Christina Birkedahl Falk‐Petersen

University of Copenhagen

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Petrine Wellendorph

University of Copenhagen

Bente Frølund

University of Copenhagen

Kasper Harpsøe

University of Copenhagen

David E. Gloriam

University of Copenhagen

Frederik Rostrup

University of Copenhagen

Louise Piilgaard

University of Copenhagen

Stine Buchleithner

University of Copenhagen

Nane Griem‐Krey

University of Copenhagen

Maria E. K. Lie

University of Copenhagen

Jacob Krall

University of Copenhagen

Cooperative Institutions

University of Copenhagen

Lundbeck (Denmark)

Rigshospitalet

Copenhagen University Hospital

Technical University of Denmark

Schools of Visual Arts, The Royal Danish Academy of Fine Arts

Novo Nordisk Foundation

Hokkaido University

Johannes Gutenberg University Mainz

TKL Research

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Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs

Journal of Medicinal Chemistry (2017)

Jacob Krall Claus Jensen Francesco Bavo Christina Birkedahl Falk‐Petersen Anne S. Haugaard

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar Ki) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Molecular model

10.1021/acs.jmedchem.7b01351

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Citations (22)

Author response for "GABA A receptor β 1 ‐subunit knock‐out mice show increased delta power in NREM sleep and decreased theta power in REM sleep"

Maria E. K. Lie Christina Birkedahl Falk‐Petersen Louise Piilgaard Nane Griem‐Krey Petrine Wellendorph

Sleep

10.1111/ejn.15267/v3/response1

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Citations (0)

Structure–Activity Studies of 3,9-Diazaspiro[5.5]undecane-Based γ-Aminobutyric Acid Type A Receptor Antagonists with Immunomodulatory Effect

Journal of Medicinal Chemistry (2021)

Francesco Bavo Heleen de-Jong Jonas Petersen Christina Birkedahl Falk‐Petersen Rebekka Löffler

The 3,9-diazaspiro[5.5]undecane-based compounds

Undecane

Moiety

Structure–activity relationship

Benzamide

10.1021/acs.jmedchem.1c00290

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Citations (4)

Synthesis and Pharmacological Evaluation of [¹¹C]4-Methoxy-N-[2-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl]benzamide as a Brain Penetrant PET Ligand Selective for the δ-Subunit-Containing γ-Aminobutyric Acid Type A Receptors

ACS Omega (2019)

Elina Tampio L’Estrade Hanne D. Hansen Christina Birkedahl Falk‐Petersen Anne S. Haugaard Nane Griem‐Krey

The α4/6βδ-containing GABAA receptors are involved in a number of brain diseases. Despite the potential of a δ-selective imaging agent, no PET radioligand is currently available for in vivo imaging. Here, we report the characterization of DS2OMe (1) as a candidate radiotracer, 11C-labeling, and subsequent evaluation of [11C]DS2OMe in a domestic pig as a PET radioligand for visualization of the δ-containing GABAA receptors.

Radioligand

Benzamide

Radioligand Assay

10.1021/acsomega.9b00434

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Development of a Robust Mammalian Cell‐based Assay for Studying Recombinant α₄β_1/3δ GABA_A Receptor Subtypes

Basic & Clinical Pharmacology & Toxicology (2017)

Christina Birkedahl Falk‐Petersen Rikke Søgaard Kenneth L. Madsen Anders B. Klein Bente Frølund

δ-Containing GABAA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABAA receptors. Inconveniently, the recombinant expression of especially α4 β1/3 δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4 β1/3 δ and binary α4 β1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn2+ had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α4 and β1/3 subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors.

HEK 293 cells

10.1111/bcpt.12778

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Citations (19)

Discovery of a new class of orthosteric antagonists with nanomolar potency at extrasynaptic GABAA receptors

Scientific Reports (2020)

Christina Birkedahl Falk‐Petersen Tsonko M. Tsonkov Malene Sofie Nielsen Kasper Harpsøe Christoffer Bundgaard

Abstract Brain GABA Α receptors are ionotropic receptors belonging to the class of Cys-loop receptors and are important drug targets for the treatment of anxiety and sleep disorders. By screening a compound library (2,112 compounds) at recombinant human α 4 β 1 δ GABA Α receptors heterologously expressed in a HEK cell line, we identified a scaffold of spirocyclic compounds with nanomolar antagonist activity at GABA Α receptors. The initial screening hit 2027 (IC 50 of 1.03 μM) was used for analogue search resulting in 018 (IC 50 of 0.088 μM). 018 was most potent at α 3,4,5 -subunit containing receptors, thus showing preference for forebrain-expressed extrasynaptic receptors. Schild analysis of 018 at recombinant human α 4 β 1 δ receptors and displacement of [ 3 H]muscimol binding in rat cortical homogenate independently confirmed a competitive profile. The antagonist profile of 018 was further validated by whole-cell patch-clamp electrophysiology, where kinetic studies revealed a slow dissociation rate and a shallow hill slope was observed. Membrane permeability studies showed that 2027 and 018 do not cross membranes, thus making the compounds less attractive for studying central GABA Α receptors effects, but conversely more attractive as tool compounds in relation to emerging peripheral GABA Α receptor-mediated effects of GABA e.g. in the immune system.

10.1038/s41598-020-66821-0

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Citations (14)

Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using 3H-HOCPCA

Neurochemistry International (2016)

Anders B. Klein Tina Bay Inge S. Villumsen Christina Birkedahl Falk‐Petersen Aleš Marek

Radioligand

Radioligand Assay

10.1016/j.neuint.2016.09.002

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Citations (12)

Molecular Determinants Underlying Delta Selective Compound 2 Activity at δ-Containing GABA_A Receptors

Molecular Pharmacology (2021)

Christina Birkedahl Falk‐Petersen Frederik Rostrup Rebekka Löffler Stine Buchleithner Kasper Harpsøe

Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit–containing GABA_A receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α₄β₁δ receptors: an α₄⁽⁺⁾δ⁽⁻⁾ interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ₂ receptors, and two sites in the transmembrane domain (TMD) - one in the α₄⁽⁺⁾β₁⁽⁻⁾ and one in the α₄⁽⁻⁾β₁⁽⁺⁾ interface, with the α₄⁽⁻⁾β₁⁽⁺⁾ site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α₄⁽⁺⁾β₁⁽⁻⁾ interface, either α₄(S303L) of the α₄⁽⁺⁾ side or β₁(I289Q) of the β₁⁽⁻⁾ side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD.

SIGNIFICANCE STATEMENT

δ-Containing GABA_A receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition, yet no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the known compound delta selective compound 2, the ground is laid for design of ligands that selectively target δ-containing GABA_A receptor subtypes, for better understanding of tonic inhibition, and ultimately, for rational development of novel drugs.

Cys-loop receptors

10.1124/molpharm.121.000266

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Citations (3)

Kinase activity simultaneously determines the constitutive and the orthosteric gating in α₄β_1/3δ GABA_A receptors in hippocampal granule cells

bioRxiv (Cold Spring Harbor Laboratory) (2018)

Nils Ole Dalby Ulrike Leurs Christina Birkedahl Falk‐Petersen Petra Scholze Jacob Krall

Abstract A subset of the GABA A receptors expressed in recombinant systems and neurons is known to exhibit both constitutive- and agonist-induced gating. Two such receptors are the δ-subunit containing GABA A receptors α 4 β 1 δ and α 4 β 3 δ, which are expressed in adult rodent hippocampal dentate gyrus granule cells (DGGCs). Here we show that the GABA A receptor mediated tonic current recorded in the presence of tetrodotoxin in adult rodent DGGCs is almost exclusively mediated by constitutively active δ-subunit containing GABA A receptors and that the constitutive current is absent in recordings at 24 °C or in recordings at 34 °C including an intracellular inhibitor of protein kinase C. These factors simultaneously govern the efficacy of an orthosteric agonist at α 4 β 1/3 δ receptors, Thio-THIP, in a reciprocal manner. In the absence of constitutive receptor activity, the efficacy of Thio-THIP was increased approximately four-fold relative to recording conditions that favors constitutive activity. Further, only under conditions of an absent constitutive current, the classified neutral antagonist gabazine (GBZ) alone, induced a tonic current in DGGCs (EC 50 2.1 μM). This effect of GBZ was not seen in recording conditions of high constitutive activity, was inhibited by picrotoxin (PTX), potentiated by DS2, completely absent in δ -/- mice and reduced in β 1 -/- mice, but could not be replicated in human α 4 β 1/3 δ receptors expressed recombinantly in HEK cells. We hypothesize that specific intracellular components in neurons interact with receptors to determine constitutive gating and receptor responsiveness to orthosteric ligands. Significance statement The presented data highlight how recording conditions for whole cell patch clamp analysis of α 4 β 1/3 δ GABA A receptors can mask important pharmacological effects. Specifically, orthosteric agonists appear with reduced efficacy, and other ligands, here exemplified with the well-known antagonist GBZ, are misinterpreted as being inactive/neutral, although they could have effect in constitutively silent receptors. Unmasking of potential hidden effects are easily done using recording conditions of reduced kinase activity in a relevant neuronal context. It follows that in pathologies with changes in phosphorylation level of δ-subunit containing GABA A receptors, the efficacy of an agonist of these receptors, measured by whole-cell recordings in vitro , will not match the efficacy of the same agonist in an unperturbed neuron in vivo .

Picrotoxin

10.1101/318543

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Silencing of spontaneous activity at α4β1/3δ GABA_A receptors in hippocampal granule cells reveals different ligand pharmacology

British Journal of Pharmacology (2020)

Nils Ole Dalby Christina Birkedahl Falk‐Petersen Ulrike Leurs Petra Scholze Jacob Krall

The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy.Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed.Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1-/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells.Kinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.

Picrotoxin

10.1111/bph.15146

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Citations (9)