Low hanging fruit for combatting SARS‐CoV‐2?

Plain language summary

Entry of SARS‐CoV‐2 into human respiratory cells, mediated by the spike protein, is absolutely dependent on the cellular receptor ACE2 (angiotensin‐converting enzyme‐2). This makes ACE2 an attractive target for therapeutic intervention in COVID‐19. In this issue, Zuo et al. discover that vitamin C, an essential nutrient and common dietary supplement, can target ACE2 for ubiquitin‐dependent degradation, resulting in the inhibition of SARS‐CoV‐2 infection (Zuo et al, 2023). The study identifies novel mechanisms of cellular ACE2 regulation and may inform the design of therapeutics targeting SARS‐2 and related coronaviruses.

Cell entry of SARS‐CoV‐2 mediated by the viral spike protein is absolutely dependent on the receptor ACE2. A study in this issue now shows that vitamin C can target ACE2 for ubiquitin‐dependent degradation, thereby inhibiting SARS‐CoV‐2 infection.

Angiotensin‐converting enzyme‐2 (ACE2) was first identified as a cellular receptor for the original SARS coronavirus in 2003 (Li et al, ²⁰⁰³) but has since been catapulted into the scientific limelight by SARS‐CoV‐2, the causative agent of COVID‐19. ACE2, in fact, acts as a receptor for a wide range of coronaviruses, including those closely related to SARS‐1/2 and more distant cousins (Hofmann et al, ²⁰⁰⁵; Starr et al, ²⁰²²; Xiong et al, ²⁰²²). As a receptor, ACE2 plays an essential role in the entry of coronavirus particles into host cells. It interacts directly with the spike protein and acts both as a foothold on the cell surface and as a molecular cue for priming and triggering the membrane fusion mechanism of spike. Without ACE2 interaction, spike will not activate and the genetic cargo of the virus particle will remain trapped inside the viral envelope. The requirement for ACE2 in this process seems non‐negotiable for SARS‐CoV‐2, as there is little to no evidence of receptor switching in any emergent variants. Consequently, there has been considerable interest in targeting ACE2 as a therapy for COVID‐19.

Prior to the emergence of SARS, ACE2 was better known for its physiological function in the renin–angiotensin system (RAS), which is responsible for the homeostatic regulation of blood pressure. In this setting, ACE2, and its counterpart ACE, perform proteolytic editing of angiotensin, a peptide hormone. This enzymatic activity had previously been targeted with small molecule inhibitors as a means of ameliorating hypertension, and early in the COVID‐19 pandemic, there was speculation whether any of these pre‐existing ACE‐targeting drugs may be effective against SARS‐CoV‐2. However, the ability of ACE2 to bind SARS‐2 and facilitate entry is independent of its enzymatic activity; moreover, interfering with RAS may also trigger feedback loops that increase ACE2 expression. Therefore, knowledge of ACE2's physiological function has yet to translate into SARS‐2 therapeutics.

An alternative approach is to physically block the interaction between spike and ACE2 using biological drugs (i.e. clinical‐grade purified proteins). In this area, two ACE2‐specific strategies are being explored. Anti‐ACE2 monoclonal antibodies, which bind the receptor and limit its availability to SARS‐2, or soluble ACE2 receptor mimetics, which occupy the receptor‐binding site of spike, neutralise the virus particle (Higuchi et al, ²⁰²¹; Chaouat et al, ²⁰²²; Monteil et al, ²⁰²²). Whilst these approaches show some promise, they present challenges, common to all biologics, around mass production and administration (typically intravenously). Consequently, there is currently no means of targeting ACE2 in SARS‐2 infection, using a simple, orally available, therapy.

It was in this context that Zuo et al (2023) screened a variety of common dietary supplements for their ability to reduce the cellular expression of ACE2, having already performed preliminary experiments to demonstrate that even a modest decrease in ACE2 may have dramatic effects on SARS‐CoV‐2 replication. All treatments, but one, had no effect on ACE2 protein levels; vitamin C (VitC) reduced ACE2 in a dose‐dependent manner and, importantly, inhibited the in vitro replication of authentic SARS‐CoV‐2. This knowledge alone would be useful, but the real strength of the investigation by Zuo et al (2023) is their thorough dissection of the molecular mechanisms underpinning this observation.

In a series of elegant, stepwise experiments, the authors revealed that VitC was interfering with a, hitherto unknown, regulatory system that controls ACE2 protein levels, independent of gene transcription or translation. The study demonstrates that in a variety of cell lines, and in primary mouse tissues including the lung, ACE2 is constitutively associated with USP50, a cellular deubiquitinase. This partnership protects ACE2 from ubiquitin‐dependent lysosomal degradation; indeed, the knockout of USP50 reduced ACE2 levels and, consequently, SARS‐CoV‐2 replication. VitC prevents ACE2‐USP50 association, resulting in the deposition of K48‐linked ubiquitin chains on ACE2, which in turn labels it for destruction (Fig 1). The authors corroborate this finding by identifying the critical lysine residue in ACE2 that is the target for ubiquitination; mutation of this residue renders ACE2 resistant to degradation, and accordingly, VitC no longer exerts inhibitory effects on ACE2 expression or SARS‐CoV‐2 replication.

Figure 1. Mechanism of ACE2 downregulation by vitamin C.

SARS‐CoV‐2 spike interaction with ACE2 is a requirement for viral entry (left). ACE2 receptors on the cell surface are constitutively associated with USP50, a deubiquitinase, which protects it from targeting lysosomal degradation. Vitamin C interferes with USP50‐ACE2 association, and this permits K48‐linked ubiquitination and degradation of ACE2 (right). The resulting loss of ACE2 from the cell surface reduces SARS‐CoV‐2 entry and replication. Figure created with BioRender.com.

To bring this discovery to its natural conclusion, Zuo et al (2023) examined the ability of VitC to interfere with infection in vivo. Using a humanised mouse model, the authors demonstrate that intraperitoneal administration of high‐dose VitC reduces ACE2 levels in a variety of tissues and inhibits infection by a surrogate reporter virus bearing the SARS‐CoV‐2 spike protein. Whilst caution should be taken when interpreting the animal and viral models used in this experiment, it suggests that the regulation of ACE2 by USP50 is a viable target for therapeutic intervention.

Changing the diet to achieve the apparent therapeutic doses of VitC necessary to tackle SARS‐CoV‐2 would require, ill‐advised, superhuman consumption of citrus fruits or dietary supplements. Nonetheless, the regulatory process identified in this work may yet provide new approaches for modulating ACE2, both in the context of its physiological function and in combatting SARS‐CoV‐2 and its receptor‐sharing relatives.

Disclosure and competing interests statement

The authors declare that they have no conflict of interest.

EMBO reports (2023) 24: e56979