key: cord-0691105-9lrakycc
authors: Almeida-Neto, Francisco Wagner Q.; Castro Matos, Maria Geysillene; Marinho, Emanuelle M.; Marinho, Márcia M.; de Menezes, Ramon R.P.P.B.; Sampaio, Tiago L.; Bandeira, Paulo N.; Celedonio Fernandes, Carla Freire; Teixeira, Alexandre M.Rodrigues; Marinho, Emmanuel S.; de Lima-Neto, Pedro; S. dos Santos, Hélcio
title: In silico study of the potential interactions of 4′-acetamidechalcones with protein targets in SARS-CoV-2
date: 2020-12-26
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2020.12.074
sha: 298dbb58d5d7e92457bd5e50db6cdc019071b271
doc_id: 691105
cord_uid: 9lrakycc

The sanitary emergency generated by the pandemic COVID-19, instigates the search for scientific strategies to mitigate the damage caused by the disease to different sectors of society. The disease caused by the coronavirus, SARS-CoV-2, reached 216 countries/territories, where about 20 million people were reported with the infection. Of these, more than 740,000 died. In view of the situation, strategies involving the development of new antiviral molecules are extremely important. The present work evaluated, through molecular docking assays, the interactions of 4′-acetamidechalcones with enzymatic and structural targets of SARS-CoV-2 and with the host's ACE2, which is recognized by the virus, facilitating its entry into cells. Therefore, it was observed that, regarding the interactions of chalcones with Main protease (Mpro), the chalcone N-(4′[(2E)-3-(4-flurophenyl)-1-(phenyl)prop-2-en-1-one]) acetamide (PAAPF) has the potential for coupling in the same region as the natural inhibitor FJC through strong hydrogen bonding. The formation of two strong hydrogen bonds between N-(4[(2E)-3-(phenyl)-1-(phenyl)-prop-2-en-1-one]) acetamide (PAAB) and the NSP16-NSP10 heterodimer methyltransferase was also noted. N-(4[(2E)-3-(4-methoxyphenyl)-1-(phenyl)prop-2-en-1-one]) acetamide (PAAPM) and N-(4-[(2E)-3-(4-ethoxyphenyl)-1-(phenyl)prop-2-en-1-one]) acetamide (PAAPE) chalcones showed at least one strong intensity interaction of the SPIKE protein. N-(4[(2E)-3-(4-dimetilaminophenyl)-1-(phenyl)-prop-2-en-1-one]) acetamide (PAAPA) chalcone had a better affinity with ACE2, with strong hydrogen interactions. Together, our results suggest that 4′-acetamidechalcones inhibit the interaction of the virus with host cells through binding to ACE2 or SPIKE protein, probably generating a steric impediment. In addition, chalcones have an affinity for important enzymes in post-translational processes, interfering with viral replication.

The health problem caused by the COVID-19 pandemic can be measured by the numbers of cases and deaths confirmed by the disease globally. In Brazil, community transmission is observed, with the collapse of the Health System in some regions. As it is an infection triggered by a new coronavirus (SARS-CoV-2), the pathophysiology of COVID-19 is little known and there is no specific treatment for the disease [1, 2] .

The search for new candidates for antiviral drugs has made great progress in recent years with the discovery of molecular targets, the development of organic synthesis and the discovery of new bioactives substances. A big number of techniques have been used in the search for new antiviral drugs. Despite the great progress, the arsenal of antiviral drugs is still small [1] . In this sense, strategies involving the development and validation of new antiviral molecules have been considered.

Chalcones, known as α, β-unsaturated ketones (1,3-diaryl-2-propene-1-one) are a class of naturally occurring compounds belonging to the flavonoid family. They can be obtained from natural sources or by synthesis, and are widely distributed in fruits, vegetables, and tea [3] . The double connection together with carbonyl group are possibly responsible for diverse biological activities such as antibacterial, antioxidant, anti-inflammatory and antiparasitic [4] . Antiviral properties of chalcones have been recorded in studies with plant viruses and human rhinoviruses [5] . Antiviral studies [6] with chalcones containing hydroxy and methoxy groups, confirm that the activity is dependent on the nature of the group and its positions in the aromatic rings. Santos [7] reports in a recent study the evaluation of the antiviral activity of hydroxychalcones and synthetic curcuminoids against infection caused by HPV in vitro.

Therefore, in this work, for the first time acetamide chalcones will be study theoretically by the Molecular Docking to characterize the inhibition power of the chalcones with the enzyme Mpro, methyltransferase, the SPIKE, and ACE2 proteins by the interaction energy and the distance of the compounds and the target protein's amino acids.

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

Using the methodological principle of synthesis the Claisen-Schmith reaction (in basic medium) [8] , chalcones were synthesized from benzaldehydes and 4-aminoacetophenone, both at a concentration of 2mmol. The reagents were added in a volumetric flask (25 mL), to which 5 mL of ethanolic NaOH solution (50%) were added. After adding the ethanol solution, the mixtures were kept under stirring for 48 hours (at room temperature). TLC (n-hexane: ethyl acetate, 2: 1) was used to monitor the progress of the reaction. After 48 h, the reaction mixture was neutralized with diluted HCl (10%) and ice water added. The products were obtained using the filtration technique under reduced pressure, washing with cold water and recrystallization in ethanol. To obtain the 4′-acetamidecalcones (1-6) (Figure 1 ), the acetylation reaction of the 4′-aminocalcones (2 mmol) with acetic anhydride (2 mmol) in buffered medium (5 mL) at pH = 5.0 with AcOH / AcONa was used [9] .

From the data available in the database, Protein Data Bank 

The interaction simulations between the selected inhibitors and proteins were performed using AutoDock Vina code (version 1.1.2), using 3-way multithreading, Lamarkian Genetic Algorithm [13] . The docking parameters: grid box sizes, centers, spacing and exhaustiveness to the proteins are given in Table S1 (Supplementary material). All grid boxes were configured to fit all the protein in the simulation for seeking the greater amplitude in the selection of molecular positions. As a standard procedure, one hundred (100) independent simulations were performed for all the target proteins, and it was obtained ten (10) positions each. As selection criteria, the simulations that showed positions with free binding energy (ΔG) below -6.0 kcal.mol -1 [14] and RMSD (Root Mean Square Deviation) values less than two thousand (2,000) [15] were analyzed.

For results analysis, image plotting, and generation of bi and tri-country maps, the Discovery Studio Visualizer [16] and UCSF Chimera [17] codes were used. For statistical analysis, the Morpheus® online server (https://software.broadinstitute.org/morpheus/) was used, in which heat maps were generated to identify the ligand-residue interaction and similarity profiles by the Pearson statistical test [18] .

Based on observations of the interactions of the molecules with the enzyme, the hydrogen bonds were plotted and classified according to previous studies that group interactions with distances between 2.5 and 3.1 Å as strong, from 3.1 to 3.55 Å as average and > 3.55 Å as weak [19] .

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

The positions shown in Figure 2A were To examine and describe the intrinsic characteristics of the interactions of 4′-acetamidechalcones molecules with the enzyme, the hydrogen bonds, and hydrophobic interactions were highlighted and compared with the reference ligands ( Figure 2B ). Since PAAPM, PAACN, and PAAPE did not show similarities with the reference ligands, those molecules were not considered for analysis. 

The interactions of chalcones with the NSP16-NSP10 are illustrated in Figure 3A .

Among the studied structures, PAACN and PAAPE did not show significant interaction with the enzyme. Besides, the other chalcones interacted in similar places. Table S4 ( The specification of the interactions of chalcones with the enzyme is illustrated in the bi-dimensional maps contained in Figure 3B . The distances of the interactions are specified in Table S4 (Supplementary material). Therefore, it is possible to understand the formation of two strong hydrogen bonds between PAAB and the methyltransferase.

The other chalcones also had two hydrogen bonds each, however, with an intermediate to weak intensity.

To study the potential of the chalcone acetamide derivatives to inhibit the 

The interactions with the amino acid residues of Spike protein heterodimer and the chalcones molecules were represented in bi-dimensional maps, corroborating with the information collected in the molecular docking simulations ( Figure 4C ). Besides, the specific interactions of the chalcones derivatives with the Spike protein are shown in Additionally, it is known that the interaction of the SPIKE protein with ACE2 is necessary for the virus to enter the host cells, hence the simulations of the interactions of the chalcone derivatives with this enzyme were performed, as shown in Figure 4B . It Those results are reinforced when characterizing the interactions of the chalcones derivatives with the ACE2 protein, as illustrated in the bi-dimensional maps ( Figure 4D ) and the interaction distances are shown in Table S5 (Supplementary material). The chalcones PAAPA and PAAPF stood out for presenting two interactions of strong hydrogen bonds. Also, the chalcones derivatives had common binding sites, such as the ILE291 residue, in which the PAAPA even had a hydrogen bond.

Overall, the present work evaluated, through molecular docking assays, the These results are relevant, as several chalcones have been described as having antiviral activity. A work performed by Park et al [20] showed that chalcones isolated from Angelica Keiskei inhibit the chymotrypsin protease (3CL (pro)) and a papain protease (PL (pro)) in SARS-CoV. Proteases are important for post-translational modifications of structural proteins in the viral particle. Therefore, the inhibition of proteases interferes with the viral replication process [20] . Literature data suggest the In fact, synthetic flavonoids and chalcones are described for potential antiviral properties. In a previous study, substituted chalcones, showed inhibition of viral translation in cells infected with hepatitis C virus (HCV) by the ablation of ribosomal protein phosphorylation 6 (rps6) [22] . Additionally, several reports in the literature demonstrate that derivatives of chalcones present antiviral activity better than the reference drugs in experimental models, including synergistic effects with these. For example, a previous study showed that thienyl-chalcone derivatives showed moderate to excellent antiviral activity, with higher in vitro potency against human cytomegalovirus compared to the standard drug Ganciclovir [23] . These data corroborate the findings of the present study, in which the chalcone derivatives interacted with the virus protease at sites and with similar affinities to the clinically used drugs and the theoretical inhibitor (MTase) and dengue virus envelope protein [24] .

Therefore, it is evident that it is feasible to study the interaction of drugs and antiviral molecules with structural proteins of these viruses, in addition to enzyme targets. Flavone derivatives, such as luteolin and semisynthetic derivatives of gallic acid have been described as potential binders to the SARS-CoV surface SPIKE protein and, therefore, may interfere with the entry of the virus into its host cells. This connection has been shown to happen with great avidity, with surface proteins linked to natural compounds being detected by frontal affinity chromatography coupled with mass spectrometry [25] .

The inhibition of the interaction of virus structural proteins with receptors in host cells is a line of research that has been highlighted. It is described that when binding the SPIKE protein to the angiotensin-converting enzyme 2 (ACE2) in the lung and intestinal cells, the virus enters the cells, releasing proteins such as the high-mobility group box 1 protein (HMGB1), allowing the occurrence of sepsis. Phenolic compounds have been described as steric inhibitors of the interaction of the viral structure with ACE2 and / or the release of HMGB1, reducing infectivity [26] .

Together, these results suggest that chalcone derivatives inhibit the interaction of the virus with host cells through binding to ACE2 or SPIKE protein, probably generating a steric impediment. In addition, chalcones have an affinity for important enzymes in post-translational processes, interfering with viral replication.

The 4′-acetamidechalcones presented inhibitory potential over the SARS-CoV-2 proteins, detaching the PAAPF that has the potential to couple to Mpro (same region as the natural inhibitor FJC through strong hydrogen bonds), PAAB that can bind to 

The authors declare no conflicts of interest. 

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