RESUMEN
The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.
Asunto(s)
Proteínas Bacterianas , Escherichia coli , Inteínas , Mycobacterium tuberculosis , Empalme de Proteína , Rec A Recombinasas , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Rec A Recombinasas/metabolismo , Rec A Recombinasas/genética , Inteínas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas/genética , Daño del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Serina EndopeptidasasRESUMEN
Coronavirus disease 2019 (COVID-19) has led to a significant number of infections and deaths worldwide, yet its pathogenesis and severity remain incompletely understood. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), play crucial roles as receptors and molecules responsible for the virus's entry into host cells, initiating the infection process. Their polymorphisms have been extensively studied in relation to COVID-19 severity. The aim of this study was to examine the association of ACE2 (rs2074192) and TMPRSS2 (rs12329760) polymorphisms with COVID-19 outcome and severity. A prospective cohort study was conducted in 2022 at Haji Adam Malik Hospital, Medan, Indonesia. We randomly recruited hospitalized adult patients with COVID-19, confirmed by real-time polymerase chain reaction (RT-PCR). The baseline demographic data, disease severity, underlying disease, comorbidities, and COVID-19 vaccination status were collected. The single-nucleotide polymorphism (SNP) was assessed using TaqMan SNP genotyping assay, and the levels of TMPRSS2 and ACE2 proteins were measured using enzyme-linked immunosorbent assay (ELISA). A total of 151 COVID-19 patients were recruited and there were significant associations between age and severity with mortality outcomes. The age, kidney and lung diseases, and vaccination status were associated with severity levels. The results showed the CC genotype of ACE2 had the highest proportion, followed by TT and CT genotypes among patients, while CT was the most prevalent genotype, followed by CC and TT for TMPRSS2. This study did not find a significant association between ACE2 and TMPRSS2 genetic variants with disease severity and outcome but highlighted a specific association of TMPRSS2 SNP with mortality within the group. In addition, ACE2 concentration was significant different between mild-moderate and severe-critical COVID-19 groups (p=0.033).
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Polimorfismo de Nucleótido Simple , Serina Endopeptidasas , Índice de Severidad de la Enfermedad , Humanos , COVID-19/genética , COVID-19/mortalidad , COVID-19/epidemiología , Serina Endopeptidasas/genética , Enzima Convertidora de Angiotensina 2/genética , Masculino , Femenino , Estudios Prospectivos , Persona de Mediana Edad , Indonesia/epidemiología , Adulto , Centros de Atención Terciaria , Anciano , SARS-CoV-2RESUMEN
Fibrous dysplasia (FD) is a mosaic skeletal disorder involving the development of benign, expansile fibro-osseous lesions during childhood that cause deformity, fractures, pain, and disability. There are no well-established treatments for FD. Fibroblast activation protein (FAPα) is a serine protease expressed in pathological fibrotic tissues that has promising clinical applications as a biomarker and local pro-drug activator in several pathological conditions. In this study, we explored the expression of FAP in FD tissue and cells through published genetic expression datasets and measured circulating FAPα in plasma samples from patients with FD and healthy donors. We found that FAP genetic expression was increased in FD tissue and cells, and present at higher concentrations in plasma from patients with FD compared to healthy donors. Moreover, FAPα levels were correlated with skeletal disease burden in patients with FD. These findings support further investigation of FAPα as a potential imaging and/or biomarker of FD, as well as a pro-drug activator specific to FD tissue.
Asunto(s)
Endopeptidasas , Displasia Fibrosa Ósea , Gelatinasas , Proteínas de la Membrana , Serina Endopeptidasas , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Femenino , Masculino , Endopeptidasas/metabolismo , Endopeptidasas/genética , Gelatinasas/metabolismo , Gelatinasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Displasia Fibrosa Ósea/metabolismo , Displasia Fibrosa Ósea/genética , Displasia Fibrosa Ósea/patología , Adulto , Adolescente , Niño , Biomarcadores/metabolismo , Biomarcadores/sangre , Osteoblastos/metabolismo , Osteoblastos/patología , Persona de Mediana EdadRESUMEN
Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688, show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline, however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307, an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71, a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.
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Antivirales , Proteínas no Estructurales Virales , Antivirales/farmacología , Antivirales/química , Humanos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Animales , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/uso terapéutico , Ensayos Clínicos como Asunto , Serina Endopeptidasas/metabolismo , Replicación Viral/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Virus Zika/efectos de los fármacos , Virus del Nilo Occidental/efectos de los fármacosRESUMEN
This study aimed to investigate the impact of different types of nasal inflammation on the regulation of entry-associated genes of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), and influenza virus, in the nasal epithelium. Subjects were classified into three groups: control, eosinophilic chronic rhinosinusitis (ECRS), and noneosinophilic CRS (NECRS) groups. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2), alanyl aminopeptidase (ANPEP), dipeptidyl peptidase 4 (DPP4), and beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1), and beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) were selected as key entry-associated genes for SARS-CoV-2, HCoV-229E, MERS-CoV, and influenza, respectively, and were evaluated. Brushing samples obtained from each group and human nasal epithelial cells cultured using an air-liquid interface system were treated for 7 days with typical inflammatory cytokines and analyzed using real-time polymerase chain reaction. Western blot analysis and confocal microscopy were performed. The entry-associated genes showed distinct regulation patterns in response to each interleukin-4 (IL-4), interleukin-13 (IL-13), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Specifically, ACE2 significantly decreased in type 2 cytokines (IL-4 and IL-13), while TMPRSS2 significantly decreased in type 1 cytokines (TNF-α and IFN-γ). ANPEP significantly decreased in both types of cytokines. Remarkably, DPP4 significantly increased in type 2 cytokines and decreased in type 1 cytokines. Moreover, ST6GAL1 and ST3GAL4 significantly increased in type 2 cytokines and decreased in type 1 cytokines, particularly IFN-γ. These findings were supported by western blot analysis and confocal imaging results, especially for ACE2 and DPP4. The findings regarding differential regulation suggest that patients with ECRS, primarily mediated by type 2 inflammation, may have lower susceptibility to SARS-CoV-2 and HCoV-229E infections but higher susceptibility to MERS-CoV and influenza infections.
Asunto(s)
Citocinas , Mucosa Nasal , Internalización del Virus , Humanos , Citocinas/genética , Citocinas/metabolismo , Mucosa Nasal/virología , Adulto , Masculino , Femenino , Persona de Mediana Edad , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Sinusitis/virología , Sinusitis/genética , Sinusitis/inmunología , SARS-CoV-2/inmunología , Rinitis/virología , Rinitis/genética , Rinitis/inmunología , Regulación de la Expresión Génica , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , COVID-19/inmunología , COVID-19/virología , Coronavirus Humano 229E/genética , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunologíaRESUMEN
The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin-a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells.
Asunto(s)
Uniones Adherentes , Antígenos CD , Cadherinas , Células Endoteliales , Proteína Reelina , Animales , Humanos , Ratones , Uniones Adherentes/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Cadherinas/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Células Endoteliales/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Uniones Intercelulares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas , Proteómica/métodos , Serina Endopeptidasas/metabolismoRESUMEN
BACKGROUND: Serum corin has been associated with stroke in observational studies, but the underlying causality is uncertain. This study examined the causal association between corin and stroke through Mendelian randomization study. METHODS AND RESULTS: In the Gusu cohort, serum corin was assayed at baseline, and stroke incidents were prospectively obtained during 10 years of follow-up. Single-nucleotide polymorphisms (SNPs) in CORIN were genotyped by MassArray for 2310 participants (mean age, 53 years; 39% men). Seventeen SNPs passed the Hardy-Weinberg test and were considered the potential instruments. Only 1 SNP (rs2271037) determined variability of serum corin was significantly associated with stroke even after adjusting for conventional risk factors (hazard ratio [HR], 1.36 [95% CI, 1.00-1.85]). The weighted genetic risk score generated from the SNP-corin associations was significantly associated with stroke (HR, 2.01 [95% CI, 1.15-3.51]). Using this genetic risk score as the instrument, 1-sample Mendelian randomization analysis found a significant HR of stroke per-SD higher log2-transformed corin (HR, 1.37 [95% CI, 1.07-1.76]). The inverse variance-weighted analysis based on the SNP-corin and SNP-stroke associations found that the HR of stroke pre-SD higher log2-transformed corin was 5.92 (95% CI, 2.23-15.72). The effect estimates stayed consistent regardless of an individual SNP being removed from the instruments. An almost identical effect estimate was also confirmed by multiple other 2-sample Mendelian randomization methods. CONCLUSIONS: Genetically determined variations of serum corin concentration were significantly associated with the risk of stroke in Chinese adults. Elevated serum corin may be a risk factor for stroke.
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Análisis de la Aleatorización Mendeliana , Polimorfismo de Nucleótido Simple , Serina Endopeptidasas , Accidente Cerebrovascular , Humanos , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/epidemiología , Femenino , Persona de Mediana Edad , Masculino , China/epidemiología , Serina Endopeptidasas/genética , Serina Endopeptidasas/sangre , Factores de Riesgo , Estudios Prospectivos , Predisposición Genética a la Enfermedad , Pueblo Asiatico/genética , Adulto , Medición de Riesgo , Biomarcadores/sangre , Anciano , Incidencia , Pueblos del Este de AsiaRESUMEN
Early in the SARS-CoV2 pandemic, in this journal, Hou et al. (BMC Med 18:216, 2020) interpreted public genotype data, run through functional prediction tools, as suggesting that members of particular human populations carry potentially COVID-risk-increasing variants in genes ACE2 and TMPRSS2 far more often than do members of other populations. Beyond resting on predictions rather than clinical outcomes, and focusing on variants too rare to typify population members even jointly, their claim mistook a well known artifact (that large samples reveal more of a population's variants than do small samples) as if showing real and congruent population differences for the two genes, rather than lopsided population sampling in their shared source data. We explain that artifact, and contrast it with empirical findings, now ample, that other loci shape personal COVID risks far more significantly than do ACE2 and TMPRSS2-and that variation in ACE2 and TMPRSS2 per se unlikely exacerbates any net population disparity in the effects of such more risk-informative loci.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Serina Endopeptidasas , Humanos , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/genética , COVID-19/epidemiología , Predisposición Genética a la Enfermedad , SARS-CoV-2/genética , Serina Endopeptidasas/genéticaRESUMEN
Elevated serum corin concentrations in patients with cardiac diseases have been associated with adverse cardiovascular events and progressive renal dysfunction. This study aimed to determine the role of serum corin levels in predicting the incidence of acute kidney injury (AKI) and mortality in critically ill patients admitted to intensive care units (ICUs). We screened 323 patients admitted to the ICU in our institution from May 2018 through December 2019. After excluding patients receiving renal replacement therapy, 288 subjects were enrolled. Cases were divided equally into high (n = 144) and low (n = 144) corin groups according to median serum corin levels, using 910 pg/mL as the cut-off point. Patient characteristics and comorbidities were collected from medical records. The primary outcome was AKI within 48 h after ICU admission, while the secondary outcome was all-cause of mortality within 1 year. Compared with the low corin group, patients in the high corin group had higher prevalence rates of diabetes, cirrhosis, and nephrotoxic agent exposure; higher Sequential Organ Failure Assessment scores, white blood cell counts, proteinuria, and serum N-terminal pro-brain natriuretic peptide levels; but had lower initial estimated glomerular filtration rates. Furthermore, elevated serum corin was associated with higher risks of AKI within 48h of ICU admission (43.1% vs. 18.1%, p < 0.001) and all-cause mortality within one year (63.9% vs. 50.0%, p = 0.024). High corin level showed strongly positive results as an independent predictor of AKI (OR 2.15, 95% CI 1.11-4.19, p = 0.024) but not for the all-cause mortality after adjusting for confounding factors in multivariate analyses. Elevated circulating corin predicted AKI in critically ill patients, but did not predict all-cause mortality within 1 year. As a key enzyme in renin-angiotensin-aldosterone system, corin expression may be regulated through a feedback loop following natriuretic peptide resistance and desensitization of natriuretic peptide receptors in different critically ill status.
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Lesión Renal Aguda , Enfermedad Crítica , Unidades de Cuidados Intensivos , Humanos , Masculino , Lesión Renal Aguda/sangre , Lesión Renal Aguda/mortalidad , Femenino , Enfermedad Crítica/mortalidad , Anciano , Persona de Mediana Edad , Serina Endopeptidasas/sangre , Biomarcadores/sangre , Factores de Riesgo , PronósticoRESUMEN
Olfactory perception is an important physiological function for human well-being and health. Loss of olfaction, or anosmia, caused by viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received considerable attention, especially in persistent cases that take a long time to recover. This review discusses the integration of different components of the olfactory epithelium to serve as a structural and functional unit and explores how they are affected during viral infections, leading to the development of olfactory dysfunction. The review mainly focused on the role of receptors mediating the disruption of olfactory signal transduction pathways such as angiotensin converting enzyme 2 (ACE2), transmembrane protease serine type 2 (TMPRSS2), neuropilin 1 (NRP1), basigin (CD147), olfactory, transient receptor potential vanilloid 1 (TRPV1), purinergic, and interferon gamma receptors. Furthermore, the compromised function of the epithelial sodium channel (ENaC) induced by SARS-CoV-2 infection and its contribution to olfactory dysfunction are also discussed. Collectively, this review provides fundamental information about the many types of receptors that may modulate olfaction and participate in olfactory dysfunction. It will help to understand the underlying pathophysiology of virus-induced anosmia, which may help in finding and designing effective therapies targeting molecules involved in viral invasion and olfaction. To the best of our knowledge, this is the only review that covered all the receptors potentially involved in, or mediating, the disruption of olfactory signal transduction pathways during COVID-19 infection. This wide and complex spectrum of receptors that mediates the pathophysiology of olfactory dysfunction reflects the many ways in which anosmia can be therapeutically managed.
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Anosmia , COVID-19 , SARS-CoV-2 , Humanos , COVID-19/metabolismo , COVID-19/complicaciones , COVID-19/fisiopatología , COVID-19/virología , Anosmia/fisiopatología , Anosmia/etiología , Anosmia/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Mucosa Olfatoria/metabolismo , Mucosa Olfatoria/virología , Transducción de Señal , Serina Endopeptidasas/metabolismo , Neuropilina-1/metabolismo , Basigina/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
The pre-fusion coronavirus HKU1 spike binds host sialoglycans and proteinaceous receptor TMPRSS2 for cell entry. In this issue of Cell, three papers by Fernández et al., McCallum et al., and Wang et al. provide structural information on HKU1 spike interactions with host receptors, providing insights into its multi-step opening.
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Serina Endopeptidasas , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Humanos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologíaRESUMEN
Introduction: Fibroblast activation protein (FAP) overexpression on cancer-associated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. Methods: In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAP-positive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results: Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Discussion: Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells.
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Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Animales , Femenino , Humanos , Ratones , Fibroblastos Asociados al Cáncer/inmunología , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Reacciones Cruzadas/inmunología , Endopeptidasas , Gelatinasas/inmunología , Gelatinasas/metabolismo , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/efectos adversos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dengue fever, prevalent in Southeast Asian countries, currently lacks effective pharmaceutical interventions for virus replication control. This study employs a strategy that combines machine learning (ML)-based quantitative-structure-activity relationship (QSAR), molecular docking, and molecular dynamics simulations to discover potential inhibitors of the NS3 protease of the dengue virus. We used nine molecular fingerprints from PaDEL to extract features from the NS3 protease dataset of dengue virus type 2 in the ChEMBL database. Feature selection was achieved through the low variance threshold, F-Score, and recursive feature elimination (RFE) methods. Our investigation employed three ML models - support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost) - for classifier development. Our SVM model, combined with SVM-RFE, had the best accuracy (0.866) and ROC_AUC (0.964) in the testing set. We identified potent inhibitors on the basis of the optimal classifier probabilities and docking binding affinities. SHAP and LIME analyses highlighted the significant molecular fingerprints (e.g. ExtFP69, ExtFP362, ExtFP576) involved in NS3 protease inhibitory activity. Molecular dynamics simulations indicated that amphotericin B exhibited the highest binding energy of -212 kJ/mol and formed a hydrogen bond with the critical residue Ser196. This approach enhances NS3 protease inhibitor identification and expedites the discovery of dengue therapeutics.
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Antivirales , Virus del Dengue , Reposicionamiento de Medicamentos , Serina Endopeptidasas , Proteínas no Estructurales Virales , Antivirales/química , Antivirales/farmacología , Virus del Dengue/enzimología , Virus del Dengue/efectos de los fármacos , Aprendizaje Automático , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad Cuantitativa , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteasas ViralesRESUMEN
West Nile virus (WNV) nonstructural protein 5 (NS5) possesses multiple enzymatic domains essential for viral RNA replication. During infection, NS5 predominantly localizes to unique replication organelles (ROs) at the rough endoplasmic reticulum (RER), known as vesicle packets (VPs) and convoluted membranes (CMs), with a portion of NS5 accumulating in the nucleus. NS5 is a soluble protein that must be in the VP, where its enzymatic activities are required for viral RNA synthesis. However, the mechanistic processes behind the recruitment of NS5 from the cytoplasm to the RER membrane remain unclear. Here, we utilize high-resolution confocal microscopy and sucrose density gradient ultracentrifugation to investigate whether the association of NS5 with other NS proteins contributes to its membrane recruitment and retention. We demonstrate that NS1 or NS3 partially influences the NS5 association with the membrane. We further demonstrate that processed NS5 is predominantly in the cytoplasm and nucleus, indicating that the processing of NS5 from the viral polyprotein does not contribute to its membrane localization. These observations suggest that other host or viral factors, such as the enwrapment of NS5 by the RO, may also be necessary for the complete membrane retention of NS5. Therefore, studies on the inhibitors that disrupt the membrane localization of WNV NS5 are warranted for antiviral drug development.
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Proteínas no Estructurales Virales , Virus del Nilo Occidental , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/fisiología , Humanos , Animales , Replicación Viral , ARN Helicasas/metabolismo , ARN Helicasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Células Vero , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fiebre del Nilo Occidental/virología , Línea Celular , Proteasas Virales , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-boxRESUMEN
Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study was to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition and dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compound's efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein-3 (NS3) might prove as a funamental target for Betanin and Glycine Betaine against Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 µM-10 µM) of selected phytochemicals. The dose-response assay illustrated downregulated expression of DENV-2 NS3 gene after administration of Betanin (IC50 = 4.35 µM) and Glycine Betaine (IC50 = 4.49 µM). Dose response analysis of Betanin (80 µM-10 µM) depicted the significant inhibition of NS3 protein expression as well. These results suggested downregulated expression of DENV-2 NS3 at mRNA and protein level portraying the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic.
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Antivirales , Betaína , Virus del Dengue , Simulación del Acoplamiento Molecular , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Humanos , Antivirales/farmacología , Células HeLa , Animales , Chlorocebus aethiops , Células Vero , Betaína/farmacología , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Betacianinas/farmacología , Células CHO , Cricetulus , Fitoquímicos/farmacología , Simulación de Dinámica Molecular , Replicación Viral/efectos de los fármacos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Dengue/tratamiento farmacológico , Dengue/virología , Proteasas ViralesRESUMEN
Fibroblast activation protein-α (FAP) has emerged as a promising target in the field of radiopharmaceuticals due to its selective expression in cancer-associated fibroblasts (CAFs) and other pathological conditions involving fibrosis and inflammation. Recent advancements have focused on developing FAP-specific radioligands for diagnostic imaging and targeted radionuclide therapy. This perspective summarized the latest progress in FAP radiopharmaceutical development, highlighting novel radioligands, preclinical evaluations, and potential clinical applications. Additionally, we analyzed the advantages and existing problems of targeted FAP radiopharmaceuticals, and discussed the key breakthrough directions of this target, so as to improve the development and conversion of FAP-targeted radiopharmaceuticals.
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Endopeptidasas , Gelatinasas , Proteínas de la Membrana , Radiofármacos , Serina Endopeptidasas , Humanos , Radiofármacos/química , Radiofármacos/farmacología , Endopeptidasas/metabolismo , Animales , Gelatinasas/metabolismo , Gelatinasas/antagonistas & inhibidores , Serina Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/antagonistas & inhibidoresRESUMEN
Microcephaly, Guillain-Barré syndrome, and potential sexual transmission stand as prominent complications associated with Zika virus (ZIKV) infection. The absence of FDA-approved drugs or vaccines presents a substantial obstacle in combatting the virus. Furthermore, the inclusion of pregnancy in the pharmacological screening process complicates and extends the endeavor to ensure molecular safety and minimal toxicity. Given its pivotal role in viral assembly and maturation, the NS2B-NS3 viral protease emerges as a promising therapeutic target against ZIKV. In this context, a dipeptide inhibitor was specifically chosen as a control against 200 compounds for docking analysis. Subsequent molecular dynamics simulations extending over 200 ns were conducted to ascertain the stability of the docked complex and confirm the binding of the inhibitor at the protein's active site. The simulation outcomes exhibited conformity to acceptable thresholds, encompassing parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), ligand-protein interaction analysis, ligand characterization, and surface area analysis. Notably, analysis of ligand angles bolstered the identification of prospective ligands capable of inhibiting viral protein activity and impeding virus dissemination. In this study, the integration of molecular docking and dynamics simulations has pinpointed the dipeptide inhibitor as a potential candidate ligand against ZIKV protease, thereby offering promise for therapeutic intervention against the virus.
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Dipéptidos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteasas , Proteínas no Estructurales Virales , Virus Zika , Virus Zika/enzimología , Virus Zika/efectos de los fármacos , Dipéptidos/química , Dipéptidos/farmacología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Antivirales/farmacología , Antivirales/química , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Humanos , Unión Proteica , Proteasas Virales , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-boxRESUMEN
OBJECTIVE: To explore the role and molecular mechanism of cancer-associated fibroblasts (CAFs) in the tumor microenvironment of gastric cancer (GC). METHODS: The expression of CAFs in GC patients was first assessed for abundance, and survival analysis was performed. Subsequently, The Cancer Genome Atlas (TCGA) data were used for differential analysis, survival analysis, and EPIC analysis, while single-cell data (GSE183904) were downloaded for differential analysis of CAFs. Clinical data pooling, univariate and multivariate Cox analysis, and immunofluorescence were carried out on clinical GC tissue samples to explore RCN3 expression within patient CAFs. Western blot and quantitative polymerase chain reaction (qPCR) were used to detect the expression of RCN3. The relationship between RCN3, PCSK6, and STAT1 was explored by chromatin immunoprecipitation (CHIP) experiments, and the effects of the genes on macrophage polarization were detected by detecting biomarkers of biological M1/M2. RESULTS: CAFs in GC were found to be significantly higher compared to the normal group. Revealing the results of TCGA differential analysis, it was observed that GC exhibited a substantial upregulation in the expression levels of RCN3. The clinical statistics indicate a positive correlation between an elevated level of RCN3 expression and the T-stage classification of tumor size. In addition, RCN3 was found to have a significant impact on the overall survival of patients with gastric cancer, acting as an independent prognostic indicator. Analysis of single-cell data showed high expression of PCSK6 in macrophages, and immunofluorescence staining of samples from GC patients showed increased expression of PCSK6 on the cell membranes of macrophages in GC tissues. The subsequent cellular experiments confirmed RCN3 protein can regulate the expression of PCSK6, and PCSK6 regulates macrophage polarization through STAT1. CONCLUSIONS: CAFs regulate macrophage polarization through the RCN3/PCSK6/STAT1 pathway in GC.
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Fibroblastos Asociados al Cáncer , Macrófagos , Neoplasias Gástricas , Microambiente Tumoral , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Microambiente Tumoral/genética , Macrófagos/metabolismo , Femenino , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Regulación Neoplásica de la Expresión Génica , Masculino , Transducción de Señal , Persona de Mediana Edad , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Activación de Macrófagos/genéticaRESUMEN
The emergence of drug-resistance-inducing mutations in Hepatitis C virus (HCV) coupled with genotypic heterogeneity has made targeting NS3/4A serine protease difficult. In this work, we investigated the mutagenic variations in the binding pocket of Genotype 3 (G3) HCV NS3/4A and evaluated ligands for efficacious inhibition. We report mutations at 14 positions within the ligand-binding residues of HCV NS3/4A, including H57R and S139P within the catalytic triad. We then modelled each mutational variant for pharmacophore-based virtual screening (PBVS) followed by covalent docking towards identifying a potential covalent inhibitor, i.e., cpd-217. The binding stability of cpd-217 was then supported by molecular dynamic simulation followed by MM/GBSA binding free energy calculation. The free energy decomposition analysis indicated that the resistant mutants alter the HCV NS3/4A-ligand interaction, resulting in unbalanced energy distribution within the binding site, leading to drug resistance. Cpd-217 was identified as interacting with all NS3/4A G3 variants with significant covalent docking scores. In conclusion, cpd-217 emerges as a potential inhibitor of HCV NS3/4A G3 variants that warrants further in vitro and in vivo studies. This study provides a theoretical foundation for drug design and development targeting HCV G3 NS3/4A.