RESUMEN
Over the last decade, for the first time, substantial efforts have been directed at the development of dedicated in silico platforms for drug repurposing, including initiatives targeting cancers and conditions as diverse as cryptosporidiosis, dengue, dental caries, diabetes, herpes, lupus, malaria, tuberculosis and Covid-19 related respiratory disease. This review outlines some of the exciting advances in the specific applications of in silico approaches to the challenge of drug repurposing and focuses particularly on where these efforts have resulted in the development of generic platform technologies of broad value to researchers involved in programmatic drug repurposing work. Recent advances in molecular docking methodologies and validation approaches, and their combination with machine learning or deep learning approaches are continually enhancing the precision of repurposing efforts. The meaningful integration of better understanding of molecular mechanisms with molecular pathway data and knowledge of disease networks is widening the scope for discovery of repurposing opportunities. The power of Artificial Intelligence is being gainfully exploited to advance progress in an integrated science that extends from the sub-atomic to the whole system level. There are many promising emerging developments but there are remaining challenges to be overcome in the successful integration of the new advances in useful platforms. In conclusion, the essential component requirements for development of powerful and well optimised drug repurposing screening platforms are discussed.
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Tratamiento Farmacológico de COVID-19 , Caries Dental , Inteligencia Artificial , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos/métodos , Humanos , Simulación del Acoplamiento MolecularRESUMEN
Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.
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Colesterol/metabolismo , Citotoxinas/efectos adversos , Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Fibroblastos/citología , Sustancias Protectoras/farmacología , Células A549 , Proteínas Bacterianas/efectos adversos , Toxinas Bacterianas/efectos adversos , Proliferación Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Células HeLa , Proteínas Hemolisinas/efectos adversos , Humanos , Estreptolisinas/efectos adversosRESUMEN
Gefitinib and erlotinib are epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) with activity against metastatic non-small cell lung cancer. Aldehyde oxidase-1 (AOX1) is a cytosolic drug-metabolizing enzyme. We conducted an experimental and molecular docking study on the effect of gefitinib, erlotinib, and select metabolites on the in vitro catalytic activity of AOX1, as assessed by carbazeran 4-oxidation, and determined the impact of AOX1 inhibition on hepatic metabolism of zaleplon and methotrexate. Gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib inhibited human hepatic cytosolic carbazeran 4-oxidation by a competitive mode, with inhibition constants in submicromolar or low micromolar concentrations. Desmethylgefitinib did not affect AOX1 catalytic activity. A similar pattern was obtained when investigated with human kidney cytosol or recombinant AOX1. The differential effect of gefitinib on human, rat, and mouse hepatic AOX1 catalytic activity suggests species-dependent chemical inhibition of AOX1. Erlotinib was considerably more potent than gefitinib in decreasing hepatic cytosolic zaleplon 5-oxidation and methotrexate 7-oxidation. Molecular docking analyses provided structural insights into the interaction between EGFR-TKIs and AOX1, with key residues and bonds identified, which provided favorable comparison and ranking of potential inhibitors. Based on the US Food and Drug Administration guidance to assess the risk of drug-drug interactions, the calculated R1 values indicate that further investigations are warranted to determine whether gefitinib and erlotinib impact AOX1-mediated drug metabolism in vivo. Overall, erlotinib desmethylerlotinib, didesmethylerlotinib, gefitinib, and desmorpholinopropylgefitinib are potent inhibitors of human AOX1 catalytic function and hepatic metabolism of zaleplon and methotrexate, potentially affecting drug efficacy or toxicity. SIGNIFICANCE STATEMENT: As epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib are first-line pharmacotherapy for metastatic non-small cell lung cancer. Our experimental findings indicate that clinically relevant concentrations of gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib, but not desmethylgefitinib, inhibit human aldehyde oxidase (AOX1) catalytic activity and hepatic cytosolic metabolism of zaleplon and methotrexate. Molecular docking analysis provide structural insights into the key AOX1 interactions with these EGFR-TKIs. Our findings may trigger improved strategies for new EGFR-TKI design and development.
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Acetamidas/metabolismo , Aldehído Oxidasa/antagonistas & inhibidores , Clorhidrato de Erlotinib/farmacología , Gefitinib/farmacología , Hígado/efectos de los fármacos , Metotrexato/metabolismo , Simulación del Acoplamiento Molecular , Pirimidinas/metabolismo , Aldehído Oxidasa/química , Aldehído Oxidasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Clorhidrato de Erlotinib/metabolismo , Gefitinib/metabolismo , Humanos , Hígado/metabolismo , Conformación ProteicaRESUMEN
Tamoxifen, raloxifene, and nafoxidine are selective estrogen receptor modulators (SERMs) reported to inhibit the catalytic activity of human aldehyde oxidase 1 (AOX1). How these drugs interact with AOX1 and whether other SERMs inhibit this drug-metabolizing enzyme are not known. Therefore, a detailed in vitro and in silico study involving parent drugs and their analogs was conducted to investigate the effect of specific SERMs, particularly acolbifene, bazedoxifene, and lasofoxifene on AOX1 catalytic activity, as assessed by carbazeran 4-oxidation, an AOX1-selective catalytic marker. The rank order in the potency (based on IC50 values) of AOX1 inhibition by SERMs was raloxifene > bazedoxifene â¼ lasofoxifene > tamoxifen > acolbifene. Inhibition of liver cytosolic AOX1 by bazedoxifene, lasofoxifene, and tamoxifen was competitive, whereas that by raloxifene was noncompetitive. Loss of 1-azepanylethyl group increased the inhibitory potency of bazedoxifene, whereas the N-oxide group decreased it. The 7-hydroxy group and the substituted pyrrolidine ring attached to the tetrahydronaphthalene structure contributed to AOX1 inhibition by lasofoxifene. These results are supported by molecular-docking simulations in terms of predicted binding modes, encompassing binding orientation and efficiency, and analysis of key interactions, particularly hydrogen bonds. The extent of AOX1 inhibition by bazedoxifene was increased by estrone sulfate and estrone. In summary, SERMs differentially inhibited human AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contributed to AOX1 inhibition, whereas those of acolbifene rendered it considerably less susceptible to AOX1 inhibition. Overall, our novel biochemical findings and molecular-docking analyses provide new insights into the interaction between SERMs and AOX1. SIGNIFICANCE STATEMENT: Aldehyde oxidase (AOX1) is a molybdo-flavoprotein and has emerged as a drug-metabolizing enzyme of potential therapeutic importance because drugs have been identified as AOX1 substrates. Selective estrogen receptor modulators (SERM), which are drugs used to treat and prevent various conditions, differentially inhibit AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contribute to AOX1 inhibition, whereas those of acolbifene render it considerably less susceptible to AOX1 inhibition. Our novel biochemical findings, together with molecular- docking analyses, provide new insights into the differential inhibitory effect of SERMs on the catalytic activity of human AOX1, how SERMs bind to AOX1, and increase our understanding of the AOX1 pharmacophore in the inhibition of AOX1 by drugs and other chemicals.
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Aldehído Oxidasa/antagonistas & inhibidores , Indoles/farmacología , Simulación del Acoplamiento Molecular , Pirrolidinas/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tetrahidronaftalenos/farmacología , Aldehído Oxidasa/química , Aldehído Oxidasa/metabolismo , Sitios de Unión , Femenino , Humanos , Hígado/enzimología , Masculino , Unión ProteicaRESUMEN
Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.
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Mutación/genética , Proteínas del Tejido Nervioso/genética , Polimicrogiria/genética , Receptores de N-Metil-D-Aspartato/genética , Animales , Niño , Preescolar , Análisis Mutacional de ADN , Agonistas de Aminoácidos Excitadores/farmacología , Salud de la Familia , Femenino , Ácido Glutámico/farmacología , Glicina/metabolismo , Glicina/farmacología , Células HEK293 , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Potenciales de la Membrana/genética , Modelos Moleculares , Mutagénesis/genética , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp , Polimicrogiria/diagnóstico por imagen , Ratas , TransfecciónRESUMEN
Mutations in alpha- and beta-tubulins are increasingly recognized as a major cause of malformations of cortical development (MCD), typically lissencephaly, pachygyria and polymicrogyria; however, sequencing tubulin genes in large cohorts of MCD patients has detected tubulin mutations in only 1-13%. We identified patients with a highly characteristic cerebellar dysplasia but without lissencephaly, pachygyria and polymicrogyria typically associated with tubulin mutations. Remarkably, in seven of nine patients (78%), targeted sequencing revealed mutations in three different tubulin genes (TUBA1A, TUBB2B and TUBB3), occurring de novo or inherited from a mosaic parent. Careful re-review of the cortical phenotype on brain imaging revealed only an irregular pattern of gyri and sulci, for which we propose the term tubulinopathy-related dysgyria. Basal ganglia (100%) and brainstem dysplasia (80%) were common features. On the basis of in silico structural predictions, the mutations affect amino acids in diverse regions of the alpha-/beta-tubulin heterodimer, including the nucleotide binding pocket. Cell-based assays of tubulin dynamics reveal various effects of the mutations on incorporation into microtubules: TUBB3 p.Glu288Lys and p.Pro357Leu do not incorporate into microtubules at all, whereas TUBB2B p.Gly13Ala shows reduced incorporation and TUBA1A p.Arg214His incorporates fully, but at a slower rate than wild-type. The broad range of effects on microtubule incorporation is at odds with the highly stereotypical clinical phenotype, supporting differential roles for the three tubulin genes involved. Identifying this highly characteristic phenotype is important due to the low recurrence risk compared with the other (recessive) cerebellar dysplasias and the apparent lack of non-neurological medical issues.
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Cerebelo/patología , Mutación , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/patología , Tubulina (Proteína)/genética , Alelos , Encéfalo/patología , Línea Celular , Vermis Cerebeloso/patología , Estudios de Cohortes , Femenino , Genotipo , Humanos , Imagen por Resonancia Magnética , Masculino , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Malformaciones del Sistema Nervioso/diagnóstico , Fenotipo , Conformación Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Tubulina (Proteína)/químicaRESUMEN
Tubulins, and microtubule polymers into which they incorporate, play critical mechanical roles in neuronal function during cell proliferation, neuronal migration, and postmigrational development: the three major overlapping events of mammalian cerebral cortex development. A number of neuronally expressed tubulin genes are associated with a spectrum of disorders affecting cerebral cortex formation. Such "tubulinopathies" include lissencephaly/pachygyria, polymicrogyria-like malformations, and simplified gyral patterns, in addition to characteristic extracortical features, such as corpus callosal, basal ganglia, and cerebellar abnormalities. Epilepsy is a common finding in these related disorders. Here we describe two unrelated individuals with infantile-onset epilepsy and abnormalities of brain morphology, harboring de novo variants that affect adjacent amino acids in a beta-tubulin gene TUBB2A. Located in a highly conserved loop, we demonstrate impaired tubulin and microtubule function resulting from each variant in vitro and by using in silico predictive modeling. We propose that the affected functional loop directly associates with the alpha-tubulin-bound guanosine triphosphate (GTP) molecule, impairing the intradimer interface and correct formation of the alpha/beta-tubulin heterodimer. This study associates mutations in TUBB2A with the spectrum of "tubulinopathy" phenotypes. As a consequence, genetic variations affecting all beta-tubulin genes expressed at high levels in the brain (TUBB2B, TUBB3, TUBB, TUBB4A, and TUBB2A) have been linked with malformations of cortical development.
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Giro Dentado/patología , Epilepsia/genética , Mutación Missense , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Epilepsia/patología , Células HEK293 , Humanos , Lactante , Imagen por Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/químicaRESUMEN
Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.
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Ascomicetos/enzimología , Proteínas Fúngicas/química , NADPH-Ferrihemoproteína Reductasa/metabolismo , Esterol 14-Desmetilasa/química , Secuencia de Aminoácidos , Ascomicetos/química , Ascomicetos/genética , Candida albicans/enzimología , Candida albicans/genética , Estabilidad de Enzimas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriales/química , Fungicidas Industriales/metabolismo , Lanosterol/análogos & derivados , Lanosterol/química , Lanosterol/metabolismo , Datos de Secuencia Molecular , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/genética , Oxidación-Reducción , Alineación de Secuencia , Esterol 14-Desmetilasa/genética , Esterol 14-Desmetilasa/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus ß subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors.
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Regulación de la Expresión Génica , Rigidez Muscular/metabolismo , Mutación Missense , Receptores de Glicina/metabolismo , Sustitución de Aminoácidos , Femenino , Humanos , Masculino , Rigidez Muscular/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Glicina/genéticaRESUMEN
Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and extended behavioural presentation. The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this family's GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and ß2 subunits in HEK 293T cells, GABA-evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism.
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Epilepsia Generalizada/genética , Fenotipo , Mutación Puntual , Receptores de GABA-A/genética , Convulsiones Febriles/genética , Adulto , Animales , Células COS , Células Cultivadas , Corteza Cerebral/fisiopatología , Niño , Preescolar , Chlorocebus aethiops , Estudios de Cohortes , Familia , Femenino , Células HEK293 , Humanos , Lactante , Masculino , Neuronas/fisiología , Células PC12 , Ratas , Receptores de GABA-A/metabolismoRESUMEN
Congenital hyperekplexia is a rare, potentially treatable neuromotor disorder. Three major genes of effect are known, and all three affect glycinergic neurotransmission. Two genes encode for subunits of the postsynaptic inhibitory glycine receptor, GLRA1 encoding the α1 subunit and GLRB encoding the ß subunit. The third, SLC6A5, encodes the cognate presynaptic glycine transporter 2. Ninety-seven individuals had a clinical diagnosis of hyperekplexia confirmed by genetic testing: 61 cases had mutations in GLRA1, 24 cases in SLC6A5 and 12 in GLRB. Detailed retrospective clinical analysis ascertained that all gene-positive cases present in the neonatal period (occasionally prenatally) and that clonazepam is the treatment of choice (95% found it to be efficacious). We confirm that hyperekplexia is predominantly a recessive condition but dominant cases are seen (16%). We found no genetic evidence for 'major' or 'minor' forms of hyperekplexia on a population basis. Thirty-five gene-negative cases were studied for comparison, their cardinal feature was presentation after the first month of life (P < 0.001). In addition to the characteristic 'stiffness, startles and stumbles' of hyperekplexia, apnoea attacks (50 of 89) and delayed development (47 of 92) were frequently reported. Patients with SLC6A5 mutations were significantly more likely to have had recurrent infantile apnoeas (RR1.9; P < 0.005) than those with GLRA1 mutations. Patients with GLRB and SLC6A5 mutations were more likely to have developmental delay (RR1.5 P < 0.01; RR1.9 P < 0.03) than those with GLRA1 mutations; 92% of GLRB cases reported a mild to severe delay in speech acquisition. Molecular modelling of pathogenic mutations demonstrates specific patterns of protein disruption that can be used to predict phenotype severity. The developmental delay in hyperekplexia, and speech acquisition in particular, may represent failure of developmental neural networks or subtle neurogenic migration defects in the absence of presynaptic glycine release. We recommend early genetic testing for symptomatic neonates and possibly preconception counselling for those at risk for GLRB and SLC6A5 mutations, because of the more challenging phenotype.
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Epilepsia/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Trastornos del Desarrollo del Lenguaje/genética , Mutación/genética , Reflejo Anormal/genética , Adolescente , Adulto , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Aprendizaje/fisiología , Masculino , Fenotipo , Receptores de Glicina/genética , Estudios Retrospectivos , Adulto JovenRESUMEN
Polymicrogyria and lissencephaly are causally heterogeneous disorders of cortical brain development, with distinct neuropathological and neuroimaging patterns. They can be associated with additional structural cerebral anomalies, and recurrent phenotypic patterns have led to identification of recognizable syndromes. The lissencephalies are usually single-gene disorders affecting neuronal migration during cerebral cortical development. Polymicrogyria has been associated with genetic and environmental causes and is considered a malformation secondary to abnormal post-migrational development. However, the aetiology in many individuals with these cortical malformations is still unknown. During the past few years, mutations in a number of neuron-specific α- and ß-tubulin genes have been identified in both lissencephaly and polymicrogyria, usually associated with additional cerebral anomalies including callosal hypoplasia or agenesis, abnormal basal ganglia and cerebellar hypoplasia. The tubulin proteins form heterodimers that incorporate into microtubules, cytoskeletal structures essential for cell motility and function. In this study, we sequenced the TUBB2B and TUBA1A coding regions in 47 patients with a diagnosis of polymicrogyria and five with an atypical lissencephaly on neuroimaging. We identified four ß-tubulin and two α-tubulin mutations in patients with a spectrum of cortical and extra-cortical anomalies. Dysmorphic basal ganglia with an abnormal internal capsule were the most consistent feature. One of the patients with a TUBB2B mutation had a lissencephalic phenotype, similar to that previously associated with a TUBA1A mutation. The remainder had a polymicrogyria-like cortical dysplasia, but the grey matter malformation was not typical of that seen in 'classical' polymicrogyria. We propose that the cortical malformations associated with these genes represent a recognizable tubulinopathy-associated spectrum that ranges from lissencephalic to polymicrogyric cortical dysplasias, suggesting shared pathogenic mechanisms in terms of microtubular function and interaction with microtubule-associated proteins.
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Genes Sobrepuestos/genética , Lisencefalia/genética , Malformaciones del Desarrollo Cortical/genética , Mutación/genética , Tubulina (Proteína)/genética , Adulto , Secuencia de Aminoácidos , Corteza Cerebral/anomalías , Corteza Cerebral/patología , Niño , Preescolar , Femenino , Humanos , Recién Nacido , Lisencefalia/diagnóstico , Masculino , Malformaciones del Desarrollo Cortical/diagnóstico , Datos de Secuencia Molecular , Tubulina (Proteína)/químicaRESUMEN
Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin-1 to have a flexible beta conformation and identifies a motif between amino acids 62-66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin-1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCV glycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.
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Claudina-1/fisiología , Hepacivirus/fisiología , Mutagénesis Sitio-Dirigida , Receptores Virales/fisiología , Tetraspanina 28/fisiología , Internalización del Virus , Animales , Línea Celular , Claudina-1/genética , Biología Computacional , Simulación por Computador , Transferencia Resonante de Energía de Fluorescencia , Hepacivirus/patogenicidad , Humanos , Modelos Moleculares , Receptores Virales/genética , Tetraspanina 28/genéticaRESUMEN
The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (K(s), 7.1 and 8.0 µM, respectively) compared to the wild-type CaCYP51 protein (K(s), 13.5 µM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher K(d) values observed.
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Inhibidores de 14 alfa Desmetilasa/farmacología , Antifúngicos/farmacología , Candida albicans/enzimología , Candida albicans/genética , Fluconazol/farmacología , Mutación Puntual/genética , Esterol 14-Desmetilasa/genética , Secuencia de Aminoácidos , Azoles/metabolismo , Unión Competitiva/efectos de los fármacos , Candida albicans/efectos de los fármacos , ADN de Hongos/genética , Cinética , Lanosterol/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas Recombinantes/biosíntesisRESUMEN
Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the ß subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.
Asunto(s)
Flavodoxina , Streptomyces coelicolor , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavodoxina/química , Flavodoxina/genética , Flavodoxina/metabolismo , Oxidación-Reducción , Suelo , Streptomyces coelicolor/metabolismoRESUMEN
BACKGROUND: The discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability. METHODS: Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene's function in cognition, Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing. RESULTS: Homozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum-anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits. CONCLUSIONS: These data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.
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Disfunción Cognitiva , Discapacidad Intelectual , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Disfunción Cognitiva/genética , Consanguinidad , Drosophila , Drosophila melanogaster , Humanos , Discapacidad Intelectual/genética , Ratones , Mutación/genéticaRESUMEN
Hyperekplexia is a rare, but potentially fatal, neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden, unexpected auditory or tactile stimuli. This disorder is primarily caused by inherited mutations in the genes encoding the glycine receptor (GlyR) alpha1 subunit (GLRA1) and the presynaptic glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of GLRA1 in 88 new unrelated human hyperekplexia patients revealed 19 sequence variants in 30 index cases, of which 21 cases were inherited in recessive or compound heterozygote modes. This indicates that recessive hyperekplexia is far more prevalent than previous estimates. From the 19 GLRA1 sequence variants, we have investigated the functional effects of 11 novel and 2 recurrent mutations. The expression levels and functional properties of these hyperekplexia mutants were analyzed using a high-content imaging system and patch-clamp electrophysiology. When expressed in HEK293 cells, either as homomeric alpha1 or heteromeric alpha1beta GlyRs, subcellular localization defects were the major mechanism underlying recessive mutations. However, mutants without trafficking defects typically showed alterations in the glycine sensitivity suggestive of disrupted receptor function. This study also reports the first hyperekplexia mutation associated with a GlyR leak conductance, suggesting tonic channel opening as a new mechanism in neuronal ligand-gated ion channels.
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Hipertonía Muscular/genética , Receptores de Glicina/genética , Reflejo Anormal/genética , Reflejo de Sobresalto/genética , Línea Celular , Femenino , Variación Genética , Humanos , Masculino , Mutación/genética , Fenotipo , TransfecciónRESUMEN
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
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Antígenos CD/fisiología , Claudinas/genética , Claudinas/fisiología , Hepacivirus/fisiología , Hepatitis/fisiopatología , Antígenos CD/metabolismo , Línea Celular , Colesterol/metabolismo , Claudina-1 , Cartilla de ADN , Transferencia Resonante de Energía de Fluorescencia/métodos , Genes Reporteros , VIH/enzimología , VIH/genética , Células Hep G2/fisiología , Humanos , Luciferasas/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Provirus/enzimología , Provirus/genética , Resonancia por Plasmón de Superficie , Tetraspanina 28 , TransfecciónRESUMEN
The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
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Ascomicetos/efectos de los fármacos , Ascomicetos/enzimología , Azoles/metabolismo , Farmacorresistencia Fúngica , Fungicidas Industriales/metabolismo , Esterol 14-Desmetilasa/metabolismo , Sustitución de Aminoácidos/genética , Ascomicetos/genética , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Triticum/microbiologíaRESUMEN
Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7ß-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3ß-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7ß-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7ß-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.