Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1

The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1S,2S,5R)-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [3H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N-methylated analog (2) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (pKB value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1β2γ2 GABAA receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABAA receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico-guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.

Epileptic Mechanisms Shared by Alzheimer’s Disease: Viewed via the Unique Lens of Genetic Epilepsy

Our recent work on genetic epilepsy (GE) has identified common mechanisms between GE and neurodegenerative diseases including Alzheimer’s disease (AD). Although both disorders are seemingly unrelated and occur at opposite ends of the age spectrum, it is likely there are shared mechanisms and studies on GE could provide unique insights into AD pathogenesis. Neurodegenerative diseases are typically late-onset disorders, but the underlying pathology may have already occurred long before the clinical symptoms emerge. Pathophysiology in the early phase of these diseases is understudied but critical for developing mechanism-based treatment. In AD, increased seizure susceptibility and silent epileptiform activity due to disrupted excitatory/inhibitory (E/I) balance has been identified much earlier than cognition deficit. Increased epileptiform activity is likely a main pathology in the early phase that directly contributes to impaired cognition. It is an enormous challenge to model the early phase of pathology with conventional AD mouse models due to the chronic disease course, let alone the complex interplay between subclinical nonconvulsive epileptiform activity, AD pathology, and cognition deficit. We have extensively studied GE, especially with gene mutations that affect the GABA pathway such as mutations in GABAA receptors and GABA transporter 1. We believe that some mouse models developed for studying GE and insights gained from GE could provide unique opportunity to understand AD. These include the pathology in early phase of AD, endoplasmic reticulum (ER) stress, and E/I imbalance as well as the contribution to cognitive deficit. In this review, we will focus on the overlapping mechanisms between GE and AD, the insights from mutations affecting GABAA receptors, and GABA transporter 1. We will detail mechanisms of E/I imbalance and the toxic epileptiform generation in AD, and the complex interplay between ER stress, impaired membrane protein trafficking, and synaptic physiology in both GE and AD.

Investigation of the Mechanisms of Neuroprotection Mediated by Lobelia Species via Computational Network Pharmacology and Molecular Modeling

BackgroundSeveral species of the medicinally valuable genus Lobelia (Campanulaceae) exhibit neuroprotection. While the neuroprotective mechanisms of some components (e.g. lobeline, lobelanine, and lobelanidine) belonging to the L. nicotianaefolia or L. inflata are extensively characterized, there remains the need to study and elucidate the mechanism of action of other species and their active components. In this work, we have studied the neuroprotective mechanism of the pharmacokinetically favorable active compounds of 17 Lobelia species.MethodsNetwork pharmacology approach and molecular modeling were employed. We have conducted drug-likeness evaluation, oral bioavailability prediction followed by the Gene Ontology (GO) terms and pathways enrichment analysis, protein-protein and protein-compound interaction network construction and analysis, and molecular docking studies. Five neurodegenerative diseases viz. Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, epilepsy, and Amyotrophic lateral sclerosis along with the common neuroprotection mechanism-associated genes were evaluated.ResultsWe revealed the neuroprotective mechanism of the active ingredients of Lobelia species. Our study strongly indicates that 12 unique active ingredients viz. luteolin, kaempferol, acacetin, chryseriol, norlobelanine, lobelanine, 2-[(2R,6S)-6-[(2R)-2-hydroxy-2-phenylethyl]-1-methylpiperidin-2-yl]-1-phenylethanone, hydroxygenkwanin, lobelanidine, quercetin, and diosmetin regulates 31 targets within multiple signaling pathways. The nitric oxide synthase, brain (NOS1), androgen receptor (ANDR), sodium- and chloride-dependent GABA transporter 1 (SC6A1), apoptosis regulator Bcl-2 (BCL2), RAC-alpha serine/threonine-protein kinase (AKT1), cellular tumor antigen p53, apoptosis regulator BAX, and tumor necrosis factor (TNFA) were identified as the majorly regulated genes. A majority of these target proteins act via several cancer-related pathways proven to have cross-talks with the pathogenesis of neurodegenerative diseases.ConclusionsThis study explains how the active ingredients of the Lobelia species exhibit their neuroprotective actions and provide a reference basis to investigate their pharmacological effects in detail.

A Proline Derivative-Enriched Fraction from Sideroxylon obtusifolium Protects the Hippocampus from Intracerebroventricular Pilocarpine-Induced Injury Associated with Status Epilepticus in Mice

The N-methyl-(2S,4R)-trans-4-hydroxy-l-proline-enriched fraction (NMP) from Sideroxylon obtusifolium was evaluated as a neuroprotective agent in the intracerebroventricular (icv) pilocarpine (Pilo) model. To this aim, male mice were subdivided into sham (SO, vehicle), Pilo (300 µg/1 µL icv, followed by the vehicle per os, po) and NMP-treated groups (Pilo 300 µg/1 µL icv, followed by 100 or 200 mg/kg po). The treatments started one day after the Pilo injection and continued for 15 days. The effects of NMP were assessed by characterizing the preservation of cognitive function in both the Y-maze and object recognition tests. The hippocampal cell viability was evaluated by Nissl staining. Additional markers of damage were studied—the glial fibrillary acidic protein (GFAP) and the ionized calcium-binding adaptor molecule 1 (Iba-1) expression using, respectively, immunofluorescence and western blot analyses. We also performed molecular docking experiments revealing that NMP binds to the γ-aminobutyric acid (GABA) transporter 1 (GAT1). GAT1 expression in the hippocampus was also characterized. Pilo induced cognitive deficits, cell damage, increased GFAP, Iba-1, and GAT1 expression in the hippocampus. These alterations were prevented, especially by the higher NMP dose. These data highlight NMP as a promising candidate for the protection of the hippocampus, as shown by the icv Pilo model.

DISTRIBUTION OF GABA TRANSPORTER (GAT1) LEVELS IN THE BÖTZINGER COMPLEX AT THE EARLY STAGES OF POSTNATAL DEVELOPMENT IN RATS WITH PRENATAL SEROTONIN DEFICIENCY

Цель работы - исследование распределения уровня GAT-транспортера ГАМК в комплексе Бетцингера на разных сроках раннего постнатального развития крыс в норме и при пренатальном дефиците серотонина. Материал и методы. Работа проведена на лабораторных крысах линии Wistar. Снижение уровня эндогенного серотонина в эмбриональный период осуществляли методом ингибирования триптофан-гидроксилазы пара-хлорфенилаланином (пХФА). Выявление транспортного белка GAТпроводили посредством иммуногистохимической реакции с использованием первичных кроличьих поликлональных антител anti-GABA transporter1 (AbCam, Великобритания). Мозг исследовали на 5-, 10-е и 20-е сутки постнатального развития. Результаты. В комплексе Бетцингера на ранних сроках постнатального развития у контрольных животных отмечено колебание уровня GAT-транспортера ГАМК. На 1-й неделе жизни уровень GATбыл высоким как в сети отростков и терминалей, так и в синапсах. В течение 2-й недели жизни уровень GATснижался, а к концу 3-й недели - повышался вновь, достигая исходного уровня. Дефицит серотонина в пренатальный период вызывал у подопытных животных существенное увеличение уровня GATв нейропиле комплекса Бетцингера на всех изученных сроках постнатального развития. Выводы. Пренатальный дефицит серотонина приводит к существенному повышению уровня GAT-транспортера ГАМК в ранние сроки постнатального развития, что может приводить к изменению трансмиссии ГАМК и, как следствие, к нарушению баланса тормозных и возбуждающих эффектов в дыхательном ядре. Objective - to study the distribution of GABA transporter 1 (GAT) levels in the Bötzinger complex at the early stages of postnatal development in rats with prenatal serotonin deficiency. Materials and methods. The work was carried out on Wistar line laboratory rats. To reduce the level of endogenous serotonin in the embryonic period, the method of tryptophan hydroxylase inhibition by para-chlorophenylalanine (PCPA) (Sigma, USA) was used. The GAT1 transport protein was detected by immunohistochemical reaction with anti-GABA transporter1 primary rabbit polyclonal antibodies (AbCam, UK). The brain was examined on the 5, 10 and 20 day of postnatal development. Results. At the early stages of postnatal development, a fluctuation in the GAT1 level of the GABA transporter was noted in the Bötzinger complex of control animals. In the first postnatal week, the GAT level was high both in the network of neuronal processes and terminals, and in synapses. During the 2 week of life, the GAT1 level decreased, and by the end of the 3 week it increased again, reaching the initial level. Deficiency of serotonin in the prenatal period caused a significant increase in the level of GAT in the neuropil of the Bötzinger complex in experimental animals at all studied stages of postnatal development. Conclusions. Prenatal deficiency of serotonin leads to a significant increase in the GAT1 level at the early stages of postnatal development, which can lead to a change in the GABA transmission, and, as a result, to a disturbance in the balance of inhibitory and stimulatory effects in the respiratory nuclei.