Coronaviruses (CoVs) are a group of RNA viruses that infect mammals and birds. In humans, CoVs mainly cause mild diseases, including the common cold. However, in the 21st century three pandemic strains of novel CoVs, highly pathogenic to humans, emerged. These three novel CoVs are zoonosis. In 2002–2003, we dealt with the Severe Acute Respiratory Syndrome coronavirus (currently, SARS-CoV) and in 2012, the Middle East Respiratory Syndrome coronavirus (MERS-CoV). The emergence of SARS-CoV-2 in December 2019 in Wuhan (Hubei province, China) marked the next introduction of a novel, highly pathogenic CoV into the human population [112]. In most cases, SARS-CoV-2 causes mild or moderate respiratory illness and the recovery does not require any special treatment. However, some infected individuals, with associated medical conditions, developed a severe disease called Coronavirus Disease 2019 (COVID-19) with such clinical manifestations as dyspnea, hypoxia, and lung lesions, being an indication for treatment in the intensive care unit. The most affected people experienced respiratory failure (ARDS, acute respiratory distress syndrome), shock, or multiorgan system dysfunction associated with cytokine storm. Such critical stage is often fatal [115, 188]. During the pandemic, millions of people around the world are infected at record speed, resulting in 823,687 deaths on August 26, 2020 (
The development of new drugs, specific for human CoVs and especially for SARS-CoV-2 needs time. Such investigations are yet undertaken, since our knowledge about SARS-CoV-2 biology is constantly increasing. In this review, we summarized the targeting strategies, used since December 2020 in COVID-19 treatment and present our current knowledge and describe their mechanism of action.
We have recently described the Betacoronavirus family and its cycle of replication and detailed the known functions of SARS-CoV-2 encoded proteins (Kwiatek and Adamczyk-Popławska,
Chloroquine (C18H26ClN3) and its derivative Hydroxychloroquine (C18H26ClN3O) are antimalarial and autoimmune disease drugs. The antiviral activity of Chloroquine was demonstrated against different viruses, including: HIV, Dengue, Hepatitis C, Chikungunya, Influenza, Ebola and novel CoVs (SARS-CoV and MERS-CoV) [150]. Both quinolones inhibit the entry of viruses into host cells. Both compounds were found to block viral infection by increasing endosomal pH and by this impeding viral envelope/endosomal membrane fusion necessary to the liberation of viral capsid into the cytoplasm of infected cell during endocytosis [148]. Studies concerning SARS-CoV demonstrated that Chloroquine, in addition to pH modulation, also interferes with the terminal glycosylation of ACE2, which is the receptor for SARS-CoV, but also for SARS-CoV-2 [74, 201]. ACE2 glycosylation may inhibit the CoV/ACE2 binding and thus abrogate the infection [174]. Chloroquine and Hydroxychloroquine were also demonstrated to bind with high affinity to sialic acids and gangliosides present on the surface of human cells. During virus endocytosis, such binding may interfere with the attachment of SARS-CoV-2 to lipid rafts and inhibit the contact between SARS-CoV-2 Spike protein and the ACE2 receptor [51].
The
Quinolones also modulate the innate immune system: Chloroquine and Hydroxychloroquine reduce the activation of the Toll-like receptor (TLR) signaling [97] and Chloroquine reduces synthesis and secretion of several proinflammatory cytokines (TNF-α, IL-1β, IL-6) [86]. This effect may be useful in control of cytokine storm associated with SARS-CoV-2 infection.
Antiviral efficacy of Chloroquine against SARS-CoV-2 was observed by several subsequent clinical trials conducted in China [56]. Hydroxychloroquine, with better clinical safety profile during long-term use, and allowing the administration of high daily dose was also tested against SARS-CoV-2 [196]. A small size pilot study, on 36 patients with different severity of COVID-19 symptoms, was conducted and the antiviral effect of Hydroxychloroquine was demonstrated by the decrease (or even disappearance) of the viral load in nasopharyngeal swabs in patients treated daily with 600 mg of Hydroxychloroquine for 3–6 days [58]. The beneficial effect was reinforced by co-administration of Azithromycin (macrolide antibiotic), [58, 116, 145].
However further studies did not confirm the beneficial effect of Chloroquine or Hydroxychloroquine in COVID-19 treatment. On May 08, 2020, at least 7 clinical trials were completed with published or pre-published results, but the beneficial effect of Chloroquine or its derivative are not conclusive [40]. Some authors even suggest, that these compounds may increase the severity of the illness [152]. Indeed, the progression of the severity of the disease was reported in one Hydroxychloroquine-treated patient and no beneficial effect of Hydroxychloroquine treatment on prognosis of 14 treated patients was observed during a recent study [36]. Another small cohort study with 11 patients also did not proved the protective efficacy of Hydroxychloroquine in COVID-19 patients and even reported one death and one adverse effect (18.1%) after administration of Hydroxychloroquine, comparing to control group [127]. An open label, randomized controlled trial (ChiCTR2000029868) tested long term and high dose (1200 mg for three days, followed by 800 mg for 2–3 weeks) of Hydroxychloroquine, administrated to patients with mild to moderate COVID-19. No effect on virus clearance, compared to standard care (supplemental oxygen, concomitant antiviral medication or antibiotics) was observed at day 28. More adverse events were observed in Hydroxychloroquine-treated group than in control group. The most common adverse event was diarrhea, reported in 10% of patients. Serious adverse events were also reported and included disease progression, upper respiratory infection and kidney injury [164]. Another report described a comparative observational study on data collected from routine care. Patients with pneumonia, who required oxygen were treated with 600 mg/day of Hydroxychloroquine. Treatment did not improved disease progression or patient survival as compared to control group without Hydroxychloroquine at day 21. Almost 10% of Hydroxychloroquine-treated patients had the drug stopped because of changes in electrocardiogram [118].
On May 27, 2020 European governments, following World Health Organization (WHO) decision from May25, acted to pause the use of quinolones to treat patients suffering from COVID-19 due to safety concerns. The decision was partially taken on the basis studies mentioned above and on a observational study, that is currently retracted due to inconsistencies in the patient data [122]. On June 3, 2020, the WHO decided to continue trials with quinolones, but the FDA retracted the EUA issued to Hydroxychloroquine for use in COVID-19 patients.
Until August 2020, several clinical trials were finished and the conclusions is that the efficacy of Chloroquine or Hydroxychloroquine (alone or in combination with azithromycin) to prevent or treat COVID-19 patients could not be established. The results of RECOVERY trial (NCT04381936) on 4674 patients did not demonstrate any clinical benefit from use of Hydroxychloroquine in hospitalized patients with COVID-19 (preprint [76]). The American College of Physicians recommends against the use of Chloroquine and its derivative [139]. On July 4, 2020, World Health Organization decided to discontinue the research concerning Hydroxychloroquine in “Solidarity” international clinical megatrial, conducted to find an effective treatment for COVID-19 (
Ivermectin (C48H74O14) is known since almost 40 years, a broad spectrum anti-parasitic drug licensed in animal and human medicine [99]. In addition to its anti-parasitic effect, Ivermectin has been also demonstrated to limit
Ivermectin was described as reducing the presence of NS5 protein, encoded by Zika or Denga viruses, in nucleus of infected cells. The drug blocked the interaction between NS5 and IMP α/β transporter [175]. Studies on novel human CoVs proteins revealed a potential role for IMPα/β1 during infection in signal-dependent nucleocytoplasmic translocation of some viral proteins. For example ORF4b of MERS-CoV inhibits the induction of IFN-β synthesis in both the cytoplasm and nucleus [195]. The N-terminal domain of SARS-CoV N protein was found in nucleus, suggesting that the N may act as a shuttle protein and have several roles during SARS-CoV replication [165]. Recent studies demonstrated that Ivermectin inhibits SARS-CoV-2 replication
Recently a pilot clinical trial evaluating Ivermectin effect on hospitalized adult patients with mild to moderate COVID-19 is terminated (NCT04343092). Sixteen patients received a single dose of Ivermectin in addition to Hydroxychloroquine and azithromycin treatment. All the patients from Ivermectin group were cured as compared to the control group, in which two person died. The mean time of hospitalization was significantly shorter (7.62±2.75 days) for Ivermectin-treated patients, comparing to controls (13.22 ± 5.90 days). The virus clearance was faster in Ivermectin group [63]. The effectiveness of Ivermectin, as drug or adjuvant in association with other compounds, is currently being evaluated in various randomized clinical trials, but there is still insufficient evidence to draw a conclusion on benefits or harms during treatment of COVID-19 patients [30].
SARS-CoV-2 exploits ACE2 receptor, recognized by S protein for entry the host cell [74, 201]. Host cell proteases are responsible for coronavirus S protein activation and are essential for viral entry by encompassing S protein cleavage at the S1/S2 and the S2’ sites. A recent report suggested that SARS-CoV-2 use the membrane-anchored serine protease TMPRSS2 for S protein priming for plasma membrane fusion [74]. The TMPRSS2 inhibitor might constitute an option for blocking fusion of viral envelope with cellular membrane and by this inhibit virus entry into host cells.
It was previously demonstrated that SARS-CoV and MERS-CoV have exhibited lower spread in TMPRSS2 deficient mice [80]. VeroE6 cells, expressing TMPRSS2, were highly susceptible to these CoVs infection [120]. TMPRSS2 protease participates also in priming of SARS-CoV-2 Spike protein [187]. Thus, inhibition of TMPRSS2 protease might constitute an option for blocking the fusion SARS-CoV-2 envelope with cellular membrane and those lowering the virus infectivity.
Camostat mesylate (C20H22N4O5) is a drug with clinically proven safety, that is licensed in Japan since 1985, for the suppression of pancreatitis-induced pain due to its ability to inhibit inflammatory proteases [162, 171]. Camostat is serine protease inhibitor and was demonstrated as blocking TMPRSS2 and by this inhibiting the activation of SARS-CoV S protein at the cell surface
Until August 10, 2020, no clinical trial, evaluating the safety and impact of Camostat mesylate in treatment of COVID-19 was finished but several trials are recruiting.
Nafamostat mesylate (C21H25N5O8S2) is an FDA-approved drug, used in Asian countries for indications unrelated to coronaviruses. Nafamostat was described to prevent S-mediated membrane fusion between cells expressing Spike protein of MERS-CoV and cells expressing MERS-CoV receptor, CD26, with TMPRSS2 [194]. Moreover Nafamostat blocked MERS-CoV infection of Calu3 lung cells
Bromhexine (C14H20Br2N2) is a widely prescribed medicine used for treatment of many respiratory conditions, associated with a disturbance of mucus secretion, and it is well tolerated and safe. In this context, Bromhexine efficacy is currently evaluated in clinical trial for chest congestion and cough in patients with suspected and mild SARS-CoV-2-associated pneumonia (NCT04273763). In fact, Bromhexine is also a potent and selective inhibitor of the TMPRSS2 [113] and was proposed previously as a candidate drug for treatment of SARS-CoV and MERS-CoV infections [155]. The inhibitory effect of Bromhexine suggest its repurposing either as a treatment or as a prophylactic agent in SARS-CoV-2 infections [117]. To date no results of clinical trial concerning Bromhexine were available (August 10, 2020).
Baricitinib (C16H17N7O2S) is an inhibitor of the release of cytokines by inhibiting the Janus-associated kinase (JAK) pathway. The drug is presently approved for treatment of rheumatoid arthritis [13]. Using artificial intelligence in a search for candidates that might have both, antiviral and anti-inflammatory activity during SARS-CoV-2 infection, Baricitinib was designed among others as acting on AP2-associated protein kinase 1 (AAK1) and on cyclin G-associated kinase (GAK), two regulators of endocytosis [138, 146]. As SARS-CoV-2 entry into ACE2 expressing cells involves, among others, clathrin-dependent endocytosis [157], Baricitinib was proposed as COVID-19 treatment, basing on prediction that this drug would reduce the ability of the virus to entry lung cells [146]. The inhibitory activity of Baricitinib was recently validated for AAK1, BIKE, GAK, and STK16 kinases [159]. The effect of Baricitinib on SARS-CoV-2 infectivity was also evaluated in 3D primary human liver spheroids. Pretreatment of spheroids with Baricitinib (400 or 800 nM) significantly reduced viral load by 30–40% without injury of liver cells [159].
Moreover, like other JAK inhibitors (Fedratinib or Ruxolitinib) Baricitinib may also reduce the effects of the increased cytokine levels that are frequently seen in patients with COVID-19.
The safety of Baricitinib therapy, combined with Lopinavir/Ritonavir, in moderate COVID-19 pneumonia patients was positively evaluated in a pilot study (NCT04358614) [25]. Baricitinib-treatment was well tolerated with no serious adverse events. In the Baricitinib-treated group, clinical and respiratory function parameters significantly improved. Authors confirm that the use of Baricitinib may limit the cytokine-release syndrome associated with COVID-19 [25]. Baricitinib administration was also associated with improvement in clinical, radiologic, and viral load parameters along with a rapid decline in CRP protein and IL-6 levels in a small case study of 4 patients with bilateral COVID-19 pneumonia [159].
However, some authors warn against using this compound. Indeed, Baricitinib may cause lymphocytopenia, neutropenia and enhance the incidence of coinfections that are one of the leading causes of mortality of COVID-19 patients [138]. By inhibiting JAK kinases, Baricitinib may also interfere with antiviral activity of interferon [52]. Baricitinib was also described as reactivating latent viral infections of such viruses as Hepatitis B, so its safety should be evaluated [68].
Arbidol (C22H25BrN2O3S) is a broad-spectrum and well-tolerated antiviral drug, which has been approved in several countries for treatment of influenza infections [19]. This compound was described as active against numerous enveloped and non-enveloped viruses as Influenza A virus, RSV (respiratory syncytial virus), Rhinovirus, Coxsackie virus and Adenovirus
The inhibitory effect of Arbidol on SARS-CoV-2 entry (75% of inhibition) was demonstrated
Application of Arbidol for treatment of COVID-19 patients took mainly place in China on small number of patients. The reports were very encouraging for the outcome of COVID-19 patients treated with Arbidol [45, 108, 185, 203]. However, a recent evidence has not demonstrated that monotherapy with Arbidol provided any benefits in mild/moderate cases outcome [105]. Another retrospective study also did not found improvement in the prognosis or acceleration of SARS-CoV-2 clearance in 45 hospitalized patients treated with Arbidol versus 36 control patients [106]. Combined administration of Arbidol and IFNα2b improved the COVID-19 associated pneumonia in mild patients, but did not accelerate the virus clearance as compared to monotherapy with IFNα2b [192]. Randomized control clinical trial assessing the efficacy of Arbidol are ongoing.
Type I interferons (IFNs) are a group of cytokines playing an important role in host defense during viral infections [143]. IFNs suppresses viral infection by interfering with replication of the virus and by inducing innate and adaptive immune responses. However, viruses have evolved many mechanisms to evade the IFN activity [143]. Currently, treatment with exogenous type I IFNs is mainly restricted to administration during chronic infections with HBV or HCV [69, 147]. IFNα is also evaluated as drug in HIV infection treatment [59].
Concerning human CoVs, IFNα treatment of VeroE6 cells effectively inhibits the replication of SARS-CoV
SNG001, an inhaled formulation of IFNβ showed promise in a recently completed COVID-19 trial (NCT04385095). According to the study, the risk of developing symptoms that required ventilation or cause death was reduced by 79% in group receiving SNG001 compared to patients who received placebo during the treatment period of 16 days (
Acute SARS-CoV-2 infection is associated with hypercytokinaemia – the upregulation of pro-inflammatory cytokines and chemokines, also known as Cytokines Release Syndrome (CRS) or cytokine storm. This aspect of COVID-19 outcome was compared to hyperinflammatory syndrome associated with haemophagocytic lymphohistiocytosis (HLH). HLH is potentially fatal disease of normal but overactive T cells and macrophages that excessively produce proinflammatory cytokines, including IFNγ. Cytokine profiles in HLH and acute COVID-19 were described as similar, including the expression of IL-1β, IL-2, IL-6, IL-7, IL-8, TNF and chemokines or its ligands (CXCL10, CCL2) [123, 176]. The management of this cytokine storm is one of the major needs regarding COVID-19 infection.
JAK are a family of intracellular tyrosine kinases involved, among others, in cytokine (including IFNs) signaling. Several cytokines employ the intracellular signaling pathway mediated by JAKs. JAK inhibitors found application in a broad spectrum of diseases concerning immune system such as autoimmune and auto-inflammatory disorders [22]. Among JAK inhibitors tested in COVID-19 treatment Ruxolitinib and, previously mentioned, Barinicitib are currently evaluated.
Ruxolitinib (C17H18N6) is a JAK inhibitor approved for patient treatment by the FDA, though not for immunological disorders, but for treatment of intermediate or high-risk myelofibrosis [111]. The beneficial effect of Ruxolitinib on HLH outcome was demonstrated on both, preclinical models and in clinical practice [204]. Ruxolitinib has been shown to reduce cytokine levels and improve outcomes in different conditions. Ruxolitinib has attenuated T-cell activation and decreased inflammation in murine model of HLH infected with LCMV [4]. Several case reports and a pilot study (on five patients) determined the activity and safety of Ruxolitinib in adults with secondary HLH [3, 158]. Cytopenia improvement was observed, allowing transfusion independence, discontinuation of corticosteroids, and hospital discharge of Ruxolitinib-treated patients [3]. Other studies also confirmed the therapeutic effect of Ruxolitinib in HLH patients [21, 177].
Recently a bioinformatic approach, involving artificial intelligence, has identified Ruxolitinib among the potential therapeutics for combining antiviral and anti-inflammatory treatments [160]. The safety of such treatment is of concern: among 5 HLH patients treated with Ruxolitinib a serious adverse event (grade 4 febrile neutropenia) was reported [3]. Another study mentioned leukopenia, thrombocytopenia, elevated transaminases, elevated bilirubin and hypertriglyceridemia in Ruxolitinib-treated HLH patients. However none of patients stopped the therapy due to its toxicity [177].
Ruxolitinib has been also used for treatment of COVID19 patients. The appearance of purpuric lesions on the skin and an erythrodermic rash were described in two COVID19 patients after administration of Ruxolitinib. However, these persons have several different treatments against SARS-CoV-2 before receiving Ruxolitinib and the effect may be the result of accumulated drugs [57]. A multicenter, single-blind, randomized controlled phase II trial, involving patients with severe coronavirus disease 2019 confirmed that levels of 7 cytokines (IL-6, nerve growth factor β, IL-12 (p40), migration inhibitory factor, MIP-1α, MIP-1β, and VEGF) were significantly decreased in the Ruxolitinib group. Ruxolitinib patients had a faster clinical improvement, chest computed tomography improvement and a faster recovery from lymphopenia [27]. A monocentric retrospective chart analysis on a subgroup of patients with severe COVID-19 that suffered from acute respiratory distress syndrome and multi organ failure also confirm the beneficial outcome of Ruxolitinib administration and the decrease of markers indicating the hyper inflammation. Side effects of short term treatment with Ruxolitinib were manageable (mild anemia and liver enzyme elevation) [98]. Data from RESPIRE Protocol (NCT04361903) have been published. This retrospective multicenter observational study concerned case series of 18 critically ill patients with COVID-19 and Acute respiratory distress syndrome (ARDS). Data collection demonstrated a rapid clinical response without evolution from non-invasive ventilation to mechanical ventilation in 16 of 18 patients treated with Ruxolitinib. After 14 days, 16 patients showed complete recovery of respiratory function. No adverse effect were observed [28]. Taken together, these data suggest that Ruxolitinib may lower the hyperinflammatory state observed in patients experiencing COVID-19-associated cytokine storm and ARDS.
In COVID-19 patients, a large number of T lymphocytes and mononuclear macrophages are activated, producing pro-inflammatory cytokines such as multifunctional interleukin-6 (IL-6). IL-6 is the key factor in acute inflammation and CRS [199]. IL-6 binds to its receptor on the target cells, causing the cytokine storm and severe inflammatory responses in lungs and other tissues and organs. Its inhibition may be of great value in reducing COVID-19-associated mortality [198, 199].
Tocilizumab is a recombinant humanized monoclonal antibody specific for IL-6 receptor. By binding IL-6 receptor Tocilizumab prevents IL-6 itself from binding and by thus blocks IL-6 transduction pathway. These IL-6 antagonist is currently administrated for the treatment of rheumatoid arthritis and systemic juvenile idiopathic arthritis [129]. Many reports described the beneficial effect of Tocilizumab administration to severe or critical COVID-19 patients. A small sample clinical trial (ChiCTR2000029765), on 21 patients with severe or critical COVID-19, has shown good efficacy of Tocilizumab administration: body temperature returned to normal, CRP (C reactive protein) decreased, oxygen intake was reduced and pulmonary lesions were absorbed [193]. Similar observation were made during treatment with Tocilizumab of 26-year-old patient in critical stage: 2 days after drug administration ventilation conditions improved and after 5 days laboratory parameters decreased (CRP returned to normal). Complete resolution of lung abnormalities was noticed on day 7 [172]. However, a recent report, describing two patients with COVID-19 and CRS complications reported progression to HLH despite treatment with Tocilizumab. One patient develop viral myocarditis [140]. So, the safety and clinical usefulness of Tocilizumab in the treatment of COVID-19-induced CRS has to be verified. Another study on 15 patients with different COVID-19 severity indicated that treatment failed in four patients but the solution may be a repeated dose of the Tocilizumab [114]. However, the more recent preliminary results from SMACORE (SMAtteo COvid19 REgistry) indicated that Tocilizumab administration did not reduce intensive care unit admission or mortality rate in a cohort of 21 patients, even with repeated treatment [42].
Similar results were obtained during a global randomized, double-blind, placebo-controlled phase III clinical trial concerning Tocilizumab administration to hospitalized patients with severe COVID-19 pneumonia (COVACTA- NCT04320615): the results did not confirm beneficial effect of treatment (
Other IL-6 inhibitors, Sarilumab [141] and Siltuximab [173], were also investigated as potential treatment of COVID-19 patients. In a recent review, 352 articles concerning IL-6 antagonists and COVID-19 were reported, but only 11 study were further analyzed [7]. The conclusion is that use of Tocilizumab may be beneficial and currently the use of Tocilizumab and other IL-6 inhibitors is intensively investigated in several clinical trials [8].
Convalescent plasma (CP) therapy is a passive immunotherapy, already applied to the prevention and treatment of several infectious diseases. CP therapy was successfully used in the treatment of different viral conditions and more recently during Ebola, SARS-CoV, MERS-CoV, and Influenza virus H1N1 pandemics with satisfactory efficacy and safety [29, 119]. Administration of convalescent plasma may be also of clinical benefit for treatment of severe acute respiratory infections of SARS-CoV-2 etiology [29, 37]. Such possibility is reinforced by several experimental studies. A case report describes CP treatment of a centenarian with laboratory confirmed SARS-CoV-2, not suitable for antiviral treatment. Patient received two CP doses (200 and 100 ml). Significant improvement of laboratory indicators and clinical symptoms was observed. SARS-CoV-2 viral load decreased after the first transfusion (from 2.55 × 104 to 1.39 × 103 copies RNA/ml) and became undetectable (13 days of hospitalization) after the second one [94]. Another study reported treatment of 10 patients, with confirmed by RT-PCR infection, treated with CP transfusion (ChiCTR2000030046) [46]. Administration of one dose of 200 ml CP was well tolerated, the clinical symptoms significantly improved with the increase of oxyhemoglobin saturation within 3 days and CRP decrease. Radiological examination showed varying degrees of absorption of lung lesions within 7 days. The neutralization of viremia was also accelerated: the viral load was undetectable after transfusion in 7 patients with previous viremia. No severe adverse effects were observed [46]. A preliminary communication about 5 patients in critical stage of COVID-19 (with acute respiratory distress syndrome) reinforces the possibility of improvement of clinical status by CP treatment. One of effects of CP administration was the decrease of viral loads: virus became undetectable within 12 days after the transfusion. 3 patients has been discharged from hospital and 2 were stabilized. No adverse events were reported [154]. The results of a randomized trial (NCT04342182) concerning CP treatment of COVID19 patients are announced as preprint [60]. The study was discontinued due to the concerns about very high antibodies titers in COVID19 patients (comparable to the levels observed in donors). No difference in mortality, hospitalization time or disease severity at day-15 was observed between CP treated patients and patients on standard care [60]. Many other trials are ongoing and results are expected.
Recently, recommendations on biological characteristics of a CP preparation were published [135]. The CP must be collected from donor who had recovered from COVID-19 for more than two weeks. CP should be tested for HIV, HCV, HBV and syphilis (nucleic acids and serology tests). A negative result of RT-PCR testing for SARS-CoV-2 is also clearly expected [135]. CP should also contain sufficient amount of SARS-CoV-2 -specific antibodies [94]. Recommendations of FDA for investigational CP treatment are also available at FDA site (
Lopinavir (C37H48N4O5)/Ritonavir (C37H48N6O5S2) treatment is used since 2000 in HIV infections in adults and children. Lopinavir is the HIV protease inhibitor, blocking HIV polyproteins maturation [33]. Molecular modeling evaluated that Lopinavir may also bind to the 3CLpro protease encoded by SARS-CoV-2 and by thus inhibit the processing of viral polyproteins [107, 130]. Recent study suggested that Lopinavir was active against SARS-CoV-2
The concern is also about Lopinavir/Ritonavir safety. ACE2 receptor is also expressed by kidney and post-mortem biopsies confirmed viral inclusions in tubular epithelial cells and podocytes. Lopinavir/Ritonavir treatment may enhance acute kidney injury in SARS-CoV-2 infected patients [16]. On July 6, 2020, WHO decided to discontinue the Lopinavir/Ritonavir arms of SOLIDARITY international trial.
Other clinical trials are completed, but results are missing and several ongoing trials of Lopinavir/Ritonavir, essentially in combination with other treatments, are currently recruiting. The lack of efficacy of Lopinavir may be due to the fact that 3CLpro is a cysteine protease while HIV protease is an aspartic protease and moreover 3CLpro, does not contain a C2-symmetric pocket in the catalytic site- the target of these HIV protease inhibitors [9, 92].
The nucleoside analogue, Remdesivir (GS-5734) (C27H35N6O8P) is a well-known broad-spectrum antiviral drug, that inhibits viral RdRp, causing termination of RNA synthesis [62]. This drug was developed in response to the Ebola outbreak in West Africa from 2014 [81]. Remdesivir had as well showed its antiviral efficacy against SARS-CoV-2
FDA has approved a EUA of Remdesivir to treat COVID-19 patients. Remdesivir should be administered intravenously. However, as pointed by FDA “Remdesivir is not yet licensed or approved anywhere globally and has not been demonstrated to be safe or effective for the treatment of COVID-19” (May 06, 2020). The use Remdesivir in patients with nephrologic or with hepatic impairment is not recommended. On 7th May 2020, Remdesivir (Veklury®) was approved for COVID-19 treatment in Japan. Remdesivir is the first medicine authorized at European Union level for treatment of COVID-19 since July 3, 2020. Several clinical trials evaluating its efficacy and safety were ongoing through USA, Europe and China and the results are expected within weeks.
Ribavirin (C8H12N4O5) is a nucleoside analogue with broad antiviral activity, approved in antiviral treatment. In Canada, for example, Ribavirin is licensed for the treatment of RSV-associated bronchiolitis and pneumonia in infants for treatment of hepatitis C infections [95, 96]. Known mechanisms of Ribavirin efficacy against RNA and DNA viruses involve polymerase inhibition, interference with RNA capping, lethal mutagenesis and inhibition of GTP synthesis [131]. Another study pointed out the boost of immune system, especially Th1 cells by Ribavirin. So, the antiviral effect may be not only due to direct inhibition of virus replication but also involves immune system activation [93, 163].
Ribavirin was extensively studied as anti- SARS-CoV and MERS-CoV drug. Although the drug activity against these CoVs was demonstrated
Ribavirin tightly binds to SARS-CoV-2 RNA-dependent RNA polymerase (RdRp)
Favipiravir (C5H4FN3O2) acts as a purine nucleoside analogue and is a competitive substrate inhibitor of the viral RdRp [55]. Unlike Ribavirin, this compound does not influence host cell nucleic acids synthesis. Favipiravir has been approved for the treatment of Influenza virus infections in Japan and is also licensed in China, but not in Europe or USA [55]. It has a broad spectrum of activity towards RNA viruses like Influenza virus, Rhinovirus or RSV [89]. Its efficacy against SARS-CoV-2 has been tested
The evidence of the clinical safety of short term use of Favipiravir seems to be well documented [137]. Generally, the drug was well tolerated, but liver toxicity, hyperuricemia and diarrhea were reported in some patients [23, 35]. Recently, a Phase I clinical trial did not revealed adverse effects of this drug in healthy persons (NCT04400682).
One clinical trials did not confirm beneficial effect of Favipiravir administration to COVID19 patients (ChiCTR 2000029544) (preprint [110]).
In March 2020, Favipiravir was approved by the National Medical Products Administration of China as the first anti-COVID-19 drug [169]. In April 2020 FDA approves clinical trials using Favipiravir in treatment of COVID-19 patients in USA. On August 12, 2020 5 trials were completed as mentioned at USA Clinical trials registry.
The predominant physiological function of SARS-CoV-2 cellular receptor-ACE2 is the maintenance of cardiovascular homeostasis: ACE2 increase blood pressure by cleaving the angiotensin I to angiotensin II (potent vasoconstrictor) and by inactivation of bradykinin (vasodilator). A possibility of blockage of virus-ACE2 interaction is the use of truncated, soluble recombinant human ACE2 (rhACE2) to block S protein, without affecting ACE2 itself [166]. Intravenous administration of active soluble ACE2 protein significantly improved the outcome of respiratory failure, induced by lipopolysaccharide, by its ability to increase the oxygenation in pigs [168]. Administration of a rhACE2 protein alleviated the severity of RSV or H5N1 virus-induced lung injury in ACE2 deficient mice [65, 205].
Soluble rhACE2 has been shown to block binding of the SARS-CoV spike S1 protein to its receptor
Other soluble form of ACE2 receptor, promising as COVID-19 treatment, is the recombinant bacterial ACE2-like soluble enzyme (rbACE2). B38-CAP obtained from
As mentioned above, SARS-CoV-2 mainly enter to host cells by endocytosis pathway and fusion occurs between viral envelope and endosomal membrane [133]. Both mechanisms involved S protein and the recognition of specific receptor present on cellular membrane: ACE2. During viral maturation, S protein is post-translationally cleaved into a S1 receptor binding unit and a S2 membrane fusion unit. The S2 subunit contain the fusion peptide and two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. Receptor binding induces conformational changes in the S2 subunit. HR1 and HR2 regions interact with each other to form a six-helix bundle (6-HB) fusion core, which in turn induces fusion by insertion of fusion peptide into the host target cell membrane, bringing viral and cellular membranes into close proximity. It was demonstrated that HR2-dervided peptide prevented cell entry (fusion entry model) of SARS-CoV, probably by interfering with the 6-HB formation. However, the endosomal pathway entry was not inhibited [170].
In studies, concerning SARS-CoV and MERS-CoV, a pan-coronavirus fusion inhibitor, named EK1, was obtained [190]. This soluble peptide, derived from the HR2 domain of HCoV-OC43, exhibited broad fusion inhibitory activity against multiple human CoVs, including SARS-CoV and MERS-CoV,
Recently, a lipopeptide derived from EK1 (EK1C4) was developed [189]. The addition of cholesterol to C-terminus of EK1 peptide improves its inhibitory activity on pseudo CoVs infection. Moreover EK1C4 blocked
Capping of 5’ termini of mRNA (addition of a 7-methyl-Gppp) is essential for efficient mRNA translation in eukaryotic cells and is also involved in pre-mRNA splicing, mRNA export from the nucleus and protection from mRNA degradation [142]. Capping is also supposed to facilitate evasion from the host’s immune response. CoVs protect their RNA with a cap moiety. However, as they replicate in the cytoplasm, CoVs don’t have access the host capping machinery, present in the nucleus, and encode their own capping and methylation apparatus. The mechanism of capping of CoVs RNA was recently described [38]. Like for CoVs that cause SARS or MERS, the mechanism of RNA capping may also be a the target in treatment of SARS-CoV-2 infection.
The nsp14 protein encoded by SARS-CoV was identified as a cap (guanine-N7)-methyltransferase (N7-MTase) and was designated as target for new antiviral drugs [38, 82]. The second viral methyltransferase involved in mRNA capping is ribose-2’-O-methyltransferase (2’-O-MTase) encoded by
A recent bioinformatic report describe the screening 123 antiviral drugs for select SARS-CoV-2 targeted inhibitors. Dolutegravir (HIV integrase inhibitor) and its derivative Bictegravir were selected as able of inhibition of 2’-O-MTase encoded by SARS-CoV-2, based on their estimated free energy of binding, the orientation of drug molecules in the active site and the interacting residues [91]. Dolutegravir was also proposed for COVID-19 treatment through a drug-target interaction deep learning model (artificial intelligence) but not as inhibitor of 2’-O-MTase but other RNA processing enzymes [14]. Currently, no studies evaluate Dolutegravir efficacy in treatment of COVID-19 patients (May 30, 2020).
CRISPR-Cas is the bacterial antiviral system that use sequence-specific crRNAs (CRISPR RNA) to inhibit bacteriophage replication by sequence-specific destruction of target viral DNA. Among different application of CRISPR, this system may allow the development of antiviral therapy targeting incurable chronic viral infections [2] such as HIV, HPV, HBV or herpesvirus or other infections as JCV (polyoma JC virus), ASF (african swine fever virus) or pseudorabies virus [101].
Such alternative antiviral approach, which relies on a CRISPR-Cas13 system was currently proposed for recognizing and specifically degrading the intracellular SARS-CoV-2 RNA genome and its mRNA [1]. The Cas13 ribonuclease could be an effective antiviral drug for single-stranded RNA viruses because it cleaves RNAs complementary to crRNA. Cas13 activity against LCV (lymphocytic choriomeningitis virus), Influenza A virus and VSV (vesicular stomatitis virus) was recently demonstrated in cell culture [54]. Currently, by testing the degradation of synthesized fragments of SARS-CoV-2 in lung epithelial cell cultures, a pool of crRNA able to direct Cas13 to viral RNA was designed. Half of these crRNAs targeted the conserved sequences of the
Our knowledge about novel CoVs and SARS-CoV-2 biology is constantly increasing, giving opportunities for the rational design of therapeutic drugs, targeting CoVs replication, as well as for elaboration of potential therapies of COVID-19 patients. Currently tested drugs against SARS-CoV-2 are shown on Figure 1. A lot of data are provided during precedent outbreaks of SARS-CoV and MERS-CoV, but many issues remains unresolved. The lack of serious preclinical studies encourage the use of different drugs, that efficacy is ambiguous even during
Unfortunately, the huge number of currently conducted case studies and clinical trials does not always turn in real know-how. In general a combination of several drugs are combined during trials. Many research was performed chaotically, trials have not always been rigorously designed and their main aim remains the assistance to hospitalized patients to save their lives. Some papers have been retracted due to methodological concern. Due to, the results are not always reliable and conclusive.
The use of Chloroquine and Hydroxychloroquine was halted in USA and Europe, due to safety issues and lack of efficacy against SARS-CoV-2. Also Lopinavir/Ritonavir and Ribavirin were removed from the COVID-19 institutional protocol. The National Institutes of Health publishes, constantly upgraded, guidelines for the medical management of COVID-19. Experts recommend against high-dose Chloroquine, against the combination of Hydroxychloroquine and Azithromycin (safety issues) and against Lopinavir/Ritonavir, or other HIV protease inhibitors (negative clinical trial data) (
In China, the management of COVID-19 included antivirals (Hydroxychloroquine, Tocilizumab, IFN), high flow oxygen, mechanical ventilation, corticosteroids, intravenous immunoglobulin and CP administration [136]. Treatments based on unproven traditional medicines were also promoted in China and in Africa, but clinical evidences for their efficacy are lacking [43, 77].
Based on available data, only the administration of Remdesivir use is accepted through an EUA issued by FDA for certain hospitalized patients requiring supplemental oxygen. Remdesivir is also accepted in European Union and Japan for treatment of COVID-19. Another inhibitor of viral polymerase, Favipiravir, also displayed encouraging results as treatment.