Journal: Cell reports

Article Title: Targeting the succinate receptor effectively inhibits periodontitis.

doi: 10.1016/j.celrep.2022.111389

Figure Lengend Snippet: Figure 4. Development of a specific antagonist of SUCNR1 and its effects in vitro (A) Schematic indicating the synthesis of compound 7a. (B) Calibration curve of cpd-7a by UV-visible (UV-vis). The absorbance of compound 7a in various concentrations was plotted against the concentration to generate the calibration curve with a goodness of fit value of 0.9993. (C) Efficacy of 7a in blocking activation of SUCNR1 by 0.5 mM succinate at the indicated concentrations in a reporter cell line. Error bars show mean ± SEM. n = 4, *p < 0.05, ***p < 0.005, ****p < 0.0001. (D and E) Representative images (D; scale bar, 150 mm) and quantitative results (E) of multinucleated (nucleus number R 10) TRAP+ primary osteoclast cultures with TRAP staining. Error bars show mean ± SEM. n = 4, *p < 0.05, **p < 0.01. (F and G) TNF-a (F) and IL-1b (G) protein levels in conditioned medium of HGF1 cells treated with LPS (5 mg/mL) for 4 and 8 h in the presence or absence of 7a (50 mM) pretreatment for 15 min as indicated. Error bars show mean ± SEM. n = 5, *p < 0.05, **p < 0.01.

Article Snippet: Global knockout of SUCNR1 (KO) mice were generated from EIIa-Cre (Lakso et al., 1996) (Jackson Laboratory, Bar Harbor, ME, USA) and SUCNR1 flox mice, which were created using a TurboKnockout gene targeting strategy by Cyagen and Taconic Companies.

Techniques: In Vitro, Concentration Assay, Blocking Assay, Activation Assay, Staining

Journal: Cell reports

Article Title: Targeting the succinate receptor effectively inhibits periodontitis.

doi: 10.1016/j.celrep.2022.111389

Figure Lengend Snippet: Figure 7. Succinate elevation correlates with periodontitis pathogens in affected individuals (A) Heatmap for the top 40 genera based on relative abundance normalized by row Z score. (B) Correlation-based network plot of the top 40 genera and succinate level in samples. (C) Schematic summary of the role of succinate in manifestation of periodontitis; targeting SUCNR1 with 7a reduces the dysbiotic microbiome, periodontal inflammation, and bone loss; created with BioRender.

Article Snippet: Global knockout of SUCNR1 (KO) mice were generated from EIIa-Cre (Lakso et al., 1996) (Jackson Laboratory, Bar Harbor, ME, USA) and SUCNR1 flox mice, which were created using a TurboKnockout gene targeting strategy by Cyagen and Taconic Companies.

Techniques:

Journal: Annals of Surgery

Article Title: Succinate Activation of SUCNR1 Predisposes Severely Injured Patients to Neutrophil-mediated ARDS

doi: 10.1097/sla.0000000000004644

Figure Lengend Snippet: FIGURE 5. PMN depletion and SUCNR1 inhibition eliminates succinate/LPS mediated lung injury and SUCNR1 inhibition abrogates PMN sequestration. Panel A: As compared to 50 µM succi- nate/NS, 50 µM succinate/LPS caused increased EBD leak into the BALF. In rats pre-treated with an anti-rat neutrophil antibody, the neutrophil depletion eliminates succinate [50 µM]/LPS induced ALI, as measured by levels of EBD extravasation into BALF fluid, and the EBD extravasation is not significant compared to NS/NS in the PMN depleted group (*P < 0.05 compared to 50 µM succinate/LPS). Panel B: As com- pared to the HSA/ NS/NS (Control, left panel), HSA/succi-nate [50 µM]/NS (middle panel) caused PMN infiltration/ sequestration and an increase in the thickness of the pulmonary alveolar membranes. Pretreatment with the GPR91-2c inhibitor, GPR91-2c/ succi- nate [50 µM]/NS decreased the num- bers of the PMNs and decreased the thickness of the pulmonary alveolar membranes (right panel). The panel is representative of experiments per- formed in duplicate. The lung sections were stained with H&E and visualized at 40X. The bar graph is the histology score, membrane thickness of the his- tology. This figure represents the quan- tification of the pulmonary alveolar thickness of 10 images from 2 separate experiments, *=P < 0.05 versus the lungs from NS/ NS controls and †=P < 0.05 versus the lungs from succinate/NS treated rats. Panel C: Pretreatment for 30 min with the GPR91-2c [30 nM] inhibitor/NS did not elicit cause ARDS, as measured by EBD leak, in rats. Rats treated with the 1.25% HSA vehicle fol- lowed by 50 µM succinate/LPS or 500 µM succinate/LPS manifested ARDS. Pretreatment with the GPR91-2c abro- gated ALI caused by 50 µM succinate/ LPS or 500 µM succi-nate/LPS (*=P < 0.05 vs GPR91-2c/NS, GPR91-2c/LPS, HSA/50 µM succinate/ NS and HSA/500 µM succinate/NS; †=P < 0.05 vs HSA/50 µM succinate/LPS and HSA/500 µM succinate/LPS, n = 5 for each bar). ARDS indicates acute respiratory distress syn- drome; LPS indicates lipopolysaccharide; HSA, human serum albumin; PMN, neutrophils; SUCNR1, succinate receptor.

Article Snippet: PMNs were incubated with NS or succinate (500–1000 μM), fixed, smeared onto slides, incubated with a GPR91/ SUCNR1 antibody (Novus Biological, Littleton, CO), and then with a species-specific fluorescent antibody followed by microscopic imaging at 100X magnification.20,34 SUCNR1 Inhibitor Synthesis and Inhibition of

Techniques: Inhibition, Control, Staining, Membrane

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Hepatic succinate levels and SUCNR1 expression increased after liver ischemia–reperfusion injury (IRI) in mice. C57BL/6 mice were subjected to 60 min of liver ischemia followed by 1, 6, or 24 h of reperfusion as indicated. Control mice were sham-operated. ( A ) Succinate levels in liver tissues increased after IRI. ( B ) Succinate levels in serum increased after IRI. ( C ) Sucnr1 mRNA expression in liver tissues was upregulated after IRI ( n = 6). ( D ) Representative western blots of liver tissues show increased SUCNR1 protein levels after IRI ( n = 3). ( E ) Representative immunofluorescence images show the co-localization of SUCNR1- and CLEC4F-positive cells in liver tissues. The double-positive cells are located within the hepatic sinusoids (indicated by the white triangle) surrounding the central venous (indicated by the white asterisk). Proportions of SUCNR + CLEC4F + cells increased after IRI ( n = 3). Bar = 50 µm. Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Expressing, Control, Western Blot, Immunofluorescence

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 alleviated liver ischemia–reperfusion injury (IRI) and hepatocyte apoptosis. Wild-type (WT) and Sucnr1 −/− mice underwent 60 min of ischemia followed by 6 h of reperfusion. ( A ) Representative images of hematoxylin–eosin (HE)-stained liver sections. Liver damage was quantified using Suzuki’s scores ( n = 6).Bar = 200 µm. ( B ) Serum ALT levels reflect liver function and hepatocellular injury ( n = 6). ( C ) Intrahepatic apoptosis was measured by TUNEL assay. Representative fluorescent sections are shown (red spots with DAPI-stained blue background represent TUNEL-positive cells) ( n = 3). Bar = 200 µm. (D) Representative western blots of Cleaved caspase 3 and Caspase 3 in liver tissues after IRI, and relative protein expression ratio of Cleaved caspase 3 to Caspase 3 was evaluated in each group ( n = 3). ( E–G ) In vivo, hepatocytes were cultured without Kupffer cells (KCs) (the Control) or co-cultured with KCs isolated from Sucnr1 −/− mice (the Sucnr1 −/− group) and WT mice (the WT group) after IRI. The percentage of apoptotic hepatocytes ( E ) was increased under co-culture with KCs from WT mice after liver IRI and alleviated under co-culture with KCs from Sucnr1 −/− mice after liver IRI ( n = 3). Relative cell viability ( F ) and relative lactate dehydrogenase (LDH) activity ( G ) of hepatocytes under co-culture with KCs from WT mice and Sucnr1 −/− mice after liver IRI ( n = 6). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, In Vivo, Cell Culture, Control, Isolation, Co-Culture Assay, Activity Assay

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: The critical role of SUCNR1 on Kupffer cells (KCs) in liver ischemia–reperfusion injury (IRI). Wild-type (WT) and Sucnr1 −/− mice were injected with clodronate or vehicle 72 h before ischemia, and samples were harvested after 6 h of reperfusion. Naïve WT mice served as control without any treatment. ( A ) Representative images of hematoxylin–eosin (HE)-stained liver sections. Immunohistochemical (IHC) staining of CLEC4F shows the efficiency of KC depletion in the liver. ( B ) Suzuki’s scores ( n = 6). Bar = 200 µm. ( C ) Ratios (%) of CLEC4F-positive cells ( n = 3). ( D ) Serum AST values from WT and Sucnr1 −/− mice were measured after 6 h of reperfusion ( n = 6). Results are presented as mean ± SEM. ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Injection, Control, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 inhibits the activation of Kupffer cells (KCs) following liver ischemia–reperfusion injury (IRI). ( A) Expressions of CD40, CD80, and MHC II in KCs from wild-type (WT) and Sucnr1 −/− mice after liver IRI were analyzed by flow cytometry ( n = 3). ( B ) Expressions of F4/80, CLEC4F, and VSIG4 in liver tissues from WT and Sucnr1 −/− mice after liver IRI was evaluated through immunohistochemical staining ( n = 3). Scale bar = 200 µm. ( C ) Primary KCs were incubated with various concentrations of succinate or stimulated with LPS (1 μg/mL) for 6 h. The mRNA expression of KC markers ( F4/80, Clec4f, and Vsig4 ) was analyzed ( n = 5). ( D ) Primary KCs from WT and Sucnr1 −/− mice stimulated with succinate or LPS (1 μg/mL) for 6 h. Relative mRNA expression of KC markers ( F4/80, Clec4f, and Vsig4 ) was analyzed ( n = 5). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Activation Assay, Flow Cytometry, Immunohistochemical staining, Staining, Incubation, Expressing

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 inhibits M1 polarization of Kupffer cell (KC) following liver ischemia–reperfusion injury (IRI). ( A ) Polarization of KCs was assessed by immunohistochemical staining for iNOS and Arg1 in Wild-type (WT) and Sucnr1 −/− mice with or without liver IRI ( n = 3). Bar = 200 µm. ( B ) Sucnr1 deficiency decreased the ratio (%) of M1 KCs (CD11c + CD206 − ) and increased that of M2 KCs (CD11c − CD206 + ) ( n = 3). ( C, D ) Relative mRNA expression of M1 markers ( iNOS and Cox2 ) and M2 markers ( Arg1 and Ym1 ) in WT and Sucnr1 −/− mice with or without liver IRI ( C ) ( n = 6) and in WT and Sucnr1 −/− KCs stimulated with succinate or LPS (1 μg/mL) for 6 h ( D ) ( n = 5). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: Deletion of Sucnr1 inhibits proinflammatory cytokine release following liver ischemia–reperfusion injury (IRI). (A) Expressions of cytokines (TNF-α, IL-6, IL-1β and IL-10) in liver tissues from WT and Sucnr1 −/− mice mice after liver IRI was analyzed by immunohistochemical staining ( n = 3). Scale bar = 200 µm. (B) Concentrations of cytokines (TNF-α, IL-6, IL-1β and IL-10) in liver tissues from WT and Sucnr1 −/− mice with or without liver IRI ( n = 6). (C) Relative mRNA expression levels of cytokines ( Tnf , Il6 , Il1b and Il10 )) genes in WT and Sucnr1 −/− KCs stimulated with succinate or LPS for 6 h ( n = 5). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Inflammation

Article Title: SUCNR1 Deficiency Alleviates Liver Ischemia–Reperfusion Injury by Regulating Kupffer Cell Activation and Polarization Through the ERK/NF-κB Pathway in Mice

doi: 10.1007/s10753-025-02290-9

Figure Lengend Snippet: SUCNR1 mediated Kupffer cell (KC) polarization following liver ischemia–reperfusion injury (IRI) via the ERK/NF-κB signaling pathway. ( A ) Protein expression was detected by Western Blot taken from KCs in Wild-type (WT) and Sucnr1 −/− mice with or without liver IRI ( n = 3). ( B-D ) KCs from WT mice were pretreated with Compound 4C (GPR91 antagonist, 5 μM) and SCH772984 (SCH; ERK inhibitor; 10 μM) 24 h prior to stimulation with succinate (1 mM), and the expression of KC protein levels was detected by Western Blot ( B ) ( n = 3), and the mRNA expression of M1-related genes ( C ) and pro-inflammatory genes ( D ) were measured by real-time polymeric chain reaction ( n = 5). Hepatocytes were co-cultured with WT mice-derived KCs that pretreated with Compound 4C and SCH772984 before stimulation with succinate. The percentage of apoptotic hepatocytes ( E ) ( n = 3), cell viability of hepatocytes ( F ) ( n = 6), and LDH activity were determined ( G ) ( n = 6). Results are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For KC detection, sections were incubated with rat anti-mouse CLEC4F (diluted 1:1000; R&D Systems) together with a rabbit polyclonal anti-SUCNR1 primary antibody (diluted 1:200; Novus Biologicals LLC).

Techniques: Expressing, Western Blot, Cell Culture, Derivative Assay, Activity Assay

Journal: Molecular cell

Article Title: Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

doi: 10.1016/j.molcel.2024.01.011

Figure Lengend Snippet: Figure 1. Pathway and dynamics for succinate binding in SUCNR1 during unbiased MD simulations (A) In the lower fragment are shown residues that succinate interacts with, from its initial binding in the extracellular vestibule in stage 1 (residues highlighted in blue) through the intermediate stage (red residues) to its final binding in the ‘‘deep orthosteric site’’ in stage 2 (green residues), all represented in the crystal structure of the humanized ratSUCNR1 (PDB: 6rnk). F280 is used as a measuring point at the bottom of the orthosteric pocket and is highlighted in white sticks. The top fragment focuses on the special network of five arginines around the inner face of the extracellular segment of TM-VI (in light brown) of SUCNR1, of which R2556.62 and R2516.58 in TM-VI form the initial catching ECV site and R2486.55 together with R953.29 and R2767.39 form the main components of the deep or- thosteric site. (B) Distance between the center of the mass of succinate and the bottom of the orthosteric pocket (center of masses of F280) throughout MD simulation ID: 33 (Table S1) with the three stages highlighted in blue, red, and green. The left insert top (stage 1) shows succinate initially caught by K2667.29 and N2697.32 and firmly bound between R2556.62 and R2516.58 in TM-VI. Middle insert top (intermediate stage)—R2516.58 is shown in two rotational conformations, illustrating how in the intermediate stage it carries the tightly bound succinate down from the ECV site to pass between D17045.52 in ECL-2b and Y2727.35 (see Figure 4 concerning water-mediated breakage of H-bond). Right, insert top (stage 2)—succinate bound between R953.29, R2486.55, R2767.39, and Y2727.35. (C) Heatmap of the number of direct and water-mediated H-bond interactions between succinate and residues in SUCNR1 throughout the MD simulation (ID #33)—with the time point of the three binding stages indicated by vertical dotted lines. (D) Long-range charge-charge interactions (atomic distance <5 A˚ ) between succinate and each of the five arginines during the MD simulation (ID: 33). In stage 1, succinate is bound between the upward-turned R2516.58 and R2556.62, in the intermediate stage, interacting with the downward-turned R2516.58 and R2767.39, and in stage 2, it is bound between R2767.39, R953.29, and R2486.55 in the deep orthosteric site.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Sodium succinate dibasic Sigma-Aldrich Product: 14160 CAS: 150-90-3 YSi SPA scintillation beads PerkinElmer Cat# RPNQ0010 Co-elenterazine h ThermoFisher Cat# C6780 CAS: 50909-86-9 HBSS Gibco Cat# 14025050 myo [3H]inositol PerkinElmer Cat# NET114A005MC DMEM 1885 Gibco Cat# 11885-084 DMEM 1966 Gibco Cat# 31966-021 PBS Substrate Department – UCPH N/A Trypsin Bioscience Cat# BE17-161E NF-56-EJ40 Targetmol Catalog# T12216 CAS:2380230-73-7 Critical commercial assays QuikChange II Site-Directed Mutagenesis Kit Agilent Cat# 200523 NucleoBond Xtra Midi kit Macherey-Nagel Cat# 740410.100 Deposited data Simulation trajectories of ligand binding to SUCNR1 https://doi.org/10.5281/zenodo.10213670 https://www.zenodo.org Experimental models: Cell lines XL1-Blue Competent Cells Agilient Cat# 200228 Cos 7 Cells ATCC ATCC CRL-1651 Recombinant DNA Murine SUCNR1 construct Origene MR204545 Human SUCNR1 construct Origene RC205888 CAMYEL Jiang et al.52 N/A Software and algorithms ICM-Pro MolSoft LLC https://www.molsoft.com/ Prime Schrödinger, Inc. https://www.schrodinger.com/ products/prime Maestro Schrödinger, Inc. https://www.schrodinger.com/products/ maestro Desmond Schrödinger, Inc. https://www.schrodinger.com/products/ desmond PyMOL Schrödinger, Inc. https://pymol.org/ VMD Humphrey et al.53 http://www.ks.uiuc.edu/Research/vmd/ Matplotlib Hunter et al.54 https://matplotlib.org/1.2.1/index.html Prism GraphPad, Inc. https://www.graphpad.com/scientific- software/prism/ MicroBeta 2 Workstation Perkin Elmer https://www.perkinelmer.com/uk/product/ microbeta2-plate-reader-with-1-detector/ MARS Data Analysis Software BMG LABTECH https://www.bmglabtech.com/en/ microplate-reader-software/ Molecular Cell 84, 955–966.e1–e4, March 7, 2024 e1

Techniques: Binding Assay

Journal: Molecular cell

Article Title: Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

doi: 10.1016/j.molcel.2024.01.011

Figure Lengend Snippet: Figure 2. The two low-energy succinate binding sites in SUCNR1 and how they can get simultaneously occupied by two succinate ligands (A) The two binding sites for succinate represented as surface in the humanized ratSUCNR1 (PDB: 6rnk): in blue, the ECV site between the outer poles of TM-VI and TM-VII indicated in the corresponding insert to the right; in green the deeper, orthosteric site between TM-III, TM-VI, and TM-VII indicated in the corre- sponding insert to the right. (B) Well-tempered metadynamics analysis of succinate binding to SUCNR1 revealing two energy minima corresponding to binding to the ECV site (blue circle) and orthosteric site (green circle), respectively. The intermediate binding state between the two low-energy binding modes is associated with an energy barrier, which must be overcome when the ligand passes from the ECV site (stage 1) to the orthosteric binding site (stage 2). Four replicates are shown, and the collective variable (CV) is the distance between the center of the masses of succinate and F280. (C) Mutational analysis of the ECV binding site in murine SUCNR1 using activation of Gq-mediated IP3 accumulations in transfected COS7 cells as a functional readout for the R2516.58A, R2556.62A, K2667.29A, and K2697.32A mutants versus WT murine SUCNR1 (n = 3, error bars represented as ± SEM). A similar mutational analysis of the orthosteric site is shown in Figures S5 and S6. (D) Simultaneous binding of two succinate molecules through a sequential mechanism. The first ligand molecule binds in the ECV site and then moves down to the orthosteric site through the stepwise mechanism described in Figure 1—this happens in 21 of the 54 unbiased MD simulations performed with either succinate or CES (Tables S1 and S2). A second succinate binds in the vacant ECV site while the first occupies the orthosteric site, which happens in six of the 21 cases; however, the second succinate in all six cases left the ECV site again before the end of the MD simulation. The occupancy plot below shows the timeline for each of the two sites with the first succinate indicated in gray and the second succinate indicated in blue for MD #27. (E) Simultaneous binding of two succinate molecules through the bypassing mechanism. The first succinate stays bound in the ECV site between R2516.58 and R2556.62, while a second succinate molecule bypasses the first succinate to get directly bound in the triple-arginine orthosteric deep site. The occupancy plot below shows the timeline for each of the two sites for MD #2. Occupancy timeline plots for all MD simulations where dual succinate occupancy was observed using PDB: 6rnk are shown in Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Sodium succinate dibasic Sigma-Aldrich Product: 14160 CAS: 150-90-3 YSi SPA scintillation beads PerkinElmer Cat# RPNQ0010 Co-elenterazine h ThermoFisher Cat# C6780 CAS: 50909-86-9 HBSS Gibco Cat# 14025050 myo [3H]inositol PerkinElmer Cat# NET114A005MC DMEM 1885 Gibco Cat# 11885-084 DMEM 1966 Gibco Cat# 31966-021 PBS Substrate Department – UCPH N/A Trypsin Bioscience Cat# BE17-161E NF-56-EJ40 Targetmol Catalog# T12216 CAS:2380230-73-7 Critical commercial assays QuikChange II Site-Directed Mutagenesis Kit Agilent Cat# 200523 NucleoBond Xtra Midi kit Macherey-Nagel Cat# 740410.100 Deposited data Simulation trajectories of ligand binding to SUCNR1 https://doi.org/10.5281/zenodo.10213670 https://www.zenodo.org Experimental models: Cell lines XL1-Blue Competent Cells Agilient Cat# 200228 Cos 7 Cells ATCC ATCC CRL-1651 Recombinant DNA Murine SUCNR1 construct Origene MR204545 Human SUCNR1 construct Origene RC205888 CAMYEL Jiang et al.52 N/A Software and algorithms ICM-Pro MolSoft LLC https://www.molsoft.com/ Prime Schrödinger, Inc. https://www.schrodinger.com/ products/prime Maestro Schrödinger, Inc. https://www.schrodinger.com/products/ maestro Desmond Schrödinger, Inc. https://www.schrodinger.com/products/ desmond PyMOL Schrödinger, Inc. https://pymol.org/ VMD Humphrey et al.53 http://www.ks.uiuc.edu/Research/vmd/ Matplotlib Hunter et al.54 https://matplotlib.org/1.2.1/index.html Prism GraphPad, Inc. https://www.graphpad.com/scientific- software/prism/ MicroBeta 2 Workstation Perkin Elmer https://www.perkinelmer.com/uk/product/ microbeta2-plate-reader-with-1-detector/ MARS Data Analysis Software BMG LABTECH https://www.bmglabtech.com/en/ microplate-reader-software/ Molecular Cell 84, 955–966.e1–e4, March 7, 2024 e1

Techniques: Binding Assay, Activation Assay, Transfection, Functional Assay

Journal: Molecular cell

Article Title: Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

doi: 10.1016/j.molcel.2024.01.011

Figure Lengend Snippet: Figure 3. Molecular dynamics simulations of antagonist NF-56-EJ40 binding to SUCNR1 (A) Free-energy landscape of NF-56-EJ40 binding to SUCNR1 (PDB: 6rnk) obtained from well-tempered metadynamics. CV, the distance between the center of masses of the antagonist and F280 (see STAR Methods for details). In all four representative experiments, the free-energy landscape demonstrates that binding occurred in a single stage with only a ‘‘shoulder’’ corresponding to the entry phase. (B) Top view of the entry poses of NF-56-EJ40 between TM-I and TM-II in close contact with E181.31 and K191.32. For comparison, a succinate molecule is shown in its very different entry position between TM-VI and -VII (see Figures 1 and 2). (C and D) Different types of molecular interactions between NF-56-EJ40 and key SUCNR1 residues during the binding process are shown in a heatmap format in (C) and in schematic form for E181.31 and K191.32 in (D). K191.32 forms cation-p interactions with two of the aromatic rings during the entry phase for the antagonist. First, E181.31 makes water-bridged H-bond interaction with the head group carboxylate, as well as with the amide linker of the antagonist and eventually shifts to making both water-mediated and ionic interactions with the terminal piperazine moiety in the final bound pose. R2767.39 and R953.29 of the orthosteric succinate binding sites make ionic interactions with the head group carboxylate of the antagonist. (E) Superimposition of a representative frame of the bound NF-56-EJ40 from the metadynamics simulations (blue) and the structure of NF-56-EJ40 with SUCNR1 (PDB: 6rnk) (pink). (F) Mutational analysis of 1.31 in the entry path for NF-56-EJ40. The top shows loss of function with respect to antagonist action in the human SUCNR1, i.e., E221.31L mutation, which is involved in both antagonist entry and in stabilizing the final pose of the antagonist (n = 3, error bars are represented as ± SEM) (E). The bottom shows the gain of function of the antagonist action in the murine SUCNR1 mediated by N18A1.31 substitution, which in the wild-type murine SUCNR1 prevents entry and function of the antagonist. In both cases, the mutants have only minimal effect on the succinate activation of the receptor.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Sodium succinate dibasic Sigma-Aldrich Product: 14160 CAS: 150-90-3 YSi SPA scintillation beads PerkinElmer Cat# RPNQ0010 Co-elenterazine h ThermoFisher Cat# C6780 CAS: 50909-86-9 HBSS Gibco Cat# 14025050 myo [3H]inositol PerkinElmer Cat# NET114A005MC DMEM 1885 Gibco Cat# 11885-084 DMEM 1966 Gibco Cat# 31966-021 PBS Substrate Department – UCPH N/A Trypsin Bioscience Cat# BE17-161E NF-56-EJ40 Targetmol Catalog# T12216 CAS:2380230-73-7 Critical commercial assays QuikChange II Site-Directed Mutagenesis Kit Agilent Cat# 200523 NucleoBond Xtra Midi kit Macherey-Nagel Cat# 740410.100 Deposited data Simulation trajectories of ligand binding to SUCNR1 https://doi.org/10.5281/zenodo.10213670 https://www.zenodo.org Experimental models: Cell lines XL1-Blue Competent Cells Agilient Cat# 200228 Cos 7 Cells ATCC ATCC CRL-1651 Recombinant DNA Murine SUCNR1 construct Origene MR204545 Human SUCNR1 construct Origene RC205888 CAMYEL Jiang et al.52 N/A Software and algorithms ICM-Pro MolSoft LLC https://www.molsoft.com/ Prime Schrödinger, Inc. https://www.schrodinger.com/ products/prime Maestro Schrödinger, Inc. https://www.schrodinger.com/products/ maestro Desmond Schrödinger, Inc. https://www.schrodinger.com/products/ desmond PyMOL Schrödinger, Inc. https://pymol.org/ VMD Humphrey et al.53 http://www.ks.uiuc.edu/Research/vmd/ Matplotlib Hunter et al.54 https://matplotlib.org/1.2.1/index.html Prism GraphPad, Inc. https://www.graphpad.com/scientific- software/prism/ MicroBeta 2 Workstation Perkin Elmer https://www.perkinelmer.com/uk/product/ microbeta2-plate-reader-with-1-detector/ MARS Data Analysis Software BMG LABTECH https://www.bmglabtech.com/en/ microplate-reader-software/ Molecular Cell 84, 955–966.e1–e4, March 7, 2024 e1

Techniques: Binding Assay, Comparison, Mutagenesis, Activation Assay

Journal: Molecular cell

Article Title: Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

doi: 10.1016/j.molcel.2024.01.011

Figure Lengend Snippet: Figure 4. Breakage of constraining H-bond and conformational changes induced in ECL-2b of SUCNR1 by succinate (agonist) passage and water cluster formation (A) The distance between the carboxylate Ca of D17045.52 and Ca of Y2727.35 throughout MD simulation of SUCNR1 (ID #20, Table S1). Left panel top, a snapshot of Y2727.35 and D17045.52 in stage 1, where the H-bond between these residues is highly stable. Middle top, snapshot from the intermediate stage where succinate in its cis conformation is passing, the H-bond is broken and D17045.52 with ECL-2b has moved away. In this frame, water molecules make H-bond bridges between the succinate carboxylates and D17045.52. Right top, stage 2 where succinate has moved down into the orthosteric site but D17045.52 and Y2727.35 stay apart as ECL-2b remains open. (B) Top view of SUCNR1 shows the conformational change of ECL2 upon agonist binding through the trajectory (from wheat to red). (C) Sequence of SUCNR1 ECL2 divided into 2a and 2b by the conserved Cys with D17045.52 highlighted in red and surrounding residues highlighted in different colors. (D) Root mean square fluctuations (RMSFs) of the side chains of the first residues of ECL-2b in SUCNR1 in the apo forms (PDB: 6ibb), with succinate, CES, or antagonist bound. The bars average four replicas per setup (Table S3). (E) H-bond versus water-mediated interactions between succinate and R2516.58, D17045.52, and Y2727.35 during the MD simulation demonstrating that succinate interacts exclusively via water molecules with D17045.52 and to a large degree with Y2727.35 as opposed to R2516.58. (F) Formation of clusters of water molecules around succinate, D17045.52, Y2727.35, and R2516.58 during the intermediate stage of succinate binding—top view of SUCNR1. Cluster A is formed between succinate in its cis conformation and D17045.52 and Y2727.35 and disappears with the increase of distance between these residues. Cluster B is formed between the downward bend R2516.58, D17045.52, and R2486.55 and gradually decreases in size as the succinate molecule reaches its final bound pose and R2516.58 swings upward.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Sodium succinate dibasic Sigma-Aldrich Product: 14160 CAS: 150-90-3 YSi SPA scintillation beads PerkinElmer Cat# RPNQ0010 Co-elenterazine h ThermoFisher Cat# C6780 CAS: 50909-86-9 HBSS Gibco Cat# 14025050 myo [3H]inositol PerkinElmer Cat# NET114A005MC DMEM 1885 Gibco Cat# 11885-084 DMEM 1966 Gibco Cat# 31966-021 PBS Substrate Department – UCPH N/A Trypsin Bioscience Cat# BE17-161E NF-56-EJ40 Targetmol Catalog# T12216 CAS:2380230-73-7 Critical commercial assays QuikChange II Site-Directed Mutagenesis Kit Agilent Cat# 200523 NucleoBond Xtra Midi kit Macherey-Nagel Cat# 740410.100 Deposited data Simulation trajectories of ligand binding to SUCNR1 https://doi.org/10.5281/zenodo.10213670 https://www.zenodo.org Experimental models: Cell lines XL1-Blue Competent Cells Agilient Cat# 200228 Cos 7 Cells ATCC ATCC CRL-1651 Recombinant DNA Murine SUCNR1 construct Origene MR204545 Human SUCNR1 construct Origene RC205888 CAMYEL Jiang et al.52 N/A Software and algorithms ICM-Pro MolSoft LLC https://www.molsoft.com/ Prime Schrödinger, Inc. https://www.schrodinger.com/ products/prime Maestro Schrödinger, Inc. https://www.schrodinger.com/products/ maestro Desmond Schrödinger, Inc. https://www.schrodinger.com/products/ desmond PyMOL Schrödinger, Inc. https://pymol.org/ VMD Humphrey et al.53 http://www.ks.uiuc.edu/Research/vmd/ Matplotlib Hunter et al.54 https://matplotlib.org/1.2.1/index.html Prism GraphPad, Inc. https://www.graphpad.com/scientific- software/prism/ MicroBeta 2 Workstation Perkin Elmer https://www.perkinelmer.com/uk/product/ microbeta2-plate-reader-with-1-detector/ MARS Data Analysis Software BMG LABTECH https://www.bmglabtech.com/en/ microplate-reader-software/ Molecular Cell 84, 955–966.e1–e4, March 7, 2024 e1

Techniques: Binding Assay, Sequencing