Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91 Trauelsen, Mette; Rexen Ulven, Elisabeth; Hjorth, Siv A; Brvar, Matjaz; Monaco, Claudia; Frimurer, Thomas M; Schwartz, Thue W Published in: Molecular Metabolism DOI: 10.1016/j.molmet.2017.09.005 Publication date: 2017 Document version Publisher's PDF, also known as Version of record Document license: CC BY-NC-ND Citation for published version (APA): Trauelsen, M., Rexen Ulven, E., Hjorth, S. A., Brvar, M., Monaco, C., Frimurer, T. M., & Schwartz, T. W. (2017). Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91. Molecular Metabolism, 6(12), 1585-1596. https://doi.org/10.1016/j.molmet.2017.09.005 Download date: 08. apr.. 2020 Original Article Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91 Mette Trauelsen 1, Elisabeth Rexen Ulven 3, Siv A. Hjorth 2, Matjaz Brvar 3, Claudia Monaco 4, Thomas M. Frimurer 1,*, Thue W. Schwartz 1,2,* ABSTRACT Objective: Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. Methods and results: Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase. Conclusions: These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91- mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflam- mation and diabetic complications such as retinopathy and nephropathy. Ó 2017 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords GPR91; SUCNR1; Metabolite receptor; GPCR; Drug discovery; Virtual screening; Chemical design 1. INTRODUCTION During normal physiological conditions, plasma levels of succinate range between 2 and 30 mM for humans and 6e17 mM for rodents It is becoming increasingly clear that key metabolites function not only as [5e8]. These levels are below the reported EC50 values for GPR91 energy sources and building blocks but can act also as extracellular activation [1]; however, metabolic stress conditions such as hyper- messengers signaling through G protein coupled receptors (GPCRs) glycemia and hypoxia cause the levels of succinate to rise both locally similarly to hormones and neurotransmitters [1].Succinate,whichhas and in the circulation, enabling activation of GPR91 [5,7,9e11]. for many years solely has been viewed as a TCA cycle intermediate, is Notably, several of the classical rodent models of metabolic dysfunc- one of these signaling metabolites. Already in 2004, succinate was tion have all displayed elevated plasma levels of succinate [7],afinding shown to be the ligand for the G-protein coupled receptor (GPCR) GPR91 also established in patients suffering from Type 2 diabetes (T2D) [12]. [2]. The receptor was predominantly found to be expressed in the kidney, GPR91 has been proposed to be involved in the development of dia- spleen, liver, and small intestine [2]. In the following years most attention betes complications including neuronal VEGF-mediated neo- was paid to the pro-hypertensive effect of GPR91 associated with renin- vascularization in diabetic retinopathy [3,13,14] as well as in diabetic release [3]. In the liver, stress-induced succinate accumulation and nephropathy [9,15]. Adipocytes also express GPR91 and activation of GPR91 activation has been linked to development of fibrosis due to its the receptor inhibits lipolysis [16]. The physiological role of GPR91 on expression in hepatic stellate cells, which, upon succinate stimulation, whole body metabolism, however, is still unclear as GPR91 deficiency produces fibrogenic factors in a GPR91-dependent manner [4,5]. in mice challenged with HFD has been reported both to give impaired 1NNF Center for Basic Metabolic Research, Section for Metabolic Receptology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark 2Laboratory for Molecular Pharmacology, Department of Biomedical Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark 3Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark 4Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Headington, OX3 7FY Oxford, UK *Corresponding authors. Novo Nordisk Foundation Center For Basic Metabolic Research, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. E-mails: thomas.frimurer@sund. ku.dk (T.M. Frimurer), [email protected] (T.W. Schwartz). Received August 23, 2017 Revision received September 14, 2017 Accepted September 25, 2017 Available online 30 September 2017 https://doi.org/10.1016/j.molmet.2017.09.005 MOLECULAR METABOLISM 6 (2017) 1585e1596 Ó 2017 The Authors. Published by ElsevierGmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1585 www.molecularmetabolism.com Original Article [17] and improved glucose tolerance [12]. McCreath and colleagues and Key Organics. Succinate and other small carboxylic acids for Ò also observed dramatic changes in WAT composition [17], something Figure 1 were purchased from SigmaeAldrich and Akos. that also was not confirmed by van Diepen et al., who notably proposed All purchased compounds were spun down and subsequently dissolved that a dramatic decrease in infiltrating macrophages was the main in 100% dimethyl sulfoxide (DMSO) to achieve a final concentration of driver of the improved glucose tolerance in GPR91 deficient mice [12]. 50 mM and left to shake overnight. Dilution rows of the 50 mM stock In fact, GPR91 does seem to be an important regulator of innate im- solutions were subsequently prepared to obtain concentrations suitable munity, particularly as an activator of dendritic cells [18] and macro- for cell-based assays. It is important to mention that all presented phages [19]. structures were confirmed using proton (1H) NMR spectra at 300/ Importantly, independent of GPR91 signaling, succinate can cause a 400 MHz and liquid chromatographyÀmass spectrometry (LC-MS) number of intracellular responses, including post translational modi- analysis confirmed a minimum purity of 95% for all compounds. fications [20], HIF-1a stabilization [21], production of mitochondrial reactive oxygen species (ROS) [11], and overall changes to cellular 2.2. Synthesis of enantiomerically pure compounds metabolism owing to its central role in the TCA cycle. Due to such non- All enantiomerically pure compounds were synthesized by the same receptor mediated effects, the endogenous metabolite succinate as general route. D-orL-aspartate protected as the corresponding such is not a reliable agent to delineate the specific physiological dimethyl esters were coupled with the appropriate carboxylic acid importance of GPR91. The aim of the present study, therefore, was to building block using fluoro-N, N, N0, N0-bis(tetramethylene)for- develop drug-like non-metabolite GPR91 agonists as potential phar- mamidinium hexafluorophosphate [22]. Lithium hydroxide-promoted macological tools by use of a receptor structure-based approach. ester hydrolysis gave the desired test compounds. The acid building blocks were either commercially available (compound 184), or syn- 2. MATERIALS AND METHODS thesized by Williamson ether synthesis (compound 131) or by Suzuki cross coupling using Buchwald’s fourth generation XPhos precatalyst 2.1. Compound acquisition and preparation [23] (compound 130), both followed by ester hydrolysis. Detailed Ligands for library 1 and 2 were purchased from a range of vendors procedures and compound characterization can be found in the including Enamines, Vitas-M Laboratories, ChemDIv, Interbioscreen, Supplementary information. Figure 1: GPR91 activation properties of TCA cycle metabolites and synthetic succinate analogs. A) The main metabolites of the TCA cycle are depicted with structures. Compounds that are able to activate GPR91 are indicated in green and inactive compounds are in gray. B) Dose response curves for the ‘active’ succinate analogs measured