Trigeminal neuropathic pain development and maintenance in rats are suppressed by a positive modulator of alpha 6 GABA(A) receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors containing the alpha 6 subunit are located in trigeminal ganglia, and their reduction by small interfering RNA increases inflammatory temporomandibular and myofascial pain in rats. We thus hypothesized that enhancing their activity may help in neuropathic syndromes originating from the trigeminal system. Here, we performed a detailed electrophysiological and pharmacokinetic analysis of two recently developed deuterated structurally similar pyrazoloquinolinone compounds. DK-I-56-1 at concentrations below 1 mu M enhanced gamma-aminobutyric acid (GABA) currents at recombinant rat alpha 6 beta 3 gamma 2, alpha 6 beta 3 delta and alpha 6 beta 3 receptors, whereas it was inactive at most GABA(A) receptor subtypes containing other alpha subunits. DK-I-87-1 at concentrations below 1 mu M was inactive at alpha 6-containing receptors and only weakly modulated other GABA(A) receptors investigated. Both plasma and brain tissue kinetics of DK-I-56-1 were relatively slow, with half-lives of 6 and 13 hr, respectively, enabling the persistence of estimated free brain concentrations in the range 10-300 nM throughout a 24-hr period. Results obtained in two protocols of chronic constriction injury of the infraorbital nerve in rats dosed intraperitoneally with DK-I-56-1 during 14 days after surgery or with DK-I-56-1 or DK-I-87-1 during 14 days after trigeminal neuropathy were already established, demonstrated that DK-I-56-1 but not DK-I-87-1 significantly reduced the hypersensitivity response to von Frey filaments. Significance Neuropathic pain induced by trigeminal nerve damage is poorly controlled by current treatments. DK-I-56-1 that positively modulates alpha 6 GABA(A) receptors is appropriate for repeated administration and thus may represent a novel treatment option against the development and maintenance of trigeminal neuropathic pain

Scientific Reports / Molecular tools for GABAA receptors : High affinity ligands for 1-containing subtypes

-Aminobutyric acid type A (GABAA) receptors are pentameric GABA-gated chloride channels that are, in mammalians, drawn from a repertoire of 19 different genes, namely 1-6, 1-3, 1-3, , , , and 1-3. The existence of this wide variety of subunits as well as their diverse assembly into different subunit compositions result in miscellaneous receptor subtypes. In combination with the large number of known and putative allosteric binding sites, this leads to a highly complex pharmacology. Recently, a novel binding site at extracellular +/ interfaces was described as the site of modulatory action of several pyrazoloquinolinones. In this study we report a highly potent ligand from this class of compounds with pronounced 1-selectivity that mainly lacks -subunit selectivity. It constitutes the most potent 1-selective positive allosteric modulatory ligand with known binding site. In addition, a proof of concept pyrazoloquinolinone ligand lacking the additional high affinity interaction with the benzodiazepine binding site is presented. Ultimately, such ligands can be used as invaluable molecular tools for the detection of 1-containing receptor subtypes and the investigation of their abundance and distribution.(VLID)460736

Towards functional selectivity for 632 GABAA receptors: a series of novel pyrazoloquinolinones

Background and Purpose The GABAA receptors are ligandgated ion channels, which play an important role in neurotransmission. Their variety of binding sites serves as an appealing target for many clinically relevant drugs. Here, we explored the functional selectivity of modulatory effects at specific extracellular +/ interfaces, using a systematically varied series of pyrazoloquinolinones. Experimental Approach Recombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABAelicited currents by the newly synthesized and reference compounds were investigated by the twoelectrode voltage clamp method. Key Results We identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at 632 GABAA receptors with nearly no residual activity at the other x32 (x = 15) subtypes. This modulation was independent of affinity for +/ interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular +/ interfaces. Conclusion and Implications These results constitute a major step towards a potential selective positive modulation of certain 6containing GABAA receptors, which might be useful to elicit their physiological role. Furthermore, these studies pave the way towards insights into molecular principles that drive positive versus negative allosteric modulation of specific GABAA receptor isoforms.(VLID)480595

Different Benzodiazepines Bind with Distinct Binding Modes to GABA_A Receptors

Benzodiazepines are clinically relevant drugs that bind to GABA_A neurotransmitter receptors at the α+/γ2– interfaces and thereby enhance GABA-induced chloride ion flux leading to neuronal hyperpolarization. However, the structural basis of benzodiazepine interactions with their high-affinity site at GABA_A receptors is controversially debated in the literature, and <i>in silico</i> studies led to discrepant binding mode hypotheses. In this study, computational docking of diazepam into α+/γ2– homology models suggested that a chiral methyl group, which is known to promote preferred binding to α5-containing GABA_A receptors (position 3 of the seven-membered diazepine ring), could possibly provide experimental evidence that supports or contradicts the proposed binding modes. Thus, we investigated three pairs of <i>R</i> and <i>S</i> isomers of structurally different chemotypes, namely, diazepam, imidazobenzodiazepine, and triazolam derivatives. We used radioligand displacement studies as well as two-electrode voltage clamp electrophysiology in α1β3γ2-, α2β3γ2-, α3β3γ2-, and α5β3γ2-containing GABA_A receptors to determine the ligand binding and functional activity of the three chemotypes. Interestingly, both imidazobenzodiazepine isomers displayed comparable binding affinities, while for the other two chemotypes, a discrepancy in binding affinities of the different isomers was observed. Specifically, the <i>R</i> isomers displayed a loss of binding, whereas the <i>S</i> isomers remained active. These findings are in accordance with the results of our <i>in silico</i> studies suggesting the usage of a different binding mode of imidazobenzodiazepines compared to those of the other two tested chemotypes. Hence, we conclude that different chemically related benzodiazepine ligands interact via distinct binding modes rather than by using a common binding mode