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。

基本信息

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
项目类别：
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

项目摘要

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.

抽象的我们研究的长期目标是开发针对 SARS-CoV-2 引发的疾病的有效疗法 Covid-19。SARS-CoV-2 刺突 (S) 蛋白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 (TMPRSSs) 协同作用，在上皮细胞中发现这些病毒进入因子的共表达。许多器官，包括口腔粘膜，最近发现口腔粘膜具有潜在的高风险。 GRHL2 作为转录因子调节上皮基因表达。 ACE2 和 TMPRSS2 在多种信号通路中强烈表达。上皮细胞，与口腔粘膜中 GRHL2 的表达模式一致，GRHL2 调节 ACE2。 GRHL2 的异位表达在正常人口腔角质形成细胞 (NHOK) 中的 TMPRSS2 表达。诱导 ACE2 和 TMPRSSs 表达，而 GRHL2 的敲除会导致这些表达水平降低 ACE2 是一种干扰素刺激基因，部分通过 IFN-α 激活 ACE2 表达。 GRHL2 的小分子抑制剂可抑制 NHOK 中的病毒进入因子表达。 ACE2和TMPRSS2在口腔粘膜上皮中广泛表达，GRHL2的敲除导致体内 ACE2 和 TMPRSS2 表达的抑制这些发现表明 GRHL2 是必需的。基于体外培养的 NHOK 和体内口腔粘膜上皮中 ACE2 和 TMPRSS 的表达。根据这些数据，我们发现靶向 GRHL2 可以抑制 SARS-CoV-2 传播至口腔这一新的假设将通过调节粘膜上皮细胞的病毒进入因子的表达来实现。通过检查 GRHL2 调节 SARS-CoV-2 表达的分子机制进行检查口腔粘膜上皮细胞中的进入因子（ACE2和TMPRSS）将采用方差分析方法。用于比较这些基因表达水平的平均值。我们还将生成一个假型病毒。水疱性口炎病毒（VSV）绿色荧光蛋白（GFP）报告病毒表达SARS- 利用这种嵌合病毒，可以用 CoV-2 S 替代天然糖蛋白，进行高通量检测。开发用于筛选 GRHL2 抑制剂，该抑制剂具有消除 SARS-CoV-2 病毒的潜力感染口腔粘膜上皮。