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.

概括 严重 呼吸系统 急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是导致全球范围内流行的新型冠状病毒肺炎 (COVID-19) 的原因。 截至撰写本文时，该流行病已在美国造成超过 52.5 万人死亡，在全球范围内造成超过 260 万人死亡。 在这里，我们建议用以下材料制作磷脂囊泡和其他相关纳米结构（液滴和胶束） 将结合并灭活 SARS-CoV-2 病毒颗粒的重组蛋白基序——我们称这些结构为 我们的inactosomes将是新型杂化材料，其中包含功能性重组蛋白。 功能化的蛋白质将是油质蛋白的变体，油质蛋白是一种天然存在的表面活性剂蛋白质。 此前，我们设计并生产了新型油质蛋白变体，可组装成囊泡膜并 由于其类似三嵌段的游离链两亲结构，我们可以很容易地形成胶束并稳定液滴。 将功能性肽基序重组整合到油质蛋白中。 SARS-CoV-2 可以通过胞吞作用和/或刺突蛋白 (S) 之间的融合进入上皮细胞。 SARS-CoV-2 和 ACE2 受体对于病毒进入上皮细胞至关重要。 或者，与 ACE2 结合后，蛋白酶 (TMPRSS2) 可以激活 S 蛋白的构象变化会导致融合进入。 可直接用于干扰 SARS-CoV-2 感染。首先，刺突蛋白结合基序家族 - 微型蛋白、单链抗体（sybodies）或 ACE2 肽模拟物 - 将重组添加到 这些基序与刺突蛋白受体结合的解离常数较低。 当这些病毒结合基序-油质蛋白是时，比大抗体更容易生产。 重组为纳米结构，结果将是多价颗粒（inactosomes），可以直接结合 接下来，我们将掺入一种呈现小颗粒的油质蛋白，并竞争性地阻止其进入。 S 蛋白的肽融合抑制剂，可防止病毒融合进入表达 TMPRSS2 的细胞系。 阻断结合和融合的肽可以组合起来制成多功能抑制性inactosome。 在目标 1 中，我们将开发并表征可防止内吞作用的 SARS-CoV-2 内切体 - 在目标 2 中，我们将开发并表征 SARS-CoV-2 内切体。 阻断融合进入，随后是具有 ACE2 阻断肽和抗- SARS-CoV-2 报告颗粒（带有 eGFP 基因）和 SARS-CoV-2 的融合肽。 inactosomes 将在共培养物中孵育，以评估对 293T、Vero A6 和 Calu- 的结合和感染 3 个细胞。该测定将用于优化 inactosomes 的化学成分（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）以最小化 然后将测试优化的inactosome 制剂对 SARS-CoV-2 感染的抑制作用。 在宾夕法尼亚州精准医学中心将 SARS-CoV-2 病毒活体植入细胞中。