Understanding subtype-selective allosteric modulation of GABA-A receptors

GABA-A Rezeptoren sind wichtige Mediatoren der hemmenden Neurotransmission im Gehirn und gehören zur Superfamilie der pentameren Liganden-gesteuerten Ionenkanäle (pLGIC). Sie sind Zielproteine vieler klinisch gebräuchlicher Medikamente wie Benzodiazepine (Bz), Barbiturate, Neurosteroide, Antikonvulsiva und allgemeiner Narkosemittel. Die Existenz einer großen Anzahl von Rezeptor-Subtypen führt zu einer sehr komplexen Pharmakologie. Strukturelle Informationen über die Bindungsstellen der Liganden sind hilfreich, um eine unselektive Bindung von Liganden an mehrere Subtypen zu verhindern. Neue strukturelle Erkenntnisse können dazu beitragen, selektive Liganden für spezifische Subtypen zu designen. Dies würde zu neuen Therapieansätzen für viele Krankheiten des zentralen Nervensystems, wie Schlaflosigkeit, Angstzustände, Epilepsie, Depression, Schizophrenie und anderen führen. Bereits bevor eine Kristallstruktur des GABA-A Rezeptors zur Verfügung stand, ermöglichten Homologiemodelle, die auf mehreren bakteriellen und eukaryotischen homologen Proteinen basieren, die Struktur des GABA-A Rezeptors besser zu verstehen (Paper 1 und 2). In Paper 1 wurden mittels eines Experiment-basierten virtuellen Screenings neue Liganden für die Bz- Bindungsstelle identifiziert. In Paper 2 wurde ein Kanalblocker, basierend auf der 5-fachen Symmetrie der Pore, designt. Die Kristallstruktur eines homopentameren β3 GABA-A Rezeptors wurde von Miller und Aricescu in 2014 veröffentlicht. Für Paper 3 wurden diese Struktur und mehrere Liganden-gebundene Kristallstrukturen homologer Proteine systematisch analysiert. Für jede beobachtete Bindetasche der Kristallstrukturen wurde beurteilt, ob diese Bindungsstellen auch für GABA-A Rezeptoren relevant sind. Das Ziel der Studie war die vielen GABA-A Rezeptor-Modelle basierend auf diversen, homologen Strukturen in Hinblick auf experimentelle Evidenzen zu untersuchen. Die Analyse ermöglichte ein vollständigeres Bild der mit Liganden gebundenen Rezeptoren zu erhalten. Diese Homologiemodelle können als Basis für die Identifizierung einer Bindungsstelle und eines Bindemodus dienen, und schließlich zu einem struktur-basierten Wirkstoffdesign führen. Experimente können auf der Grundlage dieser Modelle geplant werden, um zu prüfen, ob sie GABA-A Rezeptoren korrekt abbilden.GABA-A receptors are major mediators of inhibitory neurotransmission in the brain and belong to the superfamily of pentameric ligand-gated ion channels (pLGIC). They are targets of many clinically important drugs, such as benzodiazepines (Bz), barbiturates, neurosteroids, anticonvulsants and general anesthetics. The existence of many subtypes of these receptors results in a very complex pharmacology. Structural information on drug binding sites would be helpful for a better understanding of unselective binding of ligands to multiple subtypes, and to rationally design selective ligands for specific subtypes. This would lead to improved and novel therapeutic principles for many malfunctions of the central nervous system, such as insomnia, anxiety disorders, epilepsy, depression, schizophrenia and many more. Already before a crystal structure of the GABA-A receptor was available, comparative models based on several bacterial and eukaryotic homologues allowed some structural insights (paper 1 and 2). In the course of paper 1, novel Bz-site ligands were identified in an experiment-guided virtual screening process. In paper 2, a rational design of an open channel blocker was conducted using the 5-fold symmetry of the pore. The crystal structure of the homopentameric β3 GABA-A receptor was solved by Miller and Aricescu in 2014. A systematic analysis of this structure and of different X-ray structures of homologous proteins with interesting ligands bound was performed for paper 3. For novel binding sites, which were seen in the X-ray structures, it was assessed if those binding sites could also exist in GABA-A receptors. The aim of this study was to examine the newly generated GABA-A receptor models, which were based on several superfamily members, in the light of experimental evidence. That allowed us to derive more complete models of ligand-bound receptors. These homology models may serve as a basis for binding site confirmation, binding mode confirmation and ultimately, structure-guided drug design. Experiments can be designed on the basis of these models to test if they correctly depict GABA-A receptors.submitted by DI(FH) Roshan PuthenkalamZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)171475

Accelerated Discovery of Novel Benzodiazepine Ligands by Experiment-Guided Virtual Screening

High throughput discovery of ligand scaffolds for target proteins can accelerate development of leads and drug candidates enormously. Here we describe an innovative workflow for the discovery of high affinity ligands for the benzodiazepine-binding site on the so far not crystallized mammalian GABA_A receptors. The procedure includes chemical biology techniques that may be generally applied to other proteins. Prerequisites are a ligand that can be chemically modified with cysteine-reactive groups, knowledge of amino acid residues contributing to the drug-binding pocket, and crystal structures either of proteins homologous to the target protein or, better, of the target itself. Part of the protocol is virtual screening that without additional rounds of optimization in many cases results only in low affinity ligands, even when a target protein has been crystallized. Here we show how the integration of functional data into structure-based screening dramatically improves the performance of the virtual screening. Thus, lead compounds with 14 different scaffolds were identified on the basis of an updated structural model of the diazepam-bound state of the GABA_A receptor. Some of these compounds show considerable preference for the α₃β₂γ₂ GABA_A receptor subtype

Effect of PCCP⁻ on recombinant α₁β₂γ₂ GABA_A receptors.

<p>A, GABA_A receptors were expressed in Xenopus oocytes. The electrical currents recorded by two-electrode voltage clamp were activated with a concentration of GABA eliciting 1% of the maximal current amplitude (EC₁) and inhibited with increasing concentrations of PCCP⁻. The lower bar indicates the time of GABA application, the upper bar the time of PCCP⁻ application. The numbers indicate the concentration of PCCP⁻ in µM. At concentrations >1 µM, induces an open-channel block, characterized by an apparent desensitization of the current and an off-current. B, Averaged concentration inhibition curve by PCCP⁻. Individual curves were fitted and standardized to the current elicited by GABA. Data are shown as mean ± SEM (n = 4). Open circle: peak current amplitudes at the beginning of the drug application. Filled squares: current amplitudes at the end of the drug application. Filled circles: current amplitudes at the end of the drug application corrected for the direct effect of PCCP⁻ on membranes. C) and D) same experiment carried out at a concentration of GABA eliciting 10% of the maximal current amplitude (EC₁₀).</p

PCCP⁻ prevents the increase in PCCP⁻ sensitivity of α₁V256β₂γ₂ mediated by MTSET⁺ + GABA.

<p>GABA (EC₁₀) was applied repetitively until a stable current response was observed followed by inhibition of the channel by PCCP⁻. Subsequently 5 mM MTSET was applied in the presence of GABA. After MTSET⁺ treatment GABA was applied twice followed by a combined application of GABA and the same concentration of PCCP⁻ used before. A, Wild type receptors were not affected by this treatment. B, The treatment leads to an enhanced inhibition in α₁V256Cβ₂γ₂. C, 5 mM MTSET⁺ was applied to α₁V256C mutant receptor in presence of GABA and 1 mM PCCP⁻. PCCP⁻ prevented enhanced inhibition and therefore covalent reaction. These experiments were repeated independently three times using different oocytes.</p

Molecular model of the interaction of PCCP⁻ with GABA_A receptors.

<p>A, The side view of the PCCP⁻ docking pose from the perspective of the γ₂ subunit. The ligand and the mutated residues of the α₁ subunit, which have an impact on the affinity of the ligand, are shown in space filling representation. The 2′ valines of the α₁ subunit are rendered grey; the 6′ threonines of the α₁ subunit in green. The GABA_A receptor is displayed in ribbon representation with α₁ subunits shown in yellow, β₂ subunits in red, γ₂ subunit in blue. The complete transmembrane domain (TMD) is shown only of the α₁ and the β₂ subunits in the back. Of the subunits in front, only a segment of the transmembrane domain 2 (TMD2) is depicted. The TMD2 of the γ₂ subunit is only partly displayed to provide a “window” through which the ligand is seen. B, Top view of the pose showing the symmetric molecular interactions between ligand and receptor. PCCP⁻ (space filling) forms H-bonds (blue dashed lines) to the –OH groups of the 6′ threonines (stick representation) of each of the five subunits.</p

High concentration of PCCP⁻ induce a current in non-injected Xenopus oocytes.

<p>A, Voltage jump of 25 ms duration from −80 mV to −30 mV. A µA sized transient current flows with each voltage step. B, A small transient outward current is induced after applications of 10, 30 and 100 µM PCCP⁻ of 30 s duration to an oocyte held at a membrane potential of −80 mV.</p

<b>Pharmacological evaluation of the expressed recombinant receptors.</b>

<p>EC₅₀ for GABA, IC₅₀ for PCCP⁻, and IC₅₀ for picrotoxin are given for wild type and mutant receptors.</p><p><b>Pharmacological evaluation of the expressed recombinant receptors.</b></p

Aligned sequences of the amino acid residues in the subunits α₁β₂γ₂ of the rat GABA_A receptor.

<p>A, Alignment of 2α, 2β and 1γ subunit contributing to the formation of a GABA_A pentamer. The residues in the α₁ subunit of the GABA_A mutated to Cys are shown in boldface letters. B, α-Helical wheel representation of the rat α₁ M2 membrane-spanning domain showing the mutated residues in boldface letters.</p