Identification and pharmacological characterization of succinate receptor agonists

Abstract

Background and purpose: The succinate receptor (formerly GPR91 or SUCNR1) is described as a metabolic sensor that may be involved in homeostasis. Notwithstanding its implication in important (patho)physiological processes, the function of succinate receptors has remained ill-defined because no pharmacological tools were available. We report on the discovery of the first family of potent synthetic agonists.

Experimental approach: We screened a library of succinate analogues and analysed their activity on succinate receptors. Also, we modelled a pharmacophore and a binding site for this receptor. New agonists were identified based on the information provided by these two approaches. Their activity was studied in various bioassays, including measurement of cAMP levels, [Ca²⁺ ]_i mobilization, TGF-α shedding and recruitment of arrestin 3. The in vivo effects of activating succinate receptors with these new agonists was evaluated on rat BP.

Key results: We identified cis-epoxysuccinic acid and cis-1,2-cyclopropanedicarboxylic acid as agonists with an efficacy similar to that of succinic acid. Interestingly, cis-epoxysuccinic acid was 10- to 20-fold more potent than succinic acid on succinate receptors. For example, cis-epoxysuccinic acid reduced cAMP levels with a pEC₅₀ = 5.57 ± 0.02 (EC₅₀ = 2.7 μM), compared with succinate pEC₅₀ = 4.54 ± 0.08 (EC₅₀ = 29 μM). The rank order of potency of the three agonists was the same in all in vitro assays. Both cis-epoxysuccinic and cis-1,2-cyclopropanedicarboxylic acid were as potent as succinate in increasing rat BP.

Conclusions and implications: We describe new agonists at succinate receptors that should facilitate further research on this understudied receptor.

Figure 1

Screening of library of succinic acid analogues. We selected diverse compounds that share some characteristics with succinic acid. The compounds were grouped into three criteria: (i) nature of substituent; (ii) length of carbon backbone; and (iii) charge of the molecule. See (Supporting Information Table S1) for a complete list of molecular structures. (A) The different compounds were tested at 500 μM on succinate receptors. The agonist activity was evaluated by measuring the levels of cAMP in the presence of compounds compared with vehicle control and the response to succinic acid. No compounds showed activity on cell lines lacking succinate receptors. (B) 100 μM of compounds were added prior to the addition of 500 μM succinic acid to evaluate their antagonistic activity. Results are given as percentage of succinic acid activity for ease of comparison and as mean ± SEM of three independent experiments.

Figure 2

Effects of the nature and stereochemistry of substituents. Some of the compounds showing activity in screening at one concentration were evaluated with full concentration–response curves. Concentration–response curve for the effect on cAMP of (A) (R)‐ and (S)‐MSA; (B) BrSA and ClSA; (C) malic acid; (D) aspartic and OAA. In all experiments, succinic acid (SA) has been used as a reference compound and the data are normalized accordingly. Data are expressed as mean ± SEM of independent three experiments. (E) Model for the interaction of substituents with the binding pocket. AUC, area under curve; ctrl, control.

Figure 3

cis Conformation of the negative charges is an essential feature for succinate receptor agonists. (A) Maleic acid is a full agonist for succinate receptors, albeit with a lower potency than succinic acid (SA). Fumaric acid is completely inactive. (B) MA, MMA, EMA and mDMSA concentration–response curves on basal cAMP levels. (C) Homology modelling of the succinic acid binding pocket. succinic acid is positioned in a pseudo cis conformation. (D) Evaluation of the effect of succinic acid on basal cAMP level in several HEK293 cell lines stably transfected by GloSensor system and the succinate receptor mutants (H103A, R99A, R252A, R281A). Data are presented as mean ± SEM of three independent experiments. AUC, area under curve; ctrl, control.

Figure 4

cis‐Cyclic derivatives of succinic acid (SA) are succinate receptor agonists. (A) Model for the succinate receptor agonist pharmacophore. Yellow spheres represent exclusion volume and red spheres negative charges. (B) cCPDA fit with the pharmacophore model. (C) Evaluation of cis‐ and trans‐cyclic compounds on the inhibition of basal cAMP levels. (D) cESA concentration–response curve on the inhibition of basal cAMP level. (E, F) Kinetic measurement of the inhibition of cAMP levels followed in HEK293 cells stably expressing cAMP Glosensor and succinate receptor upon addition of the succinate receptor agonists cCPDA (E) and cESA (F).Succinic acid was used as a positive control (ctrl). Data are expressed as mean ± SEM of three independent experiments. AUC, area under curve.

Figure 5

cESA and cCPDA are agonists for all known succinate receptor pathways and do not interfere with SDH. Concentration–response curves for succinic acid, cESA and cCPDA on (A) TGF‐α shedding, (B) arrestin 3 recruitment, (C) [Ca²⁺]_i mobilization and (D) SDH activity. Data are presented as mean ± SEM of six independent experiments. AUC, area under curve; SA, succinic acid.

Figure 6

cESA and cCPDA are as active as succinic acid (SA) in vivo. (A) Repeated non‐invasive measures of BP in rats (n = 8 in each group) injected intravenously with a saline solution of the test compounds. These experiments have been performed at least three times for each condition on different animal cohorts. Data are expressed as the difference between mean BP before injection and 15 min post‐injection. Data are presented as mean ± SD. *P <0.05, significantly different as indicated: one‐way ANOVA. (B) Proposed pharmacophore for succinate derivatives as agonists.