Inhibitors of SARS-CoV-2 Entry : Current and Future Opportunities

 

Inhibitors of SARS-CoV-2 Entry:

 Current and Future Opportunities

SARS-CoV2 Viral entry into the host cell is via the viral spike protein binding with the angiotensin-converting enzyme 2 (ACE2) receptor on target cells' surface. ACE2 is expressed on the heart, testis, lungs, kidneys, brain and in other tissues.

This virus-ACE2 binding mediates viral internalization and infection. Host proteases such as TMPRSS2 are instrumental in enhancing the virus's entry by promoting viral-cell membrane fusion. It was found that both TMPRSS2 and ACE2 interact to produce their action. Specifically, TMPRSS2 produced ACE2 cleavage at the N-terminal end, which was necessary for SARS-CoV2 spike-ACE2 interaction. High TMPRSS2 expression in ACE2-expressing cells results in ACE2 cleavage.

This is blocked by TMPRSS2 inhibitor Camostat, which indicates that ACE2 is a substrate of the former enzyme. These experiments demonstrate that the ACE2 interacts on a physical level with the TMPRSS2 in the lungs and other organs . This endogenous complex does not appear to be dependent on TMPRSS2-mediated ACE2 cleavage.

Epidemiological data shows increased severity and mortality of COVID-19 suggests a potential role for androgen in SARS-CoV-2 infection. There's evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Further, Camostat – a TMPRSS2 inhibitor, blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction. Thus providing evidence for the first time a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or Camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors, androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.

Camostat alone or in combination with AR directed therapies reduces SARS-CoV-2

The researchers looked at Camostat's efficacy and other anti-AR therapies to inhibit SARS-CoV-2 entry via spike protein binding. Camostat prevented viral entry only in androgen-deprived cells, indicating that TMPRSS2 is a cofactor for its activity.

In androgen-proficient cells, Camostat, anti-androgen enzalutamide, or AR degrader ARD-69 all brought about an extremely high and observable drop in viral entry. Still, the combination of Camostat with either of the others was more effective than monotherapy. Moreover, AR-negative cells showed reduced viral entry only with Camostat.

Implications

The study may help to understand the higher risk of COVID-19 posed to men. It shows that androgen receptors regulate both TMPRSS2 and ACE2. In vitro and mouse studies showed a reduction in androgen levels to be correlated with decreased TMPRSS2 and ACE2.

Again, androgen also reduces immune function while increasing the propensity to inflammation and may increase the severity of the disease in men. Androgen induces a rise in circulating neutrophil count and IL-8 (among other cytokines), which are characteristic of severe COVID-19.

If progressive COVID-19 is due to uncontrolled replication of SARS-CoV-2, the current study's findings may prove valuable since they show the importance of androgen effects on ACE2 and TMPRSS2 expression.

Androgens may act via TMPRSS2, which in turn enhances the uptake of the virus either by interaction with or cleavage of ACE2 or by spike cleavage, which enhances viral entry into the host cell. The study demonstrates how AR-expressing lung and prostate cells can be treated with a TMPRSS2 inhibitor and anti-AR drugs to inhibit viral entry via the spike protein.

This could indicate the potential for these drug combinations in preventing the progression of COVID-19

The researchers say, “We provide the first direct evidence for the endogenous interaction between TMPRSS2 and ACE2 in human cells, and endogenous TMPRSS2 mediated cleavage of SARS-CoV-2 Spike, which could be blocked by Camostat.” This agrees with prior research showing how TMPRSS2 contributes to the activation of the spike protein of SARS-CoV2, SARS-CoV and MERS-CoV.

ACE2, TMPRSS2 and FURIN

Through whole‐exome sequencing, the frequency of the ACE2, TMPRSS2, and Furin was analyzed in relation to presence or absence of SARS‐CoV‐2 infection.

Recently, other host factors including Furin, TMPRSS4, and lyosomal cathepsins have been shown to be relevant for SARS‐CoV‐2 entry into host cells.

Furin has been implicated in the SARS‐CoV‐2 infectivity. S protein is cleaved by TMPSS2 with the collaboration of furin, which has been linked to the entry of the virus in the respiratory tract and also with an increased risk of contagion.

Bromelain inhibits SARS‐CoV‐2 infection via targeting ACE‐2, TMPRSS2, and spike protein

The new coronavirus, SARS‐CoV‐2, transmits rapidly from human‐to‐human resulting in the ongoing pandemic. SARS‐CoV‐2 infects angiotensin‐converting enzyme 2 (ACE‐2) expressing lung, heart, kidney, intestine, gall bladder, and testicular tissues of patients, leading to organ failure and sometimes death. Currently, COVID‐19 patients are treated with different agents, including favilavir, remdesivir, chloroquine, hydroxychloroquine, lopinavir, darunavir, and tocilizumab. However, the safety and efficacy of those drugs against COVID‐19 still need further confirmation by randomized clinical trials. Hence, there is an emergent need to repurpose the existing drugs or develop new virus‐based and host‐based antivirals against SARS‐CoV‐2. Bromelain is a cysteine protease isolated from pineapple stem and is used as a dietary supplement for treating patients with pain, inflammation, thrombosis, and cancer.

Recently, studies have shown that SARS‐CoV‐2 homotrimeric viral spike protein (S1) binds to the Transmembrane Serine Protease 2 (TMPRSS2) primed host cell's receptor ACE‐2 for initial entry, followed by S2‐mediated membrane fusion.8 Of several normal and cancerous cells tested, VeroE6 and Calu‐3 cells showed ACE‐2 protein expression, as well as a basal level of TMPRSS2 protein. Since ACE‐29 and TMPRSS2 (UniProtKB‐O15393) contains cysteine residues with disulfide bonds to stabilize the protein structure, we investigated the effect of bromelain on ACE‐2 and TMPRSS2 expression. Bromelain‐induced a dose‐ and time‐dependent reduction of ACE‐2 and TMPRSS2 expression in VeroE6 cells but do not alter ACE‐2 expression in Calu‐3 cells. However, bromelain reduces the expression of TMPRSS2 in Calu‐3 and ACE‐2 negative normal bronchial epithelial (BEAS‐2B) and lung adenocarcinoma (A549) cells. Cysteine protease inhibitor (E‐64) treatment further confirmed that bromelain's cysteine protease activity could cleave/reduce the expression of ACE‐2 and TMPRSS2. Surface plasmon resonance (SPR) analysis revealed that purified SARS‐CoV‐2 S‐ectodomain binds with ACE‐2 in a concentration‐dependent manner and has a comparable binding affinity as control RBD. The calculated molecular weight of the purified S‐ectodomain‐GFP protein is ∼165 kDa; however, we observed a higher molecular weight of S‐ectodomain (∼215 kDa), which may be due to heavy N‐ and O‐linked glycosylation. A serological assay showed a significantly increased median fluorescent intensity (MFI) of purified S‐ectodomain with COVID‐19 positive patients’ samples. These two results indicated that purified S‐ectodomain is a properly folded and functionally active protein.

Since bromelain digested ACE‐2 and S‐ectodomain, we investigated the effect of bromelain on the interactions of S‐ectodomain and SARS‐CoV‐2 with VeroE6 cells. Bromelain significantly reduced the binding of S‐protein to VeroE6 cells and was further confirmed by cysteine protease inhibitor (E‐64) treatment. Interestingly, bromelain pre‐treatment significantly decreased SARS‐CoV‐2 viral binding in VeroE6 cells (P = .0021). Most importantly, VeroE6 cells or SARS‐CoV‐2 or both with bromelain reduces the viral infection. Additionally, we found significantly reduced SARS‐CoV‐2 viral RNA copies in bromelain‐treated VeroE6 (P = .0010) and Calu‐3 (P = .0099) cells. Collectively, these results suggest that bromelain could inhibit SARS‐CoV‐2 binding and infection in VeroE6 and Calu‐3 cells. Studies have demonstrated that SARS‐CoV‐2 S‐protein has high homology among other coronaviruses (76% identity with SARS‐CoV) with conserved cysteine amino acids (UniProtKB: P59594). This indicates that bromelain may be used as a broad antiviral agent against SARS‐CoV‐2 and other related family members.

In conclusion, the currently used drugs against SARS‐CoV‐2 have potential side effects. Vaccine trials have started against COVID‐19, but the host immune response against SARS‐CoV‐2 is not fully understood. It differs between individuals, and also re‐infection of individuals with SARS‐CoV‐2. For the first time, our results demonstrate that bromelain can inhibit SARS‐CoV‐2 infection via targeting ACE‐2, TMPRSS2, and SARS‐CoV‐2 S‐protein. Also, thrombosis development is a significant risk factor of multiorgan failure and death in COVID‐19 patients.10 Since bromelain inhibits SARS‐CoV‐2 infection, and its profound fibrinolytic activity 6 suggests that bromelain or bromelain‐rich pineapple could be used as an antiviral against SARS‐CoV‐2 and future outbreaks of other coronaviruses.

Direct ACE2 modulators

ACE2, discovered nearly 20 years ago, is an enzyme that converts angiotensin I to angiotensin 1–9, a peptide of unknown function, and angiotensin II to angiotensin 1–7, a vasodilator. The encoded protein is a functional receptor for the S glycoprotein of the human SARS-CoV, HCoV-NL63 and SARS-CoV-2. ACE2 is the critical receptor for SARS-CoV-2 infection. Currently, it appears as a promising pharmacologic target and it has been proposed that inhibiting this interaction could be a valid strategy for treating patients with COVID-19. Although attached ACE-2 may allow SARS-CoV-2 to enter cells, its free circulating forms may inactivate SARS-CoV-2 by attaching themselves to the virus and stopping coupling to membrane ACE2 receptor and consequent entry into pulmonary endothelial cells. Recombinant soluble ACE2 (hrsACE2) has already been tested in phase 1 and phase 2 clinical trials aiming to enhance ACE2 activity in human diseases with pathologically elevated Ang1–8 and in acute respiratory distress syndrome. APN01, a rhACE2 designed to imitate the human enzyme ACE2 so that SARS-CoV-2 can no longer infect the cells, is currently in phase 2 clinical trial by the European biotech company Apeiron Biologics (NCT04335136, registered on April 6, 2020). The trial aims to compare APN01 to placebo in up to 200 severely infected COVID-19 patients at 10 sites. In addition, it was recently shown that clinical grade hrsACE2 reduces SARS-CoV-2 recovery from Vero-E6 cells by a factor of 1000–5000.

Other ACE2 modulators

Angiotensin receptor blockers

Treatment of hypertension, cardiac failure and other indications of the cardiovascular system relies on drugs targeting renin-angiotensin axis, such as ACE inhibitors (ACEi) and angiotensin II receptor blockers (ARBs). Discussion on whether or how these drugs impact COVID-19 is ongoing. A hypothesis put forward on possible upregulation of ACE2 membrane expression leading to increased susceptibility to SARS-Cov2 infection and COVID-19 disease severity in patients treated with ACEi and ARBs was not supported by findings of recent studies. In contrast, evidence is emerging pointing towards a beneficial effect of ARBs. A retrospective study in hypertensive Chinese COVID-19 patients was recently published showing a possible beneficial relation between the use of ARBs/ACEi and both severity of disease and reduced deaths. Unfortunately, no discriminative information between ARBs and ACEi was reported in this study, neither between individual ARBs. Interestingly, network proximity analysis uncovered irbesartan as the drug topping a list of 16 drugs (out of almost 3000 FDA-approved drugs that were included in the analysis) showing a significant association between irbesartan’s targets and HCoV-associated host proteins in the human interactome. Additionally, a phase 2 prospective clinical trial has been initiated by the University of Minnesota, testing losartan for its beneficial effect when prescribed in COVID-19 patients within 24 h of hospital admission or a positive test result, whichever is later (NCT04312009, registered on March 17, 2020). It should be kept in mind that the various ARBs have significant structural differences and they also have many functional differences that are not mediated by blockade of angiotensin type 1 receptor. Thus, it is necessary to study the most promising ones (discussed above) individually for their potential beneficial effect on SARS-CoV-2 infection/COVID-19.

Calmodulin antagonists

Another class of drugs that interact with ACE2 by inhibiting the shedding of its ectodomain are calmodulin antagonists. Calmodulin antagonists inhibit CALM-ACE2 interaction and increase the release of the ACE2 ectodomain in a dose- and time-dependent manner. Several marketed drugs, especially psychotropic medications, including melatonin, trifluoperazine, perphenazine, pimozide and amitriptyline, are known to inhibit calmodulin interaction with its target enzymes. It should be noted that melatonin was also identified as one of the top 16 network-predicted repurposable drugs showing a significant association between their targets and HCoV-associated host proteins in the human interactome. The potential benefits of melatonin in the attenuation of COVID-19 based on its antioxidant, anti-inflammatory and immunomodulatory properties have been recently summarized. The prophylactic use of melatonin against SARS-CoV-2 is currently assessed in healthcare workers exposed to the virus in their clinical practice [Efficacy of Melatonin in the Prophylaxis of Coronavirus Disease 2019 (COVID-19) Among Healthcare Workers. (MeCOVID), NCT04353128, registered on April 20, 2020]. Additionally, melatonin effectiveness as an add-on treatment method along with a standard antiviral drug regimen in patients with severe COVID-19 will be evaluated in an interventional clinical study due to start soon (Evaluation of Therapeutic Effects of Melatonin by Inhibition of NLRP3 Inflammasome in COVID19 Patients, NCT04409522, registered on June 1, 2020).

Evidence exists that the selective oestrogen receptor modifiers (SERMs) tamoxifen and toremifene are also calmodulin antagonists. Interestingly, both drugs have been shown to inhibit MERS-CoV and SARS-CoV, and also to exhibit antiviral activity against HIV, HCV and EBOV. In addition, in network proximity analysis, toremifene alone or in combination with emodin (an experimental drug for the treatment of polycystic kidney disease) offered a potential therapeutic approach for SARS-CoV-2. For raloxifene, another member of SERMs class, there are no reports on its effect on calmodulin. Nevertheless, it appears as the first SERM for which researchers required access to clinical trials for use in asymptomatic or COVID-19 patients with mild symptoms.

It is plausible that SERMs also affect ACE2 expression. Upon binding to the oestrogen receptors (ERs), oestrogen exerts complex tissue-specific regulation over the components of the renin-angiotensin-aldosterone system. Among ACE2 regulatory regions, there are many oestrogen receptor binding motifs. Through binding to ERα, estradiol significantly increases ACE2 expression in human atrial myocardium, whereas ACE2 expression is down-regulated in kidneys and no effect was found in the lung. Overall, no clear picture is emerging and the role (if any) of oestrogen in COVID-19 remains to be elucidated. One study went to the opposite direction hypothesising that oestrogen receptor activation with conjugated estrogens may be a good prevention and therapeutic strategy against COVID-19, on the basis of findings in animal experiments showing that oestrogen treatment silences the inflammatory reactions and decreases virus titres leading to improved survival rate.

TMPRSS2 inhibitors

TMPRSS2 gene encodes for transmembrane protease serine 2, which is essential for viral infectivity by facilitating virus-cell membrane fusions through ACE2. TMPRSS2 proteolytically cleaves and activates the S glycoproteins of human CoV and the fusion glycoproteins of other viruses such as HMPV and HPIV and is involved in proteolytic cleavage and activation of hemagglutinin (HA) protein making TMPRSS2 essential for spread and pathogenesis of influenza A virus (strains H1N1, H3N2 and H7N9). TMPRSS2 is involved in SARS-CoV-2 S protein priming, possibly promoting viral uptake.

Camostat Mesylate is a potent TMPRSS2 inhibitor and has been shown to block SARS-CoV cell entry mechanism. In BALB/c mice infected with SARS-CoV, camostat, dosed at concentrations similar to the clinically achievable concentration in humans, significantly reduced mortality following SARS-CoV infection. Hoffmann and colleagues recently confirmed that this drug effectively blocks also SARS-CoV-2 entry into lung cells. Camostat is used to treat chronic pancreatitis in Japan; it is thus readily available and represents an exciting potential therapeutic agent for respiratory coronavirus infections including SARS-CoV-2. Accordingly, a phase1/2 clinical trial has recently been initiated in Denmark, entitled “The Impact of Camostat Mesilate on COVID-19 Infection: An Investigator-initiated Randomized, Placebo-controlled, Phase IIa Trial” assessing the impact of Camostat Mesylate, on the course of COVID-19 (NCT04321096, registered on March 25, 2020). A different clinical trial (Camostat Mesylate in COVID-19 Outpatients, NCT04353284, registered on April 20, 2020) is currently recruiting patients aiming to identify whether camostat could reduce the viral burden in the upper respiratory tract and therefore forestall complications of SARS-CoV-2 infection and reduce transmission when given to outpatients within 2 days of being notified of their positive COVID-19 test result.

Another TMPRSS2 inhibitor with therapeutic potential for treatment of COVID-19 is nafamostat mesylate, an FDA-approved drug for indications unrelated to corona virus and used in Asian countries as a short-acting anticoagulant. Nafamostat was identified as a potent inhibitor of S-mediated membrane fusion of MERS-CoV and blocked MERS-CoV infection in vitro. A recent study reported that nafamostat inhibited SARS-CoV-2 entry into host cells with roughly 15-fold higher efficiency than camostat, with an EC50 in the low nanomolar range. In addition, it blocked SARS-CoV-2 infection of human lung cells with markedly higher efficiency than camostat mesylate. Thus, this drug may also be worth investigating for its ability to reduce SARS-CoV-2 infective capacity. Currently, two clinical trials are scheduled to start recruiting patients aiming to evaluate the clinical efficacy of nafamostat mesylate in adult patients hospitalized with COVID-19 pneumonia (Clinical Efficacy of Nafamostat Mesylate for COVID-19 Pneumonia, NCT04418128, registered on June 5, 2020) and its effect on lung function deterioration and need for intensive care admission in COVID-19 patients [Efficacy of Nafamostat in Covid-19 Patients (RACONA Study), NCT04352400, registered on April 20, 2020). Overall, TMPRSS2 is essential for SARS-CoV-2 infectivity and appears as a promising target for drug therapy of COVID-19 at an early stage.

Interestingly, the human TMPRSS2 gene promoter has a 15-bp androgen response element. The up-regulation of TMPRSS2 mRNA by androgens appears to be mediated by the androgen receptor. This raises the possibility that androgens may have a role in SARS-CoV-2 infectivity and COVID-19 course of disease. Some indirect evidence points to this direction. A constant characteristic of the corona virus pandemic is that more males than females get infected (with a ratio of approximately 60/40). Also, more infected males proceed to develop severe disease, get hospitalized and die. To explain the fact that males present with more severe COVID-19 symptoms, a recent study suggested a potential involvement androgens based on the preliminary epidemiologic observation of high frequency of male pattern hair loss among admitted COVID-19 patients. Stronger evidence in this direction was recently reported by a group in Italy. They extracted data from 9280 subjects (4532 males) with laboratory-confirmed SARS-CoV-2 infection from 68 hospitals in the area of Veneto. Overall, males developed more severe complications, were hospitalized more frequently and had worse clinical outcomes than females. Comparing the total number of SARS-CoV-2 positive cases, prostate cancer patients receiving androgen-deprivation therapy (ADT) had a significantly lower risk of SARS-CoV-2 infection compared to patients who did not receive ADT (OR 4.05; 95% CI 1.55–10.59). They concluded that, while cancer patients overall have an increased risk of SARS-CoV-2 infections compared to non-cancer patients, prostate cancer patients receiving ADT appear to be partially protected from SARS-CoV-2 infections.

Several classes of drugs exhibit antiadrogenic activity and are in clinical use for ADT, including androgen receptor blockers such as bicalutamide and enzalutamide, androgen synthesis inhibitors, and antigonadotropins. Furthermore, new antiandrogens that target testosterone synthesis (abiraterone acetate and seviteronel) or androgen receptor nuclear translocation (enzalutamide, apalutamide and darolutamide), as well as combined therapies (galeterone) have been recently developed and may function to better target androgen-responsive cells in combination with ADT. These agents may down-regulate TMPRSS2 mRNA and expression resulting in less entry of SARS-CoV-2 entry into cells and thus could arise as promising therapeutic tools in early SARS-CoV-2 infection and COVID-19. This hypothesis will soon be tested: two clinical trials investigating the potential protective role of the antiandrogens dutasteride and bicalutamide in SARS-CoV-2 infection are about to start [Anti-Androgen Treatment for COVID-19, NCT04446429, registered on June 24, 2020; Trial to Promote Recovery From COVID-19 With Ivermectin or Endocrine Therapy (RECOVER), NCT04374279, registered on May 5, 2020).

There is some debate as to whether inhaled corticosteroids (ICS) modify the expression of ACE2 and TMPRSS2 and could thus have a role in COVID-19 therapy. A recent study has shown that in sputum cells of asthmatic patients, the use of ICS is associated with lower expression of ACE2 and TMPRSS2, warranting prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID19 morbidity. On the contrary, another study has shown that ACE2, TMPRSS2 and furin gene expression in bronchial brush samples is not affected in patients with mild to moderate or severe asthma compared with healthy controls. This is a topic of significant interest and intense current investigation, in view also of the beneficial effect reported recently of systemic dexamethasone use in COVID-19 patients with severe disease.

ADAM-17 enhancers

The ADAMs (A Disintegrin And Metalloproteinase) is a family of transmembrane and secreted proteins. ADAMs are implicated in a variety of cellular processes, including processing of proteins, interactions with integrin receptors and with signalling molecules. In particular, one member of this family, ADAM-17, also known as TNFα-converting enzyme, appears to be an indispensible regulator of almost every cellular event from proliferation to migration. ADAM-17 is widely expressed in various tissues including bronchial epithelial cells, vascular smooth muscle cells and macrophages in the lung. The function of ADAM-17 is to cleave ectodomains of various transmembrane proteins, including ACE2. This makes it an attractive target for pharmacologic manipulation, since its stimulation could result in increased ACE2 shedding, thus higher levels of soluble ACE2 ectodomain, which could reduce the infectivity of the SARS-Cov-2.

Some evidence regarding pharmacologic up-regulation of ADAM-17 has emerged. Estradiol was shown to enhance ADAM-17 expression and protein levels in human non-small cell lung cancer. If confirmed in COVID-19 patients, this finding would suggest higher ACE2 shedding in female, providing a plausible partial explanation of the gender differences favoring females that clearly exist in SARS-CoV-2 infectivity and COVID-19 severity course. This is in agreement with the hypothesis already mentioned for a potential role of oestrogen receptor activation with conjugated estrogens as a good prevention and therapeutic strategy against COVID-19.

Also, 5-fluorouracil (5-FU), a widely used antineoplastic agent which acts as an antimetabolite, has been reported to acutely activate ADAM-17 in vitro and in vivo in models of colorectal cancer.

Conclusions

It has been presented here mechanistic-driven hypotheses supported by enourmous evidence on several pharmacologic agents (most of them already used clinically for other indications) which are known to interfere with elements of the virus entry pathway. These include human recombinant ACE2 that increases available sACE2, the angiotensin receptor blockers irbesartan and losartan, calmodulin antagonists such as melatonin and certain other psychotropic medications and the selective oestrogen receptor modifiers tamoxifen and toremifene, the TMPRSS2 inhibitors camostat mesylate, nafamostat mesylate and several antiandrogens, and finally the ADAM-17 enhancers 5-fluorouracil and conjugated estrogens. We suggest that these agents should be investigated further in appropriate preclinical and clinical disease models for their potential as promising prophylactic and therapeutic interventions in early SARS-CoV-2 infection. Since this would be a new indication of these drugs and may result from different mechanism of action, it will be necessary to establish the appropriate dose range and dosing schedules for the new indication for each of the tested agents. Finally, it should be kept in mind that all these approaches may exert benefit only if applied in the early phase of SARS-CoV-2 infection and COVID-19. Thus, such therapeutic to be effective, early detection of virus infection and drug therapy start will be required, as later in the disease course, such interventions may be overwhelmed by the inflammatory process.