S1P1 Modulator-Induced G αi Signaling and β-Arrestin Recruitment Are Both Necessary to Induce Rapid and Efficient Reduction of Blood Lymphocyte Count In Vivo

Abstract

S1P₁ (sphingosine-1-phosphate receptor 1) agonists prevent lymphocyte egress from secondary lymphoid organs and cause a reduction in the number of circulating blood lymphocytes. We hypothesized that S1P₁ receptor modulators with pathway-selective signaling properties could help to further elucidate the molecular mechanisms involved in lymphocyte trapping. A proprietary S1P₁ receptor modulator library was screened for compounds with clear potency differences in β-arrestin recruitment and G protein alpha i subunit (G _αi) protein-mediated signaling. We describe here the structure-activity relationships of highly potent S1P₁ modulators with apparent pathway selectivity for β-arrestin recruitment. The most differentiated compound, D3-2, displayed a 180-fold higher potency in the β-arrestin recruitment assay (EC₅₀ 0.9 nM) compared with the G _αi-activation assay (167 nM), whereas ponesimod, a S1P₁ modulator that is currently in advanced clinical development in multiple sclerosis, was equipotent in both assays (EC₅₀ 1.5 and 1.1 nM, respectively). Using these novel compounds as pharmacological tools, we showed that although a high potency in β-arrestin recruitment is required to fully internalize S1P₁ receptors, the potency in inducing G _αi signaling determines the rate of receptor internalization in vitro. In contrast to ponesimod, the compound D3-2 did not reduce the number or circulating lymphocytes in rats despite high plasma exposures. Thus, for rapid and maximal S1P₁ receptor internalization a high potency in both G _αi signaling and β-arrestin recruitment is mandatory and this translates into efficient reduction of the number of circulating lymphocytes in vivo.