Based on our screening strategy and already published pharmacokinetic and toxicology profiles, we identified compounds halofuginone and homoharringtonine as agents that reduce cell surface expression of TMPRSS2.
USING FLEXISIGN 12 XVIDEOS TRIAL
We developed an assay that measures protein abundance of TMPRSS2 and executed a screen of 2560 FDA-approved or current clinical trial compounds to determine their effect on TMPRSS2 abundance.
Given SARS-CoV-2′s rapid spread, coupled with the high burden of acute respiratory failure and death, there is an urgent need for therapies. These observations led us, and others 9, 17, to hypothesize that reducing or inhibiting TMPRSS2 may be an attractive strategy to mitigate SARS-CoV-2 pathogenicity. In contrast, mice lacking TMPRSS2 exhibit no discernable basal phenotype 16, but demonstrate protection from acute SARS-CoV infection 3. However, in animal models, reducing ACE2 activity appears to increase lung injury in response to SARS-CoV infection or sepsis 6, 15. Host-directed efforts to limit SARS-CoV-2 infection could logically entail strategies to manipulate either ACE2 or the S protein priming step. Additionally, SARS-CoV-2 uniquely possesses a “furin-like” cleavage site and is subject to processing during viral packaging by the intracellular proprotein convertase furin 11, 12, 13, 14. The viral entry mechanisms elucidated for SARS-CoV infection appear to be shared by SARS-CoV-2 9, 10. Two separate classes of proteases were shown to prime SARS-CoV S protein-endo-lysosomal proteases in the cathepsin family, and the plasma membrane associated protease, TMPRSS2 3, 8. S protein priming is a process wherein the spike protein is cleaved by host proteases.
A second critical process, S protein priming, is essential to complete viral entry and spread 7. To mediate cell entry, the SARS-CoV viral Spike (S) glycoprotein is recognized by the extracellular receptor angiotensin converting enzyme 2 (ACE2) on host respiratory epithelial cells 5, 6. After emergence of SARS-CoV, several groups identified the molecular and cellular pathways through which this virus attaches, enters, and replicates in host respiratory epithelial cells 2, 3, 4. SARS-CoV-2 is a coronavirus first described in Wuhan, China that shares many similarities with other pathogenic beta coronaviruses, including the SARS-CoV virus and the MERS coronavirus 1. 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 active COVID-19 infection. Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. 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).
These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. 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. SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease.
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