A G protein–biased S1P ₁ agonist, SAR247799, protects endothelial cells without affecting lymphocyte numbers

Abstract

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P1) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P1 functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P1 desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P1, could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P1, SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P1 on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P1-desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P1 functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P1. These findings demonstrate that sustained S1P1 activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability.

A label‐free impedance assay in endothelial cells differentiates the activation and desensitization properties of clinical S1P1 agonists

“…An inhibitor of b-arrestin pathway signaling (GRK inhibitor) modified the BNCI profile to one of sustained activation over 60 min, confirming that b-arrestin activation was responsible for reducing the latephase response. This finding is consistent with our previous characterization of SAR247799 relative to siponimod; SAR247799 activated G protein pathways more effectively than b-arrestin or receptor internalization and SAR247799 was more G protein-biased than siponimod [11]. Consequently, characterization of S1P 1 agonists for their ability to sustain impedance responses could be a useful tool to rapidly distinguish between the biased nature of ligands.…”

“…Furthermore, these preclinical findings showed translation to human studies, where SAR247799 showed improvement in endothelial function in type-2 diabetes patients, again at sub-lymphocyte-reducing doses [12]. SAR247799 displayed an attractive safety and tolerability profile in humans, and supratherapeutic doses were characterized by dose-dependent lymphocyte reduction, a biphasic effect consistent with that observed in preclinical studies [11,12,13].…”

“…Positive BNCI values represent a tightening of the endothelial barrier, whereas negative BNCI represents barrier disruption. Disruption of the endothelial barrier has been reported with high doses of S1P 1 -desensitizing molecules in cell-based assays as well as in animals, particularly in the lung [11,26,27,28]. It is also evidenced in clinical trials where dose-dependent lung dysfunction and macular edema have been noted with various molecules [3,4,5,29,30].…”

“…S1P 1 activation has endothelial barrier-stabilizing effects through the formation of adherens and tight junctions [9,10]. Recently, we reported the discovery of SAR247799 a G protein-biased S1P 1 selective agonist capable of S1P 1 activation while limiting receptor desensitization [11]. The biased properties of SAR247799 were associated, in rat and pig models of ischemia/reperfusion injury, with endothelial-protective properties at doses that did not show lymphocyte reduction, and lymphopenia was only evident at supratherapeutic doses [11].…”

“…A real-time cellular assay (RTCA) in human umbilical vein endothelial cells (HUVEC), utilizing a label-free electrical impedance measurement, has been shown to produce a Gi-mediated increase in impedance following activation with S1P 1 agonists [19]. We previously reported qualitative differences in the desensitization properties of SAR247799 and siponimod using such a system [11]. We now extend these observations to define the b-arrestin pathway component of the impedance profile following S1P 1 activation in HUVECs, and we report quantitative indices of the S1P 1 activation-to-desensitization ratio of various clinical molecules.…”