Photoactivatable Agonist-Antagonist Pair as a Tool for Precise Spatiotemporal Control of Serotonin Receptor 2C Signaling

Abstract

Orthogonal recreation of the signaling profile of a chemical synapse is a current challenge in neuroscience. This is due in part to the kinetics of synaptic signaling, where neurotransmitters are rapidly released and quickly cleared by active reuptake machinery. One strategy to produce a rapid rise in an orthogonally controlled signal is via photocaged compounds. In this work, photocaged compounds are employed to recreate both the rapid rise and equally rapid fall in activation at a chemical synapse. Specifically, a complementary pair of photocages based on BODIPY were conjugated to a 5-HT2C subtype-selective agonist, WAY-161503, and antagonist, N-desmethylclozapine, to generate “caged” versions of these drugs. These conjugates release the bioactive drug upon illumination with green light (agonist) or red light (antagonist). We report on the synthesis, characterization, and bioactivity testing of the conjugates against the 5-HT2C 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 in the dark for 48 h at room temperature, yet photolyze rapidly when irradiated with visible light. In live cells expressing the 5-HT2C 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 noninvasive 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 5-HT2C modulation and will allow for more detailed studies of the receptors’ signaling, interactions with other proteins, and native physiology.