Action of the Photochrome Glyght on GABAergic Synaptic Transmission in Mouse Brain Slices.

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/ß2/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.

Optical Control of GABAA Receptors with a Fulgimide-Based Potentiator.

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 (GABAA Rs) 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.

Photochromic Evaluation of 3(5)-Arylazo-1H-pyrazoles.

The desire to photocontrol molecular properties ranging from materials to pharmacology using light as an external trigger with high spatiotemporal resolution led to the development of a broad range of photochromic scaffolds. Among them, azobenzenes are synthetically well accessible and show excellent fatigue resistance. Their photochromic properties vary with the substitution pattern and for different heteroarenes. However, the photochromism of 3(5)-substituted-1H-pryazoles has not yet been investigated, although this compound class offers interesting possibilities of metal ion coordination and hydrogen bond formation via its NH moiety. Herein, we present the results of an experimental and computational investigation of arylazo-3(5)-arylazo-1H-pyrazoles. To elucidate their properties, solvent and substitution effects on their light absorption, thermal half-lives, photostationary states, fatigue, and quantum yields were determined.

Azologization of serotonin 5-HT3 receptor antagonists.

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.

Development of photoswitchable inhibitors for ß-galactosidase.

Azobenzenes are of particular interest as a photochromic scaffold for biological applications because of their high fatigue resistance, their large geometrical change between extended (trans) and bent (cis) isomer, and their diverse synthetic accessibility. Despite their wide-spread use, there is no reported photochromic inhibitor of the well-investigated enzyme ß-galactosidase, which plays an important role for biochemistry and single molecule studies. Herein, we report the synthesis of photochromic competitive ß-galactosidase inhibitors based on the molecular structure of 2-phenylethyl ß-d-thiogalactoside (PETG) and 1-amino-1-deoxy-ß-d-galactose (ß-d-galactosylamine). The thermally highly stable PETG-based azobenzenes show excellent photochromic properties in polar solvents and moderate to high photostationary states (PSS). The optimized compound 37 is a strong competitive inhibitior of ß-galactosidase from Escherichia coli and its inhibition constant (Ki) changes between 60 nM and 290 nM upon irradiation with light. Additional docking experiments supported the observed structure-activity relationship.

Solid-Phase Synthesis of an "Inaccessible" hGH-Derived Peptide Using a Pseudoproline Monomer and SIT-Protection for Cysteine.

The solid-phase peptide synthesis (SPPS) of the C-terminal sequence of hGH with one extra Tyr attached to its N-terminus (total of 16 residues with a disulfide bridge) has been accomplished for the first time by optimizing several synthetic parameters. First of all, the two Ser residues (positions 9 and 13 of the molecule) have been introduced as a single amino acid, Fmoc-Ser(ψMe,Mepro)-OH, demonstrating that the acylation of these hindered moieties is possible. This allows us to avoid the use of the corresponding dipeptides, Fmoc-AA-Ser(ψMe,Mepro)-OH, which are very often not commercially available or very costly. The second part of the sequence has been elongated via a double coupling approach using two of the most effective coupling methods (DIC-OxymaPure and HATU-DIEA). Finally, the disulfide bridging has been carried out very smoothly by a chemoselective thiol-disulfide interchange reaction between a SIT (sec-isoamyl mercaptan)-protected Cys residue and the free thiol of the second Cys. The synthesis of this short peptide has evidenced that SPPS is a multifactorial process which should be optimized in each case.

Subunit-Specific Photocontrol of Glycine Receptors by Azobenzene-Nitrazepam Photoswitcher.

Photopharmacology is a unique approach that through a combination of photochemistry methods and advanced life science techniques allows the study and control of specific biological processes, ranging from intracellular pathways to brain circuits. Recently, a first photochromic channel blocker of anion-selective GABAA receptors, the azobenzene-nitrazepam-based photochromic compound (Azo-NZ1), has been described. In the present study, using patch-clamp technique in heterologous system and in mice brain slices, site-directed mutagenesis and molecular modeling we provide evidence of the interaction of Azo-NZ1 with glycine receptors (GlyRs) and determine the molecular basis of this interaction. Glycinergic synaptic neurotransmission determines an important inhibitory drive in the vertebrate nervous system and plays a crucial role in the control of neuronal circuits in the spinal cord and brain stem. GlyRs are involved in locomotion, pain sensation, breathing, and auditory function, as well as in the development of such disorders as hyperekplexia, epilepsy, and autism. Here, we demonstrate that Azo-NZ1 blocks in a UV-dependent manner the activity of α2 GlyRs (GlyR2), while being barely active on α1 GlyRs (GlyR1). The site of Azo-NZ1 action is in the chloride-selective pore of GlyR at the 2' position of transmembrane helix 2 and amino acids forming this site determine the difference in Azo-NZ1 blocking activity between GlyR2 and GlyR1. This subunit-specific modulation is also shown on motoneurons of brainstem slices from neonatal mice that switch during development from expressing "fetal" GlyR2 to "adult" GlyR1 receptors.

Photocontrol of Endogenous Glycine Receptors In Vivo.

Glycine receptors (GlyRs) are indispensable for maintaining excitatory/inhibitory balance in neuronal circuits that control reflexes and rhythmic motor behaviors. Here we have developed Glyght, a GlyR ligand controlled with light. It is selective over other Cys-loop receptors, is active in vivo, and displays an allosteric mechanism of action. The photomanipulation of glycinergic neurotransmission opens new avenues to understanding inhibitory circuits in intact animals and to developing drug-based phototherapies.

Light-Switchable Antagonists for the Histamine H1 Receptor at the Isolated Guinea Pig Ileum.

The histamine H1 G protein-coupled receptor (GPCR) plays an important role in allergy and inflammation. Existing drugs that address the H1 receptor differ in their chemical structure, pharmacology, and side effects. Light-controllable spatial and temporal activity regulation of photochromic H1 ligands may contribute to a better mechanistic understanding and the development of improved correlations between ligand structure and pharmacologic effects. We report photochromic H1 receptor ligands, which were investigated in an organ-pharmacological assay. Initially, five photochromic azobenzene derivatives of reported dual H1 -H4 receptor antagonists were designed, synthesized, photochemically characterized, and organ-pharmacologically tested on the isolated guinea pig ileum. Among them, one compound [trans-19: (Z)-1-(4-chlorophenyl)-1-(4-methylpiperazin-1-yl)-N-(4-((E)-phenyldiazenyl)phenyl)methanimine] retained the antagonistic activity of its non-photochromic lead, and trans-cis isomerization by irradiation induced a fourfold difference in the pharmacological response. Further structural optimization resulted in two bathochromically shifted derivatives of 19 [NO2 -substituted 35 {(Z)-1-(4-chlorophenyl)-1-(4-methylpiperazin-1-yl)-N-(4-((E)-(4-nitrophenyl)diazenyl)phenyl)methanimine} and SO3- -substituted 41 {4-((E)-(4-(((Z)-(4-chlorophenyl)(4-methylpiperazin-1-yl)methylene)amino)phenyl)diazenyl)benzenesulfonate}], which do not require the use of UV light for photoisomerization and which also have improved solubility and show reduced tissue impairment. The trans isomers of both compounds showed a remarkable increase in antagonistic activity relative to their lead trans-19; furthermore, a 46-fold difference in activity on the isolated guinea pig ileum was observed between trans- and cis-35.

A photoswitchable GABA receptor channel blocker.

BACKGROUND AND PURPOSE: Anion-selective Cys-loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride-selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light-controllable modulator of GABA receptors, we constructed azobenzene-nitrazepam (Azo-NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group. EXPERIMENTAL APPROACH: The experiments were carried out on cultured cells expressing Cys-loop receptors of known subunit composition and in brain slices using patch-clamp. Site-directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo-NZ1. KEY RESULTS: At visible light, being in trans-configuration, Azo-NZ1 blocked heteromeric α1/ß2/γ2 GABAA receptors, ρ2 GABAA (GABAC ), and α2 glycine receptors, whereas switching the compound into cis-state by UV illumination restored the activity. Azo-NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans-configuration, Azo-NZ1 blocks the Cl-selective ion pore of GABA receptors interacting mainly with the 2' level of the TM2 region. CONCLUSIONS AND IMPLICATIONS: Azo-NZ1 is a soluble light-driven Cl-channel blocker, which allows photo-modulation of the activity induced by anion-selective Cys-loop receptors. Azo-NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination.