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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy Biochemical Pharmacology 85 (2013) 1534–1541 Contents lists available at SciVerse ScienceDirect Biochemical Pharmacology jo urnal homepage: www.elsevier.com/locate/biochempharm a1-Adrenoceptor and serotonin 5-HT1A receptor affinity of homobivalent 4-aminoquinoline compounds: An investigation of the effect of linker length a a a b Junli Chen , Ahsan K. Murad , Laurence P.G. Wakelin , William A. Denny , a a, Renate Griffith , Angela M. Finch * a Department of Pharmacology, School of Medical Sciences, UNSW, Sydney, NSW 2052, Australia b Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, University of Auckland, 1142, New Zealand A R T I C L E I N F O A B S T R A C T Article history: a1-adrenoceptor (a1-AR) subtype-selective ligands lacking off-target affinity for the 5-HT1A receptor (5- Received 18 January 2013 HT1A-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign Accepted 13 March 2013 prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven Available online 19 March 2013 homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2–12 atoms (C2–C12) for a1A, a1B and a1D -ARs and the 5-HT1A-R. These ligands are a1A-AR antagonists with nanomolar affinity Keywords: for a1A and a1B -ARs. They display linker-length dependent selectivity for a1A/B -ARs over a1D-AR and the GPCR 5-HT1A-R. The C2 diquinoline has the highest affinity for a1A-AR (pKi 7.60 Æ 0.26) and greater than 30-fold a1-Adrenergic receptor and 600-fold selectivity for a1A-AR over a1D-AR and 5-HT1A-R respectively. A decrease in affinity for a1-ARs is Subtype selectivity observed as the linker length increases, reaching a nadir at 5 (a1A/1B-ARs) or 6 (a1D-AR) atoms; after which Serotonin receptor Diquinoline affinity increases as the linker is lengthened, peaking at 9 (a1A/1B/1D-ARs) or 8 (5-HT1A-R) atoms. Docking Linker length studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited a1D-AR and 5-HT1A-R affinity of these compounds makes them promising leads for future drug development of a1A-AR selective ligands without a1D-AR and the 5-HT1A-R off-target activity. ß 2013 Elsevier Inc. All rights reserved. 1. Introduction subtype in the urogenital tract) [1]. Antagonism of the a1-AR prevents the contraction of the smooth muscle of the prostate a1-adrenoceptors (a1-ARs) are members of the seven-trans- gland and the bladder neck, thus decreasing lower urinary tract membrane-spanning G-protein-coupled receptor (GPCR) super- symptoms of BPH [1]. The a1-AR antagonists that are used family, and exist as three distinct subtypes: a1A, a1B and a1D. a1- currently have similar therapeutic outcomes but have different ARs respond to the endogenous catecholamines, norepinephrine side effect profiles due to the lack of subtype selectivity, and off- and epinephrine, and play a vital role in numerous physiological target affinity. For example, alfuzosin and terazosin have equal functions predominantly involving smooth muscle contraction, affinity for each of the a1-AR subtypes: this can result in which makes them a therapeutic target for several urogenital cardiovascular side effects such as hypotension mediated by the conditions, such as benign prostatic hyperplasia (BPH) and stress a1B-AR, the predominant subtype in the blood vessels of older men urinary retention [1,2]. BPH is a widespread condition in males [3]. In contrast, tamsulosin and naftopidil have higher affinity for over 60 with the incidence of this condition increasing as men age. a1A and a1D-ARs than for a1B-AR and hence reduced cardiovascu- One of the most effective treatments for BPH is therapy with a1-AR lar side effects. However, they also have nanomolar affinity for the antagonists (ideally a1A-AR antagonists, as this is the predominant serotonin 5-HT1A receptor (5-HT1A-R), and tamsulosin has nanomolar affinity for the dopamine D3 receptor (D3-R) [4,5]. The high off-target affinity of tamsulosin and naftopidil has been * Corresponding author at: School of Medical Sciences, Wallace Wurth Building, implicated in floppy iris syndrome and ejaculatory dysfunction University of New South Wales, NSW 2052, Australia. Tel.: +61 293851325. side effects [4,6]. E-mail addresses: [email protected] (J. Chen), [email protected] Previously, a series of homobivalent 4-aminoquinoline com- (A.K. Murad), [email protected] (Laurence P.G. Wakelin), pounds (diquinolines) was shown to have high affinity for rat [email protected] (W.A. Denny), r.griffi[email protected] (R. Griffith), a.fi[email protected] (A.M. Finch). a1-adrenoceptors [7,8], with tissue-specific differences. However, 0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2013.03.010 Author's personal copy J. Chen et al. / Biochemical Pharmacology 85 (2013) 1534–1541 1535 at that time the a1-AR had not been classified into the current three 2.3. Radioligand binding assay subtypes, therefore the subtype selectivity of these compounds was not determined. In this study, we find that the diquinolines The reaction mixtures for all binding experiments were show nano- to micro-molar affinity and a similar linker length- incubated at room temperature for 1 h, the reaction was affinity relationship for each a1-AR subtype. Five of the compounds terminated by the addition of PBS (4 8C) and vacuum filtration (C2, C3, C5, C6 and C12) have significantly higher affinity for a1A- through GF/B filters (Whatman, Maidstone, UK). Radioactivity was AR over a1D-AR and all compounds, with the exception of 4- measured by liquid scintillation counting. aminoquinoline, C7, and C3 amine, display significant selectivity for a1A-AR over 5-HT1A-R. 2.3.1. a1-AR binding All ligands and membranes were suspended in HEM buffer. In 2. Materials and methods saturation binding experiments, membranes containing each a1- 3 AR subtype were incubated with various concentrations of [ H] The diquinolines were prepared and isolated as their dihy- prazosin (0.125–16 nM) in a total volume of 200 mL. For drochloride salts as previously described [7,8]. C2, C3, C5 and C6 competition binding experiments, 1–4 mg of a1A and a1B -ARs diquinoline were dissolved in ultra-pure water (Millipore, Billerica, and 10–32 mg of a1D -AR membranes were incubated with 200 pM 3 MA, USA) at 10 mM, and C7, C8, C9, C10, C11 and C12 were of [ H] prazosin and increasing concentrations of test compounds solubilised at 10 mM in 90% dimethyl sulphoxide/water (DMSO). in a total volume of 200 mL. Non-specific binding was defined as These stock solutions were stored at À80 8C. Dulbecco’s modified binding in the presence of 100 mM phentolamine. Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin were purchased from Thermo Fisher 2.3.2. 5-HT1A-R binding 3 À1 Scientific (Scoresby, VIC, Australia). [ H] prazosin (85 Ci mmol ) All ligands and membranes were suspended in 50 mM Tris-HCl 3 À1 and [ H]-myo-inositol (20 Ci mmol ) were purchased from and 4 mM CaCl2, pH 7.4. In saturation binding experiments, 3 PerkinElmer (Waltham, MA, USA) and [ H]-OH-DPAT (226 Ci membranes containing 5-HT1A-R were incubated with varying À1 3 mmol ) from GE Healthcare (Uppsala, Sweden). Phentolamine concentrations of [ H]-OH-DPAT (0.5–16 nM) in a total volume of hydrochloride, serotonin hydrochloride, norepinephrine hydro- 200 mL. In competition binding experiments, 6 mg of 5-HT1A-R- 3 chloride, diethylaminoethyl-dextran (DEAE-dextran), lithium containing membranes were incubated with 1 nM of [ H]-OH- chloride (LiCl), formic acid, ammonium formate and chemicals DPAT and increasing concentrations of test compounds in a total used in buffered solutions (HEPES, EGTA and MgCl2) were bought volume of 200 mL. Non-specific binding was defined as binding in from Sigma–Aldrich (St. Louis, MO, USA), and Tris, CaCl2, NaCl, KCl, the presence of 10 mM serotonin. Na2HPO4, and KH2PO4 were purchased from Ajax Finechem (Taren Point, NSW, Australia). 2.4. Inositol phosphate accumulation assays 3 2.1. Cell culture and transient transfection Accumulation of total [ H] inositol phosphates (IPs) was 5 À1 determined as described previously [11]. Briefly, 1 Â 10 cells ml COS-1 cells (American Type Culture Collection, Manassas, VA, of transiently transfected COS-1 cells were seeded into 96 well plates USA) were cultured in DMEM supplemented with 10% (v/v) heat- and cultured overnight with DMEM supplemented with 10% À1 À1 inactivated FBS, 100 mg ml penicillin and 100 mg ml strepto- FBS. Cells were then washed with warmed PBS and labelled À1 3 mycin. COS-1 cells were maintained and passaged upon reaching overnight with 10 mCi ml [ H] myo-inositol in inositol-free confluence using standard cell culture techniques and replaced by DMEM supplemented with 5% FBS.