Inhibition of SARS-CoV-2 infections of human lungs by functional inhibitors of the acid sphingomyelinase

临床前和临床证据：几项临床前和临床研究表明，某些抗抑郁药可以防止SARS-CoV-2感染人体细胞，并防止严重症状、通气或死亡的发生。这些抗抑郁药是酸性鞘磷脂酶（FIASMA）的功能性抑制剂。我们已经表明，SARS-CoV-2感染人上皮细胞导致酸性鞘磷脂酶的激活，神经酰胺的释放和在质膜的外小叶中形成富含神经酰胺的膜平台。通过抗抑郁药抑制酸性鞘磷脂酶或使用shRNA基因敲低酸性鞘磷脂酶可防止这些生化变化并阻断SARS-CoV-2感染。反过来，用这些抗抑郁药治疗的细胞中神经酰胺的重建恢复了感染，证明了酸性鞘磷脂酶-神经酰胺系统在SARS-CoV-2感染中的重要作用。这些数据得到了三项独立研究的证实。一项涉及7,000多名患者的多中心观察性回顾性研究、两项小型和一项大型前瞻性、随机、安慰剂对照临床试验证实，抗抑郁药抑制酸性鞘磷脂酶可大大降低患者发生严重COVID-19的风险。然而，目前还不清楚抗抑郁药是否也能抑制新出现的SARS-CoV-2变异体感染人细胞，这显然是非常重要的。主要研究问题和目的：最近的研究表明，一些刺突突变体对SARS-CoV-2抗体具有抗性，并可能对疫苗具有抗性。我们将用VSV或慢病毒假型感染培养的原代和新鲜分离的人鼻上皮细胞和新鲜分离的人肺标本（支气管和肺泡组织），这些病毒假型呈现野生型或刺突病毒的B.1.1.7-、B.1.351-、P.1-或B.1.617.2-变体，或真实的SARS-CoV-2病毒（野生型和变体B.1.1.7和B.1.617.2）。我们将测试是否假型呈现野生型或变异的刺突或真正的SARS-CoV-2激活酸性鞘磷脂酶/神经酰胺系统。最重要的是，我们将确定是否抑制酸性鞘磷脂酶/神经酰胺系统，使用抗抑郁药或几种遗传系统，防止感染的人细胞和肺组织的假型呈现野生型和刺突病毒的变种和真正的野生型病毒和病毒变种的关注。我们将在抑制实验中测试酸性鞘磷脂酶的一组功能性抑制剂。抗抑郁药可以立即用于临床研究，以预防或治疗COVID-19。

Preclinical and clinical evidence: Several pre-clinical and clinical studies demonstrated that certain antidepressants prevent the infection of human cells with SARS-CoV-2 and the development of severe symptoms, ventilation or death. These antidepressants are functional inhibitors of the acid sphingomyelinase (FIASMA). We have shown that infection of human epithelial cells with SARS-CoV-2 results in activation of the acid sphingomyelinase, a release of ceramide and the formation of ceramide-enriched membrane platforms in the outer leaflet of the plasma membrane. Inhibition of the acid sphingomyelinase by antidepressants or genetic knock-down of the acid sphingomyelinase using shRNA prevents these biochemical changes and blocks infection with SARS-CoV-2. In turn, reconstitution of ceramide in cells treated with these antidepressants restores infection proving the important role of the acid sphingomyelinase-ceramide system for SARS-CoV-2 infections. These data were confirmed by three independent studies.A multicenter observational retrospective study with more than 7,000 patients, two small and one large prospective, randomized, placebo-controlled clinical trials confirmed that inhibition of the acid sphingomyelinase by antidepressants greatly reduced the risk of patients to develope severe COVID-19.However, at present it is unknown whether antidepressants also inhibit infection of human cells with newly emerging SARS-CoV-2 variants of concern, which is obviously of great importance. The present proposal aims to clarify this issue.Main research question and aims: Recent studies indicate that several spike mutants are resistant to antibodies against SARS-CoV-2 and potentially resistant to vaccines. We will infect cultured primary and freshly-isolated human nasal epithelial cells and freshly-isolated human lung specimen (bronchi and alveolar tissue) with VSV or lentiviral pseudotypes, presenting wildtype or the B.1.1.7-, B.1.351-, P.1- or B.1.617.2-variants of spike, or authentic SARS-CoV-2 virus (wildtype and the variants B.1.1.7 and B.1.617.2). We will test whether pseudotypes presenting wildtype or variants of spike or authentic SARS-CoV-2 activate the acid sphingomyelinase/ceramide system. Most importantly, we will define whether inhibition of the acid sphingomyelinase/ceramide system, using antidepressants or several genetic systems, prevents infection of human cells and lung tissue with pseudotypes presenting wildtype and variants of spike and authentic wildtype virus and virus variants of concern. We will test a panel of functional inhibitors of the acid sphingomyelinase in the inhibition experiments. Antidepressants could be immediately used in clinical studies to prevent or treat COVID-19.