Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer‐Dependency of Potent Antimitotic Bioactivity

Abstract

Druglike small molecules with photoswitchable bioactivity—photopharmaceuticals—allow biologists to perform studies with exquisitely precise and reversible, spatial and temporal control over critical biological systems inaccessible to genetic manipulation. The photoresponsive pharmacophores disclosed have been almost exclusively azobenzenes, which has limited the structural and substituent scope of photopharmacology. More detrimentally, for azobenzene reagents, it is not researchers’ needs for adapted experimental tools, but rather protein binding site sterics, that typically force whether the trans (dark) or cis (lit) isomer is the more bioactive. We now present the rational design of HOTubs, the first hemithioindigo‐based pharmacophores enabling photoswitchable control over endogenous biological activity in cellulo. HOTubs optically control microtubule depolymerisation and cell death in unmodified mammalian cells. Notably, we show how the asymmetry of hemithioindigos allows a priori design of either Z‐ or E‐ (dark‐ or lit)‐toxic antimitotics, whereas the corresponding azobenzenes are exclusively lit‐toxic. We thus demonstrate that hemithioindigos enable an important expansion of the substituent and design scope of photopharmacological interventions for biological systems.

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

“…E-SBTub2 has in fact been reported as a cytotoxic resveratrol analogue targeting tubulin, where its E-geometry was established explicitly and extensive docking simulations were performed to rationalise observed activity 37,38 . However, the E-activity stated in those reports disagrees with our experience 6,39,40 and literature understanding 16 of the requirements of the colchicine binding site. We decided therefore to pursue our paper's design logic paying particular attention to experimentally verify the isomer status and binding mode of the compound (discussion in Supporting Information).…”

“…In order to test the target-specificity of the SBTubs' bioactivities, we also designed permutation controls (designed-inactive compounds) by swapping the positions of key bioactivity-controlling substituents. We have previously reported permutation controls as a convenient and stringent method 39 to distinguish between biological disruption resulting from molecularly-specific binding to the target protein (which should be a feature of the designed-active compounds only), versus bioactivity from nonspecific interactions expected for typically hydrophobic photoswitch compounds (e.g., promiscuous binding to proteins, aggregation on proteins, compound precipitation, membrane disruption, phototoxicity, or photoswitch scaffold toxicity, which should also be observed for closely regioisomeric permutations). To this end, we permuted methoxy and hydro groups of SBTub2 to create designed-inactive SBTub1, and permuted hydroxy and methoxy groups of SBTub3 to create control SBTub4.…”

“…We attributed the weak antiproliferative activity of all SBTubs at high concentrations in the dark, and of permutation controls SBTub1 and SBTub4 also under illuminated conditions, as aggregation-dependent effects resulting from poor solubility of the compounds at high concentrations as is known for similar motifs (this is examined in more detail below). 6,39 At this stage, the light-dependent in cellulo activity matching the design principles of the active…”

Reversible Photoswitching of Isolated Ionic Hemiindigos with Visible Light

“…Other applications include light controlled peptide structure and supramolecular chemistry . These studies illustrate the broad versatility of indigoid photoswitches and their potential for use in nanotechnological and biomedical applications …”

“…[39] These studies illustrate the broad versatility of indigoid photoswitches and their potential for use in nanotechnological and biomedical applications. [40][41][42] Developing effective molecular photoswitches requires a detailed understanding of their fundamental photochemical properties and underlying photoisomerization mechanisms, best provided by solid connections between experiment and theory. Although, in principle, comparisons between theory and experiment are best achieved for molecules that are free from the complicating influence of solvent or substrate, there are challenges associated with isolating and characterising the photochemical properties of molecular photoswitches in the gas phase.…”