Emerging viruses can be defined as those whose incidence has increased in the last twenty years or whose presence has a high probability of increasing in the near future. Diseases caused by emerging viruses are one of the biggest public health threats globally. Some of the viruses that fall within this catalog are the avian influenza virus subtype H5N1, severe acute respiratory syndrome (SARS), Ebola, Zika, and MERS-CoV, to name a few ¹ . Coronaviruses (CoVs) are classified into four genera, α-CoV, β-CoV, γ-CoV, and δ-CoV2. α and β infect mammals, γ birds and δ birds and mammals, respectively ² . These viruses are of public health importance because they cause enteric, renal, and neurological respiratory diseases that range from asymptomatic to fatal ^{3, 4} .

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared in Wuhan, China, in December 2019, causing cases of SARS-like atypical pneumonia ^{5, 6} , with a clinical picture of fever, general malaise, dry cough, shortness of breath and was called the coronavirus disease 2019 (COVID-19) ⁷ . It can be asymptomatic, develop mild-to-severe symptoms, or cause death in elderly individuals or in patients with chronic diseases, such as hypertension, diabetes, and obesity ⁸ . On January 31 ^st 2020, the World Health Organization (WHO) declared COVID-19 a public health emergency of international concern and, on March 12 ^th, it was declared a global pandemic ⁹ . In Mexico, local transmission (phase 2 of transmission) was declared on March 24 ^th 2020, which resulted in the suspension of non-essential activities in the country, generating economic losses in addition to public health problems and deaths associated with the disease. As of March 1 ^st 2021, Mexico had reached 2.1 million cases of COVID-19 and 189 thousand deaths; around 63.6 million cases and 1.47 million deaths have been reported worldwide.

To date, there is no specific commercial treatment to control the infection ^{10– 12} . Measures such as early detection, blocking the route of transmission through social isolation, isolation of suspected cases, disinfection of objects, as well as frequent hand washing with soap, in addition to the use of biosafety equipment such as surgical masks for health personnel, which has been effective in reducing the transmission of COVID-19.

Coronaviruses, such as SARS-CoV-2, are positive-stranded RNA viruses enveloped on a membrane. The coronaviral genome is composed of approximately 30,000 nucleotides containing the envelope (E), membrane (M), spike (S), nucleocapsid (N) and ORFs , that encode non-structural proteins, including enzymes that appear during their in-host reproductive cycle-genes ¹³ .

This virus measures 70 to 100 nm and belongs to the genus β-CoV ¹⁴ and it has been proposed that any of the aforementioned proteins that make up CoVs may be targets for the development of vaccines or drugs ⁴ . Protein S plays an essential role on COVID-19 infection as it mediates the internalization on host cell and for the spread of the virus in the infected host ^{15, 16} . This starts when the receptor binding domain of the S1 subunit (RBD _S1) of S protein binds to the peptidase domain of angiotensin-converting enzyme 2 receptor (ACE2) ¹⁷ and it is know that disrupting the binding of S protein to ACE2 prevents the attaching an the later internalization of the virus to the host cell ¹⁸ .

This protein interaction has recently been crystallized and deposited in the Protein Data Bank database ¹⁹ , allowing us to use it as a model of study to test different strategies to counter SARS-CoV-2 infection, like blocking S glycoprotein-ACE2 interaction through the discovery of sites of potential pharmaceutical interest.

In 2019, Research and Development (R&D) spending in the pharmaceutical industry totaled 186 billion U.S. dollars globally and its projected to reach 233 billion U.S. dollars to 2026. Unfortunately, drug development takes large time and financial resources that not all countries possess, especially developing countries, like Mexico.

In this sense, drug repurposing or repositioning allow us to integrate all evidence, pharmacodynamics/kinetics, bioavailability, among other important parameters, from an existing and approved drug in order to manage emerging diseases, like COVID-19. All this translates into a considerable decrease in research time and investment of resources in R&D.

Different approaches have been taken in order to disrupt SARS-CoV-2 protein S-ACE2 interaction, as an example, many works has focus on finding potential biding sites on protein S structure, however, new variant strains has been detected worldwide, like B117 in UK, P1351 in South Africa, P1 and P2 in Brazil. All variant strains display the N501Y mutation, which is located on the RBD of the S protein, making the interaction more effective. In this sense, targeting RBD may be a transitory approach, therefore, an alternative strategy would be aiming at the ACE2 receptor. Some authors have pointed out some concerns about using drugs that targeting the renin–angiotensin signaling (RAS) pathway ^{20– 22} , but Jia and collaborators ²³ highlight current efforts of exploiting ACE2 as therapeutic target, like the use of pseudo-ligands to dominate the binding site for SARS-CoV-2 as an example. Therefore, inhibition of the SARS-CoV-2 protein S-ACE2 interaction trough aiming ACE2 receptor it is a plausible strategy. In this study, we screened a library consisting of 1300 FDA-approved drugs and acquired by Ministry of Health of Mexico in order to identify potential inhibitors of SARS-CoV-2–ACE2 interaction.

It has been established that S protein of SARS-CoV-2 virus plays a major role during viral infection. The S protein mediates receptor recognition, cell attachment and fusion of viral membrane with host cell membrane ^{24– 29} . The S protein binds to ACE2 receptor through the RBD _S1, mediating viral attachment to host cell ³⁰ . ACE2 expression it is distributed mainly in lung, intestine, heart and kidney, also alveolar epithelial type II cells had higher expression levels ²¹ . The RBD region is a critical therapeutic target (vaccines and drugs) due to its indispensable function; however, it is suggested that mutations in this region may render pharmacological or immunological therapies ineffective ^{31, 32} .

In order to block this event, we propose an in silico approach to identify potential inhibitors of the SARS-CoV-2 –ACE2 interaction aiming at the ACE2 receptor, blocking the virus accessibility to the membrane-bound ACE2. Here, we screened a drug library consisting of 1300 drugs, FDA approved and prescribed by the Mexican Public Health System, for potential SARS-CoV-2−ACE2 inhibitors, using a rigid receptor docking approach. Utilization of an FDA-approved drug library is an effective and ideal tool for drug repurposing in antiviral research ^{33, 34} , such as zika virus ³⁵ , human rhinovirus ³⁶ and hepatitis B virus ³⁷ . We identify 38 potentially inhibitor drugs of SARS-CoV-2−ACE2 interaction and these are listed on Table 2. Several of those drugs were previously reported to be used for the treatment of respiratory diseases.

Within this list of potential inhibitors of the SARS-CoV-2−ACE2 interaction, is fexofenadine, a third-generation antihistame whose therapeutic indication is the treatment of symptoms of stationary allergies through the selective blockade of H1 receptors ³⁸ . In silico evidence ^{39, 40} suggest that it may interact with the SARS-CoV-2 main protease enzyme M ^Pro, a key enzyme in viral replication ⁴¹ , acting as a potential inhibitor. Cefixime is a third-generation antibiotic derived from cephalosporin whose use is indicated for the treatment of infections in the upper and lower respiratory tract, otorhinolaryngological ⁴² and urinary tract ⁴³ , inhibiting the synthesis of the bacterial wall by binding to specific binding proteins for penicillin and is currently used as a secondary therapy to prevent opportunistic infections during the development of COVID-19 ⁴⁴ . Pitavastatin is a statin indicated for lowering blood cholesterol levels by inhibiting HMG-CoA reductase, preventing cholesterol synthesis ⁴⁵ , and it has also been observed that statin treatments can interfere with viral infectivity through inhibition of glycoprotein processing ⁴⁶ also, they modulates the inflammatory process at cellular level ⁴⁷ , which is a remarkable characteristic of the SARS-CoV-2 infection. Additionally, in silico findings suggest that could be an efficient inhibitor of SARS-CoV-2 M ^{Pro 48} and SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) ⁴⁹ thru active site binding. Lopinavir is a protease inhibitor indicated as first barrier therapy, in conjunction with Ritonavir, to treat infection caused by the HIV virus by inhibiting the HIV-1 protease ⁵⁰ in addition, studies in cell cultures have shown its effectiveness as an inhibitor of the replication of the MERS-CoV virus ⁵¹ and SARS-CoV-1 ⁵² , while in severe cases of SARS-CoV-2 infection, the results of clinical trials indicate that it is not useful ⁵³ . Formoterol and aformoterol, anenantiomer of formoterol, are long-lasting selective β agonists indicated for the treatment of chronic obstructive pulmonary disease (COPD) and bronchospasms ⁵⁴ , in the same way there is evidence of the use of these drugs as a partial inhibitor of viral replication in primary epithelial cells cultures ⁵⁵ and in silico data suggest their binding to the papain-like protease PL _pro, a coronavirus enzyme essential for viral spread ⁵⁶ . Ipratropium is a bronchodilator anticholinergic indicated for the treatment of asthma, shortness of breath, cough and tightness in the chest in patients with COPD ^{57, 58} . Inhalation therapy with ipratropium is currently in use to dilate bronchioles in COVID-19 patients to increase oxygen saturation levels (from <80% to 94%) ⁵⁹ . Pargeverine is an antispasmodic opioid alkaloid whose therapeutic indication is aimed at the treatment of painful spasms ⁶⁰ , also, acts as anticholinergic and has a moderate and non-selective blockade of muscarinic cholinergic fibers ⁶¹ . Since cholinergic activity contribute to airway narrowing, this might be a potential agent to open airway obstruction. Cholecalciferol, is a form of vitamin D (vitamin D3) that can be synthesized naturally in the skin and acts as a hormonal precursor, being converted into calcitriol, and therapeutically is used as a vitamin supplement to treat deficiencies of this vitamin ⁶² . In addition, it has been observed that vitamin D supplementation is favorable to reduce viral infections such as influenza ^{63, 64} or more aggressive cases such as HIV ⁶⁵ and it has recently been suggested that it also presents favorable effects before and during the infection caused by SARS-CoV-2 ⁶⁶ .

Likewise, it is important to take into account that ACE2 plays an important biological role since regulates cardiovascular functions and innate immune system and, therefore cautions must be taken. Another point to take into account is the delivery method of the drug, since the primary target must be smooth muscle, like the one surrounding the bronchioles, and lung epithelial cells in the airway and airspace compartments, hence, inhalable delivery would be the acceptable choice to deliver the drug in a selectively and localized manner.

Given these characteristics, the results obtained through our in silico approach, we consider that the aforementioned drugs are outlined as possible inhibitors of the RBD _S1-ACE2 interaction. These drugs are well tolerated, commonly used and affordable, hence, most of the drugs on this list can be tested in vitro, and even in vivo and, consequently, in clinical trials for the development of adjuvant therapies to treat COVID-19.

In the absence of approved therapies for treatment or prevention, drug repurposing has provided fast and valuable insight into the treatment of COVID-19. Targeting ACE2 receptor as a COVID-19 therapy it is a conceivable approach since it is essential for the viral internalization. However, this approach requires an integrative evaluation of the pros and cons by a clinical context since ACE2 is a multifunctional protein. Jia and collaborators ²³ present an extensive review for this underexplored approach to treat COVID-19, pointing that it is imperative to determine, by clinicians, the stage of the disease and comorbidities that could prove consequential for an ACE2-targeting regimen. Several drugs are currently investigated by clinical trials or are already in use to treat COVID-19 patients, like lopinavir or ipratropium. In this in silico study using structure-bases virtual screening, we identified potential inhibitors of SARS-CoV-2–ACE2 by their interaction with ACE2 receptor. We identify the uridine trisodium salt, methotrexate sodium, raltritedex, folotyn, CDP-choline(1-), cefuroxime, fexofenadine, fludarabine phosphate, cefixime, aloin, domperidone, tamsulosin, cromoglycic acid, macitentan, tafluprost -taflutan, thiopental(1-), metoprolol, irinotecan, pitavastatin(1-), amlodipine, verapamil, tolterodine, lopinavir, glimepiride, arformoterol, formoterol, ipratropium, pargeverine, pyrilamine, biperiden, orlistat, glyburide, ribociclib, ibesartan, cholecalciferol, gravignost, disopyramide phosphate and primaquine. Based on desired characteristics like pharmacokinetics, route of administration or by their background as pharmacological therapy, we propose a shortlist of drugs suitable for testing their potential RBD _S1 –ACE2 inhibitory activity: fexofenadine, cefixime, pitavastatine, lopinavir, arformoterol, formoterol, ipratropium, pargeverine and cholecalciferol. Our identification of potential inhibitors of the SARS-CoV-2–ACE2 interaction among commonly use drugs highlights their potential use for treating COVID-19. Further in vitro, in vivo or clinical trial are needed to validate their potential use as inhibitors of SARS-CoV-2–ACE2 interaction.