The Role of ACE2 in SARS‐CoV‐2 Infection, Pathogenesis, and Antiviral Interventions

ABSTRACT

The devastating clinical, psychological, and economic impact of the COVID‐19 pandemic, caused by global spread of the second Severe Acute Respiratory Syndrome Coronavirus (SARS‐CoV‐2), has engendered a massive response from the scientific community to rapidly understand the biology of SARS‐CoV‐2 and to develop interventions to prevent infection or progression to life‐threatening disease. Angiotensin converting enzyme‐2 (ACE2) and its interaction with the SARS‐CoV‐2 Spike glycoprotein, which mediates fusion of the virion envelope with the target cell membrane, have emerged as a major pharmacological target, as disruption of the Spike‐ACE2 interaction prevents cells from becoming infected and hence from producing viral progeny. Moreover, the dysregulation of ACE2 that occurs in the context of SARS‐CoV‐2 infection may have broader implications for COVID‐19 pathogenesis. Here we summarize the role of ACE2 as a physiologic regulator of human health, as a facilitator of SARS‐CoV‐2 infection, as a factor in COVID‐19 disease, and as a target for pharmacological interventions.

1. Multifacedted Role of ACE2, From Cardiovascular Regulation to SARS‐CoV‐2 Pathogenesis

Angiotensin converting enzyme‐2 (ACE2) is a 805‐amino acid‐long type I transmembrane zinc metallopeptidase glycoprotein that is well characterized for its role in the hormonally‐regulated renin‐angiotensin system (RAS), and as a receptor for Severe Acute Respiratory Syndrome Coronavirus Two (SARS‐CoV‐2) [1]. ACE2, a known homolog of angiotensin‐converting enzyme (ACE), was discovered in the early 2000s and mapped to the X chromosome at cytogenetic position Xp22.2. ACE2 consists of two domains: a large N‐terminal extracellular domain that contains the enzymatic active site, and a short C‐terminal transmembrane domain that anchors to the cell membrane with an intracellular cytoplasmic tail [2].

ACE2 expression was initially thought to be limited to the heart, kidney, and testis, and the enzyme was implicated in the manifestation of cardiovascular and renal diseases. Subsequent reports, however, indicated that ACE2 expression is ubiquitous within the adult human body, except for red blood cells. A significant functional role for ACE2 has been well established in tissues and organs, including the heart, kidneys, gastrointestinal tract, endothelial and smooth muscle cells of blood vessels, as well as the adipose tissue and epithelium of the upper and capillary‐rich lower airways of the respiratory tract [3]. In contrast, ACE2 expression is low‐to‐minimal in immune tissues such as the thymus, spleen, lymph nodes, and bone marrow [4]. It is important to note that while mRNA expression is homogeneously found in all tissues, this is not the case for protein expression [3].

Given that ACE2 is a coronavirus entry receptor and that the lung is a primary target for the virus, ACE2 expression levels in the lung became a topic of discussion [5]. ACE2 expression begins during embryogenesis, and it is required for normal lung function and development [6]. ACE2 expression levels are low during early fetal development, but gradually increase during the first years of life, starting from the growing fetus and continuing through children, adolescents, and young adults. Many studies, including bulk tissue transcriptomic and single‐cell RNA‐seq analyses, in situ RNA mapping, and immunohistochemistry, were aimed at measuring expression levels and distribution of ACE2 mRNA transcripts or protein in the lung. Surprisingly, ACE2 expression varies among individuals, making it difficult to draw direct conclusions [4, 5, 7]. However, significant differences in ACE2 expression have been observed with gender and age, with lower levels in children and women relative to adults and men, respectively [4, 8, 9]. It is plausible that location of the ACE2 gene on the X chromosome and its ability to escape X chromosome inactivation could account for differential expression, resulting in sex‐based differences [10]. Recent reports have demonstrated that ACE2 expression is upregulated by interferon gamma [11] and androgen receptor agonists, such as dihydrotestosterone [12], while ACE2 expression is downregulated by estrogen in airway epithelium [13].

1.1. ACE2 and the RAS

The RAS is a complex yet pivotal physiological pathway responsible for maintaining blood pressure homeostasis, renal function, and inflammation (Figure 1 [14]). Both ACE2 and ACE are important enzymes in the RAS pathway. Despite having some structural homology, they are functionally divergent and exhibit differences in their substrate specificity [2]. ACE2 and its metabolic by‐product angiotensin 1‐7 (Ang 1‐7), act through the MAS oncogene receptor and are referred to as the ACE2/Ang1‐7/MASR axis. They function as the classic cardiac‐protective arm of the RAS, especially in the attenuation of cardiovascular dysfunction and associated metabolic diseases [15].

Figure 1.

Overview of the RAS system involving ACE, ACE2, and multiorgan crosstalk regulating physiological function. RAS is a coordinated multiorgan enzymatic network involving the liver, lungs, kidneys, and adrenal glands that regulates blood pressure, mineral balance, and cardiovascular homeostasis. The pathway begins with the generation of Ang I, which is converted by ACE into Ang II. Ang II primarily acts through AT1R, promoting vasoconstriction, sodium retention, hypertension, and cardiovascular injury. In contrast, ACE2 counterbalances this axis by metabolizing Ang II into Ang 1–7, which signals through MasR to mediate vasodilation, anti‐inflammatory, and cardioprotective effects. ACE2 can also convert Ang I into Ang 1–9, which activates AT2R and contributes additional protective responses. Together, these interconnected pathways highlight the dynamic balance between the classical ACE/Ang II/AT1R axis and the protective ACE2/Ang 1–7/MasR and Ang 1–9/AT2R axes. The schematic illustrates the enzymatic conversions and receptor‐mediated effects that maintain normal physiological balance or drive disease when dysregulated. Red circles and arrows, pro‐inflammatory, vasoconstrictive ACE/Ang II/AT1R pathway; green circles and arrows, anti‐inflammatory, vasodilatory, and cardiovascular‐protective ACE2/Ang 1–7/MasR and Ang 1–9/AT2R pathways; solid arrows, high‐affinity binding and strong effects; dashed arrows, low‐affinity binding and weak or secondary effects.

ACE removes a carboxy‐terminal dipeptide from angiotensin I (Ang I) to form angiotensin II (Ang II), which is mainly found in the lungs [16]. Ang II is a potent vasoconstrictor that binds to the G protein‐coupled receptor angiotensin II type 1 receptor (AT1R), thereby affecting blood pressure homeostasis and stimulating the release of aldosterone, a hormone that regulates sodium and potassium balance [17]. Under pathological conditions, such as chronic Ang II stimulation or hyperaldosteronism, aldosterone activates vascular mineralocorticoid receptors (MR) in endothelial and smooth‐muscle cells, leading to oxidative stress, reduced nitric‐oxide bioavailability, and activation of pro‐inflammatory signaling, which together drive endothelial dysfunction and vascular inflammation [18, 19]. Consistent with this mechanism, MR antagonists such as eplerenone and spironolactone have been shown to improve endothelial function in humans [20].

In contrast, ACE2 cleaves Ang II into inactive fragments and Ang 1‐7, a vasodilatory molecule that counterbalances the effect of Ang II and regulates blood pressure and inflammation [21, 22]. Additionally, ACE2 can convert Ang I to Ang 1–9, which is subsequently processed to Ang 1–7 by other enzymes, further opposing the ACE/Ang II/AT1R axis. By facilitating Ang II→Ang 1–7 and Ang I→Ang 1–9, ACE2 helps maintain the balance between vasoconstriction and vasodilation and supports blood‐pressure regulation [23, 24].   Collectively, these data underscore the dynamic balance between the ACE/Ang II/AT1R and ACE2/Ang 1–7/MASR axes in maintaining cardiovascular homeostasis [25, 26, 27] (Figure 1).

The SARS‐CoV‐1 outbreak in 2002‐2004 uncovered a critical functional alteration between the two arms of the RAS pathway. Following ACE2‐mediated viral internalization, ACE2 is either degraded or cleaved by the transmembrane proteinase A Disintegrin and Metalloprotease 17 (ADAM17), mediated by Ang II [28], leading to its downregulation on the cell surface [25]. This reduction of ACE2 disrupts the balance of the RAS pathway, decreasing Ang 1–7 levels while leaving Ang II unopposed at the tissue level (Figure 2A). Consequently, the harmful ACE/Ang II/AT1R axis predominates over the protective ACE2/Ang 1–7/MASR axis, particularly in the lungs and heart [29]. In patients with COVID‐19, ACE2 mediates viral entry, and its subsequent downregulation contributes to cardiovascular complications such as thrombosis, myocardial injury, and heart failure via oxidative stress, endothelial dysfunction, and inflammation [30]. Therefore, although viral invasion is initially facilitated by ACE2, its postinfection downregulation likely contributes to oxidative stress, robust inflammation, and multiorgan dysfunction due to tissue damage [31]. Furthermore, loss of ACE2 may amplify the hyperinflammatory response, contributing to the cytokine storm observed in severe COVID‐19 cases, characterized by elevated IL‐6, TNF‐α, and other pro‐inflammatory mediators, which further drive tissue injury and organ failure [32]. Postinfection, the loss of ACE2 disrupts the balance between the RAS and ACE2/Ang1‐7/MASR axes, exacerbating oxidative stress, inflammation, and severe clinical outcomes [27]. Further investigation is warranted to understand the link between ACE2 regulation after viral entry, the RAS pathway, and COVID‐19 pathogenesis (Figure 2A).

Figure 2.

Schematic illustration of ACE2 role in SARS‐CoV‐2 infection and RAS dysregulation in normal versus and CF airway epithelium. (A) S protein binds to ACE2 on epithelial cells, facilitated by HS proteoglycans (HSPG) and TMPRSS2. Viral entry through receptor internalization and ADAM17‐mediated cleavage downregulates surface ACE2, disrupting the RAS balance. Loss of ACE2 impairs conversion of Ang I to Ang 1–9 and Ang II to Ang 1–7, suppressing MasR–mediated protective signaling, while Ang II accumulation drives excessive AT1R activation. Unopposed AT1R signaling promotes reactive oxygen species (ROS) generation, NF‐κB–dependent cytokine and chemokine transcription, leukocyte recruitment, vascular inflammation, and endothelial dysfunction, creating an inflammatory loop that contributes to cytokine storm, pulmonary edema, acute lung injury, and multiorgan failure in severe COVID‐19. (B) In CF airways, elevated NE cleaves membrane‐bound ACE2 on epithelium to generate sACE2, which acts as a decoy receptor to block S protein engagement with cell‐surface ACE2, thereby attenuating infection and partially protecting people with CF from SARS‐CoV‐2–induced RAS dysregulation. The schematic highlights the dual role of ACE2 as both a viral entry receptor and a key regulator of RAS signaling. Red circles and arrows, pro‐inflammatory, vasoconstrictive ACE/Ang II/AT1R pathway; green circles and arrows, anti‐inflammatory, vasodilatory, and cardiovascular‐protective ACE2/Ang 1–7/MasR and Ang 1–9/AT2R pathways; solid arrows, high‐affinity binding and strong effects; dashed arrows, low‐affinity binding and weak or secondary effects.

1.2. ACE2 and SARS‐CoV‐2 Pathogenesis

Over the years, numerous studies have focused on the role of ACE2 in the context of cardiovascular functions of the RAS system [33]. In contrast, SARS‐CoV‐2 has redefined the functional importance of ACE2 expression in the lungs due to the presence of ACE2 expression in the respiratory system, which serves as a cellular doorway/receptor for SARS‐CoV‐2 entry into the human body [34, 35]. Interactions between the virus and host cells are critical for the onset of infection. SARS‐CoV‐2 uses ACE2 as a host cellular entry receptor in airway epithelia, which is the point of initial entry of the virus [36, 37, 38, 39, 40]. The ACE2 protein is localized to the apical domain of the plasma membrane of epithelial cells and is expressed in the nasal epithelia, the large airway epithelia, and type 1 and type 2 alveolar epithelial cells [7].

Interestingly, endothelial cells also express ACE2 and are infected by SARS‐CoV‐2, which may be one mechanism for multiorgan infection, injury, and failure [41]. Viral pathogenesis and the entry of SARS‐CoV‐2 into host cells are facilitated by the viral trimeric Spike (S) protein that protrudes from the viral envelope [42]. SARS‐CoV‐2 entry into susceptible target cells is a complex process that requires the concerted action of S protein binding to the cell receptor and proteolytic processing of S protein to promote virus‐cell membrane fusion [43]. The S protein is composed of two functional subunits, S1 and S2. Within the S1 subunit is a Receptor Binding Domain (RBD) that is responsible for the initial binding to the host cell and contributes to the stabilization of the membrane‐anchored complex, while the S2 subunit contains the fusion machinery necessary for fusion between the virion envelope and cell membrane [44].

Membrane fusion depends on S protein cleavage by host cell membrane serine proteases such as Transmembrane serine protease 2 (TMPRSS2) or Furin at the S1/S2 interface, which results in S protein activation for viral envelope‐cell membrane fusion, followed by internalization of the complex by the host cell [45]. Both SARS‐CoV‐1 and SARS‐CoV‐2 initiate entry into target cells via S protein attachment to ACE2. Entry of the virus primarily occurs via ACE2‐expressing nasal, bronchial, or alveolar epithelial cells of the upper or lower airway. Following local replication of the virus in these cells, viral dissemination is possibly mediated via endothelial ACE2 on blood vessels or capillaries and renal tubular cells [46]. Therefore, a better understanding of ACE2‐mediated fusion and signaling at the molecular level is important for developing preventative strategies involving the modulation of ACE2 levels, binding, and signaling [47]. Since the emergence of SARS‐CoV‐2 in late 2019, the virus has continually evolved through point mutations, particularly in the S protein, giving rise to genetically distinct variants with altered biological and epidemiological properties. Certain variants have demonstrated increased transmissibility, immune escape, or clinical impact, leading the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) to designate them as variants of concern (VOCs). To illustrate their molecular and clinical characteristics, Table 1 summarizes the key S protein mutations, their effects on viral entry, transmissibility, immune evasion, disease severity, and the current classification status of major SARS‐CoV‐2 VOCs [49, 50, 51].

Table 1.

Summary of SARS‐CoV‐2 VOCs^† and their characteristics^‡.

Variant	First reported (location)	PANGO lineage	Spike Protein Mutations		ACE2 interaction entry efficiency	Epidemiologic impact	Immunological impact	Clinical severity	Current status
			S1	S2
WT	Dec. 2019 (China)	Wuhan‐Hu‐1	WT	WT	baseline	baseline	baseline	asymptomatic to severe	no longer circulating
alpha	Sep. 2020 (United Kingdom)	B.1.1.7	N501Y Δ69‐70	P681H	↑ACE2 affinity ↑entry ↑cleavage	⇑transmissibility	↓neutralization vaccines protective	↑hospitalization ↑mortality	former VOC, de‐escalated
beta	Oct. 2020 (S. Africa)	B.1.351	E484K K417N N501Y		↑ACE2 affinity	moderate transmissibility not globally dominant	⇓neutralization	no clear severity increase; vaccines protective	displaced by delta
gamma	Nov. 2020 (Manaus, Brazil)	P.1 (B.1.1.28.1)	E484K K417N N501Y		↑ACE2 affinity	regional surges outcompeted by delta	↑reinfections ↓neutralization (E484K)	severe outcomes in naïve populations	former VOC, de‐escalated
delta	Oct. 2020 (India)	B.1.617.2	L452R P681R T478K	P681R	↑infectivity ↑entry	⇑transmissibility globally dominant (2021)	partial immune escape	↑hospitalization ↑severity vaccines protective	displaced by omicron
omicron + subvariants	Nov. 2021 (S. Africa, Botswana)	B.1.1.529 sub‐lineages BA.1 to BA.5	K417N E484A N501Y	sub‐lineage‐specific mutations	↑transmissibility immune escape altered entry/tropism	⇑transmissibility rapid global replacement through multiple sub‐lineages	↓neutralization immune escape booster vaccine restored protection	milder than delta high case numbers strained hospitals vaccines protected	under active WHO/CDC monitoring

^†data from the World Health Organization [48].

^‡↑↓ moderate increase or decrease;

⇑⇓ large increase or decrease.

In airway epithelial cells, the extracellular domain of ACE2 has been shown to be constitutively clipped by an unidentified proteolytic activity in a process called shedding, resulting in an active soluble ACE2 form (sACE2) that lacks the membrane anchor [52]. Further, phorbol ester‐ or ionomycin‐induced clipping of the ACE2 ectodomain from epithelial cell surfaces is known to be mediated by ADAM17 (also known as TNF‐α‐converting enzyme) (Figure 2B) [52]. Th sACE2 is circulated in the blood stream and eventually excreted in urine. Increased sACE2 levels in plasma and urine have been characterized as a biomarker associated with cardiovascular and renal diseases [53]. TMPRSS2 also cleaves the ACE2 ectodomain at specific residues to enhance viral uptake and cleaves the S protein to activate membrane fusion. However, cleavage of ACE2 by ADAM17 does not affect viral entry, despite competing with TMPRSS2. The TMPRSS2 and ADAM17 cleavage sites in ACE2 are distinct, with cleavage by TMPRSS2 promoting viral entry [54]. Furthermore, Ang II also mediates ACE2 clipping via ADAM17 in the myocardium, resulting in a positive feedback mechanistic loop in the RAS [28]. Interestingly, the interaction between ACE2 and S protein increases the activity of ADAM17, which can lead to the shedding of sACE2. However, the role of ADAM17 and ACE2 shedding is still unclear and remains to be understood in the context of SARS‐CoV‐2 infection.

Recent reports suggest a protective role for recombinant sACE2, which acts as a decoy ligand to sequester SARS‐CoV‐2 S protein and prevent infection (Figure 2B) [34, 35]. The sACE2 released by ADAM17 still contains the virus binding domain. Therefore, sACE2 present either in the airway or in circulation can act as a decoy receptor and block the virus from binding to membrane‐bound ACE2, resulting in an inhibitory effect on viral infection [55]. Furthermore, sACE2 released by ADAM17 into the blood circulation retains its enzymatic activity, so it can also cleave angiotensin II to Ang 1‐7, increasing the protective effects of the ACE2/Ang1‐7/MASR axis (Figure 1) [29, 56]. These reports imply a protective role for sACE2 in acute lung injury. Thus, there is contradictory evidence suggesting both negative and positive effects of ADAM17 in COVID‐19 pathogenesis.

Patients with chronic lung diseases are generally at increased risk for acute respiratory viral infections. Interestingly, reports have shown that, compared to the general population, cystic fibrosis (CF) patients have a lower incidence of SARS‐CoV‐2 infections [57], despite their increased susceptibility to other respiratory viral infections and their higher levels of ACE2 expression [7]. This phenomenon was initially attributed to factors such as the younger age of CF patients or the more aggressive adoption of disease prevention strategies, such as social distancing, vaccination, and mask‐wearing. However, recent studies have demonstrated that neutrophil elastase (NE), a serine proteinase present in higher concentrations in the airways of CF patients, may influence SARS‐CoV‐2 infections. NE might prevent viral entry by cleaving the ectodomain of the ACE2 on epithelial cells [58] or by impairing the virus's early lifecycle through cleavage of the S protein [59]. These findings suggest that NE's proteolytic activity may act as an antiviral mechanism in CF patients, potentially lessening SARS‐CoV‐2 severity by removing the ACE2 ectodomain from airway epithelial cells and cleaving the S protein (Figure 2B).

Further research is needed to better understand the interactions between NE and COVID‐19 susceptibility in CF patients. It is important to explore how ACE2 regulation affects inflammatory responses, as ACE2 cleavage may influence the RAS. Ongoing studies should aim to elucidate the mechanistic details of NE's antiviral effects and its role in modulating inflammation, as well as to investigate therapeutic strategies to enhance protection against respiratory viral infections in vulnerable populations.

2. Antivirals Targeting S Protein/ACE2 Interactions

Fueled by the early identification of ACE2 as the SARS‐CoV‐2 receptor, coupled with advances in structural biology and the analysis of protein interactions both in vitro and in silico, a vast array of novel and innovative strategies have been explored to develop prophylactic and therapeutic interventions targeting the binding of S protein to ACE2. The various classes of SARS‐CoV‐2 inibitors are summarized in Table 2 and discussed in more detail below.

Table 2.

Summary of SARS‐CoV‐2 inhibitors that target Spike‐ACE2 interactions.

Category	Mechanism	Inhibitor	Comments	References/clinical trials
repurposed drugs	disruption of RBD‐ACE2 interaction	ceftazidime		[60, 61]
		dalbavancin		[62]
neutralizing antibodies	competitively bind RBD	numerous	EUAs withdrawn due to resistance	[63]
		Pemgarda™	EUA for pre‐exposure prophylaxis in immunocompromised subjects	[64]
peptides	competitively bind RBD	alpha‐defensin ATN‐161		[65]
	competitively bind ACE2	numerous	may be less subject to resistance	[66, 67, 68]
soluble ACE2	competitively bind RBD	APN01	inhaled delivery under study	[69, 70] NCT04335136, NCT05065645
		CTB‐ACE2	chewing gum formulation	[71] NCT05433181
DARPins	competitively bind RBD	Ensovibep	reduced viral loads and time to recovery in outpatients, no benefit to hospitalized patients	[72, 73, 74, 75] NCT04501978, NCT04828161
aptomers	competitively bind either S protein or ACE2	numerous	single‐stranded DNA	[76, 77, 78, 79]
heparin, heparin‐mimetics	competitively bind S protein	heparin sodium	intranasal/nebulized delivery	[80, 81, 82] NCT04490239, NCT04490239
		marine sulfated glycans	reduced anticoagulant activity vs heparin	[83, 84]
		Iota‐Carrageenan	nasal spray reduced infections in hospital personnel	[85] NCT04521322
		ProLectin‐M	oral delivery in phase 2	NCT05733780
carbohydrate binding proteins	competitively bind carbohydrate components of proteoglycans	Q‐Griffithsin	plant lectin	NCT05122260, NCT05437029
		bovine lactoferrin	intranasal delivery reduced duration of SARS‐CoV‐2 PCR positivity	[86, 87] NCT04475120
metal‐based charged coordination complexes	competitively bind HS	DiplatinNC TriplatinNC	broad spectrum may be less subject to resistance	[88, 89] author's unpublished data

2.1. Repurposed Drugs

Early in the COVID‐19 pandemic screening of drugs that were approved by the Food and Drug Administration (FDA) for other conditions was used to identify existing drugs that exhibit in vitro activity against SARS‐CoV‐2 [60, 90, 91]. Several cephalosporin antibiotics, including the third‐generation ceftazidime, were among the hits [90]. An independent screen based on disruption of the RBD‐ACE2 interaction also identified ceftazidime, and follow‐up studies demonstrated in vitro inhibition of SARS‐CoV‐2 [61]. Consequently, a small study in Egypt compared the effectiveness of third‐generation ceftazidime and the fourth‐generation cephalosporin cefepime, in combination with dexamethasone, for the management of patients with moderate to severe COVID‐19, versus the contemporary standard of care, a complex regimen of colchicine and dexamethasone, anticoagulants, and four antivirals (favipiravir, remdesivir, hydroxyl chloroquine, and ivermectin). That either ceftazidime or cefepime with dexamethasone performed as well as the standard of care suggested that the cephalosporins were as effective as the antiviral cocktail in use at that time [62]. Similarly, in silico screening of an FDA‐approved peptide drug library identified the antibiotic dalbavancin as potentially disrupting RBD‐ACE2 interactions [63]. Follow‐up studies confirmed that dalbavancin blocks the S protein‐ACE2 interaction and inhibits SARS‐CoV‐2 in vitro, and in both mouse and rhesus macaque models of SARS‐CoV‐2 infection dalbavancin greatly reduced lung viral loads and prevented lung histopathological damage [63].

2.2. Neutralizing Antibodies

Among the earliest successful but unfortunately short‐lived therapeutics were monoclonal antibodies (mAbs) that neutralize SARS‐CoV‐2 infectivity by binding to the RBD of the S protein, thereby disrupting its ability to interact with ACE2 and mediate fusion of the virion envelope with the target cell membrane. While several mAb cocktails were effective for preventing hospitalization and progression to severe illness if administered before the onset of serious symptoms, the emergence of new SARS‐CoV‐2 variants with mutations within the RBD progressively conferred resistance to these neutralizing mAbs such that, for a time, no mAb‐based therapeutics remained effective [64]. However, in March 2024 the FDA issued an Emergency Use Authorization for the mAb pemivibart (Pemgarda™) for pre‐exposure prophylaxis in immunocompromised subjects who might respond inadequately to COVID‐19 vaccination, contingent on the frequency of variants with “substantially reduced susceptibility to pemivibart remaining less than or equal to 90%” [92]. Other recent efforts are focused on identifying broadly neutralizing mAbs targeting conserved regions that may be less likely to be subject to resistance (reviewed in [65]). An alternative approach, which may be much less subject to resistance, is to develop mAbs that bind to ACE2 and thereby protect cells from SARS‐CoV‐2 infection [65].

2.3. Peptides and Soluble ACE2

A similar strategy has been to identify peptides that disrupt RBD‐ACE2 interactions. Naturally occurring peptides, such as the fibronectin derivatives alpha‐defensin and ATN‐161, were found to disrupt RBD‐ACE2 interactions in screening assays, while fragments and peptides derived from ACE2 can bind competitively to the RBD to disrupt its binding to authentic ACE2 [66]. Additional peptides have been developed using structure‐based in silico methods. Some target RBD, while others are designed to target ACE2, and thereby be broad‐spectrum and less subject to resistance. In general, peptides have good safety profiles and are easy to manufacture, however, their limited half‐life in vivo is a potential limitation [67, 68, 69].

Soluble recombinant ACE2 or subdomains of ACE2 have also been explored as potential SARS‐CoV‐2 therapeutics [66]. APN01, a recombinant ACE2 developed by Apeiron Biologics AG, was evaluated in a phase 2 study for treatment of patients hospitalized with COVID‐19 (NCT04335136). While intravenous delivery of APN01 did not reduce mortality, a press release by Apeiron Biologics AG noted that patients who received APN01 had reduced viral loads and required fewer days on a mechanical ventilator compared to those who received the placebo [70]. Inhaled delivery of aerosolized APN01 is currently under study. A phase 1 trial (NCT05065645) determined that both single and multiple inhaled doses were safe and well tolerated [71]. Also in development is a chewing gum that releases a recombinant ACE2, CTB‐ACE2 [72]. However, a phase 1/2 trial to determine if use of the gum reduces viral load in the saliva of nonhospitalized COVID‐19 patients (NCT05433181), was never opened and has been withdrawn for administrative reasons.

2.4. DARPins

Design Ankyrin Repeat Proteins, or “DARPins”, are based on a generic scaffold comprised of a consensus sequence from proteins that contain ankyrin repeats, which in eukaryotic cells function to tether membrane‐bound proteins to the cytoskeleton (reviewed in [73]). Combinatorial libraries and in vitro selection can then be used to optimize the scaffold DARPin for specific binding to target proteins. This flexibility allows for periodic re‐optimization to maintain binding as target proteins acquire resistance mutations. DARPin domains with different binding specificities can be linked.

Ensovibep, developed by Novartis and Molecular Partners, is the first antiviral DARPin to reach clinical phase. It contains DARPin domains that enhance in vivo half‐life by binding to human serum albumin, while three additional domains target the binding of Ensovibep to the S protein RBD. In phase 1 trials Ensovibep was generally safe and well tolerated, and exhibited a 2‐week half‐life in vivo. In outpatients it reduced viral loads and time to recovery but had no benefit to hospitalized patients (NCT04501978, NCT04828161) [73, 74, 75, 76]. In 2022 the FDA declined to issue an emergency use authorization, citing need for additional clinical data.

2.5. Aptomers

Small single‐stranded DNA molecules called “aptomers” have been developed that specifically bind to S protein or ACE2 and disrupt S protein‐RBD binding [77, 78, 79, 93]. Therapeutic aptomers targeting SARS‐CoV‐2 have not entered the clinical phase. However, an aptomer targeting TRL4, originally developed as a neuroprotective therapeutic [94, 95, 96], has been explored as a treatment to prevent the cytokine storm associated with severe COVID‐19 (NCT05293236).

2.6. Heparin and Heparin‐Mimetics

In addition to RBD‐ACE2 interactions, S protein binds to heparan sulfate (HS) proteoglycans on cell surfaces. As these interactions are important for attachment, engagement of ACE2, and subsequent entry of the virus [80, 97], heparin and other “heparin‐mimetics” (generally complex polysaccharides or anionic polymers) can disrupt SARS‐CoV‐2 infection by binding to S protein and competitively inhibiting its ability to interact with HS on target cell surfaces [81, 82].

Heparin has been explored as a potential antiviral therapeutic. While heparin's anticoagulant activity complicates its systemic use, no serious adverse events were associated with daily intranasal delivery of heparin sodium for up to 2 weeks (NCT04490239; NCT04490239) [98]. In 2021 a phase 2 study (NCT04842292) was initiated to evaluate nebulized heparin for treating COVID‐19 patients with acute respiratory failure and on mechanical ventilation; however, the study was terminated after 4 months due to lack of enrollment.

Numerous studies have explored various heparin‐mimetics as SARS‐CoV‐2 attachment and entry inhibitors. These include natural products derived from animals or marine organisms, as well as synthetic polyanionic compounds [82, 83, 99, 100, 101, 102]. Compared to heparin, some marine sulfated glycans have antiviral activity yet lack anticoagulant activity [84], or antiviral and anticoagulant activity can be segregated by hydrolysis and isolation of lower molecular weight species [85]. A number of heparin‐mimetics are in the clinical phase development [100]. For example, a nasal spray containing Iota‐Carrageenan significantly reduced the acquisition of SARS‐CoV‐2 infections by hospital personnel caring for COVID‐19 patients (NCT04521322) [103], and a phase 1b/2a trial to evaluate oral delivery of ProLectin‐M for treatment of patients with mild to moderately‐severe COVID‐19, planned for 2023/2024, has not begun recruiting (NCT05733780).

2.7. Carbohydrate Binding Proteins

Proteins such as plant lectins and lactoferrin can bind to carbohydrate components of proteoglycans and thereby inhibit S protein‐ACE2 interactions and viral entry [86, 104, 105]. Phase 1 studies of an intranasal spray containing the lectin Q‐Griffithsin have been completed (NCT05122260, NCT05437029), while phase 2 studies in asymptomatic subjects or COVID‐19 patients with mild‐to‐moderate symptoms found that oral bovine lactoferrin or oral and intranasal liposomal bovine lactoferrin reduced the duration of SARS‐CoV‐2 PCR positivity (NCT04475120) [87, 106].

2.8. Metal‐Based Charged Coordination Complexes

Polynuclear platinum‐based compounds such as DiplatinNC and TriplatinNC, initially developed as anticancer drugs [107], were found to bind tightly to HS on cell surface proteoglycans, partly due to their strong positive charge [88, 108]. This led us to hypothesize that they could block viral entry by competitively disrupting viral‐HS interactions. Thus far DiplatinNC and TriplatinNC have been shown to inhibit entry of cytomegalovirus, adenovirus, metapneumovirus, and enterovirus 71 [89, 109], as well as SARS‐CoV‐2 (Zoepfl et al., manuscript in preparation). Consistent with the predicted mechanism of action, time‐of‐addition studies confirmed that these compounds must be present at the time virus is added to cells, while add‐and‐remove studies suggested that they act by binding to factors on the host cell surface rather than to the virion (Zoepfl et al., manuscript in preparation).

3. The Future

Although the global burden of COVID‐19 has declined substantially since reaching a peak of 526,000 hospitalizations and 99,000 deaths per week in early 2021, the current 2025 hospitalization rate of ~11,500/month indicates that COVID‐19 remains a significant public health burden [110]. In the US the CDC estimates that 78,000 to 130,000 hospitalizations and 8,900 to 15,000 deaths will be attributable to COVID‐19 during the 2024‐2025 season [111], which is comparable to the morbidity and mortality caused by influenza [112].

While SARS‐CoV‐2 continues to mutate to produce new variants, ACE2 remains the key receptor for viral entry. Future research on the role of ACE2 in COVID‐19 should focus on the regulation of ACE2 in different tissues, genetic variations across populations, and the role of ACE2 in immune responses, including the systemic cytokine storm postinfection. Investigating ACE2's distribution and involvement in long COVID may provide insights into chronic disease symptoms. Additionally, the regulation of blood pressure and inflammation by ACE2 is critical, as its dysregulation can impact cardiovascular, pulmonary, and renal health. Thus ongoing research into ACE2 will be crucial for advancing COVID‐19 treatments.

As antiviral interventions have thus far had limited efficacy once disease has reached a stage requiring hospitalization, future antiviral development will likely emphasize prevention of SARS‐CoV‐2 infections or the abrogation of progression to symptomatic disease. Thus, for example, additional mAbs may be needed for prophylaxis of immunocompromised subjects who fail to respond to vaccination [113], and there is potential for the discovery of broader spectrum mAbs targeting either the S protein or ACE2. A number of novel interventions have reached the latter stages of clinical development and may prove useful for prophylaxis (e.g., aerosolized heparin), prevention of transmission (e.g., chewing gum), or treatment of high‐risk patients with early mild‐to‐moderate symptoms. In a larger context, the COVID‐19 pandemic has driven the development of many novel and innovative antiviral technologies, which in the future may prove valuable for combating other viral infections or outbreaks of pandemic potential.

McVoy M. A., and Kummarapurugu A. B., “The Role of ACE2 in SARS‐CoV‐2 Infection, Pathogenesis, and Antiviral Interventions,” Journal of Medical Virology 97 (2025): 1‐12, 10.1002/jmv.70721.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.