Sphingosine-1-phosphate (S1P) is a pleiotropic phospholipid that is synthesized intracellularly via the action of sphingosine kinase-1, which is activated by ERK1. S1P is transported to the extracellular environment by SPNS2, an ion channel specific for S1P translocation. S1P mediates several of its biological effects via nanomolar affinity for five S1PR, which act as class A GPCR and work through one of three distinct GTP-binding proteins to mediate a cell signaling cascade. Unique amongst these S1PR is S1P₂, which is upregulated in several tumor cell lines, yet when activated by a selective agonist appears to inhibit cell migration, invasiveness, and angiogenesis by activation the G_12/13cascade, which involves activation of Rho, Rho kinase (ROCK) and PTEN, which antagonizes the effects of PREX2. This inhibition of PREX2 results in the inhibition of PI3K, Akt and Rac. To date, only two S1P₂-selective agonists have been described, and no FDA-approved agonists have migrated from bench to bedside. Previous homology modeling of active S1P₂ has resulted in incomplete validation, but a recent cryo EM crystal structure of S1P₂ co-crystallized with S1P has placed a long-sought tool to elucidate the agonist binding pocket of this enigmatic receptor. In this current work we describe several novel amino acid interactions that serve to stabilize S1P and as such, constitute residues that line the established binding pocket. In addition, we have performed numerous in-silico mutations in junction with 1 microsecond molecular dynamics simulations to more effectively predict biological site-directed mutagenesis results previously published by three different laboratories. Using the molecular operating environment (MOE) fragment search tool in addition to chemical intuition, we have designed, docked, and analyzed a series of 300 compounds and have proposed the most effective lead compound for organic synthesis. Finally, we have docked and performed 1 microsecond molecular dynamics to the most promising computational lead compound in all five S1PR active conformations to analyze potential selectivity, and assert that ZOE-I-2 represents a unique S1P₂-selective agonist capable of binding in the same volume of S1P occupation in this receptor.

Sofia Alzugaray Orellana

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