Targeting Grainyhead-Like 2 Suppresses Entry Factors of SARS-CoV-2 in Epithelial Cells of Oral Mucosa.

靶向 Grainyhead-Like 2 可抑制口腔粘膜上皮细胞中 SARS-CoV-2 的进入因子。

• 批准号: 10453095
• 负责人: WEI CHEN
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453095
• 关键词: 2019-nCoV; 3-Dimensional; ACE2; Analysis of Variance; Binding; Binding Proteins; Biological Assay; COVID-19; COVID-19 pandemic; COVID-19 patient; CRISPR/Cas technology; Cell Differentiation process; Cell Proliferation; Cell membrane; Cells; Containment; Data; Ectopic Expression; Epigenetic Process; Epithelial; Epithelial Cells; Foundations; Functional disorder; Gene Expression; Genes; Genetic Transcription; Gingiva; Glycoproteins; Goals; Green Fluorescent Proteins; Health; Human; In Vitro; Infection; Interferon-alpha; Interferons; Knock-out; Light; Mediating; Methods; Modeling; Molecular; Mus; Oral; Oral cavity; Oral mucous membrane structure; Organ; Outcome; Pattern; Peptide Hydrolases; Play; Predisposition; Process; Public Health; Publishing; Reporter; Research; Role; Route; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; SARS-CoV-2 spike protein; SARS-CoV-2 transmission; Salivary Glands; Serine Protease; Signal Pathway; Site; TMPRSS2 gene; Testing; Tissues; Transcriptional Regulation; Transduction Gene; Tropism; Vesicular stomatitis Indiana virus; Viral; Virus; Virus Diseases; base; coronavirus disease; effective therapy; experimental study; gain of function; high risk; high throughput screening; in vivo; inhibitor; keratinocyte; knock-down; knockout gene; monolayer; mouse model; new therapeutic target; novel; oral cavity epithelium; promoter; receptor; screening; small molecule inhibitor; transcription factor; translational potential; viral entry inhibitor

Abstract The long-term goal of our research is to develop effective therapies against SARS-CoV-2-elicited disease Covid-19. SARS-CoV-2 spike (S) protein binds to its cognate receptor, angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine proteases (TMPRSSs), promotes cellular entry. Co-expressions of these viral entry factors are found in the epithelial cells of many organs, including oral mucosa. Recently, oral mucosa has been found to be a potentially high-risk route of SARS-CoV-2 infection. GRHL2, as a transcriptional factor, modulates epithelial gene expression and plasticity through multiple signaling pathways. ACE2 and TMPRSS2 are strongly expressed in epithelial cells, coinciding with the pattern of GRHL2 expression in oral mucosa. GRHL2 regulates ACE2 and TMPRSS2 expression in normal human oral keratinocytes (NHOK). Ectopic expression of GRHL2 induces ACE2 and TMPRSSs expression, while knockdown of GRHL2 leads to decreased levels of these entry factors. ACE2 is an interferon-stimulated gene. IFN-α activates ACE2 expression in part through regulating GRHL2. Small-molecule inhibitors of GRHL2 suppress viral entry factor expression in NHOK. ACE2 and TMPRSS2 are widely expressed in oral mucosal epithelium, knockout of GRHL2 leads to suppression of ACE2 and TMPRSS2 expression in vivo. These findings indicate that GRHL2 is required for ACE2 and TMPRSS expression in NHOK cultured in vitro and oral mucosal epithelium in vivo. Based on these data, we hypothesize that targeting GRHL2 inhibits SARS-CoV-2 transmission into oral mucosal epithelium through regulating viral entry factor expression. This novel hypothesis will be tested by examining the molecular mechanism underlying GRHL2 regulates expression of SARS-CoV-2 entry factors (ACE2 and TMPRSSs) in oral mucosal epithelial cells. Analysis of variance methods will be used to compare means of these gene expression levels. We will also generate a pseudotyped virus in which vesicular stomatitis virus (VSV) green fluorescent protein (GFP) reporter virus expressing SARS- CoV-2 S in replacement of native glycoprotein. Utilizing this chimeric virus, a high throughput assay will be developed to screen GRHL2 inhibitors, which possess the potentials to abrogate SARS-CoV-2 virus infection into oral mucosal epithelium.

摘要 我们研究的长期目标是开发有效的治疗SARS-CoV-2引发的疾病的方法。 新冠肺炎。SARS-CoV-2刺突蛋白(S)与其同源受体血管紧张素转换酶2结合 (ACE2)，并与宿主蛋白酶，主要是跨膜丝氨酸蛋白酶(TMPRSS)协同工作， 促进手机进入。这些病毒进入因子的共同表达在肺腺癌的上皮细胞中被发现。 许多器官，包括口腔粘膜。最近，口腔粘膜被发现是一种潜在的高危因素。 SARS-CoV-2的感染途径。Grhl2作为转录因子调控上皮细胞基因表达 以及通过多条信号通路的可塑性。ACE2和TMPRSS2在 上皮细胞，与GRHL2在口腔粘膜中的表达模式一致。Grhl2调节血管紧张素转换酶2 TMPRSS2在正常口腔角质形成细胞(NHOK)中的表达。GRHL2基因的异位表达 诱导ACE2和TMPRSS表达，而GRHL2基因敲除导致这些表达水平下降 进入因素。ACE2是一种干扰素刺激基因。干扰素-α部分通过激活血管紧张素转换酶2的表达 调节GRHL2。GRHL2小分子抑制剂抑制NHOK中病毒进入因子的表达。 ACE2和TMPRSS2在口腔粘膜上皮细胞中广泛表达，GRHL2基因敲除导致 体内抑制ACE2和TMPRSS2的表达。这些发现表明GRHL2是必需的 在体外培养的NHOK和体内口腔粘膜上皮中检测ACE2和TMPRSS的表达。基座 根据这些数据，我们假设靶向GRHL2抑制SARS-CoV-2经口传播 粘膜上皮细胞通过调节病毒进入因子的表达。这一新的假设将是 GRHL2调控SARS-CoV-2表达的分子机制研究 口腔黏膜上皮细胞的进入因子(ACE2和TMPRSSs)差异分析方法将是 用于比较这些基因表达水平的平均值。我们还将在 表达SARS的水疱性口炎病毒(VSV)绿色荧光蛋白(GFP)报告病毒 冠状病毒2型S中天然糖蛋白的替代。利用这种嵌合病毒，高通量检测将 为筛选具有消灭SARS-CoV-2病毒潜力的GRHL2抑制剂而开发 感染口腔粘膜上皮。