Antiviral Activity of Halogenated Compounds Derived from L-Tyrosine Against SARS-CoV-2

Abstract

Introduction: Currently, there are no effective medications for treating all the clinical conditions of patients with COVID-19. We aimed to evaluate the antiviral activity of compounds derived from L-tyrosine against the B.1 lineage of SARS-CoV-2 in vitro and in silico.

Methodology: The cytotoxicities of 15 halogenated compounds derived from L-tyrosine were evaluated in Vero-E6 cells by the MTT assay. The antiviral activity of the compounds was evaluated using four strategies, and viral quantification was performed by a plaque assay and qRT-PCR. The toxicity of the compounds was evaluated by ADMET predictor software. The affinity of these compounds for viral or cellular proteins and the stability of their conformations were determined by docking and molecular dynamics, respectively.

Results: TODC-3M, TODI-2M, and YODC-3M reduced the viral titer >40% and inhibited the replication of viral RNA without significant cytotoxicity. In silico analyses revealed that these compounds presented low toxicity and binding energies between -4.3 and -5.2 Kcal/mol for three viral proteins (spike, Mpro, and RdRp). TODC-3M and YODC-3M presented the most stable conformations with the evaluated proteins.

Conclusions: The most promising compounds were TODC-3M, TODI-2M, and YODC-3M, which presented low in vitro and in silico toxicity, antiviral potential through different strategies, and favorable affinities for viral targets. Therefore, they are candidates for in vivo studies.

Keywords: COVID-19; L-tyrosine; SARS-CoV-2; antiviral activity; halogenated compounds.

Figure 1

Classification of halogenated compounds derived from L-tyrosine. Fifteen halogenated compounds derived from L-tyrosine were evaluated and classified into halotyrosine and halotyramine derivatives. These groups were further subdivided into compounds with free phenolic OH and methylated phenolic OH (methyl ether derivatives). Finally, each subgroup was disrupted into compounds classified by halogen type (bromine [Br], chlorine [Cl], and iodine [I]) and by the number of methylations in the amino group.

Figure 2

Halogenated compounds derived from L-tyrosine did not have significant cytotoxic effects on Vero-E6 cells. Vero-E6 cells were treated with different concentrations (from 9.4 μM to 300 μM for 48 h) of halotyrosine derivative compounds (A) and halotyramine derivative (B) compounds. The percentage of viability (% cell viability) was calculated relative to a control of cells without treatment (control of cell viability). The results are shown as the mean ± standard deviation (SD), n = 8. The dotted line indicates 80% cell viability. The CC50 (50% cytotoxic concentration) values for each compound were included.

Figure 3

The halotyrosine derivatives TODC-3M and TODI-2M and the halotyramine derivatives YDB-3M, YDI-3M, and YODC-3M reduced the viral titer of SARS-CoV-2. The viral titers (PFU/mL) obtained after combined strategy with the five halogenated compounds were quantified. The figure shows the inhibition percentages calculated from the comparison of viral titers of each treatment with respect to an untreated control (control of infection without treatment). Chloroquine (CQ) was used as a positive control of viral inhibition (50 μM). Four replicates and two experimental units were used for each concentration. Significance was calculated according to the normality of the data and with respect to the untreated control. Symbol meaning: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

TODC-3M, TODI-2M, and YODC-3M showed significant effectiveness against SARS-CoV-2 through individual strategies. The viral titers after antiviral evaluation of TODC-3M (A), TODI-2M (B), and YODC-3M (C) using three strategies (pre-, post-, and co-treatment) were quantified by plaque assay. The bars show the inhibition percentages calculated by comparing each treatment with an untreated control (control of infection without treatment). Heparin (50 µM) was selected as a positive control of viral inhibition in pre-treatment, while CQ was used for post-treatment and co-treatment (50 µM and 100 μM, respectively). The significance was calculated according to the data distribution and concerning an untreated control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 5

TODC-3M, TODI-2M, and YODC-3M reduced the amount of intracellular SARS-CoV-2 RNA when individual strategies were used. Vero-E6 monolayers infected with lineage B.1 of SARS-CoV-2 and treated with TODC-3M (A) and TODI-2M (B) by post- and co-treatment, and with YODC-3M through co-treatment (C), were used to quantify the number of copies of the SARS-CoV-2 E gene by qRT-PCR. The bars show the percentages of inhibition of the intracellular E gene after each treatment in comparison with an untreated control (monolayers infected without treatment). The positive control of viral inhibition used for post- and co-treatment was CQ (50 µM and 100 µM, respectively). Significances were calculated according to the data normality. Symbol meaning: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 with respect to an untreated control.

Figure 6

In silico interactions between halogenated compounds and viral and cellular proteins. The figure shows the heatmap of the binding free energies between the ligands with three SARS-CoV-2 proteins (spike, RdRp, Mpro) and the cellular protein ACE2. Data were obtained from a molecular docking analysis using AutodockVina®. Three docking simulations with the same settings for each complex were performed, and the average was plotted on a color scale (A). The 2D visualization between ligands and target proteins is shown. The spheres represent the interacting amino acids in the following complexes: Mpro-TODC-3M (B), Spike-TODC-3M (C), Mpro-TODI-2M (D), and Spike-YODC-3M (E). The amino acids of the spike protein with a blue border are those that interact with the ACE2 receptor. For Mpro, they represent the amino acids associated with their catalytic function. Discovery Studio was used for visualization of the complexes.

Figure 7

Molecular dynamics analysis of the TODC-3M ligand with the target proteins. The RMSD is from the molecular dynamic trajectories for TODC-3M, ACE2 (red), Mpro (blue), RdRp (green), and spike (magenta) (A). PCA-based FEL of the complex; blue indicates the minimum energy (stable conformation), and red indicates the maximum energy (unstable conformation) (B). Clustering of the more representative conformations during 100 ns: DP, MRS, ACE2 (C), Mpro (D), RdRp (E), spike (F). TODC-3M is represented in yellow.

Figure 8

Molecular dynamics analysis of the TODI-2M compound with the target proteins. RMSD from the molecular dynamic trajectories for TODI-2M, ACE2 (red), Mpro (blue), RdRp (green), and spike (magenta) (A). PCA-based FEL of the complex; blue indicates the minimum energy (stable conformation), and red indicates the maximum energy (unstable conformation) (B). Clustering of the more representative conformations during 100 ns: DP, MRS, ACE2 (C), Mpro (D), RdRp (E), and spike (F). TODI-2M is represented in yellow.

Figure 9

Molecular dynamics analysis of the YODC-3M compound with the target proteins. RMSD from the molecular dynamic trajectories for YODC-3M, ACE2 (red), Mpro (blue), RdRp (green), and spike (magenta) (A). PCA-based FEL of the complex; blue indicates the minimum energy (stable conformation), and red indicates the maximum energy (unstable conformation) (B). Clustering of the more representative conformations during 100 ns: DP, MRS, ACE2 (C), Mpro (D), RdRp (E), and spike (F). YODC-3M is represented in yellow.

Figure 10

Possible mechanism of the antiviral action of promising compounds against SARS-CoV-2. Halogenated compounds as L-tyrosine derivatives may exert a multimodal action that deserves further investigation. Based on our results, we propose some hypothesis about their antiviral mechanisms against SARS-CoV-2: The compounds TODC-3M, YODC-3M, and TODI-2M exhibited a potential virucidal effect that could be due to the interaction with RBD of spike protein and, consequently, interfere with the viral entry into cells. Additionally, TODC-3M and TODI-2M demonstrated the potential to inhibit later stages of viral entry, which could be related to interference with viral genome replication and/or other downstream steps such as translation, assembly, or release of infectious viral particles. According to in silico analysis, some proposed viral targets of these compounds that could be considered for additional in vitro and in vivo studies are the non-structural proteins Mpro and RdRp of SARS-CoV-2.