key: cord-336585-19vwpjkt
authors: Adem, Şevki; Eyupoglu, Volkan; Sarfraz, Iqra; Rasul, Azhar; Zahoor, Ameer Fawad; Ali, Muhammad; Abdalla, Mohnad; Ibrahim, Ibrahim M; Elfiky, Abdo A
title: Caffeic acid derivatives (CAFDs) as inhibitors of SARS-CoV-2: CAFDs-based functional foods as a potential alternative approach to combat COVID-19
date: 2020-08-22
journal: Phytomedicine
DOI: 10.1016/j.phymed.2020.153310
sha: 
doc_id: 336585
cord_uid: 19vwpjkt

BACKGROUND: : SARS-CoV-2, an emerging strain of coronavirus, has affected millions of people from all the continents of world and received worldwide attention. This emerging health crisis calls for the urgent development of specific therapeutics against COVID-19 to potentially reduce the burden of this emerging pandemic. PURPOSE: : This study aims to evaluate the anti-viral efficacy of natural bioactive entities against COVID-19 via molecular docking and molecular dynamics simulation. METHODS: : A library of 27 caffeic-acid derivatives was screened against 5 proteins of SARS-CoV-2 by using Molegro Virtual Docker 7 to obtain the binding energies and interactions between compounds and SARS-CoV-2 proteins. ADME properties and toxicity profiles were investigated via www.swissadme.ch web tools and Toxtree respectively. Molecular dynamics simulation was performed to determine the stability of the lead-protein interactions. RESULTS: : Our obtained results has uncovered khainaoside C, 6-O-Caffeoylarbutin, khainaoside B, khainaoside C and vitexfolin A as potent modulators of COVID-19 possessing more binding energies than nelfinavir against COVID-19 M(pro), Nsp15, SARS-CoV-2 spike S2 subunit, spike open state and closed state structure respectively. While Calceolarioside B was identified as pan inhibitor, showing strong molecular interactions with all proteins except SARS-CoV-2 spike glycoprotein closed state. The results are supported by 20 ns molecular dynamics simulations of the best complexes. CONCLUSION: : This study will hopefully pave a way for development of phytonutrients-based antiviral therapeutic for treatment or prevention of COVID-19 and further studies are recommended to evaluate the antiviral effects of these phytochemicals against SARS-CoV-2 in in vitro and in vivo models.

A severe respiratory coronavirus disease 2019 caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has emerged as a pandemic at the end of 2019 Zhu et al., 2020) . By July 2020, SARS-CoV-2 has affected more than 200 territories, infecting more than 13 million individuals and causing more than 0.5 million deaths (WHO, . COVID-19 has marked the history with third life-threatening coronavirus epidemic into the human population during 21 st century . By 7 th January, 2020, Chinese scientists released the sequenced SARS-CoV-2 genome for the identification and development of potential candidates against COVID-19 by computational methods and other therapeutic techniques .

The SARS-CoV-2 is a β-coronavirus, enveloped and positive sense-RNA virus, with ~30 kb genome . SARS-CoV-2 genome possesses a complex organization encoding various structural as well as non-structural proteins (Nsps) (Kim et al., 2020a) . Majority part of viral genome (replicase ORF1ab encompassing Nsps) is translated into two overlapping polyproteins known as pp1a and pp1ab (Amoretti et al., 2002) . These polypeptides also codes for ~306 amino acid long main protease (M pro ) , which digests polypeptides at various conserved sites yielding 16 functional viral Nsps possessing multiple enzymatic activities especially in viral replication (Amoretti et al., 2002; Kim et al., 2020a) . One of such enigmatic protein, Nsp15, is an endoribonuclease known to be indispensable for protein interference during innate immune response (Kim et al., 2020a) . Due to functional importance of M pro and Nsp15 in viral replication and survival, both could be potential therapeutic drug targets to combat COVID-19.

In addition to Nsps, SARS-CoV-2 genome also consists of structural protein encoding genes including S (spike) gene, E gene (viral envelop protein), and N (nucleo-capsid protein) gene (Khan et al., 2020) . Viral spike proteins possess strong affinity with the human ACE2 (angiotensin-converting enzyme 2) receptor by which virus fuses with target membrane to gain entry into human cells (Hussain et al., 2020) . SARS-CoV-2 fusion potential and ACE2 affinity is much greater as compared to SARS-CoV, suggesting that the SARS-CoV-2 fusion machinery is a novel target for coronavirus fusion inhibitors. S protein binds ACE2 receptor via its S1 subunit, while its S2 subunit interacts to form fusion core, which brings viral and target cell membranes into proximity for efficient fusion and subsequent infection (Xia et al., 2020) . Thus, SARS-CoV-2 S2 subunit could be a potential target for coronavirus fusion inhibitors. Moreover, S1 trimeric crowns removal or opening is expected to be essential for exposure of receptor binding domain (RBD) to ACE2 receptor and S2 conformational changes which enable binding and membrane fusion (Walls et al., 2020b) suggesting that SARS-CoV-2 spike ectodomain structure and SARS-CoV-2 spike closed state glycoprotein structure might be novel therapeutic targets to develop anti-COVID-19 drugs.

Growing evidences have established the worth of polyphenols as lead compounds for drug discovery against various human diseases (Dos Santos et al., 2018) . Recent studies reported that polyphenol have potential to combat with COVID-19 (Adem et al., 2020) . Caffeic acids are one of the abundant plant-based polyphenols possessing 2 phenolic hydroxyl moieties and commonly found in coffee, fruits and vegetables (Magnani et al., 2014) . Caffeic acids have been reported for their potent virucidal activity against herpes simplex virus (Langland et al., 2018) , SFTS (severe fever with thrombocytopenia syndrome) virus (Ogawa et al., 2018) , and influenza virus (Utsunomiya et al., 2014) . Based upon these results, we have screened a library of caffeic acid derivatives (CAFDs) (Figure 1 ) for the identification of novel natural anti-COVID-19 compounds against various SARS-CoV-2 drug targets including COVID-19 M pro (6LU7), SARS-CoV-2 S2 subunit (6LXT), Nsp15 endoribonuclease (6VWW), SARS-CoV-2 spike ectodomain open state structure (6VYB), and SARS-CoV-2 spike closed state glycoprotein structure (6VXX). Our results present in silico-based identification of khainaoside C, 6-O-Caffeoylarbutin, khainaoside B, khainaoside C and vitexfolin A as potent modulators of COVID-19 M pro , Nsp15, coronavirus fusion protein, spike open state and closed state structure respectively. Our findings will provide valuable data for exploration and development of caffeic acid-derivatives as lead structures, novel therapeutic and prophylaxis agents against COVID-19 in the near future.

To obtain binding interactions between CAFDs and binding pockets of 5 different proteins of SARS-CoV-2, five independent docking analyses were performed by using Molegro Virtual Docker (MVD) software in a computer cluster system provided by EXPER, model-FQC-01266 running Intel Core i3-2100 CPU @3.10GHz Processor, 64 BİT, 4 GB RAM, 1TB hard disk, and NVIDIA GeForce GT 630 Graphic card. The crystal structures of the following SARS-CoV-2 proteins were retrieved from the protein data bank web site (http://www.rcsb.org/pdb): SARS-CoV-2 M pro (PDB ID: 6LU7: Resolution 2.16 Å) (Jin et al., 2020) , Nsp15 endoribonuclease (PDB ID: 6VWW) (Kim et al., 2020b) , coronavirus fusion protein (PDB ID: 6LXT) (Xia et al., 2020) , SARS-CoV-2 spike ectodomain (PDB ID: 6VYB) and spike glycoprotein (open state) (PDB ID: 6VXX) (Walls et al., 2020a) . The selected cavity of 6LU7 is the binding site of natural inhibitor N3. Nelfinavir, utilized for the cure of the HIV (human immunodeficiency virus), was utilized as a positive control. For other targets, the docking cavities of target proteins were 

In silico ADME analysis was conducted to investigate physicochemical properties of potent hits, such as water solubility, lipophilicity and pharmacokinetics by using following website http://www.swissadme.ch (Daina et al., 2017) . Absorption (% ABS) of potent hits from intestine was evaluated by: % ABS = 109 × (0.345xTPSA). Toxicity analysis was performed using offline software Toxtree 3.1 application (Zhao et al., 2002) .

The structures of the best-docked complex for each protein are selected for in-depth molecular dynamics simulation (MDS) study for a period of 20 ns. NAMD software was utilized to conduct the MDS with CHARMM 36 force field (Huang and MacKerell, 2013; Phillips et al., 2005) .

VMD is used to prepare the complexes for the MDS (Humphrey et al., 1996) . Complexes are subjected to equilibration using the CHARMM GUI web server after that a production run for 20 ns is performed on Shaheen supercomputer of King Abdullah University of Science and Technology (KAUST) under the project number k1482 (Jo et al., 2008) . The equilibration is done on the protein-small molecule solvated in the TIP3P water model and 0.154 M NaCl solution at 310 K temperature and pH 7 (Mark and Nilsson, 2001) . VMD is utilized in trajectories analysis, while the Chimera software of UCSF is used for cluster analysis (Mark and Nilsson, 2001; Pettersen et al., 2004) . After trajectory clustering, the five most populous clusters are represented by a conformation and tested for its binding to the protein. AutoDock Vina software is used in the binding energy calculations using 40 Å × 40 Å × 40 Å box dimensions (Morris et al., 2009; Trott and Olson, 2010) .

The MolDock Scores obtained from the docking studies of CAFDs and 6LU7 are summarized in Table 1 . Based on these in-silico results, khainaoside C, calceolarioside B, vitexfolin A, calceolarioside C and scrophuloside B exhibited best binding potential with COVID-19 virus Figure 2B represents residual wise van der Waals interactions, pi- Nelfinavir which possess MolDock score of -148.413 The interactions of these compounds with amino acid residues of target protein are shown in Figure 3A . Ligand plot representing residual wise van der Waals interaction, pi-alkyl interactions, pi-cation and pi-anion interactions are presented in Figure 3B . Figure 4A . Figure 4B shows residual wise van der Waals interactions, pi-alkyl interactions, pi-pi interactions, amide-pistacked interactions and pi-anion interactions of potent hits with 6LXT. The binding energies obtained from the docking analysis of 6VXX with ligands are presented in Table 5 . Vitexfolin A, chicoric acid, eutıgosıde A exhibited the best potential against spike glycoproteins of SARS-CoV-2. According to in silico results, 7 of the compounds have a better affinity to bind with spike glycoproteins than Nelfinavir (Table 5 ). The ligand-protein interactions are shown in Figure 6A . Amino acid residues of 6VXX interact with CAFDs via van der Waals interaction, pi-alkyl interactions, pi-pi interactions and pi-sigma interactions are ( Figure 6B ). 

The eight compounds with high-binding affinity against COVID-19 were analysed for ADME properties using SwissADME web tool. The results of eight compounds with high activity potential are presented in Figure 8 . Eutigoside A meets all criteria for oral use. Physicochemical, pharmacokinetics and drug-likeness properties of potent hits are presented in Table 6 . All of the compounds are water soluble. They do not cross the blood brain barrier, do not interact with interaction of main enzymes of Cytochromes P450, and have P-gp substrate properties. Toxicity assessment according to the chemical structure was carried out using Toxtree software. Figure 9B shows the stability of the residues during the simulation through per residue Root Mean Square Fluctuation (RMSF) in Å. Dashed-red lines indicate the positions of the active site residues. Figure 9C shows the average binding energies obtained for the representative conformation for each cluster after the MDS run for 20 ns on the protein-ligand complexes. Post-dynamics average binding energies (in kcal/mol) calculated for the complexes using AutoDock Vina. Error bars represent the standard deviations.

Polyphenols possess various beneficial properties against viral diseases such as modulation of immune system (Jasso-Miranda et al., 2019) , inhibiting viral replication (Lalani and Poh, 2020) and reduction of viral uptake by target membrane (Vazquez-Calvo et al., 2017) . CAFDs are polyphenolic compounds which possess prominent antiviral activity especially against hepatitis B virus (HBV) (Zhang et al., 2014) and HIV (Pluymers et al., 2000) . Keeping in view the potential of CAFDs against viral diseases (Figure 1) These groups specifically -OH group serves as excellent candidate for hydrogen bonding and exhibits tendency to form strong interactions with amino acid residues of target protein. As reported in Figure 9 , the 20 ns MDS runs were enough for the protein-ligand complexes. RMSD for each system show equilibration, while SASA and RoG both show stabilities during the MDS.

The per-residues RMSF profiles in Figure 9B indicate the stability of the binding since the interacting residues (H41, C149 in Mpro, K90, T167 in NSP15, R185 in the post-fusion core, (Luecha et al., 2009 ) and possess anti-diabetic potential (Somtimuang et al., 2018) . Vitexfolin A is an important constituent of Vitex rotundifolia and possesses strong analgesic effect (Okuyama et al., 1998) . Scrophuloside B has been identified in traditional Chinese medicinal plants including Picrorhiza scrophulariiflora (Wang et al., 2013) and Radix scrophulariae (Jing et al., 2011) . Calceolarioside B has been isolated from Fraxinus sieboldiana and Forsythia suspensea plants both of which possess anti-inflammatory and anti-viral properties (Kim et al., 2002; Wang et al., 2009) . While some of potent CAFDs are found in vegetables and fruits such as: Lettuce (Lactuca sativa), a widely consumed leafy vegetable, is found to be enriched with various CAFDs such as chicoric acid and chlorogenic acid (Abu-Reidah et al., 2013) . Chicoric acid has been long known as potent anti-viral agent against HIV (Lin et al., 1999) and its mechanism of action involves deactivation of HIV-1 integrase, increased Tlymphoblastoid viability, and downregulation of reverse transcriptase of HIV-1 . 6'-O-caffeoylarbutin is abundantly found in Vaccinium dunalianumas. V. dunalianumas is a commonly cultivated blueberry species in China and its dried leaf buds as herbal tea while its leaves are used as folk medicine (Li et al., 2016; Luo et al., 2015) . Sunflower (Helianthus annuus) sprouts (nutritious sunflower lettuce) found to be enriched with cynarin (Sun et al., 2012) , recommending these foods as beneficial food choice for COVID-19 patients.

Among all these potent compounds, calceolarioside B is identified as pan-inhibitor of SARS-CoV-2 having potential to target SARS-CoV-2 M pro , Nsp15, coronavirus fusion protein, as well as spike ectodomain which brought excitement about calceolarioside B's potential against COVID-19. Interestingly, calceolarioside B is also an important constituent of Akebia trifoliata fruit which is popularized in Asia due to its nutritional values as well as delicious taste and also used as dietary supplement for its various health benefits including anti-microbial and antiinflammation , suggesting that Akebia trifoliata fruit could potentially help COVID-19 patients to fight this disease. Calceolarioside B has potential to inhibit gp4l transmembrane glycoproteins of HIV (human immunodeficiency virus) (Kim et al., 2002) . These glycoproteins play critical role in virus cell fusion with target membrane to enable viral entry into the host cell (Buzon et al., 2010) . As calceolarioside B is identified as potent binder of SARS-CoV-2 spike glycoprotein during our study, the results from both of these studies propose calceolarioside B as viral and target membrane fusion inhibitor further strengthening its role against COVID-19.

In addition, calceolarioside B exhibits good anti-RSV (respiratory syncytial virus) effects (Dong et al., 2017) which recommend that calceolarioside B rich foods could be a potential alternative approach for the prevention and treatment of COVID-19. In addition to its anti-viral properties, it has potential to inhibit IL-6 production to exert it anti-inflammatory properties (Jin et al., 2014) .

COVID-19 patients suffer with severe inflammatory response in the later stages of infection, thus, dual anti-viral/anti-inflammatory properties of this compound makes it ideal candidate for drug development for prevention and treatment of COVID-19. As coronaviruses mutations make it difficult to develop vaccine, so does the single-target drugs. Single-target drugs might encounter low efficacy as the virus mutates. Thus, complex diseases like COVID-19 are more likely to be alleviated or healed though simultaneous modulation of multiple targets. Based on the above discussion, in our personal opinion, efficacy of CAFDs-enriched foods against COVID-19 as well as safety suggests that their adoption in daily diets might help prevent the onset of COVID-19 in an alternative and nonpharmacological approach.

Conclusively, this study provides scientific basis for the possible utilization of CAFDs as drug leads to develop anti-COVID-19 therapeutics. Since there is an urgent and timely need to find out effective and specific anti-viral treatment for COVID-19, this study will hopefully lay the foundation to work forward on small scale studies for the determination of 1) efficacy of CAFDs in reducing viral load and shortening the infectious period, 2) optimal dosing regimen based on impact on viremia, 3) impact on antibody production, inflammatory signaling and oxidative stress in COVID-19 patients. It seems that CAFDs have better absorption and good safety profiles as many studies pointed out the better absorption of CAFDs and CAFDs are important constituent of dietary foods. However, it would be worthwhile to conduct pharmacological studies to determine whether the bioavailability of CAFDs is good enough or it is better to administer in combinations. Once we understand the best way to deal with the CAFDs, it will be a reasonable starting point for therapeutic interventions based on the pharmacokinetic and pharmacodynamic studies on CAFDs. Despite excitement about CAFD's potential from antiviral and anti-inflammatory researches, there is an urgent and timely need to fill the room of knowledge for validation of CAFDs potential against COVID-19.

Reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry as a powerful tool for metabolic profiling of vegetables: Lactuca sativa as an example of its application

Identification of potent COVID-19 main protease (Mpro) inhibitors from natural polyphenols: An in silico strategy unveils a hope against CORONA

Production and detection of cold antihydrogen atoms

Crystal structure of HIV-1 gp41 including both fusion peptide and membrane proximal external regions

SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules

Polyphenols as New Leads in Drug Discovery: Biological Activity and Mechanisms

Predictors of Mortality for Patients with COVID-19 Pneumonia Caused by SARS-CoV-2: A Prospective Cohort Study. The European respiratory journal

The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak -an update on the status

CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data

Journal of molecular graphics 14

Structural Variations in Human ACE2 may Influence its Binding with SARS-CoV-2 Spike Protein

Antiviral and immunomodulatory effects of polyphenols on macrophages infected with dengue virus serotypes 2 and 3 enhanced or not with antibodies

Three new lignan glycosides with IL-6 inhibitory activity from Akebia quinata

Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

CHARMM-GUI: a web-based graphical user interface for CHARMM

Identification of Dietary Molecules as Therapeutic Agents to Combat COVID-19 Using Molecular Docking Studies

HIV gp41 binding phenolic components from Fraxinus sieboldiana var. angustata

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2. Protein science : a publication of the

Flavonoids as Antiviral Agents for Enterovirus A71 (EV-A71)

Antiviral activity of metal chelates of caffeic acid and similar compounds towards herpes simplex, VSV-Ebola pseudotyped and vaccinia viruses

Fungal endophytes of South China blueberry (Vaccinium dunalianum var. urophyllum)

Chicoric acid analogues as HIV-1 integrase inhibitors

Potential inhibitors against 2019-nCoV coronavirus M protease from clinically approved medicines

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Antiestrogenic constituents of the Thai medicinal plants Capparis flavicans and Vitex glabrata

HPLC simultaneous determination of arbutin, chlorogenic acid and 6'-O-caffeoylarbutin in different parts of Vaccinium dunalianum Wight

Caffeic acid: A review of its potential use in medications and cosmetics

Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility

Caffeic acid, a coffee-related organic acid, inhibits infection by severe fever with thrombocytopenia syndrome virus in vitro

Pharmacologically active components of viticis fructus (Vitex rotundifolia). II. The components having analgesic effects

The Bioactive Effects of Chicoric Acid As a Functional Food Ingredient

UCSF Chimera--a visualization system for exploratory research and analysis

Scalable molecular dynamics with NAMD

Viral entry as the primary target for the anti-HIV activity of chicoric acid and its tetra-acetyl esters

Evaluation of In Vitro alpha-Amylase and alpha-Glucosidase Inhibitory Potentials of 14 Medicinal Plants Constituted in Thai Folk Antidiabetic Formularies

Cynarin-rich sunflower (Helianthus annuus) sprouts possess both antiglycative and antioxidant activities

AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading

Inhibition by caffeic acid of the influenza A virus multiplication in vitro

Antiviral Properties of the Natural Polyphenols Delphinidin and Epigallocatechin Gallate against the Flaviviruses West Nile Virus, Zika Virus, and Dengue Virus

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

2020b. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell

New phenylethanoid glycosides from the fruits of forsythia suspense (thunb.) vahl

Chemical investigation of an antimalarial Chinese medicinal herb Picrorhiza scrophulariiflora

The profiling of bioactives in Akebia trifoliata pericarp and metabolites, bioavailability and in vivo antiinflammatory activities in DSS-induced colitis mice

A new coronavirus associated with human respiratory disease in China

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Evaluation of hepatocyteprotective and anti-hepatitis B virus properties of Cichoric acid from Cichorium intybus leaves in cell culture

Rate-limited steps of human oral absorption and QSAR studies

A Novel Coronavirus from Patients with Pneumonia in China

Shaheen supercomputer of King Abdullah University of Science and Technology (KAUST) is used to perform the MDS study (under the project number k1482). We thank Dr. Bahaa Mostafa for using his computational power unit during the analysis of the MDS data.