Photoswitchable microtubule inhibitors enabling robust, GFP-orthogonal optical control over the tubulin cytoskeleton

Abstract

Here we present GFP-orthogonal optically controlled reagents for reliable and repetitive in cellulo modulation of microtubule dynamics and its dependent processes. Optically controlled reagents ("photopharmaceuticals") have developed into powerful tools for high-spatiotemporal-precision control of endogenous biology, with numerous applications in neuroscience, embryology, and cytoskeleton research. However, the restricted chemical domain of photopharmaceutical scaffolds has constrained their properties and range of applications. Styrylbenzothiazoles are an as-yet unexplored scaffold for photopharmaceuticals, which we now rationally design to feature potent photocontrol, switching microtubule cytoskeleton function off and on according to illumination conditions. We show more broadly that this scaffold is exceptionally chemically and biochemically robust as well as substituent-tolerant, and offers particular advantages for intracellular biology through a range of desirable photopharmaceutical and drug-like properties not accessible to the current classes of photoswitches. We expect that these reagents will find powerful applications enabling robust, high precision, optically controlled cell biological experimentation in cytoskeleton research and beyond. Introduction:Molecular photoswitches have been used to install optical control over a broad range of phenomena, with applications from material sciences 1,2 through to reversible photocontrol of ligand binding affinities 3 and manipulation of diverse cellular processes in chemical biology 4,5 . For studies of temporally-regulated and spatially anisotropic biological systems, particularly those that simultaneously support several cellular functions, photoswitchable inhibitors conceptually enable a range of powerful studies not accessible with other tool systems. [6][7][8] A prime example of such spatiotemporally regulated, multifunctional systems is the microtubule (MT) cytoskeleton. This complex cellular network plays central roles in nearly all directed mechanical processes, such as intracellular transport and cell motility; its crucial function in cell proliferation has also made it a central anticancer drug target. 9-11 Yet, whereas cytoskeleton research typically aims to study a subset of MT-dependent processes that are spatially and/or temporally localised, nearly all MT inhibitors reported as tool compounds for biological research are drugs that are active wherever they are distributed, including at sites and at times where drug activity is not desired. 12 This restricts the scope of applications and utility of these inhibitors for selective research into the various, highly dynamic, anisotropic processes dependent on MTs. 13 The structure of the colchicine site MT inhibitor combretastatin A-4 (CA4; Fig 1a) 14 has recently inspired photoswitchable solutions to the problem of achieving spatiotemporal control over MT inhibition. CA4 is a stilbene whose Z-isomer (cis) binds tubulin, acts as a low nanomolar cytotoxin in cellulo, and reached Phase III trials as an ...

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

“…Photocontrol over protein function is an attractive method to study anisotropic, multifunctional cellular systems, since it can deliver the spatiotemporal specificity required to focus on specific roles or aspects within these complex biological systems. Small molecule photopharmaceuticals have already proven valuable photocontrol tools because of their ability to address such targets that are not directly accessible to optogenetics, such as the MT cytoskeleton (for which a range of photoswitchable depolymerising agents have recently been reported 16,28,30 ). Here we have expanded the scope of photopharmaceutical MT reagents to demonstrate the first photoswitchable MT stabilising agents.…”

“…Second, red-shifting photoswitch absorption wavelengths is also likely to be counterproductive for microscopy, since there are few fluorescent proteins with significant excitation efficiency at laser lines above 561 nm (typically the next wavelength available is 647 nm). Therefore, maintaining orthogonality to the widest possible range of imaging wavelengths by blue-shifting is in our opinion and experience more advantageous, since it can keep other imaging channels vacant for multiparameter, photoorthogonal studies 28 . However, a key property that should be readily tunable to the advantage of this system is the E→Z photoisomerisation efficiency at 405 nm, which is usually the only microscopy laser available in the 350-440 nm range.…”

“…The azobenzene-based Photostatins (PSTs), which can be reversibly photoswitched by low-intensity visible light between their biologically inactive E-isomers and their MT-destabilising colchicinelike Z-isomers, were first developed in 2014 16,[25][26][27] . MTdestabilising photopharmaceuticals based on two different families of molecular photoswitch-styrylbenzothiazoles (SBTubs) 28 and hemithioindigos (bi-active HOTubs 29 and darkactive HITubs 30 )-have since been developed, delivering increased metabolic robustness in the intracellular environment and alternative optical switching profiles (all-visible switching with hemithioindigos, GFP-orthogonal switching with SBTubs). All three reagent families have enabled spatiotemporally precise optical control over endogenous MT network integrity, MT polymerisation dynamics, cell division and cell death.…”

Photoswitchable microtubule stabilisers optically control tubulin cytoskeleton structure and function

“…Redshifting photoswitch absorption wavelengths is also likely to be counterproductive for a microscopy reagent, since there are few fluorescent proteins with significant excitation efficiency at laser lines above 561 nm (typically the next wavelength available is 647 nm); maintaining orthogonality to the widest possible range of imaging wavelegnths by blueshifting is probably more advantageous. 20 However, a key property that should be readily tunable to the advantage of this system is the E→Z photoisomerisation efficiency at 405 nm, which is usually the only microscopy laser available in the 350-440 nm range. Here we consider that improved performance for AzTax-like reagents will depend on optimising photoconversion at the wavelength/s that will in practice be used for their photocontrol.…”

“…The azobenzene-based Photostatins (PSTs), which can be reversibly photoswitched by low-intensity visible light between their biologically inactive E-isomers and their MT-inhibiting, colchicine-like Z isomers, were first reported in 2014. 9,[17][18][19] MT destabilising photopharmaceuticals based on two different families of molecular photoswitch -styrylbenzothiazoles (SBTubs) 20 and hemithioindigos (bi-active HOTubs 21 and dark-active HITubs) 22 -have since been developed, delivering increased metabolic robustness in the intracellular environment as well as alternative optical switching profiles (all-visible switching with hemithioindigos, and GFP-orthogonal switching with SBTubs). All three reagent families have enabled highly spatiotemporally precise optical control over endogenous MT network integrity, MT polymerisation dynamics, cell division and cell death; and the PSTs have already been used in animals to resolve outstanding questions e.g.…”