Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase

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.     

Optical Modulation of Antibiotic Resistance by Photoswitchable Cystobactamids

The rise of antibiotic resistance causes a serious health care problem, and its counterfeit demands novel, innovative concepts. The combination of photopharmacology, enabling a light-controlled reversible modulation of drug activity, with antibiotic drug design has led to first photoswitchable antibiotic compounds derived from established scaffolds. In this study, we converted cystobactamids, gyrase-inhibiting natural products with an oligoaryl scaffold and highly potent antibacterial activities, into photoswitchable agents by inserting azobenzene in the N-terminal part and/or an acylhydrazone moiety near the C-terminus, yielding twenty analogs that contain mono- as well as double-switches. Antibiotic and gyrase inhibition properties could be modulated 3.4-fold and 5-fold by light, respectively. Notably, the sensitivity of photoswitchable cystobactamids towards two known resistance factors, the peptidase AlbD and the scavenger protein AlbA, was light-dependent. While irradiation of an analog with an N-terminal azobenzene with 365 nm light led to less degradation by AlbD, the AlbA-mediated inactivation was induced. This provides a proof-of-principle that resistance towards photoswitchable antibiotics can be optically controlled.     

Optical Control of Acetylcholinesterase with a Tacrine Switch

Photochromic ligands have been used to control a variety of biological functions, especially in neural systems. Recently, much effort has been invested in the photocontrol of ion channels and G‐protein coupled receptors found in the synapse. Herein, we describe the expansion of our photopharmacological approach toward the remote control of an enzyme. Building on hallmark studies dating from the late 1960s, we evaluated photochromic inhibitors of one of the most important enzymes in synaptic transmission, acetylcholinesterase (AChE). Using structure‐based design, we synthesized several azobenzene analogues of the well‐known AChE inhibitor tacrine (THA) and determined their effects on enzymatic activity. One of our compounds, AzoTHA, is a reversible photochromic blocker of AChE in vitro and ex vivo with high affinity and fast kinetics. As such, AzoTHA can be used to control synaptic transmission on the neuromuscular endplate based on the light‐dependent clearance of a neurotransmitter.     

A Photoswitchable Agonist for the Histamine H3 Receptor,a Prototypic Family A G‐Protein‐Coupled Receptor

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 G_i 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.     

Photoswitching the Efficacy of a Small‐Molecule Ligand for a Peptidergic GPCR: from Antagonism to Agonism

For optical control of GPCR function, we set out to develop small‐molecule ligands with photoswitchable efficacy in which both configurations bind the target protein but exert distinct pharmacological effects, that is, stimulate or antagonize GPCR activation. Our design was based on a previously identified efficacy hotspot for the peptidergic chemokine receptor CXCR3 and resulted in the synthesis and characterization of five new azobenzene‐containing CXCR3 ligands. G protein activation assays and real‐time electrophysiology experiments demonstrated photoswitching from antagonism to partial agonism and even to full agonism (compound VUF16216). SAR evaluation suggests that the size and electron‐donating properties of the substituents on the inner aromatic ring are important for the efficacy photoswitching. These compounds are the first GPCR azo ligands with a nearly full efficacy photoswitch and may become valuable pharmacological tools for the optical control of peptidergic GPCR signaling.     

Transformation of Receptor Tyrosine Kinases into Glutamate Receptors and Photoreceptors

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 photoreceptors.     

Optical Control of Insulin Secretion Using an Incretin Switch

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.     

A Photochromic Agonist for μ‐Opioid Receptors

Opioid receptors (ORs) are widely distributed in the brain, the spinal cord, and the digestive tract and play an important role in nociception. All known ORs are G‐protein‐coupled receptors (GPCRs) of family A. Another well‐known member of this family, rhodopsin, is activated by light through the cis/trans isomerization of a covalently bound chromophore, retinal. We now show how an OR can be combined with a synthetic azobenzene photoswitch to gain light sensitivity. Our work extends the reach of photopharmacology and outlines a general strategy for converting Family A GPCRs, which account for the majority of drug targets, into photoreceptors.     

Taking Photochromism beyond Visible: Direct One‐Photon NIR Photoswitches Operating in the Biological Window

The success of photopharmacology is inevitably tied to the availability of photoswitches, which can be operated within the biological window (λ=650–1450 nm) to maximize penetration in tissue. A general design strategy has been devised and a dihydropyrene derivative is described here that displays negative T‐type photochromism, allowing for efficient and nearly quantitative (95 %) switching induced by NIR light λ>800 nm. The thermal half‐life of the decolored ring‐open meta‐cyclophanediene isomer ranges from minutes to hours, depending on the solvent polarity and hence serves as a probe of the local environment. Due to the rather subtle geometrical differences between the two isomers, suitably modified NIR photoswitches are potential candidates for switching when bound in the pocket of the biological target, in principle allowing for reversible light‐induced inhibitor deactivation as an alternative approach to externally regulate biological functions.     

Recent Implementations of Molecular Photoswitches into Smart Materials and Biological Systems

Light is a nearly ideal stimulus for molecular systems. It delivers information encoded in the form of wavelengths and their intensities with high precision in space and time. Light is a mild trigger that does not permanently contaminate targeted samples. Its energy can be reversibly transformed into molecular motion, polarity, or flexibility changes. This leads to sophisticated functions at the supramolecular and macroscopic levels, from light-triggered nanomaterials to photocontrol over biological systems. New methods and molecular adapters of light are reported almost daily. Recently reported applications of photoresponsive systems, particularly azobenzenes, spiropyrans, diarylethenes, and indigoids, for smart materials and photocontrol of biological setups are described herein with the aim to demonstrate that the 21st century has become the Age of Enlightenment—“Le siècle des Lumières”—in molecular sciences.