Potent hemithioindigo-based antimitotics photocontrol the microtubule cytoskeleton in cellulo

Abstract

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.

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

“…Therefore, for potency optimisation, we tested both adding methyl groups to SBTub3 and "walking" them around the scaffold south ring (9-13); and also deleting the north ring hydroxy/methoxy substituent pair from 1 and replacing them with smaller methyls (14)(15)(16)(17)(18). Note that the SBT Z-isomers orient their sulfur towards the inner face of the molecule 16 so we did not rely on rotations around the alkene-benzothiazole single bond to 'reflect' substituents into similar positions (e.g.…”

“…South ring derivatives 9-13 did not approach the potency of 1-5, although 10-11 still have excellent performance (~1 µM Z-potency, ~100-fold photoswitchability of bioactivity). However, north ring derivative SBTub2M (15) in which the methyl group replaces the spacefilling OMe group, was six times as potent as its methoxy homologue 3 (Z-IC50 35 nM) while having excellent photoswitchability of bioactivity (ca. 200-fold; Fig 2c).…”

“…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 .…”

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

“…Intriguingly, these compounds seem to show much slower diffusion than comparable small molecules. [15,23,24] Their solubility is moderate at best; and they display at best only ca. 10-fold ratio of bioactivity between the isomers, whereas PSTs and SBTubs can have ratios ≥200 making the latter much simpler to use.…”

Photoswitchable epothilone-based microtubule stabilisers allow GFP-imaging-compatible, optical control over the microtubule cytoskeleton

“…However, only one family of photoswitchable MT stabilisers has 3/15 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 Principal epothilones.…”