Kidney Microphysiological Analysis Platforms (MAP) to Explore SARS-CoV-2 Receptors and Inhibitors. A supplement to Parent Grant: Kidney Microphysiological Analysis Platforms (MAP) to Optimize Function

摘要 冠状病毒病2019年(新冠肺炎)已达到大流行的程度。肺脏和 肾脏疾病是感染SARS-CoV-2病毒的高度流行的严重后果。肾 损伤分子-1(Kim-1)是由Bonventre和Ichiura博士鉴定的最上调的分子 受损的肾脏近端小管中的蛋白质。Kim-1，也称为Tim-1，是肝炎的受体 A、埃博拉、登革热，可能还有SARS-CoV1病毒。我们假设Kim-1是一种受体 SARS-CoV-2在肾小管上皮细胞和呼吸道上皮细胞中的表达 JB1，我们新发现的KIM-1的小分子抑制剂，和/或纳米盘- 掺入的KIM-1胞外结构域可作为新冠肺炎的预防和治疗药物。 我们还假设我们可以利用Kim-1和ACE2与 为病毒创造新的诊断设备。在具体目标1中，我们将描述 Kim-1在促进SARS-CoV-2通过肾脏进入肾和肺上皮细胞中的作用 将微生理分析平台(MAP)集成到芯片上，并开发了一种用于 高通量评价潜在的SARS-CoV-2结合抑制剂。Kim-1和ACE2 将比较SARS-CoV-2仿生病毒(病毒体)在肾脏中介导的内吞作用 和肺上皮细胞。我们将评估Kim-1介导的Spike蛋白或 仿生病毒细胞黏附和摄取激活旁分泌因子的产生 血管内皮细胞使用肾-肺图。为了了解结合和/或摄取动力学 针对SARS-CoV-2和表征潜在的抑制剂，我们将开发一种超灵敏的 一种基于纳米等离子体三联体的快速侧向流动快速抑制芯片 使用肾-肺图进行化验。此方法也可用于护理点诊断 检测病毒。在特定目标2中，我们将评估JB1、可溶性KIM-1的疗效 胞外区和纳米盘掺入的Kim-1或ACE2，以抑制结合和内化 利用肺-肾综合图谱研究肾和肺细胞的SARS-CoV-2仿生病毒 在芯片上。潜在的抑制剂将接受测试，以评估它们是否与S竞争-蛋白质和/或 仿生病毒结合，减少IL-6的产生。KIM-1的结合亲和力和动力学 变异体或ACE2作为游离的胞外结构域或被掺入纳米盘和Spike蛋白 将使用微型热电泳法(MST)和生物层干涉测量法进行测量。

Abstract Coronavirus disease 2019 (COVID-19) has reached pandemic proportions. Pulmonary and kidney disease are highly prevalent serious consequences of infection with SARS-CoV-2. Kidney Injury Molecule-1 (KIM-1) was identified by Drs Bonventre and Ichimura as the most upregulated protein in the injured kidney proximal tubule. KIM-1, also called TIM-1, is a receptor for hepatitis A, Ebola, Dengue and possibly SARS-CoV1 viruses. We hypothesize that KIM-1 is a receptor for SARS-CoV-2 both in renal tubule epithelial cells and in airway epithelial cells and that JB1, our newly discovered small molecule inhibitor of KIM-1, and/or nanodisc- incorporated KIM-1 ectodomain can be prophylactic and therapeutic agents for COVID-19. We also hypothesize that we can use the high-affinity binding of KIM-1 and ACE2 to the virus to create novel diagnostic devices for the virus. In Specific Aim 1 we will characterize the role of KIM-1 in promoting SARS-CoV-2 entry into kidney and lung epithelia using kidney microphysiological analysis platforms (MAPs) on chip and develop an ultrasensitive chip for the high-throughput evaluation of potential SARS-CoV-2 binding inhibitors. KIM-1 and ACE2 mediated endocytosis of SARS-CoV-2 biomimetic viruses (virosomes) will be compared in kidney and lung epithelial cells. We will evaluate the effects of KIM-1-mediated spike proteins or biomimetic virus cellular adhesion and uptake on production of paracrine factors which activate endothelial cells using a kidney-lung MAP. In order to understand binding and/or uptake kinetics of SARS-CoV-2 and characterize potential inhibitors we will develop an ultrasensitive nanoplasmonic triplets-based rapid lateral flow diagnostic chip for a rapid and sensitive inhibition assay using the kidney-lung MAP. This approach can also be used for point of care diagnostic testing for the virus. In Specific Aim 2 we will evaluate the efficacy of JB1, soluble KIM-1 ectodomain and nanodisc-incorporated KIM-1 or ACE2 to inhibit binding and internalization of SARS-CoV-2 biomimetic viruses by kidney and lung cells using an integrated lung-kidney MAP on chip. Potential inhibitors will be tested to evaluate whether they compete with S-protein and/or biomimetic virus binding and reduce IL-6 production. Binding affinity and kinetics between KIM-1 variants or ACE2 either as free ectodomains or incorporated into nanodiscs and the Spike protein will be measured using MicroScale Thermophoresis (MST) and Biolayer Interferometry.