A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton.

Optically controlled chemical reagents, termed "photopharmaceuticals," are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.

Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton.

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.

Potent hemithioindigo-based antimitotics photocontrol the microtubule cytoskeleton in cellulo.

Background: Hemithioindigo is a promising molecular photoswitch that has only recently been applied as a photoswitchable pharmacophore for control over bioactivity in cellulo. Uniquely, in contrast to other photoswitches that have been applied to biology, the pseudosymmetric hemithioindigo scaffold has allowed the creation of both dark-active and lit-active photopharmaceuticals for the same binding site by a priori design. However, the potency of previous hemithioindigo photopharmaceuticals has not been optimal for their translation to other biological models. Results: Inspired by the structure of tubulin-inhibiting indanones, we created hemithioindigo-based indanone-like tubulin inhibitors (HITubs) and optimised their cellular potency as antimitotic photopharmaceuticals. These HITubs feature reliable and robust visible-light photoswitching and high fatigue resistance. The use of the hemithioindigo scaffold also permitted us to employ a para-hydroxyhemistilbene motif, a structural feature which is denied to most azobenzenes due to the negligibly short lifetimes of their metastable Z-isomers, which proved crucial to enhancing the potency and photoswitchability. The HITubs were ten times more potent than previously reported hemithioindigo photopharmaceutical antimitotics in a series of cell-free and cellular assays, and allowed robust photocontrol over tubulin polymerisation, microtubule (MT) network structure, cell cycle, and cell survival. Conclusions: HITubs represent a powerful addition to the growing toolbox of photopharmaceutical reagents for MT cytoskeleton research. Additionally, as the hemithioindigo scaffold allows photoswitchable bioactivity for substituent patterns inaccessible to the majority of current photopharmaceuticals, wider adoption of the hemithioindigo scaffold may significantly expand the scope of cellular and in vivo targets addressable by photopharmacology.