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Nafamostat mesylate blocks activation of SARS-CoV-2: New treatment option for COVID-19

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Abstract

The currently unfolding coronavirus pandemic threatens health systems and economies worldwide.…

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... Recently, an increasing number of studies have shown the potent antiviral activities of nafamostat on coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [7][8][9][10][11]. There are also several clinical trials ongoing to evaluate the efficacy of nafamostat in patients with COVID-19 (ClinicalTrials.gov ...
... Nafamostat is a TMPRSS2 inhibitor. Nafamostat has been shown to inhibit SARS-CoV-2 entry into host cells and block SARS-CoV-2 infection of human lung cells [7]. Nafamostat is expected to bind spontaneously and stably to the catalytic center of TMPRSS2 and inhibit its proteolytic processing of the S protein [7,10,11,14,15]. ...
... Nafamostat has been shown to inhibit SARS-CoV-2 entry into host cells and block SARS-CoV-2 infection of human lung cells [7]. Nafamostat is expected to bind spontaneously and stably to the catalytic center of TMPRSS2 and inhibit its proteolytic processing of the S protein [7,10,11,14,15]. However, the effect of nafamostat on COVID-19 is still controversial. ...
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Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and 13C6-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.
... Nafamostat is a serine protease inhibitor that is used in Japan for the treatment of acute pancreatitis and disseminated intravascular coagulation [9,10]. With regard to SARS-CoV-2, it has been demonstrated that nafamostat inhibits viral entry into cells by blocking the activity of TMPRSS2 [11], a cellular protease that primes the SARS-CoV-2 spike protein [11][12][13]. A recent study published in Viruses [14] showed that Nafamostat also inhibited −1 programmed ribosomal frameshifting (−1PRF) of SARS-CoV-2, a process that is required for expression of the viral ORF1b ( Figure 1a) protein and viral replication [15,16]. ...
... Nafamostat is a serine protease inhibitor that is used in Japan for the treatment of acute pancreatitis and disseminated intravascular coagulation [9,10]. With regard to SARS-CoV-2, it has been demonstrated that nafamostat inhibits viral entry into cells by blocking the activity of TMPRSS2 [11], a cellular protease that primes the SARS-CoV-2 spike protein [11][12][13]. A recent study published in Viruses [14] showed that Nafamostat also inhibited −1 programmed ribosomal frameshifting (−1PRF) of SARS-CoV-2, a process that is required for expression of the viral ORF1b ( Figure 1a) protein and viral replication [15,16]. ...
... Our previous studies showed that Nafamostat inhibits SARS-CoV-2 entry by blocking TMPRSS2 and established the concept that Nafamostat should only exert anti-SARS-CoV-2 activity in cells for which viral entry depends on TMPRSS2 activity [11]. However, our studies were mainly carried out with a surrogate system that measures SARS-CoV-2 entry but not the subsequent steps in viral replication. ...
Article
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has been reported to have caused 18 [...]
... SARS-CoV-2 uses the same host receptor as SARS-CoV, the human angiotensin converting enzyme 2 (ACE2), which is a membraneanchored carboxypeptidase, highly expressed by the respiratory epithelium, including the type I and type II alveolar epithelium (Hoffmann et al. 2020;Zhou et al. 2020a, b, c;Perrota et al. 2020). After binding to ACE2, protein S undergoes a process of cleavage by host proteases, resulting in the formation of two distinct functional subunits, S1 and S2 (Fig. 4.7). ...
... This process is similar to the key-lock fitting (Uzunian 2020). This process depends on two proteases: transmembrane protease/serine subfamily member 2 (TMPRSS2), which cleaves and activates the S protein, allowing the virus to bind to the ACE2 receptor, adhering to the cell membrane allowing its internalization in the endosomes (Hoffmann et al. 2020), and Furina, which has a similar role to TMPRSS2 (Liu and Saif 2020). The role of TMPRSS2 in the adhesion and internalization of SARS-CoV-2 has been demonstrated by Danish researchers who used nafamostat mesylate, a short-acting anticoagulant, used to treat pancreatitis and during hemodialysis to prevent fibrinogen proteolysis (Al-Horani and Desai 2014; Sadahiro et al. 2018), and also a TMPRSS2 inhibitor. ...
... The internalization of SARS-CoV-2 is also facilitated by a priming reaction of the viral S protein by a cellular protein, the transmembrane protease/serine subfamily member 2 (TMPRSS2) (Hoffmann et al. 2020;Sungnak et al. 2020). Scientific investigations still reveal that SARS-CoV-2 determines the appearance of at least three virulence factors that induce the release of virions from host cells that inhibit the immune response (Canrong et al. 2020). ...
Chapter
Despite the recent announcement of the new pathogenic coronavirus to man, SARS-CoV2, a large number of publications are presented to the scientific community. An organized and systematic review of the epidemiological, etiological, and pathogenic factors of COVID-19 is presented. This is a systematic review using the databases MEDLINE, EMBASE, Web of Science, SCIELO; the descriptors coronavirus, SARS-CoV-2, etiology, epidemiology, pathophysiology, pathogenesis, COVID-19, with publications from December 2019 to January 2021, resulting in more than 800 publications and 210 selected. The data suggest that COVID-19 is associated with SAR-CoV-2 infection, with the transmission of contagion by fomites, salivary droplets, and other forms, such as vertical and fecal–oral. The bat and other vertebrates appear to be reservoirs and part of the transmission chain. The virus uses cell receptors to infect human cells, especially ACE2, like other coronaviruses. Heat shock proteins have different roles in the infection, sometimes facilitating it, sometimes participating in more severe conditions, when not serving as a therapeutic target. The available data allow us to conclude that COVID-19 is a pandemic viral disease, behaving as a challenge for public health worldwide, determining aggressive conditions with a high mortality rate in patients with risk factors, without treatment, but with the recent availability of the first vaccines.
... Previous experimental data (14,15) has established that the SARS-CoV-2 spike (S) protein uses the host cell factors angiotensin-converting enzyme 2 (ACE2) to bind to target cells, and that the host cell surface transmembrane protease serine 2 (TMPRSS2) cleavage of S protein enables entry into target cells (Fig. 1A). ...
... Camostat mesylate and the related molecule, nafamostat, are approved in some countries (but not in the United States) for the treatment of pancreatitis and esophagitis (16,17). Both molecules block TMPRSS2 priming of S protein in vitro, a mechanism that has been shown to be both necessary and sufficient for viral entry into respiratory epithelial cells (14,15,18). Camostat and nafamostat was shown to block SARS-CoV-2 infection of primary human lung epithelial cells (18) and nafamostat was found to inhibit SARS-CoV-2 spread and pathogenesis in mice (19). ...
Article
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Strategies to repurpose already-approved drugs for the treatment of COVID-19 has been attractive since the beginning of the pandemic. Camostat mesylate, a serine protease inhibitor approved in Japan for the treatment of acute exacerbations of chronic pancreatitis, inhibits TMPRSS1, a host cell surface serine protease essential for SARS-CoV-2 viral entry. In vitro experiments provided data suggesting that camostat might be effective in the treatment of COVID-19.
... However, viral mutations may jeopardize the efficiency of these drugs in anti-SARS-CoV-2 treatment. Targeting host proteases, on the other hand, should be less subject to evasion, and show promising results [6,46,47]. TMPRSS2 is one of these host proteases. Our results clearly showed that SMYD2 deficiency or inhibition significantly decreased TMPRSS2 levels in multiple cell lines. ...
... Interestingly, SMYD2 inhibition showed strong antiviral effects only in TMPRSS2-sufficient Caco-2 cells but not TMPRSS2-deficient Vero cells, suggesting that SMYD2 may regulate SARS-CoV-2 infection via a TMPRSS2-dependent pathway. Compared to classical TMPRSS2 inhibitors, such as camostat mesylate and nafamostat, which inhibits its protease activity [6,47], SMYD2 inhibition targets TMPRSS2 protein levels. This may provide a more efficient way to block TMPSS2 function, or at least provide an alternative strategy regarding SARS-CoV-2 treatment. ...
Article
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The COVID-19 pandemic caused by SARS-CoV-2 has lasted for more than two years. Despite the presence of very effective vaccines, the number of virus variants that escape neutralizing antibodies is growing. Thus, there is still a need for effective antiviral treatments that target virus replication independently of the circulating variant. Here, we show for the first time that deficiency or pharmacological inhibition of the cellular lysine-methyltransferase SMYD2 decreases TMPRSS2 expression on both mRNA and protein levels. SARS-CoV-2 uses TMPRSS2 for priming its spike protein to infect target cells. Treatment of cultured cells with the SMYD2 inhibitors AZ505 or BAY598 significantly inhibited viral replication. In contrast, treatment of Vero E6 cells, which do not express detectable amounts of TMPRSS2, had no effect on SARS-CoV-2 infection. Moreover, by generating a recombinant reporter virus that expresses the spike protein of the Delta variant of SARS-CoV-2, we demonstrate that BAY598 exhibits similar antiviral activity against this variant of concern. In summary, SMYD2 inhibition downregulates TMPRSS2 and blocks viral replication. Targeting cellular SMYD2 represents a promising tool to curtail SARS-CoV-2 infection.
... To ensure that this interaction leads to serpin-specific inhibition of TMPRSS2-mediated cleavage of the viral S-protein, an in vitro cleavage assay was performed. Recombinant TMPRSS2 and viral S-protein were incubated with or without recombinant SERPINA1, SERPINE1, and SERPINC1 protein or the positive control, nafamostat mesylate (20), followed by detection of SARS-CoV-2 S-protein and its cleavage products by Western blotting. In the presence of TMPRSS2, several cleavage products of the S-protein were detected, and these were reduced when adding the protease inhibitor nafamostat mesylate ( Fig. 4E and F). ...
... Interestingly, both A1AT and ATIII have been shown to be biologically active following administration via inhalation in different model systems (30,31). Another way to prevent TMPRSS2mediated SARS-CoV-2 entry is by using chemical protease inhibitors, such as camostat mesylate or nafamostat mesylate (13,20), also currently in clinical trials against COVID-19 (ClinicalTrials.gov registration no. ...
Article
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Identification of host factors affecting individual SARS-CoV-2 susceptibility will provide a better understanding of the large variations in disease severity and will identify potential factors that can be used, or targeted, in antiviral drug development. With the use of an advanced lung cell model established from several human donors, we identified cellular protease inhibitors, serpins, as host factors that restrict SARS-CoV-2 infection.
... 5 This method of cell entry is shared by other coronaviruses including SARS-CoV-2, and in-vitro studies have confirmed activity against SARS-CoV-2. 6,7 Nafamostat has shown to significantly reduce weight loss and lung tissue SARS-CoV-2 titres in murine models. 8 Nafamostat has a short half-life 9,10 and poor oral bioavailability, 11 which necessitates intravenous administration, limiting the potential use of the current formulation outside of a hospital setting. ...
... Nafamostat has been reported to inhibit the activity of TMPRSS2 with an IC50 of between 5 and 55 nM. 7 It is known that the plasma half-life (t1/2) of nafamostat is approximately 8À23 min 9,23 and thus a continuous infusion is required to achieve a steady-state concentration sufficient to inhibit therapeutic targets. In this trial we used a dose of 0.2 mg/kg/h which is the current clinically licensed dose of nafamostat for DIC and pancreatitis in Japan. ...
Article
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Background Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. Methods We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. Findings Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 – 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43–18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55–95% CI 0.31–0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. Interpretation In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events.
... Inhibition of TMPRSS2 activity has been suggested as a promising approach against viral infection [11]. For example, Camostat mesylate, a protease inhibitor that inhibits TMPRSS2 and is approved for use in other clinical indications, was shown to block SARS-CoV-2 entry into primary human lung cells [12,13]. In addition, cell lines expressing TMPRSS2 were shown to be highly susceptible to SARS-CoV-2 infection [14]. ...
... Additionally, overexpression of human ACE2 in a mouse model of SARS-CoV brought enhanced disease severity [22]. However, while ACE2 and TMPRSS2 have both been suggested as putative therapeutic targets for COVID-19 [13,23,24], their circulatory levels have only recently and scarcely been evaluated as potential biomarkers for disease severity and progression. ...
Article
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One of the major challenges for healthcare systems during the Coronavirus-2019 (COVID-19) pandemic was the inability to successfully predict which patients would require mechanical ventilation (MV). Angiotensin-Converting Enzyme 2 (ACE2) and TransMembrane Protease Serine S1 member 2 (TMPRSS2) are enzymes that play crucial roles in SARS-CoV-2 entry into human host cells. However, their predictive value as biomarkers for risk stratification for respiratory deterioration requiring MV has not yet been evaluated. We aimed to evaluate whether serum ACE2 and TMPRSS2 levels are associated with adverse outcomes in COVID-19, and specifically the need for MV. COVID-19 patients admitted to an Israeli tertiary medical center between March--November 2020, were included. Serum samples were obtained shortly after admission (day 0) and again following one week of admission (day 7). ACE2 and TMPRSS2 concentrations were measured with ELISA. Of 72 patients included, 30 (41.6%) ultimately required MV. Serum ACE2 concentrations >7.8 ng/mL at admission were significantly associated with the need for MV (p = 0.036), inotropic support, and renal replacement therapy. In multivariate logistic regression analysis, elevated ACE2 at admission was associated with the need for MV (OR = 7.49; p = 0.014). To conclude, elevated serum ACE2 concentration early in COVID-19 illness correlates with respiratory failure necessitating mechanical ventilation. We suggest that measuring serum ACE2 at admission may be useful for predicting the risk of severe disease.
... Thus, TMPRSS2 inhibitors have become a promising target for inhibiting virus infection. [275][276][277] Camostat and Nafamostat mesylate are oral TMPRSS2 inhibitors, and both enter phase III clinical trials with previously reported applications on SARS-CoV and MERS-CoV infection. 278 Furthermore, Nafamostat was reported superior to Camostat in specificity and effectiveness. ...
... 261,279 Research reported that these two drugs could effectively block the virus infection of ancestral SARS-CoV-2. 261,275,276 Moreover, another novel small molecule compound targeting TMPRSS2, N-0385, exerted equivalent potency against four VOCs, Alpha, Beta, Gamma, and Delta variants, with EC 50 ranging from 2.1 to 13.9 nM from SARS-CoV-2 nucleocapsid staining assay, and 2.6-26.5 nM from dsRNA staining assay. 251,280 Chloroquine is a widely used antimalaria and anti-autoimmune drug by modulating endosomal pH and disturbing the Clathrindependent endocytosis to inhibit pathogen entry into the host cell, for which it manifested broadly anti-pathogen activity. ...
Article
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The persistent COVID-19 pandemic since 2020 has brought an enormous public health burden to the global society and is accompanied by various evolution of the virus genome. The consistently emerging SARS-CoV-2 variants harboring critical mutations impact the molecular characteristics of viral proteins and display heterogeneous behaviors in immune evasion, transmissibility, and the clinical manifestation during infection, which differ each strain and endow them with distinguished features during populational spread. Several SARS-CoV-2 variants, identified as Variants of Concern (VOC) by the World Health Organization, challenged global efforts on COVID-19 control due to the rapid worldwide spread and enhanced immune evasion from current antibodies and vaccines. Moreover, the recent Omicron variant even exacerbated the global anxiety in the continuous pandemic. Its significant evasion from current medical treatment and disease control even highlights the necessity of combinatory investigation of the mutational pattern and influence of the mutations on viral dynamics against populational immunity, which would greatly facilitate drug and vaccine development and benefit the global public health policymaking. Hence in this review, we summarized the molecular characteristics, immune evasion, and impacts of the SARS-CoV-2 variants and focused on the parallel comparison of different variants in mutational profile, transmissibility and tropism alteration, treatment effectiveness, and clinical manifestations, in order to provide a comprehensive landscape for SARS-CoV-2 variant research.
... Camostat mesilate, a protease inhibitor developed for the treatment of pancreatitis in Japan in the 1980s, was reported to inhibit TMPRSS2 and block the entry of SARS-CoV and SARS-CoV-2 into host cells [21]. Previous studies have demonstrated that camostat mesilate suppresses viruscell membrane fusion in vitro and in vivo, thus, viral replication [22,23]. Another study revealed that camostat mesilate blocks the entry of the SARS-CoV-2 virus into the lungs [24]. ...
... Nafamostat mesilate was also found to suppresses TMPRSS2 and virus-cell fusion, and thus block the entry of SARS-CoV-2 virus into host cells [26,27]. Notely, nafamostat mesilate displayed a much more potent inhibiting effect compared with camostat mesilate on TMPRSS2 [23]. In present study, we further demonstrated that nafamostat mesilate can block the interaction of the coronavirus S protein and its host ACE2 receptor. ...
Article
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Background The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social. The ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health and economic stability worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. Methods We first established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus spike protein and its host receptor ACE2. Two compound libraries of 2,864 molecules were screened with this platform. Selected candidate compounds were validated by SARS-CoV-2_S pseudotyped lentivirus and ACE2-overexpressing cell system. Molecular docking was used to analyze the interaction between S protein and compounds. Results We identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S protein and the human cell ACE2 receptor. Additionally, tannic acid showed moderate inhibitory effect on the interaction of S protein and ACE2. Conclusion This platform is a rapid, sensitive, specific, and high throughput system, and available for screening large compound libraries. TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.
... Although some percentage of the viral entry was found to be carried out by endosomal cysteine proteases, CatB/L (cathepsin B and L), camostat mesylate, a drug approved in Japan for pancreatic cancer, blocked the viral entry significantly by TMPRSS2 inhibition [31]. Subsequent study by the same group mentions that nafamostat mesylate, another TMPRSS2 inhibitor, was more effective in preventing viral entry in both spike-protein and whole cell-based assays than camostat [36]. A small percentage of entry which was still taking place was attributed again to the CatB/L-mediated entry [36]. ...
... Subsequent study by the same group mentions that nafamostat mesylate, another TMPRSS2 inhibitor, was more effective in preventing viral entry in both spike-protein and whole cell-based assays than camostat [36]. A small percentage of entry which was still taking place was attributed again to the CatB/L-mediated entry [36]. ...
Article
The objective of this research work is to identify molecules through an advanced computational screening technique from a database of approved drugs/nutraceuticals that would inhibit transmembrane protease serine 2 (TMPRSS2) and thereby prevent SARS-CoV-2’s entry into human host cells. A homology model was built for TMPRSS2 and the standard inhibitors nafamostat and camostat were docked on the model. Ligand-based screening, flexible ligand docking and induced-fit docking followed by free binding energy calculations were carried out as part of the screening technique to generate hits. Eventually, molecular dynamics (MD) simulation was done for all the hits, and the results were compared with that of the standard inhibitors to validate our claims. From our computational study, we determined that streptomycin, doxorubicin and tetrahydrofolic acid are potential inhibitors of TMPRSS2. By analysing the MD simulation results, we also propose that streptomycin had the highest potential to inhibit TMPRSS2 among the three molecules. The three molecules we identified are most likely to show the efficacy when tested in vitro by prevention of entry of SARS-CoV-2 into human cells. These molecules can be taken further for clinical trials, and we expect fast processing since they are already approved by FDA and EMA for other diseases.
... 259,415,416 Nafamostat mesylate, which is FDA-approved for indications unrelated to coronavirus infection, inhibits viral entry with roughly 15-fold higher efficiency than camostat mesylate, but requires intravenous dosing. 417 Chloroquine and hydroxychloroquine may elevate endosomal pH and hinder viral entry and RNA release process. 418 However, two The mechanism underlying extrapulmonary complications of the coronavirus. . . ...
Article
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The coronavirus disease 2019 (COVID-19) is a highly transmissible disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that poses a major threat to global public health. Although COVID-19 primarily affects the respiratory system, causing severe pneumonia and acute respiratory distress syndrome in severe cases, it can also result in multiple extrapulmonary complications. The pathogenesis of extrapulmonary damage in patients with COVID-19 is probably multifactorial, involving both the direct effects of SARS-CoV-2 and the indirect mechanisms associated with the host inflammatory response. Recognition of features and pathogenesis of extrapulmonary complications has clinical implications for identifying disease progression and designing therapeutic strategies. This review provides an overview of the extrapulmonary complications of COVID-19 from immunological and pathophysiologic perspectives and focuses on the pathogenesis and potential therapeutic targets for the management of COVID-19.
... Nafamostat mesylate can be considered as a safer alternative to camostat mesylate because nafamostat has more efficient antiviral activity and a favorable safety profile. 110 Besides, disseminated intravascular coagulation (DIC) with increased fibrinolysis shown in COVID-19 patients is also treated with nafamostat mesylate. 49 ...
Article
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The rising outbreak of SARS-CoV-2 continues to unfold all over the world. The development of novel effective antiviral drugs to fight against SARS-CoV-2 is a time cost. As a result, some specific FDA-approved drugs have already been repurposed and authorized for COVID-19 treatment. The repurposed drugs used were either antiviral or non-antiviral drugs. Accordingly, the present review thoroughly focuses on the repurposing efficacy of these drugs including clinical trials experienced, the combination therapies used, the novel methods followed for treatment, and their future perspective. Therefore, drug repurposing was regarded as an effective avenue for COVID-19 treatment. Recently, molnupiravir is a prodrug antiviral medication that was approved in the United Kingdom in November 2021 for the treatment of COVID-19. On the other hand, PF-07321332 is an oral antiviral drug developed by Pfizer. For the treatment of COVID-19, the PF-07321332/ritonavir combination medication is used in Phase III studies and was marketed as Paxlovid. Herein, we represented the almost history of combating COVID-19 from repurposing to the recently available oral anti-SARS-CoV-2 candidates, as a new hope to end the current pandemic.
... Initially, APR was used to treat acute pancreatitis, then complex surgical interventions, such as heart and liver surgeries [14][15][16]. Since the early 1980s, APR has been actively studied as an anti-influenza drug [17][18][19][20][21], and, in recent years, protease inhibitors, including APR, have been actively studied as anti-coronavirus drugs [12,22,23]. APR is a nonspecific inhibitor that competitively and reversibly inhibits the activity of several different serine proteases, especially trypsin, chymotrypsin, plasmin, and kallikrein. ...
Article
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The efficacy of aprotinin combinations with selected antiviral-drugs treatment of influenza virus and coronavirus (SARS-CoV-2) infection was studied in mice models of influenza pneumonia and COVID-19. The high efficacy of the combinations in reducing virus titer in lungs and body weight loss and in increasing the survival rate were demonstrated. This preclinical study can be considered a confirmatory step before introducing the combinations into clinical assessment.
... To increase the potency of the drug they could both be given in combination with one another. 39 Nafamostat is a potent inhibitor of the Middle East Respiratory Syndrome (MERS) coronavirus which broke out in the Arabian Peninsula (2012). The results concluded that Nafamostat had inhibited the activity of the dual split protein (DSP) assay (more than 98% inhibition) by its interference with MERS-S-mediated membrane fusion. ...
Article
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The recent outbreak of severe acute respiratory syndrome (SARS) belongs to a broad family of viruses known as Coronaviridae. SARSCoV- 2 is an emerging global pandemic with a relatively low mortality rate. The virus has been mutated in a unique manner thus prolonging its search for its vaccine and drug therapy. SARS-CoV-2 is an enveloped virus consisting of many spike (S) proteins, which mediates its fusion to the membrane of the host cell. Its ‘crown-like’ appearance under an electron microscope has led to its name. The clinical symptoms that patients experience would be due to their central immune response to the infection. Pro-inflammatory cytokines play an essential role in cell growth and regulation of the immune system. However, its abundance could contribute to pathological conditions which can cause further injury and possible death. This brief review discusses the pathogenesis of the SARS-CoV-2 along with receptors that can be potentially targeted by therapeutic strategies, inhibiting the membrane fusion, genome replication and immune response.
... In addition, SARS-CoV-2 completes its entry into host cells when the S protein bound to the ACE2 receptor is degraded by the cell surface enzyme TMPRSS2, but nafamostat inhibits the function of TMPRSS2 and thus acts to block this step [130][131][132][133]. ...
Article
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Coronavirus Disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is frequently complicated by thrombosis. In some cases of severe COVID-19, fibrinolysis may be markedly enhanced within a few days, resulting in fatal bleeding. In the treatment of COVID-19, attention should be paid to both coagulation activation and fibrinolytic activation. Various thromboses are known to occur after vaccination with SARS-CoV-2 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) can occur after adenovirus-vectored vaccination, and is characterized by the detection of anti-platelet factor 4 antibodies by enzyme-linked immunosorbent assay and thrombosis in unusual locations such as cerebral venous sinuses and visceral veins. Treatment comprises high-dose immunoglobulin, argatroban, and fondaparinux. Some VITT cases show marked decreases in fibrinogen and platelets and marked increases in D-dimer, suggesting the presence of enhanced-fibrinolytic-type disseminated intravascular coagulation with a high risk of bleeding. In the treatment of VITT, evaluation of both coagulation activation and fibrinolytic activation is important, adjusting treatments accordingly to improve outcomes.
... ACE2 (angiotensin converting enzyme receptor 2) receptors are one of the important receptors in a human body, it is spread all over the internal organs viz. lungs, gallbladder, kidney, heart etc. SARS-CoV-2enters a human body through openings of nose, mouth and ears, subsequently they easily attack the ACE2 receptors [14][15][16][17][18]. The type II alveolar cell produces the natural pulmonary surfactant [19] (Fig. 2). ...
Article
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Our world is under serious threat of environmental degradation, climate change and in association with this the out breaks of diseases as pandemics. The devastating impact of the very recent COVID-19, The sharp increase in cases of Cancer, Pulmonary failure, Heart health has triggered questions for the sustainable development of pharmaceutical and medical sciences. In the search of inclusive and effective strategies to meet today's demand, improvised methodologies and alternative green chemical, bio-based precursors are being introduced by scientists around the globe. In this extensive review we have presented the potentiality and Realtime applications of both synthetic and bio-based surfactants in bio-medical and pharmaceutical fields. For their excellent unique amphoteric nature and ability to solubilise in both organic and inorganic drugs, surfactants are one of the most potential candidates for bio-medicinal fields such as dermatology, drug delivery, anticancer treatment, surfactant therapy, vaccine formulation, personal hygiene care and many more. The self-assembly property of sur-factants is a very powerful function for drug delivery systems that increases the bio-availability of the poorly aqueous soluble pharmaceutical products by influencing their solubility. Over the decades many researchers have reported the antimicrobial, anti-adhesive, antibiofilm, anti-inflammatory, antioxidant activities of surfactants regarding its utility in medicinal purposes. In some reports surfactants are found to have spermicidal and laxative activity too. This comprehensive report is targeted to enlighten the versatile applications of Surfactants in drug delivery, vaccine formulation, Cancer Treatment, Therapeutic and cosmetic Pharmaceutical Sciences and prevention of pulmonary failure due to COVID-19.
... This result is in agreement with other studies reporting gabexate is inactive in cell-based assays testing TMPRSS2 inhibition. 27,28 Notably, bortezomib appeared to demonstrate a highly potent inhibition of viral entry (IC50 <50 nM); however, the cytotoxicity counter-assay revealed it was due to high cytotoxicity of the Calu-3 cells, which again demonstrates the importance and necessity of appropriate counterassays in designing an assay pipeline . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ...
Preprint
SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allowed for rapid movement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.
... 6 In order to inhibit the entry of SARS-CoV-2 into the host, screening of inhibitors targeting TMPRSS2 has been conducted, and nafamostat mesylate (NM) has been identified as a candidate. 7,8 Basic studies suggest that NM inhibits SARS-CoV-2 entry into lung epithelium-derived calu-3 cells by inhibiting TMPRSS2. 7,9 In addition, NM has been reported to be effective against COVID-19 in clinical practice, 10,11 and its use in combination with antiviral drugs may improve the therapeutic outcome because of its ability to inhibit the entry of SARS-CoV-2 into host cells. ...
Article
What is known and objective: Nafamostat mesylate (NM) is used clinically in combination with antiviral drugs to treat coronavirus disease (COVID-19). One of the adverse events of NM is hyperkalaemia due to inhibition of the amiloride-sensitive sodium channels (ENaC). The incidence and risk factors for hyperkalaemia due to NM have been studied in patients with pancreatitis but not in COVID-19. COVID-19 can be associated with hypokalaemia or hyperkalaemia, and SARS-CoV-2 is thought to inhibit ENaC. Therefore, frequency and risk factors for hyperkalaemia due to NM may differ between COVID-19 and pancreatitis. Hyperkalaemia may worsen the respiratory condition of patients. The objective of this study was to determine the incidence and risk factors for hyperkalaemia in COVID-19 patients treated with favipiravir, dexamethasone and NM. Methods: This retrospective study reviewed the records of hospitalized COVID-19 patients treated with favipiravir and dexamethasone, with or without NM, between March 2020 and January 2021. Multivariable logistic regression analysis was performed to identify the risk factors for hyperkalaemia. Results and discussion: Of 45 patients who received favipiravir and dexamethasone with NM for the treatment of COVID-19, 21 (47%) experienced hyperkalaemia. The duration of NM administration was a significant predictor of hyperkalaemia (odds ratio: 1.55, 95% confidence interval: 1.04-2.31, p = 0.031). The receiver-operating characteristic curve analysis determined that the cut-off value for predicting the number of days until the onset of hyperkalaemia was 6 days and the area under the curve was 0.707. What is new and conclusion: This study revealed that the incidence of hyperkalaemia is high in patients treated for COVID-19 with NM, and that the duration of NM administration is a key risk factor. When NM is administered for the treatment of COVID-19, it should be discontinued within 6 days to minimize the risk of hyperkalaemia.
... Nafamostat, a potent inhibitor of various serine proteases, was initially authorized as a short-acting anticoagulant and used for the treatment of pancreatitis in Japan and Korea for more than 20 years with a well-established safety profile [20]. Previous studies have established that serine protease inhibitors targeting TMPRSS2, such as nafamostat, can block SARS-CoV-2 entry and has been demonstrated in vitro and using animal models [21][22][23]. It is currently in the clinical trials for the treatment of COVID-19, including a phase 3 clinical trial. ...
Article
Pediatric patients with coronavirus disease 2019 (COVID-19) are increasing, and severe cases such as multisystem inflammatory syndrome are being reported. Nafamostat, a repurposing drug, is currently being explored for the treatment of COVID-19 in adults. However, the data supporting its exposure in pediatrics remains scarce. Physiologically-based pharmacokinetic (PBPK) modeling enables the prediction of drug exposure in pediatrics based on ontogeny of metabolic enzymes and age dependent anatomical and physiological changes. The study aimed to establish a PBPK model of nafamostat in adults, then scale the adult PBPK model to children for predicting pediatric exposures of nafamostat and an optimal weight-based nafamostat dose in pediatric population. The developed model adequately described adult exposure data in healthy volunteers following i.v. administration with three doses (10, 20, and 40 mg). Scaling adult PBPK models to five pediatric groups predicted that as age advances from neonate to adult, the exposure of nafamostat slightly increased from neonate to infant, steadily decreased from infant to child, and then increased from child to adult after the administration of 0.2 mg/kg/h for 14 days, a dosing regimen being conducted in a clinical trial for COVID-19. Based on the fold change of predicted area under the curve for the respective pediatric group over those of adults, weight-based dosages for each pediatric group may be suggested. The novel PBPK model described in this study may be useful to investigate nafamostat pharmacokinetics in a pediatric subgroup further.
... Submit a manuscript: https://www.tmrjournals.com/tmr [54,55] Monoclonal antibodies COV2-2196 and COV2-2130 [56] Antiviral peptides CR3022, OC43-HR2P, EK1C4 and EK1 [57][58][59] Arbidol [36] E, M and N proteins CVL218 (a PARP1/2 inhibitor) [37] siRNA drug [60] Hexamethylene amiloride [61] PJ34 (a PARP inhibitor) [62] ACE2 NAAE [63] ACE2 [64] Chloroquine [39] CD147 Humanized antibody meplazumab [65] DDP4 DPP4 monoclonal antibody [66] TMPRSS2 Nafamostat, gabexate [67,68] Camostat [40] Cysteine K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfonephenyl) [64] Teicoplanin [69] Endocytosis Chlorpromazine [70] Targeting virus replication ...
... There is a global quest for a regimen that can be administered for the prevention and control of COVID-19, a disease of public health emergency of international concern which has devastated the global community since the first reported case in Wuhan, China in December 2019. 1 Different supportive regimens have been used in different countries for the management of the SARS-Cov-2 patients. Chloroquine, 3 hydroxychloroquine, 3 Lopinavir/ Ritonavir, 4 Nafamostat and Camostat, 5 famotidine, 6 interferon, 7 convalescent plasma 8 monoclonal or polyclonal antibodies 9 and other therapies including multivitamins and antimicrobial agents 10 have been used for the treatment of SARS-Cov-2 by clinicians in different part of the world. Most of these therapies are in different stages of clinical trials. ...
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BACKGROUND: Ivermectin has been a popular anti-parasitic drug since the late 1970s. The promising result of in-vitro studies on the antiviral activity of the drug has led clinicians in many countries to use this drug to treat COVID-19 patients. This study determined and compared the mean number of days at clinical recovery for mild to moderate cases of COVID -19 treated with Lopinavir/Ritonavir (Alluvia) and Ivermectin at the Kaduna State Infectious Disease Control Centres. METHODS: This was a comparative cross-sectional study conducted among 300 mild to moderate COVID- 19 cases enrolled for the study. The outcome variables were the time required for the resolution of symptoms from the onset and at commencement of the treatment regimens. Data were collected from patient folders using a questionnaire. Data were analysed with the IBM SPSS Version 25.0 and STATA/SE 13. Statistical significance was set at p<0.05. RESULTS: The mean recovery time (MRT) from symptom onset was significantly lower for Covid-19 patients treated with ivermectin (7.15±4.18 days) compared to lopinavir/ritonavir (9.7±5.3 days), 95%CI=7.37–9.62. Multivariate logistic regression showed that there was no significant relationship between the patients age (AOR=0.36, 95%CI=0.09–1.49), sex (AOR=0.34,95%CI=0.54–5.93), educational status (AOR=1.04, 95%CI=0.3–3.57), marital status (AOR=0.55,95%CI=0.14–2.11) place of treatment (AOR=1.66, 95%CI=0.54–5.11) and MRT. There was also no significant relationship between patients’ comorbid chronic illness (AOR=0.83, 95%CI=0.27–2.61) and MRT. CONCLUSION: The mean recovery time for COVID-19 patients managed with ivermectin was slightly lower than for the lopinavir/ ritonavir regimen. RECOMMENDATION: Clinical trials to further prove the efficacy of Ivermectin as a supportive therapy in clinical management of mild to moderate cases of COVID-19 in this setting should be carried out.
... Naf suppresses transmembrane protease serine 2 (TMPRSS2), an enzyme that cleaves and activates the spike protein, and blocks the invasion of SARS-CoV-2 into host cells. 28,33 It is used for the treatment of DIC and acute pancreatitis because it binds to the active center (serine) of enzymes such as thrombin, XIIa, Xa, VIIa, kallikrein, plasmin and trypsin, and suppresses the coagulation/fibrinolytic system. Doi et al. have reported the efficacy of Naf 0.2 mg/kg/h in combination with Fav for COVID-19 when administered as a continuous intravenous infusion for an average of 14 days, 34 but the optimal administration method has not yet been established. ...
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BACKGROUND In COVID-19 pneumonia, cases of severe hypoxemia in the early stage and cases of sudden deterioration in respiratory status due to silent hypoxia leading to death, have been reported. CASE SUMMARY A 70-year-old Japanese man with essential hypertension, dyslipidemia, chronic kidney disease and emphysema was hospitalized with the novel coronavirus disease. He had hypoxemia that was disproportionate to the severity of pneumonia indicated by computed tomography (CT), along with coagulation abnormalities. We speculated that there was a high possibility that he had developed ventilation and blood flow imbalance due to pulmonary intravascular coagulopathy (PIC) or hypoxic pulmonary vasoconstriction (HPV). In this case, early, short-term combination therapy with remdesivir, nafamostat mesylate and low-dose dexamethasone (Dex) was successful. CONCLUSION In COVID-19 patients with multiple comorbidities who have hypoxemia and coagulation abnormalities that are disproportionate to the severity of pneumonia on CT, it is important to commence antiviral and anticoagulant therapy as soon as possible, followed by use of a low dose of Dex.
... Similarly, the expression of Cath L is up-regulated during chronic inflammation as observed with cytokine storm in COVID-19 infection and is also implicated in extracellular matrix degradation, a critical process for SARS-CoV-2 viral entry into the host cell [11]. Generally, the host cell proteins are genetically more stable than viral structural proteins, which are also typical druggable targets on the viral genome [12,13]. Hence, the development of novel phytotherapeutics that can modulate viral entry or replication through inhibition of hACE2, TMPRSS2, and Cath L would be of immense benefit to mankind as we grapple with the COVID-19 pandemic. ...
Article
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The coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is highly pathogenic and transmissible. It is mediated by the binding of viral spike proteins to human cells via entry and replication processes involving human angiotensin converting enzyme-2 (hACE2), transmembrane serine protease (TMPRSS2) and cathepsin L (Cath L). The identification of novel therapeutics that can modulate viral entry or replication has been of research interest and would be germane in managing COVID-19 subjects. This study investigated the structure-activity relationship inhibitory potential of 99 phytocompounds from selected African botanicals with proven therapeutic benefits against respiratory diseases focusing on SARS-CoV-2's human cell proteins (hACE2, TMPRSS2, and Cathepsin L) as druggable targets using computational methods. Evaluation of the binding energies of the phytocompounds showed that two compounds, Abrusoside A (−63.393 kcal/mol) and Kaempferol-3-O-rutinoside (−58.939 kcal/mol) had stronger affinity for the exopeptidase site of hACE2 compared to the reference drug, MLN-4760 (−54.545 kcal/mol). The study further revealed that Verbascoside (−63.338 kcal/mol), Abrectorin (−37.880 kcal/mol), and Friedelin (−36.989 kcal/mol) are potential inhibitors of TMPRSS2 compared to Nafamostat (−36.186 kcal/mol), while Hemiphloin (−41.425 kcal/mol), Quercetin-3-O-rutinoside (−37.257 kcal/mol), and Myricetin-3-O-galactoside (−36.342 kcal/mol) are potential inhibitors of Cathepsin L relative to Bafilomycin A1 (−38.180 kcal/mol). The structural analysis suggests that these compounds do not compromise the structural integrity of the proteins, but rather stabilized and interacted well with the active site amino acid residues critical to inhibition of the respective proteins. Overall, the findings from this study are suggestive of the structural mechanism of inhibitory action of the identified leads against the proteins critical for SARS-CoV-2 to enter the human host cell. While the study has lent credence to the significant role the compounds could play in developing potent SARS-CoV-2 candidate drugs against COVID-19, further structural refinement, and modifications of the compounds for subsequent in vitro as well as preclinical and clinical evaluations are underway.
... Recent evidence indicates that camostat mesylate is active against TMPRSS2, a transmembrane enzyme activating S protein [35]. Nafamostat mesylate blocked human lung cell SARS-CoV-2 infection with markedly greater efficacy than camostat mesylate, although both compounds were not active against infection with vesicular stomatitis virus [36]. There have been documented incidences of nafamostat adverse reactions such as hyperkalemia, agranulocytosis, anaphylaxis, and cardiac arrest in dialysis patients [37]. ...
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Numerous drugs have been repurposed to effectively tackle the COVID-19 pandemic as scientists and pharmaceutical firms compete to produce vaccines and antivirals without dwelling on the toxicity aspects. This paper explores the toxicity of several major medications used in the treatment of patients with COVID-19. Relevant literature from PubMed and Google scholar were reviewed. Several toxicities such as hepatic function disorder, damage to organs, Muscle problems, skin rash, seizures, lack of appetite, vision problems, low levels of blood cells, diarrhea, hyperkalemia renal damage, and other adverse reaction were found to be associated with drugs use for COVID-19 pandemic. The current race to produce therapeutics and vaccines must be advance with caution to avoid future consequences.
... The surface S1 and transmembrane S2 subunits contain the receptor-binding domain and the heptad repeat region and fusion region, respectively [79,80]. The receptor-bound SARS-CoV-2 particle invades into the cell following the proteolytic cleavage of the S proteins by cell-derived proteases including transmembrane serine protease 2 (TMPRSS2), which is widely expressed on the surface of human lung cells and is thought to play a critical role for virus entry [77,81,82]. Besides other coronaviruses, SARS-CoV and SARS-CoV-2 possess a poly basic amino acid insertion at the junction of the S1-S2 subunits (RRAR), which enables the cleavage of the S protein by the host furin like ubiquitous protease [83]. ...
Article
Respiratory viruses have sometimes resulted in worldwide pandemics, with the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) being major participants. Long-term efforts have made it possible to control the influenza virus, but seasonal influenza continues to take many lives each year, and a pandemic influenza virus sometimes emerges. Although vaccines for coronavirus disease 2019 (COVID-19) have been developed, we are not yet able to coexist with the SARS-CoV-2. To overcome such viruses, it is necessary to obtain knowledge about international surveillance systems, virology, ecology and to determine that immune responses are effective. The information must then be transferred to drugs. Delivery systems would be expected to contribute to the rational development of drugs. In this review, virologist and drug delivery system (DDS) researchers discuss drug delivery strategies, especially the use of lipid-based nanocarriers, for fighting to respiratory virus infections.
... Nafamostat mesylate. Nafamostat mesylate activity was around 15-fold more significant than that of camostat mesylate, with an EC50 of 5 nM, and it could hinder TMPRSS2-dependent cell membrane entrance (Hoffmann et al., 2020b). Furthermore, Docking on US-FDA licensed drugs that target the targets TMPRSS2 and ACE2 shows that lopinavir and valrubicin have the ability to block SARS-CoV-2 entry into the host by inhibiting both targets (Baby et al., 2021). ...
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Coronavirus disease 2019 (COVID-19) is a remarkably contagious and pathogenic viral infection arising from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first appeared in Wuhan, China. For the time being, COVID-19 is not treated with a specific therapy. The Food and Drug Administration (FDA) has approved Remdesivir as the first drug to treat COVID-19. However, many other therapeutic approaches are being investigated as possible treatments for COVID-19. As part of this review, we discussed the development of various drugs, their mechanism of action, and how they might be applied to different cases of COVID-19 patients. Furthermore, this review highlights an update in the emergence of new prophylactic or therapeutic vaccines against COVID-19. In addition to FDA or The World Health Organization (WHO) approved vaccines, we intended to incorporate the latest published data from phase III trials about different COVID-19 vaccines and provide clinical data released on the networks or peer-review journals.
... The use of a cell fusion assay to explore the effect of Ang II on the SARS-CoV-2 spike protein-mediated cell entry confirmed the results obtained by the viral infection assay and is supported by multiple observations: (i) the lack of detectable luminescence signal in control conditions, i.e., when Calu-3 cells expressing DSP1 were co-incubated with Hela cells expressing only DSP2; (ii) the markedly blunted fusion efficiency with exposure to different inhibitors of ACE2-and spike protein-mediated cell entry, including the potent inhibitor of TMPRSS2 nafamostat mesylate [36][37][38], a polyclonal antibody targeting ACE2, and a soluble ACE2 recombinant protein ( Figure 5, panel a). ...
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Blockers of the renin-angiotensin system (RAS) have been reported to increase the angiotensin converting enzyme (ACE)2, the cellular receptor of SARS-CoV-2, and thus the risk and course of COVID-19. Therefore, we investigated if angiotensin (Ang) II and RAS blockers affected ACE2 expression and SARS-CoV-2 infectivity in human epithelial bronchial Calu-3 cells. By infectivity and spike-mediated cell–cell fusion assays, we showed that Ang II acting on the angiotensin type 1 receptor markedly increased ACE2 at mRNA and protein levels, resulting in enhanced SARS-CoV-2 cell entry. These effects were abolished by irbesartan and not affected by the blockade of ACE-1-mediated Ang II formation with ramipril, and of ACE2- mediated Ang II conversion into Ang 1-7 with MLN-4760. Thus, enhanced Ang II production in patients with an activated RAS might expose to a greater spread of COVID-19 infection in lung cells. The protective action of Angiotensin type 1 receptor antagonists (ARBs) documented in these studies provides a mechanistic explanation for the lack of worse outcomes in high-risk COVID-19 patients on RAS blockers.
... Deep Docking docked all the compounds and provided a list of potential drug hits, that could be used to repurpose for COVID-19 [69]. Several studies have implemented random-walk algorithms to identify drug repurposing candidates for COVID-19 [70][71][72][73]. The pathogenic mechanisms instigated by the spike protein using the information from all related coronaviruses were also explored and random walk algorithms were implemented on the molecular networks to recognize the most relevant Fig. 3 The latest developments and scope of computational medicinal chemistry in the treatment of COVID-19. ...
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The unprecedented outbreak of the severe acute respiratory syndrome (SARS) Coronavirus-2, across the globe, triggered a worldwide uproar in the search for immediate treatment strategies. With no specific drug and not much data available, alternative approaches such as drug repurposing came to the limelight. To date, extensive research on the repositioning of drugs has led to the identification of numerous drugs against various important protein targets of the coronavirus strains, with hopes of the drugs working against the major variants of concerns (alpha, beta, gamma, delta, omicron) of the virus. Advancements in computational sciences have led to improved scope of repurposing via techniques such as structure-based approaches including molecular docking, molecular dynamic simulations and quantitative structure activity relationships, network-based approaches, and artificial intelligence-based approaches with other core machine and deep learning algorithms. This review highlights the various approaches to repurposing drugs from a computational biological perspective, with various mechanisms of action of the drugs against some of the major protein targets of SARS-CoV-2. Additionally, clinical trials data on potential COVID-19 repurposed drugs are also highlighted with stress on the major SARS-CoV-2 targets and the structural effect of variants on these targets. The interaction modelling of some important repurposed drugs has also been elucidated. Furthermore, the merits and demerits of drug repurposing are also discussed, with a focus on the scope and applications of the latest advancements in repurposing.
... Chen et al. discovered six inhibitors against S-protein (Cepharanthine, Abemacicilib, Osimertinib, Trimipramine, Colforsin, and Ingenol) that were tested against pseudotyped particles SARS-S and MERS-S . MM3122, Camostat Mesylate, Nafamostat, MNP10 (marine natural product 10) are some of the potential inhibitors of TMPRSS2 (Gunst et al., 2021;Hoffmann et al., 2020Hoffmann et al., , 2021Ko et al., 2021;Mahmudpour et al., 2021;Mahoney et al., 2021;Uno, 2020;Zhuravel et al., 2021). ...
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The SARS-CoV-2 virus has caused high-priority health concerns at a global level. Vaccines have stalled the proliferation of viruses to some extent. Yet, the emergence of newer, potentially more infectious, and dangerous mutants such as Delta and Omicron are among the major challenges in finding a more permanent solution for this pandemic. The effectiveness of antivirals Molnupiravir and Paxlovid, authorized for emergency use by the FDA, are yet to be assessed on a larger population. Patients with a high risk of disease progression have received treatment with antibody-cocktail. Most of the mutations leading to the new lineage of SARS-CoV-2 are found in the spike protein of this virus that plays a key role in facilitating host entry. The current study has investigated how to modify a promising peptide-based inhibitor of spike protein, LCB3, against common mutations, N501Y and K417N in the target protein so that it retains its efficacy against the spike protein. LCB3 being a prototype for protein based inhibitors is an ideal testing system to learn about protein-based inhibitors. This study proposes the substitutions of amino acid residues of LCB3 inhibitor using a structure-based approach that considers free energy decomposition of residues, the distance between atoms, and the interaction among amino acids. The binding free energy calculations suggest a possible improvement in the binding affinity of existing inhibitor LCB3 to the mutant forms of the S-protein using simple substitutions at specific positions of the inhibitor. This approach, being general, can be used in different inhibitors and other mutations and help in fighting against SARS-CoV-2. ARTICLE HISTORY
... For instance, nafamostat was found to prevent the entry of viruses via the inhibition of transmembrane protease serine 2 (TMPRSS2), leading to the blockage of S protein-mediated membrane fusion (Hoffmann et al., 2020). A case study on three patients (above 60 years) with COVID-19 pneumonia revealed that nafomostat can promisingly alleviate the clinical symptoms (Jang & Rhee, 2020). ...
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The current COVID‐19 pandemic is severely threatening public healthcare systems around the globe. Some supporting therapies such as remdesivir, favipiravir, and ivermectin are still under the process of a clinical trial, it is thus urgent to find alternative treatment and prevention options for SARS‐CoV‐2. In this regard, although many natural products have been tested and/or suggested for the treatment and prophylaxis of COVID‐19, carotenoids as an important class of natural products were underexplored. The dietary supplementation of some carotenoids was already suggested to be potentially effective in the treatment of COVID‐19 due to their strong antioxidant properties. In this study, we performed an in silico screening of common food‐derived carotenoids against druggable target proteins of SARS‐CoV‐2 including main protease, helicase, replication complex, spike protein and its mutants for the recent variants of concern, and ADP‐ribose phosphatase. Molecular docking results revealed that some of the carotenoids had low binding energies toward multiple receptors. Particularly, crocin had the strongest binding affinity (−10.5 kcal/mol) toward the replication complex of SARS‐CoV‐2 and indeed possessed quite low binding energy scores for other targets as well. The stability of crocin in the corresponding receptors was confirmed by molecular dynamics simulations. Our study, therefore, suggests that carotenoids, especially crocin, can be considered an effective alternative therapeutics and a dietary supplement candidate for the prophylaxis and treatment of SARS‐CoV‐2. Practical applications In this study, food‐derived carotenoids as dietary supplements have the potential to be used for the prophylaxis and/or treatment of SARS‐CoV‐2. Using in silico techniques, we aimed at discovering food‐derived carotenoids with inhibitory effects against multiple druggable sites of SARS‐CoV‐2. Molecular docking experiments against main protease, helicase, replication complex, spike protein and its mutants for the recent variants of concern, and ADP‐ribose phosphatase resulted in a few carotenoids with multitarget inhibitory effects. Particularly, crocin as one of the main components of saffron exhibited strong binding affinities to the multiple drug targets including main protease, helicase, replication complex, mutant spike protein of lineage B.1.351, and ADP‐ribose phosphatase. The stability of the crocin complexed with these drug targets was further confirmed through molecular dynamics simulations. Overall, our study provides the preliminary data for the potential use of food‐derived carotenoids, particularly crocin, as dietary supplements in the prevention and treatment of COVID‐19.
... Even though another small molecule protease inhibitor Gabexate is reported to inhibit TMPRSS2, the results of its antiviral activity remain inconsistent [107]. SARS-CoV-2 S-mediated entry in lung cells was not affected by Gabexate [108]. As a result, further experiments need to be conducted to establish Gabexate's influence on protease activity and viral replication. ...
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Since the end of 2019, the whole world has been struggling with the life-threatening pandemic amongst all age groups and geographic areas caused by Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). The Coronavirus Disease 2019 (COVID-19) pandemic, which has led to more than 468 million cases and over 6 million deaths reported worldwide (as of 20 March 2022), is one of the greatest threats to human health in history. Meanwhile, the lack of specific and irresistible treatment modalities provoked concentrated efforts in scientists around the world. Various mechanisms of cell entry and cellular dysfunction were initially proclaimed. Especially, mitochondria and cell membrane are crucial for the course of infection. The SARS-CoV-2 invasion depends on angiotensin converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), and cluster of differentiation 147 (CD147), expressed on host cells. Moreover, in this narrative review, we aim to discuss other cell organelles targeted by SARS-CoV-2. Lastly, we briefly summarize the studies on various drugs.
... Accordingly, a drug that inhibits TMPRSS2 activity is highly likely to be an effective therapeutic drug for COVID-19. In addition, when the activity of TMPRSS2 was blocked, membrane fusion between the virus and human cells was inhibited, virus invasion was blocked, and the pathogenicity and infectivity of the virus could be minimized by anti-plasmin activity [6][7][8][9][10][11]. Among the drugs that strongly inhibit TMPRSS2, nafamostat mesylate has the strongest antiviral effect against COVID-19, and its IC 50 value is approximately 600 times higher than that of remdesivir, the standard treatment for COVID-19 [12]. ...
Article
We aimed to develop nafamostat mesylate immediate-release tablets for the treatment of COVID-19 through drug repositioning studies of nafamostat mesylate injection. Nafamostat mesylate is a serine protease inhibitor known to inhibit the activity of the transmembrane protease, serine 2 enzyme that affects the penetration of the COVID-19 virus, thereby preventing the binding of the angiotensin-converting enzyme 2 receptor in vivo and the spike protein of the COVID-19 virus. The formulation was selected through a stability study after manufacturing by a wet granulation process and a direct tableting process to develop a stable nafamostat mesylate immediate-release tablet. Formulation issues for the selected processes were addressed using the design of experiments and quality-by-design approaches. The dissolution rate of the developed tablet was confirmed to be >90% within 30 min in the four major dissolutions, except in the pH 6.8 dissolution medium. Additionally, an in vivo pharmacokinetic study was performed in monkeys, and the pharmacokinetic profiles of nafamostat injections, oral solutions, and tablets were compared. The half-life during oral administration was confirmed to be significantly longer than the reported literature value of 8 min, and the bioavailability of the tablet was approximately 25% higher than that of the oral solution.
... This result is in agreement with other studies that reported gabexate is inactive in cell-based assays testing TMPRSS2 inhibition. 27,28 Notably, bortezomib appeared to demonstrate a highly potent inhibition of viral entry (IC 50 < 50 nM); however, the cytotoxicity counter assay revealed it was due to high cytotoxicity of the Calu-3 cells, which again demonstrates the importance and necessity of appropriate counter assays in designing an assay pipeline ( Figure 4F). Additional evidence that confirms 7-hydroxycoumarin is a false positive identified from the fluorogenic assay is its lack of activity within the cell-based assay evaluating viral entry ACS Infectious Diseases pubs.acs.org/journal/aidcbc ...
Article
SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allows for rapid movement of the existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites by TMPRSS2. Therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.
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Previously, we showed that coagulation factors directly cleave SARS-CoV-2 spike and promote viral entry (Kastenhuber et al., 2022). Here, we show that substitutions in the S1/S2 cleavage site observed in SARS-CoV-2 variants of concern (VOCs) exhibit divergent interactions with host proteases, including factor Xa and furin. Nafamostat remains effective to block coagulation factor-mediated cleavage of variant spike sequences. Furthermore, host protease usage has likely been a selection pressure throughout coronavirus evolution, and we observe convergence of distantly related coronaviruses to attain common host protease interactions, including coagulation factors. Interpretation of genomic surveillance of emerging SARS-CoV-2 variants and future zoonotic spillover is supported by functional characterization of recurrent emerging features.
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Importance Early treatment of mild SARS-CoV-2 infection might lower the risk of clinical deterioration in COVID-19. Objective To determine whether oral camostat mesylate would reduce upper respiratory SARS-CoV-2 viral load in newly diagnosed outpatients with mild COVID-19, and would lead to improvement in COVID-19 symptoms. Design From June, 2020 to April, 2021, we conducted a randomized, double-blind, placebo-controlled phase 2 trial. Setting Single site, academic medical center, outpatient setting in Connecticut, USA. Participants Of 568 COVID-19 positive potential adult participants diagnosed within 3 days of study entry and assessed for eligibility, 70 were randomized and 498 were excluded (198 did not meet eligibility criteria, 37 were not interested, 265 were excluded for unknown or other reasons). The primary inclusion criteria were a positive SARS-CoV-2 nucleic acid amplification result in adults within 3 days of screening regardless of COVID-19 symptoms. Intervention Treatment was 7 days of oral camostat mesylate, 200 mg po four times a day, or placebo. Main Outcomes and Measures The primary outcome was reduction of 4-day log 10 nasopharyngeal swab viral load by 0.5 log 10 compared to placebo. The main prespecified secondary outcome was reduction in symptom scores as measured by a quantitative Likert scale instrument, Flu-PRO-Plus modified to measure changes in smell/taste measured using FLU-PRO-Plus. Results Participants receiving camostat had statistically significant lower quantitative symptom scores (FLU-Pro-Plus) at day 6, accelerated overall symptom resolution and notably improved taste/smell, and fatigue beginning at onset of intervention in the camostat mesylate group compared to placebo. Intention-to-treat analysis demonstrated that camostat mesylate was not associated with a reduction in 4-day log 10 NP viral load compared to placebo. Conclusions and relevance The camostat group had more rapid resolution of COVID-19 symptoms and amelioration of the loss of taste and smell. Camostat compared to placebo was not associated with reduction in nasopharyngeal SARS-COV-2 viral load. Additional clinical trials are warranted to validate the role of camostat mesylate on SARS-CoV-2 infection in the treatment of mild COVID-19. Trial registration: Clinicaltrials.gov, NCT04353284 (04/20/20) ( https://clinicaltrials.gov/ct2/show/NCT04353284?term=camostat+%2C+yale&draw=2&rank=1 ) Key Points Question Will early treatment of COVID-19 with a repurposed medication, camostat mesylate, improve clinical outcomes? Findings In this phase 2 randomized, double-blind placebo-controlled clinical trial that included 70 adults with early COVID-19, the oral administration of camostat mesylate treatment within 3 days of diagnosis prevented the loss of smell/taste and reduced the duration of illness. Meaning In the current COVID-19 pandemic, phase III testing of an inexpensive, repurposed drug for early COVID-19 is warranted.
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Type II transmembrane serine proteases (TTSPs) are a family of trypsin-like membrane-anchored serine proteases that play key roles in the regulation of some crucial processes in physiological conditions, including cardiac function, digestion, cellular iron homeostasis, epidermal differentiation, and immune responses. However, some of them, in particular TTSPs expressed in the human airways, were identified as host factors that promote the proteolytic activation and spread of respiratory viruses such as influenza virus, human metapneumovirus, and coronaviruses, including SARS-CoV-2. Given their involvement in viral priming, we hypothesized that members of the TTSP family may represent targets of positive selection, possibly as the result of virus-driven pressure. Thus, we investigated the evolutionary history of sixteen TTSP genes in mammals. Evolutionary analyses indicate that most of the TTSP genes that have a verified role in viral proteolytic activation present signals of pervasive positive selection, suggesting that viral infections represent a selective pressure driving the evolution of these proteases. We also evaluated genetic diversity in human populations and we identified targets of balancing selection in TMPRSS2 and TMPRSS4. This scenario may be the result of an ancestral and still ongoing host–pathogen arms race. Overall, our results provide evolutionary information about candidate functional sites and polymorphic positions in TTSP genes.
Chapter
Prior 2019 to work date entire world is seriously influenced by an appalling illness called COVID sickness [Coronavirus disease-2019 (COVID-19)] which is brought about by another strain of coronavirus known as severe acute respiratory syndrome-Coronavirus-2. This pandemic was first seen in the Hubei area in Wuhan city of China. To date above 170 million individuals have been influenced by this infection and more than 3 million individuals died. The race of finding specific therapeutic drugs and efficacious vaccine candidates is still going on to tackle the pandemic-associated morbidities. This chapter discussed different clinically accessible medications (remdesivir, hydroxychloroquine, azithromycin, etc.) and immunizations (mRNA-1273, Sputanik, Pfizer, etc.) which are either in use or under trial for the treatment of COVID-19.
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Introduction. Recently, humanity has faced a new public health threat – the spread of the new coronavirus in 2019 (SARS-CoV-2). To address the problems caused by the high REVIEW ARTICLE Epidemiological aspects in COVID-19 infection based on current evidences: article of narrative synthesis What is not known yet, about the topic COVID-19 infection is an emerging disease that has a number of unclear epidemiological features, such as the primary sources of human infection, the duration of the patient’s contagiousness, the possibility of transmitting the infection from mother to foetus, and the specific prophylaxis of infection. Research hypothesis Publications addressing the epidemiological features of COVID-19 infection could contribute to the development of non-pharmaceutical measures for the prevention and early localization of outbreaks of SARS-CoV-2 in conditions of community spread. Article’s added novelty on this scientific topic The article summarizes the recently published articles on the features of the spread of COVID-19 infection according to the sources of infection, infectivity rate, routes of transmission, risk factors related to patients age, gender, associated comorbidities, and last but not least, trends in the development of the effective vaccine in combating the spread of SARS-CoV-2 virus. morbidity and mortality caused by this virus, a series of researches, have been carried out. The aim of these researches is to implement measures based on scientific evidence that will help to reduce the medical, social and economic impact worldwide. Material and methods. The existing bibliographic source on the PubMed platform was analysed, 251 articles, that are related to the objectives of our research, were pre-selected, and after excluding articles focused on clinic, treatment and diagnosis, 72 articles relevant to the proposed research topic were selected. Results. The analysed bibliographic sources allowed the highlighting of the inter-human transmission of the virus; incubation period is 2-14 days; the reproduction index varies from 2 to 6 infected people, depending on the prevention measures applied; the sources of infection can be asymptomatic persons, the most vulnerable to infection are the elderly with co-morbidities; the virus is sensitive to the action of hydrogen peroxide, sodium hypochlorite and alcohol over 60%. At the same time, based on the large number of infected people, the strategy for the development of effective vaccines against SARS-CoV-2 is initiated. Conclusions. At the current stage, COVID-19 infection has caused a very high medical, social and economic impact, becoming a pressing public health problem that needs to be solved. Assessing the features of the epidemic process with highlighting the epidemiological aspects, risk factors and specific prevention measures, will contribute to reducing the cases of infection and stabilizing the situation in the world. Despite the fact that a number of scientific publications mention the epidemiological features of the infection spread, there are some moments in studies, based on outbreaks of infection recorded under the conditions of community transmission, that need to be elucidated. Key words: COVID-19 infection, SARS-CoV-2 virus, source of infection, reproduction rate, routes of transmission, risk factors, vaccine.
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The outbreak of SARS-CoV-2 infections around the world has prompted scientists to explore different approaches to develop therapeutics against COVID-19. This study focused on investigating the mechanism of inhibition of clioquinol (CLQ) and its derivatives (7-bromo-5-chloro-8-hydroxyquinoline (CLBQ), 5, 7-Dichloro-8-hydroxyquinoline (CLCQ)) against the viral glycoprotein, and human angiotensin-converting enzyme-2 (hACE-2) involved in SARS-CoV-2 entry. The drugs were docked at the exopeptidase site of hACE-2 and receptor binding domain (RBD) sites of SARS-CoV-2 Sgp to calculate the binding affinity of the drugs. To understand and establish the inhibitory characteristics of the drugs, molecular dynamic (MD) simulation of the best fit docking complex performed. Evaluation of the binding energies of the drugs to hACE-2 after 100 ns MD simulations revealed CLQ to have the highest binding energy value of -40.4 kcal/mol close to MLN-7640 (-45.4 kcal/mol), and higher than the exhibited values for its derivatives: CLBQ (-34.5 kcal/mol) and CLCQ (-24.8 kcal/mol). This suggests that CLQ and CLBQ bind more strongly at the exopeptidase site than CLCQ. Nevertheless, the evaluation of binding affinity of the drugs to SARS-CoV-2 Sgp showed the drugs are weakly bound at the RBD site, with CLBQ, CLCQ, CLQ exhibiting relatively low energy values of -16.8 kcal/mol, -16.34 kcal/mol, -12.5 kcal/mol, respectively compared to the reference drug, Bisoxatin (BSX), with a value of -25.8 kcal/mol. The structural analysis further suggests decrease in systems stability and explain the mechanism of inhibition of clioquinol against SARS-CoV-2 as reported in previous in vitro study.Communicated by Ramaswamy H. Sarma.
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Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-CoV-2. Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) protein, enabling virus–host membrane fusion and infection of the airways. We present here a recombinant production strategy for enzymatically active TMPRSS2 and characterization of its matured proteolytic activity, as well as its 1.95 Å X-ray cocrystal structure with the synthetic protease inhibitor nafamostat. Our study provides a structural basis for the potent but nonspecific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that explain specificity. TMPRSS2 cleaved SARS-CoV-2 S protein at multiple sites, including the canonical S1/S2 cleavage site. We ranked the potency of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.4 nM to 120 µM and determined inhibitor mechanisms of action, providing the groundwork for drug development efforts to selectively inhibit TMPRSS2.
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Coronavirus disease 2019 (COVID-19) expanded dramatically all over the world. Nafamostat mesylate inhibits the intracellular entry of novel severe acute respiratory syndrome coronavirus 2 and it has been thought as the therapeutic potential of treatment for patients with COVID-19. In the present study, patients with moderate COVID-19 who were admitted to our hospital were retrospectively analyzed. 31 patients received nafamostat mesylate monotherapy and 33 patients received conservative treatment. Nafamostat mesylate was initiated by continuous intravenous infusion for 4.5 days on average. Comparing with conservative treatment group, nafamostat mesylate did not improve the outcomes and laboratory data 5 days after admission. There were also no significant differences in the laboratory data 5 days after admission and outcomes in high-risk patients. Incidence of hyperkalemia was significantly higher in the nafamostat mesylate group, however, none of the patients required additional treatment for hyperkalemia. In conclusion, nafamostat mesylate monotherapy did not improve clinical outcomes in patients with moderate COVID-19. The therapeutic potential of the combination of nafamostat mesylate and other antiviral agents has not been examined in the present study, and further investigation is required. Because of high incidence of hyperkalemia, regular laboratory tests are required during the use of nafamostat mesylate.
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Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases as well as coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.
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COVID-19 (novel coronavirus disease 2019), caused by the SARS-CoV-2 virus, has various clinical manifestations and several pathogenic pathways. Although several therapeutic options have been used to control COVID-19, none of these medications have been proven to be a definitive cure. Transmembrane serine protease 2 (TMPRSS2) is a protease that has a key role in the entry of SARS-CoV-2 into host cells. Following the binding of the viral spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptors of the host cells, TMPRSS2 processes and activates the S protein on the epithelial cells. As a result, the membranes of the virus and host cell fuse. Bromhexine is a specific TMPRSS2 inhibitor that potentially inhibits the infectivity cycle of SARS-CoV-2. Moreover, several clinical trials are evaluating the efficacy of bromhexine in COVID-19 patients. The findings of these studies have shown that bromhexine is effective in improving the clinical outcomes of COVID-19 and has prophylactic effects by inhibiting TMPRSS2 and viral penetration into the host cells. Bromhexine alone cannot cure all of the symptoms of SARS-CoV-2 infection. However, it could be an effective addition to control and prevent the disease progression along with other drugs that are used to treat COVID-19. Further studies are required to investigate the efficacy of bromhexine in COVID-19.
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COVID-19 (novel coronavirus disease 2019), caused by the SARS-CoV-2 virus, has various clinical manifestations and several pathogenic pathways. Although several therapeutic options have been used to control COVID-19, none of these medications have been proven to be a definitive cure. Transmembrane serine protease 2 (TMPRSS2) is a protease that has a key role in the entry of SARS-CoV-2 into host cells. Following the binding of the viral spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptors of the host cells, TMPRSS2 processes and activates the S protein on the epithelial cells. As a result, the membranes of the virus and host cell fuse. Bromhexine is a specific TMPRSS2 inhibitor that potentially inhibits the infectivity cycle of SARS-CoV-2. Moreover, several clinical trials are evaluating the efficacy of bromhexine in COVID-19 patients. The findings of these studies have shown that bromhexine is effective in improving the clinical outcomes of COVID-19 and has prophylactic effects by inhibiting TMPRSS2 and viral penetration into the host cells. Bromhexine alone cannot cure all of the symptoms of SARS-CoV-2 infection. However, it could be an effective addition to control and prevent the disease progression along with other drugs that are used to treat COVID-19. Further studies are required to investigate the efficacy of bromhexine in COVID-19. For citation: Bahadoram S., Keikhaei B., Bahadoram M., Mahmoudian-Sani M.-R., Hassanzadeh S., Saee-di-Boroujeni A., Alikhani K. Bromhexine is a potential drug for COVID-19; From hypothesis to clinical trials. Problems of Virology (Voprosy Virusologii). 2022; 67(2): 126-132.
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Citation: Derruau, S.; Bouchet, J.; Nassif, A.; Baudet, A.; Yasukawa, K.; Lorimier, S.; Prêcheur, I.; Bloch-Zupan, A.; Pellat, B.; Chardin, H.; et al. COVID-19 and Dentistry in 72 Questions: An Overview of the Literature.
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Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein–protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein–protein interaction that is common to COVID-19 aggravation and heart failure.
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Since the outbreak of Coronavirus Disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, global public health and the economy have suffered unprecedented damage. Based on the increasing related literatures, the characters and pathogenic mechanisms of the virus, epidemiological and clinical features of the disease are rapidly discovered. The spike glycoprotein (S protein), as a key antigen of SARS‐CoV‐2 for developing vaccines, antibodies, and drug targets, has been shown to play an important role in viral entry, tissue tropism, and pathogenesis. In this review, we summarize the molecular mechanisms of interaction between S protein and host factors, especially receptor‐mediated viral modulation of host signaling pathways, and highlight the progression on potential therapeutic targets, prophylactic and therapeutic agents for prevention and treatment of SARS‐CoV‐2 infection. This article is protected by copyright. All rights reserved.
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Introduction: The ongoing epidemic of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) creates a massive panic worldwide due to the absence of effective medicines. Developing a new drug or vaccine is time-consuming to pass safety and efficacy testing. Therefore, repurposing drugs have been introduced to treat COVID-19 until effective drugs are developed. Area covered: A detailed search of repurposing drugs against SARS-CoV-2 was carried out using the PubMed database, focusing on articles published 2020 years onward. A different class of drugs has been described in this article to target hosts and viruses. Based on the previous pandemic experience of SARS-CoV and MERS, several antiviral and antimalarial drugs are discussed here. This review covers the failure of some repurposed drugs that showed promising activity in the earlier CoV-pandemic but were found ineffective against SARS-CoV-2. All these discussions demand a successful drug development strategy for screening and identifying an effective drug for better management of COVID-19. The drug development strategies described here will serve a new scope of research for academicians and researchers. Expert opinion: Repurposed drugs have been used since COVID-19 to eradicate disease propagation. Drugs found effective for MERS and SARS may not be effective against SARS-CoV-2. Drug libraries and artificial intelligence are helpful tools to screen and identify different molecules targeting viruses or hosts.
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Broad-spectrum antiviral drugs against highly pathogenic coronaviruses and other emerging viruses are desirable to enable a rapid response to pandemic threats. Transmembrane protease serine type 2 (TMPRSS2), a protease belonging to the type II transmembrane serine protease family, cleaves the coronavirus spike protein, making it a potential therapeutic target for coronavirus infections. Here, we examined the role of TMPRSS2 using animal models of SARS-CoV and MERS-CoV infection. The results suggest that lack of TMPRSS2 in the airways reduces the severity of lung pathology after infection by SARS-CoV and MERS-CoV. Taken together, the results will facilitate development of novel targets for coronavirus therapy.
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Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS corona virus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dpendent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably co-expressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1000 drugs approved for use by the US Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.
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The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation, but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface, and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. By contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and EST, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells which derived from lung but these characters differ from mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.
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The novel human coronavirus EMC (hCoV-EMC), which recently emerged in Saudi Arabia, is highly pathogenic and could pose a significant threat to public health. The elucidation of hCoV-EMC interactions with host cells is critical to our understanding of the viral pathogenesis and to the identification of targets for antiviral intervention. Here, we investigated the viral and cellular determinants governing hCoV-EMC entry into host cells. We found that the spike-protein of hCoV-EMC (EMC-S) is incorporated into lentiviral particles and mediates transduction of human cell lines derived from different organs, including lung, kidney and colon, as well as primary human macrophages. Expression of known coronavirus receptors, ACE2, CD13 and CEACAM1, did not facilitate EMC-S-driven transduction, suggesting that hCoV-EMC uses a novel receptor for entry. Directed protease expression and inhibition analyses revealed that TMPRSS2 and endosomal cathepsins activate EMC-S for virus-cell fusion and constitute potential targets for antiviral intervention. Finally, EMC-S-driven transduction was abrogated by serum from an hCoV-EMC infected patient, indicating that EMC-S-specific neutralizing antibodies can be generated in patients. Collectively, our results indicate that hCoV-EMC uses a novel receptor for protease-activated entry into human cells and might be capable of extrapulmonary spread. In addition, they define TMPRSS2 and cathepsins B and L as potential targets for intervention and suggest that neutralizing antibodies contribute to the control of hCoV-EMC infection.
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The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.
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The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.
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Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.
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The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.
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Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.
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Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2−/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2−/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2−/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2−/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.
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The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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p-Guanidinobenzoate derivates were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r and C1 esterase were examined. Among the various inhibitors tested, 6'-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(beta-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein. plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r and C1 esterase, and then strongly inhibited complement-mediated hemolysis.
SARS-CoV-2 Cell Entry 62
  • M Hoffmann
  • H Kleine-Weber
  • S Schroeder
Hoffmann M, Kleine-Weber H, Schroeder S, et al. 2020. SARS-CoV-2 Cell Entry 62