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

In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

“…enzymatic hydrolysis of cages). In the MT field, recent photoswitchable analogues of taxane 12 , epothilone 13 and colchicinoid 5,[14][15][16][17][18][19] MT inhibitors have all been applied to cellular studies. These photopharmaceuticals have enabled noninvasive, reversible optical control over MT dynamics and MT-associated downstream effects, with cell-specific spatial precision and sub-second-scale temporal precision, and have been brought to bear on research in embryology 20 , neuroscience 21 , and cytoskeleton 5 .…”

“…However, the typical photoswitch scaffolds introduce problems particularly for in vivo application, three of which this paper will focus on: (1) Metabolic stability: azobenzenes, particularly as their Z-isomers, are reductively degraded by cellular glutathione (GSH). [22][23][24] This reduces photoswitchability as well as potency, and on timescales typical for in vivo studies (> hours) the degradation byproducts are likely to give off-target effects, particularly in metabolically active multiorgan systems (tested below, and also discussed elsewhere 16 ). (2) Orthogonal photocontrol and imaging: azobenzenes, and the less common hemithioindigos, are isomerised by excitation of the typical imaging labels used in biological studies (GFP/fluorescein at 490 nm, YFP at 514 nm); some also isomerise with RFP/rhodamine imaging at 561 nm (at focussed laser intensities).…”

“…(2) Orthogonal photocontrol and imaging: azobenzenes, and the less common hemithioindigos, are isomerised by excitation of the typical imaging labels used in biological studies (GFP/fluorescein at 490 nm, YFP at 514 nm); some also isomerise with RFP/rhodamine imaging at 561 nm (at focussed laser intensities). 16,25 Typically this leaves only the 647 nm laser channel available for orthogonal imaging during photoswitching, yet this laser typically only addresses small molecule probes. These scaffolds' inability to allow orthogonal fluorescent-protein-based imaging during photocontrol is particularly problematic for in vivo research that extensively relies on two-or three-channel imaging with fluorescent protein fusions to resolve processes with biological specificity.…”

“…This either requires high-potency compounds or chemically robust solubilising strategies: neither of which are often seen with typically hydrophobic azobenzene or hemithioindigo drug analogues. To minimise illumination while maximising bioactivity explicitly requires tuning the photoswitch scaffold's photoresponse at those laser wavelength/s that are in practice used for photoconversion in the biological study 16 (aim: high efficiency of isomerisation E(λ) 5 and high photostationary state): since photoswitch performance at optimal but unavailable wavelengths (e.g. 260-380 nm) is irrelevant.…”

“…SBTub3 could photocontrol microtubule dynamics, organization, and MT-dependent processes in live cells with reversible temporal patterning, and with cellular and even sub-cellular spatial precision. 16 The metabolic stability and imaging-orthogonal photocontrol of SBTub3 were excellent features towards in vivo use, but practical limitations remained, which we determined to address in this study. Primarily, while their parent colchicinoid inhibitor combretastatin A-4 (CA4) has ca.…”