Non-selective Ligands Research Articles

Antagonism of the brain P2X7 ion channel attenuates repeated social defeat induced microglia reactivity, monocyte recruitment and anxiety-like behavior in male mice

Chronic stress is linked to increased anxiety. Repeated social defeat (RSD) in mice causes anxiety that is dependent on activated neurons, reactive microglia, and accumulation of monocytes in the brain. This response requires interactions between the immune system and central nervous system (CNS). Neuronal activation within threat appraisal regions is a key response to RSD, however, it is unclear how microglia become activated. One potential explanation is that microglia express a purinergic non-selective ligand gated adenosine-triphosphate (ATP) receptor 7 (P2X7). Activation of P2X7 promotes the release of chemokines and cytokines, and recruitment of monocytes to the brain. Thus, the purpose of this study was to determine if a novel P2X7 antagonist blocked neuronal and microglia interactions and the corresponding anxiety following RSD. Male mice were administered (i.p.) a P2X7 antagonist, JNJ-54471300, prior to each cycle of RSD. Fourteen hours after RSD, behavioral deficits including social avoidance and anxiety-like were determined. Moreover, several immune parameters were assessed. RSD caused neuronal activation in stress-responsive regions, monocyte production and release, splenomegaly, and social avoidance. These parameters were unaffected by P2X7 antagonism. RSD-associated proportional area of Iba-1+ microglia, monocyte accumulation in the brain, IL-1β mRNA expression in enriched myeloid cells, plasma IL-6, and anxiety-like behavior were ameliorated by P2X7 antagonism. Gene expression analysis in the hippocampus and amygdala showed regional specific responses to RSD and some were reversed with P2X7 antagonism. Overall, blocking P2X7 activation attenuated RSD-induced microglia reactivity with corresponding reduction in neuroinflammation, monocyte accumulation, and anxiety-like behavior in male mice.

Platinum(IV)-based prodrugs as an alternative to Pt(II)-based drugs: synthesis and biological action

The chemotherapy with cisplatin and its analogues widely used in medical practice is associated with undesirable side effects caused by non-selective ligand exchange and binding of the complexes to various biomolecules in the body. An alernative to classical platinum(II)-based drugs are platinum(IV) prodrugs, that is, platinum(II) complexes additionally modified with diverse biologically active axial ligands, including known pharmaceutical products. In recent years, quite a few studies devoted to the design of effective Pt(IV) prodrugs have been published, with some of the developed agents being markedly superior to clinically used cisplatin and carboplatin in therapeutic efficacy. This review summarizes the synthetic approaches to the design of Pt(IV) prodrugs and modification of the axial ligands. The second part of the review is devoted to the biological activity of Pt(IV) prodrugs reported in the period from 2018 to 2023 and comparison of various approaches to the design of effective anticancer agents based on these compounds. The bibliography includes 239 references.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Structures of the σ2 receptor enable docking for bioactive ligand discovery.

The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probesthrough structure-based screens of ultra large libraries, enabling study ofunderexplored areas of biology.

Tolerance and dependence following chronic alprazolam treatment in rhesus monkeys: Role of GABAA receptor subtypes

BackgroundTo assess GABAA receptor subtypes involved in benzodiazepine tolerance and dependence, we evaluated the ability of subtype-selective and non-selective ligands to substitute for (i.e., produce “cross-tolerance”) or precipitate withdrawal during chronic alprazolam treatment. MethodsFour female rhesus monkeys (Macaca mulatta) were implanted with chronic intravenous catheters and administered alprazolam (1.0 mg/kg every 4 h). Following 14+ days of chronic alprazolam, acute administration of selected doses of non-selective and subtype-selective ligands were substituted for, or administered with, alprazolam, followed by quantitative behavioral observations. The ligands included alprazolam and midazolam (positive modulators, non-selective), zolpidem (positive modulator, preferential affinity for α1-containing GABAA receptors), HZ-166 (positive modulator, preferential efficacy at α2- and α3-containing GABAA receptors), and βCCT (antagonist, preferential affinity for α1-containing GABAA receptors). ResultsAcutely, alprazolam and midazolam both induced observable ataxia along with a mild form of sedation referred to as “rest/sleep posture” at a lower dose (0.1 mg/kg, i.v.), whereas at a higher dose (1.0 mg/kg, i.v.), induced deep sedation and observable ataxia. With chronic alprazolam treatment, observable ataxia and deep sedation were reduced significantly, whereas rest/sleep posture was unchanged or emerged. Zolpidem showed a similar pattern of effects, whereas no behaviors engendered by HZ-166 were changed by chronic alprazolam. Administration of βCCT, but not HZ-166, resulted in significant withdrawal signs. ConclusionsThese results are consistent with a role for α1-containing GABAA receptor subtypes in tolerance and dependence observed with chronic alprazolam, although other receptors may be involved in the withdrawal syndrome.

Development of template systems for ligand interactions of CYP3A5 and CYP3A7 and their distinctions from CYP3A4 template

Starting from established CYP3A4 Template (DMPK. 2019, and 2020), CYP3A5 and CYP3A7 Templates have been constructed to be reliable tools for verification of their distinct catalytic properties. A distinct occupancy was observed on CYP3A4-selective ligands, but not on the non-selective ligands, in simulation experiments. These ligands often invade into Bay-1 region during the migration from Entrance to Site of oxidation in simulation experiments. These results offered an idea of the distinct localization of Bay-1 residue on CYP3A5 Template, in which the Bay-1 residue stayed closely to Template border. The idea also accounted for the higher oxidation rates of CYP3A5, than of CYP3A4, of noscapine and schisantherin E through their enhanced sitting-stabilization. Typical CYP3A7 substrates such as zonisamide and retinoic acids took their placements without occupying a left side region of Template for their metabolisms. In turn, the occupancies of the left-side region were inevitably observed among poor ligands of CYP3A7. Altered extent of IJK-Interaction or localization of a specific residue at the left-side would thus explain distinct catalytic properties of CYP3A7 on Template. These data suggest the alteration of each one of Template region, from CYP3A4 Template, led to the distinct catalytic properties of CYP3A5 and CYP3A7 forms.

Abstract 5687: Selective, chemically-induced degradation of BRM (SMARCA2) enables in vivo efficacy in BRG1 (SMARCA4)-deficient xenograft tumor models

Abstract The mammalian SWI/SNF complex catalyzes the remodeling of chromatin through the helicase activity of two mutually-exclusive, paralogous subunits, BRG1 and BRM. BRG1 is frequently mutated in cancer and its inactivation results in a cellular dependence on BRM. Despite the attractiveness of BRM as a synthetic lethal therapeutic target, the selective inhibition of BRM represents a considerable challenge due to the high degree of homology between BRM and BRG1. Furthermore, published data indicate that achieving such selectivity is likely essential to afford an acceptable therapeutic index. We sought to mimic the synthetic lethality observed in BRG1-mutant cancers by identifying proteolysis-targeting chimera (PROTAC®) molecules capable of selectively degrading BRM via trimeric complex formation with the von Hippel-Lindau (VHL) E3 ligase. In this disclosure, we report our initial discovery of potent and selective chimeric BRM-degrader molecules which exhibit BRM DC50 values <1 nM and BRG1/BRM DC50 ratios >25. Importantly, selective BRM degradation can be achieved in the absence of selective PROTAC® binding (BRG1/BRM Kd ratios <2). Global ubiquitin mapping and proteome profiling reveal no unexpected off-target activity of the selective BRM PROTAC® degraders. Treatment of a panel of NSCLC cell lines with a representative degrader molecule resulted in enhanced growth inhibition in BRG1-mutant relative to BRG1-wild-type cell lines. We also demonstrate that intermittent intravenous administration of an optimized BRM PROTAC® degrader exhibited strong in vivo modulation of pharmacodynamic biomarkers and afforded tumor growth inhibition in several BRG1-mutant xenograft models. Our study thus highlights the ability to transform a non-selective BRM-binding ligand into a selective and efficacious in vivo BRM PROTAC® degrader. Citation Format: Michael Berlin, Jennifer Cantley, Jing Wang, Mark Bookbinder, Gregory Cadelina, Emily Chan, Huifen Chen, Xin Chen, Kim Davenport, Tharu Fernando, Debbie Gordon, Brian Hamman, Roy Haskell, Alexey Ishchenko, Donald S. Kirkpatrick, Jonathan Maher, Mark Merchant, John Moffat, Alicia Morgan, An Nguyen, Jennifer Pizzano, Connor Quinn, Christopher M. Rose, Emma Rousseau, Vijay Sethuraman, Leanna Staben, Catherine Wilson, Xiaofen Ye, Fabio Broccatelli, Robert Yauch, Peter S. Dragovich. Selective, chemically-induced degradation of BRM (SMARCA2) enables in vivo efficacy in BRG1 (SMARCA4)-deficient xenograft tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5687.

Virtual Screening of C. Sativa Constituents for the Identification of Selective Ligands for Cannabinoid Receptor 2.

The selective targeting of the cannabinoid receptor 2 (CB2) is crucial for the development of peripheral system-acting cannabinoid analgesics. This work aimed at computer-assisted identification of prospective CB2-selective compounds among the constituents of Cannabis Sativa. The molecular structures and corresponding binding affinities to CB1 and CB2 receptors were collected from ChEMBL. The molecular structures of Cannabis Sativa constituents were collected from a phytochemical database. The collected records were curated and applied for the development of quantitative structure-activity relationship (QSAR) models with a machine learning approach. The validated models predicted the affinities of Cannabis Sativa constituents. Four structures of CB2 were acquired from the Protein Data Bank (PDB) and the discriminatory ability of CB2-selective ligands and two sets of decoys were tested. We succeeded in developing the QSAR model by achieving Q2 5-CV > 0.62. The QSAR models helped to identify three prospective CB2-selective molecules that are dissimilar to already tested compounds. In a complementary structure-based virtual screening study that used available PDB structures of CB2, the agonist-bound, Cryogenic Electron Microscopy structure of CB2 showed the best statistical performance in discriminating between CB2-active and non-active ligands. The same structure also performed best in discriminating between CB2-selective ligands from non-selective ligands.

Short-term outcomes of mirogabalin in patients with peripheral neuropathic pain: a retrospective study

BackgroundMirogabalin, which is approved for the treatment of peripheral neuropathic pain in Japan, is a ligand for the α2δ subunit of voltage-gated calcium channels. Both pregabalin and mirogabalin act as nonselective ligands at the α2δ-1 and α2δ-2 subunits. Mirogabalin has a unique binding profile and long duration of action. Pregabalin has been reported to produce intolerable adverse effects in some patients. This study investigated outcomes associated with mirogabalin administration in patients with peripheral neuropathic pain who ceased treatment with pregabalin.MethodsWe retrospectively assessed peripheral neuropathic pain using the neuropathic pain screening questionnaire (NeP score) in 187 patients (58 men, 129 women) who were treated with mirogabalin. All patients had switched from pregabalin to mirogabalin due to lack of efficacy or adverse events. Differences in the treatment course (i.e., numeric rating scale (NRS) scores) were compared using one-way analysis of variance with Bonferroni post hoc tests.ResultsThe mean age of the patients was 72.3 years (range, 30–94 years), and the mean duration of disease was 37 months (range, 3–252 months). After treatment with mirogabalin for 1 week, NRS scores significantly decreased compared with baseline and continued to decrease over time. After 8 weeks, NRS scores improved by ≥ 30% from baseline in 113 patients (69.3%). Twenty-four patients (12.8%) stopped mirogabalin treatment due to adverse events. Somnolence (26.7%), dizziness (12.3%), edema (5.9%), and weight gain (0.5%) were noted as adverse events of mirogabalin.ConclusionsThe results of this investigation indicate that mirogabalin is safe and effective for reducing peripheral neuropathic pain.

ПРОГНОСТИЧЕСКАЯ МОДЕЛЬ РАННЕЙ ДИАГНОСТИКИ ПРОГРЕССИРОВАНИЯ ХРОНИЧЕСКОЙ ИШЕМИИ МОЗГА

Цель – определить диагностическую значимость функциональной активности рецепторов тромбоцитов при прогрессировании хронической ишемии мозга и разработать модель прогнозирования риска развития 3-й стадии заболевания. Исследование носило проспективный характер и включало 107 пациентов с клиническими и нейро-визуализационными признаками ХИМ. Обследование проводилось до начала лечения и включало клинико-неврологическое и нейропсихологическое исследование; ультразвуковую допплерографию брахиоцефальных артерий, компьютерную и магнитно-резонансную томографию головного мозга. Для изучения функциональной активности рецепторов тромбоцитов использовали агонисты рецепторов, участвующих в патогенезе ХИМ, в частности АДФ (лиганд пуриновых Р2Y-рецепторов), ангиотензин-2 (лиганд АТ1-рецептора), фактор активации тромбоцитов (лиганд ФАТ-рецептора), адреналин (неселективный лиганд α2-адренорецепторов) коллаген IV типа (лигпанд GPVI-рецептора). Оценку агрегации тромбоцитов проводили турбидиметрическим методом на анализаторе ChronoLog (США) Для анализа связи стадий ХИМ со значением активности рецепторов тромбоцитов использованы методы парного корреляционного анализа и построения моделей линейной регрессии. Выявлены 7 значимых факторов риска, тесно связанных со стадией ХИМ. В наибольшей степени риск 3-й стадии хронической ишемии мозга был связан с уровнем АДФ-индуцированной агрегации тромбоцитов (активностью Р2Y-рецептора). Площадь под кривой операционных характеристик для модели, рассчитанной на основании значений данного индикатора, достигала 0,968 (AUC=0,968; 95% ДИ 0,92-0,99), что свидетельствует о высокой диагностической значимости предлагаемого теста. Для выбранного критического порога АДФ-индуцированной агрегации тромбоцитов чувствительность предлагаемого диагностического теста составляет 88,8% (95% ДИ 75,9-96,3) и специфичность – 91,9% (95% ДИ 82,2-97,3). Выявление лиц высокого риска прогрессирования ХИМ на более ранней стадии с использованием модели может привести к улучшению профилактических и терапевтических методов лечения пациентов с цереброваскулярной патологией.

Therapeutic senescence via GPCR activation in synovial fibroblasts facilitates resolution of arthritis

Rheumatoid arthritis affects individuals commonly during the most productive years of adulthood. Poor response rates and high costs associated with treatment mandate the search for new therapies. Here we show that targeting a specific G-protein coupled receptor promotes senescence in synovial fibroblasts, enabling amelioration of joint inflammation. Following activation of the melanocortin type 1 receptor (MC1), synovial fibroblasts acquire a senescence phenotype characterized by arrested proliferation, metabolic re-programming and marked gene alteration resembling the remodeling phase of wound healing, with increased matrix metalloproteinase expression and reduced collagen production. This biological response is attained by selective agonism of MC1, not shared by non-selective ligands, and dependent on downstream ERK1/2 phosphorylation. In vivo, activation of MC1 leads to anti-arthritic effects associated with induction of senescence in the synovial tissue and cartilage protection. Altogether, selective activation of MC1 is a viable strategy to induce cellular senescence, affording a distinct way to control joint inflammation and arthritis.

Signal Transduction and Pathogenic Modifications at the Melanocortin-4 Receptor: A Structural Perspective.

The melanocortin-4 receptor (MC4R) can be endogenously activated by binding of melanocyte-stimulating hormones (MSH), which mediates anorexigenic effects. In contrast, the agouti-related peptide (AgRP) acts as an endogenous inverse agonist and suppresses ligand-independent basal signaling activity (orexigenic effects). Binding of ligands to MC4R leads to the activation of different G-protein subtypes or arrestin and concomitant signaling pathways. This receptor is a key protein in the hypothalamic regulation of food intake and energy expenditure and naturally-occurring inactivating MC4R variants are the most frequent cause of monogenic obesity. In general, obesity is a growing problem on a global scale and is of social, medical, and economic relevance. A significant goal is to develop optimized pharmacological tools targeting MC4R without adverse effects. To date, this has not been achieved because of inter alia non-selective ligands across the five functionally different MCR subtypes (MC1-5R). This motivates further investigation of (i) the three-dimensional MC4R structure, (ii) binding mechanisms of various ligands, and (iii) the molecular transfer process of signal transduction, with the aim of understanding how structural features are linked with functional-physiological aspects. Unfortunately, experimentally elucidated structural information is not yet available for the MC receptors, a group of class A G-protein coupled receptors (GPCRs). We, therefore, generated MC4R homology models and complexes with interacting partners to describe approximate structural properties associated with signaling mechanisms. In addition, molecular insights from pathogenic mutations were incorporated to discriminate more precisely their individual malfunction of the signal transfer mechanism.

Enhancing Actinide(III) over Lanthanide(III) Selectivity through Hard-by-Soft Donor Substitution: Exploitation and Implication of Near-Degeneracy-Driven Covalency.

Soft donor ligands often provide higher selectivity for actinides(III) over chemically similar lanthanides(III), e.g., in the AmIII-EuIII pair. Frequently, the origin of such selectivity is associated with an increased covalency in actinide-ligand bonding. However, the relationship between the degree of covalency and ion selectivity has yet to reach general consensus. Further, it is unclear whether the enhanced covalency leads to a thermodynamic stabilization of the complex or not. Using relativistic density functional theory, we have addressed these outstanding issues by analyzing the subtle change of metal-ligand interactions from a hard donor ligand to a mixed soft-hard one. The present comparative study on the structure of and binding in Am3+ and Eu3+ complexes with 3,4,3-LI(1,2-HOPO) (L) and its mixed-donor variant (LS) shows that the introduction of sulfur as a soft donor atom into the metal coordination sphere indeed infuses an Am3+ selectivity into the otherwise nonselective ligand L but also leads to a significant reduction of the metal-binding Gibbs free energies. Natural population analysis, charge decomposition analysis, and its extended version point to the critical role of ligand-to-metal charge transfer in the overall thermodynamic stability of the complexes. A detailed energy decomposition analysis combining the extended transition state with the natural orbitals chemical valence method reveals an enhancement of the covalency upon switching to the soft-hard donor ligand because of the different nature of the metal-ligand interaction. The ligand L predominantly binds the metal via π donation, whereas the ligand LS prefers σ donation. Molecular orbital and quantum theory of atoms in molecules analyses as well as a comparison to a simple model system show that the covalency occurs as a result of orbital mixing and is near-degeneracy-driven in nature. This enhanced covalency, however, fails to thermodynamically compensate for the loss of strong electrostatic interaction and thus does not lead to an additional stabilization of the metal-LS complexes.

The Pharmacology of Bile Acids and Their Receptors.

This review provides a historical perspective of bile acids and their receptors as therapeutic targets. Bile acids are atypical steroids generated by the liver from cholesterol and have been used for almost half a century for treating liver and biliary disorders. Since the early 1970s of the last century, chenodeoxycholic acid (CDCA), a primary bile acid, and ursodeoxycholic acid (UDCA), a secondary bile acid and the 7βepimer of CDCA, have been shown effective in promoting the dissolution of cholesterol gallstones. However, lack of activity and side effects associated with the use of CDCA, along with the advent of laparoscopic cholecystectomy, have greatly reduced the clinical relevance of this application. At the turn of the century, however, the discovery that bile acids activate specific receptors, along with the discovery that those receptors are placed at the interface of the host and intestinal microbiota regulating physiologically relevant enterohepatic and entero-pancreatic axes, has led to a "bile acid renaissance." Similarly to other steroids, bile acids bind and activate both cell surface and nuclear receptors, including the bile acid sensor farnesoid X receptor (FXR) and a G-protein-coupled bile acid receptor, known as GPBAR1 (TGR5). Both receptors have been proved druggable, and several highly potent, selective, and nonselective ligands for the two receptors have been discovered in the last two decades. Currently, in addition to obeticholic acid, a semisynthetic derivative of CDCA and the first in class of FXR ligands approved for clinical use, either selective or dual FXR and GPBAR1 ligands, have been developed, and some of them are undergoing pre-approval trials. The effects of FXR and GPBAR1 ligands in different therapeutic area are reviewed.

3D similarities between the binding sites of monoaminergic target proteins.

The study of binding site similarities can be relevant to understand the interaction of different drugs at several molecular targets. The increasing availability of protein crystal structures and the development of novel algorithms designed to evaluate three-dimensional similarities, represent a great opportunity to explore the existence of electronic and shape features shared by clinically relevant proteins, which could assist drug design and discovery. Proteins involved in the recognition of monoaminergic neurotransmitters, such as monoamine transporters or monoamine oxidases (MAO) have been related to several psychiatric and neurological disorders such as depression or Parkinson’s disease. In this work, we evaluated the possible existence of similarities among the binding sites of the serotonin transporter (SERT), the dopamine transporter (DAT), MAO-A and MAO-B. This study was carried out using molecular simulation methodologies linked to the statistical algorithm PocketMatch, which was modified in order to obtain similarities profiles. Our results show that DAT and SERT exhibit a high degree of 3-D similarities all along the pathway that is presumably involved in the substrate transport process. Distinct differences, on the other hand, were found both at the extracellular and the intracellular ends of the transporters, which might be involved in the selective initial recognition of the corresponding substrate. Similarities were also found between the active (catalytic) site of MAO-A and the extracellular vestibule of SERT (the S2 binding site). These results suggest some degree of structural convergence for these proteins, which have different functions, tissue distribution and genetic origin, but which share the same endogenous ligand (serotonin). Beyond the functional implications, these findings are valuable for the design of both selective and non-selective ligands.

Binding of anticancer drug daunomycin to a TGGGGT G-quadruplex DNA probed by all-atom molecular dynamics simulations: additional pure groove binding mode and implications on designing more selective G-quadruplex ligands.

DNA G-quadruplex structures are emerging cancer-specific targets for chemotherapeutics. Ligands that bind to and stabilize DNA G-quadruplexes have the potential to be anti-cancer drugs. Lack of binding selectivity to DNA G-quadruplex over DNA duplex remains a major challenge when attempting to develop G-quadruplex ligands into successful anti-cancer drugs. Thorough understanding of the binding nature of existing non-selective ligands that bind to both DNA quadruplex and DNA duplex will help to address this challenge. Daunomycin and doxorubicin, two commonly used anticancer drugs, are examples of non-selective DNA ligands. In this study, we extended our early all-atom binding simulation studies between doxorubicin and a DNA duplex (d(CGATCG)2) to probe the binding between daunomycin and a parallel DNA quadruplex (d(TGGGGT)4) and DNA duplex. In addition to the end stacking mode, which mimics the mode in the crystal structure, a pure groove binding mode was observed in our free binding simulations. The dynamic and energetic properties of these two binding modes are thoroughly examined, and a detailed comparison is made between DNA quadruplex binding modes and DNA duplex binding modes. Implications on the design of more selective DNA quadruplex ligands are also discussed. Graphical abstract Top stacking and groov binding modes from the MD simulations.

The Search for a Subtype-Selective PET Imaging Agent for the GABAA Receptor Complex: Evaluation of the Radiotracer [11C]ADO in Nonhuman Primates.

The myriad physiological functions of γ-amino butyric acid (GABA) are mediated by the GABA-benzodiazepine receptor complex comprising of the GABAA, GABAB, and GABAC groups. The various GABAA subunits with region-specific distributions in the brain subserve different functional and physiological roles. For example, the sedative and anticonvulsive effects of classical benzodiazepines are attributed to the α1 subunit, and the α2 and α3 subunits mediate the anxiolytic effect. To optimize pharmacotherapies with improved efficacy and devoid of undesirable side effects for the treatment of anxiety disorders, subtype-selective imaging radiotracers are required to assess target engagement at GABA sites and determine the dose–receptor occupancy relationships. The goal of this work was to characterize, in nonhuman primates, the in vivo binding profile of a novel positron emission tomography (PET) radiotracer, [11C]ADO, which has been indicated to have functional selectivity for the GABAA α2/α3 subunits. High specific activity [11C]ADO was administrated to 3 rhesus monkeys, and PET scans of 120-minute duration were performed on the Focus-220 scanner. In the blood, [11C]ADO metabolized at a fairly rapid rate, with ∼36% of the parent tracer remaining at 30 minutes postinjection. Uptake levels of [11C]ADO in the brain were high (peak standardized uptake value of ∼3.0) and consistent with GABAA distribution, with highest activity levels in cortical areas, intermediate levels in cerebellum and thalamus, and lowest uptake in striatal regions and amygdala. Tissue kinetics was fast, with peak uptake in all brain regions within 20 minutes of tracer injection. The one-tissue compartment model provided good fits to regional time–activity curves and reliable measurement of kinetic parameters. The absolute test–retest variability of regional distribution volumes (V T) was low, ranging from 4.5% to 8.7%. Pretreatment with flumazenil (a subtype nonselective ligand, 0.2 mg/kg, intravenous [IV], n = 1), Ro15-4513 (an α5-selective ligand, 0.03 mg/kg, IV, n = 2), and zolpidem (an α1-selective ligand, 1.7 mg/kg, IV, n = 1) led to blockade of [11C]ADO binding by 96.5%, 52.5%, and 76.5%, respectively, indicating the in vivo binding specificity of the radiotracer. Using the nondisplaceable volume of distribution (V ND) determined from the blocking studies, specific binding signals, as measured by values of regional binding potential (BP ND), ranged from 0.6 to 4.4, which are comparable to those of [11C]flumazenil. In conclusion, [11C]ADO was demonstrated to be a specific radiotracer for the GABAA receptors with several favorable properties: high brain uptake, fast tissue kinetics, and high levels of specific binding in nonhuman primates. However, subtype selectivity in vivo is not obvious for the radiotracer, and thus, the search for subtype-selective GABAA radiotracers continues.

Evaluation of FAAH‐ and MGL‐Inhibition in Cannabinoid Drug Discrimination

The discriminative‐stimulus (SD) effects of CB1 ligands can be reproduced in mice by non‐selective inhibition of the anandamide‐(AEA‐) and 2‐arachidinoyl glycerol‐(2‐AG‐) inactivating enzymes, respectively, fatty‐acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MGL). Additionally, we have shown that nonselective FAAH/MGL inhibitors produce a CB1‐like SD effect in squirrel monkeys. The present studies with selective and nonselective ligands [FAAH (URB597; AM2018), MGL (AM2036); and FAAH/MGL‐mixed (AM2129)] were conducted to further evaluate the effects of FAAH and MGL inhibition in squirrel monkeys trained to discriminate the CB1 agonist AM4054 from vehicle (n=4). Results indicate that: 1) co‐administration of selective FAAH‐ and MGL‐inhibitors, but not FAAH‐ or MGL‐selective inhibitors alone, produces CB1‐related SD effects;2) AEA, but not 2‐AG, produces CB1‐like SD effects after either FAAH or mixed FAAH/MGL inhibition; and 3) CB1 receptor antagonism effectively and blocks the CB1‐like effects of combined FAAH/MGL inhibition; this effect was insurmountable. The present data strengthen the idea that complementary, but not individual, actions of the endocannabinoids AEA and 2‐AG produce CB1‐related SD, and perhaps subjective, effects in primate species.

Serotonergic system and its role in epilepsy and neuropathic pain treatment: a review based on receptor ligands.

The serotonergic system is involved in pathomechanisms of both epilepsy and neuropathic pain. So far, participation in the epileptogenesis and maintenance of epilepsy was proved for 5-HT1A, 5-HT2C, 5-HT3, 5-HT4 and 5-HT7 receptors as well as 5-HTT serotonin transporter. Depending on the receptor type or its localization, its stimulation may increase or decrease neuronal excitability. According to the available data, neuropathic pain mechanisms involve 5-HT1A/1B/1D, 5-HT2A/2B/2C, 5-HT3, 5-HT4, 5-HT6, 5-HT7 receptors and 5-HTT serotonin transporter. Changes in their expression modulate pain mainly by affecting the transmission through serotonergic descending pathways. Several compounds, whose mechanisms of action base on influence on the serotonergic system, are already in use. These are 5-HT3 agonists (triptans) in case of migraine, tricyclic antidepressants or monoamine reuptake inhibitors in neuropathic pain treatment. In addition, selective and non-selective ligands are tested for their anticonvulsant or analgesic properties. Some ED50 values have been already obtained in such animal models as maximal electroshock (MES)-induced seizures (epilepsy), spinal nerve ligation (SNL), chronic constriction injury (CCI) or formalin (neuropathic pain). This review shows that in case of drug discovery within the serotonergic system one must take into account special significance of factors such as: the species, the type of model, the route of administration, and the dose range.

Novel 5-HT5A receptor antagonists ameliorate scopolamine-induced working memory deficit in mice and reference memory impairment in aged rats

Despite the human 5-HT5A receptor being cloned in 1994, the biological function of this receptor has not been extensively characterized due to a lack of specific ligands. We recently reported that the selective 5-HT5A receptor antagonist ASP5736 ameliorated cognitive impairment in several animal models of schizophrenia. Given that areas of the brain with high levels of 5-HT5A receptor expression, such as the hippocampus and cerebral cortex, have important functions in cognition and memory, we evaluated the chemically diverse, potent and brain-penetrating 5-HT5A receptor antagonists ASP5736, AS2030680, and AS2674723 in rodent models of cognitive dysfunction associated with dementia. Each of these compounds exhibited a high affinity for recombinant 5-HT5A receptors that was comparable to that of the non-selective ligand of this receptor, lysergic acid diethylamide (LSD). Although each compound had a low affinity for other receptors, 5-HT5A was the only receptor for which all three compounds had a high affinity. Each of the three compounds ameliorated scopolamine-induced working memory deficit in mice and improved reference memory impairment in aged rats at similar doses. Further, ASP5736 decreased the binding of LSD to 5-HT5A receptors in the olfactory bulb of rats in a dose-dependent manner and occupied 15%–50% of brain 5-HT5A receptors at behaviorally effective doses. These results indicate that the 5-HT5A receptor is involved in learning and memory and that treatment with 5-HT5A receptor antagonists might be broadly effective for cognitive impairment associated with not only schizophrenia but also dementia.