Biology subjects

Kotova, P. D.

Publications and source records attributed to Kotova, P. D..

3 recordsLinked to original sources

bioRxiv2025

Serotonin 5-HT2C 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 Ca2+ signaling in cells of various types independently of PI3K activity. Here we studied serotonin-induced Ca2+ signaling in HEK293 cells expressing the recombinant mouse 5-HT2C receptor and analyzed the effects of LY294002 and LY303511 on cell responsiveness. As shown with Ca2+ imaging, both LY294002 and LY303511 affected intracellular Ca2+ 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-HT2C receptor. In turn, LY303511 itself triggered Ca2+ transients in 5-HT2C-positive cells, exhibiting traits of a 5-HT2C agonist. In support of these findings, molecular docking and molecular dynamics simulations validated the binding of both LY294002 and LY303511 to the 5-HT2C 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-HT2C receptor. This expands the list of non-specified cellular targets of LY294002 and LY303511 with 5-HT2C subtype of serotonin receptors.

pharmacology and toxicology↗

bioRxiv2024

PI3 kinase-unrelated effects of LY294002 and LY303511 on serotonin-induced Ca2+ and cAMP signaling

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied effects of PI3K inhibitor LY294002 and its inactive analogue LY303511 on Ca2+ and cAMP signals initiated by serotonin. In the present study several monoclonal HEK293 cell lines were used, in particular, monitoring of Ca2+ signals were carried out on Fura-2 loaded cells expressed recombinant serotonin 5-HT2C receptors, cAMP signals were studied on cells expressed the genetically encoded cAMP sensor Pink Flamindo and recombinant 5-HT4 receptors, for monitoring PI3K activity cells stably expressed the genetically encoded PIP3 sensor PH(Akt)-Venus were used. It turned out that LY294002 suppressed Ca2+ signals initiated by activation 5-HT2C receptors irrespectively of PI3K inhibition, but did not affect cAMP responses initiated by 5-HT4 receptors. In turn LY303511 suppressed cAMP signals initiated by 5-HT4 receptors, and elicited Ca2+ transients exclusively in cells expressed 5-HT2C receptors. Based on these facts and the results of the inhibitory analysis, we hypothesize that the described effects may be due to the activity of LY294002 and LY303511 on the serotonin 5-HT2C and 5-HT4 receptors.

pharmacology and toxicology↗

bioRxiv2024

PI3 kinase inhibitor PI828 uncouples aminergic GPCRs and Ca2+ mobilization irrespectively of its primary target

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca2+ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca2+ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca2+ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca2+ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP3 sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca2+ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca2+ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.

pharmacology and toxicology↗