Serotonin 5-HT _2C receptor as a cellular target of PI3K inhibitor LY294002 and its analog LY303511

The inhibitory analysis of intracellular signaling pathways is widely employed to gain insight into molecular mechanisms underlying diverse physiological processes. Unfortunately, the essential drawback of this basically effective methodology is that many, if not all, inhibitors, antagonists, modulators, and blockers can affect cellular functions not only acting through specified cellular targets, but also causing off-target effects. In particular, the class I phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and its PI3K-inactive structural analog LY303511 have been shown to affect agonist-induced Ca ²⁺ signaling in cells of various types independently of PI3K activity. Here we studied serotonin-induced Ca ²⁺ signaling in HEK293 cells expressing the recombinant mouse 5-HT _2C receptor and analyzed the effects of LY294002 and LY303511 on cell responsiveness. As shown with Ca ²⁺ imaging, both LY294002 and LY303511 affected intracellular Ca ²⁺ but via distinct mechanisms. LY294002 suppressed responsiveness of assayed cells to serotonin in a manner suggesting that this substance acted as a competitive antagonist of the 5-HT _2C receptor. In turn, LY303511 itself triggered Ca ²⁺ transients in 5-HT _2C -positive cells, exhibiting traits of a 5-HT _2C agonist. In support of these findings, molecular docking and molecular dynamics simulations validated the binding of both LY294002 and LY303511 to the 5-HT _2C receptor and occupying its orthosteric site. Altogether, physiological findings and computational data suggested that the observed effects of these compounds were most likely mediated by extracellular mechanisms associated with the direct interaction of both with the 5-HT _2C receptor. This expands the list of non-specified cellular targets of LY294002 and LY303511 with 5-HT _2C subtype of serotonin receptors.