Keep out! SARS-CoV-2 entry inhibitors: their role and utility as COVID-19 therapeutics

COVID-19, the disease caused by the novel coronavirus SARS-CoV-2, was declared a pandemic and global emergency shortly after it began in late 2019. As of today, the disease has claimed about 3 million lives and cost the world trillions of dollars [1, 2]. Even though the majority of people recover from the disease and experience only mild or no symptoms, the pathogenicity and transmissibility of the virus grants COVID-19 a higher burden of mortality than seen in other ongoing viral diseases such as the seasonal flu caused by influenza [3, 4]. Given this fatality rate and the novelty of the virus, the science surrounding COVID-19 has been a rapidly evolving field. Developing and validating treatment strategies has required a delicate balance between rigor in scientific evaluation and expediency in developing therapies that help to slow down the rampage of the pandemic. The scientific community continues to unravel the nature of SARS-CoV-2, though we now know much more about SARS-CoV-2 and the consequences of infection than when it was first discovered [5‐7].

SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA from the betacoronavirus genus [6, 7]. The major entry point is the nasal passage, from which infection begins following exposure [8‐10]. Specifically, the virus enters the nasal epithelial cells through the binding of the viral Spike (S) glycoprotein to its cellular receptor known as angiotensin-converting enzyme-2 (ACE2). Following binding, the virus will gain access into the cell via endocytosis or fusion with surface cell membrane. After the virus has unloaded its genome into the cytoplasm, it hijacks the host translational machinery and directs the production of large polyproteins from which essential proteins such as RNA dependent RNA polymerase and helicase are made via viral proteolytic cleavage. The replication proteins will generate genomic RNA as well as sub-genomic RNA that become templates for the synthesis of accessory and structural proteins [3, 7, 11]. The genomic RNA and the proteins are then assembled into virions that exit the cell via exocytosis and infect new cells (Fig. 1). Later on, the virus may spread to the lower respiratory system through aspiration and/or infection of cells in conducting airways [3, 7‐10]. Through a timely and balanced immune response involving the innate and adaptive response, viral propagation is managed and mostly confined to upper airways, resulting in mild symptoms being observed in the majority of patients [12, 13].

However, some individuals will go on to develop more severe symptoms, likely due to successful immune evasion by the virus and/or delayed and impaired responses by their immune systems [9, 12, 13]. It has been shown that a balanced response involving innate immunity, B cells, CD4⁺ T cells, and CD8⁺ T cells is needed to control SARS-CoV-2 [12]. In the absence of this balanced response, the virus will eventually reach the pulmonary gas exchange units and infect type II alveolar cells [9]. A dampened initial immune response will allow the virus to replicate and spread to new cells. The infected cells will undergo apoptosis and die, with their death leading not only to alveolar damage but also an excessive production of pro-inflammatory cytokines such as IL-6, IFN-γ, IL-8, TNF-α, and IL-1β [3, 9, 11, 14, 15]. The release of these cytokines by host cells (also known as cytokine storm) will further distort the antiviral immune response and usually coincides with recruitment of immune cells such as monocytes and neutrophils [12, 16]. An interplay of these events may lead to a vicious cycle in severe cases, marked by complications such as increased vascular permeability, pulmonary edema, acute respiratory distress syndrome (ARDS), multi-organ failure and death [3, 11, 15].

These emerging concepts regarding SARS-CoV-2 and the pattern of clinical progression of COVID-19 have clarified several issues. For example, we now know that there are two phases in the pathogenesis of COVID-19, and that treatment of the disease requires two distinct strategies. Specifically, in the early phase of COVID-19, viral growth and propagation are the primary determinants driving disease progression or resolution. In the later phases, a hyperinflammatory response by the host is much more important in driving the disease than is viral replication [11, 17]. This understanding has now translated into current approaches to treat COVID-19. Treatment for outpatients diagnosed early with mild to moderate COVID-19, but who are at risk of hospitalization due to comorbidities or other factors, often involves administration of neutralizing anti-SARS-CoV-2 monoclonal antibodies, as discussed below. However, patients that are hospitalized may be given redemsevir, dexamathosone, bariticinib or a combinatorial regimen comprising these, depending on whether the patient requires supplemental oxygen and ventilation [18]. Importantly, targeting the virus during the early phase of infection means that significant benefit can be gained from rapid viral testing, as a quick diagnosis can capture a therapeutic window of opportunity before an exuberant host-mediated immune response leads to potentially fatal complications such as pneumonia, ARDS, multi-organ system dysfunction and hypercoagulation [19]. Such early treatment of COVID-19 can shorten disease recovery rates, prevent hospitalizations and be a more cost-effective way to manage COVID-19 [11, 17]. Thus far, monoclonal antibodies have been approved for early management of COVID-19. Studies have shown significant neutralization potency and efficacy, providing proof of principle that targeting viral entry can be an effective way to treat COVID-19, at least in the early stages [18, 20]. These encouraging results have not, however, adequately addressed the potential of other types of entry inhibitors. As the mission of a vaccine-driven end to this pandemic faces new challenges due to the rise of mutant variants, new questions are emerging about how these other potential therapeutics may help to alleviate these problems.

In this review we summarize the development of anti-SARS-CoV-2 neutralizing monoclonal antibodies for clinical use and the impact they have had on early management of mild-to-moderate COVID-19. These concepts are discussed in the context of the emerging evidence showing that currently available vaccines are challenged by the rise of new SARS-CoV-2 variants. We also highlight the challenges that antibodies are facing as they deal with SARS-CoV-2 mutants and how, together with a multitude of other emerging candidates for entry inhibition, they can overcome those challenges. Finally, we discuss and evaluate the potential of entry inhibitors as prophylactic agents, as well as the role they can play against emerging SARS-CoV-2 lineages and future coronavirus outbreaks.

The availability of several effective vaccines against SARS-CoV-2 has given hope to billions of people across the globe [21, 22]. Since the pandemic started, vaccination has appropriately been viewed as the long term and most sustainable solution to deal with COVID-19. It is therefore encouraging that currently available vaccines have been shown to induce both humoral and cellular immunity and to provide substantial protection to vaccinees in clinical trials [12, 23]. Simultaneous with the continued rollout of vaccines across the globe, more and more people will also acquire protective immunity due to infection and recovery from the disease. Nevertheless, it is critical to note that the availability of the vaccine does not obviate the need for available therapies nor for the development of new and more effective therapies. First, the available vaccines work effectively in people who are not yet infected, and not in those already infected. Secondly, there are challenges associated with reaching adequate levels of vaccine coverage. Experts estimate it will require vaccination rates of about 80 to 85% for the US population to be adequately protected against COVID-19 related hospitalizations and deaths [24‐26]. It will take time to vaccinate enough people to reach herd immunity or to eradicate the disease, especially given the 2-dose regimen recommended for some of the current vaccines. Additionally, there has been inequitable global distribution of vaccines according to WHO, with high and upper middle-income countries receiving the lion’s share of the available doses in comparison to developing countries. More initiatives such as COVAX, created by WHO and earmarked for provision of vaccines to the developing world, are needed to continue to improve equitable access to vaccines in the future [27‐30]. Another issue arises from individuals who will not be vaccinated even when given access, including immunocompromised patients who cannot take the vaccine, as well as those who choose to defer or delay vaccination due to vaccine hesitancy. Recent reports show that a significant fraction of Americans is unwilling to take the vaccine, and the recent pauses of Janssen and Astrazeneca vaccines could worsen this hesitancy and derail plans to reach herd immunity [24, 31‐33]. These unvaccinated populations will inevitably become reservoirs and factories for ‘fitter’ virus species to evolve and emerge, potentially undermining the efforts to fight or eradicate the disease.

Perhaps of greatest concern is the emergence of new variants with reduced sensitivity to neutralization by vaccine-induced antibodies, as these variants represent the greatest threat to protective efficacy of current vaccines. As of the time of writing, there are 4 major variants of concern (VOC) in the world, namely Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2). Additional variants of interest are currently under surveillance including Lambda (C.37) [34‐36]. Importantly, VOCs beta, gamma and delta harbor mutations in their Spike protein, such as E484K and L452R, that confer higher virulence, re-infection rates and resistance to sera from individuals vaccinated with Pfizer, Moderna and other major available vaccines [36‐45]. Recently, reports by WHO and Eurosurveillance have demonstrated that the aforementioned VOCs have spread from their origins to hundreds of countries across the globe, with some instances showing that they can rapidly become the dominant source of new infections over the original SARS-CoV-2 variant [36, 46, 47]. The emergence of these new strains evokes fear of more deadly viral diseases breaking out in the future.

Therefore, there remains an urgent need for additional therapeutic strategies that work alongside these vaccines. Early translational efforts targeted against COVID-19 focused primarily on identifying treatments for the critically ill, as evidenced by several drug repurposing campaigns that tested antivirals and immune modulators in hospitalized patients. Most of the antivirals tested targeted the more advanced checkpoints of the virus life cycle, such as translation and RNA replication. Redemsivir is one agent that emerged from these studies, and is now used to treat hospitalized patients who require supplemental oxygen [48‐50]. However, we have also learned that treating unhospitalized COVID-19 patients early improves prognosis. This highlights the importance of identifying more therapies for early management. Furthermore, some outpatients who eventually recover without medical intervention will experience long term-effects such as fatigue, loss of taste and smell, as well as cognitive impairment and cardiopulmonary dysfunction [19, 51]. These patients are not eligible for remdesivir treatment, and developing therapies for this population has the potential to improve recovery and quality of life. Antibodies and other viral entry inhibitors are suitable candidates for addressing these concerns, as they have the potential to minimize hospitalizations, prevent chronic late effects, and slow down spread by shortening the period of infectiousness and reducing mortality. While vaccines help to prevent new infections, entry inhibitors complement these gains by fighting COVID-19 in the unvaccinated population and the vaccinated who develop breakthrough infections. Moreover, the use of antibody cocktails and other viral entry inhibitors is also showing potential in combating new variants and other coronavirus clades. Below, we discuss the potential of inhibiting SARS-CoV-2 entry with antibodies and with alternative entry inhibitors such as recombinant human soluble ACE2, miniproteins, peptides and small molecules.

SARS-CoV-2 Spike is a homotrimeric protein found on the surface of the viral membrane [52, 53]. Each monomer consists of two subunits, S1 and S2, located at the N- and C- termini, respectively. S1 consists of the N terminal domain (NTD) and receptor binding domain (RBD). Within the RBD is a sub-domain called the receptor binding motif (RBM) [54, 55]. The virus makes first contact with target cells that express ACE2 by way of a direct contact between the S1 RBD and ACE2 [56]. This receptor binding and virus attachment is the first step of viral entry into the cells, and therefore an attractive therapeutic target (Fig. 1). A subsequent step involves fusion with cell membrane to facilitate the release of the viral genome into the cell cytoplasm, and is mediated by the S2 subunit. S2 consists of the fusion peptide (FP), two α-helical heptad repeats (HR1 and HR2), a loop region, a transmembrane (TM) domain and cytoplasmic tail (CT). Binding of ACE2 to S1 induces a conformational change and processing that allows the FP to insert into the host cell membrane [54, 55]. The HR1 repeats, one from each subunit of the trimer, will refold together with the HR2 repeats in an anti-parallel fashion to form a coil-coil fusion core called the six-helix bundle (6HB). This structure brings the viral and cellular membranes together and drives cell fusion and release of the viral contents into the cytoplasm [54, 55]. This step is crucial and requires completion in order for the virus to achieve productive infection. Therefore, it represents another entry checkpoint that can be selectively targeted. The domain structure of the Spike glycoprotein and binding motifs of ACE2 are shown in Fig. 2. Various therapeutic proteins and their derivatives and small molecules have been employed to target these two distinct steps of SARS-CoV-2 entry as discussed in the following sections.

Although S1 and S2 mediate viral attachment and membrane fusion to enable the virus to unload its genetic cargo, function of these two subunits is enabled by the participation of at least 3 types of host proteases: furins, cathepsins and surface serine proteases. Viral entry generally occurs either through direct fusion of the virus with the surface membrane or endocytic uptake, and what determines which proteases will dominate in facilitating fusogenic activity is the entry pathway utilized [54, 55, 108, 144]. The SARS-CoV-2 Spike protein has two cleavage sites, S1/S2’ and S2’. For non-endocytic entry, the S1/S2’ site is cleaved primarily by the furin proprotein convertase. This cleavage may then help to reveal the S2’ site more fully to the surface trypsin-like serine proteases such as TMPRSS2. The S2’ site is immediately upstream of the fusion peptide (FP), and its cleavage by TMPRSS2 exposes the hydrophobic peptide (FP) for insertion into the membrane (Fig. 2a) and subsequent formation of the 6HB as already described. Conversely, if the virus takes the endocytic route, cathepsins will play a more dominant role [54, 55, 108, 144]. Specifically, the cathepsin L isoform has been shown to be more important in S2’ cleavage for coronaviruses. Cathepsin L is a lysosomal cysteine protease and its function, like that of many other cathepsins, is pH-dependent, with optimal pH activity ranging from 3–6.5 [114, 144, 145]. Without cleavage of Spike by these proteases, the virus would not be able to fuse with the lysosomal or autolysosomal membrane to release its genome into the cytoplasm (Fig. 1). Therefore, all the three different classes of proteases noted above represent rational targets for COVID-19 therapeutic intervention.

COVID-19 is now understood as a biphasic illness, with an early viral phase and a more dangerous host-immune response phase. This knowledge has shaped our translational and clinical therapeutic strategies to find treatments for those infected. The ongoing antigenic drift of SARS-CoV-2 is also shaping the fight against COVID-19. Four major variant strains have now been identified, which have generally shown increased transmissibility and resistance to the efficacy of vaccines and monoclonal antibodies [36, 37]. Vaccines, particularly those that are mRNA-based, have shown that they offer some protection against the variants, albeit with reduced effectiveness, and multiple doses of the vaccines, including booster shots, may be necessary in the future [165‐168]. Health officials will also continue to monitor variants of interest that have already been identified. In addition, the emergence of SARS-CoV-2 has also renewed fears that another zoonotic spillover will occur and cause an even more deadly outbreak. These fears are not unfounded, given that we experienced more than 10 serious outbreaks from emerging RNA viruses in the last 20 years alone [169]. Each of these aspects have subjected the counter-measures currently in place to increased attention, asking how such measures can be made more effective based on available evidence. In addition, and also of critical importance, is the development of future plans for dealing with mutant strains and potential outbreaks. In this review, we have highlighted the utility of vaccines and the gaps they leave in fighting COVID-19. We then demonstrated that the mechanism of action of entry inhibitors makes them suitable agents for early management of COVID-19 to help cover some of the gaps, and shown why continued research on such inhibitors is crucial. The monoclonal antibody entities are farthest along the drug development pipeline, with some already approved for use (EUA) and several more in advanced stages of clinical trials [66]. Cocktails of monoclonal antibodies, multivalent nanobodies and recombinant soluble ACE2 have also demonstrated therapeutic effect against mutant strains, including those currently in circulation, as well as broad, cross-family coronavirus efficacy. The antibody species’ ability to limit resistance and deliver broad activity comes from their targeting of more than one epitope, including some from the more conserved regions of Spike. For agents based on recombinant ACE2, similar efficacy comes from their similarity with the endogenous receptor, which makes it difficult for mutant strains to arise without also losing infectivity. In addition to these valuable therapeutic effects and their potential as agents to treat future outbreaks, these protein-based antivirals have also demonstrated they can be useful when given prophylactically, even several days before exposure. As noted earlier, various subgroups of people will benefit from prophylactic treatment using these agents. As for the miniprotein, peptide and small molecule therapeutics, current literature suggests that they are not as advanced in terms of clinical development as are the antibodies or recombinant ACE2. However, their utility is in their size and ability to be more readily developed into therapeutic formulations that can be self-administered either as oral pills or inhalants. More research is therefore still needed, as researchers and decision makers continue to evaluate the potential use of entry inhibitors for outpatient prophylaxis. Also, given that the nasal passage is the most dominant and initial site of infection, aerosolization can potentially be beneficial in preventing viral spread to the lungs through use of nasal sprays [8]. Finally, more investment in the development of entry inhibitor therapeutics as well as other antivirals and therapies directed against the host immune response is needed, as their availability will impact our options in responding not only to future SARS-CoV-2 lineages, but also to future coronavirus pandemics. Recent events have made it abundantly clear that it is both more impactful and cost effective to prevent or prepare for a pandemic like the one caused by COVID-19, than to encounter such a pandemic without preparation. For this reason, current proposals by the US and international community to invest more into pro-active and pre-emptive countermeasures against future outbreaks are commendable, as this development will shorten the time between an outbreak and an effective therapeutic response [170, 171].