key: cord-0699810-169mcbp9
authors: Bailly, Christian; Vergoten, Gérard
title: Glycyrrhizin: An alternative drug for the treatment of COVID-19 infection and the associated respiratory syndrome?
date: 2020-06-24
journal: Pharmacol Ther
DOI: 10.1016/j.pharmthera.2020.107618
sha: 5c2fdcbe9e832c4652d1637f15c258edd06052a9
doc_id: 699810
cord_uid: 169mcbp9

Safe and efficient drugs to combat the current COVID-19 pandemic are urgently needed. In this context, we have analyzed the anti-coronavirus potential of the natural product glycyrrhizic acid (GLR), a drug used to treat liver diseases (including viral hepatitis) and specific cutaneous inflammation (such as atopic dermatitis) in some countries. The properties of GLR and its primary active metabolite glycyrrhetinic acid are presented and discussed. GLR has shown activities against different viruses, including SARS-associated Human and animal coronaviruses. GLR is a non-hemolytic saponin and a potent immuno-active anti-inflammatory agent which displays both cytoplasmic and membrane effects. At the membrane level, GLR induces cholesterol-dependent disorganization of lipid rafts which are important for the entry of coronavirus into cells. At the intracellular and circulating levels, GLR can trap the high mobility group box 1 protein and thus blocks the alarmin functions of HMGB1. We used molecular docking to characterize further and discuss both the cholesterol- and HMG box-binding functions of GLR. The membrane and cytoplasmic effects of GLR, coupled with its long-established medical use as a relatively safe drug, make GLR a good candidate to be tested against the SARS-CoV-2 coronavirus, alone and in combination with other drugs. The rational supporting combinations with (hydroxy)chloroquine and tenofovir (two drugs active against SARS-CoV-2) is also discussed. Based on this analysis, we conclude that GLR should be further considered and rapidly evaluated for the treatment of patients with COVID-19.

6 erythrodermic psoriasis (Si et al., 2014; . But one of the most frequent inflammatory skin diseases treated with GLR (at least in Japan and Korea) is atopic dermatitis (AD). Recently, it was reported that GLR inhibited the release of cytokines IL-4 and IL-13 in a murine model of AD (Lee, Bae et al., 2019) and another study showed that, through inhibition of the protein HMGB1 (High-Mobility Group protein B1), GLR can ameliorate the symptoms of AD in a mouse model with the drug injected daily intraperitoneally . By sequestering HMGB1 (see mechanism below), GLR modulates the production of inflammatory cytokines such as IL-18, to prevent contact dermatitis induced by contact allergens (Galbiati et al., 2014) . The activity of GLR against cutaneous inflammation should be kept in mind considering the cutaneous manifestations in COVID-19-positive patients (Torres et al., 2020) .

The drug is considered as having a good safety and economical profile. Its clinical use is increasing. In recent years, we noted the development of different clinical trials to investigate the benefit of GLR in multiple pathologies such as depression , Parkinson's disease (Petramfar et al., 2020) and different cancers, such as hepatocellular carcinoma and pancreatic cancer (Kwon et al., 2020) .

Over the past thirty years, the effects of GLR against a variety of human viruses have been reported (Fig. 2) . The anti-HIV1 activity of GLR is well documented (Baba and Shigeta, 1987; Wolkerstorfer et al., 2009; Michaelis et al., 2010 Michaelis et al., , 2011 Lin et al., 2003 Lin et al., , 2008 Bentz et al., 2019; Sakai-Sugino et al., 2017; Crance et al., 1990 Crance et al., , 1994 Ashfaq et al., 2011; Matsumoto et al., 2013; Baltina et al., 2019; Sun et al., 2019; Briolant et al., 2004) . GLR dose-dependently inhibits virus-cell binding and the replication be a convenient system to permit a prolonged release of the active principle (Marianecci et al., 2014) . Improvements have been proposed to further ameliorate the stability, skin permeability and capacity of these niosomes to deliver the drug, with the use of gel -forming polysaccharides (Coviello et al., 2015) GLR-containing vesicles can serve to transport and deliver GLR, but also to facilitate the transportation and delivery of co-loaded drugs. Indeed, GLR is viewed as a multifunctional carrier for a variety of hydrophobic molecules, by virtue of its amphiphilic properties (Fi. 1, Table 1 ). The Solvent Accessible Surface Area (SASA) for GLR is large, offering a significant potential to interact with both hydrophilic and hydrophobic molecules, and with multiple H-bond donor/acceptor sites (Table 1) . GL complexes or micelles with many drugs have been reported, for examples with anticancer drugs such as paclitaxel, podophyllotoxin and camptothecin (Yang et al., 2015; Wang et al., 2016; Cai et al., 2019) . Not only GLR can enhance the solubility of poorly soluble drugs but it can increase also their passive diffusion through cell membranes. The capacity of GLR to interact with phospholipids-based membrane models and to cause transient pore formation has been elegantly modeled. GLR was shown to embark a few water molecules within the membrane and to make the membrane thinner locally (Selyutina et al., 2016) but apparently it does not simply induce transient local pores in the membrane; the mechanism is more complex and seems to involve a drug-induced decrease in cholesterol within the lipid bilayer (Shelepova et al., 2018) . Therefore, the drug can be used as nonselective delivery vector for a variety of drugs (Su et al., 2017; Selyutina et al., 2019) . A nice example has been reported recently with the GLR-mediated enhanced delivery of the anti-helminthic drug praziquantel, showing that the self-association of GLR molecules plays an important role in The antiviral activity of GLR relies on both cytoplasmic and membrane effects. The drug inhibits hepatitis A virus penetration of the plasma membrane (Crance et al., 1994) and suppresses the secretion of viral antigens, such as the hepatitis B surface antigen. GLR inhibits the sialylation of the antigen and intracellular transport to induce its accumulation in cytoplasmic vacuoles in the Golgi apparatus (Takahara et al., 1994; Sato et al., 1996) . The saponin reduces the movement of molecules within the membrane and thus impedes the formation of fusion pores necessary for the entry of viruses. In fact, the drug lowers the membrane fluidity and thus suppresses infection by different viruses, such as HIV-1, HTLV-1, influenza A virus and vesicular stomatitis virus, but not by poliovirus (Harada, 2005) . It is very important to underline that GLR is a non-lytic saponin. In contrast to other saponins like digitonin, GLR shows weak permeabilizing effects and thus has a very low hemolytic profile (Gilabert-Oriol et al., 2013) . It does not destroy the whole membrane integrity and induces very little leakage from liposomes compared to other saponins (Malabed et al., 2017) . In the context of the Herpes simplex virus (HSV), it has been shown that GLR reduced adhesion force and stress between cerebral capillary vessel endothelial cells and polymorphonuclear leukocytes, thereby attenuating the inflammatory responses to HSV (Huang et al., 2012) . In the context of HIV-1, the drug has been shown to bind to the conserved core sequence of V3 loop in the su rface glycoprotein gp120 with a sub-micromolar affinity (Li, Zhao et al., 2015) .

At least a part of the membrane effects of GLR can be linked to an interaction with cholesterol (Fig. 5 ) and the reduction in cholesterol domain in membrane. The interaction of saponins with cholesterol is a main element of their mechanism of action, and their capacity to induce membrane disorganization and in some cases disruption. For example, cholesterol recognition by the anticancer saponin OSW-1 is essential for the subsequent membrane permeabilization (Malabed et al., 2017) .

Cholesterol is known as a very important modifier of the dynamics and structural properties of lipid membranes. Compared to other saponins like digitonin, OSW-1 and Quillaja saponin, GLR shows very weak membrane permeabilizing effects on membranes from lysosomes or red blood cells (Gilabert -Oriol et al., 2013) . Nevertheless, GLR interacts with cholesterol and this interaction modifies the membrane permeability to ions and small molecules (Selyutina et al., 2016). A study using artificial J o u r n a l P r e -p r o o f Journal Pre-proof membranes prepared at 1.5:1 cholesterol-to-phospholipid mole ratio has shown that GLR reduced cholesterol domain formation by 54. 9% (Mason et al., 2016) . Not only GLR decreases the level of cholesterol of lipid rafts but also inhibits the translocation of TLR-4 to lipid the rafts (Fu et al., 2014a (Fu et al., , 2014b . This mode of action is distinct from that of conventional cholesterol -lowering statins.

Consequently, combinations of GLR and the statins atorvastatin and simvastatin ( leading to combo named atorvaglyzin and simvaglyzin) have been proposed to reinforce the cholesterol-lowering effect Ragino et al., 2008; Stakhneva et al., 2013) .

We have used molecular modeling to compare the interaction with cholesterol of GLR and platycodin D, a bidesmosic saponin known to exhibit cholesterol-lowering effects (Zhao et al., 2006) . As shown in Fig. 5 , the modeling analysis shows that GLR can form stable complexes with cholesterol, mainly via hydrophobic interaction with the sapogenin moiety of GLR. The potential energy of interaction is lower for the complex of GLR with cholesterol (ΔE = -19.5 kcal/mol) compared to that measured for the platycodin D-cholesterol complex (ΔE = -24.3 kcal/mol) and this is in agreement with the very weak hemolytic capacity of GLR. However, the interaction is significant, stronger than with GA and sufficient to induce plasma membrane perturbations. It has been shown that the drug can move within the membrane, with exchange of GLR molecules from solution to the hydrophobic interior of the lipid bilayer (Selyutina et al., 2016) . Normally, cholesterol makes the bilayer more rigid but the interaction with GLR reduces the rigidification of the membrane, and thus possibly increases membrane permeability. This interaction contributes to the regulation of the size of lipid raft domains observed in the presence of GLR (Sakamoto et al., 2013) . GA was found to interact even more strongly with a raft monolayer model than GLR (Sakamoto et al., 2013 (Sakamoto et al., , 2015 . The disorganization of cholesterol-containing lipid rafts by GLR is an important element to consider in the GLR may further inhibit the entry of the virus, via a membrane destabilizing effect. Recently, inhibition of viral lipid-dependent attachment of the virus envelope of the SARS-COV-2 to host cell plasma membrane in vitro, with natural products such as cyclodextrin and sterols, has been proposed as a strategy for reducing SARS-COV-2 infectivity (Baglivo et al., 2020) . Our proposal is totally in-line with this view.

Lipid rafts play an important role in the life cycle of SARS-coronavirus. The depletion of cholesterol (with methyl-β-cyclodextrin) inhibits the production of coronavirus particles released from infected cells (Li et al., 2007) . Different studies suggest that lipid rafts serve as an entry port for SARS-CoV (Choi et al., 2005; Lu et al., 2008; Wang et al., 2008) . Similarly, the depletion of plasma membrane cholesterol with methyl-β-cyclodextrin or a statin drug considerably reduces the infection by the coronavirus infectious bronchitis virus (IBV) in vitro, presumably impairing the attachment of the virus to the cell surface (Guo, Huang et al., 2017) . PEDV and human metapneumovirus (hMPV) can also enter cells through lipid raft-mediated endocytosis (Chen, He et al., 2019; Wei et al., 2020) . In fact, lipid rafts play a key role in the endocytosis process of many viruses and, as a corollary, interfering with lipid raft organization is a mechanism to control virus infection. Moreover, this is probably the reason why the impairment of cellular cholesterol metabolism and lipid raft functionality have been evoked as a co-morbidity factor in some viral diseases, such as HIV infection (Sviridov et al., 2019) .

The cholesterol lowering effect of GLR can explain its antiviral effect against the porcine virus PRRSV, at least partially. Indeed, it has been demonstrated that cell membrane cholesterol is required for porcine nidovirus entry into cells and a drug able to induce cholesterol depletion dose-dependently suppressed the replication of the nidoviruses PRRSV and PEDV ( Sun et al., 2011; Jeon and Lee, 2017) .

Similarly, GLR reduces membrane cholesterol and, as discussed above, it is active against those two porcine nidoviruses (Duan et al., 2015; Tong et al., 2020) . Other viruses also partly depend on membrane cholesterol for entry into cells. This is the case of another coronavirus, the highly neurovirulent porcine hemagglutinating encephalomyelitis virus (PHEV). Here also, a perturbation of (Song et al., 2017 Zhang et al., 2019) and other viruses . It would be interesting to evaluate the antiviral activity of a combination of GLR and 25HC.

The pharmacological action of GLR is not limited to the plasma membrane. The drug displays a marked anti-inflammatory activity and modulates the immune system, via an action on multiple pathways such as MAPK and Toll-like receptors signaling pathways (Zhao et al., 2016) . The signaling activities of GLR likely derive from its binding to high-mobility group proteins B1 (HMGB1 and possibly HMGB2), thereby inhibiting the DNA-binding and phosphorylation of the protein (Sakamoto et al., 2001) . Structural studies have revealed that GLR can physically bind to HMGB1 and a modeling study suggested that the drug can also bind to the nuclear HMGB1-DNA complex (Yamaguchi et al., 2012) . GLR shows a modest affinity for HMGB1 (K d 150 M, an abundant protein) via a binding on the shallow concave surface formed by the two arms of the HMGB protein, as represented in Fig. 6 . GLR derivatives bearing amino acid residues on the carbohydrate chain have been shown also to bind to HMGB1 and block its activity, but they were not significantly more potent than GLR itself (Du et al., 2013) . GLR does not distort the protein structure upon binding but forms stable kissing complexes with the HMG protein. The stability of the drug-protein complex is mainly assured by favorable hydrophobic and electrostatic interactions between the triterpene scaffold of GLR and several key amino acid residues of the proteins (such as Y15, F37, A16 and V19) (Mollica et al., 2007) .

We have used molecular modeling to compare the overall binding of GLR and GA to HMGB1, on the basis of the crystallographic structure of the HMG box motif in the B domain of HMG1 (PDB code: 1HME) (Weir et al., 1993) . It is an L-shaped small protein (77 amino acids) with three alpha-helices J o u r n a l P r e -p r o o f Journal Pre-proof defining two arms (Fig. 6 ). Our molecular docking analysis indicates that GLR can form much more stable complexes with this HMG box domain than GA. The potential energy of interaction is 2.4 times better (more negative) with GLR than with GA. The Gibbs free energy of hydration (free enthalpy of hydration) is also much more favorable for GLR compared to GA ( Table 2) . The difference illustrates the major contribution of the carbohydrate moiety of GLR to the HMG interaction. A more detailed analysis of the binding shows the important contribution of both H-bonds and hydrophobic drugprotein interactions (Fig. 7) . GLR, with a much higher number of H-bond donor/acceptor atoms (Table 1) , is better adapted to fit into the concave side of the HMG box structure. The disaccharide motif of GLR sits on the L-structure, interacting with two alpha-helices. The contact map in Fig. 8 illustrates the higher number of interactions observed with the GLR-HMG structure, compared to the GA-HMG one, with in particular the major molecular contacts provided by the glycosyl moiety. In both cases, the aglycone hydrophobic core interacts about similarly with the protein (implicating residues Leu-16, Leu-63 and Lys-8, Lys-66 for both ligands) but in the case of GLR, the two glucuronic acid residues provide extra attachment sites to the protein (in particular with Lys-24, Lys-59 and Glu-20 residues). There is no doubt that GLR is a very well adapted HMG ligand. Our docking analysis is coherent with the published NMR structure of GLR bound to HMGB1 (Mollica et al., 2007) and highlights the contribution of the glycoside moiety of GLR to the protein interaction.

The binding of GLR to HMGB1 is not extremely tight but sufficiently strong to perturb the various physiological activities of the protein, notably its interaction with other proteins such as the receptor the protein and downregulates the expression of inflammatory cytokines. Consequently, GLR interferes with several HMGB1-mediated pathological conditions, notably a variety of neurological disorders including traumatic brain injury, epileptic seizures, multiple sclerosis, and Alzheimer and Parkinson diseases (Paudel et al., 2020) . In fact, the successfully inhibition of HMGB1 by GLR translates into a variety of effects depending on the cell system and its environment. By modulating atherosclerosis , (vii) attenuates chronic inflammatory pain .

Given the central roles of HMGB1 both as a transcription regulator in the nucleus and as a circulating damage-associated molecular pattern (DAMP), the targeting of HMGB1 is considered a valid strategy in many diseases: cancer, autoimmune diseases, inflammatory heart diseases, neurological diseases, trauma (Musumeci et al., 2014; Venereau et al., 2016; Ugrinova and Pasheva, 2017) . Indeed, PEDV infection induces HMGB1 transcription and its subsequent release. GLR can be used to counteract this effect (Huan et al., 2016 (Huan et al., , 2017 . Therefore, we could expect a similar advantage using GLR to alleviate the effects of COVID-19. A beneficial effect on the respiratory distress syndrome could be expected also because HMGB1 has a significant role in the development and progression of acute respiratory distress syndrome (ARDS), through the regulation of cytokines such as IL-33. As mentioned above, inhibition of HMGB1 release by GLR is associated with a decrease of the HMGB1induced up-regulation of IL-33 expression in a mouse model of LPS-induced lung inflammation/injury (Fu et al., 2016) .

A few other HMGB1 inhibitors have revealed interesting antiviral activities such as acteoside (a phenylpropanoid glycoside from Kuding Tea) which blocks HMGB1 release (Seo et al., 2013) and displays antiviral effects, presumably via its capacity to induce IFN-γ production (Song et al., 2016) .

Similarly, the non-narcotic alkaloid papaverine has been identified recently as a direct inhibitor of the HMGB1/RAGE interaction and a suppressor of the HMGB1-mediated production of pro-inflammatory cytokines (Tamada et al., 2019) and it is also active against influenza viruses and paramyxoviruses (Aggarwal et al., 2020) . But in the present context, our favorite example is chloroquine which has been shown previously to inhibit HMGB1 release in different cell types (macrophages, monocytes, and endothelial cells) in a mouse model of endotoxemia or sepsis (Yang et al., 2013) . Chloroquine downregulates the expression of HMGB1 and reduces the level of serum HMGB1 in a model of chemical-induced acute liver injury (Dai et al., 2018) .

In addition to HMG proteins, GLR has also been shown to bind to other proteins such as serum albumin GLR is a multi-target compound and novel potential targets are regularly disclosed. For example, in silico molecular docking has predicted that GLR binds to the Nrf2 (Nuclear factor-E2 related factor 2) peptide binding site on Keap-1 (Kelch like ECH-associated protein 1) and thus can possibly function as a Nrf2 stimulator (Kamble et al., 2017) . GLR exerts Nrf2-dependent activities (Mou et al., 2019; but thus far a direct, physical interaction with Keap-1 has not been evidenced. Another recent study revealed that GLR (as well as its analogues GA and carbenoxolone (18β-glycyrrhetinic acid 3β-O-hemisuccinate) are selective and competitive inhibitors of kynurenine aminotransferase 2 (KAT2), the enzyme which catalyzes the conversion of kynurenine to kynurenic acid (Yoshida et al., 2019) . This inhibitory effect may contribute to the anti-Parkinson activity of GLR (Wang, Lian et al., 2018) . GLR can also bind very weakly to nucleic acids, both DNA and RNA (Nafisi et al., 2012a (Nafisi et al., , 2012b .

GLR can be easily combined with many types of drugs, either to promote solubilization an d bioavailability of the co-transported product as discussed above, or to complement its mechanism of action leading to synergistic effects in some cases . We will not review here all combinations previously reported, but only focus on two combinations useful in the context of coronavirus infections, with the antiviral drugs chloroquine and tenofovir.

As mentioned above, chloroquine downregulates HMGB1 expression and reduces HMGB1 serum level in a model of acute liver injury (Dai et al., 2018) . Given the therapeutic potential of chloroquine J o u r n a l P r e -p r o o f against SARS- CoV-2 (Fantini et al., 2020; Cortegiani et al., 2020) , it could make sense to consider both GLR and chloroquine for the treatment of the current pandemia. Human viruses exploit the autophagy pathway to help viral propagation and escape immune response (Abdoli et al., 2018) . In particular, coronavirus infection has been demonstrated to induce autophagy (Maier and Britton, 2012; Cong et al., 2017) , notably through the membrane-associated papain-like protease PLP2 (PLP2-TM) acting as an autophagy-inducing protein, via a direct interaction with the key autophagy regulators LC3 and Beclin1 (Chen et al., 2014) . For example, the porcine viruses PEDV and PHEV both induce autophagy to benefit their replication Ding et al., 2017) . As another example, the mycotoxin ochratoxin A induces autophagy to promote porcine circovirus type 2 (PCV2) replication, whereas inhibitors of autophagy such as 3-methylademine and chloroquine significantly attenuate PCV2 replication (Qian et al., 2017) . Consequently, interfering with the autophagy process could perturb coronavirus infection. Autophagy inhibition can be induced by chloroquine (or hydroxychloroquine), leading to inhibition of virus replication. For example, chloroquine -induced inhibition of autophagy suppresses the replication of the hepatitis C virus (Mizui et al., 2010) . GLR has been shown also to modulate autophagy in different cell systems, although both inhibition (Jeon et al., 2019) and activation Umar et al., 2019; Qu et al., 2019) of autophagy have been reported. However, it has been shown that HMGB1 translocation and release induce autophagy in lung macrophages and this process can be attenuated via the blockade of HMGB1 with GLR (Le et al., 2020) . Moreover, the use of an autophagy inhibitor enhances the anticancer activity of GA (Shen et al., 2017; . Thus, we believe that the combination of GLR and (hydroxy)chloroquine could be useful to inhibit coronavirus replication. Chloroquine is hydrophilic compound, relatively well absorbed orally and with good bioavailability. It exhibits linear absorption and clearance (Zhao et al., 2014; Rainsford et al., 2015) . However, the entrapment of chloroquine into multilamellar vesicles has been shown to enhance drug delivery (Fotoran et al., 2019) .

Tenofovir (Viread®, TDF, Fig. 9 ) is a nucleotide analog reverse-transcriptase inhibitor widely used as the first-line therapy to inhibit hepatitis B virus replication. It is also a recommended first-line drug for HIV treatment. The combination of tenofovir and GLR has been investigated in a pilot clinical J o u r n a l P r e -p r o o f Journal Pre-proof study with a cohort of patients with severe acute exacerbation of chronic hepatitis B and showed that the early introduction of GLR can be safe and beneficial for those patients difficult to manage (Hung et al., 2017) . The molecular basis of this benefit is not known but a hypothesis can be advanced because it was shown GA increases distribution in the cytoplasm and nucleus of liver cells of the related antiviral drug entecavir . On the other hand, a recent molecular modeling study has suggested that tenofovir binds tightly to the RNA-dependent RNA polymerase of the SARS-CoV-2 virus and thus could be a useful antiviral agent (Elfiky et al., 2020) . A clinical trial including tenofovir and other antiviral agents is on-going in China (Zhai et al., 2020; Zhu et al., 2020) .

The convergence of these pieces of information led us to consider also the combination of GLR and tenofovir as a potential anti-coronavirus approach.

The membrane-perturbating effects of GLR have been also observed with other structurally related saponins such as platycodin D (Fig. 9 ) and -escin (Bailly and Vergoten, 2020). These two natural products interact with cholesterol thereby modulating the organization of lipid rafts in membranes.

This activity likely contributes to their biological activity, in particular to the antiviral effects reported with both products (Kim et al., 2017) and their adjuvant properties to increase immunogenicity of proteins and vaccines (Xie et al., 2009; . Escin has been recently proposed as add-on therapy in acute lung injury related to COVID-19 infection (Gallelli et al., 2020) . Platycodin D has been characterized as a potent inhibitor of PPRSV infection in vitro, directly inhibiting the virus replication and reducing the production of different virus-induced cytokines . A recent modeling study indicated that this saponin derivative presents a high binding affinity to papain-like protease of SARS-CoV-2 . We must also mention the saikosaponins, structurally close al., 2015) and saikosaponins A and D showed interesting activities agai nst porcine circovirus 2 (PCV2) , as well as PPRSV infections, enhancing the immune responses and decreasing the incidence and severity of PRRSV-induced immunopathological damages in vivo (Hu et al., 2020) . We consider this information as indirect element to support the potential use of a saponin like GLR for the treatment of coronavirus infections. GLR is a relatively safe product, well tolerated, inducing limited undesirable effects and used for a long time in medicine, whereas saponins like platycodin D and saikosaponin A are only laboratory tools, not drugs. Moreover, the HMGB-1 binding activity is specific to GLR, therefore making this natural product a unique drug to be further considered.

GLR is a well-established oriental phytomedicine used for a long time to treat hepatic disorders. The production of the drug is securitized, and drug products of good quality can be found easily. Our scientific analysis highlighted the following main characteristics (Fig. 10) :

-GLR is considered a safe natural product, with a long-established use in Human as a hepatoprotecting agent. Adverse effects are relatively rare and manageable (Nazari et al., 2017) .

-GLR is used in Human for the treatment of chronic hepatitis (and other liver diseases) and has shown marked activity against some coronaviruses such as the porcine virus PEDV. It is also used to treat cutaneous inflammation.

-The anti-inflammatory activity of GLR could be useful to alleviate the respiratory distress syndrome associated to the viral infection.

-There is no obstacle to the combination of GLR with a variety of drugs and its combination with antiviral drugs such as chloroquine or tenofovir could be beneficial. GLR, which can be administered iv and orally, can serve as a codrug to increase the bioavailability of poorly soluble products. It makes sense also to combine GLR with other antiviral agents having different mechanisms to reinforce the antiviral response (combination of cytoplasmic and membrane effects).

-GLR induces cholesterol-dependent disorganization of lipid rafts which are important for the entry of coronavirus into cells. These membrane effects, also observed with other amphiphilic J o u r n a l P r e -p r o o f Journal Pre-proof saponins, likely play an important role in the antiviral activity. GLR is considered a non-hemolytic saponin.

-GLR is an efficient binder of HMGB1. The glycoside moiety of GLR plays a major role in the interaction with the HMG box protein. Considering the multiple functions of HMGB1 in viral infections and replication, the trapping of HMGB1 by GLR could contribute significantly to a diminution of the virus-induced excessive inflammatory response and the viral replication.

For all these reasons, we believe that GLR should be rapidly tested as an anti -SARS-CoV-2 agent, alone and in combination with other drugs (notably CQ/HCQ and tenofovir) to combat the current COVID-19 pandemic.

While this manuscript was reviewed, three groups also proposed the use of GLR, alone or in combination with other drugs, to treat coronavirus infections Luo et al., 2020; Zhao et al., 2020) . Notably, one study underlined the capacity of GLR to bind to the angiotensin converting enzyme 2 (ACE2) which represents a SARS-CoV-2 receptor. Therefore, the targeting of ACE2 could be very useful to inhibit the virus from diffusing out of infected cells and to enter new cells (Luo et al., 2020) . Even more recently, a study reported interesting clinical data for a patient with severe COVID-19 who recovered upon treatment with diammonium glycyrrhizinate (Ding et al., 2020) . These data are encouraging and support our proposal to clinically evaluate GLR as a drug to treat SARS-CoV-2 infections, considering notably that the drug and derivatives have shown activity against other SARS-coronavirus (However et al., 2005) . The risk of hypertension induced by GLR, due Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People's Republic of China) who kindly provided the illustrations of GLR nanofibrils presented in Fig. 4 . Table 2 . Fig. 7 . Views of the GLR-and GA-HMG complexes with a focus on the H-bond and hydrophobic surfaces exposed implicated in the protein complex formation. Note the specific contribution of the carbohydrate moiety of GLR to the protein interaction. In both cases, the specific color code is indicated. 10 . Summary of the main characteristics and properties of GLR, supporting its potential activity against the SARS-CoV-2 coronavirus. The drug is safe, used for a long time to treat viral hepatitis (iv and oral formulations available). GLR binds to cholesterol, thereby affecting the organization of lipid rafts that are essential for the entry of the virus into cells. GLR forms stable complexes with HMGB1 protein, thereby blocking the propagation of the danger signals. GLR displays antiviral activities against multiple viruses, including hepatitis virus A-B-C and some coronavirus. according to published procedures (Vergoten et al., 2003; Lagant et al., 2004) . 

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Investigation of interfacial behavior of glycyrrhizin with a lipid raft model via a Langmuir monolayer study

Glycyrrhetinic acid alleviates hepatic inflammation injury in viral hepatitis disease via a HMGB1-TLR4 signaling pathway

Erythrodermic psoriasis with bullous pemphigoid: combination treatment with methotrexate and compound glycyrrhizin

Treatment with HMGB1 inhibitors diminishes CTL-induced liver disease in HBV transgenic mice

The antiviral effects of acteoside and the underlying IFN -γ-inducing action

25-Hydroxycholesterol provides antiviral protection against highly pathogenic porcine reproductive and respiratory syndrome virus in swine

Cholesterol 25-hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection

Antiviral and immune stimulant activities of glycyrrhizin against duck hepatitis virus

Effects of simvaglyzin and atorvaglyzin on the expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase in rat liver

Glycyrrhizic acid: A promising carrier material for anticancer therapy

Advances in the use of chloroquine and hydroxychloroquine for the treatment of COVID-19

Glycyrrhizin amel iorates inflammatory pain by inhibiting microglial activation-mediated inflammatory response via blockage of the HMGB1-TLR4-NF-kB pathway

Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells

Research Progress of Glycyrrhizic Acid on

A highly sensitive LC-MS/MS method for simultaneous determination of glycyrrhizin and its active metabolite glycyrrhetinic acid: Application to a human pharmacokinetic study after oral administration

Co-morbidities of HIV infection: role of Nef-induced impairment of cholesterol metabolism and lipid raft functionality. products interaction suppresses high mobility group box 1-mediated inflammatory responses

Licorice root extract and magnesium isoglycyrrhizinate protect against triptolide-induced hepatotoxicity via up-regulation of the Nrf2 pathway

Efficacy and safety of addition of minor bloodletting (petit phlebotomy) in hepatitis C virus-infected patients receiving regular glycyrrhizin injections

Clinical spectrum of acute sporadic hepatitis E and possible benefit of glycyrrhizin therapy

High-Mobility Group Box 1 Protein Signaling in Painful Diabetic Neuropathy

Glycyrrhizic-Acid-Based Carbon Dots with High Antiviral Activity by Multisite Inhibition Mechanisms

Managing Cutaneous Immune-Mediated Diseases During the COVID-19

HMGB1 Protein: A Therapeutic Target Inside and Outside the Cell

Glycyrrhizic Acid Prevents Oxidative Stress Mediated DNA Damage Response through Modulation of Autophagy in Ultraviolet-B-Irradiated Human Primary Dermal Fibroblasts

HMGB1 as biomarker and drug target

The SPASIBA force field as an essential tool for studying the structure and dynamics of saccharides

Comparative study of selective in vitro and in silico BACE1 inhibitory potential of glycyrrhizin together with its metabolites, 18α-and 18β-glycyrrhetinic acid, isolated from Hizikia fusiformis

Thermoresponsive structured emulsions based on the fibrillar self-assembly of natural saponin glycyrrhizic acid

Responsive Emulsion Gels with Tunable Properties Formed by Self-Assembled Nanofibrils of Natural Saponin Glycyrrhizic Acid for Oil Structuring

Long-Lived and Thermoresponsive Emulsion Foams Stabilized by Self-Assembled Saponin Nanofibrils and Fibrillar Network

Glycyrrhizic acid ameliorates the kynurenine pathway in association with its antidepressant effect. Comprehensive Review for Phytochemical, Pharmacological, and Biosynthesis Studies on Glycyrrhiza spp

Characterization of binding interaction between magnesium isoglycyrrhizinate and human serum albumin

Efficient production of glycyrrhetinic acid in metabolically engineered Saccharomyces cerevisiae via an integrated strategy

Preparative separation of liquiritigenin and glycyrrhetic acid from Glycyrrhiza uralensis Fisch using hydrolytic extraction combined with high-speed countercurrent chromatography

SARS coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway

Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1

Glycyrrhizin ameliorates atopic dermatitis-like symptoms through inhibition of HMGB1

Formulation and evaluation of novel glycyrrhizic acid micelles for transdermal delivery of podophyllotoxin

In vitro antiviral activity and underlying molecular mechanisms of dipotassium glycyrrhetate against porcine reproductive and respiratory syndrome virus

PEDV enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway

Structure of the HMG box motif in the B-domain of HMG1

Glycyrrhizin inhibits influenza A virus uptake into the cell

Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods

Glycyrrhizin Suppresses the Growth of Human NSCLC Cell Line HCC827 by Downregulating HMGB1 Level

Platycodin D is a potent adjuvant of specific cellular and humoral immune responses against recombinant hepatitis B antigen

Platycodin D improves the immunogenicity of newcastle disease virusbased recombinant avian influenza vaccine in mice

Structural insight into the ligandreceptor interaction between glycyrrhetinic acid (GA) and the high-mobility group protein B1 (HMGB1)-DNA complex

Protective effect of glycyrrhizic acid on cerebral ischemia/reperfusion injury via inhibiting HMGB1-mediated TLR4/NF-κB pathway

Effects and cost of glycyrrhizin in the treatment of upper respiratory tract infections in members of the Japanese maritime self -defense force: Preliminary report of a prospective, randomized, double-blind, controlled, parallel-group, alternate-day treatment assignment clinical trial

Bioavailability enhancement of paclitaxel via a novel oral drug delivery system: paclitaxel-loaded glycyrrhizic acid micelles

Glycyrrhizic Acid Alleviates 6-Hydroxydopamine and Corticosterone-Induced Neurotoxicity in SH-SY5Y Cells Through Modulating Autophagy

Antiviral and Immunoregulatory Role Against PCV2 in Vivo of Chinese Herbal Medicinal Ingredients

Chloroquine inhibits HMGB1 inflammatory signaling and protects mice from lethal sepsis

In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Multi-Evaluating Strategy for Siji-kangbingdu Mixture: Chemical Profiling, Fingerprint Characterization, and Quantitative Analysis

Selective and competitive inhibition of kynurenine aminotransferase 2 by glycyrrhizic acid and its analogues

Compound glycyrrhizin plus conventional therapy for psoriasis vulgaris: a systematic review and meta-analysis of randomized controlled trials

Engagement of cellular cholesterol in the life cycle of classical swine fever virus: its potential as an antiviral target

Purification of high -purity glycyrrhizin from licorice using hydrophilic interaction solid phase extraction coupled with preparative reversed-phase liquid chromatography

Critical roles of platelets in lipopolysaccharide-induced lethality: effects of glycyrrhizin and possible strategy for acute respiratory distress syndrome

System optimisation quantitative model of on-line NIR: a case of Glycyrrhiza uralensis Fisch extraction process

The epidemiology, diagnosis and treatment of COVID-19

Synergistic protection of Schizandrin B and Glycyrrhizic acid against bleomycin-induced pulmonary fibrosis by inhibiting TGF-β1/Smad2 pathways and overexpression of NOX4

Platycodin D Suppresses Type 2 Porcine Reproductive and Respiratory Syndrome Virus In Primary and Established Cell Lines

Cholesterol 25-hydroxylase negatively regulates porcine intestinal coronavirus replication by the production of 25-hydroxycholesterol

HMGB1 mediates the development of tendinopathy due to mechanical overloading

Cholesterol-lowering effect of platycodin D in hypercholesterolemic ICR mice

Population pharmacokinetics of azithromycin and chloroquine in healthy adults and paediatric malaria subjects following oral administration of fixed-dose azithromycin and chloroquine combination tablets

Analysis of the susceptibility to COVID-19 in pregnancy and recommendations on potential drug screening

Acknowledgments. The authors thank Prof. Xiao-Quan Yang (Research and Development Centre of