Potent Hemithioindigo-Based Antimitotics Photocontrol the Microtubule Cytoskeleton in Cellulo

Abstract

<div> <div> <p>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. </p> <p>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-hydroxy hemistilbene 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 are 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 network structure, cell cycle and cell survival.</p><div><div><div> <p>We conclude that HITubs represent a powerful addition to the growing toolbox of photopharmaceutical reagents for microtubule 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 cellular and in vivo targets addressable by photopharmacology. </p></div></div></div> </div> </div>

Isoquinoline-Based Biaryls as a Robust Scaffold for Microtubule Inhibitors

“…2 and 3; Fig. 1b) but the south ring has also been reported to tolerate replacement with bicyclics, 10 which our recent experience [18][19][20] has supported. We therefore determined to synthesise and evaluate both "south" and "north" ring replacement types.…”

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

“…2 ). The apparent cytotoxicity differential seen for AzTax3DMA might reflect reduced availability to the cytosol rather than differential binding of the isomers 30 although these results do not allow further conjecture.…”

“…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.…”

“…The azobenzene-based Photostatins ( PST s), which can be reversibly photoswitched by low-intensity visible light between their biologically inactive E -isomers and their MT-destabilising colchicine-like Z -isomers, were first developed in 2014 16 , 25 – 27 . MT-destabilising photopharmaceuticals based on two different families of molecular photoswitch—styrylbenzothiazoles ( SBTubs ) 28 and hemithioindigos (bi-active HOTubs 29 and dark-active 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 epothilone-based microtubule stabilisers allow GFP-imaging-compatible, optical control over the microtubule cytoskeleton

“…However, only one family of photoswitchable MT stabilisers has been reported; the azobenzene-based “AzTax” taxane analogues, which allowed fast, spatiotemporally precise, reversible control of MT dynamics, with a degree of subcellular control in neurons. 15 In contrast, work on photoswitchable colchicinoid MT depolymerisers has explored a diversity of photoswitch scaffolds, from azobenzene (PST reagents), 3,16 , to hemithioindigo (HOTub/HITub), 17,18 , spiropyran-merocyanin 19 ), and styrylbenzothiazole or “SBT” (SBTub reagents), each with their own drawbacks and advantages ( Fig 1a ). 20…”