RESUMEN
Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds-termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.
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Acrilamida , Proteómica , Humanos , Acrilamida/química , Estereoisomerismo , Triptaminas/química , Triptaminas/farmacología , Proteoma/metabolismo , Línea Celular Tumoral , Unión Proteica , CarbolinasRESUMEN
Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these 'photo-stereoprobes' interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.
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5-Methylcytosine (m5 C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1-7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C5 position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell-active inhibitors of these enzymes are lacking. Here, we use cysteine-directed activity-based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross-reactivity with other human NSUNs and exhibit good proteome-wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2-tRNA interactions in cancer cells, leading to a global reduction in tRNA m5 C content. Our findings thus highlight the potential to create isotype-selective and cell-active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.
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Azetidinas , Inhibidores Enzimáticos , Metiltransferasas , ARNt Metiltransferasas , Humanos , Acrilamidas , Cisteína/metabolismo , Metilación , Metiltransferasas/antagonistas & inhibidores , Proteómica , ARN de Transferencia/química , ARNt Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacologíaRESUMEN
Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.
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Cisteína , Neoplasias , Humanos , Cisteína/química , Proteómica , Edición Génica , Proteoma/química , Neoplasias/genética , Proteínas NuclearesRESUMEN
Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.
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Proteómica , Factores de Transcripción , Humanos , Proteómica/métodos , Cisteína/metabolismo , LigandosRESUMEN
SUMMARY: Since June 2019, under the umbrella of the national health insurance system, Japan has started cancer genomic medicine (CGM) with comprehensive genomic profiling (CGP) tests. The Ministry of Health, Labour and Welfare (MHLW) of Japan constructed a network of CGM hospitals (a total of 233 institutes as of July 1, 2022) and established the Center for Cancer Genomics and Advanced Therapeutics (C-CAT), the national datacenter for CGM. Clinical information and genomic data from the CGP tests are securely transferred to C-CAT, which then generates "C-CAT Findings" reports containing information of clinical annotation and matched clinical trials based on the CGP data. As of June 30, 2022, a total of 36,340 datapoints of clinical/genomic information are aggregated in C-CAT, and the number is expected to increase swiftly. The data are now open for sharing with not only the CGM hospitals but also other academic institutions and industries.
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Medicina Genómica , Neoplasias , Humanos , Japón , Genómica , Neoplasias/genéticaRESUMEN
OBJECTIVE: The R value is adopted as a metric for the effectiveness of the respiratory waveform in the Advanced Motion Free implemented in the PET scanner as the data-driven respiratory gating (DDG) algorithm. The effects of changes in various factors on R values were evaluated by phantom analysis. METHODS: We used a programmable respiratory motion phantom QUASAR with a sphere filled with an 18F solution. Respiratory motion simulation was performed by changing the sphere diameter, radioactivity concentration, amplitude, respiratory cycle, and respiratory waveform shape. Three evaluations were performed. (1) The power spectra calculated from the input waveforms were evaluated. (2) The effects of changes in the factors on the R value were evaluated. (3) DDG waveforms and inspiratory peak intervals were compared with the input waveform data set. RESULTS: The R values were increased and converged to a certain value as sphere diameter, radioactivity concentration, and amplitude gradually increased. The respiratory cycle showed the highest R value at 7.5 s, and the graph showed an upward convex pattern. The R value of the sinusoid waveform was higher than that of the typical waveform. There was a relationship between the power spectrum of the input waveform and R value. The visual score was also lower in the condition with a lower R value. In cases of no sphere, radioactivity, or motion, and a fast respiratory cycle, peak intervals were not accurately acquired. CONCLUSIONS: Factors affecting the R value were sphere diameter, radioactivity concentration, amplitude, respiratory cycle, and respiratory waveform shape.
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Algoritmos , Tomografía Computarizada por Rayos X , Movimiento (Física) , Fantasmas de ImagenRESUMEN
The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.
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Endocannabinoides , Enfermedades del Sistema Nervioso , Humanos , Niño , Fenotipo , Enfermedades del Sistema Nervioso/genética , Heterocigoto , Síndrome , Proteínas MutantesRESUMEN
Monoacylglycerol lipase (MAGL) constitutes a serine hydrolase that orchestrates endocannabinoid homeostasis and exerts its function by catalyzing the degradation of 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA). As such, selective inhibition of MAGL represents a potential therapeutic and diagnostic approach to various pathologies including neurodegenerative disorders, metabolic diseases and cancers. Based on a unique 4-piperidinyl azetidine diamide scaffold, we developed a reversible and peripheral-specific radiofluorinated MAGL PET ligand [18F]FEPAD. Pharmacokinetics and binding studies on [18F]FEPAD revealed its outstanding specificity and selectivity towards MAGL in brown adipose tissue (BAT) - a tissue that is known to be metabolically active. We employed [18F]FEPAD in PET studies to assess the abundancy of MAGL in BAT deposits of mice and found a remarkable degree of specific tracer binding in the BAT, which was confirmed by post-mortem tissue analysis. Given the negative regulation of endocannabinoids on the metabolic BAT activity, our study supports the concept that dysregulation of MAGL is likely linked to metabolic disorders. Further, we now provide a suitable imaging tool that allows non-invasive assessment of MAGL in BAT deposits, thereby paving the way for detailed mechanistic studies on the role of BAT in endocannabinoid system (ECS)-related pathologies.
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Endocannabinoides , Monoacilglicerol Lipasas , Endocannabinoides/metabolismo , Tejido Adiposo Pardo/diagnóstico por imagen , Tejido Adiposo Pardo/metabolismo , Tomografía de Emisión de Positrones/métodos , Ligandos , Inhibidores Enzimáticos/farmacologíaRESUMEN
The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.
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Química Clic , Imagen Óptica , Animales , Encéfalo , Sistemas de Liberación de Medicamentos , Mamíferos , Ratones , Imagen Óptica/métodosRESUMEN
Monoacylglycerol lipase (MAGL) is a 33 kDa serine protease primarily responsible for hydrolyzing 2-arachidonoylglycerol into the proinflammatory eicosanoid precursor arachidonic acid in the central nervous system. Inhibition of MAGL constitutes an attractive therapeutic concept for treating psychiatric disorders and neurodegenerative diseases. Herein, we present the design and synthesis of multiple reversible MAGL inhibitor candidates based on a piperazinyl azetidine scaffold. Compounds 10 and 15 were identified as the best-performing reversible MAGL inhibitors by pharmacological evaluations, thus channeling their radiolabeling with fluorine-18 in high radiochemical yields and favorable molar activity. Furthermore, evaluation of [18F]10 and [18F]15 ([18F]MAGL-2102) by autoradiography and positron emission tomography (PET) imaging in rodents and nonhuman primates demonstrated favorable brain uptakes, heterogeneous radioactivity distribution, good specific binding, and adequate brain kinetics, and [18F]15 demonstrated a better performance. In conclusion, [18F]15 was found to be a suitable PET radioligand for the visualization of MAGL, harboring potential for the successful translation into humans.
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Azetidinas/farmacología , Monoacilglicerol Lipasas/antagonistas & inhibidores , Tomografía de Emisión de Positrones , Radiofármacos/farmacología , Animales , Azetidinas/síntesis química , Azetidinas/química , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Haplorrinos , Ligandos , Modelos Moleculares , Estructura Molecular , Monoacilglicerol Lipasas/metabolismo , Radiofármacos/síntesis química , Radiofármacos/química , Ratas , Relación Estructura-ActividadRESUMEN
As a serine hydrolase, monoacylglycerol lipase (MAGL) is principally responsible for the metabolism of 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS), leading to the formation of arachidonic acid (AA). Dysfunction of MAGL has been associated with multiple CNS disorders and symptoms, including neuroinflammation, cognitive impairment, epileptogenesis, nociception and neurodegenerative diseases. Inhibition of MAGL provides a promising therapeutic direction for the treatment of these conditions, and a MAGL positron emission tomography (PET) probe would greatly facilitate preclinical and clinical development of MAGL inhibitors. Herein, we design and synthesize a small library of fluoropyridyl-containing MAGL inhibitor candidates. Pharmacological evaluation of these candidates by activity-based protein profiling identified 14 as a lead compound, which was then radiolabeled with fluorine-18 via a facile SNAr reaction to form 2-[18F]fluoropyridine scaffold. Good blood-brain barrier permeability and high in vivo specific binding was demonstrated for radioligand [18F]14 (also named as [18F]MAGL-1902). This work may serve as a roadmap for clinical translation and further design of potent 18F-labeled MAGL PET tracers.
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PURPOSE: Quantitative analysis using a standardized uptake value (SUV) has become possible for single-photon emission computed tomography-computed tomography (SPECT-CT) of bone. However, previous research was targeted to the trunk area, and there are few studies for the head and neck region. Therefore, the purpose of this study was to determine the optimal image reconstruction conditions for bone SPECT of the head and neck using a phantom study. METHOD: The radioactivity concentration of the 99mTc solution enclosed in the cylindrical phantom was set to the same count rate as in clinical cases, and six hot spheres (10, 13, 17, 22, 28, 37 mm) with four times the concentration were placed within it. The image reconstruction was 3D-OSEM, and the reconstruction conditions were varied by the number of iterative updates and the width of the Gaussian filter. Quantitative evaluations of the image quality were performed using the % contrast, background variability, and SUV for the hot spheres and background. A visual evaluation was performed by four observers to determine the optimal image reconstruction conditions for bone SPECT of the head and neck region. RESULT: The concentration of the 99mTc solution enclosed in the phantom was 6.95 (kBq/ml). Based on the results of the quantitative and visual evaluations, the optimal image reconstruction conditions were iterative updates=60 (subset: 10, iteration: 6) and a Gaussian filter of 7.8 mm. CONCLUSION: The optimal image reconstruction conditions were subset=10, iterations=6, and a Gaussian filter of 7.8 mm.
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Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Tomografía Computarizada de Emisión de Fotón Único , Procesamiento de Imagen Asistido por Computador , Cuello/diagnóstico por imagen , Fantasmas de ImagenRESUMEN
The endocannabinoid system (ECS) is involved in a wide range of biological functions and comprises cannabinoid receptors and enzymes responsible for endocannabinoid synthesis and degradation. Over the past 2 decades, significant advances toward developing drugs and positron emission tomography (PET) tracers targeting different components of the ECS have been made. Herein, we summarized the recent development of PET tracers for imaging cannabinoid receptors 1 (CB1R) and 2 (CB2R) as well as the key enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), particularly focusing on PET neuroimaging applications. State-of-the-art PET tracers for the ECS will be reviewed including their chemical design, pharmacological properties, radiolabeling, as well as preclinical and human PET imaging. In addition, this review addresses the current challenges for ECS PET biomarker development and highlights the important role of PET ligands to study disease pathophysiology as well as to facilitate drug discovery.
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Endocannabinoides/metabolismo , Tomografía de Emisión de Positrones/métodos , Amidohidrolasas/antagonistas & inhibidores , Animales , Biomarcadores/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Receptores de Cannabinoides/metabolismoRESUMEN
Cannabinoids are part of an endogenous signaling system found throughout the body, including the eye. Hepler and Frank showed in the early 1970s that plant cannabinoids can lower intraocular pressure (IOP), an effect since shown to occur via cannabinoid CB1 and GPR18 receptors. Endocannabinoids are synthesized and metabolized enzymatically. Enzymes implicated in endocannabinoids breakdown include monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), but also ABHD12, NAAA, and COX-2. Inhibition of MAGL activity raises levels of the endocannabinoid 2-arachidonoyl glycerol and substantially lowers IOP. Blocking other cannabinoid metabolizing enzymes or cannabinoid transporters may similarly contribute to lowering IOP and so serve as therapeutic targets for treating glaucoma. We have tested blockers for several cannabinoid-metabolizing enzymes and transporters (FABP5 and membrane reuptake) for their ability to alter ocular pressure in a murine model of IOP. Of FAAH, ABHD12, NAAA, and COX2, only FAAH was seen to play a role in regulation of IOP. Only the FAAH blocker URB597 lowered IOP, but in a temporally, diurnally, and sex-specific manner. We also tested two blockers of cannabinoid transport (SBFI-26 and WOBE437), finding that each lowered IOP in a CB1-dependent manner. Though we see a modest, limited role for FAAH, our results suggest that MAGL is the primary cannabinoid-metabolizing enzyme in regulating ocular pressure, thus pointing towards a role of 2-arachidonoyl glycerol. Interestingly, inhibition of cannabinoid transport mechanisms independent of hydrolysis may prove to be an alternative strategy to lower ocular pressure.
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Endocannabinoides/metabolismo , Presión Intraocular/fisiología , Hipertensión Ocular/metabolismo , Animales , Modelos Animales de Enfermedad , Transporte Iónico , Ratones , Ratones Endogámicos C57BL , Hipertensión Ocular/fisiopatologíaRESUMEN
Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.
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Activadores de Enzimas/química , Activadores de Enzimas/farmacología , Lisofosfolipasa/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Descubrimiento de Drogas , Activadores de Enzimas/farmacocinética , Polarización de Fluorescencia , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Resistencia a la Insulina , Lisofosfolipasa/química , Lisofosfolipasa/genética , Masculino , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/metabolismo , Ratones Endogámicos C57BL , Ratones Obesos , Simulación de Dinámica Molecular , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Relación Estructura-ActividadRESUMEN
N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.
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Conducta Animal/efectos de los fármacos , Inhibidores Enzimáticos/química , Metabolismo de los Lípidos/efectos de los fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipasa D/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Cannabinoides/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Miedo/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Receptores de Cannabinoides/metabolismo , Transducción de SeñalRESUMEN
PHARC (polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa, and cataract) is a human neurological disorder caused by deleterious mutations in the ABHD12 gene, which encodes an integral membrane lyso-phosphatidylserine (lyso-PS) lipase. Pharmacological or genetic disruption of ABHD12 leads to higher levels of lyso-PS lipids in human cells and the central nervous system (CNS) of mice. ABHD12 loss also causes rapid rewiring of PS content, resulting in selective increases in the level of arachidonoyl (C20:4) PS and decreases in the levels of other PS species. The biochemical basis for ABHD12-dependent PS remodeling and its pathophysiological significance remain unknown. Here, we show that genetic deletion of the lysophospholipid acyltransferase LPCAT3 blocks accumulation of brain C20:4 PS in mice lacking ABHD12 and concurrently produces hyper-increases in the level of lyso-PS in these animals. These lipid changes correlate with exacerbated auditory dysfunction and brain microgliosis in mice lacking both ABHD12 and LPCAT3. Taken together, our findings reveal that ABHD12 and LPCAT3 coordinately regulate lyso-PS and C20:4 PS content in the CNS and point to lyso-PS lipids as the likely bioactive metabolites contributing to PHARC-related neuropathologies.
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1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Monoacilglicerol Lipasas/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Fosfatidilserinas/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/deficiencia , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Animales , Ratones , Ratones Noqueados , Estructura Molecular , Monoacilglicerol Lipasas/deficiencia , Monoacilglicerol Lipasas/genéticaRESUMEN
Despite a continuous increase in R&D spending on potential new medicines, the success rate of drug development has not improved. The pharmaceutical industry is now facing a major challenge. As a college student who was studying pharmaceutical sciences in Japan, I became passionate about developing a new technology that would allow us to efficiently discover novel drug targets and selective chemical ligands for these targets. This realization encouraged me to join the PhD program at The Scripps Research Institute (TSRI) in 2013, where I carried out thesis research focusing on ligand discovery for poorly characterized metabolic enzymes for lipid signaling under the guidance of Prof. Benjamin Cravatt. TSRI is a unique place where researchers with different backgrounds collaborate frequently to conduct highly interdisciplinary research with the goal of translating cutting-edge research into clinical use. In this column, I am sharing my experiences as a PhD student at TSRI. I hope this column will be a useful source of information for younger students considering going abroad for a PhD degree.
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Biofarmacia/educación , Sistemas de Liberación de Medicamentos , Descubrimiento de Drogas , Educación de Postgrado en Farmacia , Ligandos , Análisis por Matrices de Proteínas , Investigación , Descubrimiento de Drogas/educación , Descubrimiento de Drogas/métodos , Humanos , Japón , Análisis por Matrices de Proteínas/métodos , Estados UnidosRESUMEN
Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.