Gonzalez-Cabrera Pedro J, Jo Euijung, Sanna M Germana, Brown Steven, Leaf Nora, Marsolais David, Schaeffer Marie-Therese, Chapman Jacqueline, Cameron Michael, Guerrero Miguel, Roberts Edward, Rosen Hugh
Departments of Chemical Physiology & Immunology, Scripps Research Institute Molecular Screening Center, La Jolla, CA 92037, USA.
Mol Pharmacol. 2008 Nov;74(5):1308-18. doi: 10.1124/mol.108.049783. Epub 2008 Aug 15.
Strong evidence exists for interactions of zwitterionic phosphate and amine groups in sphingosine-1 phosphate (S1P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S1P receptors. The contribution of Arg(120) and Glu(121) for high-affinity ligand-receptor interactions is essential, because single-point R(120)A or E(121)A S1P(1) mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly selective S1P(1) agonist [2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino) ethanol (CYM-5442)] that does not require Arg(120) or Glu(121) residues for activating S1P(1)-dependent p42/p44 mitogen-activated protein kinase phosphorylation, which defines a new hydrophobic pocket in S1P(1). CYM-5442 is a full agonist in vitro for S1P(1) internalization, phosphorylation, and ubiquitination. It is noteworthy that CYM-5442 was a full agonist for induction and maintenance of S1P(1)-dependent blood lymphopenia, decreasing B lymphocytes by 65% and T lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose- and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S1P(1) activation by CYM-5442 were noncompetitively inhibited by a specific S1P(1) antagonist [(R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146)], competitive for S1P, 2-amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720-P), and 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2, 4-oxadiazole (SEW2871). In addition, lymphopenia induced by CYM-5442 was reversed by W146 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S1P(1)-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket separate from the orthosteric site of S1P binding that is headgroup-dependent.
有充分证据表明，鞘氨醇-1-磷酸（S1P）中的两性离子磷酸基团和胺基与S1P受体第三个跨膜结构域细胞外表面保守的精氨酸（Arg）和谷氨酸（Glu）残基之间存在相互作用。精氨酸（120）和谷氨酸（121）对高亲和力配体-受体相互作用的贡献至关重要，因为单点R（120）A或E（121）A S1P（1）突变体既不结合S1P，也不转导S1P功能。由于S1P受体具有治疗意义，因此鉴定具有不同结合模式和体内疗效的强效选择性激动剂具有重要的药理学意义。在此，我们描述了一种适度水溶性的高选择性S1P（1）激动剂[2-（4-（5-（3,4-二乙氧基苯基）-1,2,4-恶二唑-3-基）-2,3-二氢-1H-茚-1-基氨基）乙醇（CYM-5442）]，其激活S1P（1）依赖性p42/p44丝裂原活化蛋白激酶磷酸化不需要精氨酸（120）或谷氨酸（121）残基，这在S1P（1）中定义了一个新的疏水口袋。CYM-5442在体外是S1P（1）内化、磷酸化和泛素化的完全激动剂。值得注意的是，CYM-5442是诱导和维持S1P（1）依赖性血液淋巴细胞减少的完全激动剂，使B淋巴细胞减少65%，T淋巴细胞减少85%（相对于赋形剂）。CYM-5442诱导的淋巴细胞减少具有剂量和时间依赖性，血清浓度需要在50 nM范围内。CYM-5442对S1P（1）的体外激活作用被特异性S1P（1）拮抗剂[（R）-3-氨基-（3-己基苯基氨基）-4-氧代丁基膦酸（W146）]非竞争性抑制，对S1P、2-氨基-2-（4-辛基苯乙基）丙烷-1,3-二醇（FTY720-P）和5-[4-苯基-5-（三氟甲基）-2-噻吩基]-3-[3-（三氟甲基）苯基]-1,2,4-恶二唑（SEW2871）具有竞争性。此外，W146给药或药代动力学激动剂清除后，CYM-5442诱导的淋巴细胞减少得以逆转。小鼠体内的药代动力学也表明，CYM-5442在中枢神经组织中有显著分布。这些数据表明，CYM-5442在体外激活S1P（1）依赖性途径，并通过一个与S1P结合的正构位点不同的疏水口袋在体内达到完全疗效水平，该疏水口袋依赖于头部基团。
