Tipping the Balance of Ubiquitination toward a Therapy for COVID-19

Scientists have been racing to identify approaches to modulate ACE2 (angiotensin-converting enzyme 2) for the treatment of coronavirus disease (COVID-19) ever since the discovery that ACE2 is the primary receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1, 2). Neutralizing monoclonal antibodies that bind to the spike protein of SARS-CoV-2 are effective in part by preventing viral attachment to ACE2. Although monoclonal antibodies were efficacious for treatment of symptomatic disease early in the pandemic, there are no monoclonal antibodies with activity against more recent SARS-CoV-2 variants such as Omicron. Human recombinant soluble ACE2 (hrsACE2) is capable of binding SARS-CoV-2 (3) and has been proposed as a “decoy” therapy that might lower effective viral load and thus limit cellular engagement (1). Clinical trials testing this approach are ongoing (www.clinicaltrials.gov ID NCT04335136); however, there remains concern that protein instability and insufficient viral–ligand interactions will limit the efficacy of hrsACE2. Another approach to preventing cellular infection with SARS-CoV-2 is to reduce cell-surface ACE2 density by increasing ACE2 shedding (4) or by augmenting ACE2 cellular internalization (5). One advantage of therapies based on modifying host cells compared with direct antiviral approaches is that these treatments might be less susceptible to resistance due to viral mutations.

Modulating the ubiquitin/proteasome system offers a novel approach to treating COVID-19 by decreasing ACE2 cell-surface density in the setting of SARS-CoV-2 infection. Ubiquitination is a highly regulated posttranslational modification that marks cellular proteins for degradation by the proteasome (6). Conversely, ubiquitinated proteins can also be rapidly deubiquitinated by deubiquitylating enzymes. As such, the dynamic balance between ubiquitylating ligases and deubiquitinases (DUBs) is a major regulator of cellular protein abundance and function. The degree to which ACE2 cell-surface protein abundance, and ultimately SARS-CoV-2 viral entry, is modulated by ubiquitination and deubiquitination is an unanswered question.

In this issue of the Journal (7), Bednash and colleagues (pp. 566–576) build on previous work showing that ACE2 is regulated by ubiquitination during SARS-CoV-2 infection (8–10). Using their cell-culture model, they first validated that SARS-CoV-2 spike protein ligation increases ACE2 cell-surface protein abundance and that ubiquitination modulates ACE2 stability. They next used an siRNA screen targeting all known 96 human DUBs in epithelial cells infected with SARS-CoV-2 spike protein to show, for the first time, that SARS-CoV-2 spike particle uptake is influenced by DUBs. UCHL1 and UCHL3 were the two DUBs (both from the ubiquitin carboxy-terminal hydrolase subfamily) whose inhibition was associated with the largest reduction in SARS-CoV-2 spike protein uptake. The authors go on to establish that UCHL1 modulates ACE2 protein stability by performing a series of knockdown, gain-of-function, and coimmunoprecipitation experiments between UCHL1 and ACE2. Finally, the authors show that a small-molecule inhibitor of UCHL1 (LDN-57444) decreases ACE2 protein abundance as well as SARS-CoV-2 antigen uptake into Calu-3 cells and human bronchial epithelial cells. These experiments strongly suggest that the association between inhibiting UCHL1 and decreased SARS-CoV-2 spike protein uptake is mechanistically linked through UCHL1–ACE2 interactions.

The report by Bednash and colleagues adds significant mechanistic clarity by identifying a causal role for SARS-CoV-2 spike protein entry into human bronchial epithelial cells via DUBs interacting with ACE2. This finding opens up an entire pathway and a new set of targets that might be leveraged to modulate ACE2 cell-surface protein abundance. The analysis of the small molecule inhibitor is very interesting and could lead to more important studies. Prior work has demonstrated ubiquitination of ACE2 by Skp2, UBR4, and MDM2 (8–10); however, none of these studies explored therapeutically targeting the ubiquitin/proteasome system to modulate SARS-CoV-2 viral entry. Indeed, the major strength of this study is that it fills mechanistic knowledge gaps associated with a logical therapeutic target (ACE2) for COVID-19.

This study also leaves some unanswered questions. First, the authors identified multiple DUBs besides UCHL1 and UCHL3 that were associated with SARS-CoV-2 spike particle uptake in their siRNA screen. It is possible that some of these DUBs may play a key role in ACE2 ubiquitylation, and they should be studied in the future. Second, the off-target consequences of inhibiting DUBs in epithelial cells infected with SARS-CoV-2 are not known. The ubiquitin/proteasome system plays a key role in most protein degradation pathways, including key antiviral and inflammatory cellular programs. A comprehensive functional assessment of epithelial cells in the presence of DUB inhibitors should be performed as part of a complete preclinical exploration of this potential therapeutic approach. Finally, although some DUBs have been tested in clinical trials (6), additional preclinical target-validation experiments of the various DUBs reported in this study are needed.

What are the broader implications of the findings reported by Bednash and colleagues for COVID-19 and our understanding of lung biology in general? These studies demonstrate that an existing small molecule can decrease ACE2 protein abundance and inhibit SARS-CoV-2 viral infection in vitro. Theoretical advantages of this approach include drug delivery (small molecules are not as susceptible to degradation as proteins, which is advantageous for aerosolized drug administration) and efficacy for multiple strains of SARS-CoV-2 that might evade protection from vaccination or prior infection. This study also supports the general concept of “tipping the balance” of ubiquitination as a therapeutic approach for other pulmonary diseases. There are many existing compounds that regulate the ubiquitin/proteasome system (6). The ubiquitin/proteasome system is a rational potential drug target for other inflammatory and infectious diseases of the lung considering the central role the ubiquitin/proteasome system plays in cellular protein abundance and function.