Identification of the Atypical L-Type Ca²⁺ Channel Blocker Diltiazem and Its Metabolites As Ghrelin Receptor Agonists

Abstract

Using a high-throughput functional screen, the atypical L-type Ca 2ϩ channel blocker diltiazem was discovered to be an agonist at the human ghrelin (GHSR1a) receptor. In cellular proliferation, Ca 2ϩ mobilization, and bioluminescence resonance energy transfer (BRET-2) assays, diltiazem was a partial agonist at GHSR1a receptors, with 50 to 80% relative efficacy compared with the GHSR1a peptide agonist GHRP-6, and high nanomolar to low micromolar potency, depending upon the assay. Seven of the known primary metabolites of diltiazem were synthesized, and three of them (M A , M 1 , and M 2 ) were more efficacious and/or more potent than diltiazem at GHSR1a receptors, with a rank order of agonist activity of M 2 Ͼ M 1 Ͼ M A Ͼ diltiazem, whereas M 4 and M 6 metabolites displayed weak agonist activity, and the M 8 and M 9 metabolites were inactive. Binding affinities of diltiazem and these metabolites to GHSR1a receptors followed a similar rank order. In vivo tests showed that diltiazem and M 2 each stimulated growth hormone release in male Sprague-Dawley neonatal rats, although to a lesser degree than GHRP-6. Thus, diltiazem and chemical analogs of diltiazem represent a new class of GHSR1a receptor agonists. The possible contributions of GHSR1a receptor activation to the clinical actions of diltiazem are discussed in the context of the known beneficial cardiovascular effects of ghrelin.Receptors that mediate the effects of growth hormone secretagogues (GHSs), including modified enkephalin peptides (Bowers et al., 1984) as well as small molecules (Smith et al., 1993), each capable of stimulating GH release, were only recently cloned (Howard et al., 1996). Two isoforms (A and B) were identified, but only growth hormone secretagogue receptor type 1a (GHSR1a) binds and responds to GHSs (Howard et al., 1996). Both isoforms are widely expressed, with the highest levels of GHSR1a found in the arcuate nucleus of the hypothalamus, and lower levels in cardiac tissue (Guan et al., 1997;Gnanapavan et al., 2002). Subsequently, a naturally occurring GHSR1a agonist was identified called ghrelin (Kojima et al., 1999).A great deal of evidence exists linking ghrelin, GHSR1a receptors, food intake, and obesity in both animal models (Tschöp et al., 2000;Nakazato et al., 2001;Asakawa et al., 2003;Shearman et al., 2006) and humans (Cummings et al., 2001(Cummings et al., , 2002Wren et al., 2001). However, ghrelin also regulates a variety of other endocrine and metabolic processes, including gut motility, energy homeostasis, cellular proliferation, and hormone production (for review, see van der Lely et al., 2004). It is noteworthy that ghrelin, and other GHS ligands produce a number of beneficial effects on cardiovasArticle, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.034298. ABBREVIATIONS: GHS

Ghrelin Through GHSR1a and OX1R Heterodimers Reveals a Gαs–cAMP-cAMP Response Element Binding Protein Signaling Pathway in Vitro

“…Fluorescence resonance energy transfer (FRET) assays were performed as described previously ( Ma et al, 2007 ; Cai et al, 2017 ). The EYFP-GHSR1a and ECFP-OX1R plasmids were co-transfected into HEK293 cells as the FRET system.…”