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.

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.

<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>

<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>