Photo/photoredox-switchable ligands as chemical tools for optogenetics

合成的小分子试剂是目标蛋白质的可光切换配体(“光药物”)，它在重要方面扩大了药物操作的范围以及空间和时间的精确度。特别是，当蛋白质是带有反应结合位点的基因工程跨膜受体(通常是胞外半胱氨酸)，而配体具有互补反应基团时，这种方法被证明在光遗传学研究中特别成功地创建了光致可逆的光驱动离子通道和GPCR(最初被称为光化学遗传学)。然而，被确认为所有这些可光切换配体的基础的偶氮苯分子支架通常只有一个很窄的光可逆性指数，通常具有大约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表现出高度非线性响应的蛋白质；尽管如此，从短期细胞应用的原则证明过渡到一般细胞或体内生物学研究的几个困难。本项目旨在为光制药特别是光化学遗传学研究提供一个新的分子概念：具有大约2个数量级动态光可逆性的光/光氧化还原可切换分子结构(目标1)。更宽的动态范围将使这些工具能够比现有技术更广泛和更可靠地应用于工程跨膜受体(AIM 2)。我们还希望将这种方法应用于细胞内，这是一个迄今具有挑战性的目标，需要在感兴趣的蛋白质上安装高度特异的反应基序。我们将使用遗传密码扩展来创建具有生物正交性特定标记位置和最小功能/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，我们希望检查自由扩散的光/光氧化还原开关配体作为内源性受体的高灵敏人工光敏剂的性能--特别是视网膜神经节细胞中的离子通道，它有可能恢复退化、失明的视网膜的敏感视力(目标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).