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: 10370745
• 负责人: Daniel A Hammer
• 金额: $ 24.1万
• 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/10370745
• 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; Epithelial Cells; 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; Seeds; 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; nanobodies; nanoparticle; nanovesicle; novel; novel coronavirus; pandemic disease; particle; peptidomimetics; precision medicine; prevent; receptor; receptor binding; reconstitution; respiratory; self assembly; 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.

摘要 严重者 呼吸性 急性呼吸综合征冠状病毒2(SARS-CoV-2)是新冠肺炎的病原体，是世界性的 截至撰写本文时，大流行在美国造成超过52.5万人死亡，在全球造成超过260万人死亡。 在这里，我们建议用以下物质来制造磷脂微囊和其他相关的纳米结构(液滴和胶束) 结合和灭活SARS-CoV-2病毒粒子的重组蛋白基序-我们称之为这些结构 无性体。我们的无性体将是一种新型的杂交材料，功能重组蛋白将进入其中 重新组合。这种功能化的蛋白质将是油球蛋白的变体，油球蛋白是一种天然存在的表面活性蛋白质。 此前，我们已经设计和生产了新的油球蛋白变体，它们可以组装成泡泡膜和 由于其具有三嵌段的自由链两亲性结构，因此可以稳定胶束并稳定液滴。我们可以很容易地 将功能性多肽基序重组到油球蛋白中。 SARS-CoV-2可以通过内吞和/或融合进入上皮细胞。尖峰蛋白之间的结合(S) 在SARS-CoV-2和ACE2受体上，ACE2受体对于病毒进入上皮细胞是必不可少的。ACE2 受体可以介导内吞作用。或者，在与ACE2结合后，蛋白水解酶(TMPRSS2)可以激活 S蛋白的构象变化导致融合基因进入。我们设想了一些化学物质，它们将 在干预SARS-CoV-2感染方面直接有用。首先，一系列刺激性蛋白结合基序- 迷你蛋白、单链抗体(抗体)或ACE2多肽模拟物-将被重组添加到 油球蛋白的亲水性末端。这些基序与刺激性蛋白受体结合的解离常数很低。 并且比大的抗体更容易产生。当这些病毒结合基序油球蛋白 重组成纳米结构，结果将是一个多价粒子(无性体)，可以直接结合到 SARS-CoV-2，并竞争性地阻止其进入。接下来，我们将结合一种油球蛋白，它呈现一个小的 S蛋白的多肽融合抑制物以防止病毒融合进入表达TMPRSS2的细胞系。 阻断结合和融合的多肽可以结合在一起形成多功能抑制性无性体。 在目标1中，我们将开发和表征SARS-CoV-2无性体，以防止内吞- 通过中介进入细胞。在目标2中，我们将开发和表征SARS-CoV-2无性体 阻断融合进入，随后是同时拥有ACE2封闭肽和抗ACE2的无性体。 融合多肽。SARS-CoV-2报告颗粒(携带EGFP基因)与SARS-CoV-2的结合 无性体将在共培养中孵化，以评估与293T、Vero A6和Calu-2的结合和感染。 3个细胞。本试验将用于优化无性体的化学组成(病毒结合基序的类型， 纳米颗粒结构、总蛋白密度、病毒结合基序与融合抑制基序的比率)来最小化 SARS-CoV-2的进入。然后将对优化的无性体配方进行测试，以抑制感染 将SARS-CoV-2活病毒送入宾夕法尼亚大学精密医学中心的细胞中。