SUCNR1 (or GPR91) belongs to the family of G protein-coupled receptors (GPCR), which represents the largest group of membrane proteins in human genome. The majority of marketed drugs targets GPCRs, directly or indirectly. SUCNR1 has been classified as an orphan receptor until a landmark study paired it with succinate, a citric acid cycle intermediate.According to the current paradigm, succinate triggers SUCNR1 signaling pathways to indicate local stress that may affect cellular metabolism. SUCNR1 implication has been well documented in renin-induced hypertension, ischemia/reperfusion injury, inflammation and immune response, platelet aggregation and retinal angiogenesis. In addition, the SUCNR1-induced increase of blood pressure may contribute to diabetic nephropathy or cardiac hypertrophy.The understanding of SUCNR1 activation, signaling pathways and functions remains largely elusive, which calls for deeper investigations. SUCNR1 shows a high potential as an innovative drug target and is probably an important regulator of basic physiology. In order to achieve the full characterization of this receptor, more specific pharmacological tools such as small-molecules modulators will represent an important asset. In this review, we describe the structural features of SUCNR1, its current ligands and putative binding pocket. We give an exhaustive overview of the known and hypothetical signaling partners of the receptor in different in vitro and in vivo systems. The link between SUCNR1 intracellular pathways and its pathophysiological roles are also extensively discussed.

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We report here the synthesis of 7-phenoxy-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides and their evaluation as AMPA receptor positive allosteric modulators (AMPApams). The impact of substitution on the phenoxy ring and on the nitrogen atom at the 4-position was examined. At GluA2(Q) expressed in HEK293 cells (calcium flux experiment), the most potent compound was 11m (4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, EC50=2.0 nM). The Hill coefficient in the screening and the shape of the dimerization curve in small angle X-ray scattering (SAXS) experiments using isolated GluA2 ligand-binding domain (GluA2-LBD) is consistent with binding of one molecule of 11m per dimer interface, contrary to most benzothiadiazine dioxides developed to date. This observation was confirmed by the X-ray structure of 11m bound to GluA2-LBD and by NMR. This is the first benzothiadiazine dioxide AMPApam to reach the nanomolar range.

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

G protein-coupled receptors are the most important drug targets for human diseases. An important number of them remain devoid of confirmed ligands. GPR27 is one of these orphan receptors, characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. In addition, it has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in -arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of-arrestin 2 to a GPR27V chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds [-[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro--{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a -phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology-arrestin 2 assay, imaging of green fluorescent protein-tagged -arrestin 2, and PathHunter-arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. Our study provides the first selective surrogate agonists for the orphan GPR27.

Many chemotherapeutic drugs exert their cytotoxicity through the formation of DNA modifications (adducts), which interfere with DNA replication, an overactive process in rapidly dividing cancer cells. Side effects from the therapy are common, however, because these drugs also affect rapidly dividing noncancerous cells. Hypoxia-activated prodrugs (HAPs) have been developed to reduce these side effects as they preferentially activate in hypoxic environments, a hallmark of solid tumors. CP-506 is a newly developed DNA-alkylating HAP designed to exert strong activity under hypoxia. The resulting CP-506-DNA adducts can be used to elucidate the cellular and molecular effects of CP-506 and its selectivity toward hypoxic conditions. In this study, we characterize the profile of adducts resulting from the reaction of CP-506 and its metabolites CP-506H and CP-506M with DNA. A total of 39 putative DNA adducts were detected in vitro using our high-resolution/accurate-mass (HRAM) liquid chromatography tandem mass spectrometry (LC−MS 3 ) adductomics approach. Validation of these results was achieved using a novel strategy involving 15 N-labeled DNA. A targeted MS/MS approach was then developed for the detection of the 39 DNA adducts in five cancer cell lines treated with CP-506 under normoxic and hypoxic conditions to evaluate the selectivity toward hypoxia. Out of the 39 DNA adducts initially identified, 15 were detected, with more adducts observed from the two reactive metabolites and in cancer cells treated under hypoxia. The presence of these adducts was then monitored in xenograft mouse models bearing MDA-MB-231, BT-474, or DMS114 tumors treated with CP-506, and a relative quantitation strategy was used to compare the adduct levels across samples. Eight adducts were detected in all xenograft models, and MDA-MB-231 showed the highest adduct levels. These results suggest that CP-506-DNA adducts can be used to better understand the mechanism of action and monitor the efficacy of CP-506 in vivo, as well as highlight a new role of DNA adductomics in supporting the clinical development of DNA-alkylating drugs.

Hypoxia is a characteristic of many solid tumors and is defined as a low level or absence of oxygen due to an insufficient vascularization of the tumor or a transient blockage of blood vessels. Hypoxia activates a survival response within the tumor cells driving cancer progression and is associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are anti-neoplastic agents that can solely be activated in hypoxic areas allowing the targeted delivery of cytotoxic compounds in the hypoxic tumor niches. Convert Pharmaceuticals is developing CP-506, a novel generation of HAP presenting a strong bystander effect. A key step in the activation of CP-506 is its conversion by one-electron reductases to an oxygen-sensing intermediate. Under normoxic conditions, the intermediate is rapidly re-oxidized to the pro-drug. However, under severe hypoxic conditions, further reduction steps lead to the generation of the active cytotoxic metabolite, CP-506M. In an in vitro cytotoxicity assay, CP-506 significantly inhibited the viability of several tumor cell lines specifically under anoxic conditions. In spheroid models, CP-506 demonstrates a strong bystander effect i.e. the ability of the active metabolite to locally diffuse beyond the hypoxic core and induce tumor cell death. CP-506 was administered intraperitoneally at a dose of 600 mg/kg once a day for 5 consecutive days and showed a significant inhibition of tumor growth in four tumor xenograft models (triple negative breast cancer, lung and pancreatic cancers). In all tumor models, CP-506 treatment significantly increased the survival of treated mice assessed by the time to reach four times the start volume as a surrogate endpoint for survival. Different dosing regimens for CP-506 were investigated and all demonstrated a significant tumor growth inhibition compared to the vehicle group. The most profound anti-tumor effect was observed for the repeated cycles of daily consecutive administrations. Finally, no cumulative toxicity was observed for any of the repeated dosing schedules as indicated by bodyweight change. The successful development of CP-506 will require combination with treatments targeting well-oxygenated cells and the careful selection of patients predicted to benefit from a HAP treatment. To this end, we are currently identifying key parameters (presence of tumor hypoxia, expression of reductases and DNA repair capacity) in the activation of CP-506 to define predictive gene signatures in liquid biopsies enabling patient selection. Our current pre-clinical data strongly suggest that CP-506 is a potent, highly selective HAP with a favorable pharmacokinetic profile and the addition of predictive gene signatures or imaging biomarkers for patient selection holds great promise for the treatment of cancer. Citation Format: sophie thiolloy, Sofie Deschoemaeker, Nicolas Ongenae, Julie Gilissen, Ludwig Dubois, Ala Yaromina, Amir Ashoorzadeh, Jeff Smaill, Adam Patterson, Philippe Lambin, Arne Heyerick. CP-506, a next generation hypoxia-activated prodrug, as promising novel anti-cancer therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4959.