Modulation of lysosomal function as a therapeutic approach for coronaviral infections (preprint)

The endo-lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis – TFEB – is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin-mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks viral-mediated TFEB degradation and protects against viral-induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit viral-triggered TFEB degradation and demonstrate broad anti-viral activities including attenuating in vivo SARS-CoV-2 infection. Together, these results delineate a viral-triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo-lysosomal, host-directed, anti-viral therapies.

Investigation of the Impact of SARS-CoV-1 Infection on the Immunologic Status and Lung Function After 15 Years (preprint)

Background: To investigate the long-term effects of SARS-CoV-1 on patients’ lung and immune systems 15 years post-infection. Methods: We enrolled 58 health care workers with confirmed SARS in Peking University People’s Hospital in 2003. We evaluated lung damage by mMRC score, pulmonary function tests, and chest CT. Immune function was assessed by their serum levels of globin, complete components, and peripheral T cell subsets. ELISA was used to detect SARS-CoV-specific IgG antibodies in sera. Results: After 15 years of disease onset, 19 (36.5%), 8 (34.6%), and 19 (36.5%) subjects had impaired DL(CO), RV, and FEF25-75, respectively. 17 (30.4%) subjects had an mMRC score ≥ 2. Fourteen (25.5%) cases had residual CT abnormalities. T regulatory cells were a bit higher in the SARS survivors. IgG antibodies against SARS S-RBD protein and N protein were detected in 11 (18.97%) and 12 (20.69%) subjects, respectively. Subgroup analysis revealed that small airway dysfunction and CT abnormalities were more common in the severe group than in the non-severe group (57.1% vs 22.6%, 54.5% vs 6.1%, respectively, p < 0.05). Conclusions: SARS-CoV-1 could cause permanent damage to the lung, which requires early pulmonary rehabilitation. The long-lived immune memory response against coronavirus requires further studies to assess the potential benefit.Trail registration: ClinicalTrials.gov, NCT03443102. Registered prospectively on 22/02/2018.

A high throughput screen for TMPRSS2 expression identifies FDA-approved and clinically advanced compounds that can limit SARS-CoV-2 entry (preprint)

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identified small molecules that can reduce surface expression of TMPRSS2 using a 2,700 FDA-approved or current clinical trial compounds. Among these, homoharringtonine and halofuginone appear the most potent agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrated marked resistance to SARS-CoV-2 pseudoviral infection. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat COVID-19 infection.