Photo/photoredox-switchable ligands as chemical tools for optogenetics

合成的小分子试剂是感兴趣的蛋白质的光切换配体（“光药物”），在重要的方面扩展了药理学操作的范围和空间和时间精度。特别是，当蛋白质是一种基因工程跨膜受体，具有反应性附着位点（通常是细胞外的Cys），配体具有互补反应基团时，这种方法已被证明在创建光可逆光驱动离子通道和光遗传学研究（最初称为“光化学遗传学”）的gpcr方面特别成功。然而，作为所有这些可光切换配体基础的偶氮苯分子支架通常只有一个狭窄的光可逆性指数，通常约为5倍的动态范围。因此，这种方法在很大程度上依赖于识别对有效配体浓度变化具有高度非线性响应的蛋白质；尽管如此，从短期细胞应用的原理证明过渡到一般的细胞或体内生物研究使用，经历了一些困难。该项目旨在为光制药和光化学遗传学研究提供一个新的分子概念：具有约2个数量级光可逆性动态范围的光/光氧化还原可切换分子结构（Aim 1）。与现有技术相比，更宽的动态范围将使这些工具能够更广泛、更可靠地应用于工程跨膜受体（Aim 2）。我们进一步希望将这种方法转化为细胞内应用，这是一个迄今为止具有挑战性的目标，需要在感兴趣的蛋白质上安装高度特异性的反应性基序。我们将使用遗传密码扩展来创建具有生物正交特异性标记位点和最小功能/结构扰动的蛋白质，并评估生物正交标记光/光氧化还原开关控制跨膜（Aim 3）和细胞内（Aim 4）靶标的能力。最后，我们希望研究自由扩散的光/光氧化还原开关配体作为内源性受体（特别是视网膜神经节细胞中的离子通道）的高度敏感的人工光敏剂的性能，它有可能恢复退化的失明视网膜的敏感视力（Aim 5）。

Synthetic small-molecule reagents that are photoswitchable ligands for proteins of interest (“Photopharmaceuticals”) have extended the reach and the spatial and temporal precision of pharmacological manipulations in important ways. In particulary, when the protein is a genetically engineered transmembrane receptor bearing a reactive attachment site (typically an extracellular Cys) and the ligand features the complementary reactive group, this approach has proven particularly successful in creating photoreversibly light-actuated ion channels and GPCRs for optogenetics studies (initially called “Optochemical Genetics”). However, the azobenzene molecular scaffolds identified as the basis for all these photoswitchable ligands usually have only a narrow photoreversibility index, with typically ca. 5-fold dynamic range. Therefore, this approach has substantially relied on identifying proteins exhibiting a highly nonlinear response to changes of effective ligand concentration; and nonetheless has experienced several difficulties transitioning from proof-of-principle in short-term cellular applications, to cell or in vivo biological research use in general.This project aims at a new molecular concept for photopharmaceutical and especially optochemical genetics studies: photo/photoredox-switchable molecular constructs with ca. 2 orders of magnitude dynamic range of photoreversibility (Aim 1). The wider dynamic range will enable these tools to be applied more broadly and more reliably to engineered transmembrane receptors (Aim 2) than existing techniques. We further want to translate this method to intracellular applications, a hitherto challenging goal that will require installing highly specific reactive motifs on the proteins of interest. We will use genetic code expansion to create proteins with bioorthogonally specific labelling sites and minimal functional/structural perturbation, and assess the ability of bioorthogonal-label photo/photoredox switches to control both transmembrane (Aim 3) and intracellular (Aim 4) targets. Finally, we wish to examine the performance of freely diffusing photo/photoredox-switch ligands as highly sensitive artificial photosensitisers of endogenous receptors - in particular of ion channels in retinal ganglion cells, which has the potential to restore sensitive visual acuity to the degenerated, blind retina (Aim 5).