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Staff member

Galyna Malieieva
Staff member publications

Castagna R, Maleeva G, Pirovano D, Matera C, Gorostiza P, (2022). Donor-Acceptor Stenhouse Adduct Displaying Reversible Photoswitching in Water and Neuronal Activity Journal Of The American Chemical Society 144, 15595-15602

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.

JTD Keywords: behavior, receptor, visible-light, wavelength, Optical control


Castagna, R, Kolarski, D, Durand-de Cuttoli, R, Maleeva, G, (2022). Orthogonal Control of Neuronal Circuits and Behavior Using Photopharmacology Journal Of Molecular Neuroscience 72, 1433-1442

Over the last decades, photopharmacology has gone far beyond its proof-of-concept stage to become a bona fide approach to study neural systems in vivo. Indeed, photopharmacological control has expanded over a wide range of endogenous targets, such as receptors, ion channels, transporters, kinases, lipids, and DNA transcription processes. In this review, we provide an overview of the recent progresses in the in vivo photopharmacological control of neuronal circuits and behavior. In particular, the use of small aquatic animals for the in vivo screening of photopharmacological compounds, the recent advances in optical modulation of complex behaviors in mice, and the development of adjacent techniques for light and drug delivery in vivo are described.

JTD Keywords: Architecture, Azobenzene photoswitches, Brain circuits, Channels, Circadian rhythm, In vivo photomodulation, In vivo technology, Light, Modulator, Neuronal receptors, Optical control, Optogenetics, Pharmacology, Photopharmacology, Receptors, Systems


Maleeva G, Nin-Hill A, Rustler K, Petukhova E, Ponomareva D, Mukhametova E, Gomila AM, Wutz D, Alfonso-Prieto M, König B, Gorostiza P, Bregestovski P, (2021). Subunit-specific photocontrol of glycine receptors by azobenzene-nitrazepam photoswitcher Eneuro 8,

© 2021 Maleeva et al. 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 a2 GlyRs (GlyR2), while being barely active on a1 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.

JTD Keywords: brain slices, glycine receptors, hypoglossal motoneurons, molecular modelling, patch-clamp, photopharmacology, Brain slices, Glycine receptors, Hypoglossal motoneurons, Molecular modelling, Patch-clamp, Photopharmacology


Rustler, Karin, Gomila, Alexandre, Maleeva, Galyna, Gorostiza, Pau, Bregestovski, Piotr, König, Burkhard, (2020). Optical control of GABAA receptors with a fulgimide-based potentiator Chemistry - A European Journal 26, (56), 12722-12727

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 (GABA A 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.

JTD


Gomila, Alexandre M. J., Rustler, Karin, Maleeva, Galyna, Nin-Hill, Alba, Wutz, Daniel, Bautista-Barrufet, Antoni, Rovira, Xavier, Bosch, Miquel, Mukhametova, Elvira, Petukhova, Elena, Ponomareva, Daria, Mukhamedyarov, Marat, Peiretti, Franck, Alfonso-Prieto, Mercedes, Rovira, Carme, König, Burkhard, Bregestovski, Piotr, Gorostiza, Pau, (2020). Photocontrol of endogenous glycine receptors in vivo Cell Chemical Biology 27, (11), 1425-1433.e7

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.

JTD Keywords: Glycine receptors, Photopharmacology, Optopharmacology, Inhibitory neurotransmission, CNS, Photoswitch


Maleeva, Galyna, Nin-Hill, Alba, Rustler, Karin, Petukhova, Elena, Ponomareva, Daria, Mukhametova, Elvira, Gomila-Juaneda, Alexandre, Wutz, Daniel, Alfonso-Prieto, Mercedes, König, Burkhard, Gorostiza, Pau, Bregestovski, Piotr, (2020). Subunit-specific photocontrol of glycine receptors by azobenzene-nitrazepam photoswitcher eneuro 8, (1), 0294-20

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, 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 modelling 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 alpha2 GlyRs (GlyR2), while being barely active on alpha1 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 "foetal" GlyR2 to "adult" GlyR1 receptors. Significance Statement Photochromic molecules are becoming widely used for studying and modulating various biological processes. Successful application of these compounds, whose activity can be controlled with light, potentially provides a promising tool for future therapeutic approaches. The main advantage of such compounds is their precise spatial and temporal selectivity, a property that favours specific drug action and diminishes their side effects. In the present study, we describe in detail the interaction of the novel azobenzene-nitrazepam-based photochromic compound (Azo-NZ1) with glycine receptors (GlyRs) and determine its subunit-specific blocking activity in the Cl-selective pore of GlyRs. This compound offers a new strategy for specific control of glycinergic circuits and stepping stone for design of new GlyR-active drugs.

JTD Keywords: Brain slices, Glycine receptors, Hypoglossal motoneurons, Molecular modelling, Patch-clamp, Photopharmacology


Nin-Hill, Alba, Maleeva, Galyna, Gomila-Juaneda, Alexandre, Wutz, Daniel, Rustler, Karin, Bautista-Barrufet, Antoni, Rovira, Xavier, Bosch, Miquel, Scholze, Petra, Peiretti, Franck, Rovira, Carme, König, Burkhard, Gorostiza, Pau, Bregestovski, Piotr, Prieto, Mercedes Alfonso, (2020). Photomodulation of inhibitory neurotransmission. Insights from molecular modeling Biophysical Journal Biophysical Society 64th Annual Meeting , CellPress (San Diego (USA)) 118, (3), 325a-326a

Photoswitches are molecules that change their conformation with light of specific wavelength. These light-regulated molecules can be designed to target ion channels, thus providing a unique tool for precise spatial and temporal control of ion channel functioning. Recently, we have applied a multidisciplinary approach to design, synthesize and functionally characterize two of such photoswitches, azo-NZ1 [Maleeva et al. Br. J. Pharmacol. 2019] and Glyght [Gomila-Juaneda et al. BioRxiv 2019], targeting GABA and glycine receptors, respectively. Using homology modeling and molecular docking, we have provided a molecular explanation of the light-dependent effect of these two photoswitchable ligands, as observed in in vitro electrophysiology experiments and in vivo tadpole behavioral assays. Azo-NZ1 is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group, yet it has an inhibitory effect on GABA A receptors under visible light and also inhibits benzodiazepine-insensitive GABA C (rho2) receptors. Molecular modeling, combined with electrophysiology and mutagenesis experiments, shows that addition of the sulfonyl azobenzene unexpectedly converts the ligand into a pore blocker. Glyght is also an azobenzene-containing benzodiazepine, yet it acts selectively on glycine receptors as a negative modulator and its inhibitory action increases under UV light. Molecular modeling suggests that Glyght binds to a novel allosteric site located at the interface between the extracellular and transmembrane domains. The two aforementioned photoswitches pave the way towards photomanipulation of inhibitory (gabaergic and glycinergic) neurotransmission, with potential applications in understanding inhibitory circuits in intact animals and in development of drug-based phototherapies

JTD


Maleeva, Galyna, Wutz, Daniel, Rustler, Karin, Nin-Hill, Alba, Rovira, Carme, Petukhova, Elena, Bautista-Barrufet, Antoni, Gomila-Juaneda, Alexandre, Scholze, Petra, Peiretti, Franck, Alfonso-Prieto, Mercedes, König, Burkhard, Gorostiza, Pau, Bregestovski, Piotr, (2019). A photoswitchable GABA receptor channel blocker British Journal of Pharmacology 176, (15), 2661-2677

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/β2/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.

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Bregestovski, Piotr, Maleeva, Galyna, Gorostiza, Pau, (2018). Light-induced regulation of ligand-gated channel activity British Journal of Pharmacology 175, (11), 1892-1902

The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field.

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