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1.
J Enzyme Inhib Med Chem ; 37(1): 1077-1082, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35418253

RESUMO

Despite a huge effort by the scientific community to determine the animal reservoir of SARS-CoV-2, which led to the identification of several SARS-CoV-2-related viruses both in bats and in pangolins, the origin of SARS-CoV-2 is still not clear. Recently, Temmam et al. reported the discovery of bat coronaviruses with a high degree of genome similarity with SARS-CoV-2, especially concerning the RBDs of the S protein, which mediates the capability of such viruses to enter and therefore infect human cells through a hACE2-dependent pathway. These viruses, especially the one named BANAL-236, showed a higher affinity for the hACE2 compared to the original strain of SARS-CoV-2. In the present work, we analyse the similarities and differences between the 3CL protease (main protease, Mpro) of these newly reported viruses and SARS-CoV-2, discussing their relevance relative to the efficacy of existing therapeutic approaches against COVID-19, particularly concerning the recently approved orally available Paxlovid, and the development of future ones.


Assuntos
Quirópteros , Proteases 3C de Coronavírus , Coronavirus , Animais , Quirópteros/virologia , Coronavirus/enzimologia , SARS-CoV-2
2.
Theriogenology ; 185: 109-120, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35395587

RESUMO

Matrix metalloproteinases (MMPs) are a family of enzymes that degrade extracellular matrix (ECM) molecules, playing a vital role in tissue remodeling under physiological and pathological conditions. Their expression and/or activity are regulated by specific tissue inhibitors of MMPs named TIMPs. Recently, an imbalance in the MMP/TIMP system has been found in human and bovine ovarian cysts, but its role in porcine cyst pathogenesis is unknown. This study examined mRNA expression, protein abundance and localization for selected members of the MMP/TIMP system in follicular cysts of sows. Based on histological analysis, we have assessed follicular (FC) and follicular lutein (FLC) cysts with preovulatory follicles (PF) used as a control. Regarding the pattern of MMP expression, increased MMP2, MMP7 and MMP9 mRNA levels were observed in FLC. Furthermore, both pro- and active forms of MMP-2 and MMP-9 proteins were more abundant in FLC. In FC, the abundance of latent and active forms of MMP-9 and the active form of MMP-2 were greater when compared with PF. In relation to TIMPs, TIMP-2 mRNA and protein expression were increased in FLC, whereas TIMP-3 was up-regulated in both FC and FLC only at the protein level. Using immunofluorescence, MMP-2, MMP-7, TIMP-2 and TIMP-3 were detected in granulosa and theca compartments of FC and within the entire luteinized wall of FLC. Notably, MMP-9 occurred weakly in the granulosa layer of FC, but abundantly in the theca compartment of FC and in the luteinized FLC. Taken together, our findings indicate altered expression of the MMP/TIMP system, suggestive of increased ECM degradation, in sow follicular cysts. These components may be involved in the pathogenesis of porcine ovarian cysts through the ECM remodeling.


Assuntos
Doenças dos Bovinos , Cisto Folicular , Metaloproteinases da Matriz , Cistos Ovarianos , Doenças dos Suínos , Inibidores Teciduais de Metaloproteinases , Animais , Bovinos , Feminino , Cisto Folicular/veterinária , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Cistos Ovarianos/enzimologia , Cistos Ovarianos/veterinária , RNA Mensageiro , Suínos , Doenças dos Suínos/enzimologia , Doenças dos Suínos/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(16): e2117465119, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35412890

RESUMO

SignificanceInoculation of cereals with diazotrophic (N2-fixing) bacteria offers a sustainable alternative to the application of nitrogen fertilizers in agriculture. While natural diazotrophs have evolved multilayered regulatory mechanisms that couple N2 fixation with assimilation of the product NH3 and prevent release to plants, genetic modifications can permit excess production and excretion of NH3. However, a lack of stringent host-specificity for root colonization by the bacteria would allow growth promotion of target and nontarget plants species alike. Here, we exploit synthetic transkingdom signaling to establish plant host-specific control of the N2-fixation catalyst nitrogenase in Azorhizobium caulinodans occupying barley roots. This work demonstrates how partner-specific interactions can be established to avoid potential growth promotion of nontarget plants.


Assuntos
Azorhizobium caulinodans , Grão Comestível , Hordeum , Fixação de Nitrogênio , Nitrogenase , Raízes de Plantas , Azorhizobium caulinodans/enzimologia , Azorhizobium caulinodans/genética , Grão Comestível/microbiologia , Hordeum/microbiologia , Inositol/análogos & derivados , Inositol/genética , Inositol/metabolismo , Nitrogenase/genética , Nitrogenase/metabolismo , Raízes de Plantas/microbiologia , Simbiose
4.
Ecotoxicol Environ Saf ; 236: 113486, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35397445

RESUMO

Methanethiol is a widely existing malodorous pollutant with health effects on the human population. However, the cytotoxicity mechanism of methanethiol in vitro and its metabolic transformation (bioactivation or detoxification) have not been fully elucidated. Herein, the metabolites of methanethiol during cell culture and the cytotoxicity of methanethiol in human bronchial epithelial (16HBE) cells were investigated. Results indicate that methanethiol (10-50 µM) was partially converted into dimethyl sulfide, mainly catalyzed by thiol S-methyltransferase in the 16HBE cells, and then it induced potent cytotoxicity and cell membrane permeability. Moreover, methanethiol induced intracellular reactive oxygen species (ROS) up to 50 µM and further activated the tumor necrosis factor (TNF) signaling pathway, which eventually led to the decline in the mitochondrial membrane potential (MMP) and cell necrosis. However, all these effects were significantly alleviated with gene silencing of the methyltransferase-like protein 7B (METTL7B). These results indicate that methanethiol may induce cell necrosis in human respiratory tract cells mainly mediated by S-methyltransferase with interfering TNF and ROS induction. Non-target metabolomics results suggest that methanethiol potently affects expression of endogenous small molecule metabolites in 16HBE cells. To some extent, this work shows the possible conversion path and potential injury mechanism of human respiratory tract cells exposed to methanethiol.


Assuntos
Brônquios , Proteínas de Transporte , Metiltransferases , Compostos de Sulfidrila , Brônquios/efeitos dos fármacos , Brônquios/enzimologia , Brônquios/patologia , Proteínas de Transporte/metabolismo , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Necrose , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/farmacologia
5.
J Med Chem ; 65(8): 6231-6249, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35439007

RESUMO

Enzymes involved in RNA capping of SARS-CoV-2 are essential for the stability of viral RNA, translation of mRNAs, and virus evasion from innate immunity, making them attractive targets for antiviral agents. In this work, we focused on the design and synthesis of nucleoside-derived inhibitors against the SARS-CoV-2 nsp14 (N7-guanine)-methyltransferase (N7-MTase) that catalyzes the transfer of the methyl group from the S-adenosyl-l-methionine (SAM) cofactor to the N7-guanosine cap. Seven compounds out of 39 SAM analogues showed remarkable double-digit nanomolar inhibitory activity against the N7-MTase nsp14. Molecular docking supported the structure-activity relationships of these inhibitors and a bisubstrate-based mechanism of action. The three most potent inhibitors significantly stabilized nsp14 (ΔTm ≈ 11 °C), and the best inhibitor demonstrated high selectivity for nsp14 over human RNA N7-MTase.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/tratamento farmacológico , COVID-19/virologia , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/química , Humanos , Metiltransferases , Simulação de Acoplamento Molecular , RNA Viral/genética , S-Adenosilmetionina , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química
6.
Proc Natl Acad Sci U S A ; 119(14): e2107994119, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35363566

RESUMO

SignificanceMannitol biosynthesis is essential for Acinetobacter baumannii to cope with osmotic stress. Currently, only Pseudomonas putida, Acinetobacter baylyi, and A. baumannii are able to de novo synthesize mannitol by a structurally unique bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). The molecular mechanism of reduction and dephosphorylation of fructose-6-phosphate to mannitol is highly dependent on the substrate shuffling from one protomer to the other protomer by a unique helix-loop-helix domain-mediated dimer formation, thus ensuring unidirectional and efficient biosynthesis of mannitol. These observations support an evolutionary adaptation of AbMtlD by fusion of dehydrogenase and phosphatase domains to facilitate efficient unidirectional enzymatic production of mannitol, unifying regulatory control and minimizing the intracellular concentration of toxic mannitol-1-phosphate during salt stress.


Assuntos
Acinetobacter baumannii , Sequências Hélice-Alça-Hélice , Manitol , Desidrogenase do Álcool de Açúcar , Acinetobacter baumannii/enzimologia , Manitol/metabolismo , Pressão Osmótica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Estresse Salino , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo
7.
Sci Rep ; 12(1): 5664, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35383201

RESUMO

Genetic variants that regulate lipid phosphate phosphatase 3 (LPP3) expression are risk factors for the development of atherosclerotic cardiovascular disease. LPP3 is dynamically upregulated in the context of vascular inflammation with particularly heightened expression in smooth muscle cells (SMC), however, the impact of LPP3 on vascular pathology is not fully understood. We investigated the role of LPP3 and lysophospholipid signaling in a well-defined model of pathologic aortic injury and observed Angiotensin II (Ang II) increases expression of PLPP3 in SMCs through nuclear factor kappa B (NF-κB) signaling Plpp3 global reduction (Plpp3+/-) or SMC-specific deletion (SM22-Δ) protects hyperlipidemic mice from AngII-mediated aneurysm formation. LPP3 expression regulates SMC differentiation state and lowering LPP3 levels promotes a fibroblast-like phenotype. Decreased inactivation of bioactive lysophosphatidic acid (LPA) in settings of LPP3 deficiency may underlie these phenotypes because deletion of LPA receptor 4 in mice promotes early aortic dilation and rupture in response to AngII. LPP3 expression and LPA signaling influence SMC and vessel wall responses that are important for aortic dissection and aneurysm formation. These findings could have important implications for therapeutics targeting LPA metabolism and signaling in ongoing clinical trials.


Assuntos
Angiotensina II , Aneurisma da Aorta Abdominal , Miócitos de Músculo Liso , Fosfatidato Fosfatase , Animais , Aneurisma da Aorta Abdominal/patologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/enzimologia , Fosfatidato Fosfatase/metabolismo
8.
Proc Natl Acad Sci U S A ; 119(16): e2200476119, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35412887

RESUMO

SignificanceThe receptor tyrosine kinase Alk was originally discovered as an oncogenic fusion protein generated in anaplastic large cell lymphoma. A variety of oncogenic Alk fusion proteins were subsequently identified as key drivers of subsets of different cancers, including non-small cell lung cancer patients, large B cell lymphomas, and inflammatory myofibroblast tumors. In addition, activating oncogenic somatic mutations were identified in populations of pediatric neuroblastoma patients. Crizotininb, lorlatinib, and other drugs that inhibit the tyrosine kinase activity of Alk were successfully applied for the treatment of patients harboring oncogenic Alk mutants. In this report, we present experiments demonstrating that the physiological ligands of Alk function in the hypothalamus to control body weight, offering new therapeutic treatments for metabolic diseases and cancer.


Assuntos
Quinase do Linfoma Anaplásico , Peso Corporal , Citocinas , Hipotálamo , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Hipotálamo/metabolismo , Ligantes , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Magreza/genética
9.
Sci Rep ; 12(1): 6367, 2022 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-35430596

RESUMO

The identification of acquired resistance mutations has been essential in non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) active mutations. Rebiopsy plays a pivotal role in selecting the optimal treatment for patients who develop resistance to initial EGFR-tyrosine kinase inhibitors (EGFR-TKIs). This multicenter, observational study was conducted to investigate the details of rebiopsy in Japanese clinical practice. The primary endpoints were the implementation rate of rebiopsy and the concordance rate for T790M mutation detection between histological and cytological specimens using the cobas EGFR Mutation Test, version 2. One hundred ninety-four patients with EGFR-mutant NSCLC were enrolled, and 120 patients developed acquired resistance to EGFR-TKIs. The median age was 68 years (range 20-87), and 52.5% of the patients were women. Rebiopsy was performed in 109 patients, and the implementation rate of rebiopsy was 90.8%. The success rates of rebiopsy in the total, histology, cytology and liquid biopsy populations were 67.9%, 81.3%, 66.7% and 43.8%, respectively. The positive percent agreement and the negative percent agreement in the detection of the T790M mutation between the histological and cytological specimens were both 90.9%. Obtaining histological or cytological tissue samples at rebiopsy may contribute to improving the detection rate of the T790M mutation (trial registration number: UMIN000026019).


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Adulto Jovem
11.
Proc Natl Acad Sci U S A ; 119(15): e2100361119, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35394876

RESUMO

SignificanceLand plants produce numerous terpenoids that regulate development and mediate environmental interactions. Thus, how typical plant terpene synthase (TPS) genes originated and evolved to create terpenoid diversity is of fundamental interest. By investigating TPSs from the genomes and transcriptomes of diverse taxa of green plants, it was demonstrated here that the ancestral TPS gene originated in land plants after divergence from green algae and encoded a bifunctional ent-kaurene synthase for phytohormone biosynthesis. This ancestral TPS then underwent gene duplication at least twice early in land plant evolution, leading to three ancient TPS lineages reflecting sub-functionalization of class I and II activities for phytohormone biosynthesis and neo-functionalization from primary to secondary metabolism, followed in each case by dynamic functional divergence.


Assuntos
Alquil e Aril Transferases , Embriófitas , Evolução Molecular , Proteínas de Plantas , Alquil e Aril Transferases/classificação , Alquil e Aril Transferases/genética , Embriófitas/enzimologia , Embriófitas/genética , Duplicação Gênica , Filogenia , Reguladores de Crescimento de Plantas , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Terpenos/metabolismo
12.
Proc Natl Acad Sci U S A ; 119(15): e2113310119, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35377817

RESUMO

SignificanceThis study demonstrates that maternal inflammation during pregnancy perturbs blood-brain barrier formation via cyclooxygenase activation in fetal microglia, leading to abnormal cerebrovascular function and chronic brain inflammation persisting across the offspring life span. Therefore, developmental disruption of blood-brain barrier formation could be an important etiological factor contributing to the pathology of neurodevelopmental disorders.


Assuntos
Barreira Hematoencefálica , Ciclo-Oxigenase 2 , Encefalite , Troca Materno-Fetal , Microglia , Efeitos Tardios da Exposição Pré-Natal , Animais , Barreira Hematoencefálica/anormalidades , Barreira Hematoencefálica/fisiopatologia , Celecoxib/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Encefalite/imunologia , Feminino , Deleção de Genes , Troca Materno-Fetal/imunologia , Camundongos , Microglia/enzimologia , Poli I-C/imunologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/imunologia
13.
Proc Natl Acad Sci U S A ; 119(15): e2116844119, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35377811

RESUMO

SignificanceIt is well known that silent memory engrams in pathological or artificial conditions can be artificially switched into the latent state for retrieval by natural recall cues. Thus, physiological strategies that depend on the underlying molecular mechanisms for switching between silent state and latent state are a subject for investigation. Here, we show that social experiences stimulated switching between latent and silent engrams to achieve flexible memory accessibility and also reveal the basic molecular mechanism of: 1) social reward turning silent engram to latent via suppression of Rac1 activity in CA1 neurons of the hippocampus; and 2) social stress switching latent memory engram into silent through activating Rac1. Together, this work demonstrates emotion-driven bidirectional switching between latent and silent engrams.


Assuntos
Região CA1 Hipocampal , Rememoração Mental , Comportamento Social , Proteínas rac1 de Ligação ao GTP , Animais , Região CA1 Hipocampal/enzimologia , Sinais (Psicologia) , Rememoração Mental/fisiologia , Camundongos , Neurônios/enzimologia , Recompensa , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
Proc Natl Acad Sci U S A ; 119(16): e2117142119, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35380892

RESUMO

SignificanceCOVID-19 is a deadly rampaging infectious disease with over 480 million cases worldwide. Unfortunately, effective therapies remain very limited. Novel antiviral agents are urgently needed to combat this global healthcare crisis. Here, we elucidate the structural basis for replicase polyprotein cleavage and substrate specificity of SARS-CoV-2 main protease (Mpro). Through analyzing a series of high-resolution structures of SARS-CoV-2 Mpro throughout the proteolytic process, we demonstrate the molecular mechanism of Mpro in proteolytic processing that confers substrate specificity. Substrate selectivity is revealed using structures of the H41A mutant in complex with six individual native cleavage substrates. Our study underscores the mechanistic function of Mpro in the viral life cycle, which provides structural insights to develop effective inhibitors against this essential target of SARS-CoV-2.


Assuntos
Proteases 3C de Coronavírus , RNA-Polimerase RNA-Dependente de Coronavírus , SARS-CoV-2 , Antivirais/química , Proteases 3C de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Poliproteínas/química , Conformação Proteica , Proteólise , SARS-CoV-2/enzimologia , Especificidade por Substrato/genética
15.
PLoS One ; 17(4): e0267509, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35452491

RESUMO

ß-Mannans are a heterogeneous group of polysaccharides with a common main chain of ß-1,4-linked mannopyranoside residues. The cleavage of ß-mannan chains is catalyzed by glycoside hydrolases called ß-mannanases. In the CAZy database, ß-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.


Assuntos
Firmicutes , Glicosídeo Hidrolases , Mananas , Firmicutes/enzimologia , Firmicutes/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Mananas/química , Manose , Especificidade por Substrato , beta-Manosidase/metabolismo
16.
Biochem Biophys Res Commun ; 608: 52-58, 2022 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-35390672

RESUMO

Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.


Assuntos
Retículo Endoplasmático , Neoplasias , Sialiltransferases , Processos de Crescimento Celular , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Gangliosídeos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Sialiltransferases/metabolismo
18.
Lancet Neurol ; 21(5): 417-427, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35429480

RESUMO

BACKGROUND: CDKL5 deficiency disorder (CDD) is a rare, X-linked, developmental and epileptic encephalopathy characterised by severe global developmental impairment and seizures that can begin in the first few months after birth and are often treatment refractory. Ganaxolone, an investigational neuroactive steroid, reduced seizure frequency in an open-label, phase 2 trial that included patients with CDD. We aimed to further assess the efficacy and safety of ganaxolone in patients with CDD-associated refractory epilepsy. METHODS: In the double-blind phase of this randomised, placebo-controlled, phase 3 trial, done at 39 outpatient clinics in eight countries (Australia, France, Israel, Italy, Poland, Russia, the UK, and the USA), patients were eligible if they were aged 2-21 years with a pathogenic or probably pathogenic CDKL5 variant and at least 16 major motor seizures (defined as bilateral tonic, generalised tonic-clonic, bilateral clonic, atonic, or focal to bilateral tonic-clonic) per 28 days in each 4-week period of an 8-week historical period. After a 6-week prospective baseline period, patients were randomly assigned (1:1) via an interactive web response system to receive either enteral adjunctive ganaxolone or matching enteral adjunctive placebo (maximum dose 63 mg/kg per day for patients weighing ≤28 kg or 1800 mg/day for patients weighing >28 kg) for 17 weeks. Patients, caregivers, investigators (including those analysing data), trial staff, and the sponsor (other than the investigational product manager) were masked to treatment allocation. The primary efficacy endpoint was percentage change in median 28-day major motor seizure frequency from the baseline period to the 17-week double-blind phase and was analysed (using a Wilcoxon-rank sum test) in all patients who received at least one dose of trial treatment and for whom baseline data were available. Safety (compared descriptively across groups) was analysed in all patients who received at least one dose of trial treatment. This study is registered with ClinicalTrials.gov, NCT03572933, and the open-label extension phase is ongoing. FINDINGS: Between June 25, 2018, and July 2, 2020, 114 patients were screened for eligibility, of whom 101 (median age 6 years [IQR 3 to 10]) were randomly assigned to receive either ganaxolone (n=50) or placebo (n=51). All patients received at least one dose of a study drug, but seizure frequency for one patient in the ganaxolone group was not recorded at baseline and so the primary endpoint was analysed in a population of 100 patients. There was a median percentage change in 28-day major motor seizure frequency of -30·7% (IQR -49·5 to -1·9) in the ganaxolone group and of -6·9% (-24·1 to 39·7) in the placebo group (p=0·0036). The Hodges-Lehmann estimate of median difference in responses to ganaxolone versus placebo was -27·1% (95% CI -47·9 to - 9·6). Treatment-emergent adverse events occurred in 43 (86%) of 50 patients in the ganaxolone group and in 45 (88%) of 51 patients in the placebo group. Somnolence, pyrexia, and upper respiratory tract infections occurred in at least 10% of patients in the ganaxolone group and more frequently than in the placebo group. Serious adverse events occurred in six (12%) patients in the ganaxolone group and in five (10%) patients in the placebo group. Two (4%) patients in the ganaxolone group and four (8%) patients in the placebo group discontinued the trial. There were no deaths in the double-blind phase. INTERPRETATION: Ganaxolone significantly reduced the frequency of CDD-associated seizures compared with placebo and was generally well tolerated. Results from what is, to our knowledge, the first controlled trial in CDD suggest a potential treatment benefit for ganaxolone. Long-term treatment is being assessed in the ongoing open-label extension phase of this trial. FUNDING: Marinus Pharmaceuticals.


Assuntos
Síndromes Epilépticas , Pregnanolona , Espasmos Infantis , Criança , Pré-Escolar , Método Duplo-Cego , Síndromes Epilépticas/tratamento farmacológico , Síndromes Epilépticas/enzimologia , Humanos , Lactente , Pregnanolona/análogos & derivados , Estudos Prospectivos , Convulsões/tratamento farmacológico , Convulsões/enzimologia , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/enzimologia , Resultado do Tratamento
19.
Molecules ; 27(7)2022 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-35408682

RESUMO

A new dicoumarin, jusan coumarin, (1), has been isolated from Artemisia glauca aerial parts. The chemical structure of jusan coumarin was estimated, by 1D, 2D NMR as well as HR-Ms spectroscopic methods, to be 7-hydroxy-6-methoxy-3-[(2-oxo-2H-chromen-6-yl)oxy]-2H-chromen-2-one. As the first time to be introduced in nature, its potential against SARS-CoV-2 has been estimated using various in silico methods. Molecular similarity and fingerprints experiments have been utilized for 1 against nine co-crystallized ligands of COVID-19 vital proteins. The results declared a great similarity between Jusan Coumarin and X77, the ligand of COVID-19 main protease (PDB ID: 6W63), Mpro. To authenticate the obtained outputs, a DFT experiment was achieved to confirm the similarity of X77 and 1. Consequently, 1 was docked against Mpro. The results clarified that 1 bonded in a correct way inside Mpro active site, with a binding energy of -18.45 kcal/mol. Furthermore, the ADMET and toxicity profiles of 1 were evaluated and showed the safety of 1 and its likeness to be a drug. Finally, to confirm the binding and understand the thermodynamic characters between 1 and Mpro, several molecular dynamics (MD) simulations studies have been administered. Additionally, the known coumarin derivative, 7-isopentenyloxycoumarin (2), has been isolated as well as ß-sitosterol (3).


Assuntos
Artemisia , Proteases 3C de Coronavírus , Cumarínicos , Inibidores de Proteases , SARS-CoV-2 , Artemisia/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Cumarínicos/química , Cumarínicos/farmacologia , Dicumarol/química , Dicumarol/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
20.
Molecules ; 27(7)2022 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-35408716

RESUMO

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.


Assuntos
Arabidopsis , Fosfolipases A1 , Proteínas de Plantas , Arabidopsis/enzimologia , Capsicum/enzimologia , Dimerização , Ésteres , Fosfolipases A1/química , Proteínas de Plantas/química
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