Abstract
Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. ...However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.
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.
Switch me off, switch me on: The first use of the hemithioindigo scaffold as a photoswitchable inhibitor pharmacophore is reported for the preparation of photopharmaceuticals to control endogenous cytoskeleton structure and cell cycle. Hemithioindigos allow a priori design of either Z‐ or E‐ (dark‐ or lit‐) bioactives, so they may address the problem of undesirable dark activity in the chemistry of cellular photopharmaceuticals.
Optically controlled chemical reagents, termed “photopharmaceuticals,” are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or ...optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.
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•SBTub3 photocontrols microtubule dynamics, organization, and dependent processes•Microtubule photocontrol is cell and sub-cellularly precise and temporally reversible•SBT photocontrol is orthogonal to GFP/YFP imaging and SBTs are metabolically stable•The SBT scaffold is promising for photopharmaceuticals for other protein targets
Photocontrollable reagents have unique potential as high spatiotemporal precision modulators of biological systems. Here, Gao et al. demonstrate a GFP-orthogonal and metabolically stable photoswitch that allows optical control over microtubule dynamics and architecture with subcellular resolution. The photoswitch scaffold also offers new possibilities for photopharmaceutical design against other targets.
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.
Inspired by the structure of tubulin-inhibiting indanones, we created hemithioindigo-based indanone-like tubulin inhibitors (
) and optimised their cellular potency as antimitotic photopharmaceuticals. These
feature reliable and robust visible-light photoswitching and high fatigue resistance. The use of the hemithioindigo scaffold also permitted us to employ a
-hydroxyhemistilbene motif, a structural feature which is denied to most azobenzenes due to the negligibly short lifetimes of their metastable
-isomers, which proved crucial to enhancing the potency and photoswitchability. The
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.
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.
We here report the discovery of isoquinoline-based biaryls as a new scaffold for colchicine domain tubulin inhibitors. Colchicinoid inhibitors offer highly desirable cytotoxic and vascular disrupting ...bioactivities, but their further development requires improving in vivo robustness and tolerability: properties that both depend on the scaffold structure employed. We have developed isoquinoline-based biaryls as a novel scaffold for high-potency tubulin inhibitors, with excellent robustness, druglikeness, and facile late-stage structural diversification, accessible through a tolerant synthetic route. We confirmed their bioactivity mechanism in vitro, developed soluble prodrugs, and established safe in vivo dosing in mice. By addressing several problems facing the current families of inhibitors, we expect that this new scaffold will find a range of in vivo applications towards translational use in cancer therapy.
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•Isoquinoline-based biaryls were developed as colchicine domain tubulin inhibitors.•The combretastatin-like biaryls are potent, robust, and tolerated in vivo.•Microtubule structure and tubulin-dependent processes are inhibited in live cells.•Scaffold synthesis is straightforward and diversifiable.•Live cell confocal microscopy reveals their cellular target is rapidly engaged.
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