Photoactivable WAY-161503 and Desmethylclozapine for Tight Regulation of Serotonin Receptor 2C Signaling.

30 December 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Recreating the signaling profile a chemical synapse to analyze serotonin receptor activation is a challenge. This is due in part to the kinetics of the synapse, where neurotransmitters are rapidly released and quickly cleared by active reuptake machinery. One strategy to produce a rapid rise in a bio-orthogonally controlled signal is via photocaged compounds. In this work, a complementary pair of BODIPY photoremovable protecting groups was conjugated to a 5HT2C subtype selective agonist, WAY-161503, and antagonist, N-desmethylclozapine, to generate “caged” versions of these drugs. These conjugates can release their bioactive drug upon stimulation with green light (agonist) or red light (antagonist). We report on the synthesis, characterization, and bioactivity testing of the conjugates against the 5HT2C receptor. We then characterize the kinetics of photolysis quantitatively using HPLC and qualitatively in cell culture conditions stimulating live cells. The compounds are shown to be stable under dark conditions for 48 hours at room temperature, yet photolyze readily when irradiated with visible light. In live cells expressing the 5HT2C receptor, precise spatiotemporal control of the degree and length of calcium signaling is demonstrated. By loading both compounds in tandem and leveraging spectral multiplexing as a non-invasive method to control local small molecule drug availability, we can reproducibly initiate and suppress intracellular calcium flux on a timescale not possible by traditional methods of drug dosing. These tools enable a greater spatiotemporal control of 5HT2C modulation and will allow for more detailed studies of the receptors signaling, interactions with other proteins, and native physiology.

Keywords

Photocaging
Serotonin
GPCR
Synapse
Calcium Signaling
5HT2C

Supplementary materials

Title
Description
Actions
Title
Supporting Information for Photoactivable WAY-161503 and Desmethylclozapine for Tight Regulation of Serotonin Receptor 2C Signaling.
Description
synthetic procedures, biological protocols, and NMR spectra
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