key: cord-329010-n0mz098o
authors: McKee, Dwight L.; Sternberg, Ariane; Stange, Ulrike; Laufer, Stefan; Naujokat, Cord
title: Candidate drugs against SARS-CoV-2 and COVID-19
date: 2020-04-29
journal: Pharmacol Res
DOI: 10.1016/j.phrs.2020.104859
sha: 
doc_id: 329010
cord_uid: n0mz098o

Outbreak and pandemic of coronavirus SARS-CoV-2 in 2019/2020 will challenge global health for the future. Because a vaccine against the virus will not be available in the near future, we herein try to offer a pharmacological strategy to combat the virus. There exists a number of candidate drugs that may inhibit infection with and replication of SARS-CoV-2. Such drugs comprise inhibitors of TMPRSS2 serine protease and inhibitors of angiotensin-converting enzyme 2 (ACE2). Blockade of ACE2, the host cell receptor for the S protein of SARS-CoV-2 and inhibition of TMPRSS2, which is required for S protein priming may prevent cell entry of SARS-CoV-2. Further, chloroquine and hydroxychloroquine, and off-label antiviral drugs, such as the nucleotide analogue remdesivir, HIV protease inhibitors lopinavir and ritonavir, broad-spectrum antiviral drugs arbidol and favipiravir as well as antiviral phytochemicals available to date may prevent spread of SARS-CoV-2 and morbidity and mortality of COVID-19 pandemic.

an exopeptidase expressed on epithelial cells of the respiratory tract, may constitute a pharmacological target to limit cell entry of SARS-CoV-2. The established antimalarial drugs chloroquine and hydroxychloroquine have been shown to inhibit terminal phosphorylation of ACE2 and to elevate the pH in endosomes, respectively. Chloroquine and hydroxychloroquine constitute candidate drugs against SARS-CoV infection and COVID-19 disease, and are now investigated for their therapeutic efficacy in international clinical trials with COVID-19 patients (i. e. SOLIDARITY Trial).

The glycosylated S protein of SARS-CoV is highly immunogenic to the host immune system, and murine polyclonal antibodies against SARS-Co-V S protein potently inhibit SARS-CoV-2 S-mediated cell entry, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination [9] . Similar to the earlier SARS and MERS beta coronaviruses, SARS-CoV-2 primarily infects alveolar epithelial cell of the lung, leading to a severe bilateral peripheral pneumonia with ground glass opacity in CT images (COVID-19 disease), with a mortality rate of 2 % to 5 % [10] .

SARS-CoV-2 also can contribute to multiple organ failure, affecting heart, liver, kidney, central nervous system and gastrointestinal tract [11] . Epidemiology thus far suggests that SARS-CoV-2 is more contagious than SARS-CoV or MERS-CoV [12] . Multiple mechanisms now identified in the infective and replication processes of SARS-CoV-2 offer targets for pharmacological interventions. Infection of pneumocytes, macrophages and pulmonary mast cells requires viral S protein. This invasion process which involves attachment of S protein to the ACE2 receptor is facilitated by host cell derived serine protease TMPRSS211 [8] . Agents that inhibit TMPRSS211, such as camostat mesilate, may be useful in blocking viral host cell entry. After host cell entry, the viral single-stranded positive RNA, is released for replication of virus RNA and translation of virus polyproteins that are finally cleaved into mature effector proteins by virus proteases [13] . The S protein interaction with ACE2 on host cell cytoplasmic membrane initiates viral infection. Strategies capable of disrupting S protein interaction with ACE2 could be of significant therapeutic value, because the binding affinity of SARS-CoV-2 S protein to ACE2 is 10-20-fold higher than for the S protein of SARS-CoV which may contribute to the higher contagiousness of SARS-CoV-2 as compared to SARS-CoV [12] . Although SARS-CoV and SARS-CoV-2 have only 79% genomic sequence similarity, they share a highly conserved receptor binding domain for their S proteins [1] There is also potential for targeting other highly conserved proteins associated with SARS-CoV and SARS-Co-V-2, including RdRP and 3Clpro (also termed Mpro), which share over 95% similarity between the two viruses, despite only 79% genomic sequence sharing.

RdRP is an RNA-dependent RNA polymerase required for replicating the viral genome within the host cell. 3Clpro and Plpro are both viral proteases which break down viral polyprotein into functional units within host cells that are finally assembled into new viruses. The 3Clpro sequences between the two viruses are 96% similar, the Plpro sequence identity is 83%, and their active sites show a high degree of conservation [14] . Drugs that have recently been shown to target MERS-CoV in mice [15] , and to inhibit Ebola virus RdRP and SARS-CoV-2 proteases in humans, such as remdesivir and ritonavir/lopinavir, also constitute candidate drugs against SARS-CoV-2 and are now investigated for their therapeutic efficacy in COVID-19 patients in 2 international clinical trials (SOLIDARITY Trial and DisCoVeRy Trial). Finally, certain phytochemicals and natural products with high antiviral activity should be considered for treatment of SARS-CoV-2 infection and COVID-19 disease. 

Results from previous studies reveal that diverse viruses, including Ebola virus, SARS-coronavirus (SARS-CoV), MERS-coronavirus (MERS-CoV) and influenza virus employ host cell proteases for activation of their envelope glycoproteins [16] [17] [18] . Cleavage and activation of the spike protein (S protein) of SARS-CoV that is required for membrane fusion and host cell entry is mediated by transmembrane protease/serine subfamily member 2 (TMPRSS2), an airway and alveolar cell serine protease [19] [20] [21] . Pöhlmann and coworkers recently demonstrated that SARS-CoV-2 also employs TMPRSS2 for SARS-CoV-2 S protein priming and S protein-driven cell entry [8] . Using camostat mesilate, a clinically proven and commercial serine protease inhibitor that partially blocks infection by [26, 27] , and chronic pancreatitis [28] [29] [30] [31] . Camostat mesilate (NI-03) is manufactured as an oral drug by Nichi-Iko Pharmaceutical Co., Ltd., and Ono Pharmaceutical, Japan, with a three times daily dose recommendation of 100 mg to 300 mg [30, 31] . In a clinical trial investigating camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease, 95 patients received 200 mg camostat mesilate three times daily for 2 weeks and showed only mild, but no severe adverse effects [28] , indicating that camostat mesilate is a well-tolerated drug.

Nafamostat mesilate (Buipel TM ), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (FUT-175), (CAS number: 81525-10-2), is a clinical proven and synthetic serine protease inhibitor approved in Japan for the treatment of acute pancreatitis, disseminated intravascular coagulation and for anticoagulation in extracorporeal circulation [32] [33] [34] . In a screening approach of about 1,100 drugs approved by the FDA, nafamostat mesilate has been identified to inhibit MERS-CoV S protein-J o u r n a l P r e -p r o o f mediated viral membrane fusion with TMPRSS2-expressing lung Calu-3 host cells by inhibiting TMPRSS2 protease activity [35] . Since the S proteins of MERS-Cov and SARS-CoV-2 share considerable amino acid sequence homology [1, 9] , nafamostat mesilate may also inhibit cell entry of SARS-CoV-2. In cell culture experiments with simian Vero E6 cells infected with SARS-CoV-2, nafamostat mesilate was shown to be inhibitive against SARS-CoV-2 infection at EC50 of 22.50 µM [36] , suggesting that nafamostat mesilate is able to prevent SARS-CoV-2 infection. In a multicenter, randomized, open-label, phase 2 trial in 19 patients with severe acute pancreatitis, nafamostat mesilate was administered intravenously at a daily dose of 240 mg for 5 days without severe adverse effects [34] .

SARS-CoV and related coronaviruses directly interact via their S proteins with angiotensin-converting enzyme 2 (ACE2), a host cell exopeptidase and metallocarboxypeptidase that catalyses the conversion of angiotensin I to the nonapeptide angiotensin and the conversion of angiotensin II to angiotensin 1-7, to initiate S protein-mediated cell entry [37] [38] [39] . It was demonstrated recently that also SARS-CoV-2 uses ACE2 as a receptor for S protein-driven host cell entry [8, 9] . Therefore, ACE2 constitute a molecular target to inhibit cell entry of SARS-CoV-2. Unfortunately, ACE inhibitors as standard drugs for the treatment of hypertension and chronic heart failure fail to inhibit ACE2 [40] , but a number of other drugs and compounds have been shown to inhibit ACE2.

Chloroquine phosphate (Resochin TM ) and its derivative hydroxychloroquine (Quensyl TM , Plaquenil TM , Hydroquin TM , Dolquine TM , Quinoric TM ) have been used for decades for the prophylaxis and treatment of malaria and for the treatment of chronic Q fever and various autoimmune diseases [41] , and have recently been demonstrated as potential broad-spectrum antiviral drugs [42, 43] . Chloroquine phosphate inhibits terminal phosphorylation of ACE2, and hydroxychloroquine elevates the pH in endosomes which are involved in virus cell entry [44, 45] , both mechanisms constitute relevant antiviral mechanisms of chloroquine and hydroxychloroquine. In vivo, hydroxychloroquine is metabolized into chloroquine. Chloroquine phosphate has previously been shown to inhibit SARS-CoV infection and spread in vitro [44, 46] , and results from very recent studies reveal that chloroquine phosphate and, more effectively, hydroxychloroquine also inhibit replication of SARS-CoV-2 in simian Vero cells [46, 47] . By using a physiologically-based pharmacokinetic model for chloroquine phosphate and hydroxychloroquine in human lung fluid, it was demonstrated that the concentrations of hydroxychloroquine recommended for treatment of SARS-CoV-2 infection comprise an oral loading dose of 400 mg twice daily at day 1, followed by an oral maintenance dose of 200 mg twice daily for 4 J o u r n a l P r e -p r o o f days [47] . These results were deduced from in vitro data obtained from SARS-CoV-2-infected Vero cells treated with hydroxychloroquine [47] . A recent pilot trial conducted in more than 10 hospitals in Wuhan, Jingzhou, Guangzhou, Bejing, Shanghai, Chongqing and Ningbo, China, with more than 100 patients with COVID-19 disease demonstrated that treatment with chloroquine phosphate is superior to control treatment in inhibiting the exacerbation of pneumonia, improving lung imaging findings, promoting laboratory virus-negative conversion, and shortening the course of COVID-19 disease [48] .

Chloroquine phosphate should be administered as an oral daily dose of 250 mg until clinical convalescence [49] . Thus, in view of these results and the urgent clinical demand regarding SARS-CoV-2/COVID-19 pandemia, chloroquine phosphate should be recommended to treat COVID-19 associated pneumonia in larger populations [48] . A recent open-label non-randomized clinical trial conducted in March 2020 in France with 20 COVID-19 patients treated with daily 600 mg hydroxychloroquine for 6 days demonstrated at day 6 a negative viral load (negative nasopharyngeal PCR) in 57% of the hydroxychloroquine-treated patients, as compared to negative viral load in 12.5% of untreated COVID-19 patients (control group, n=16) [50] . In a randomized clinical trial conducted in February 2020 in Wuhan, China, sixty two COVID-19 patients were randomized to receive either daily 400 mg hydroxychloroquine for 5 days (n=31) or no pharmacological treatment (n=31) [51] .

Improvement and absorption of pneumonia as analyzed in chest CT at day 6 was observed in 80.6% of the hydroxychloroquine-treated patients vs. 54.8% in the untreated patients [51] . The results from these small studies therefore strongly suggest that hydroxychloroquine has therapeutic efficacy in

Thus, a considerable number of clinical trials investigating therapeutic efficacy of chloroquine phosphate and hydroxychloroquine in patients with SARS-CoV-2 infection and COVID-19 disease have been initiated in China, Great Britain, Spain and Thailand [52] [53] [54] [55] [56] .

The triple combination of cepharanthine (an anti-inflammatory alkaloid from Stephania cepharantha 

GX_P2V/2017/Guangxi (GX_P2V), whose S protein shares 92.2% amino acid identity with that of SARS-CoV-2 [60] . Further, it was demonstrated that GX_P2V also uses ACE2 as the receptor for viral cell entry [60] . Two libraries of 2,406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V, and only the combination of cepharanthine, selamectin and mefloquine hydrochloride was identified as candidate drug combination against SARS-

J o u r n a l P r e -p r o o f

Shortly after the identification of the angiotensin-converting enzyme 2 (ACE2), a metallocarboxypeptidase that mediates various cardiovascular and renal functions, peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [61] .

The most potent inhibitor, termed DX600, with the amino acid sequence of Ac- Vero-E 6 cells, and inhibits infection of engineered human capillary organoids and kidney organoids by SARS-CoV-2 isolated from a nasopharyngeal sample of a patient with confirmed COVID-19 disease [74] , suggesting that hrsACE2 can block host cell entry of SARS-CoV-2 and early stages of SARS-CoV-2 infections.

Remdesivir (GS-5734), (CAS number: 1809249-37-3), is a novel small-molecule adenine nucleotide analogue antiviral drug that has shown efficacy against Ebola virus in rhesus monkeys. Once-daily intravenous administration of 10 mg kg(-1) remdesivir for 12 days resulted in profound suppression of Ebola virus replication and protected 100% of Ebola virus-infected animals against lethal disease [75] .

Remdesivir displays antiviral activity against other single stranded RNA viruses, including filoviruses, pneumoviruses, paramyxoviruses, and the coronaviruses MERS-CoV and SARS-CoV [76] [77] [78] .

Remdesivir is a prodrug that is metabolized into its active form GS-441524, an adenine nucleotide analogue that interferes with the activity of viral RNA polymerase and that promotes evasion of J o u r n a l P r e -p r o o f proofreading by viral exoribonuclease, leading to inhibition of viral RNA synthesis [78] . Remdesivir acts early in infection, and decreases viral RNA levels in a dose-dependent manner that parallels impairment of viral load in vitro [78] . These and related mechanisms of action of remdesivir have been demonstrated in vitro for SARS-CoV [78] , Ebola virus [79] and MERS-CoV [80] . A recent study demonstrates in cell culture experiments with simian Vero E6 cells infected with SARS-CoV-2 that remdesivir is inhibitive against SARS-CoV-2 infection at EC90 of 1.76 µM, a concentration achieved in vivo in nonhuman primate models [36] . It was further shown that remdesivir efficiently inhibited SARS-CoV-2 infection of human liver cancer Huh-7 cells, which are sensitive to SARS-CoV-2 infection [36] . [87, 88] , in the USA [89] , and in France [90, 91] .

Lopinavir (ABT-378) is a highly potent inhibitor of the human immunodeficiency virus (HIV) protease essential for intracellular HIV assembly that was developed in 1998 to circumvent HIV resistance towards the protease inhibitor ritonavir (ABT-538), caused by mutation of valine at position 82 (Val 82) in the active site of HIV protease in response to ritonavir therapy [92] . Because the metabolism of lopinavir is strongly inhibited by ritonavir, concomitant oral administration of lopinavir and ritonavir exceeded the in vitro antiviral EC50 of lopinavir by >50-fold after 8 h in rat, dog, and monkey plasma J o u r n a l P r e -p r o o f [92] . Coadministration of 400 mg lopinavir with 50 mg ritonavir enhanced in healthy human volunteers the area under the concentration curve of lopinavir in plasma by 77-fold over that observed after dosing with lopinavir alone, and mean concentrations of lopinavir exceeded the EC50 for >24 h [92] .

Therefore, the combination of lopinavir and ritonavir (Kaletra TM ) has been established as an effective oral drug for the treatment of HIV-infected individuals when used in combination with other antiretroviral agents [93, 94] .An initial study in 2003 demonstrated that lopinavir at 4 µg/ml inhibited the cytopathic effect in a plaque reduction assay with fetal rhesus kidney-4-cells infected with SARS-CoV (HKU-39849 isolate) [95] . In this study, newly diagnosed SARS patients infected with SARS-CoV were treated with the combination of lopinavir (400 mg)/ritonavir (100 mg) orally every 12 hours for 14 days.

At day 21, SARS patients treated with lopinavir/ritonavir had a milder disease course in terms of diarrhea, recurrence of fever, worsening of chest radiographs and reduction of viral load, compared to a historical control group [95] . In a nonhuman primate model of common marmosets infected with MERS-CoV, lopinavir/ritonavir-treated animals displayed an improved clinical outcome compared to untreated animals, with improved weight loss, lung imaging and pathological findings, and lower mean viral loads in necropsied lung and extrapulmonary tissues [96] . In response to these findings, an ongoing randomized control trial (MIRACLE Trial) was initiated to determine the therapeutic efficacy of lopinavir/ritonavir combined with interferon β-1b in patients infected with MERS-CoV [97] . In a recent [98] . Treatment with lopinavir/ritonavir was not associated with a difference from standard care in the time to clinical improvement, and mortality at 28 days was similar in the lopinavir/ritonavir group and the standard-care group [98] .

Moreover, treatment with lopinavir/ritonavir treatment did not reduce viral RNA loads or duration of viral RNA detectability as compared with standard supportive care. SARS-CoV-2 RNA was still detected in 40.7% of the patients in the lopinavir/ritonavir group at the end of the trial at day 28 [98] .

However, the numbers of lopinavir/ritonavir recipients who had serious complications (acute kidney injury and secondary infections) or requiring noninvasive or invasive mechanical ventilation for respiratory failure were fewer than in those not receiving lopinavir/ritonavir treatment [98] . These results and observations require additional studies to determine whether treatment with lopinavir/ritonavir given at a certain disease stage can reduce some complications in Covid-19 patients [98] .

China [99] [100] [101] [102] , Hong Kong [103] , Republic of Korea [104] , and in Europe (DisCoVeRy Trial), investigating remdesivir, lopinavir/ritonavir, and lopinavir/ritonavir plus interferon β-1a [91] .

Umifenovir [112] . In view of these promising clinical results, clinical trials with umifenovir alone or in combination with lopinavir/ritonavir, chloroquine phosphate or carrimycin have been recently initiated in China [113] [114] [115] [116] . the human interleukin-6 receptor, tocilizumab [123] or favipiravir in combination with chloroquine phosphate and the viral neuramidase inhibitor oseltamivir [124] have been initiated recently in China.

3Clpro (also termed Mpro) constitutes the main protease of beta coronaviruses that is essential for processing of polyproteins translated from the viral RNA [125] . Recently, the X-ray structures of the unligated SARS-CoV-2 3Clpro and its complex with α-ketoamides designed as specific inhibitors of 3Clpro were reported [126] . Two pyridine-containing α-ketoamides, designated 13a and 13b, displayed favorable pharmacokinetic properties in mice and were detected at sufficient concentrations in lung tissue and broncheo-alveolar lavage fluid within 4 hours to 24 hours after subcutaneous administration, demonstrating lung tropism of the compounds [126] . Besides subcutaneous administration, inhalation of nebulized 13b by mice resulted in high and long-lasting (24 hours) concentrations in lung tissue, without any adverse effects [126] , pointing out a role of pyridinecontaining α-ketoamides in COVID-19 therapy. In a recent study that employed combined structureassisted drug design, virtual drug screening and high-throughput screening, a mechanism-based inhibitor of Mpro, termed N3, was identified by computer-aided drug design [127] . N3, a Michael acceptor inhibitor that can inhibit the Mpros of SARS-CoV and MERS-CoV, was shown to form a covalent bond with and to be an irreversible inhibitor of SARS-CoV-2 Mpro [127] . Further, in a highthroughput screening approach for identifying inhibitors of SARS-CoV-2 Mpro, ebselen, an organoselenium compound with anti-inflammatory, anti-oxidant and cytoprotective properties, was identified [127] . In a plaque-reduction assay with simian Vero cells infected with SARS-CoV-2, N3 and ebselen displayed antiviral and cell protection efficacy at EC50 values of 16.77 µM and 4.67 µM, respectively [127] , ultimately demonstrating their antiviral potential against SARS-CoV-2.

Natural products can inhibit various steps in viral infection and replication, and many of them have broad-spectrum antiviral effects, the mechanisms of which have not been fully characterized. They also can function as immunomodulators, suppressing inflammatory reaction responsible for the major morbidity and mortality of SARS-CoV-2 infection. Phytochemicals, especially flavonoids, which are widely distributed in food plants and botanicals, have been shown to interfere with NLRP3

inflammasome signaling [128] . The respiratory distress syndrome associated with SARS coronaviruses develops in part due to viral activation of the NLRP3 inflammasome within activated macrophages and T helper-1 lymphocytes, which causes increased production of inflammatory cytokines [129] . Several flavonoids that interfere with activation of the NLRP3 inflammasome may modulate inflammatory response to SARS beta coronaviruses: luteolin [130] , myricetin [131] , apigenin J o u r n a l P r e -p r o o f [132] , quercetin [133] kaempferol [134] , baicalin [135] , and wogonoside [136] . These flavonoids have been shown to be active against a wide variety of viruses, via multiple mechanisms [137, 138] , and are available as nutraceutical supplements at a daily dose ranging from 100 mg to 500 mg. Emodin (6methyl-1,3,8-trihydroxyanthraquinone) (CAS number: 518-82-1) is an anthraquinone compound found in various Chinese herbs and is also produced by many species of fungi, including members of the genera Aspergillus, Pyrenochaeta, and Pestalotiopsis. Emodin has been shown to inhibit the interaction of SARS-CoV S protein with its receptor ACE2 in a dose-dependent manner [139] . [140] , suggesting that resveratrol may also be effective against SARS-CoV-2 infection.

The emergence of the novel beta coronavirus SARS-CoV-2 from Wuhan, Hubei province, China in December 2019 rapidly led to a pandemic involving more than 2,500,000 infected persons and more proven drugs such as camostat mesilate which prevents virus host cell entry by inhibiting TMPRSS2 [8] , and chloroquine phosphate which inhibits terminal phosphorylation of ACE2, or hydroxychloroquine which is metabolized in vivo to chloroquine [44] . For the treatment of ordinary and severe COVID-19 pneumonia, and to lower the mortality rate of COVID-19 disease, the antiviral drugs remdesivir, favipiravir, umifenovir, and lopinavir/ritonavir plus interferon β-1a should be administered, in particular after the consideration of (preliminary) results from the recent ongoing clinical trials SOLIDARITY and DisCoVeRy [141, 91] 

CN and DLM conceived the original idea and wrote large parts of the manuscript. AS and US wrote section 3 and prepared Table 1 and Figure 1 . SL finished the manuscript.

None.

We have no conflicts of interest.

Sincerely, 

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We are grateful to all of the colleagues who have given critical comments on this work.J o u r n a l P r e -p r o o f

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f