Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death.

Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

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.

Controlling the Covalent Reactivity of a Kinase Inhibitor with Light.

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and become effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.

Oxidative Approach Enables Efficient Access to Cyclic Azobenzenes.

Azobenzenes are versatile photoswitches that have found widespread use in a variety of fields, ranging from photopharmacology to the material sciences. In addition to regular azobenzenes, the cyclic diazocines have recently emerged. Although diazocines have fascinating conformational and photophysical properties, their use has been limited by their synthetic accessibility. Herein, we present a general, high-yielding protocol that relies on the oxidative cyclization of dianilines. In combination with a modular substrate synthesis, it allows for rapid access to diversely functionalized diazocines on gram scales. Our work systematically explores substituent effects on the photoisomerization and thermal relaxation of diazocines. It will enable their incorporation into a wide variety of functional molecules, unlocking the full potential of these emerging photoswitches. The method can be applied to the synthesis of a new cyclic azobenzene with a nine-membered central ring and distinct properties.

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.

Multiply nitrated high-energy dense oxidizers derived from the simple amino acid glycine.

Various energetic polynitro esters, carbamates, and nitrocarbamates that were derived from the amino acid glycine were fully characterized by single-crystal X-ray diffraction, vibrational spectroscopy (IR and Raman), multinuclear NMR spectroscopy, elemental analysis, and differential scanning calorimetry (DSC). Owing to their positive oxygen balance, the suitability of these compounds as potential oxidizers in energetic formulations was investigated and discussed. In addition, the heats of formation of the products were calculated by using the Gaussian 09 program package at the CBS-4M level of theory. From these values and the calculated densities (from the X-ray data), several detonation parameters, such as detonation pressure, velocity, energy, and temperature, were computed by using the EXPLO5 code. Furthermore, their sensitivities towards impact, friction, and electrostatic discharge were tested by using a drop hammer, a friction tester (both BAM certified), and a small-scale electrical-discharge device, respectively.

Photoswitchable Molecular Glues Enable Optical Control of Transcription Factor Degradation.

Immunomodulatory drugs (IMiDs), which include thalidomide and its derivatives, have emerged as the standard of care against multiple myeloma. They function as molecular glues that bind to the E3 ligase cereblon (CRBN) and induce protein interactions with neosubstrates, including the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). The subsequent ubiquitylation and degradation of these transcription factors underlies the antiproliferative activity of IMiDs. Here, we introduce photoswitchable immunomodulatory drugs (PHOIMiDs) that can be used to degrade Ikaros and Aiolos in a light-dependent fashion. Our lead compound shows minimal activity in the dark and becomes an active degrader upon irradiation with violet light. It shows high selectivity over other transcription factors, regardless of its state, and could therefore be used to control the levels of Ikaros and Aiolos with high spatiotemporal precision.

Photoactivated Protein Degrader for Optical Control of Synaptic Function.

Hundreds of proteins determine the function of synapses, and synapses define the neuronal circuits that subserve myriad brain, cognitive, and behavioral functions. It is thus necessary to precisely manipulate specific proteins at specific sub-cellular locations and times to elucidate the roles of particular proteins and synapses in brain function. We developed PHOtochemically TArgeting Chimeras (PHOTACs) as a strategy to optically degrade specific proteins with high spatial and temporal precision. PHOTACs are small molecules that, upon wavelength-selective illumination, catalyze ubiquitylation and degradation of target proteins through endogenous proteasomes. Here, we describe the design and chemical properties of a PHOTAC that targets Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα), which is abundant and crucial for the baseline synaptic function of excitatory neurons. We validate the PHOTAC strategy, showing that the CaMKIIα-PHOTAC is effective in mouse brain tissue. Light activation of CaMKIIα-PHOTAC removed CaMKIIα from regions of the mouse hippocampus only within 25 µm of the illuminated brain surface. The optically controlled degradation decreases synaptic function within minutes of light activation, measured by the light-initiated attenuation of evoked field excitatory postsynaptic potential (fEPSP) responses to physiological stimulation. The PHOTACs methodology should be broadly applicable to other key proteins implicated in synaptic function, especially for evaluating their precise roles in the maintenance of long-term potentiation and memory within subcellular dendritic domains.

Optical control of targeted protein degradation.

Molecular glues and proteolysis targeting chimeras (PROTACs) have emerged as small-molecule tools that selectively induce the degradation of a chosen protein and have shown therapeutic promise. Recently, several approaches employing light as an additional stimulus to control induced protein degradation have been reported. Here, we analyze the principles guiding the design of such systems, provide a survey of the literature published to date, and discuss opportunities for further development. Light-responsive degraders enable the precise temporal and spatial control of protein levels, making them useful research tools but also potential candidates for human precision medicine.

PHOTACs Enable Optical Control of Protein Degradation.

Proteolysis Targeting Chimeras (PROTACs) are a promising technology to degrade specific target proteins. As bifunctional small molecules, PROTACs induce the ternary complex formation between an E3 ligase and a protein of interest (POI), leading to polyubiquitylation and subsequent proteasomal degradation of the protein in a catalytic fashion. We have developed a strategy to control PROTACs with the spatiotemporal precision of light, which led to light-activated versions, termed PHOTACs (PHOtochemically TArgeted Chimeras). By incorporating an azobenzene photoswitch into the PROTAC, we can reversibly control degradation of the POI, as demonstrated for BRD2-4 and FKBP12. Here, we describe our modular approach and the application of PHOTACs for the optical control of protein levels in detail. PHOTACs hold promise as both research tools and precision pharmaceutics.

PHOTACs enable optical control of protein degradation.

PROTACs (PROteolysis TArgeting Chimeras) are bifunctional molecules that target proteins for ubiquitylation by an E3 ligase complex and subsequent degradation by the proteasome. They have emerged as powerful tools to control the levels of specific cellular proteins. We now introduce photoswitchable PROTACs that can be activated with the spatiotemporal precision that light provides. These trifunctional molecules, which we named PHOTACs (PHOtochemically TArgeting Chimeras), consist of a ligand for an E3 ligase, a photoswitch, and a ligand for a protein of interest. We demonstrate this concept by using PHOTACs that target either BET family proteins (BRD2,3,4) or FKBP12. Our lead compounds display little or no activity in the dark but can be reversibly activated with different wavelengths of light. Our modular approach provides a method for the optical control of protein levels with photopharmacology and could lead to new types of precision therapeutics that avoid undesired systemic toxicity.