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The interest in the photochromism and functional applications of donor-acceptor Stenhouse adducts (DASAs) soared in recent years owing to their outstanding advantages and flexible design. However, their low solubility and irreversible conversion in aqueous solutions hampered exploring DASAs for biology and medicine. It is notably unknown whether the barbiturate electron acceptor group retains the pharmacological activity of drugs such as phenobarbital, which targets γ-aminobutyric acid (GABA)-type A receptors (GABAARs) in the brain. Here, we have developed the model compound DASA-barbital based on a scaffold of red-switching second-generation DASAs, and we demonstrate that it is active in GABAARs and alters the neuronal firing rate in a physiological medium at neutral pH. DASA-barbital can also be reversibly photoswitched in acidic aqueous solutions using cyclodextrin, an approved ingredient of drug formulations. These findings clarify the path toward the biological applications of DASAs and to exploit the versatility displayed in polymers and materials science.

G-protein-coupled receptors (GPCRs) are a super-family of membrane proteins and major targets for drug development. However, many GPCRs drug candidates suffer from a lack of selectivity. Photopharmacology gives the possibility of modulating GPCR activity with an unprecedented local and temporal precision with the use of light. In this review, we compile and classify the different strategies in photopharmacology for GPCRs, we revise the different methods of analysis and characterization of light-regulated molecules used for GPCRs, and we give perspective of the impact of photopharmacology in research applications and in the development of new drugs.

International audience; Supporting information for this article is given via a link at the end of the document.

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes. This research was funded by EU Horizon 2020 Framework Programme for Research and Innovation: Human Brain Project WaveScalES, Specific Grant Agreement 2 No. 785907 and Specific Grant Agreement 3 No. 945539; NEUROPA project, Grant Agreement No. 863214; DEEPER project ICT-36-2020-101016787; European Union Regional Development Fund within the framework of the ERDF Operational Program of Catalonia 2014-2020: CECH project; Ministry of Science and Innovation DEEP RED grant PID2019-111493RB-I00 funded by MCIN/AEI/10.13039/501100011033; Ministerio de Economía y Competitividad and European Regional Development Funds: Grant no. SAF2017-87629-R to V.C., SAF2016-80726-R to M.V.P., SAF2017-88076-R to M.M.; AGAUR/Generalitat de Catalunya: CERCA Programme; Generalitat de Catalunya: Grant no. 2017-SGR-1442 to P.G., 2017-SGR-1497 to V.C., 2017-SGR-210 to M.V.P., and 2017-SGR-00465 to J.H.; Fundaluce and “la Caixa” foundations: ID 100010434, grant agreement LCF/PR/HR19/52160010.

Classical psychedelics are a group of hallucinogens which trigger non-ordinary states of consciousness through activation of the 5-HT2A receptor (5-HT2AR) in the brain. However, the exact mechanism of how 5-HT2AR agonism alters perception remains elusive. When studying receptor signaling, tools which work at the same spatiotemporal resolution as the receptor are exceptionally useful. To create such a tool, we designed a set of photoswitchable ligands based on the classical psychedelic N,N-dimethyltryptamine (DMT). By incorporation of the DMT-indole ring into the photoswitchable system, we obtained red-shifted ligands which can be operated by visible light. Among these azo-DMTs, compound 2 h ("Photo-DMT") stands out as its cis isomer exhibits DMT like activity while the trans isomer acts as weak partial agonist. Such a cis-on "efficacy switch" substantially expands the pharmacological toolbox to investigate the complex mechanisms of 5-HT2AR signaling.

Pain is a common symptom that impairs the quality of life for people around the world. Local anesthetics widely used for pain relief have a number of side effects, which makes the development of both new drugs and new ways to control their activity particularly important. Photopharmacology makes it possible to reduce the side effects of an anesthetic and control its biological activity in the body. The purpose of this work was to create a new light-controlled local anesthetic and study its biological activity in animals. A compound with a simple scheme of synthesis was chosen to shift the UV-Vis absorption band towards the visible range of the spectrum and was synthesized for the first time. Some computer calculations were performed to make sure that the aforementioned changes would not lead to loss of biological activity. The micellar form of the new compound was prepared, and in vivo biological studies were carried out in rabbits. The existence of a local anesthetic effect, which disappeared almost completely on irradiation with light (λ = 395 nm), was shown using the surface anesthesia model. Moreover, the possibility of multiple reversible changes in the biological activity of ethercaine under the action of light was demonstrated. The latter compound manifests no local irritating effect, either. The data obtained indicate the prospects for the development of new compounds based on azobenzene for light-controlled local anesthesia.

Cell‐cycle interference by small molecules has widely been used to study fundamental biological mechanisms and to treat a great variety of diseases, most notably cancer. However, at present only limited possibilities exist for spatio‐temporal control of the cell cycle. Here we report on a photocaging strategy to reversibly arrest the cell cycle at metaphase or induce apoptosis using blue‐light irradiation. The versatile proteasome inhibitor MG132 is photocaged directly at the reactive aldehyde function effectively masking its biological activity. Upon irradiation reversible cell‐cycle arrest in the metaphase is demonstrated to take place in vivo. Similarly, apoptosis can efficiently be induced by irradiation of human cancer cells. With the developed photopharmacological approach spatio‐temporal control of the cell cycle is thus enabled with very high modulation, as caged MG132 shows no effect on proliferation in the dark. In addition, full compatibility of photo‐controlled uncaging with dynamic microscopy techniques in vivo is demonstrated. This visible‐light responsive tool should be of great value for biological as well as medicinal approaches in need of high‐precision targeting of the proteasome and thereby the cell cycle and apoptosis., Blue‐light control of the proteasome is achieved using a new photocaging approach. Photolabile protection of the versatile proteasome inhibitor MG132 at the reactive aldehyde function abolished proteasome binding. After irradiation MG132 activity is restored resulting in cell‐cycle arrest at metaphase or apoptosis. This visible‐light‐responsive tool opens up new avenues for spatiotemporal control of the proteasome in basic and medicinal research.

International audience; Receptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand-binding domain of metabotropic glutamate receptor type 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate-based photoswitch via a self-labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light-controlled human insulin receptor (LihIR) and light-controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photo-receptors.

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.

Glyght is a new photochromic compound described as an effective modulator of glycine receptors at heterologous expression, in brain slices and in zebrafish larvae. Glyght also caused weak inhibition of GABAA-mediated currents in a cell line expressing α1/β2/γ2 GABAA receptors. However, the effects of Glyght on GABAergic transmission in the brain have not been analysed, which does not allow a sufficiently comprehensive assessment of the effects of the compound on the nervous system. Therefore, in this study using whole-cell patch-clamp recording, we analysed the Glyght (100 µM) action on evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in mice hippocampal slices. Two populations of cells were found: the first responded by reducing the GABAergic eIPSCs’ amplitude, whereas the second showed no sensitivity to the compound. Glyght did not affect the ionic currents’ amplitude induced by GABA application, suggesting the absence of action on postsynaptic GABA receptors. Additionally, Glyght had no impact on the paired-pulse modulation of GABAergic eIPSCs, indicating that Glyght does not modulate the neurotransmitter release mechanisms. In the presence of strychnine, an antagonist of glycine receptors, the Glyght effect on GABAergic synaptic transmission was absent. Our results suggest that Glyght can modulate GABAergic synaptic transmission via action on extrasynaptic glycine receptors.

Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.

Neuropathic pain is a common health problem resulting in exacerbated response to noxious and non noxious stimuli, as well as impaired emotional and cognitive responses. Unfortunately, neuropathic pain is also one of the most difficult pain syndromes to manage, highlighting the importance of better understanding the brain regions and neuromodulatory mechanisms involved in its regulation. Among the many interconnected brain areas which process pain, the amygdala is known to play an important role in the integration of sensory and emotional pain signals. Here we questioned the ability of a recently identified neuromodulatory mechanism associated with the metabotropic glutamate receptors mGlu4 in the amygdala to modulate neuropathic pain. In a murine model of peripheral mononeuropathy, we demonstrate that pharmacological activation of amygdala mGlu4 efficiently alleviates sensory and depressive-like symptoms in both male and female mice. Moreover, we reveal a differential modulation of these symptoms. Activating mGlu4 in the contralateral amygdala relative to the side of the mononeuropathy, is necessary and sufficient to relieve both sensory and depressive-like symptoms, while ipsilateral activation solely reduces depressive-like symptoms. Furthermore, using photopharmacology, a recent strategy allowing precise photocontrol of endogenous proteins, we further demonstrate the dynamic alleviation of neuropathic pain through light-dependent facilitation of mGlu4 by a photoswitchable positive allosteric modulator. Finally, coupling photopharmacology and analgesic conditioned place preference, we show a significant pain-reducing effect of mGlu4 activation. Taken together, these data highlight the analgesic potential of enhancing amygdala mGlu4 activity to counteract neuropathy reinforcing its therapeutic interest for the treatment of pathological pain., This work was supported by the International Emerging Action (IEA) from the Centre National de la Recherche Scientifique (CNRS) to CG and AL and by grants from the French National Research Agency (ANR): ANR-16-CE16–0010 to CG and ANR-17-NEU3–0001 under the frame of Neuron Cofund to CG and AL.

Microtubules are key components of the eukaryotic cytoskeleton with essential roles in cell division, intercellular transport, cell morphology, motility, and signal transduction. They are composed of protofilaments of heterodimers of α-tubulin and β-tubulin organized as rigid hollow cylinders that can assemble into large and dynamic intracellular structures. Consistent with their involvement in core cellular processes, affecting microtubule assembly results in cytotoxicity and cell death. For these reasons, microtubules are among the most important targets for the therapeutic treatment of several diseases, including cancer. The vast literature related to microtubule stabilizers and destabilizers has been reviewed extensively in recent years. Here we summarize recent experimental and computational approaches for the identification of novel tubulin modulators and delivery strategies. These include orphan small molecules, PROTACs as well as light-sensitive compounds that can be activated with high spatio-temporal accuracy and that represent promising tools for precision-targeted chemotherapy.

The optical control and investigation of neuronal activity can be achieved and carried out with photoswitchable ligands. Such compounds are designed in a modular fashion, combining a known ligand of the target protein and a photochromic group, as well as an additional electrophilic group for tethered ligands. Such a design strategy can be optimized by including structural data. In addition to experimental structures, computational methods (such as homology modeling, molecular docking, molecular dynamics and enhanced sampling techniques) can provide structural insights to guide photoswitch design and to understand the observed light-regulated effects. This review discusses the application of such structure-based computational methods to photoswitchable ligands targeting voltage- and ligand-gated ion channels. Structural mapping may help identify residues near the ligand binding pocket amenable for mutagenesis and covalent attachment. Modeling of the target protein in a complex with the photoswitchable ligand can shed light on the different activities of the two photoswitch isomers and the effect of site-directed mutations on photoswitch binding, as well as ion channel subtype selectivity. The examples presented here show how the integration of computational modeling with experimental data can greatly facilitate photoswitchable ligand design and optimization. Recent advances in structural biology, both experimental and computational, are expected to further strengthen this rational photopharmacology approach.

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 μL, 3–10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial‐magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light‐mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug‐based light‐mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide‐Azo‐Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations—as in slow wave sleep—is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans., Brain pathologies often require drug treatments, however drugs act all over the central nervous system. Wouldn't it be good to determine where/when a drug should be active? Drugs can be made sensitive to light, to be activated at specific times and locations. This study demonstrates that a light‐activated cholinergic drug can effectively modulate activity in the cerebral cortex network.

The spatial and temporal control of bioactivity of small molecules by light (photopharmacology) constitutes a promising approach for study of biological processes and ultimately for the treatment of diseases. In this study, we investigated two different ‘caged’ antibiotic classes that can undergo remote activation with UV-light at λ=365 nm, via the conjugation of deactivating and photocleavable units through a short synthetic sequence. The two widely used antibiotics vancomycin and cephalosporin were thus enhanced in their performance by rendering them photoresponsive and thus suppressing undesired off-site activity. The antimicrobial activity against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, S. aureus ATCC 43300 (MRSA), Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 could be spatiotemporally controlled with light. Both molecular series displayed a good activity window. The vancomycin derivative displayed excellent values against Gram-positive strains after uncaging, and the next-generation caged cephalosporin derivative achieved good and broad activity against both Gram-positive and Gram-negative strains after photorelease.

[eng] The main aim of this thesis is setting up and using in vivo screening techniques to identify and characterize new photopharmacological compounds targeting endogenous proteins. In order to achieve that aim, the thesis has been structured in three specific objectives, which are developed in the corresponding parts and chapters indicated below: Objective I. Photomanipulation of cardiac activity and organism development (part I). - Establish a procedure to identify photoswitchable compounds affecting zebrafish larvae development based on phenotypic outcomes during embryogenesis and hatching (Chapter 2). - Identification of relevant morphological, anatomical, and organotypic traits that are affected by the departure compound of photoswitchable drugs (Chapter 2). - Study of phenotypic markers as a function of photoswitchable drug isoforms (Chapter 2). - Developing a strategy for real-time recording of early stage tadpoles’ cardiac function under controlled illumination (Chapter 3). - Developing a computational approach for video extracting readouts and analysis of cardiac parameters (Chapter 3). - Identification of photoswitchable compounds with reversible effects on tadpoles’ cardiac function (Chapter 3). Objective II. Photomanipulation of locomotion (part II). - Establish an experimental and analytical procedure to categorise differences in locomotion outcomes under two wavelengths illumination (Chapter 4). - Identify swimming profiles related to inhibitory neurotransmission pathways of zebrafish larvae in darkness and illumination (Chapter 4). - Devise a strategy to screen photoswitchable compounds using zebrafish larvae activity profiles and their alteration under varying light conditions (Chapter 4). - Identification and characterization of photoswitchable GABAAR drugs using zebrafish locomotion assays (Chapter 5). - Identify swimming profiles related to inhibitory neural transmission pathways present during locomotion in Xenopus tropicalis tadpoles under long relaxed dark periods and light stimuli (Chapter 6). - Identification and characterization of a photoswitchable potentiator of glycinergic transmission using tapole swimming assays (Chapter 6). - Identification and characterization of a photoswitchable ligands of adrenergic receptors using zebrafish swimming assays (Chapter 7). - Identification and characterization of a photoswitchable ligands of dopaminergic receptors using zebrafish swimming assays (Chapter 8). Objective III. Photomanipulation of visual function (part III). - Develop a model of acute blinding in zebrafish larvae by photoinduced retinal degeneration in order to test vision restoration drugs (Chapter 9). - Identification and characterization of locomotive responses in blinded larvae (Chapter 9). - Development and validation of an optokinetic visual-motor reflex assay to test vision restoration drugs (Chapter 9).

Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene‐based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto‐unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light., Photocontrollable antibiotic: There is growing interest in the spatiotemporal control of drugs for research and medical applications. To this end, a photocontrollable azobenzene‐based inhibitor of tryptophan synthase, an essential metabolic enzyme complex in bacteria, was designed. Thorough biochemical, crystallographic, and computational analyses elucidated the noncompetitive mode of action of this promising antibiotic lead compound.

The human serotonin transporter (hSERT) terminates serotonergic signaling through reuptake of neurotransmitter into presynaptic neurons and is a target for many antidepressant drugs. We describe here the development of a photoswitchable hSERT inhibitor, termed azo-escitalopram, that can be reversibly switched between trans and cis configurations using light of different wavelengths. The dark-adapted trans isomer was found to be significantly less active than the cis isomer, formed upon irradiation.

Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide‐mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub‐micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long‐chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo‐based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell‐based activity assay., Switched into action: Besides fine‐tuning molecular geometry for enzyme inhibition, photoinduced isomerization of azopyridine‐based nicotinamide mimics results in improved aqueous solubility, enforcing the effect on target protein sirtuin 2. The resulting Sirt2 inhibitor could be used to study binding events in cellular systems with spatiotemporal control by two independent molecular and supramolecular effects.

Optogenetic and photopharmacological tools to manipulate neuronal inhibition have limited efficacy and reversibility. We report the design, synthesis, and biological evaluation of Fulgazepam, a fulgimide derivative of benzodiazepine that behaves as a pure potentiator of ionotropic γ‐aminobutyric acid receptors (GABAARs) and displays full and reversible photoswitching in vitro and in vivo. The compound enables high‐resolution studies of GABAergic neurotransmission, and phototherapies based on localized, acute, and reversible neuroinhibition., Fulgazepam: Herein, the design, synthesis, and biological evaluation of Fulgazepam, a fulgimide derivative of benzodiazepine that behaves as a pure potentiator of ionotropic γ‐aminobutyric acid receptors (GABAARs) and displays full and reversible photoswitching in vitro and in vivo, is reported.

This study evaluates the embryotoxicity of dithienylethene-modified peptides upon photoswitching, using 19 analogues based on the β-hairpin scaffold of the natural membranolytic peptide gramicidin S. We established an in vivo assay in two variations (with ex vivo and in situ photoisomerization), using larvae of the model organism Danio rerio, and determined the toxicities of the peptides in terms of 50% lethal doses (LD50). This study allowed us to: (i) demonstrate the feasibility of evaluating peptide toxicity with D. rerio larvae at 3–4 days post fertilization, (ii) determine the phototherapeutic safety windows for all peptides, (iii) demonstrate photoswitching of the whole-body toxicity for the dithienylethene-modified peptides in vivo, (iv) re-analyze previous structure–toxicity relationship data, and (v) select promising candidates for potential clinical development.

International audience; G-protein-coupled receptor (GPCR) pharmacology tends to be complex and at times poorly understood. This has led to the development of GPCR-targeting agents that often demonstrate poor pharmacokinetic properties and poor selectivity for their target receptors. One approach that is emerging as a means of addressing these limitations is the use of molecules whose activity can be controlled by light. Photopharmacology involves the incorporation of a photoswitch into the structure of a given compound, cage or linker and following irradiation with light, undergoes a structural rearrangement, which changes its biological activity. The use of light-regulated ligands offers the opportunity to modulate and understand GPCR signaling in a more spatiotemporal manner than classical pharmacological approaches. In this chapter we will discuss some of the advancements that have been made in photopharmacology, particularly in developing photoswitchable ligands that target class A GPCRs, e.g., muscarinic acetylcholine receptors, class B GPCRs, e.g., glucagon-like peptide-1 receptor, and class C GPCRs, e.g., metabotrobic glutamate receptors. Given the intricacy of GPCR pharmacology, this chapter will also discuss some of the challenges the field faces when designing photopharmacological tools. Furthermore, it will propose that it is with a full appreciation of the spectrum of pharmacological and pharmacokinetic properties of photoswitchable ligands that research will be better placed to develop ligands with a reduced risk of failure during preclinical progression. This will likely enable photopharmacological approaches to continue to find novel applications and offer new perspectives in understanding (patho)physiology to ultimately inform future GPCR drug discovery efforts.

A multiphotochromic hybrid system is presented in which a light-driven overcrowded alkene-based molecular rotary motor is connected to a dithienylethene photoswitch. Ring closing of the dithienylethene moiety, using an irradiation wavelength different from the wavelength applied to operate the molecular motor, results in inhibition of the rotary motion as is demonstrated by detailed 1H-NMR and UV/Vis experiments. For the first time, a light-gated molecular motor is thus obtained. Furthermore, the excitation wavelength of the molecular motor is red-shifted from the UV into the visible-light region upon attachment of the dithienylethene switch.

In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chemical and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacology that aim at establishing such control over protein activity through bioactive molecules with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacology to reach its maturity as a chemical biology research tool.

Molecular photoswitches comprise chromophores that can be interconverted reversibly with light between two states with different photochemical and physicochemical properties. This feature renders them useful for diverse applications, ranging from materials science, biology (specifically photopharmacology) to supramolecular chemistry. With new and more challenging systems to control, especially extending towards biomedical applications, using visible or near-infrared light for photoswitch activation becomes vital. Donor-acceptor Stenhouse adducts are a novel class of visible light-responsive negative photochromes that provide a possible answer to current limitations of other photoswitch classes in the visible and NIR window. Their rapid development since their discovery in 2014, together with first successful examples of applications, demonstrate both their potential and areas where improvements are needed. A better understanding of DASA characteristics and its photoswitching mechanism has revealed that they are in fact a subset of a more general structural class of photochromes, namely Stenhouse photoswitches. This tutorial review aims at providing an introduction and practical guide on DASAs: it focuses on their structure and synthesis, provides fundamental insights for understanding their photoswitching behavior and demonstrates guiding principles for tailoring these switches for given applications.

We describe two water‐soluble ruthenium complexes, [1]Cl2 and [2]Cl2, that photodissociate to release a cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a low dose (21 J cm−2) of red light in an oxygen‐independent manner. Using a specific NAMPT activity assay, up to an 18‐fold increase in inhibition potency was measured upon red‐light activation of [2]Cl2, while [1]Cl2 was thermally unstable. For the first time, the dark and red‐light‐induced cytotoxicity of these photocaged compounds could be tested under hypoxia (1 % O2). In skin (A431) and lung (A549) cancer cells, a 3‐ to 4‐fold increase in cytotoxicity was found upon red‐light irradiation for [2]Cl2, whether the cells were cultured and irradiated with 1 % or 21 % O2. These results demonstrate the potential of photoactivated chemotherapy for hypoxic cancer cells, in which classical photodynamic therapy, which relies on oxygen activation, is poorly efficient.

The effect of a minor difference in ligand structure is shown to have a large effect on the photochemical pathways followed by two ruthenium(II) polypyridyl based complexes [Ru(CH3CN) (LL)](2+), 1 and 2, where LL is MeN4Py (1,1-di(pyridin-2-yl)-N,N-bis (pyridin-2-yl-methyl) ethan-1-amine) or N4Py (1,1-di (pyri din-2-yl)-N,N-bis (pyridin-2-yl-m ethyl) methanamine), respectively. In our earlier report we demonstrated near completely reversible two-way photochromism of 1, in which a pyridyl ring dissociated on irradiation with visible light to form the thermally stable 1P, [Ru(CH3CN)(2)(MeN4Py)](2+). Complex 1 was recovered upon irradiation in the near-UV. Here, we show that the methyl group in the ligand backbone is critical to the reversibility by impeding the dissociation of one of the two sets of pyridyl rings. Irradiation of 2, which does not bear the methyl group, with visible light results in formation of two thermally stable isomers 2a and 2b, which are characterized by UV-vis absorption, FTIR, H-1 NMR spectroscopy, ESI mass spectrometry, and X-ray crystallography. In contrast to 1P, in both 2a and 2b, a different pyridyl moiety is dissociated. Whereas UV irradiation returns 2a to its original state (2), the overall reversibility is limited by the relative stability of 2b. The changes to the structure of 2 made possible by the increased freedom for all four pyridyl moieties to dissociate allows access to coordination modes that are not accessible thermally opening opportunities toward new catalysts for oxidation chemistry, photochromism and photoswitching.

Photopharmacology relies on ligands that change their pharmacodynamics upon photoisomerization. Many of these ligands are azobenzenes that are thermodynamically more stable in their elongated trans-configuration. Often, they are biologically active in this form and lose activity upon irradiation and photoisomerization to their cis-isomer. Recently, cyclic azobenzenes, so-called diazocines, have emerged, which are thermodynamically more stable in their bent cis-form. Incorporation of these switches into a variety of photopharmaceuticals could convert dark-active ligands into dark-inactive ligands, which is preferred in most biological applications. This “pharmacological sign-inversion” is demonstrated for a photochromic blocker of voltage-gated potassium channels, termed CAL, and a photochromic opener of G protein-coupled inwardly rectifying potassium (GIRK) channels, termed CLOGO.

International audience; Neuronal communication involves a multitude of neurotransmitters and an outstanding diversity of receptors and ion channels. Linking the activity of cell surface receptors and ion channels in defined neural circuits to brain states and behaviors has been a key challenge in neuroscience, since cell targeting is not possible with traditional neuropharmacology. We review here recent technologies that enable the effect of drugs to be restricted to specific cell types, thereby allowing acute manipulation of the brain’s own proteins with circuit specificity. We highlight the importance of developing cell-specific neuropharmacology strategies for decoding the nervous system with molecular and circuit precision, and for developing future therapeutics with reduced side effects.

The serotonin 5-hydroxytryptamine 3 receptor (5-HT3R) plays a unique role within the seven classes of the serotonin receptor family, as it represents the only ionotropic receptor, while the other six members are G protein-coupled receptors (GPCRs). The 5-HT3 receptor is related to chemo-/radiotherapy provoked emesis and dysfunction leads to neurodevelopmental disorders and psychopathologies. Since the development of the first serotonin receptor antagonist in the early 1990s, the range of highly selective and potent drugs expanded based on various chemical structures. Nevertheless, on-off-targeting of a pharmacophore’s activity with high spatiotemporal resolution as provided by photopharmacology remains an unsolved challenge bearing additionally the opportunity for detailed receptor examination. In the presented work, we summarize the synthesis, photochromic properties and in vitro characterization of azobenzene-based photochromic derivatives of published 5-HT3R antagonists. Despite reported proof of principle of direct azologization, only one of the investigated derivatives showed antagonistic activity lacking isomer specificity.

To better understand the relationship between kinase activity and cellular behaviour, the use of light as an external stimulus to modulate kinase activity with high spatiotemporal resolution has gained increasing interest of late. Herein we highlight the progress made towards the development of light‐responsive kinase enzymes and small molecule inhibitors.

L’objectiu principal de la neurociència és l’estudi i el control dels sistemes neuronals. Actualment, aquesta àrea està sent revolucionada per l’ús de petites molècules fotoactives, un camp conegut com fotofarmacologia. Mitjançant l’activació remota de fàrmacs amb llum, la fotofarmacologia tracta d’afrontar alguns dels principals reptes de la farmacologia convencional, com la pobra selectivitat dels fàrmacs i els efectes secundaris.1 Tres estratègies principals s’han derivat en aquest camp: els photocaged ligands (CL), profàrmacs que difonen lliurement, l’efecte des quals s’activa mitjançant l’eliminació dels grups protectors fotolàbils amb il·luminació; els photochromic ligands (PCL), que permeten la modulació reversible de la resposta de molècules bioactives a través de la fotoisomerització de grups annexats a aquestes que responen a la llum; i photoswitched tethered ligands (PTL), un cas especial de PCLs que s'uneixen covalentment al receptor d’interès. Tot i que aquestes aproximacions han demostrat tenir èxit per a una gran varietat d’objectius terapèutics, tant in vitro com in vivo, encara resten diversos reptes en el camp de la fotofarmacologia. Per tant, en aquest treball hem abordat la investigació de noves estratègies fotofarmacològiques que superin algunes de les debilitats de les eines desenvolupades fins ara. Ens hem centrat especialment en el fotocontrol dels receptors ionotròpics de glutamat (iGluRs), els quals tenen un paper clau en la modulació de l’excitabilitat neuronal. Les noves eines fotofarmacològiques desenvolupades al llarg d’aquesta tesi consisteixen en: (i) un PTL basat en fotointerruptors basats en azobenzè substituïts de forma push-pull que responen a l'excitació amb dos fotons amb llum NIR. (ii) un lligand engabiat no destructiu que permeti la conversió irreversible i quantitativa de l'estat inactiu a l'estat actiu, actuant d'una manera similar als CLs, però sense generació de subproductes. (iii) un PCL basat en azobenzens amb un pont C2 que presenta una forma inactiva termodinàmicament estable que es converteix selectivament en l’estat biològicament actiu al ser irradiat. Aquesta tesi presenta la síntesi, la caracterització fotoquímica i l’avaluació de l’activitat biològica d’aquestes tres noves eines fotofarmacològiques. The main objective of neuroscience is the study and control of neuronal systems. Nowadays, this area is being revolutionised by the use of photoresponsive small molecules, a field known as photopharmacology. By enabling remote activation of drugs with light, photopharmacology seeks to tackle some of the main challenges faced by conventional pharmacology, such as poor drug selectivity and side effects.1 Three main strategies have been derived in this area: photocaged ligands (CL), freely diffusing prodrugs the effect of which is triggered by removing photolabile protecting groups upon illumination; photochromic ligands (PCL), which allow reversible modulation of the response of bioactive molecules through photoisomerisation of appended light-responsive moieties; and photoswitched tethered ligands (PTL), a special case of PCLs that are covalently attached to the therapeutic receptor. Although these approaches have proven to be successful for a variety of therapeutic targets in vitro and in vivo, several challenges still remain in the field of photopharmacology. Therefore, in this work we have aimed at the investigation of new photopharmacological strategies that overcome some of the weaknesses of the tools developed so far. We have particularly focused on the photocontrol of ionotropic glutamate receptors (iGluRs), which play a key role in the modulation of neuronal excitability. The novel photopharmacological tools developed along this thesis consist in: (i) a PTL based on push-pull-substituted azobenzene photoswitches that responds to two-photon excitation with NIR light. (ii) a non-destructive caged ligand that enable irreversible and quantitative conversion from the inactive to the active state, thus performing in a similar fashion as CLs but without by-product generation. (iii) a PCL based on C2-bridged azobenzenes which displays a thermodynamically stable inactive form that selectively turns into the biologically-active state when irradiated. This thesis reports the synthesis, photochemical characterisation and biological activity evaluation of these three novel photopharmacological approaches.

The field of photopharmacology uses molecular photoswitches to establish control over the action of bioactive molecules. It aims to reduce systemic drug toxicity and the emergence of resistance, while achieving unprecedented precision in treatment. By using small molecules, photopharmacology provides a viable alternative to optogenetics. We present here a critical overview of the different pharmacological targets in various organs and a survey of organ systems in the human body that can be addressed in a non-invasive manner. We discuss the prospects for the selective delivery of light to these organs and the specific requirements for light-activatable drugs. We also aim to illustrate the druggability of medicinal targets with recent findings and emphasize where conceptually new approaches have to be explored to provide photopharmacology with future opportunities to bring smart molecular design ultimately to the realm of clinical use.

Pain afflicts billions of people worldwide, who suffer especially from long-term chronic pain. This gruelling condition affects the nervous system at all levels: from the brain to the spinal cord, the Dorsal Root Ganglia (DRG) and the peripheral fibres innervating the skin. The nature of the different molecular and cellular components of the somatosensory modalities, as well as the complexity of the peripheral and central circuitry are yet poorly understood. Light-based techniques such as optogenetics, in concert with the recent advances in single-cell genetic profiling, can help to elucidate the role of diverse neuronal sub-populations in the encoding of different sensory and painful stimuli by switching these neurons on and off via optically active proteins, namely opsins. Recently, photopharmacology has emerged from the efforts made to advance optogenetics. The introduction of azo-benzene-based light-sensitive molecular switches has been applied to a wide variety of molecular targets, from ion channels and receptors to transporters, enzymes and many more, some of which are paramount for pain research and therapy. In this Review, we summarise the recent advances in the fields of optogenetics and photopharmacology and we discuss the use of light-based techniques for the study of acute and chronic pain physiology, as well as their potential for future therapeutic use to improve pain treatment.

Spatiotemporal control over biochemical signaling processes involving G protein‐coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H3 receptor (hH3R). Upon illumination, key compound 65 decreases its affinity for the hH3R by 8.5‐fold and its potency in hH3R‐mediated Gi protein activation by over 20‐fold, with the trans and cis isomer both acting as full agonist. In real‐time two‐electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light‐induced modulation of hH3R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.

New technologies for spatial and temporal remote control of G protein-coupled receptors (GPCRs) are necessary to unravel the complexity of GPCR signalling in cells, tissues and living organisms. An effective approach, recently developed, consists on the design of light-operated ligands whereby light-dependent GPCR activity regulation can be achieved. In this context, the use of light provides an advantage as it combines safety, easy delivery, high resolution and it does not interfere with most cellular processes. In this review we summarize the most relevant successful achievements in GPCR photopharmacology. These recent findings constitute a significant advance in research studies on the molecular dynamics of receptor activation and their physiological roles in vivo. Moreover, these molecules hold potential toward clinical uses as light-operated drugs, which can overcome some of the problems of conventional pharmacology. © 2019 Elsevier B.V., We acknowledge financial support from the ERANET Neuron LIGHTPAIN and MAGNOLIA projects, the Spanish Ministry of Science, Innovation and Universities (CTQ2014-57020-R and CTQ2017-89222-R), cofinanced by the European Regional Development Fund and the Catalan Government (2017SGR01604).

Gramicidin S is a naturally occurring antimicrobial cyclic peptide. Herein, we present a series of cyclic peptides based on gramicidin S that contain an azobenzene photoswitch to reversibly control secondary structure and, hence, antimicrobial activity. H-1 NMR spectroscopy and density functional theory calculations revealed a beta-sheet/beta-turn secondary structure for the cis configuration of each peptide, and an ill-defined conformation for all associated trans structures. The cis-enriched and trans-enriched photostationary states (PSSs) for peptides 1-3 were assayed against Staphylococcus aureus to reveal a clear relationship between well-defined secondary structure, amphiphilicity and optimal antimicrobial activity. Most notably, peptides 2 a and 2 b exhibited a fourfold difference in antimicrobial activity in the cis-enriched PSS over the trans-enriched equivalent. This photopharmacological approach allows antimicrobial activity to be regulated through photochemical control of the azobenzene photoswitch, thereby opening new avenues in the design and synthesis of future antibiotics. Refereed/Peer-reviewed

Application of molecular photochromic ion channel blockers to recover the visual function of a degenerated retina is one of the promising trends in photopharmacology. To this day, several photochromic azobenzene-based compounds have been proposed and their functionality has been demonstrated on cell lines and knockout mouse models. Further advance necessitates testing of the physiological activity of a great number of new compounds. The goal of this study is to propose animal models of photoreceptor degeneration that are easier to obtain than knockout mouse models but include the main features required for testing the physiological activity of molecular photoswitches. Two amphibian-based models were proposed. The first model was obtained by mechanical deletion of the photoreceptor outer segments. The second model was obtained by intraocular injection of tunicamycin to induce the degeneration of rods and cones. To test our models, we used 2-[(4-{(E)-[4-(acryloylaminophenyl]diazenyl}phenyl)amino]-N,N,N-triethyl-2-oxoethanammonium chloride (AAQ), one of the compounds that have been studied in other physiological models. The electroretinograms recorded from our models before and after AAQ treatment are in agreement with the results obtained on knockout mouse models and reported in other studies. Hence, the proposed models can be used for primary screening of molecular photochromic ion channel blockers.

Altres ajuts: Fundació la Marató de TV3 (Grant 20152031) G protein-coupled adenosine receptors are promising therapeutic targets for a wide range of neuropathological conditions, including Parkinson's disease (PD). However, the ubiquity of adenosine receptors and the ultimate lack of selectivity of certain adenosine-based drugs have frequently diminished their therapeutic use. Photopharmacology is a novel approach that allows the spatiotemporal control of receptor function, thus circumventing some of these limitations. Here, we aimed to develop a light-sensitive caged adenosine A receptor (A R) antagonist to photocontrol movement disorders. We synthesized MRS7145 by blocking with coumarin the 5-amino position of the selective A R antagonist SCH442416, which could be photoreleased upon violet light illumination (405 nm). First, the light-dependent pharmacological profile of MRS7145 was determined in A R-expressing cells. Upon photoactivation, MRS7145 precluded A R ligand binding and agonist-induced cAMP accumulation. Next, the ability of MRS7145 to block A R in a light-dependent manner was assessed in vivo. To this end, A R antagonist-mediated locomotor activity potentiation was evaluated in brain (striatum) fiber-optic implanted mice. Upon irradiation (405 nm) of the dorsal striatum, MRS7145 induced significant hyperlocomotion and counteracted haloperidol-induced catalepsy and pilocarpine-induced tremor. Finally, its efficacy in reversing motor impairment was evaluated in a PD animal model, namely the hemiparkinsonian 6-hydroxydopamine (6-OHDA)-lesioned mouse. Photo-activated MRS7145 was able to potentiate the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (L-DOPA). Overall, MRS7145 is a new light-operated A R antagonist with potential utility to manage movement disorders, including PD.

Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.

Exposure to low‐intensity radiation in the near‐infrared (NIR) spectral region matching the optically transparent “phototherapeutic window” of biological tissues can be applied to directly populate spin‐restricted excited states of light‐responsive compounds. This unconventional and unprecedented approach is introduced herein as a new strategy to overcome some of the major unresolved problems observed in the rapidly emerging fields of photopharmacology and molecular photomedicine, where practical applications in living cells and organisms are still limited by undesired side reactions and insufficient light penetration. Water‐soluble and biocompatible metal complexes with a significant degree of spin–orbit coupling were identified as target candidates for testing our new hypothesis. As a first example, a dark‐stable manganese carbonyl complex acting as a visible‐light‐triggered CO‐releasing molecule (Photo‐CORM) is shown to be photoactivated by NIR radiation, although apparently no spectroscopically evident absorption bands are detectable in this low‐energy region. This quite remarkable effect is ascribed to a strongly restricted, but obviously not completely forbidden optical population of the lowest triplet excited state manifold of the diamagnetic complex from the singlet ground state.

Singlet oxygen generated in a type II photodynamic action, due to its limited lifetime (1 μs) and reactive distance (< 10 nm), could regulate live cell function nanoscopically. The genetically-encoded protein photosensitizers (engineered fluorescent proteins such as KillerRed, TagRFP, and flavin-binding proteins such as miniSOG, Pp2FbFPL30M) could be expressed in a cell type- and / or subcellular organelle-specific manner for targeted protein photo-oxidative activation / desensitization. The newly emerged active illumination technique provides an additional level of specificity. Typical examples of photodynamic activation include permanent activation of G protein-coupled receptor CCK1 and photodynamic activation of ionic channel TRPA1. Protein photosensitisers have been used to photodynamically modulate major cellular functions (such as neurotransmitter release and gene transcription) and animal behavior. Protein photosensitisers are increasingly used in photon-driven nanomanipulation in cell physiology research.

The field of dynamic functional molecular systems has progressed enormously over the past few decades. By coupling the mechanical properties of molecular switches and motors to chemical and biological processes, exceptional control of function has been attained. Overcrowded alkene-based light-driven molecular motors are very attractive in this respect owing to their unique multistate photochemically and thermally induced switching processes and their helical chirality inversion in each switching step. However, extending our control over properties from the molecular scale to larger length scales is still a fundamental challenge. In this Perspective, we discuss recent developments that address this challenge, ranging from the application of these motors in catalysis and synthetic materials to the control of biological properties. We may now be positioned at the dawn of a new era in which artificial molecular motors are able to perform programmed tasks and dynamic functions akin to the biological machines that are found in daily life. Light-driven artificial molecular switches and motors afford dynamic control of various molecular systems, ranging from catalytic to biological. This Perspective provides insight into the challenges that must be addressed to transition the field from the proof-of-concept stage to realization of its myriad applications.

Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitch embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. While both classes of inhibitor show excellent inhibitory activity (nM IC50 values) of the target enzymes and promising differential activity of the switchable E- and Z-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the Z‑isomer). We also report protein-ligand crystal structures of the E-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding, but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily.

Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca2+, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R.

Incretin mimetics are set to become a mainstay of type 2 diabetes treatment. By acting on the pancreas and brain, they potentiate insulin secretion and induce weight loss to preserve normoglycemia. Despite this, incretin therapy has been associated with off‐target effects, including nausea and gastrointestinal disturbance. A novel photoswitchable incretin mimetic based upon the specific glucagon‐like peptide‐1 receptor (GLP‐1R) agonist liraglutide was designed, synthesized, and tested. This peptidic compound, termed LirAzo, possesses an azobenzene photoresponsive element, affording isomer‐biased GLP‐1R signaling as a result of differential activation of second messenger pathways in response to light. While the trans isomer primarily engages calcium influx, the cis isomer favors cAMP generation. LirAzo thus allows optical control of insulin secretion and cell survival.