N-(3-Cyclohexylbutanoyl)-N-[3-(1H-imidazol-4- yl)propyl]guanidine (UR-AK57), a Potent Partial Agonist for the Human Histamine H1- and H2- Receptors

Both the histamine H1-receptor (H1R) and H2-receptor (H2R) exhibit pronounced species selectivity in their pharmacological properties; i.e., bulky agonists possess higher potencies and efficacies at guinea pig (gp) than at the corresponding human (h) receptor isoforms. In this study, we examined the effects of N-acylated imidazolylpropylguanidines substituted with a single phenyl or cyclohexyl substituent on H1R and H2R species isoforms expressed in Sf9 insect cells. N-(3-Cyclohexylbutanoyl)-N-[3-(1H-imidazol-4-yl)propyl]guanidine (UR-AK57) turned out to be the most potent hH2R agonist identified so far (EC50 of 23 nM in the GTPase assay at the hH2R-Gs fusion protein expressed in Sf9 insect cells). UR-AK57 was almost a full-hH2R agonist and only slightly less potent and efficacious than at gpH2R-Gs . Several N -acylated imidazolylpropylguanidines showed similar potency at hH2R and gpH2R. Most unexpectedly, UR-AK57 exhibited moderately strong partial hH1R agonism with a potency similar to that of histamine, whereas at gpH1R, UR-AK57 was only a very weak partial agonist. Structure/activity relationship studies revealed that both the alkanoyl chain connecting the aromatic or alicyclic substituent with the guanidine moiety and the nature of the carbocycle (cyclohexyl versus phenyl ring) critically determine the pharmacological properties of this class of compounds. Collectively, our data show that gpH1R and gpH2R do not necessarily exhibit preference for bulky agonists compared with hH1R and hH2R, respectively, and that UR-AK57 is a promising starting point for the development of both potent and efficacious hH1R and hH2R agonists. Histamine (HIS) (1) (see Fig. 1) is a neurotransmitter and autacoid and acts through H1-, H2-, H3-, and H4-receptors (H1R–H4R) (Hill et al., 1997; Hough, 2001; Bakker et al., 2002). The H1R couples to Gq-proteins to mediate phospholipase C activation and plays a role in the regulation of alertness and as mediator of type 1 allergic reactions (Hill et al., 1997; Bakker et al., 2002). The H2R couples to Gs-proteins to mediate adenylyl cyclase activation and regulates H secretion in gastric parietal cells, cardiac contractility, and various myeloid cell functions (Klinker et al., 1996; Hill et al., 1997; Bakker et al., 2002). It has been difficult to establish relevant native test systems for the analysis of the human H1R (hH1R) and human H2R (hH2R), because there are unexplained pharmacological differences in the properties of hH1R and hH2R in native cells relative to standard guinea pig test organs (Burde et al., 1990; Seifert et al., 1994; Klinker et al., 1996). To facilitate the comparison of histamine receptors under identical experimental conditions, we established expression systems for the H1R and H2R in Sf9 insect cells (Kelley et al., 2001; Houston et al., 2002). Sf9 cells express the H1R and H2R at high levels and can be cultured in large quantities. GPCR/Gprotein coupling in Sf9 membranes is monitored with high sensitivity using the steady-state GTPase assay. This assay This work was supported by the National Institutes of Health COBRE Award 1 P20 RR15563 and matching support from the State of Kansas and the University of Kansas (to R.S. and Q.-Z.Y.) and the Graduate Training Program (Graduiertenkolleg GRK 760, “Medicinal Chemistry: Molecular Recognition— Ligand-Receptor Interactions”) of the Deutsche Forschungsgemeinschaft (to R.S., S.E., and A.B.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.102897. ABBREVIATIONS: HIS, histamine; UR-AK57, N-(3-cyclohexylbutanoyl)-N-[3-(1H-imidazol-4-yl)propyl]guanidine; AIPG, N-acylated imidazolylpropylguanidine; GPCR, G-protein-coupled receptor; gpH1R, guinea pig histamine H1-receptor; gpH2R, guinea pig histamine H2-receptor; gpH2R-Gs S, fusion protein of the guinea pig histamine H2-receptor and the short splice variant of Gs ; hH1R, human histamine H1-receptor; hH2R, human histamine H2-receptor; hH2R-Gs S, fusion protein of the human histamine H2-receptor and the short splice variant of Gs ; HIS, histamine; RGS, regulator of G-protein signaling; TM, transmembrane domain. 0022-3565/06/3173-1262–1268$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 317, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 102897/3117058 JPET 317:1262–1268, 2006 Printed in U.S.A. 1262 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from assesses GPCR/G-protein coupling at a proximal point of the signaling cascade, avoiding potential bias introduced by assessing more downstream events such as effector activation or changes in gene expression. For the H1R, coupling of the GPCR to insect cell Gq-proteins is determined, and the GTPase signal is amplified by RGS proteins (Houston et al., 2002; Seifert et al., 2003). For the H2R, fusion proteins of GPCR and mammalian Gs proteins ensure defined 1:1 stoichiometry of the coupling partners and their efficient interaction (Seifert et al., 1999; Kelley et al., 2001). By measuring GTP hydrolysis, potencies and efficacies of H2R agonists are assessed in an expression level-independent manner (Seifert et al., 1999; Kelley et al., 2001; WenzelSeifert et al., 2001). Both H1R and H2R agonists are important pharmacological tools for studying the role of the H1R and H2R, respectively, in (patho)physiological processes (Bakker et al., 2002; Dove et al., 2004; Pertz et al., 2004). H1R agonists are divided into three classes: 1) small agonists derived from HIS, such as 2-methylhistamine and 2-(2-thiazolyl)ethanamine, 2) HIS derivatives with a bulkier aromatic substituent at position 2 of the imidazole ring, such as 2-(3-bromophenyl)histamine, and 3) the histaprodifens (Bakker et al., 2002; Pertz et al., 2004). Unfortunately, bulky H1R agonists exhibit considerably lower potency and efficacy at the hH1R than at the guinea pig H1R (gpH1R), limiting their usefulness as tools for studying the hH1R (Seifert et al., 2003). The molecular basis for the differences in pharmacological properties between hH1R and gpH1R has recently been elucidated (Bruysters et al., 2005). A further complication is that, at concentrations in the range of 10 M to 1 mM, 2-phenylhistamines may activate G-proteins directly, i.e., in a receptor-independent manner (Seifert et al., 1994; Hagelüken et al., 1995; Klinker et al.,