A photo-SAR study of photoswitchable azobenzene tubulin-inhibiting antimitotics identifying a general method for near-quantitative photocontrol

Here, we performed photo-SAR studies in a series of photoswitchable tubulin inhibiting antimitotics, and discovered that fluorescent antenna motifs permit their single-photon photoisomerisation in biological conditions at up to >600 nm; we were also able to visualise the temporal onset of their long-term photopharmacological effects, using large-field-of-view microscopy. Previously, azobenzene analogues of the tubulin polymerisation inhibitor combretastatin A4 (PSTs) had been developed to optically control microtubule dynamics in living systems, with subsecond response time and single-cell spatial precision, by reversible in situ photoswitching of their bioactivity with near-UV/visible light. First-generation PSTs were sufficiently potent and photoswitchable for use in live cells and embryos. However, the link between their seconds-scale and hours-scale bioactivity remained untested. Here, we now used tandem photoswitching/microscopy to reveal the timing of onset of their long-term bulk cytostatic effects. Since the scope for modifications to tune their photo-structure-activity-relationship or expand PST function had been unknown, we then synthesised a panel of novel PSTs exploring structural variations that tune photoresponse wavelengths and lipophilicity, identifying promising blue-shifted analogues that are better-compatible with GFP/YFP imaging. Taken together, these results can guide new design and applications for photoswitchable microtubule inhibitors. Finally, we also identified tolerated sites for linkers to attach functional cargos, and tested them with fluorescent "antennas" as reporters. Serendipitously, we found that antennas can greatly enhance long-wavelength single-photon photoisomerisation, by a hitherto un-explored mechanism. This final result will drive progress towards near-quantitative long-wavelength photoswitching of photopharmaceuticals in living systems, with minimal molecular redesign and general application scope.