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.

总结 严重 呼吸 急性呼吸道综合征冠状病毒2（SARS-CoV-2）是COVID-19的病因，是全球范围内 截至本文撰写时，这场大流行在美国造成了超过52.5万人死亡，在全球造成了超过260万人死亡。 在这里，我们建议用磷脂囊泡和其他相关的纳米结构（液滴和胶束）， 重组蛋白基序，将结合并包裹SARS-CoV-2病毒粒子-我们称这些结构 不乳糖体。我们的非乳糖体将是一种新型的杂交材料，功能性重组蛋白将被整合到其中。 重组。功能化蛋白质将是油质蛋白的变体，油质蛋白是天然存在的表面活性剂蛋白质。 以前，我们已经设计和生产了新的油质蛋白变体，它们组装成囊泡膜， 胶束，并由于其三嵌段样自由链两亲结构而可以稳定液滴。我们可以很容易 将功能性肽基序重组并入油质蛋白中。 SARS-CoV-2可以通过内吞和/或融合进入上皮细胞。刺突蛋白（S）之间的结合 SARS-CoV-2和ACE 2受体的结合对于病毒进入上皮细胞至关重要。述ace 2 受体介导内吞作用。或者，在与ACE 2结合后，蛋白酶（TMPRSS 2）可以激活ACE 2。 导致融合进入的S蛋白的构象变化。我们设想了一些化学物质， 直接用于干扰SARS-CoV-2感染。首先，一个刺突蛋白结合基序家族- 微蛋白、单链抗体（sybodies）或ACE 2肽模拟物-将被重组地添加到 油质蛋白的亲水末端。这些基序与刺突蛋白受体结合的解离常数较低 结构域，并且比大抗体更容易产生。当这些病毒结合基序-油质蛋白被 重构成纳米结构，结果将是多价颗粒（非乳糖体），其可以直接结合到 SARS-CoV-2并竞争性地阻止其进入。接下来，我们将加入一种油质蛋白， S蛋白的肽融合抑制剂，以防止病毒融合进入表达TMPRSS 2的细胞系。 阻断结合和融合的肽可以组合以制备多功能抑制性非乳糖体。 在目标1中，我们将开发和表征SARS-CoV-2非乳糖体，防止内吞作用- 介导进入细胞。在目标2中，我们将开发和表征SARS-CoV-2非乳糖体， 阻断融合进入，随后是具有ACE 2阻断肽和抗- 融合肽SARS-CoV-2报告颗粒（携带eGFP基因）和SARS-CoV-2的组合 将在共培养物中孵育非乳糖体，以评估结合和感染293 T、Vero A6和Calu。 3个细胞。该测定将用于优化非乳糖体的化学组成（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），以最小化 SARS-CoV-2的进入。然后将测试优化的非乳糖体制剂对感染的抑制。 在宾夕法尼亚大学精准医学中心将SARS-CoV-2活病毒植入细胞。