrate containing an amine at the third residue of a ghrelin mimetic peptide, resulting in an octanamide modification (Taylor et al., 2015). Regions of ghrelin downstream of the N-terminal ‘GSSF’ sequence may also interact with GOAT, although these downstream interactions appear to play a much smaller role in ghrelin binding to GOAT (Darling et al., 2015; Taylor et al., 2015). Utilizing the GOAT substrate selectivity preferences defined in these studies, bioinformatics analysis of the human proteome established that it is likely that ghrelin serves as the only substrate for GOAT in humans (Darling et al., 2015).

While ghrelin is predominantly modified by octanoic acid (Hosoda et al., 2003; Kojima et al., 1999), the mouse and human forms of GOAT can accept a range of acyl CoA substrates (Gutierrez et al., 2008; Nishi et al., 2005; Ohgusu et al., 2009; Yoh et al., 2011). Acyl donors with long carbon chains do not serve as efficient substrates for GOAT (Ohgusu et al., 2009; Yang et al., 2008b), unlike other MBOAT family members (Buglino & Resh, 2012; Chang & Magee, 2009; Hofmann, 2000). The preference of GOAT for medium-chain fatty acids is unique among MBOAT family members and provides a potential element for increasing GOAT inhibitor specificity. Inhibition studies using a series of acylated ghrelin mimetic peptides suggest that the acyl chain binding pocket within GOAT is composed of two distinct regions, with GOAT most strongly binding a peptide bearing an octanoyl group (Darling et al., 2015).

While the location and nature of the active site and substrate binding sites within GOAT remain undefined, several lines of investigation have provided insights into the regions and residues within GOAT that are essential for catalytic function. Sequence conservation analysis of MBOAT family members suggested conserved and potentially required catalytic residues essential for enzyme function, with subsequent studies of GOAT and Hhat indicating a shared membrane topology within this C-terminal region (Konitsiotis et al., 2015; Matevossian & Resh, 2015; Taylor et al., 2013). Photocrosslinking studies using acylated ghrelin mimetic peptides similarly localize ghrelin binding to the C-terminal half of GOAT (Barnett et al., 2010; Taylor et al., 2013, 2015). At the amino acid level, Asn 307 is highly conserved and His 338 is absolutely conserved within the MBOAT superfamily and mutation of either residue abrogates acylation activity (Gutierrez et al., 2008; Hofmann, 2000; Taylor et al., 2015; Yang et al., 2008b). However, the predicted topology of GOAT places these two residues on opposite sides of the ER membrane, suggesting that both cannot be directly involved in catalysis (Taylor et al., 2013). A recent study provided evidence that GOAT contains a functionally essential cysteine residue, as N-ethylmaleimide treatment inhibits the human isoform of GOAT (McGovern-Gooch et al., 2017). Our understanding of the structural and mechanistic basis for GOAT-catalyzed ghrelin octanoylation has expanded rapidly in the last decade, but many of the aspects of GOAT enzymatic function remain to be determined.

Towards modulating ghrelin signaling for therapeutic effect: Desacyl ghrelin mimics and GOAT inhibitors

Ghrelin signaling has been linked to multiple physiological functions impacting health and disease, including diabetes, obesity, symptoms of Prader-Willi syndrome, psychological stress and anxiety, depression, aging and neuroprotection, taste sensitivity and reward-seeking behavior, sleep regulation and deprivation, cardiac health and function, gastric mobility and acid secretion, and protection against muscle atrophy (Anderwald et al., 2003; Andrews et al., 2009; Barim et al., 2009; Broglio et al., 2001; Celi et al., 2005; Chuang & Zigman, 2010; Cummings et al., 2002; Erdmann et al., 2005; Falken et al., 2010; Heppner et al., 2012; Kamegai et al., 2001; Kurt et al., 2007; Kweh et al., 2015; Lutter et al., 2008; Mager et al., 2006; Moon et al., 2009; Murray et al., 2005; Muller et al., 2015; Poykko et al., 2003; Shiiya et al., 2002; Spencer et al., 2015; Tack et al., 2006; Tschop et al., 2000; Tong et al., 2010; Van der Ploeg et al., 2014). Modulating the ghrelin pathway presents a potential therapeutic avenue for treating these disorders and diseases. While significant work has focused on controlling ghrelin signaling using molecules targeting the GHS-R1a receptor (Cameron et al., 2014; McGovern et al., 2016), recent studies have shifted focus to ghrelin and ghrelin acylation as potential therapeutic targets. We discuss two approaches currently being explored for altering ghrelin signaling – analogs of desacyl ghrelin and GOAT inhibitors.

Analogs of desacyl ghrelin: AZP-531 and CF801

Although initially thought to play no biological role, desacyl ghrelin demonstrates an antagonistic effect with ghrelin in improving insulin sensitivity and decreasing availability of free fatty acids (Barazzoni et al., 2007; Benso et al., 2012; Broglio et al., 2004; Cederberg et al., 2012; Delhanty et al., 2013, 2015; Delhanty & van der Lely, 2011; Heppner et al., 2014; Hosoda et al., 2000; Lear et al., 2010; Stevanovic et al., 2014; Togliatto et al., 2015). Microarray expression studies also support desacyl ghrelin involvement in lipid metabolism and regulation through gene regulation in fat, muscle, and liver cells (Delhanty et al., 2010). Investigation of truncated analogs of desacyl ghrelin led to the development of a biologically active linear peptide composed of residues 6–13 of desacyl ghrelin, with circularization of this sequence generating a compound with increased biostability and improved pharmacokinetics named AZP-531 (Figure 3) (Delhanty et al., 2013; Julien et al., 2012). Mice treated with AZP-531 exhibited increased insulin sensitivity, reduced glucose intolerance, and reduced fat accumulation when challenged with a high-fat diet (Delhanty et al., 2013). In a Phase I clinical trial, AZP-531 was well-tolerated and resulted in metabolic improvements. Subjects with impaired glucose tolerance treated with AZP-531 exhibited decreased glucose concentrations, and obese subjected exhibited greater weight loss compared to placebo controls, despite receiving identical meals (Allas et al., 2016). In a 2016 press release describing the top-line results of phase II clinical trial investigating AZP-531 as a treatment for patients with Prader-Willi syndrome, Alizè Pharma reported patients treated with AZP-531 reported reduced appetite and showed improved glucose control compared to those in the placebo group.

Figure 3. Desacyl ghrelin mimics. (a) The CF801 inhibitor consists of the first 10 amino acids of ghrelin with an S3A mutation appended to an HIV Tat sequence. (b) The Alizè pharmaceutical AZP-531 is a circularized peptide consisting of ghrelin residues 6–13.

In another approach to develop an agent capable of lowering ghrelin levels in circulation, Wellman and co-workers designed the desacyl ghrelin analog CF801 (Figure 3) (Wellman et al., 2015). This analog consists of the first 10 amino acids of ghrelin with an alanine mutation at the octanoylation site and an attached Tat sequence to enable cell permeability. Intriguingly, CF801 does not serve as a GOAT inhibitor. It does not block ghrelin octanoylation in recombinant enzyme-based assays but CF801 treatment decreased acylated ghrelin levels in SG-1 cells (Wellman et al., 2015; Zhao et al., 2010). Administration of CF801 also decreased weight gain in mice fed a high-fat diet. The biological target of this peptide is not clear, but the beneficial effects observed with both AZP-531 and CF801 treatment provides support for examining desacyl ghrelin analogs for potential therapeutic applications.

GOAT inhibitors

As noted above, ghrelin must be octanoylated in order to bind and active the GHS-R1a receptor. Therefore, blocking GOAT-catalyzed ghrelin octanoylation presents an attractive option for modulating ghrelin signaling. With ghrelin predicted to be the only substrate for GOAT within the human proteome (Darling et al., 2015), inhibition of GOAT acylation activity also carries a reduced likelihood of impacting the modification of other proteins leading to undesired off-target side-effects. The lack of structural and mechanistic information about the GOAT active site and substrate binding sites has made rational design of GOAT inhibitors problematic, but several classes of GOAT inhibitors have been described in the scientific and patent literature in recent years. The reported GOAT inhibitors can be sorted into three classes which will be discussed in detail below: acylated ghrelin/product mimetics, bisubstrate analogs, and small molecules.

Product-mimetic inhibitors: [Dap³]octanoyl-ghrelin(1–5)-NH_2, [Dap³]octanoyl-ghrelin(1–28)-NH₂ and triazole-linked acylated ghrelin analogs

Immediately following the initial identification of GOAT as the enzyme responsible for octanoylating ghrelin, Yang and co-workers developed potent GOAT inhibitors by replacing the hydrolytically susceptible ester linkage of octanoylated ghrelin with an amide linkage (Yang et al., 2008b). In these molecules, the Ser-3 residue of both full-length ghrelin (amino acids 1–28) and a truncated form of ghrelin (amino acids 1–5) were replaced with octanoylated (S)-2, 3-diaminopropionic acid (Dap) (Figure 4). In assays measuring radiolabeled [H³]octanoate transfer to His-tagged proghrelin by GOAT in the microsomal fraction, both [Dap³]octanoyl-ghrelin(1-28)-NH₂ and [Dap³]octanoyl-ghrelin(1-5)-NH₂ effectively inhibited proghrelin octanoylation. Using variations of the shorter [Dap³]octanoyl-ghrelin(1-5)-NH₂ inhibitor, Darling and co-workers demonstrated that an 8-carbon acyl group is required for maximum potency against GOAT (Darling et al., 2015). While useful for investigating GOAT activity in enzyme-based assays (Darling et al., 2013, 2015; Yang et al., 2008b), these inhibitors have limited therapeutic potential due to two factors: (1) as peptides, these molecule are unlikely to exhibit high cell permeability; and (2) mimics of octanoylated ghrelin are likely to bind to and activate GHS-R1a, thus contravening the rationale for inhibiting ghrelin acylation. The receptor requires only the first four residues of ghrelin and a hydrophobic moiety at position 3 for equivalent receptor agonism to full-length ghrelin (Bednarek et al., 2000).

Figure 4. Peptide mimetic GOAT inhibitors.

The lipid chain mimicking acyl ghrelin’s octanoyl modification is important for GOAT inhibitor potency (Darling et al., 2015; Yang et al., 2008b). Inspired by the [Dap³]-ghrelin(1-5)-NH₂ inhibitor, Zhao and co-workers replaced the amide linkage to the lipid chain with a bioisosteric 1,2,3-triazole linkage to an phenyl group through a short alkyl chain (Figure 4) (Zhao et al., 2015). Triazoles, in addition to being stable against hydrolysis, also allow for the synthesis of a diverse panel of inhibitor candidates using ‘click’ chemistry (Holub & Kirshenbaum, 2010). In a microsomal GOAT activity assay, these phenylalkyl triazole peptidomimetics inhibited GOAT activity, with the most potent exhibiting an IC₅₀ of 0.7 μM. Varying the alkyl chain length yielded a similar structure-activity profile to that observed with amide-linked acylated ghrelin mimics (Darling et al., 2015).

Bisubstrate analog: GO-CoA-Tat

Barnett and co-workers designed a bisubstrate analog combining chemical aspects of ghrelin and octanoyl-CoA as a potential GOAT inhibitor based on the success of similar approaches in creating inhibitors targeting histone acyl transferase (Barnett et al., 2010; Lau et al., 2000; Parang et al., 2001). The bisubstrate analog, named GO-CoA-Tat, couples the first 10 amino acids of ghrelin to octanoyl CoA via a hydrolytically stable amide linkage (Figure 4). GO-CoA-Tat also includes an 11-amino acid Tat peptide appended to the C-terminus of the ghrelin sequence to increase cell permeability. Importantly, despite including the first 10 residues of ghrelin and an octanoyl chain GO-CoA-Tat does not activate the GHS-R1a receptor (Barnett et al., 2010).

In both microsomal enzyme assays and cell studies, GO-CoA-Tat inhibited production of acylated ghrelin at micromolar or lower concentrations. In animal studies, mice treated with GO-CoA-Tat show decreased levels of serum acyl ghrelin, resistance to weight gain when fed a medium-chain triglyceride-rich high-fat diet and a significant increase of insulin in response to a glucose challenge (Barnett et al., 2010). In subsequent studies, GO-CoA-Tat treatment decreased fasting-induced food foraging and hoarding in hamsters and reduced meal frequency in rats (Teubner et al., 2013; Teuffel et al., 2015). While the in vivo utility and pharmaceutical potential of GO-CoA-Tat is limited by its susceptibility to proteolytic degradation and its high molecular weight (∼3600 Da), studies using GO-CoA-Tat remain the best reported data supporting inhibition of ghrelin octanoylation as a therapeutic avenue for treating conditions impacted by ghrelin signaling.

Small molecule GOAT inhibitors

The first small molecule inhibitors of GOAT were identified by Garner and Janda in 2011 using a screen of small library of lipidated molecules in a microsomal GOAT activity assay (Figure 5) (Garner & Janda, 2010, 2011). The most potent of these molecules exhibited half-maximal inhibitory concentrations in the single to tens of micromolar concentration range. The presence of medium-chain length alkyl chains in these compounds suggests their inhibitory activity could derive from competing for the octanoyl CoA binding site. While these inhibitors presaged the potential for small molecule GOAT inhibitors, no studies of their effectiveness in cells or animal studies have been reported.

Figure 5. Small molecule GOAT inhibitors. Small molecule GOAT inhibitors currently reported in the scientific and patent literature; molecules in the top row are described in peer-reviewed publications and molecules in the bottom row are reported in patent applications or issued patents.

A number of small-molecule GOAT inhibitors have been reported by both academic and industrial research groups in the patent literature. Working from a proposed mechanism for ghrelin acylation including a classical tetrahedral intermediate, Harran and co-workers proposed a series of small molecule transition state analogs mimicking this structure (Harran et al., 2012). While originally intending to mimic the side chains of the serine and phenylalanine residues flanking the serine octanoylation site within ghrelin, their most potent inhibitor BK1114 lacks these functionalities (Figure 5). BK1114 inhibits GOAT activity at micromolar concentrations in a previously reported microsomal assay (Yang et al., 2008b).

Industrial researchers have also contributed to the small molecule GOAT inhibitor patent literature. In patents assigned to Takeda Pharmaceuticals, a number of small molecules featuring multiple aromatic rings are reported to potently inhibit GOAT activity using an ELISA-based high throughput assay for detection of acyl ghrelin (Figure 5) (Takakura et al., 2013). Two recently published patents assigned to Eli Lilly report substituted piperidyl-ethyl-pyrimidine derived GOAT inhibitors with demonstrated potency in enzyme-, cell-, and animal-base studies. The lead compound in this class was discovered using a high-throughput ELISA-based GOAT activity screening assay, and optimized inhibitors were reported to inhibit GOAT in the mid-nanomolar range in in vitro GOAT activity assays (Galka et al., 2016; Martinez-Grau, 2014).

In the most recent report of small molecule GOAT inhibitors, a class of molecules derived from synthetic triterpenoids was found to inhibit the human GOAT (hGOAT) ortholog (McGovern-Gooch et al., 2017). In screening a NIH-provided library of structurally diverse small molecules, two derivatives of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) were identified as hGOAT inhibitors with IC₅₀ values in the low micromolar range as assessed by a microsomal hGOAT activity assay (Figure 5) (Darling et al., 2013). Structure-activity analysis of these inhibitors revealed the α-cyanoenone Michael acceptor moiety in ring A was essential for inhibition. Further studies of these compounds and simpler derivatives containing Michael addition electrophiles support a covalent reversible inhibition mechanism for these molecules, implicating the presence of a functionally essential cysteine residue within hGOAT. Surprisingly, the mouse isoform of GOAT did not demonstrate the same susceptibility to these cysteine-modifying inhibitors, indicating an unexpected but important distinction between these two closely related enzyme orthologs.

These CDDO derivatives have been studied as potential therapeutics for inflammation and oxidative stress in multiple cell signaling pathways including the Nrf2 and NF-κB pathways (Liby & Sporn, 2012). In rodent and human studies, treatment with CDDO derivatives was reported to produce side-effects such as weight loss, reduced insulin resistance, and improved glucose tolerance (Camer et al., 2015; de Zeeuw et al., 2013; Dinh et al., 2015; Saha et al., 2010). These side-effects would be consistent with altered ghrelin signaling resulting from GOAT inhibition by CDDO derivatives, supporting future studies of the physiological impact of CDDO derivatives and similar molecules on ghrelin signaling.

Conclusions

Ghrelin is a unique peptide hormone impacting multiple physiological processes, playing a central role in energy metabolism while exerting effects on a wide range of systems within the body. Following its production and secretion into the bloodstream, ghrelin can interact with a number of different proteins including lipoproteins, autoantibodies, and ghrelin esterases prior to binding the GHS-R1a receptor. Many of the physiological effects of ghrelin are dependent upon activation of the ghrelin receptor GHS-R1a, which requires that ghrelin be acylated with a medium-chain fatty acid. A growing body of evidence supports a biological role for desacyl ghrelin as well, although less is known about the specific signaling pathways affected by desacyl ghrelin.

The enzyme responsible for ghrelin acylation, ghrelin O-acyltransferase (GOAT), is a member of the MBOAT family of enzymes including fellow protein modifying members PORCN and Hhat. With ghrelin as the unique substrate for GOAT within the human proteome, development of GOAT inhibitors has been identified as an exciting approach to modulating ghrelin-dependent signaling. Ranging from ghrelin-mimetic peptides to bisubstrate inhibitors to small molecules, increasing numbers of reported GOAT inhibitors in recent years justifies a growing enthusiasm in the ghrelin-GOAT system as a potential therapeutic target. Ongoing mechanistic and structural investigation of GOAT-catalyzed ghrelin acylation will further accelerate rational design of small molecule inhibitors for preclinical studies.

Ghrelin signaling is proposed to be connected to an ever-expanding list of diseases and disorders. Modulation of this pathway through GOAT inhibition is a promising strategy in treating these diseases. Defining the scope of ghrelin’s interactions within the body will help to identify possible approaches for controlling ghrelin signaling. Through a combination of these approaches, the ghrelin-GOAT system has great potential as a novel treatment avenue for diabetes, obesity, and other diseases impacted by ghrelin.

Acknowledgements

We thank members of the Hougland research group for discussions and helpful comments.

Disclosure statement

JLH has patent interests in the use of multiple compounds reported herein to target ghrelin signaling and associated health conditions. The other authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

We gratefully acknowledge funding support from the American Diabetes Association to JLH [1-16-JDF-042].