Subscriber access provided by UNIV OF OREGON
Article
Super-agonist, full agonist, partial agonist and antagonist actions of arylguanidines at h5-HT3A receptors Katie Alix, Shailesh Khatri, Philip D. Mosier, Samantha Casterlow, Dong Yan, Heather L Nyce, Michael M. White, Marvin K. Schulte, and Malgorzata Dukat ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00196 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 20, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
Super-agonist, full agonist, partial agonist and antagonist actions of arylguanidines at h5-HT3A receptors
Katie Alix,† Shailesh Khatri,‡ Philip D. Mosier,† Samantha Casterlow,† Dong Yan,§ Heather L. Nyce,§ Michael M. White,§ Marvin K. Schulte,‡ and Małgorzata Dukat*,†
†
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University,
Richmond, VA 23298, USA ‡
Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of Sciences,
Philadelphia, PA 19104, USA §
Department of Biochemistry and Molecular Biology Drexel University College of Medicine,
Philadelphia, PA 19102, USA
*Corresponding author. Tel.: 8048285256; Fax: 8048287625. E-mail address:
[email protected] (M. Dukat) Address: Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, PO Box 980540, Richmond, VA 23298-540, USA
ACS Chemical Neuroscience_ Revised _August 16, 2016
1
ACS Paragon Plus Environment
ACS Chemical Neuroscience
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Abstract Introduction of minor variations to the substitution pattern of arylguanidine 5-HT3 ligands resulted in a broad spectrum of functionally-active ligands from antagonist to super-agonist. For example: i) introduction of an additional Cl-substituent(s) to our lead full agonist N-(3chlorophenylguanidine (mCPG) (2; efficacy % = 106) yielded super-agonists 7–9 (efficacy % = 186, 139, and 129, respectively); ii) a positional isomer of 2, p-Cl analog 11, displayed partial agonist actions (efficacy % = 12); iii) replacing the halogen atom at the meta or para position with an electron donating OCH3 group or a stronger electron withdrawing (i.e., CF3) group resulted in antagonists 13–16. We posit based on combined mutagenesis, crystallographic, and computational analyses that for the 5-HT3 receptor, the arylguanidines that are better able to simultaneously engage the primary and complementary subunits, thus keeping them in close proximity, have greater agonist character while those that are deficient in this ability are antagonists.
Keywords: Arylguanidines, 5-HT3A receptors, functional activities, binding affinities, 3D-graphic models, site-directed mutagenesis, SAR
2
ACS Paragon Plus Environment
Page 2 of 36
Page 3 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
INTRODUCTION The homomeric 5-HT3A receptor is a member of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily. To date, five distinct 5-HT3 receptor subunits, A-E, are known. Antagonists of 5HT3 receptors are currently used in the treatment of chemotherapy- and radiation-associated nausea and vomiting. Partial agonists are being investigated for potential treatment of irritable bowel syndrome (IBS), whereas a therapeutic role for 5-HT3 receptor agonists has not yet been established.1 N-(3-Chlorophenyl)biguanide (mCPBG; 1) was one of the first selective 5-HT3 receptor agonists demonstrating utility for characterizing this receptor population. Nearly 20 years ago we showed that the structure of 1 could be truncated to N-(3-chlorophenyl)guanidine (mCPG; 2), and that mCPG (2) and a naphthyl counterpart, N-(2-naphthyl)guanidine (3), possessed 5-HT3 receptor agonist character.2 For example, both 2 and 3 were active in a von Bezold–Jarisch assay and rabbit bladder assay, and their actions were blocked using a 5-HT3 receptor antagonist.2 mCPG (2) also served as a training drug in drug discrimination studies using rats. The mCPG stimulus, generalized to compounds 1 and 3 as well as several other 5-HT3 receptor agonists, and was blocked by pretreatment of the animals with 5-HT3 receptor antagonists.3
Additional arylguanidines were prepared in order to optimize affinity and formulate structure–affinity relationships. Affinities of the analogs, depending upon the aryl substitution pattern and other structural
3
ACS Paragon Plus Environment
ACS Chemical Neuroscience
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
modifications, were found to span a >10,000-fold range.2,4,5 These arylguanidines were also employed in QSAR studies4,5 to better understand how they might interact at 5-HT3 receptors. Although a distinct possibility, there is no reason to suspect that structurally–related agonists and antagonists bind differently at a receptor. Given the premise that structurally–related agonists might, perhaps, have a greater likelihood of binding in a manner more similar to one another than to that of antagonists, and vice versa, it was important to determine the functional activity of 2-like agents. That is, subsequent structure–activity studies could focus on agonist analogs and antagonist analogs individually; this could provide useful information with regard to what structural feature(s) contribute to which action. Accordingly, we examined the functional activity of sixteen (2–17; Tables 1 and 2) arylguanidine analogs at 5-HT3A receptors expressed in Xenopus oocytes using a two–electrode voltage clamp assay. Here we show that by making minor variations to the substitution pattern of a small, unconstrained arylguanidine core scaffold, ligands with a broad spectrum of functional activity from antagonist to superagonist are obtained. We postulate, based on combined mutagenesis, crystallographic, and computational analyses, that for the 5-HT3 receptor, the arylguanidines that are better able to simultaneously engage the primary and complementary subunits, thus keeping them in close proximity, have greater agonist character while those that are deficient in this ability are antagonists.
RESULTS and DISCUSSION Ligand Synthesis. mCPBG (1) and arylguanidines 2, 3, 7–9 and 11–17 (Tables 1 and 2) were resynthesized as previously reported by us.2,4 The nitrate salt of arylguanidine 4 (Table 1) was prepared according to a literature procedure.6 Arylguanidine 5 (Table 1; Scheme 1) was prepared using our standard protocol for the synthesis of arylguanidines as depicted in Scheme 1. The free base of 6 (Table 1) was synthesized following the literature procedure7 and then converted to its nitrate salt. In short, the corresponding commercially available anilines (for 4–6) were converted to their HCl salts and heated at reflux in a condensation reaction with cyanamide in ethanol. The hydrochloride salts of the obtained 4
ACS Paragon Plus Environment
Page 4 of 36
Page 5 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Neuroscience
arylguanidines were converted to the nitrate salts (4–6) with ammonium nitrate. N-(3-Chloro-4hydroxyphenyl)guanidine hydrochloride (10) (Table 1) was synthesized by the literature method outlined in Scheme 1. The nitro intermediate, 19, was obtained by sonication of 2-chlorophenol (18) with nitric acid (9% aqueous).8 The nitro group was then reduced using sodium hydrosulfite and aqueous sodium hydroxide.9 An ethanolic solution of the HCl salt of aniline 20 was heated at reflux with cyanamide (50% aqueous) to give 10.10 Table 1 and 2 Effects of arylguanidines on 5-HT3 receptor currents. Both the endogenous ligand 5-HT, and mCPBG (1), produced concentration-dependent inward currents in Xenopus oocytes expressing h5-HT3A receptors (EC50 = 2.34 and 0.96 µM, respectively) (Table 1). The examined arylguanidines can be divided into two major groups: agonists and antagonists. Compounds 2–11, similar to 5-HT and mCPBG (1), produced concentration-dependent inward currents (Table 1; Figure S1). That is, all meta-substituted heavy halogen (Cl, Br, I; 2, 5 and 6, respectively) arylguanidine derivatives mimicked the action of 5-HT and mCPBG (1) with comparable efficacy (Table 1), whereas introduction of additional chloro-groups as seen in arylguanidines 7–9 resulted in substantially more-efficacious agonists, i.e. “super-agonists” (Table 1). A “super-agonist” is a ligand that elicits a maximal effect greater than that of the endogenous ligand. This phenomena was reported for one of the oldest and most studied LGICRs, nAChRs11,12 as well as for GPCRs.13 Table 3 shows the relative efficacy for each of the compounds 7−10 compared to the endogenous agonist 5-HT. Using unpaired t tests, 7− −9 were all shown to produce statistically significant increases in efficacy compared to 5-HT (p
