A High-Quality Photoswitchable Probe that Selectively and Potently Regulates the Transcription Factor RORγ.

Retinoic acid receptor-related orphan receptor γ (RORγ) is a nuclear hormone receptor with multiple biological functions. As an experimental therapeutic target in inflammation and immunity, there is great interest in spatially-localised RORγ inhibition; and its cyclic temporal role in circadian rhythms also makes it an intriguing target for time-resolved pharmacology. To create tools that can study RORγ biology with appropriate spatial and temporal resolution, we designed light-dependent inverse RORγ agonists by building azobenzene photoswitches into ligand consensus structures. Optimizations gave photoswitchable RORγ inhibitors with a large degree of potency photocontrol, plus remarkable on-target potency, plus excellent selectivity over related off-target receptors. This still-rare, but urgently-needed combination of performance features, distinguishes them as high quality photopharmaceutical probes; and they can now serve as high precision tools to study the spatial and dynamic intricacies of RORγ action in signaling and in inflammatory disorders.

A photo-SAR study of photoswitchable azobenzene tubulin-inhibiting antimitotics identifying a general method for near-quantitative photocontrol.

Azobenzene analogues of the tubulin polymerisation inhibitor combretastatin A4 (PSTs) were previously developed to optically control microtubule dynamics in living systems, with subsecond response time and single-cell spatial precision, by reversible in situ photoswitching of their bioactivity with near-UV/visible light. First-generation PSTs were sufficiently potent and photoswitchable for use in live cells and embryos. However, the link between their seconds-scale and hours-scale bioactivity remained untested. Furthermore, the scope for modifications to tune their photo-structure-activity-relationship or expand their function was unknown. Here, we used large-field-of-view, long-term tandem photoswitching/microscopy to reveal the temporal onset of cytostatic effects. We then synthesised a panel of novel PSTs exploring structural variations that tune photoresponse wavelengths and lipophilicity, identifying promising blue-shifted analogues that are better-compatible with GFP/YFP imaging. Taken together, these results can guide new design and applications for photoswitchable microtubule inhibitors. We also identified tolerated sites for linkers to attach functional cargos; and we tested fluorophores, aiming at RET isomerisation or reporter probes. Instead we found that these antennas greatly enhance long-wavelength single-photon photoisomerisation, by an as-yet un-explored mechanism, that can now drive general progress towards near-quantitative long-wavelength photoswitching of photopharmaceuticals in living systems, with minimal molecular redesign and broad scope.

A Photocaged Microtubule-Stabilising Epothilone Allows Spatiotemporal Control of Cytoskeletal Dynamics.

The cytoskeleton is essential for spatial and temporal organisation of a wide range of cellular and tissue-level processes, such as proliferation, signalling, cargo transport, migration, morphogenesis, and neuronal development. Cytoskeleton research aims to study these processes by imaging, or by locally manipulating, the dynamics and organisation of cytoskeletal proteins with high spatiotemporal resolution: which matches the capabilities of optical methods. To date, no photoresponsive microtubule-stabilising tool has united all the features needed for a practical high-precision reagent: a low potency and biochemically stable non-illuminated state; then an efficient, rapid, and clean photoresponse that generates a high potency illuminated state; plus good solubility at suitable working concentrations; and efficient synthetic access. We now present CouEpo, a photocaged epothilone microtubule-stabilising reagent that combines these needs. Its potency increases approximately 100-fold upon violet/blue irradiation to reach low-nanomolar values, allowing efficient photocontrol of microtubule dynamics in live cells, and even the generation of cellular asymmetries in microtubule architecture and cell dynamics. CouEpo is thus a high-performance tool compound that can support high-precision research into many microtubule-associated processes, from biophysics to transport, cell motility, and neuronal physiology.

Merged Molecular Switches Excel as Optoacoustic Dyes: Azobenzene-Cyanines Are Loud and Photostable NIR Imaging Agents.

Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents, which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches with cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (>3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury.

Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies in vivo. Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.e., cannot delineate damaged volumes in vivo nor image non-nucleated cell types or compartments). Here, we develop modular fluorogenic probes that reveal the whole cytosolic volume of damaged cells, with near-zero background fluorescence so that no washing is needed. We identify a specific disulfonated fluorogenic probe type that only enters cells with damaged membranes, then is enzymatically activated and marks them. The esterase probe MDG1 is a reliable tool to reveal live cells that have been permeabilized by biological, biochemical, or physical membrane damage, and it can be used in multicolor microscopy. We confirm the modularity of this approach by also adapting it for improved hydrolytic stability, as the redox probe MDG2. We conclude by showing the unique performance of MDG probes in revealing axonal membrane damage (which DNA fluorogens cannot achieve) and in discriminating damage on a cell-by-cell basis in embryos in vivo. The MDG design thus provides powerful modular tools for wash-free in vivo imaging of membrane damage, and indicates how designs may be adapted for selective delivery of drug cargoes to these damaged cells: offering an outlook from selective diagnosis toward therapy of membrane-compromised cells in disease.

Piperazine-Fused Cyclic Disulfides Unlock High-Performance Bioreductive Probes of Thioredoxins and Bifunctional Reagents for Thiol Redox Biology.

We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis- and trans-piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis-disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while trans-disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols' potential as high-performance reducing agents.

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers.

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Inhibition of Cell Motility by Cell-Penetrating Dynamic Covalent Cascade Exchangers: Integrins Participate in Thiol-Mediated Uptake.

Integrins are cell surface proteins responsible for cell motility. Inspired by the rich disulfide exchange chemistry of integrins, we show here the inhibition of cell migration by cascade exchangers (CAXs), which also enable and inhibit cell penetration by thiol-mediated uptake. Fast-moving CAXs such as reversible Michael acceptor dimers, dithiabismepanes, and bioinspired epidithiodiketopiperazines are best, much better than Ellman's reagent. The implication that integrins participate in thiol-mediated uptake is confirmed by reduced uptake in integrin-knockdown cells. Although thiol-mediated uptake is increasingly emerging as a unifying pathway to bring matter into cells, its molecular basis is essentially unknown. These results identify the integrin superfamily as experimentally validated general cellular partners in the dynamic covalent exchange cascades that are likely to account for thiol-mediated uptake. The patterns identified testify to the complexity of the dynamic covalent networks involved. This work also provides chemistry tools to explore cell motility and expands the drug discovery potential of CAXs from antiviral toward antithrombotic and antitumor perspectives.

Cyclic Dichalcogenides Extend the Reach of Bioreductive Prodrugs to Harness Thiol/Disulfide Oxidoreductases: Applications to seco-Duocarmycins Targeting the Thioredoxin System.

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 thiol-manifold oxidoreductases, targeting the thioredoxin (Trx) system. The Trx system is a critical cellular redox axis that is tightly linked to dysregulated redox/metabolic states in cancer, yet it cannot be addressed by current bioreductive prodrugs, which mainly cluster around oxidized nitrogen species. We instead harnessed Trx/TrxR-specific artificial dichalcogenides to gate the bioactivity of 10 "off-to-on" reduction-activated duocarmycin prodrugs. The prodrugs were tested for cell-free and cellular reductase-dependent activity in 177 cell lines, establishing broad trends for redox-based cellular bioactivity of the dichalcogenides. They were well tolerated in vivo in mice, indicating low systemic release of their duocarmycin cargo, and in vivo anti-tumor efficacy trials in mouse models of breast and pancreatic cancer gave promising indications of effective tumoral drug release, presumably by in situ bioreductive activation. This work therefore presents a chemically novel class of bioreductive prodrugs against a previously unaddressed reductase chemotype, validates its ability to access in vivo-compatible small-molecule prodrugs even of potently cumulative toxins, and so introduces carefully tuned dichalcogenides as a platform strategy for specific bioreduction-based release.

Microtubule depolymerization contributes to spontaneous neurotransmitter release in vitro.

Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.

Correction to "40 Years of Duocarmycins: A Graphical Structure/Function Review of Their Chemical Evolution, from SAR to Prodrugs and ADCs".

[This corrects the article DOI: 10.1021/jacsau.2c00448.].

[5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity.

Helicenes are high interest synthetic targets with unique conjugated helical structures that have found important technological applications. Despite this interest, helicenes have had limited impact in chemical biology. Herein, we disclose a first-in-class antimitotic helicene, helistatin 1 (HA-1), where the helicene scaffold acts as a structural mimic of colchicine, a known antimitotic drug. The synthesis proceeds via sequential Pd-catalyzed coupling reactions and a π-Lewis acid cycloisomerization mediated by PtCl2. HA-1 was found to block microtubule polymerization in both cell-free and live cell assays. Not only does this demonstrate the feasibility of using helicenes as bioactive scaffolds against protein targets, but also suggests wider potential for the use of helicenes as isosteres of biaryls or cis-stilbenes-themselves common drug and natural product scaffolds. Overall, this study further supports future opportunities for helicenes for a range of chemical biological applications.

Exhaustive Catalytic ortho-Alkoxylation of Azobenzenes: Flexible Access to Functionally Diverse Yellow-Light-Responsive Photoswitches.

We develop the first method for catalytic, exhaustive ortho-alkoxylation of azobenzene photoswitches. Alkoxylation is known to improve the photoswitch properties that control azobenzenes' success in chemical biology or materials sciences, e.g., better completeness of both E → Z and Z → E photoisomerizations and >100 nm red shift of photoresponse. Our method enables straightforward late-stage diversification of photoswitches with interesting functional handles. We showcase four applications: using it to rationally tune lipophilicity, prepare isotopic tracers for metabolism studies, install full water solubility without ionic charges, and efficiently access previously difficult mixed-substituent photoswitches. We also identified a previously unexplored mixed-substituent tetra-ortho family, difluoro-dialkoxy-azobenzenes, whose photoresponse can outperform previous 'gold standard' tetrafluoro-, dichloro-difluoro-, and tetrachloro-azobenzenes in significant ways. We thus expect that both the scaffolds we showcase and the method we develop will impact broadly on photochemistry and photopharmacology.

Next Generation Opto-Jasplakinolides Enable Local Remodeling of Actin Networks.

The natural product jasplakinolide is widely used to stabilize F-actin. Based on extensive structure-activity relationship studies, we have developed a new generation of photoswitchable jasplakinolides that feature rationally designed red-shifted azobenzene photoswitches. Our lead compound, nOJ, can be activated with longer wavelengths in the visible range (e.g. 440-475 nm) and rapidly returns to its inactive state through thermal relaxation. nOJ enables the reversible control of F-actin dynamics, as shown through live-cell imaging, cell migration, and cell proliferation assays. Short, local irradiation with blue light resulted in highly localized and reversible actin aggregation with subcellular precision. Our optical tool can be useful in diverse fields to study actin dynamics with excellent spatiotemporal resolution.

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

Novel photoswitches offering features complementary to the well-established azobenzenes are increasingly driving high-precision research in cellular photopharmacology. Styrylthiazolium (StyTz) and styrylbenzothiazolium (StyBtz) are cellularly untested E/Z-isomerisation photoswitches which are nearly isosteric to azobenzenes, but have distinct properties: including ca. 60 nm red-shifted π → π* absorption, self-reporting fluorescence, Z → E relaxation on typical biological timescales, and decent solubility (positive charge). We tested StyTz and StyBtz for their potential as photopharmaceutical scaffolds, by applying them to photocontrol microtubule dynamics. They light-specifically disrupt microtubule network architecture and block cell proliferation: yet, testing lead compound StyBtz2 for its molecular mechanism of action showed that it did not inhibit microtubule dynamics. Using its self-reporting fluorescence, we tracked its localisation in live cells and observed accumulation of E-StyBtz2 into mitochondria; during prolonged illumination, it was released into the cytosol, and blebbing and cell death were observed. We interpret this as light-dependent rupturing of mitochondria on acute timescales. We conclude that StyTz/StyBtz can be interesting photopharmaceutical scaffolds for addressing mitochondrial, rather than cytosolic, targets.

Selective cellular probes for mammalian thioredoxin reductase TrxR1: rational design of RX1, a modular 1,2-thiaselenane redox probe.

Quantifying the activity of key cellular redox players is crucial for understanding physiological homeostasis, and for targeting their perturbed states in pathologies including cancer and inflammatory diseases. However, cellularly-selective probes for oxidoreductase turnover are sorely lacking. We rationally developed the first probes that selectively target the mammalian selenoprotein thioredoxin reductase (TrxR), using a cyclic selenenylsulfide oriented to harness TrxR's unique selenolthiol chemistry while resisting the cellular monothiol background. Lead probe RX1 had excellent TrxR1-selective performance in cells, cross-validated by knockout, selenium starvation, knock-in, and chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors, which indicated that tempered SNAr electrophiles may be more selective TrxR drugs than the classical electrophiles used hitherto. The RX1 design thus sets the stage for in vivo imaging of the activity of this key oxidoreductase in health and disease, and can also drive TrxR1-inhibitor drug design.

BTDAzo: A Photoswitchable TRPC5 Channel Activator.

Photoswitchable reagents can be powerful tools for high-precision biological control. TRPC5 is a Ca2+ -permeable cation channel with distinct tissue-specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally-resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5-modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. BTDAzos are also the first reported azo-benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two-colour photoswitching. BTDAzo's ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents.

Microtubule dynamics can be inhibited with sub-second temporal resolution and cellular-scale spatial resolution, by using precise illuminations to optically pattern where and when photoswitchable microtubule-inhibiting chemical reagents exert their latent bioactivity. The recently available reagents (SBTub, PST, STEpo, AzTax, PHTub) now enable researchers to use light to reversibly modulate microtubule-dependent processes in eukaryotes, in 2D and 3D cell culture as well as in vivo, across a variety of model organisms: with applications in fields from cargo transport to cell migration, cell division, and embryonic development.Here we give an introduction to using these photoswitchable microtubule inhibitors in cells. We describe the theory of small molecule photoswitching, and the unique performance features, usage requirements, and limitations that photoswitchable chemical reagents have; then we summarize the major classes of photoswitchable microtubule inhibitors that are currently available, with the properties that suit them to different applications, and troubleshooting measures for avoiding common mistakes. We outline workflows to establish cellular assays where they are used to optically control microtubule dynamics in a temporally reversible fashion with spatial specificity down to a single selected cell within a field of view. The methods in this chapter also equip the reader to tackle advanced uses of photoswitchable chemical reagents, in 3D culture and in vivo.

Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically.

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as "TRFS" probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes' complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.