Photo/photoredox-switchable ligands as chemical tools for optogenetics

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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).