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.
摘要:
化学突触的信号传导谱的正交再现是神经科学中当前的挑战。这部分是由于突触信号的动力学,神经递质通过主动再摄取机制迅速释放并迅速清除。在正交控制的信号中产生快速上升的一种策略是通过光老化的化合物。在这项工作中,光老化的化合物被用来在化学突触中重现快速上升和同样快速下降的激活。具体来说,一对基于BODIPY的互补光致抗体与5-HT2C亚型选择性激动剂缀合,WAY-161503和拮抗剂,N-去甲基氯氮平,生成这些药物的“笼中”版本。这些缀合物在用绿光(激动剂)或红光(拮抗剂)照射时释放生物活性药物。我们报告了综合情况,表征,和针对5-HT2C受体的缀合物的生物活性测试。然后,我们使用HPLC定量表征光解的动力学,并在刺激活细胞的细胞培养条件下定性表征。该化合物在室温下在黑暗中稳定48小时,然而,当用可见光照射时,光解迅速。在表达5-HT2C受体的活细胞中,证明了钙信号传导的程度和长度的精确时空控制。通过串联加载两种化合物并利用光谱复用作为一种非侵入性方法来控制局部小分子药物的可用性,我们可以在传统给药方法无法实现的时间范围内重复启动和抑制细胞内钙流动。这些工具能够对5-HT2C调节进行更多的时空控制,并将允许对受体信号进行更详细的研究。与其他蛋白质的相互作用,和原生生理学。