key: cord-0690873-1c4vqaqr
authors: Benítez-Cardoza, Claudia Guadalupe; Vique-Sánchez, José Luis
title: Potential inhibitors of the interaction between ACE2 and SARS-CoV-2 (RBD), to develop a drug
date: 2020-06-15
journal: Life Sci
DOI: 10.1016/j.lfs.2020.117970
sha: e643182cf895ca02c685d87a3b0b25535251e2e2
doc_id: 690873
cord_uid: 1c4vqaqr

AIMS: The COVID-19 disease caused by the SARS-CoV-2 has become a pandemic and there are no effective treatments that reduce the contagion. It is urgent to propose new treatment options, which are more effective in the interaction between viruses and cells. In this study was to develop a search for new pharmacological compounds against the angiotensin-converting enzyme 2 (ACE2), to inhibit the interaction with SARS-CoV-2. MATERIALS AND METHODS: Docking, virtual screening using almost 500,000 compounds directed to interact in the region between the residues (Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353, and Arg357) in ACE2. The average of ΔG(binding), the standard deviation value and the theoretical toxicity from compounds were analyzed. KEY FINDINGS: 20 best compounds directed to interact in ACE2 with a high probability to be safe in humans, validated by web servers of prediction of ADME and toxicity (ProTox-II and PreADMET), to difficult the interaction between ACE2 and region binding domain (RBD) of SARS-CoV-2. SIGNIFICANCE: In this study, 20 compounds were determined by docking focused on the region of interaction between ACE2 and RBD of SARS-CoV-2 was carried out. The compounds are publicly available to validate the effect in in vitro tests.

Currently, the pandemic that has developed has some antecedents related to the SARS-CoV 2002 outbreak, of which several works have been carried out to develop new drugs directed to specific regions of the coronavirus (SARS-CoV 2002). The disease caused by SARS-CoV-2 generates a wide range of signs and symptoms, causing respiratory, gastrointestinal and even death diseases [1] [2] . In the first reports on the SARS-CoV-2 (COVID- 19) outbreak in China reported that the average age was 47 years, with an incubation period of 4 days, 41.9 % were women, with fever 88.7 % and cough the 67.8 % of the patients in the study, accompanied by lymphocytopenia in 83.2 %. It should be noted that there was no specific treatment for SARS-CoV-2, the treatment was based on antibiotics in 58.0 % and antivirals (Oseltamivir) in 36.2 % [2] . Recently, new antivirals have been developed, focusing on RNA-Dependent RNA Polymerase (RdRp), Polyproteins (3CLpro and PLpro), Spike Protein (S-Protein) [3] [4] and membrane fusion inhibitors (HR1 and HR2 of S-Protein) [5] [6] [7] from SARS-CoV-2. Without a treatment that demonstrating an advantage therapeutic, which demonstrates the urgent need for the development of specific drugs against a selective target that alters the evolution of this disease.

There are works related to SARS-CoV, for the development of a specific drug, which have reported the development of peptides related to the key protein for the interaction between the SARS-CoV and the host cell; the angiotensin-converting enzyme 2 (ACE2), reporting amino acids sequence that was essential for drug development (Glu22, Glu23, Lys26, Asp30, Lys31, His34, Glu35, Glu37, Asp38, Glu56 and Glu57) [8] . As well as another work that reported the important amino acids between the region binding domain (RBD) of the S-Protein SARS-CoV with the ACE2 (Gln24, Thr27, Lys31, His34, Glu37, Asp38, Tyr41, Gln42, Leu45, Leu79, Met82, Tyr83, Asn90, Gln325, Glu329, Asn330, Lys353 and Gly354 in ACE2) [9] . Currently, has been reported the crystallographic structure of the interaction between SARS-CoV2-RBD and ACE2 (Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353, and Arg357 in ACE2) [10] , which we used in this study.

There are reports of the development of pharmacological compounds that have an effect on the interaction of SARS-CoV and ACE2 [11] [12] , as well as focused on important J o u r n a l P r e -p r o o f proteins in the SARS-CoV such as the Main protease (Mpro), which propose synthesized aromatic compounds [13] .

Although there is a great similarity between SARS-CoV and SARS-CoV-2 sequence (sequence identity of almost 80 %) [14] , the same results of compounds or antibodies that were tested in SARS-CoV are not presented in SARS-CoV-2; the differences in the residues found in SARS-CoV-2 explain the resistance generates against compounds and antibodies (S230) [15] [16] .

The affinity of SARS-CoV-2 with ACE2 has been determined to be up to 20 times higher than that reported in SARS-CoV in 2002, which may help explain why the complications that develop are more serious, and the probability of contagion is greater, having a great impact on the health of the population [17] . It was determined that in SARS-CoV, ACE2 plays a very important role so that it can cross the cell membrane and be able to replicate the SARS-CoV; taking into account that SARS-CoV-2 also interacts with ACE2. Recently, an important protein for the interaction of ACE2 with SARS-CoV2, TMPRSS2, was identified, where it is demonstrated that if it is limited to this protein, the interaction of the virus with the cell can be affected [18] .

Some works for the development of new drugs against SARS-CoV-2, propose epitopes as potential sites of interaction [19] , as well as using Docking and compound libraries, as well as looking for a repositioning of drugs [5] , to search for compounds that interact with some SARS-CoV-2 region and thus be able to prevent interaction with ACE2 [20] . A drug that was proposed to interact in ACE2, is Arbidol, which recently reported the crystallographic structure, demonstrating that Arbidol interacts in S-protein (domain S2) from SARS-CoV-2 [21] , which demonstrates the low existence of drugs that are directly interacting with ACE2.

We use the amino acids reported in the crystallographic structure of the interaction between the S-protein-RBD of SARS-CoV-2 and ACE2 (Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353 and Arg357 in ACE2) [10] [22] , therefore, using the crystallographic structure of ACE2 (PDB 1R42), we carried out a Docking directed to these mentioned residues using a library of compounds (EXPRESS-pick Collection from Chembridge Corp.) to select the best compounds, and that these can affect the interaction between ACE2 and SARS-CoV-2, making these results an important contribution to establishing the foundations that allow the development of a drug that optimizes the resolution of this pandemic. Atomic coordinates of Angiotensin converting enzyme 2 (ACE2) were obtained from the Protein Data Bank (PDB: 1R42). The structure was used as protein targets for docking procedures. The protonation and energy minimization of PDB file was performed using Molecular Operating Environment (MOE) software with the default parameters and the CHARMM27 force field [23] [24] . We select one region to interaction in ACE2 (Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353 and Arg357) [10] .

The EXPRESS-pick Collection Stock of the small molecule screening library from Chembridge

Copr. was used for docking [25] . This collection of small molecule screening compounds contains over 500,000 chemical compounds that fulfill the druggable properties of Lipinski's rules [26] [27] and cover a broad area of chemical space.

For docking, the receptors were kept rigid, while the ligand atoms were released to move to a maximal number of rotatable bonds. All crystallographic water molecules were deleted from the initial structures. High-throughput virtual molecular docking was carried out [25] [28] by means of the software AutoDock and MOE, using default parameters (Placement: Triangle Matcher, Rescoring 1: London ΔG, Refinement: Forcefield, Rescoring 2: London ΔG, for each ligand up to 20 conformations were generated and saved).

The binding affinity of each complex (Ligand-protein) was estimated with the ratio of General Born vs Volume Integral (GB/VI), using parameters in MOE [29] [30] . General Born or nonbonded interaction energies comprise Van der Waals, Coulomb electrostatic interactions and implied solvent interaction energies [30] .

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

Each compound was simulated with up to 50 conformations, to select the best compounds, the average of the ΔG binding interaction value of up to 20 conformers, the description of chemical properties by PhysChem -ACD/Labs [31], the theoretical toxicity [32] , carcinogenicity and mutagenicity [33] were considered. The calculated interactions between ACE2 and compounds were visualized with Ligand-interaction interactions implemented in MOE.

Among the interactions in ACE2 (Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353 and Arg357) with compounds ( Figure 1 ), the selection criteria of the top poses, out of almost 500,000 compounds from Chembridge library, were the frequency of the conformers of each compound and the ΔG binding values between -6.0 to -7.3 kcal/mol -1 . We made the choice of compounds based on the average of the score from up to 20 conformers per compound and better probability to be safe in humans. We selected 20 compounds depicted here as C1 to C20 (Table 1) 

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

For selection of the best compounds, the analysis from Docking´s results was carried out, taking into account the average of the interaction ΔG binding (15 to 20 conformers) was determined, as well as the standard deviation for each compound. Subsequently, the theoretical toxicity were evaluated with two website (ProTox-II -Prediction of TOXicity and

PreADMET web server, prediction of carcinogenicity and mutagenicity, Table 2 ). Besides, we determined 30 compounds with good results, but with significant theoretical toxicity effects, we

show them in Table S21 .

The description of the chemical properties of each compound (C1 -C20, Table S22 ), ADME (Table S23 ) and theoretical toxicity (Table S24) , are shown in the supplemental material.

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

To propose the probable interaction sites between each compound (C1 -C20) with ACE2 we analyzed up to 20 conformers of each compound that showed the better ΔG binding values of interaction in amino acids Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353 and Arg357 ( Figure 1 ). From docking´s result (Table S1 -S20), we determined the main amino acids in ACE2 to interact with the 20 compounds, these are Lys26, His34, Glu37, Asp38, Tyr41, Gln96, Gln325, Asn330, Lys353, Arg357, Ala386, Ala387, Pro389 and Arg393 ( Table   2 ). The interaction of each compound and its conformers in ACE2 are shown in the supplementary material ( Figure S1 -S20).

Several 

Arg393 are important for the majority of the compounds that we propose to interact in ACE2 (Table 1) . Furthermore, we propose that the interaction site in ACE2 presents little change in the structural conformation when the S-protein-RBD is present, since we perform an alignment and superposition of the three-dimensional structures, of the apo-ACE2 (PDB: 1R42) and the ACE2 with RBD (PDB: 6M17) and there is an RMSD between them of 2.4 Å ( Figure S21 ), which shows that the interaction of ACE2 with RBD does not affect the three-dimensional conformation, moreover, the amino acids that we take into account to do the Docking (amino [7] , in which they seek to prevent the HR1 region of S-Protein ( Figure S22 ) from interacting with HR2 and its ligand in the ACE2 membrane, hindering the process of fusing the viral membrane and blocking the introduction of viral genetic material, there are currently very promising results of this type of drug, with evaluated doses with an IC50s between 1.3 and 15.8 nM against SARS-CoV-2 [7] , it shows that its development is viable.

We determined that there are very few studies of new drugs against ACE2, previously

Arbidol was thought to have ACE2 as a selective target, but the description of the interaction of Arbidol with S2-Domain in S-Protein of SARS-CoV-2 was made [21] .

Therefore, the development of antivirals against COVID-19, still in development, shows that there are no specific drugs against SARS-CoV-2, since several of the drugs that are using, they have been developed against other diseases, such as ebola (Remdisivir) [4] , influenza (Arbidol) [21] , SARS and MERS [13] [14] , searching a drug repurposing [38] . Besides,

proposing combinations of drugs, with different mechanisms of action (such as those mentioned), will be used for the pandemic that is occurring, in addition, will be necessary to develop selective drugs against ACE2 ( Figure S23) , which may be able to prevent interaction with SARS -CoV-2.

Carrying out the selection of the compounds, taking into account the results of between 15 to 20 conformers of each compound, gives us a greater probability of choosing the compounds that could be selective in the amino acids sequence Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353 and Arg357 in ACE2 (Figure 1 ), subsequently validate them by two toxicity prediction web servers ( Table 2 and Table S22 ), obtaining important characteristics such as a lethal dose 50 (LD50) within acceptable values as well as a very low probability of toxicity; which must be fulfilled by each compound for its selection. Thus, this could reduce the time that must be waited for to be used in humans, therefore we propose compounds (C1 -C20), with a high probability to be safe in humans. In addition, we show the next 30 compounds, which have some probability of generating side effects, such as carcinogenicity, hepatotoxicity and immunotoxicity mainly; these compounds are on the supplementary material (Table S21 ) which could be tested in in vitro tests with ACE2 -SARS-CoV-2

interaction.

It will be necessary to evaluate by in vivo tests, the effect of these compounds could to generate when interacting with ACE2 in humans, since the ACE2 functions on angiotensin and its effects at the cardiovascular system level [39] [40] [41] [42] [43] , they would have to be considered to determine the therapeutic effect and the degree of impact that they could have on the health-disease process of COVID-19 and/or some alteration in the functions of ACE2.

Most of these proposed compounds do not have any specific use registered, nor a scientific article or registered patent, all the compounds are available to purchase or J o u r n a l P r e -p r o o f systematize them, to carry out in vitro assays for the interaction of SARS-CoV-2 with ACE2, and in this way, be able to develop a new drug that helps combat this pandemic. Furthermore, as already reported, SARS-CoV have an affinity for the same ACE2 region, which could help in the future to prevent new viruses that have an affinity for this region of interaction in ACE2.

We propose 20 compounds that have a high probability of interacting in a specific region in ACE2 (Gln24, Asp30, His34, Tyr41, Gln42, Met82, Lys353 and Arg357), and thus hinder interaction with the RBD of SARS-CoV-2. Furthermore, these 20 compounds have a high probability to be safe in humans, since they were validated by the ProTox-II and PreADMET server (ADME and Toxicity Predictor). These 20 compounds are available from Chembridge

Corp. (Table 1) J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Table S23 : ADME -PreADMET | Prediction of ADME/Tox of compounds C1 -C20. Table S24 : Toxicity -PreADMET | Prediction of ADME/Tox of compounds C1 -C20.

Declarations Life Sciences require that the corresponding author, signs on behalf of all authors, a declaration of conflicting interests. If you have nothing to declare in any of these categories then this should be stated.

A conflicting interest exists when professional judgment concerning a primary interest (such as patient's welfare or the validity of research) may be influenced by a secondary interest (such as financial gain or personal rivalry). It may arise for the authors when they have financial interest that may influence their interpretation of their results or those of others. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding.

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