Functionalized lipid inactosomes to bind and clear SARS-CoV-2

功能化脂质内切体结合并清除 SARS-CoV-2

• 批准号: 10611896
• 负责人: Daniel A Hammer
• 金额: $ 20.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-20 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10611896
• 关键词: 2019-nCoV; ACE2; Acute; Amino Acid Motifs; Amino Acids; Antibodies; Bacteria; Binding; Biological Assay; Biotechnology; COVID-19; Cell Line; Cell membrane; Cells; Cessation of life; Chemicals; Chemistry; Cholesterol; Coculture Techniques; Coronavirus; Development; Disease; Dissociation; Endocytosis; Engineering; Epithelial Cells; Epithelium; Family; Fat Body; Formulation; Future; Genes; Human; Hybrids; Hydrophobicity; In Vitro; Incubated; Infection; Infectious Agent; Integral Membrane Protein; Lecithin; Length; Lipids; Mediating; Membrane; Membrane Lipids; Micelles; Microfluidic Microchips; Microfluidics; Molecular; Molecular Conformation; Nanostructures; Nature; Peptide Hydrolases; Peptides; Phospholipids; Plants; Protein Binding Domain; Proteins; Pulmonary Surfactant-Associated Protein A; Recombinant Proteins; Recombinants; Reporter; Respiratory distress; SARS coronavirus; SARS-CoV-2 infection; Serine Protease; Severe Acute Respiratory Syndrome; Structure; Surface; Syndrome; TMPRSS2 gene; Tertiary Protein Structure; Testing; Therapeutic; Vaccines; Variant; Vesicle; Viral; Virion; Virus; Virus Diseases; Water; Work; Writing; amphiphilicity; biosafety level 3 facility; density; design; dimer; hydrophilicity; inhibitor; innovation; interfacial; mimetics; monomer; nanobodies; nanoparticle; nanovesicle; novel; novel coronavirus; pandemic disease; particle; peptidomimetics; precision medicine; prevent; protein reconstitution; receptor; receptor binding; reconstitution; respiratory; self assembly; success; surfactant; viral entry inhibitor

Summary Severe respiratory acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19, a world-wide pandemic causing over 525K deaths in the U.S. and over 2.6M deaths world-wide as of this writing. Here, we propose to make phospholipid vesicles and other related nanostructures (droplets and micelles) with recombinant protein motifs that will bind and inactivate the SARS-CoV-2 virion – we call these structures inactosomes. Our inactosomes will be novel hybrid materials into which functional recombinant proteins are reconstituted. The functionalized protein will be variants of oleosin, a naturally occurring surfactant protein. Previously, we have designed and produced novel oleosin variants that assemble into vesicle membranes and micelles and can stabilize droplets due to their triblock-like free-chain amphiphilic structure. We can readily incorporate functional peptide motifs into oleosin recombinantly. SARS-CoV-2 can enter epithelial cells via endocytosis and/or fusion. Binding between the spike protein (S) on SARS-CoV-2 and the ACE2 receptor is essential for the entry of the virus into the epithelium. The ACE2 receptor can mediate endocytosis. Alternatively, after binding to ACE2, a protease (TMPRSS2) can activate a conformational change in the S protein leading to fusogenic entry. We envision a number of chemistries that will be directly useful at interfering with infection by SARS-CoV-2. First, a family of spike protein binding motifs – mini-proteins, single chain antibodies (sybodies), or ACE2 peptide mimetics - will be recombinantly added to the hydrophilic ends of oleosin. These motifs have low dissociation constants with the spike protein receptor binding domain and are much easier to produce than large antibodies. When these virus binding motif-oleosins are reconstituted into nanostructures, the result will be a multivalent particle (inactosomes) that can bind directly to SARS-CoV-2 and competitively blocks its entry. Next, we will incorporate an oleosin that presents a small peptidic fusion inhibitor of the S protein to prevent fusogenic entry of the virus on cell lines expressing TMPRSS2. Peptides that block either binding and fusion can be combined to make multi-functional inhibitory inactosomes. In Aim 1, we will develop and characterize SARS-CoV-2 inactosomes that prevent endocytosis- mediated entry into cells. In Aim 2, we will develop and characterize the SARS-CoV-2 inactosomes that block fusogenic entry, followed by inactosomes which possess both ACE2 blocking peptides and anti- fusogenic peptides. Combinations of SARS-CoV-2 reporter particles (bearing an eGFP gene) and SARS-CoV-2 inactosomes will be incubated in a co-culture to assess the binding and infection into 293T, Vero A6, and Calu- 3 cells. This assay will be used to optimize the chemical composition of inactosomes (type of virus binding motif, nanoparticle structure, total protein density, ratio of virus binding motifs to fusion inhibitory motifs) to minimize the entry of SARS-CoV-2. The optimized inactosome formulations will then be tested for inhibiting infection of live SARS-CoV-2 virus into cells at the Penn Center for Precision Medicine.

概括 严重 呼吸道 急性呼吸综合征冠状病毒2（SARS-COV-2）是Covid-19的原因，这是世界范围内的 在撰写本文时，大流行在美国造成超过52.5万人死亡，全球死亡人数超过260万。 在这里，我们建议将磷脂蔬菜和其他相关纳米结构（液滴和胶束）与 重组蛋白基序将结合并灭活SARS-COV-2病毒体 - 我们称这些结构称为 无效。我们的无活性将是新型混合材料，功能重组蛋白是 重构。功能化的蛋白将是一种天然存在的表面活性剂蛋白的含油蛋白的变体。 以前，我们设计和生产了新型油胶质变体，这些变体组装成囊泡膜和 由于胶束像三嵌段一样的自由链两亲性结构，可以稳定液滴。我们很容易 将功能性肽基序掺入油蛋白蛋白重组中。 SARS-COV-2可以通过内吞和/或融合进入上皮细胞。峰值蛋白之间的结合 在SARS-COV-2和ACE2受体上，对病毒进入上皮是必不可少的。 ACE2 受体可以介导内吞作用。另外，在与ACE2结合后，蛋白酶（TMPRSS2）可以激活A S蛋白的构象变化导致融合的进入。我们设想了许多化学成分 在干扰SARS-COV-2感染时直接有用。首先，一个尖峰蛋白结合基序 - 微型蛋白质，单链抗体（Sybodies）或ACE2 Pepper Mimetics - 将重组添加到 油脂的亲水末端。这些基序与峰值蛋白受体结合具有低解离常数 域，比大型抗体容易得多。当这些病毒结合基序蛋白是 结果重构为纳米结构，结果将是一个多价粒子（不活跃），可以直接结合到 SARS-COV-2并有竞争力阻止其进入。接下来，我们将掺入一个呈现小的油脂 S蛋白的肽融合抑制剂，以防止病毒在表达TMPRSS2的细胞系上的融合进入。 可以将阻断结合和融合的肽组合起来，以使多函数抑制性不活跃。 在AIM 1中，我们将开发并表征SARS-COV-2无效体，以防止内吞作用 - 介导的进入细胞。在AIM 2中，我们将开发并描述SARS-COV-2的不作用 阻塞融合的进入，其次是无活性体，既有ACE2封闭肽又有抗 - fusogenic petides。 SARS-COV-2报告基因颗粒（带有EGFP基因）和SARS-COV-2的组合 无活性体将在共培养中孵育，以评估与293t，Vero A6和Calu-的结合和感染 3个细胞。该测定法将用于优化非活性体的化学组成（病毒结合基序的类型， 纳米颗粒结构，总蛋白质密度，病毒结合基序与融合抑制基序的比率），以最大程度地减少 SARS-COV-2的进入。然后，将测试优化的无效公式的抑制感染 活体SARS-COV-2病毒进入宾夕法尼亚州精密医学中心的细胞。