GABAA receptor-mediated seizure liabilities: a mixed-methods screening approach.

GABAA receptors, members of the pentameric ligand-gated ion channel superfamily, are widely expressed in the central nervous system and mediate a broad range of pharmaco-toxicological effects including bidirectional changes to seizure threshold. Thus, detection of GABAA receptor-mediated seizure liabilities is a big, partly unmet need in early preclinical drug development. This is in part due to the plethora of allosteric binding sites that are present on different subtypes of GABAA receptors and the critical lack of screening methods that detect interactions with any of these sites. To improve in silico screening methods, we assembled an inventory of allosteric binding sites based on structural data. Pharmacophore models representing several of the binding sites were constructed. These models from the NeuroDeRisk IL Profiler were used for in silico screening of a compiled collection of drugs with known GABAA receptor interactions to generate testable hypotheses. Amoxapine was one of the hits identified and subjected to an array of in vitro assays to examine molecular and cellular effects on neuronal excitability and in vivo locomotor pattern changes in zebrafish larvae. An additional level of analysis for our compound collection is provided by pharmacovigilance alerts using FAERS data. Inspired by the Adverse Outcome Pathway framework, we postulate several candidate pathways leading from specific binding sites to acute seizure induction. The whole workflow can be utilized for any compound collection and should inform about GABAA receptor-mediated seizure risks more comprehensively compared to standard displacement screens, as it rests chiefly on functional data.

Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness.

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity.

Molecular Mingling: Multimodal Predictions of Ligand Promiscuity in Pentameric Ligand-Gated Ion Channels.

Background: Human pentameric ligand-gated ion channels (pLGICs) comprise nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine type 3 receptors (5-HT3Rs), zinc-activated channels (ZAC), γ-aminobutyric acid type A receptors (GABAARs) and glycine receptors (GlyRs). They are recognized therapeutic targets of some of the most prescribed drugs like general anesthetics, anxiolytics, smoking cessation aids, antiemetics and many more. Currently, approximately 100 experimental structures of pLGICs with ligands bound exist in the protein data bank (PDB). These atomic-level 3D structures enable the generation of a comprehensive binding site inventory for the superfamily and the in silico prediction of binding site properties. Methods: A panel of high throughput in silico methods including pharmacophore screening, conformation analysis and descriptor calculation was applied to a selection of allosteric binding sites for which in vitro screens are lacking. Variant abundance near binding site forming regions and computational docking complement the approach. Results: The structural data reflects known and novel binding sites, some of which may be unique to individual receptors, while others are broadly conserved. The membrane spanning domain, comprising four highly conserved segments, contains ligand interaction sites for which in vitro assays suitable for high throughput screenings are critically lacking. This is also the case for structurally more variable novel sites in the extracellular domain. Our computational results suggest that the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) can utilize multiple pockets which are likely to exist on most superfamily members. Conclusion: With this study, we explore the potential for polypharmacology among pLGICs. Our data suggest that ligands can display two forms of promiscuity to an extent greater than what has been realized: 1) Ligands can interact with homologous sites in many members of the superfamily, which bears toxicological relevance. 2) Multiple pockets in distinct localizations of individual receptor subtypes share common ligands, which counteracts efforts to develop selective agents. Moreover, conformational states need to be considered for in silico drug screening, as certain binding sites display considerable flexibility. In total, this work contributes to a better understanding of polypharmacology across pLGICs and provides a basis for improved structure guided in silico drug development and drug derisking.

α6-Containing GABAA Receptors: Functional Roles and Therapeutic Potentials.

GABAA receptors containing the α6 subunit are highly expressed in cerebellar granule cells and less abundantly in many other neuronal and peripheral tissues. Here, we for the first time summarize their importance for the functions of the cerebellum and the nervous system. The cerebellum is not only involved in motor control but also in cognitive, emotional, and social behaviors. α6ßγ2 GABAA receptors located at cerebellar Golgi cell/granule cell synapses enhance the precision of inputs required for cerebellar timing of motor activity and are thus involved in cognitive processing and adequate responses to our environment. Extrasynaptic α6ßδ GABAA receptors regulate the amount of information entering the cerebellum by their tonic inhibition of granule cells, and their optimal functioning enhances input filtering or contrast. The complex roles of the cerebellum in multiple brain functions can be compromised by genetic or neurodevelopmental causes that lead to a hypofunction of cerebellar α6-containing GABAA receptors. Animal models mimicking neuropsychiatric phenotypes suggest that compounds selectively activating or positively modulating cerebellar α6-containing GABAA receptors can alleviate essential tremor and motor disturbances in Angelman and Down syndrome as well as impaired prepulse inhibition in neuropsychiatric disorders and reduce migraine and trigeminal-related pain via α6-containing GABAA receptors in trigeminal ganglia. Genetic studies in humans suggest an association of the human GABAA receptor α6 subunit gene with stress-associated disorders. Animal studies support this conclusion. Neuroimaging and post-mortem studies in humans further support an involvement of α6-containing GABAA receptors in various neuropsychiatric disorders, pointing to a broad therapeutic potential of drugs modulating α6-containing GABAA receptors. SIGNIFICANCE STATEMENT: α6-Containing GABAA receptors are abundantly expressed in cerebellar granule cells, but their pathophysiological roles are widely unknown, and they are thus out of the mainstream of GABAA receptor research. Anatomical and electrophysiological evidence indicates that these receptors have a crucial function in neuronal circuits of the cerebellum and the nervous system, and experimental, genetic, post-mortem, and pharmacological studies indicate that selective modulation of these receptors offers therapeutic prospects for a variety of neuropsychiatric disorders and for stress and its consequences.

Two Distinct Populations of α1α6-Containing GABAA-Receptors in Rat Cerebellum.

GABAA receptors are pentameric GABA-gated chloride channels. The existence of 19 different subunits (six α, three ß, three γ, δ, ε, θ, π, and three ρ) in mammalian systems gives rise to an enormous theoretical diversity of GABAA receptor subtypes with distinct subunit composition and unique pharmacological properties. These receptors are already now the drug targets of several clinically used compounds, such as benzodiazepines, anesthetics, and many more. There is a constant quest to identify novel molecules and possible future drug targets: Currently, α6-containing GABAA receptors are being discussed in the context of treating sensorimotor gating deficits in neuropsychiatric disorders, such as tic disorders and schizophrenia. Therefore, we aim to learn more about α6-containing GABAA receptors. They are mostly expressed in the cerebellar granule cell layer, where they form the following subtypes: α6ßxγ2, α1α6ßxγ2, α6ßxδ, and α1α6ßxδ. In former studies, α1α6ßxγ2-containing GABAA receptors were considered a single receptor population. In the current study, we investigate the possibility, that this population can consist of two subgroups with alternative arrangements depending if α1 neighbors γ2 (forming a "diazepam-sensitive" receptor), or if α6 neighbors γ2 (forming a "diazepam-insensitive" receptor) and aimed to prove the existence of both subtypes in native tissue. We performed immunoprecipitation experiments on rat cerebellar lysates using α1- or α6 subunit-specific antibodies followed by radioligand binding assays with either 3H-flunitrazepam or 3H-Ro 15-4513. Indeed, we were able to prove the existence of two distinct populations of α1α6-containing GABAA-receptors and could quantify the different receptor populations: α1ßxγ2 receptors constitute approximately 60% of all γ2-containing receptors in the rat cerebellum, α6ßxγ2 about 20%, and both isoforms of α1α6ßxγ2 9-15% each. The simple classification of GABAA-receptors into αx-containing subtypes seems not to reflect the complexity of nature; those receptors are more diverse than previously thought.

Structure-Guided Computational Methods Predict Multiple Distinct Binding Modes for Pyrazoloquinolinones in GABAA Receptors.

Pyrazoloquinolinones (PQs) are a versatile class of GABAA receptor ligands. It has been demonstrated that high functional selectivity for certain receptor subtypes can be obtained by specific substitution patterns, but so far, no clear SAR rules emerge from the studies. As is the case for many GABAA receptor targeting chemotypes, PQs can interact with distinct binding sites on a given receptor pentamer. In pentamers of αßγ composition, such as the most abundant α1ß2γ2 subtype, many PQs are high affinity binders of the benzodiazepine binding site at the extracellular α+/γ2- interfaces. There they display a functionally near silent, flumazenil-like allosteric activity. More recently, interactions with extracellular α+/ß- interfaces have been investigated, where strong positive modulation can be steered toward interesting subtype preferences. The most prominent examples are functionally α6-selective PQs. Similar to benzodiazepines, PQs also seem to interact with sites in the transmembrane domain, mainly the sites used by etomidate and barbiturates. This promiscuity leads to potential contributions from multiple sites to net modulation. Developing ligands that interact exclusively with the extracellular α+/ß- interfaces would be desired. Correlating functional profiles with binding sites usage is hampered by scarce and heterogeneous experimental data, as shown in our meta-analysis of aggregated published data. In the absence of experimental structures, bound states can be predicted with pharmacophore matching methods and with computational docking. We thus performed pharmacophore matching studies for the unwanted sites, and computational docking for the extracellular α1,6+/ß3- interfaces. The results suggest that PQs interact with their binding sites with diverse binding modes. As such, rational design of improved ligands needs to take a complex structure-activity landscape with branches between sub-series of derivatives into account. We present a workflow, which is suitable to identify and explore potential branching points on the structure-activity landscape of any small molecule chemotype.

Trigeminal neuropathic pain development and maintenance in rats are suppressed by a positive modulator of α6 GABAA receptors.

γ-Aminobutyric acid type A (GABAA ) receptors containing the α6 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 µM enhanced γ-aminobutyric acid (GABA) currents at recombinant rat α6ß3γ2, α6ß3δ and α6ß3 receptors, whereas it was inactive at most GABAA receptor subtypes containing other α subunits. DK-I-87-1 at concentrations below 1 µM was inactive at α6-containing receptors and only weakly modulated other GABAA 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 α6 GABAA receptors is appropriate for repeated administration and thus may represent a novel treatment option against the development and maintenance of trigeminal neuropathic pain.

Towards functional selectivity for α6ß3γ2 GABAA receptors: a series of novel pyrazoloquinolinones.

BACKGROUND AND PURPOSE: The GABAA receptors are ligand-gated 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 α+/ß- interfaces, using a systematically varied series of pyrazoloquinolinones. EXPERIMENTAL APPROACH: Recombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABA-elicited currents by the newly synthesized and reference compounds were investigated by the two-electrode 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 α6ß3γ2 GABAA receptors with nearly no residual activity at the other αxß3γ2 (x = 1-5) subtypes. This modulation was independent of affinity for α+/γ- interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular α+/ß- interfaces. CONCLUSION AND IMPLICATIONS: These results constitute a major step towards a potential selective positive modulation of certain α6-containing 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.

Photocatalytic degradation with immobilised TiO(2) of three selected neonicotinoid insecticides: imidacloprid, thiamethoxam and clothianidin.

This research focused on photocatalytic degradation of imidacloprid, thiamethoxam and clothianidin employing a tailor-made photoreactor with six polychromatic fluorescent UVA (broad maximum at 355 nm) lamps and immobilised titanium dioxide (TiO(2)) on glass slides. The disappearance was followed by high pressure liquid chromatography (HPLC-DAD) analyses, wherein the efficiency of mineralization was monitored by measurements of total organic carbon (TOC). Within 2h of photocatalysis, all three neonicotinoids were degraded following first order kinetics with rate constants k=0.035 ± 0.001 min(-1) for imidacloprid, k=0.019 ± 0.001 min(-1) for thiamethoxam and k=0.021 ± 0.000 min(-1) for clothianidin. However, the rate of mineralization was low, i.e. 19.1 ± 0.2% for imidacloprid, 14.4 ± 2.9% for thiamethoxam and 14.1 ± 0.4% for clothianidin. This indicates that several transformation products were formed instead. Some of them were observed within HPLC-DAD analyses and structures were proposed according to the liquid chromatography-electro spray ionization tandem mass spectrometry analyses (LC-ESI-MS/MS). The formation of clothianidin, as thiamethoxam transformation product, was reported for the first time.