Recreating a chemical synapse for the purpose of kinetic analysis of serotonin receptor activation is a challenge. Neurotransmitters are rapidly released and rapidly 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 spectrally 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 payload upon stimulation with red light (antagonist) or green light (agonist). Here, we first 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 are able to reproducibly control calcium signaling dwell times 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.

Photoactivable WAY-161503 and desmethylclozapine for tight regulation of neuron signaling

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