Acta Pharmacol Sin 2008 Jun; 29 (6): 752–758 Full-length article High-throughput screening of novel antagonists on melanin-concentrat- ing hormone receptor-11 Jian-hua YAN2, Qun-yi LI2, Jean A BOUTIN3, M Pierre RENARD3, Yi-xiang DING4, Xiao-jiang HAO5, Wei-min ZHAO2, Ming- wei WANG2,6 2The National Center for Drug Screening and the State Key Laboratory of New Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 3Les Laboratoires Servier, Neuilly-sur-Seine 92200, France; 4Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China; 5Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China Key words Abstract melanin-concentrating hormone; melanin- Aim: To find new antagonists on human melanin-concentrating hormone recep- concentrating hormone receptor-1; antagonist tor-1 (MCHR-1) through high-throughput screening (HTS) of a diverse com- 3 1Project supported in part by the Shanghai pound library. Methods: MCHR-1, [ H]SNAP7941, and FlashBlue G-protein- Municipality Science and Technology coupled receptor beads were used to measure the receptor-binding activities of Development Fund (No 06DZ22907 and various compounds based on scintillation proximity assay (SPA) technology. 07DZ22920), the Ministry of Science and 35 35 Technology (No 2004CB518902), and The guanosine 5' (γ-[ S]thio) triphosphate ([ S]GTPγS) binding assay was sub- Servier Beijing Pharmaceutical Research and sequently applied to functionally characterize the “hits” identified by the HTS Development Co, Ltd. campaign. Results: Of the 48 240 compounds screened with the SPA method, 6Correspondence to Dr Ming-wei WANG. Phn 86-21-5080-1313. 12 hits were confirmed to possess MCHR-1 binding activities, 8 were function- 35 Fax 86-21-5080-0721. ally studied subsequently with the [ S]GTPγS binding assay, and only 1 com- E-mail [email protected] pound (NC127816) displayed moderate human MCHR-1 binding affinity (K =115.7 nmol/L) and relatively potent antagonism (K =23.8 nmol/L). This Received 2008-01-22 i B Accepted 2008-03-13 compound shares a novel scaffold (1-ethoxy-2H-2-aza-1-phospha-naphthalene 1-oxide) with 3 other analogs in the group. Conclusion: Considering the marked doi: 10.1111/j.1745-7254.2008.00800.x difference in molecular shape and electrostatic status between NC127816 and the structures reported elsewhere, we anticipate that its derivatives may repre- sent a new class of potent MCHR-1 modulators. Introduction feeding and energy balance[4-6], sexual behavior[7], stress re- Melanin-concentrating hormone (MCH) is a 17-amino sponses[8], neuroendocrine functions[9-11], anxiety[7,12], sei- acid cyclic peptide originally isolated and sequenced from zure[13], memory and/or learning[14,15], grooming and locomo- the salmon pituitary in 1983. It was regarded as a regulator tor activities[16], and arousal regulation[17]. of the pigmentary changes in background adaptation[1]. Rat In 1999, 5 independent groups almost simultaneously MCH, purified from the hypothalamus, is identical to that of reported the identification of a MCH receptor [MCHR; humans, and is 19 amino acids in length, 2 amino acids longer MCHR-1, MCH-1R, MCH1, MCH1, or SLC-1 (orphan soma- than its salmon counterpart[2]. In mammalian species, MCH tostatin-like receptor 1)] through a reverse pharmacology is predominately synthesized in 2 brain centers, namely the approach[18-22]. A second MCH receptor (MCHR-2, MCH- perikarya of the lateral hypothalamus and zona incerta, and 2R, MCH2, MCH2, or SLT) was identified subsequently based becomes widely distributed throughout the central nervous on the sequence homology to MCHR-1[23-27]. However, the system (CNS). The extensive terminal distribution suggests functions of MCHR-2 remain unclear at present due to its that MCH may serve as a neurotransmitter or modulator in species-specific expression and the lack of non-rodent in regulating brain functions[3]. Previous studies have revealed vivo models for behavioral studies[28,29]. Therefore, the fo- that the biological effects of MCH are complex, including cus of the current research is directed towards MCHR-1. 752 ©2008 CPS and SIMM Http://www.chinaphar.com Yan JH et al The stimulation of MCHR-1 leads to the elevation of the proportional to the amount of bound radiolabeled ligands. intracellular Ca2+ level, inhibition of forskolin-stimulated cy- In this study, we describe a SPA-based HTS campaign in- clic adenosine monophosphate (cAMP) production, and volving a diverse library of 48 240 synthetic and natural activation of the mitogen-activated protein kinase cascade, compounds. Using the SPA technology, human MCHR-1 indicating that MCHR-1 is a G-protein-coupled receptor (hMCHR-1) binding affinities were determined with [3H] [30] (GPCR) linked to both Gi/o and Gq proteins . MCHR-1 is SNAP7941 competitive displacement. The functionality different from the limited expression of MCH in the lateral (agonist or antagonist activities) was subsequently assessed hypothalamus and zona incerta, as it is widely distributed by a guanosine 5' (γ-[35S]thio) triphosphate ([35S]GTPγS) bind- throughout the CNS, including the cerebral cortex, caudate- ing assay[41]. As a result, a novel hMCHR-1 antagonist was putamen, hippocampal formation, subiculum, the shell of the discovered together with a series of ligands previously un- nucleus accumbens, amygdala, hypothalamus and thalamus, reported for this receptor. the locus coeruleus of the brainstem, various nuclei of the mesencephalon and rhombencephalon, as well as most ana- Materials and methods tomical areas implicated in the control of olfaction, with the Reagents The radioligand [3H]SNAP7941 (specific activity: exception of the main olfactory bulb[31,32]. Based upon a se- 2.26 TBq/mmol) and [35S]GTPγS (specific activity: 37 TBq/ ries of studies using MCHR-1 antagonists and knock-out mice mmol) were purchased from Amersham (Buckinghamshire, UK). lacking MCHR-1, it has been established that MCHR-1 medi- FlashBlue GPCR beads were obtained from Perkin–Elmer ates feeding behavior and energy expenditure[33-35]. Experi- (Boston, MA, USA). Guanosine diphosphate (GDP), GTPγS, mental evidence also demonstrates that MCHR-1 plays an saponin, HEPES, glutamine, bovine serum albumin (BSA), important role in the regulation of mood and stress responses. and MCH are the products of Sigma–Aldrich (St Louis, MO, Furthermore, the pharmacological properties of MCHR-1 an- USA). Fetal bovine serum (FBS) was bought from Hyclone tagonists in rodent models of stress-related disorders (such [35,36] (Logan, UT, USA). Dulbecco’s modified Eagle’s medium as depression and anxiety) have been elucidated . (DMEM) with high glucose was the product of GIBCO BRL The discovery of the MCH receptors has prompted phar- (Grand island, NY, USA). SNAP7941 was provided by Servier macological scientists and behavioral biologists to evaluate (Neuilly-sur-Seine, France). their potential therapeutic applications. Both peptidic and Membrane preparation Human embryonic kidney cells non-peptidic MCHR-1 ligands have been reported, and the (HEK293) stably expressing the hMCHR-1 receptor were effects of more than 50 MCH analogs have been investi- cultured in DMEM with high glucose supplemented with gated to understand their structure–activity relationships 10% FBS, 2 mmol/L glutamine, 1×105 IU/L penicillin, 100 mg/L (SAR)[37]. Intracerebroventricular injection of these MCH streptomycin, and 400 mg/L G418. The cells were grown at analogs led to a rapid and significant increase in food intake, confluence, harvested in phosphate-buffered saline, and cen- the efficacy of which was strongly correlated with their po- trifuged at 1000×g for 5 min (4 °C). The resulting pellet was tency at MCHR-1[38]. The results of this study clearly sug- suspended in isotonic buffer (5 mmol/L Tris/HCl, 0.2 mmol/L gest that MCHR-1 mediates the orexigenic effects of MCH[38]. MgCl , and 0.25 mol/L sucrose, pH 7.4) and homogenized us- Some non-peptidic MCHR-1-selective antagonists have been 2 ing the BioNeb Cell Disruption System (Terre Haute, IN, USA). identified, such as T-226296 and SNAP7941, both of which The homogenate was then centrifuged (20 000×g for 30 min at have anorectic effects[35,39]. 4 °C), and the resulting pellet was resuspended in the binding The scintillation proximity assay (SPA) uses homoge- buffer 1 (50 mmol/L Tris/HCl, 120 mmol/L NaCl, 5 mmol/L KCl, neous and radioisotopic technology that does not involve 1 mmol/L MgCl , 2.5 mmol/L CaCl , pH 7.4). Protein content post-reaction liquid handling steps, and is well-suited to 2 2 was determined using the Bradford assay[42]. Aliquots of mem- automation and high-throughput screening (HTS)[40] . In the brane preparations were stored at –80 °C until use. SPA system, membranes that express a particular receptor Homogeneous hMCHR-1 binding assay The membranes are attached to a microbead coated with wheat germ agglutinin. were incubated overnight in binding buffer 1 containing 2.5 An isotope (e.g. [3H]) is brought very close to the scintillant- nmol/L [3H]SNAP7941, 0.75 g/L FlashBlue GPCR beads, vari- impregnated microbead by binding to its surface. Because ous titrations of SNAP7941 from a stock solution of 40 µmol/L, the emitted particles can only travel short distances in the β and the library compounds with an average concentration of bulk solution, the microbead preferentially captures electrons 6.7 µmol/L (final volume: 100 µL). Non-specific binding was from the bound radiolabeled ligand. The amount of light defined with 1 µmol/L SNAP7941. Data were analyzed with emitted from the scintillant in the microbead is thus directly GraphPad PRISM (GraphPad Software, San Diego, CA, USA). 753 Yan JH et al Acta Pharmacologica Sinica ISSN 1671-4083 The competitive inhibition constant (Ki) was calculated ac- ratio of 6.59 and a coefficient of variation (CV) value of 4.7%.