key: cord-0791376-uu0tnnst
authors: Braz, Helyson Lucas Bezerra; de Moraes Silveira, João Alison; Marinho, Aline Diogo; de Moraes, Maria Elisabete Amaral; de Moraes Filho, Manoel Odorico; Monteiro, Helena Serra Azul; Jorge, Roberta Jeane Bezerra
title: In silico Study of Azithromycin, Chloroquine and Hydroxychloroquine and their Potential Mechanisms of Action against SARS-CoV-2 Infection
date: 2020-07-30
journal: Int J Antimicrob Agents
DOI: 10.1016/j.ijantimicag.2020.106119
sha: 6ba5afa67d79e7e5f3bcb1302e008daa1ed14082
doc_id: 791376
cord_uid: uu0tnnst

The coronavirus disease 19 (COVID-19) is a highly transmissible viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical trials have reported an improved outcome resulting from effective reduction or absence of viral load when patients were treated with chloroquine or hydroxychloroquine. In addition, these drugs had their effects improved by simultaneous azithromycin administration. The Receptor-Binding Domain of SARS-CoV Spike Protein (RBD of S Protein) binds to the cell surface angiotensin-converting enzyme 2 (ACE2) receptors allowing viral entry and replication into the host cells. The viral Main Protease (Mpro) and the Cathepsin L (CTSL) are among the proteolytic systems involved in S Protein activation by SARS-CoV-2. Hence, molecular docking studies were performed to test the binding performance of these three drugs against four targets. Our finding showed azithromycin affinity scores (∆G) with strong interactions with ACE2, CTSL, Mpro and RBD. Chloroquine affinity scores showed 3 low energy results (less negative) with ACE2, CTSL and RBD; and a firm bond score with Mpro. With hydroxychloroquine, two results (ACE2 and Mpro) were firmly bound to the receptors; however, CTSL and RBD showed low interaction energies. The differences in better interactions and affinity between hydroxychloroquine and chloroquine with ACE2 and Mpro were probably due to structural differences between the drugs. Azithromycin, on other hand, not only showed more negative (better) values in affinity, but also in the number of interactions in all targets. Nevertheless, further studies are needed to investigate the antiviral properties of these drugs against SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the highly transmissible coronavirus disease [1] . This new coronavirus, firstly reported in Wuhan, China, in the beginning of December 2019, rapidly emerged worldwide. Following the implementation of human mobility control measures, such as extensive travel bans and quarantine in China, surveys showed that social restriction limited the spread of COVID-19 [2] , but it remains a Public Health Emergency of International Concern (PHEIC).

Scientists from many countries are striving to understand, to track, and to contain COVID-19 pandemic. The spread of COVID-19 is increasing worldwide, with 14,043,176 confirmed cases and 597,583 deaths in less than four months (data from July 18, 2020 by the WHO). Although drug repurposing has some limitations, the repositioning of some drugs has been considered or suggested as potential candidates for treatment of the novel coronavirus disease [3, 4, 5] .

Recent preliminaries clinical trials conducted, until now, in China and France, reported improved diseases outcomes with Chloroquine (CQ) or Hydroxychloroquine (HCQ) as shown by the evidence of their effectiveness in reducing or eliminating the viral load of COVID-19 patients in critical condition [6, 7] . Besides that, their effects can be improved through their combination with Azithromycin (AZM) [5, 8] . Thus, CQ phosphate is already among the drugs with antiviral activity included in the latest in version of the Treatment Guidelines issued by the National Health Commission (NHC) of the People's Republic of China for the tentative treatment of Novel Coronavirus-induced Pneumonia [4] . Some studies have discussed the possible mechanisms of AZM/ CQ/ HCQ against SARS-CoV-2 [9, 10, 11, 12] . It is known that the Receptor-Binding Domain of Spike Protein (RBD of S Protein) from virus binds to the cell surface angiotensin-converting enzyme 2 4 (ACE2) receptors, allowing the virus entry and replication inside the host cells [9, 10] . A recently published update has shown that the Main Protease (Mpro) might represent a suitable target for drugs inhibiting viral replication [11] . Preliminary data indicates that CQ interferes with SARS-CoV-2 promoting increase of endosomal pH, the compartment where the cleavage of S Protein is facilitated by host protease cathepsin L (CTSL), which requires low pH, impairing virus-endosome fusion, and consequently prevents the release into the cytosol [12, 13, 14] .

Nevertheless, recently, some studies have raised questions about clinical efficacy of above-mentioned drugs. A multicenter, open label, randomized controlled clinical trial did not show additional benefits in the virus elimination by HCQ in association with specifically standard of care in patients with mild to moderate COVID-19. It also promoted increased frequency of adverse events [15] . Others studies with patients that received HCQ and/or AZM were not significantly associated with in-hospital mortality [16, 17] . Meanwhile, a retrospective study demonstrated that addition of HCQ, on top of the basic treatments, reduced death risk in severe COVID-19 patients [18] . Therefore, the aim of this work is to evaluate the molecular interactions of AZM, CQ and HCQ through ACE2, CTSL, Mpro and RBD using molecular docking. Hence, we also aim to provide results that can be useful in other studies on the mechanism of action of these drugs in the COVID-19 therapeutic approach.

The ligands AZM (CSID: 10482163), CQ (CSID: 2618) and HCQ (CSID: 3526) were obtained from the virtual repository Chemspider (http://www.chemspider.com/) in .mol 5 format. ChemSpider is a free chemical structure database that provides quick access to more than 67 million structures, properties and associated information [19] . Using the Avogadro Software ® 1.2.0, the molecules were optimized, calculated with force field using MMFF94 type and converted into the .pdb format [20] .

For CoVs, a single region of the Spike Protein called the Receptor-Binding Domain (RBD) mediates the interaction with the host cell receptor [21] . Thus, in the Protein Data Bank ® (PDB) (https://www.rcsb.org) the ACE2 receptors and the RBD region of the same structure were obtained (PDB: 6VW1obtained by X-ray diffraction, 2.68 Å resolution), through structural separation using SWISSMODEL ® (https://swissmodel.expasy.org/), denominated ACE2 (PDB: 6VW1, chain A) and RBD (PDB: 6VW1, chain B) by the software itself. The structures PDB: 2XU3, obtained by X-ray diffraction with 0.9 Å resolution and PDB: 6Y2E, also obtained by X-ray diffraction with 1.75 Å resolution, were used for CTSL and Mpro receptors, respectively. The structures were obtained in .pdb format for use in molecular docking.

The AutoDock Vina ® v.1.1.3 software was used in all docking experiments [22] .

In the Autodock Vina, the proteins were optimized, by removing water and other residues not important for the study, and then a polar hydrogen group was added to all structures. The 

Before the docking, the structures of ligands were prepared using their optimized form. At this stage, the ligands showed 10 pre-established conformations for AZM, 7 for CQ and 8 for HCQ. Figure 1 shows the values of the fitting score (binding affinity) for ACE2

(column 1), CTSL (column 2), Mpro (column 3) and RBD (column 4) and their ligands (above the columns).

AZM is a macrolide antibiotic generally used to treat infections such as pneumonia and upper respiratory tract ones. Its antibacterial mechanism of action is bacterial protein synthesis inhibition through binding with the 50s ribosomal subunit and blocking the messenger RNA-directed polypeptide synthesis [23] . Moreover, it has also been used for the treatment of cancer, autoimmune and inflammatory diseases [24] . We found that AZM affinity scores showed strong interactions of -10.5 kcal/mol (ACE2), -9.6 kcal/mol (CTSL), -8.2 kcal/mol (Mpro) and -7.0 kcal/mol (RBD).

Although the AZM mechanism of action is still unclear in some previously tested viral infections, studies have shown anti-Zika virus activity in vitro, by inhibiting viral replication [25, 26] . This antibiotic was administered intranasally to infected mice and reduced the viral load in the lungs (in vivo study) against influenza A virus (H1N1). It also showed, in an in vitro study, with the same virus, effective blockade of viral internalization and inactivation of the endocytic activity of host cell progeny virus [27] . Therefore, our results suggest that AZM affects the internalization of the virus, as well as its binding on the host cell surface. Another research about the respiratory syncytial virus (RSV), found in common colds, hypothesized that macrolides may reduce the expression of activated CoV) and SARS-CoV-1 [12, 30] . Furthermore, there is an ongoing randomized controlled clinical trial using HCQ and AZM combination in 630 hospitalized and noncritical patients with COVID-19 infection, which is expecting results [31] .

Based on our data, we conclude that, while the interactions of CQ and HCQ showed better results with one and two receptors, respectively, AZM showed strong binding with all tested receptors, demonstrating a great binding potential in several biological processes related to viral replication of SARS-CoV-2. These drugs with a score above -6.0 kcal/mol were able to firmly bind to the structures [32] that perform the SARS-CoV-2 8 molecular replication process and, therefore, are potential candidates for inhibiting processes and reinforcing those currently showing promising results in clinical trials.

To analyze the possible reason for the differences in binding energies, the interactions formed after coupling were assessed using Biovia Discovery Studio v.4.5. Figure   2 The angiotensin-converting enzyme 2 (ACE2) is widely distributed in the heart, kidneys, lungs and testicles, and plays a vital role in renin-angiotensin-aldosterone system (RAAS) and homeostasis [33] . In silico and in vivo studies have suggested a potential deleterious effect of the RAAS [34, 10] , and a pilot trial using soluble human recombinant ACE2 (APN01) has been recently initiated in patients with COVID-19 (Clinicaltrials.gov #NCT04287686) [35, 36] .

The mRNA SARS-CoV-2 encodes essential proteins, 4-structural (small envelope (E) protein, matrix (M) protein, nucleocapsid (N) protein, and spike (S) glycoprotein) and 16 non-structure proteins (NSPs). The M and E proteins play a role in particle assembly and release [10, 13] . The S glycoprotein is responsible for a critical step for virus entry, as it binds to host cell receptors (ACE2) and fuses the viral membrane with the cell membrane. This glycoprotein has two subunits, S1 (key function domain -RBD, cell tropism) and S2

(mediates virus-cell membrane fusion through heptad repeats 1 -HR1and HR2 domains) [37] .

However, a recent research has shown that SARS-CoV-2 interaction with ACE2 alone is not sufficient to allow host cell entry, and preliminaries studies aim to identify 9 proteolytic systems involved in S Protein activation by SARS-CoV-2 [38] . The pH-dependent endosomal cell factors, such as cysteine protease, are determinant to SARS-CoV fusion membrane, especially CTSL [39] . CTSL is ubiquitously expressed, unlike other proteases; the site it was reported is close to predicted S1/S2 boundary, a critical site for proteolysis [40] .

Since it is most commonly associated with the activation of viral glycoproteins (MERS-CoV,

HCoV-229E and MHV-2) [41] , several researches have suggested that cathepsin inhibitors are possibly virus therapeutic targets and might have a broad applicability [42, 43] .

Another important step for SARS-CoV-2 replication is cysteine protease is the Main Protease (Mpro). It participates in proteolysis process, cleaving polyproteins, which are encoded by the coronavirus genome to mature NSPs, assisting the viral replication and transcription [44] . Mpro (or Nsp5) cleaves the polyprotein at 11 conserved sites [45] . During the infection, the replication/transcription complex is anchored to double-membrane vesicles (from endoplasmic reticulum or lysosomal membrane) [46] . Chain B: ASN52). In addition, the fittings showed other types of bonds, such as the carbonhydrogen bond, π-sigma, alkyl and Van der Waals forces. Figure 5 shows the threedimensional (3D) bonds of Azithromycin between the residues of the receptor structures. The affinity value of each coupling is also shown.

As for CQ, only two couplings showed H-bonds, a single interaction with CTSL (ASN66) and an interaction with Mpro (ILE249). In this coupling, 8 alkyl bonds were verified, due to the capacity of several saturated carbons in the structures. In the two dockings that did not show an H connection (ACE2 and RBD), many interactions of the carbon-hydrogen type, π-sigma and Van der Waals forces were also observed. With HCQ, 4

couplings showed H-bonds, two interactions with ACE2 (ASN376, SER29) and a π-π stacked interaction. That latter occurs when two aromatic rings interact with each other, in this case, the two HCQ rings bonded with the TRP331 ring [47] . Furthermore, π-anion, π-cation and carbon-hydrogen types of bonds were observed.

The structures of CQ and HCQ that showed the highest binding affinity are shown in Figure 5 in a three-dimensional (3D) format. The HCQ has one hydroxyl group more than CQ. This difference allows HCQ to have a greater role of regioselectivity and binding character in molecular simulations, because oxygen is an atom with greater regioselectivity [48] . This may explain the difference in docking results between these drugs.

The modulation of autophagy may be the mechanism responsible for the success of preliminary studies against SARS-CoV-2 [49] . It was reported that HCQ and CQ are lysosomotropic agents. Their effects of inhibition of autophagy is due to the impact of lysosomal acidification, inhibiting autophagosome-lysosome fusion and inactivating enzymes that several viruses require for replication, in the case of the SARS-CoV-2 may be affect Mpro [50] . During infection, autophagy can play either a pro-viral or antiviral role, depending on the virus, the cell type, and the cell environment [51, 52] . In case of the SARS-CoV-1, the autophagy inhibition is necessary for the success of treatment [52] .

However, understanding these molecular details require further investigation. In fact, this assumption has been investigated in relation to other viral infections [49] . A recent study showed that Mpro has 96% homology to SARS-CoV-1 [53] . Among the targets related to coronavirus diseases, a greater number of patents of SARS-CoV-1 Mpro inhibitor complexes has been registered and more potential drug candidates are emerging [54] . Some of these inhibitors (peptidic or peptidomimetic) have been used to attain covalent binding to the active-site cysteine of the Mpro [55, 56] .

Several published studies with inhibitor of viral proteases have supported the theory that the Mpro of SARS-CoV-2 could be a good target to therapeutic agents [54, 57, 58, 59, 60] . Furthermore, Remdesivir and Chloroquine, alone and/or in combination, are under investigation, showing they significantly blocked SARS-CoV-2 replication and patients were declared to be clinically recovered [61] . These data also can be useful to research potentials inhibitors of this protease, aiming to block viral replication in COVID-19 [62] .

The quinoline-based drugs, such as CQ and HCQ, accumulate in the acid lysosomes, aggravating endoplasmatic reticulum (ER) stress [50] . In this context, the proteasomes and inhibitors of sarcoplasmic/ER calcium ATPase (SERCA), might be a strategy [63] , a theory that was recently reinforced by Wang et al. [14] , by showing the CQ effectively inhibits SARS-CoV-2 in vitro. Zhou et al. [64] , showed that teicoplanin blocks the Ebolavirus entry by specifically inhibiting the activity of CTSL, a tetrahydroquinoline oxocarbazate, on Ebola and SARS-CoV-1.

A recent work using molecular modeling showed that CQ and HCQ bind to sialic acid-containing gangliosides, a site responsible for viral primary attachment factors along the respiratory tract in Coronavirus diseases, besides ACE2, which strengthens the hypothesis that these drugs could act as antiviral (against SARS-CoV-2) [65] . The same study demonstrated that HCQ is more potent than CQ. Similar results were found in other studies with SARS-CoV and SARS-CoV-2, respectively [66, 67] . Thus, our results corroborate these findings, showing that HCQ had better interactions and affinity when was compared to the same target (ACE2 and Mpro).

The CQ and HCQ association has been reported in an early clinical trial conducted in COVID-19 Chinese patients, and has shown to be efficient against SARS-CoV-12 2 [4, 5, 7] . Another preliminary study also suggested promising results of the HCQ and AZM combination. At day 6 post-inclusion (HCQ and AZM combination), 100% of patients treated were virologically cured, comparing with 57.1% of patients treated with HCQ only, and 12.5% in untreated ones [8] . Consistent with this idea, our molecular modeling study has simultaneously identified the binding of ACE2, Mpro, CTSL and RBD (to AZM/ CQ/ HCQ) against SARS-CoV-2, to surmise the molecular mechanisms underlying the antiviral mechanisms. Interestingly, our simulations indicated that AZM has better affinity than HCQ, and a possible association with this drug might increase antiviral activity of HCQ against SARS-CoV-2. Further studies will help clarify this point.

To date, neither drug or vaccine has been approved to be clinically used as an Ethical Approval: Not required.

The raw data that support the findings of this study are available from the corresponding author upon reasonable request. 

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Residues are identified by abbreviations/numbering in each field and the fitting scores are listed in each complex

Interaction between Azithromycin and CTSL, (C) Interaction between Azithromycin and Mpro, (D) Interaction between Azithromycin and RBD, (E) Interaction between Chloroquine and Mpro, (F) Interaction between Hydroxychloroquine and ACE2, (G) Interaction between Hydroxychloroquine and Mpro