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

Abstract

Optical methods to modulate microtubule stability and dynamics are promising approaches to reach the micron- and millisecond-scale resolution needed to decrypt the diverse roles of the microtubule cytoskeleton in biology. However, such optical methods have until now focussed nearly exclusively on microtubule destabilisation. Here, we introduce "STEpos" as light-responsive epothilone reagents, designed to photoswitchably bind to tubulin and stabilise lateral contacts in the microtubule lattice. Using a novel styrylthiazole photoswitch, designed to allow the hydrogen-bonding that is key to epothilone potency, we have created the first set of GFP-orthogonal photoswitchable microtubule stabilisers. The STEpos can photocontrol microtubule polymerisation, cell division, and cellular microtubule dynamics with micron- and second-scale spatiotemporal precision. STEpos offer substantial improvements of potency, solubility, and ease-of-use compared to the only previous photopharmaceuticals for microtubule stabilisation. The intriguing structure-photoswitching activity relationship insights from this work will also assist future developments of improved STEpo reagents, and we anticipate that these will contribute greatly to high-precision cytoskeleton research across the fields of biophysics, cargo transport, cell motility, cell division, development, and neuroscience.

Self-reporting styrylthiazolium photopharmaceuticals: mitochondrial localisation as well as SAR drive biological activity

“…We hoped that the high-Stokes shift fluorescence of the StyBtz would allow imaging their biodistribution directly in live cells, towards identifying its targets. We first performed confocal imaging of live HeLa cells transfected with EB3-GFP, a marker for the plus tips of growing MTs which reveals them as “comets” speeding around the cell, 41 after treatment with StyBtz. (Although we no longer expected StyBtz to inhibit tubulin, MT dynamics also provide a useful readout for cell shape and metabolic health).…”

Recent Approaches to the Identification of Novel Microtubule-Targeting Agents

“…Unfortunately, the SBT-Taxane (SBTax) compounds show poor solubility, low bioactivity and modest photocontrol on the biological process. The extension approach has been also applied with styrylthiazole (ST) to epothilones (STEpos, photoactivated at the 405 nm laser line), which are a class of MSA targeting the same Taxane binding site ( Gao et al, 2021a ).…”

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

“…STEpos have only been demonstrated in cell-free and 2D cell culture studies so far. They appear to have rather slow diffusional reversibility (halftime >5 min), which may be related to their binding potency and their mechanism of action, and this may be beneficial for long-term localised inhibition [13]. Azobenzene-based taxol derivatives, AzTax (best compound AzTax3MP), are moderate-potency MT stabiliser photopharmaceuticals which have moderate diffusional reversibility (halftime ca.…”

“…Note 2. Drug Scaffolds: Pharmaceuticals that have already been used as part of MT-inhibiting photopharmaceuticals include taxol [14] and epothilone [13] for MT-stabilising photopharmaceuticals AzTax and STEpo; and colchicinoids (analogues of colchicine-binding-site drugs like colchicine, colcemid, nocodazole or combretastatin) have been used in MT-destabilising photopharmaceuticals e.g. PST, SBTub and PHTub (Fig 5) [2,7,9,15,23,24].…”