RESUMO
BACKGROUND: A comprehensive understanding of the genetic basis of rare diseases and their regulatory mechanisms is essential for human molecular genetics. However, the genetic mutant spectrum of pathogenic genes within the Chinese population remains underrepresented. Here, we reported previously unreported functional ABHD12 variants in two Chinese families and explored the correlation between genetic polymorphisms and phenotypes linked to PHARC syndrome. METHODS: Participants with biallelic pathogenic ABHD12 variants were recruited from the Chinese Deafness Genetics Cohort. These participants underwent whole-genome sequencing. Subsequently, a comprehensive literature review was conducted. RESULTS: Two Han Chinese families were identified, one with a compound heterozygous variant and the other with a novel homozygous variant in ABHD12. Among 65 PHARC patients, including 62 from the literature and 3 from this study, approximately 90% (57 out of 63) exhibited hearing loss, 82% (50 out of 61) had cataracts, 82% (46 out of 56) presented with retinitis pigmentosa, 79% (42 out of 53) experienced polyneuropathy, and 63% (36 out of 57) displayed ataxia. Seventeen different patterns were observed in the five main phenotypes of PHARC syndrome. A total of 33 pathogenic variants were identified in the ABHD12. Compared with other genotypes, individuals with biallelic truncating variants showed a higher incidence of polyneuropathy (p = 0.006), but no statistically significant differences were observed in the incidence of hearing loss, ataxia, retinitis pigmentosa and cataracts. CONCLUSIONS: The diagnosis of PHARC syndrome is challenging because of its genetic heterogeneity. Therefore, exploring novel variants and establishing genotype-phenotype correlations can significantly enhance gene diagnosis and genetic counseling for this complex disease.
Assuntos
Ataxia , Catarata , Estudos de Associação Genética , Monoacilglicerol Lipases , Linhagem , Fenótipo , Polineuropatias , Retinose Pigmentar , Humanos , Masculino , Feminino , Ataxia/genética , Catarata/genética , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Polineuropatias/genética , Monoacilglicerol Lipases/genética , Mutação , Adulto , Criança , Adolescente , GenótipoRESUMO
Monoacylglycerol lipase (MAGL) is the key enzyme for the hydrolysis of endocannabinoid 2-arachidonoylglycerol (2-AG). The central role of MAGL in the metabolism of 2-AG makes it an attractive therapeutic target for a variety of disorders, including inflammation-induced tissue injury, pain, multiple sclerosis, and cancer. Previously, we reported LEI-515, an aryl sulfoxide, as a peripherally restricted, covalent reversible MAGL inhibitor that reduced neuropathic pain and inflammation in preclinical models. Here, we describe the structure-activity relationship (SAR) of aryl sulfoxides as MAGL inhibitors that led to the identification of LEI-515. Optimization of the potency of high-throughput screening (HTS) hit 1 yielded compound ±43. However, ±43 was not metabolically stable due to its ester moiety. Replacing the ester group with α-CF2 ketone led to the identification of compound ±73 (LEI-515) as a metabolically stable MAGL inhibitor with subnanomolar potency. LEI-515 is a promising compound to harness the therapeutic potential of MAGL inhibition.
Assuntos
Inibidores Enzimáticos , Monoacilglicerol Lipases , Sulfóxidos , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Relação Estrutura-Atividade , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Sulfóxidos/química , Sulfóxidos/farmacologia , Sulfóxidos/síntese química , Animais , Microssomos Hepáticos/metabolismo , Ensaios de Triagem em Larga EscalaRESUMO
The endocannabinoid system, known for its regulatory role in various physiological processes, relies on the activities of several hydrolytic enzymes, such as fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), monoacylglycerol lipase (MAGL), and α/ß-hydrolase domains 6 (ABHD6) and 12 (ABHD12), to maintain homeostasis. Accurate measurement of these enzymes' activities is crucial for understanding their function and for the development of potential therapeutic agents. Fluorometric assays, which offer high sensitivity, specificity, and real-time monitoring capabilities, have become essential tools in enzymatic studies. This review provides a comprehensive overview of the principles behind these assays, the various substrates and fluorophores used, and advances in assay techniques used not only for the determination of the kinetic mechanisms of enzyme reactions but also for setting up kinetic assays for the high-throughput screening of each critical enzyme involved in endocannabinoid degradation. Through this comprehensive review, we aim to highlight the strengths and limitations of current fluorometric assays and suggest future directions for improving the measurement of enzyme activity in the endocannabinoid system.
Assuntos
Amidoidrolases , Endocanabinoides , Ensaios Enzimáticos , Endocanabinoides/metabolismo , Humanos , Ensaios Enzimáticos/métodos , Amidoidrolases/metabolismo , Amidoidrolases/antagonistas & inibidores , Hidrólise , Monoacilglicerol Lipases/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Animais , Fluorometria/métodos , Fluorescência , Cinética , Corantes Fluorescentes/química , Inibidores Enzimáticos/farmacologiaRESUMO
We present a new computational approach, named Watermelon, designed for the development of pharmacophore models based on receptor structures. The methodology involves the sampling of potential hotspots for ligand interactions within a protein target's binding site, utilising molecular fragments as probes. By employing docking and molecular dynamics (MD) simulations, the most significant interactions formed by these probes within distinct regions of the binding site are identified. These interactions are subsequently transformed into pharmacophore features that delineates key anchoring sites for potential ligands. The reliability of the approach was experimentally validated using the monoacylglycerol lipase (MAGL) enzyme. The generated pharmacophore model captured features representing ligand-MAGL interactions observed in various X-ray co-crystal structures and was employed to screen a database of commercially available compounds, in combination with consensus docking and MD simulations. The screening successfully identified two new MAGL inhibitors with micromolar potency, thus confirming the reliability of the Watermelon approach.
Assuntos
Inibidores Enzimáticos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Estrutura Molecular , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Monoacilglicerol Lipases/química , Ligantes , Relação Estrutura-Atividade , Simulação de Dinâmica Molecular , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Citrullus/químicaRESUMO
Background: Human immunodeficiency virus (HIV) affects nearly 40 million people globally, with roughly 80% of all people living with HIV receiving antiretroviral therapy. Antiretroviral treatment suppresses viral load in peripheral tissues but does not effectively penetrate the blood-brain barrier. Thus, viral reservoirs persist in the central nervous system and continue to produce low levels of inflammatory factors and early viral proteins, including the transactivator of transcription (Tat). HIV Tat is known to contribute to chronic neuroinflammation and synaptodendritic damage, which is associated with the development of cognitive, motor, and/or mood problems, collectively known as HIV-associated neurocognitive disorders (HAND). Cannabinoid anti-inflammatory effects are well documented, but therapeutic utility of cannabis remains limited due to its psychotropic effects, including alterations within brain regions encoding reward processing and motivation, such as the nucleus accumbens. Alternatively, inhibiting monoacylglycerol lipase (MAGL) has demonstrated therapeutic potential through interactions with the endocannabinoid system. Methods: The present study utilized a reward-related operant behavioral task to quantify motivated behavior in female Tat transgenic mice treated with vehicle or MAGL inhibitor MJN110 (1 mg/kg). Brain tissue was collected to assess dendritic injury and neuroinflammatory profiles, including dendritic microtubule-associated protein (MAP2ab) intensity, microglia density, microglia morphology, astrocyte density, astrocytic interleukin-1ß (IL-1ß) colocalization, and various lipid mediators. Results: No significant behavioral differences were observed; however, MJN110 protected against Tat-induced dendritic injury by significantly upregulating MAP2ab intensity in the nucleus accumbens and in the infralimbic cortex of Tat(+) mice. No or only minor effects were noted for Iba-1+ microglia density and/or microglia morphology. Further, Tat increased GFAP+ astrocyte density in the infralimbic cortex and GFAP+ astrocytic IL-1ß colocalization in the nucleus accumbens, with MJN110 significantly reducing these measures in Tat(+) subjects. Lastly, selected HETE-related inflammatory lipid mediators in the striatum were downregulated by chronic MJN110 treatment. Conclusions: These findings demonstrate anti-inflammatory and neuroprotective properties of MJN110 without cannabimimetic behavioral effects and suggest a promising alternative to cannabis for managing neuroinflammation.
Assuntos
HIV-1 , Monoacilglicerol Lipases , Doenças Neuroinflamatórias , Animais , Feminino , Humanos , Camundongos , Complexo AIDS Demência/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/virologia , Encéfalo/patologia , Modelos Animais de Doenças , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/etiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Over the past decade, the idea of targeting the endocannabinoid system to treat anxiety disorders has received increasing attention. Previous studies focused more on developing cannabinoid receptor agonists or supplementing exogenous cannabinoids, which are prone to various adverse effects due to their strong pharmacological activity and poor receptor selectivity, limiting their application in clinical research. Endocannabinoid hydrolase inhibitors are considered to be the most promising development strategies for the treatment of anxiety disorders. More recent efforts have emphasized that inhibition of two major endogenous cannabinoid hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), indirectly activates cannabinoid receptors by increasing endogenous cannabinoid levels in the synaptic gap, circumventing receptor desensitization resulting from direct enhancement of endogenous cannabinoid signaling. In this review, we comprehensively summarize the anxiolytic effects of MAGL and FAAH inhibitors and their potential pharmacological mechanisms, highlight reported novel inhibitors or natural products, and provide an outlook on future directions in this field.
Assuntos
Amidoidrolases , Ansiolíticos , Endocanabinoides , Inibidores Enzimáticos , Monoacilglicerol Lipases , Humanos , Ansiolíticos/farmacologia , Ansiolíticos/química , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Animais , Endocanabinoides/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Transtornos de Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/metabolismoRESUMO
BACKGROUND: Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol. RESULTS: In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect. CONCLUSIONS: In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.
Assuntos
Gossypium , Monoacilglicerol Lipases , Gossypium/genética , Gossypium/enzimologia , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Alelos , Família Multigênica , Estudo de Associação Genômica Ampla , Genoma de Planta , Variação Genética , Filogenia , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , HaplótiposRESUMO
Retinitis pigmentosa (RP) is an inherited retinal dystrophy caused by the loss of photoreceptors and retinal pigment epithelial atrophy, leading to severe visual impairment or blindness. RP can be classified as nonsyndromic or syndromic with complex clinical phenotypes. Three unrelated Polish probands affected with retinitis pigmentosa coexisting with cerebellar ataxia were recruited for this study. Clinical heterogeneity and delayed appearance of typical disease symptoms significantly prolonged the patients' diagnostic process. Therefore, many clinical and genetic tests have been performed in the past. Here, we provide detailed clinical and genetic analysis results of the patients. Whole-exome sequencing (WES) and targeted NGS analysis allow the identification of four novel and two previously reported variants in the following genes: ABHD12, FLVCR1, and PNPLA6. The use of next-generation sequencing (NGS) methods finally allowed for confirmation of the clinical diagnosis. Ultra-rare diseases such as PHARC, PCARP, and Oliver-McFarlane syndromes were diagnosed in patients, respectively. Our findings confirmed the importance of the application of next-generation sequencing methods, especially in ultra-rare genetic disorders with overlapping features.
Assuntos
Sequenciamento do Exoma , Retinose Pigmentar , Humanos , Retinose Pigmentar/genética , Retinose Pigmentar/diagnóstico , Masculino , Feminino , Linhagem , Sequenciamento de Nucleotídeos em Larga Escala , Adulto , Ataxia Cerebelar/genética , Ataxia Cerebelar/diagnóstico , Proteínas de Membrana Transportadoras/genética , Monoacilglicerol Lipases/genética , Mutação , Ataxia/genética , Ataxia/diagnóstico , Fenótipo , Aciltransferases , Catarata , Fosfolipases , PolineuropatiasRESUMO
The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.
Assuntos
Amidoidrolases , Peptídeo Relacionado com Gene de Calcitonina , Hiperalgesia , Gânglio Trigeminal , Animais , Masculino , Hiperalgesia/tratamento farmacológico , Ratos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Amidoidrolases/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/sangue , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Ratos Sprague-Dawley , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Endocanabinoides/metabolismo , Nitroglicerina/farmacologia , Modelos Animais de Doenças , Citocinas/metabolismo , Citocinas/sangue , Transtornos de Enxaqueca/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Oligopeptídeos , Proteínas e Peptídeos SalivaresRESUMO
BACKGROUND AND PURPOSE: Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall. EXPERIMENTAL APPROACH: 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRABeCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. KEY RESULTS: 2-AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant-GRABeCB2.0. ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase ß activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/ß-hydrolase domain containing 6 (ABHD6). CONCLUSIONS AND IMPLICATIONS: Considering that P2X7R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.
Assuntos
Ácidos Araquidônicos , Endocanabinoides , Glicerídeos , Neurônios , Receptores Purinérgicos P2X7 , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Ácidos Araquidônicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animais , Camundongos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Monoacilglicerol Lipases/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Receptor CB1 de Canabinoide/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Linhagem Celular TumoralRESUMO
The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.
Assuntos
Ácidos Araquidônicos , Encefalomielite Autoimune Experimental , Endocanabinoides , Glicerídeos , Camundongos Endogâmicos C57BL , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Glicerídeos/metabolismo , Camundongos , Endocanabinoides/metabolismo , Ácidos Araquidônicos/farmacologia , Ácidos Araquidônicos/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/patologia , Sinapses/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismoRESUMO
Biological studies on the endocannabinoid system (ECS) have suggested that monoacylglycerol lipase (MAGL), an essential enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), is a novel target for developing antidepressants. A decrease of 2-AG levels in the hippocampus of the brain has been observed in depressive-like models induced by chronic stress. Herein, employing a structure-based approach, we designed and synthesized a new class of (piperazine-1-carbonyl) quinolin-2(1H)-one derivatives as potent, reversible and selective MAGL inhibitors. And detailed structure-activity relationships (SAR) studies were discussed. Compound 27 (IC50 = 10.3 nM) exhibited high bioavailability (92.7%) and 2-AG elevation effect in vivo. Additionally, compound 27 exerted rapid antidepressant effects caused by chronic restraint stress (CRS) and didn't show signs of addictive properties in the conditioned place preference (CPP) assays. Our study is the first to report that reversible MAGL inhibitors can treat chronic stress-induced depression effectively, which may provide a new potential therapeutic strategy for the discovery of an original class of safe, rapid antidepressant drugs.
Assuntos
Inibidores Enzimáticos , Monoacilglicerol Lipases , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Monoacilglicerol Lipases/metabolismo , Depressão/tratamento farmacológico , Monoglicerídeos , Relação Estrutura-Atividade , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , EndocanabinoidesRESUMO
AIMS: Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). METHODS: Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. RESULTS: In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased, and bladder MAGL was decreased after MAGL inhibitor treatment. CONCLUSIONS: MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.
Assuntos
Camundongos Endogâmicos C57BL , Monoacilglicerol Lipases , Traumatismos da Medula Espinal , Bexiga Urinária , Urodinâmica , Animais , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/metabolismo , Feminino , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiopatologia , Urodinâmica/efeitos dos fármacos , Camundongos , Modelos Animais de Doenças , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Receptores de Canabinoides/metabolismo , Receptores de Canabinoides/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Endocanabinoides/metabolismo , Citocinas/metabolismo , Bexiga Urinaria Neurogênica/tratamento farmacológico , Bexiga Urinaria Neurogênica/fisiopatologia , Bexiga Urinaria Neurogênica/etiologia , Sintomas do Trato Urinário Inferior/tratamento farmacológico , Sintomas do Trato Urinário Inferior/fisiopatologia , Sintomas do Trato Urinário Inferior/etiologia , Carbamatos , SuccinimidasRESUMO
Intervertebral disc degeneration (IVDD) stands as the primary cause of low back pain (LBP). A significant contributor to IVDD is nucleus pulposus cell (NPC) senescence. However, the precise mechanisms underlying NPC senescence remain unclear. Monoacylglycerol lipase (MAGL) serves as the primary enzyme responsible for the hydrolysis of 2-arachidonoylglycerol (2-AG), breaking down monoglycerides into glycerol and fatty acids. It plays a crucial role in various pathological processes, including pain, inflammation, and oxidative stress. In this study, we utilized a lipopolysaccharide (LPS)-induced NPC senescence model and a rat acupuncture-induced IVDD model to investigate the role of MAGL in IVDD both in vitro and in vivo. Initially, our results showed that MAGL expression was increased 2.41-fold and 1.52-fold within NP tissues from IVDD patients and rats induced with acupuncture, respectively. This increase in MAGL expression was accompanied by elevated expression of p16INK4α. Following this, it was noted that the suppression of MAGL resulted in a notable decrease in the quantity of SA-ß-gal-positive cells and hindered the manifestation of p16INK4α and the inflammatory factor IL-1ß in NPCs. MAGL inhibition promotes type II collagen (Col-2) expression and inhibits matrix metalloproteinase 13 (MMP13), thereby restoring the balance of extracellular matrix (ECM) metabolism both in vitro and in vivo. A significant role for STING has also been demonstrated in the regulation of NPC senescence by MAGL. The expression of the STING protein was reduced by 57% upon the inhibition of MAGL. STING activation can replicate the effects of MAGL and substantially increase LPS-induced inflammation while accelerating the senescence of NPCs. These results strongly indicate that the inhibition of MAGL can significantly suppress nucleus pulposus senescence via its interaction with STING, consequently restoring the balance of ECM metabolism. This insight provides new perspectives for potential treatments for IVDD.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Animais , Humanos , Ratos , Inflamação/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Lipopolissacarídeos/farmacologia , Monoacilglicerol Lipases/metabolismoRESUMO
OBJECTIVES: Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase considered a potential novel drug target for the treatment of CNS disorders including epilepsy. Here we examined MAGL levels in a rat model of epilepsy. METHODS: Autoradiography has been used to validate the binding properties of the MAGL radiotracer, [3H]T-401, in the rat brain, and to explore spatial and temporal changes in binding levels in a model of temporal lobe epilepsy model using unilateral intra-hippocampal injections of kainic acid (KA) in rats. RESULTS: Specific and saturable binding of [3H]T-401 was detected in both cortical grey and subcortical white matter. Saturation experiments revealed a KD in the range between 15 nM and 17 nM, and full saturation was achieved at concentrations around 30 nM. The binding could be completely blocked with the cold ligand (Ki 44.2 nM) and at higher affinity (Ki 1.27 nM) with another structurally different MAGL inhibitor, ABD 1970. Bilateral reduction in [3H]T-401 binding was observed in the cerebral cortex and the hippocampus few days after status epilepticus that further declined to a level of around 30% compared to the control. No change in binding was observed in either the hypothalamus nor the white matter at any time point. Direct comparison to [3H]UCB-J binding to synaptic vesicle glycoprotein 2 A (SV2A), another protein localized in the pre-synapse, revealed that while binding to MAGL remained low in the chronic phase, SV2A was increased significantly in some cortical areas. SIGNIFICANCE: These data show that MAGL is reduced in the cerebral cortex and hippocampus in a chronic epilepsy model and indicate that MAGL inhibitors may further reduce MAGL activity in the treatment resistant epilepsy patient.
Assuntos
Epilepsia , Estado Epiléptico , Humanos , Ratos , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Monoacilglicerol Lipases , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/metabolismo , Epilepsia/metabolismo , Inibidores Enzimáticos/farmacologiaRESUMO
Monoacylglycerol lipase (MAGL) plays a crucial role in the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids in the brain. Inhibiting MAGL could lead to increased levels of 2-AG, which showed beneficial effects on pain management, anxiety, inflammation, and neuroprotection. In the current study, we report the characterization of an enantiomerically pure (R)-[11C]YH132 as a novel MAGL PET tracer. It demonstrates an improved pharmacokinetic profile compared to its racemate. High inâ vitro MAGL specificity of (R)-[11C]YH132 was confirmed by autoradiography studies using mouse and rat brain sections. In vivo, (R)-[11C]YH132 displayed a high brain penetration, and high specificity and selectivity toward MAGL by dynamic PET imaging using MAGL knockout and wild-type mice. Pretreatment with a MAGL drug candidate revealed a dose-dependent reduction of (R)-[11C]YH132 accumulation in WT mouse brains. This result validates its utility as a PET probe to assist drug development. Moreover, its potential application in neurodegenerative diseases was explored by inâ vitro autoradiography using brain sections from animal models of Alzheimer's disease and Parkinson's disease.
Assuntos
Monoacilglicerol Lipases , Doenças Neurodegenerativas , Ratos , Camundongos , Animais , Monoacilglicerol Lipases/metabolismo , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/tratamento farmacológico , Tomografia por Emissão de Pósitrons/métodos , Inflamação , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologiaRESUMO
Rationale: Renal fibrosis, with no therapeutic approaches, is a common pathological feature in various chronic kidney diseases (CKD). Tubular cell injury plays a pivotal role in renal fibrosis. Commonly, injured tubular cells exhibit significant lipid accumulation. However, the underlying mechanisms remain poorly understood. Methods: 2-arachidonoylglycerol (2-AG) levels in CKD patients and CKD model specimens were measured using mass spectrometry. 2-AG-loaded nanoparticles were infused into unilateral ureteral obstruction (UUO) mice. Lipid accumulation and renal fibrosis were tested. Furthermore, monoacylglycerol lipase (MAGL), the hydrolyzing enzyme of 2-AG, was assessed in CKD patients and models. Tubular cell-specific MAGL knock-in mice were generated. Moreover, MAGL recombination protein was also administered to unilateral ischemia reperfusion injury (UIRI) mice. Besides, a series of methods including RNA sequencing, metabolomics, primary cell culture, lipid staining, etc. were used. Results: 2-AG was increased in the serum or kidneys from CKD patients and models. Supplement of 2-AG further induced lipid accumulation and fibrogenesis through cannabinoid receptor type 2 (CB2)/ß-catenin signaling. ß-catenin knockout blocked 2-AG/CB2-induced fatty acid ß-oxidation (FAO) deficiency and lipid accumulation. Remarkably, MAGL significantly decreased in CKD, aligning with lipid accumulation and fibrosis. Specific transgene of MAGL in tubular cells significantly preserved FAO, inhibited lipid-mediated toxicity in tubular cells, and finally retarded fibrogenesis. Additionally, supplementation of MAGL in UIRI mice also preserved FAO function, inhibited lipid accumulation, and protected against renal fibrosis. Conclusion: MAGL is a potential diagnostic marker for kidney function decline, and also serves as a new therapeutic target for renal fibrosis through ameliorating lipotoxicity.
Assuntos
Monoacilglicerol Lipases , Insuficiência Renal Crônica , Animais , Humanos , Camundongos , beta Catenina , Fibrose , RimRESUMO
The mechanisms of ß-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: ß-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.
Assuntos
Analgesia , Ácidos Araquidônicos , Endocanabinoides , Glicerídeos , Sesquiterpenos Policíclicos , Animais , Ratos , Endocanabinoides/farmacologia , Glicerol , Isótopos , Monoacilglicerol Lipases , Receptor CB2 de CanabinoideRESUMO
This study aimed to evaluate (R)-[18F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on (R)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, (R)-[18F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of (R)-[18F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume (V T) of (R)-[18F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using (R)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of (R)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.