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Small molecule prodrug approaches that can activate cancer therapeutics selectively in tumors are urgently needed. Here, we developed the first antitumor prodrugs designed for activation by the thioredoxin (Trx) oxidoreductase system. This critical cellular disulfide redox axis is tightly linked to dysregulated redox/metabolic states in cancer, yet it cannot be addressed by current bioreductive prodrugs, which mainly cluster around oxidised nitrogen species. We instead harnessed Trx/TrxR-specific artificial dichalcogenides to gate the bioactivity of a series of 10 “off-to-on” reduction-activated duocarmycin prodrugs. The prodrugs were tested for cell-free and cellular activity dependent on reducing enzyme systems in 177 cell lines, to establish broad trends for redox-based cellular bioactivity of the dichalcogenides. They were well toleratedin vivoin mice, indicating low systemic release of their duocarmycin cargo, andin vivoanti-tumor efficacy trials in mouse models of breast and pancreatic cancer gave promising initial results indicating effective tumoral drug release, presumably byin situbioreductive activation. This work therefore presents a chemically novel class of bioreductive prodrugs against a previously unaddressed reductase type, validates its ability to accessin vivocompatible small-molecule prodrugs even of potently cumulative toxins, and so introduces carefully tuned dichalcogenides as a platform strategy for specific bioreduction-based release.

Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential forin vivotranslation across a variety of research fields and for all eukaryotes. However, photopharmacology’s reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or toin vivoapplications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes.Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminationsin vivoto photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar,in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing generalin vivophotopharmacology one step closer to productive realisation.

Small molecule inhibitors provide a versatile method for studies in microtubule cytoskeleton research, since tubulin is not readily amenable to functional control using genetics. However, traditional chemical inhibitors do not allow spatiotemporally precise applications on the length and time scales appropriate for selectively modulating microtubule-dependent processes. We have synthesised a panel of taxane-based light-responsive microtubule stabilisers, whose tubulin hyperpolymerisation activity can be induced by photoisomerisation to their thermodynamically metastable state. These reagents can be isomerised in live cells, optically controlling microtubule network integrity, cell cycle repartition, and cell survival, and offering biological response on the timescale of seconds and spatial precision to the level of individual cells. These azobenzene-based microtubule stabilisers offer the possibility of noninvasive, highly spatiotemporally precise modulation of the microtubule cytoskeleton in live cells, and can prove powerful reagents for studies of intracellular transport, cell motility, and neurodegeneration.Abstract Figure