key: cord-1018804-c0gfuv6d
authors: Heimfarth, Luana; dos Santos, Mario Adriano; Barreto-Filho, José Augusto; Barreto, André Sales; Macedo, Fabrício Nunes; Araújo, Adriano Antunes de Souza; Martins-Filho, Paulo; Scotti, Marcus Tullius; Scotti, Luciana; Quintans-Júnior, Lucindo José
title: Insights into the actions of angiotensin-1 receptor (AT1R) inverse agonists: Perspectives and implications in COVID-19 treatment
date: 2021-02-08
journal: EXCLI J
DOI: 10.17179/excli2021-3412
sha: 520e4caa668b3efe6e5923361dfbf917b6d36582
doc_id: 1018804
cord_uid: c0gfuv6d

New coronavirus SARS-CoV-2 (COVID-19) has caused chaos in health care systems. Clinical manifestations of COVID-19 are variable, with a complex pathophysiology and as yet no specific treatment. It has been suggested that the renin-angiotensin-aldosterone system has a possible role in the severity of cases and the number of deaths. Our hypothesis is that drugs with inverse agonist effects to the angiotensin-1 receptor can be promising tools in the management of patients with COVID-19, possibly avoiding complications and the poor evolution in some cases. Any risk factors first need to be identified, and the most appropriate time to administer the drugs during the course of the infection also needs to be established. Several angiotensin receptor blockers (ARB) have a favorable profile and are important candidates for the treatment of COVID-19. In this review we discussed a set of compounds with favorable profile for COVID-19 treatment, including azilsartan, candesartan, eprosartan, EXP3174, olmesartan, telmisartan, and valsartan. They are effective as inverse agonists and could reduce the “cytokine storm” and reducing oxidative stress. As COVID-19 disease has several evolution patterns, the effectiveness of ARB therapy would be related to infection "timing", patient risk factors, previous use of ARBs, and the specific molecular effects of an ARB. However, controlled studies are needed to identify whether ARBs are beneficial in the treatment of patients with COVID-19.

Sudden or emerging pathogen outbreaks have always been a challenge to public health worldwide. A recent outbreak of coronavirus, identified as SARS-CoV-2, started in Wuhan, China and quickly spread to almost all other countries. According to the WHO, 76,250,431 cases have been confirmed globally, with 1,699,230 deaths up to December, 22, 2020 (WHO, 2020 . Most non-survivors were older (>80 years old) with severe other diseases and more likely to develop acute organ dysfunction (Guo et al., 2021) . In addition, extremely low lethality was observed in young patients (Flacco et al., 2020) . During the last five months, various studies about coronavirus disease 2019 (COVID-19, the disease caused by the SARS-CoV-2 virus) have described its clinical features, reported laboratory findings and provided diagnostic evaluations of the disease. COVID-19 mortality rates vary by country, and are also influenced by the clinical profile of patients, and the presence of other comorbidities (Carlino et al., 2020; Scholz et al., 2020) . No proven effective drug against SARS-CoV-2 has yet been found and treatment is based on symptom management and life support, although some advances have been made with the use of anticoagulants and dexamethasone-type corticosteroid drugs. Thus, the lethality of the disease for patients with comorbidities, older adults and those with secondary infections is still a challenge in those who develop the severe form of COVID-19 (Iaccarino et al., 2020) .

The clinical manifestations of COVID-19 are extremely complex and variable. SARS-CoV-2 infection may be asymptomatic or cause mild symptoms such as fever, dry cough, shortness of breath, headache, dyspnea, diarrhea and vomiting (Huang et al., 2020) . In some patients, the SARS-CoV-2 infection can have a worse prognosis, requiring hospitalization (Bloomgarden 2020; Cossarizza et al., 2020) . In severe cases, COVID-19 may progress to acute respiratory distress syndrome (ARDS), followed by refractory metabolic acidosis, coagulation dysfunction, septic shock, multiple organ failure and, consequently, death (She et al., 2020; Wang et al., 2020) . Despite the tropism of SARS-CoV-2 for the respiratory airways , several other organs are also affected by the virus, explaining the cases of multiorgan failure. The cardiovascular and renal systems appear to have complex interactions with COVID-19, making patients more predisposed to severe cardiovascular damage or renal failure (Deep et al., 2020; Tomasoni et al., 2020) . Recent retrospective clinical findings have established an association between the incidence of vascular thrombosis or thromboembolic events and COVID-19 severity (Huang et al., 2020; Tang et al., 2020) .

In addition, some biological mechanisms that occur during SARS-CoV-2 infection are thought to be pathogenic and could be associated with poor prognosis of the disease. With disease progression, infiltration of neutrophils, macrophages, and red blood cells (Matthay et al., 2019) and the accumulation of protein-rich edema in the alveoli occurs, causing local production of pro-inflammatory cytokines associated with adaptive and innate immune cells. These factors contribute to the exaggerated recruitment of inflammatory cells, protease release, cytokine production and oxidative stress, that are responsible for the disruption of the blood-alveolar barrier, intrapulmonary hemorrhage, pulmonary edema, and the dangerous impairment of gas exchange (Channappanavar and Perlman 2017) , provoking massive injuries in lung microvascular endothelial and epithelial cells (Matthay et al., 2019) . This excessive uncontrolled inflammatory response seems to be related to ARDS, multiorgan failure and even the death of patients with COVID-19.

The role of the renin-angiotensin-aldosterone system (RAS), particularly the role of ACE2 (angiotensin-converting enzyme 2) receptors, has been studied in SARS-CoV infections and appears to be associated with the progression of COVID-19 (McMillan and Uhal 2020; Ni et al., 2020) . ACE2 is an enzyme that catalyzes the conversion of angiotensin I to angiotensin (1-9) and angiotensin II to angiotensin (1-7) and plays a key role in the renin-angiotensin-aldosterone system (RAS) (Voors et al., 1998) . Ang II, the main active RAS component, acts on angiotensin-II type 1 receptors (AT1R), exerting its physiological (Keidar et al., 2007) and antagonistic effects through the angiotensin-II type 2 receptors (AT2R). SARS-CoV binds to the ACE2 receptor to achieve intracellular invasion and infectivity (Ni et al., 2020; Tang et al., 2020) with the virus using S protein priming by the transmembrane protease serine 2 (TMPRSS2) for cell infection. TMPRSS2 activity is crucial for viral spread and pathogenesis in the infected host and seems to be responsible for the ease of transmissibility of SARS-CoV-2 (Matsumoto et al., 2003; Hoffmann et al., 2020) .

ACE2 is also highly expressed in the kidneys, heart, respiratory tract, and gastrointestinal tract tissues, with lower expression levels in the brain and blood cells (Hamming et al., 2004; Gkogkou et al., 2020) . The expression of ACE2 is responsible for the susceptibility of human cells to SARS-CoV-2 (Hardenberg and Luft 2020). In addition, ACE2 is expressed at the same sites where pro-inflammatory cytokines released following SARS-CoV infection are produced (He et al., 2006) . It is associated with the pulmonary tropism of the viruses found in diseases caused by other similar coronaviruses . Thus, the modulation of ACE2 activity and its secondary effects may exert a protective role against cardiac and/or lung injury, ARDS and other COVID-19 complications. However, the correlations between the different stages of SARS-CoV-2 infection and the RAS system remain to be clarified (Vitiello and Ferrara, 2020) .

The detrimental actions of the Ang II AT1R-mediated inflammatory response have been demonstrated in various models of ARDS SARS-CoV-induced acute respiratory failure Kuba et al., 2005) . Moreover, some studies have shown that experimentally ARDS and lung fibrosis can be prevented by administration of Angiotensin-II receptor antagonists (ARBs), limiting pulmonary disease progression (Wösten-van Asperen et al., 2011) . This implies that ARBs could act in SARS-CoV-2 infection, modifying disease progression (Dublin et al., 2020) . In addition, ARBs possess inverse agonist properties that give them an additional pharmacological effect and improves drug efficacy . Thus, drugs that act on the AT1R have been proposed as a treatment for COVID-19 (Gurwitz, 2020) . Interestingly, Zhang et al. (2020) found that among COVID-19 patients hospitalized with hypertension, inpatient treatment with ACEI/ ARB was related to a lower risk of all-cause mortality.

Although the role of the RAS has been extensively studied in COVID-19 patients, there are unquestionable gaps of information in relation to this topic, especially regarding the role of AT1-inverse agonists and their action in SARS-CoV-2 infection. We, therefore, performed a review of the AT1-inverse agonists that could be of benefit in the treatment of patients with severe COVID-19. Based on our examination of basic and clinical studies, we hypothesize that angiotensin-1 receptor (AT1R) inverse agonist drugs may be a promising tool in the management of COVID-19 patients, avoiding complications and the development of the severe phase of the disease, and the subsequent poor outcomes.

An inverse agonist is defined as a ligand that binds to the same receptor as the agonist but induces opposite effects . While the agonist molecule stabilizes receptor conformations that increase signaling through G proteins, the inverse agonists promote other conformations that decrease the basal, agonist-independent level of signaling and modulate the response, besides suppressing stimuli from the agonist (Milligan 2003; Akazawa et al., 2009) . The agonist is a molecule that binds and activates a receptor to produce a biological response, whereas the antagonist inhibits the action of the agonist (Figure 1 ). In this context, we can highlight AT1-inverse agonists that may exhibit enhanced therapeutic effects for various disease states (Takezako et al., 2017) . Studies have shown that several G protein-coupled receptors (GPCRs), including the AT1R, show spontaneous activity, even in the absence of an agonist ). AT1-inverse agonists regulate several important signaling pathways, leading to anti-inflammatory (Gupta et al., 2020) , anti-apoptotic (Kanamori et al., 2007) , antioxidant (Occhieppo et al., 2020) , and immunomodulatory (Yuan et al., 2016) effects, and consequently opposing the effects of angiotensin II. This evidence supports the potential actions of AT1-inverse agonists in the fight against COVID-19.

Several ARBs have shown inverse agonist effects and favorable profiles for COVID-19 treatment, including azilsartan (Takezako et al., 2017) , candesartan (Miura et al., 2006; Qin et al., 2009a) , eprosartan (Takezako et al., 2017) , EXP3174 (Miura et al., 2006; Qin et al., 2009) , olmesartan (Miura et al., 2006; Qin et al., 2009) , telmisartan (Takezako et al., 2017) , and valsartan (Takezako et al., 2017) , with each drug having different features and efficacy as inverse agonists (Takezako et al., 2018) . Table 1 and 2 summarize the main molecular and physiologic actions of AT1-inverse agonists, and Figure 1 shows their chemical structure.

Azilsartan is a drug approved worldwide for the treatment of hypertension, either as a prodrug (azilsartan medoxomil) or a primary compound (Pradhan et al., 2019) and has more affinity to the AT1R than to the AT2 receptor. It is one of the most effective approved ARBs tested to date in relation to the reduction of blood pressure (Al-Majed et al., 2020) . Azilsartan could provide an effective treatment against COVID-19 by ameliorating the deleterious effects of angiotensin II, such as cardiac hypertrophy, fibrosis, and insulin resistance (Arumugam et al., 2016) . In addition, it increases left ventricular diastolic function (Sakamoto et al., 2015) , reduces cardiovascular sympathetic activity (Kusuyama et al., 2014) , and restores endothelial function (Matsumoto et al., 2003) . Therefore, these cardiovascular protective actions could help in the complications that have been found in SARS-CoV-2 patients (Table 1) .

Moreover, the modulation of Ang II could be a key element in the immunomodulatory and anti-inflammatory effects of azilsartan, making it an attractive candidate for mitigating the inflammatory condition observed in SARS-CoV-2 patients. This drug attenuates the release of IL1-β, TNF-α and IL-6 pro-inflammatory cytokines, as well as increasing the production of the important anti-inflammatory molecule IL-10. Azilsartan also downregulates ROS formation, protecting the tissue from oxidative damage (Liu et al., 2016; Gupta et al., 2020) , and it is able to inhibit the apoptotic pathway by blocking caspase activation (Gupta et al., 2020) (Table  2) . Azilsartan, therefore, presents anti-inflammatory, antioxidant and anti-apoptotic profiles that could contribute to the suppression of multiorgan failure, and disseminated intravascular coagulation, reducing the severity of SARS-CoV-2 infection.

Candesartan is an oral selective AT1R blocker available as a pro-drug, candesartan cilexetil, which undergoes hydrolysis in the gastrointestinal tract during absorption to its active form. Candesartan also acts as an inverse agonist, decreasing the basal activity of the AT1R. Accumulating evidence has demonstrated that candesartan could be a very attractive candidate for COVID-19 treatment due to its anti-inflammatory (Yu et al., 2019) , 2019), anti-apoptotic (Goel et al., 2018) and antioxidant (Occhieppo et al., 2020) activities.

257 Zhang et al. (2020) and Khuman et al. (2016) showed that candesartan protects the cardiovascular system from stroke, myocardial infarction, atherosclerosis, and hypertension by modulating vascular remodeling, thereby preventing the vascular damage-induced by SARS-CoV-2. Furthermore, intermediate to high doses of candesartan ameliorate the progression of nephropathy, showing renoprotective properties (Callera et al., 2016) . According to Kim and Im (2019) candesartan ameliorates pulmonary injury by attenuating eosinophil infiltration and inhibiting mucin production in the lung after damage.

In addition, candesartan modulates several inflammatory pathways (Gong et al., 2019; Kim and Im, 2019) and could reduce the cytokine storm induced by SARS-CoV-2 infection. Kim and Im (2019) and Gong et al. (2019) reported that candesartan attenuates IL1-β, IL-2, IL-4, IL-5, IL-6, IL13 and IFN-γ production and upregulates IL-10 secretion. Moreover, this ARB could block NKκB (Ahmed and Mohamed, 2019) and COX-2 activation, as well as inhibit iNOS (Gong et al., 2019) , decreasing the production of inflammatory mediators.

Candesartan could also mitigate oxidative imbalance by reducing ROS formation (Occhieppo et al., 2020), attenuating oxidative stress, and restoring cellular homeostasis. The reduction in ROS generation causes a decrease in lipid oxidation and, consequently, protects cell membrane integrity and ion transportation across them. Moreover, candesartan inhibits the apoptotic pathway (Goel et al., 2018) and MAPK activation (Ahmed and Mohamed, 2019), attenuating cell death and cellular homeostasis disruption. Thus, the maintenance of oxidative and cellular homeostasis, as well as the reduction in inflammatory process, induced by candesartan could be associated with a reduction in the severity of COVID-19.

Eprosartan is a nonbiphenyl nontetrazole AT1R blocker routinely used in the treatment of hypertension. Although various ARBs have inverse agonism, eprosartan could be considered a better therapeutic option than other ARBs for the treatment of diseases due to its capacity to act in the active state of the AT1R (Takezako et al., 2018) . Studies have shown that eprosartan may protect the cardiovascular, renal and pulmonary systems from inflammatory (Mukaddam-Daher et al., 2009) , oxidative (Morsy et al., 2015) and apoptotic (Mukaddam-Daher et al., 2009) damage. Barone et al. (2001) reported that eprosartan reduces cardiac hypertrophy and heart failure and preserves cardiac and renal structural integrity and maintains normal function of the heart and kidneys. Furthermore, this ARB significantly decreased platelet activation and endothelial dysfunction in patients, protecting against organ failure.

According to Mukaddam-Daher et al. (2009) and Labiós et al. (2008) , eprosartan mitigates inflammatory and oxidative conditions, respectively. This AT1R blocker reduces the production of the inflammatory mediators IL1-β, TNF-α (Mukaddam-Daher et al., 2009) and oxide nitric (Morsy et al., 2015) . Furthermore, eprosartan decreases ROS formation by upregulating enzymatic and non-enzymatic antioxidant defenses, attenuating tissue lipoperoxidation and protecting the cell from apoptosis (Mukaddam-Daher et al., 2009; Morsy et al., 2015) . Therefore, eprosartan could be suggested for the treatment of COVID-19 patients due to its capacity to mitigate the "cytokine storm" by modulating the production of cytokines, mainly IL-6, and reducing oxidative stress. Increased cytokine levels have been closely correlated with ARDS severity and a worse prognosis in COVID-19.

Losartan is metabolized by cytochrome P450 enzymes to active 5-carboxylic acid derivative EXP3174. This compound is an active metabolite of losartan, with a higher affinity to AT1 and a longer half-life than losartan (Stearns et al., 1995; Wong et al., 1996) . Thus, the pharmacological activities of losartan are predominantly mediated by EXP3174 (Wong et al., 1996) . Similar to other ARBs, losartan and its active metabolite could reduce organ failure, and protect tissues from damage caused by SARS-CoV-2-infection (AlSaad et al., 2020; Xin et al., 2020) . It has also been found that EXP3174 plays a role in preventing heart failure, as well as reducing hypertension (Timmermans, 1999 ). In addition, losartan and EXP3174 have been reported to inhibit platelet aggregation, showing antithrombotic activity (Matys et al., 2003) . According to Poisner et al. (2018) losartan, and probably its metabolite EXP3174, prevented vasculitis and inflammatory processes in the lung, showing that these compounds could protect against acute respiratory distress syndrome and severe COVID-19.

Additionally, EXP3174 modulates IL-6, IL-1β, TNF-α, IFN-γ and TGF-β secretion (Lin et al., 2014; Wang et al., 2019a; AlSaad et al., 2020) , and downregulates MAPK and NFκB pathway activation (Wang et al., 2019a) . Moreover, losartan and EXP3174 were able to ameliorate organ injury by increasing catalase, glutathione peroxidase and superoxide dismutase activity, and consequently decreasing ROS generation and oxidative tissue damage (Lin et al., 2014; AlSaad et al., 2020) . According to Xin et al. (2020) losartan can also prevent organ failure by inhibiting the activation of the intrinsic apoptotic cascade, thereby reducing cell death. Therefore, Exp3174 could be an important candidate in the management of COVID-19 patients due to its capacity to block the fulminant response by the immune system and suppress multiple organ failure, contributing to a reduction in the severity of cases.

Olmesartan is a 3 rd generation angiotensin-II receptor blocker approved by the FDA for the treatment of mild to severe hypertension. It has been shown that olmesartan prevents ventricular hypertrophy and fibrosis in hypertensive rats with advanced heart failure (Yoshida et al., 2004) , and inhibits vascular remodeling, immune cell infiltration, and endothelial dysfunction, attenuating the severity of the heart injury (Chen et al., 2020) . Moreover, studies have reported that olmesartan exerts renoprotective effects (Si et al., 2014) and ameliorates lung damage by the modulation of the pro-fibrogenic cytokine TGF-β1, and by its antioxidant effect (Sharaf El-Din and Abd Allah, 2016).

In addition, olmesartan might also be a promising therapeutic approach in SARS-CoV-2 infection due to its capacity to modulate ACE2 expression (Araújo et al., 2018) and to regulate inflammatory, oxidative and apoptotic signaling pathways (Kanamori et al., 2007; Lakshmanan et al., 2012; Saber et al., 2019b) , leading to a reduction in multiorgan failure. Studies suggest that olmesartan can mitigate the "cytokine storm" since this drug decreases the production of IL1-β and TNF-α, and particularly IL-6 and TGF-β1. It also elevates anti-inflammatory cytokine IL-10 levels (Sharaf El-Din and Abd Allah, 2016; Saber et al., 2019b) . In fact, this ARB inhibits important inflammatory molecules that play a pivotal role in the inflammatory cascade, including the MAPK family and the transcriptional factor NFκB (Lakshmanan et al., 2012; Araujo et al., 2018) , blocking the inflammatory process, and, consequently, inflammatory disease progression. Moreover, this ARB was able to restore redox homeostasis by upregulating the cellular antioxidant system, and consequently suppressing reactive oxygen species (ROS) generation and oxidative damage (Saber et al., 2019b) . Olmesartan may inhibit the inflammation and oxidative stress induced by SAR-CoV-2 infection, reducing cell death and, consequently, the multiorgan failure observed in severe cases of COVID-19. This is supported by the results of a study by Kanamori et al. (2007) that found that olmesartan downregulates the extrinsic and intrinsic apoptotic pathways by inhibiting caspase activation and reducing BAX and FAS expression. Therefore, olmesartan could inhibit the fulminant response by the immune system and suppress multiorgan failure, contributing to a reduction in the severity of the disease.

Recently, Gurwitz (2020) proposed the use of telmisartan as an alternative option for the management of COVID-19 patients prior to development of ARDS. This ARB produces marked suppression of inflammation, oxidative stress and apoptosis, leading to a decrease in cellular damage and multiorgan injury (Takeuchi et al., 2013; Saber et al., 2019a; Zhang et al., 2019) .

Telmisartan is widely used for the treatment of patients with hypertension with concomitant diabetes mellitus (Wang et al., 2019b) . This drug has a cardioprotective effect due to its capacity to inhibit inflammatory markers, myocardial apoptosis, and oxidative and endoplasmic reticulum stress, which results in an improvement in myocardial function (Sukumaran et al., 2011) . Moreover, telmisartan prevents pulmonary ischemia and reperfusion injury, decreases pulmonary vascular resistance and improves cough symptoms (Fan et al., 2016) , protecting the lung from oxidative and inflammatory injury. According to Zhang et al. (2019) it reduces renal apoptosis and autophagy, ameliorating the renal impairment caused by chronic intermittent hypoxia.

Compelling evidence suggests that telmisartan possesses an anti-inflammatory and antioxidant profile (Takeuchi et al., 2013; Choe et al., 2019; Saber et al., 2019a) . Saber et al. (2019a) showed that this ARB reduces IL1-β, TNF-α and IL-6 levels, and elevates IL-10 production, attenuating the inflammatory process by downregulating NFκB nuclear translocation/activation. Furthermore, telmisartan blocks myeloperoxidase activation and consequently reduces the infiltration of inflammatory cells into the injured organ (Saber et al., 2019a) , mitigating oxide nitric production (Choe et al., 2019) . Additionally, telmisartan also stimulates the endogenous antioxidant system, reducing ROS formation (Saber et al., 2019a) and oxidative tissue damage. According to Takeuchi et al. (2013) and Zhang et al. (2019) , this drug mitigates tissue injury by downregulating caspase pathway activation, reducing the number of apoptotic cells and death. Table 1 and 2 summarize the main molecular and physiologic actions of telmisartan. Taken together, the evidence suggests that telmisartan may inhibit inflammation, oxidative stress and the apoptotic signaling pathway induced by SARS-CoV-2 infection, thereby reducing cell death and the multiorgan failure observed in the most severe cases of COVID-19.

Valsartan is an angiotensin-II-receptor antagonist with specificity for the AT1R subtype and is commonly used for the treatment of cardiovascular disease. It demonstrates antihypertensive effects and slows the progression of chronic heart failure (Croom and Keating, 2004) . This ARB protects against cardiovascular, pulmonary and renal injury, mitigating organ failure through its ability to modulate the RAS system and inflammatory and oxidative mediators . Valsartan modulates the mRNA expression of ACE and AT1R , resulting in RAS inhibition and the prevention of renal and cardiac damage (Ulutas et al., 2021) . Moreover, studies have shown that valsartan attenuates pulmonary fibrosis in rats by blocking the NFκB signaling pathway and regulating the ratio of antifibrotic/profibrotic cytokines, resulting in an improvement in pathological changes in lung tissue (Mojiri-Forushani et al., 2018) .

In this context, Mohany et al. (2020) showed that rats treated with valsartan presented a substantial reduction in the levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and decreased activation of NF-kB. Moreover, the same authors reported that this drug restores antioxidant enzyme levels, indicating that the antioxidant effect of valsartan could mitigate cellular oxidative damage. Finally, some studies have reported that this ARB can modulate the expression of antiapoptotic and pro-apoptotic proteins, decreasing caspase activation, and, consequently, cell death (Sakr et al., 2016) . Table 1 and 2 summarize the main molecular and physiologic actions of valsartan, showing the main effects that could contribute to mitigating the effects of COVID-19, based on the significant anti-inflammatory and antioxidant profile of the drug. Valsartan could, therefore, block the "cytokine storm" and consequently suppress the fulminant response by the immune system and multiple organ failure, and be a promising possible treatment in the alleviation of COVID-19 symptoms.

Computational molecular docking is a commonly used tool in drug repurposing studies and performs a structure-based computational analysis based on the binding efficiency predicted between a drug and its target molecule (Elfiky, 2020) . Molecular docking was performed to determine the binding efficiency between the ACE2-AT1-inverse agonists ligands and the AT1 receptor using the Molegro Virtual Docker v. 6.0.1 (MVD) (Thomsen and Christensen, 2006) . The structures of the receptors were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The receptors investigated and respective ID PDB are ACE2AT1 -6M17 (Yan et al., 2020) . For the analysis, all water compounds were deleted from the receptors and the default parameter settings were used with the same software: GRID of 15 Å of radius. The Moldock search algorithm was used, and the Moldock score [GRID] algorithm was used as the score function. The chemical structure of the AT1-inverse agonists studied are represented in Figure 2 .

Molecular docking predicted that all the AT1-inverse agonists tested possess high affinity with the AT1R. The AT1-inverse agonists exhibited binding energies for the AT1R between -133.224 (Valsartan) and -174.52 (Telmisartan) kcal/mol, with telmisartan> exp3174 > eprosartan > azilsartan > olmesartan > ibelsartan > candesartan > valsartan.

Interestingly, nitro functional groups in ARBs reinforce the nature of this group as one of the most important pharmacophorics for drugs which modulate this large family of receptors (Qin et al., 2009) . The presence of amide and ester groups in ARB drugs are responsible for their high affinity for the AT1R, and the effects they produce. Tetrazolate anions (nitrogen-rich five-membered heterocycles, a common pharmacophoric group of some ARB drugs) are more lipophilic than carboxylates, which promotes the passage of drug molecules through cell membranes, and makes them more resistant to metabolic degradation pathways, with a longer duration of action (Aziz et al., 2018) . The tetrazolate pharmacophoric group is common in AT1 inverse agonists (tetrazole ARB drugs) and the binding of the tetrazole moiety with the AT1R involves multiple binding through contact with residues of lysine and histamine that constitute the same subsite of the ligand binding pocket (Noda et al., 1995) .

The AT1R structures share the common architecture of having seven plasma membrane-spanning domains, or transmembrane domains (TMs) -which is why this family of proteins are known as 7TM receptors, connected to each other with three extracellular (ECL) and three intracellular loops (ICL), a disulfide bridge between ECL 2 and TM 3, and a cytoplasmic C-terminus containing an a-helix (Hx8) parallel to the cell membrane (Liapakis et al., 2012) . In the docking study, losartan (an AT1R), EXP3174 (the main losartan metabolite and a compound with inverse agonist activity) and olmesartan (an AT1 inverse agonist) clearly demonstrated their pharmacophoric binding with tetrazole and carboxyl groups (Matsoukas et al., 2013) . Interestingly, they demonstrated an additional interaction with Tyr87 2.63 (a key side chain in the binding pocket of the secondary/tertiary structures at the seven-helical-bundle receptor domain) which was not contained in the pharmacophoric groups, so the hydroxyl group of Tyr87 2.63 forms either a halogen bond interaction with the -Cl atom of losartan and EXP3174, or a hydrogen bond interaction with the hydroxyl group of olmesartan (Matsoukas et al., 2013; Wilcken et al., 2013) .

Here, EXP3174 and olmesartan demonstrated similar binding energy, but they have different affinities for the AT1R, and consequently different pharmacological efficacy that seems to benefit the modulation of the response mediated by this pathway (Bonde et al., 2010) . Takezako et al. (2015) provided evidence of the essential role of the ECL2 (the second extracellular loop) residues Glu173 and Phe182 in the regulation of the conformational states of the AT1R, suggesting a potential strategy for developing new ARBs that directly target the ECL2, a key target for ligand binding and receptor activation. Moreover, the authors of the study reported that substitution of Val108 TM3 , Ala163 TM4 , Asn295 TM7 , and Phe182 ECL2 in the constitution of the AT1R switched efficacy toward agonism for the ARBs in the activated state, but not in the ground state, although the link with ECL2 seems to be crucial. Furthermore, drugs such as azilsartan and eprosartan that are suggested to have greater inverse agonist activity in respect of the AT1R are not those with higher binding energies as suggested by our docking study, although the strong action on ECL2 seems to be one of the pillars of this activity when compared with binding energy Takezako et al., 2018) .

Telmisartan had the strongest binding affinity to the AT1R in our docking study (Figure 3) . However, eprosartan, an inverse agonist of the active state of the AT1R, despite having a lower binding energy, seems to be a better ARB drug because of its properties in respect of the ECL2 and receptor ligand binding pocket (Takezako et al., 2018) .

SARS-CoV-2 has affected millions of people worldwide; the disease caused by the virus is associated with inflammatory processes that can lead to severe pneumonia, cardiovascular failure, the dysfunction of several organs, and in severe cases can result in death (She et al., 2020; Watkins, 2020) . As there is as yet no available specific and effective antiviral therapy to treat COVID-19 patients, a drug repurposing strategy is a rational response to the pandemic. In this context, among the wide range of anti-inflammatory and other drugs that should be considered, the AT1-inverse agonists present an interesting therapeutic option for COVID-19 treatment. Drugs with inverse agonism exhibit high efficacy and strong therapeutic effects for various disease states , with actions that go beyond their antagonistic effects, giving them great potential as possible treatment options for SARS-CoV-2 infection.

AT1-inverse agonists exhibit anti-inflammatory (Saber et al., 2019a) and antioxidant (Saber et al., 2019b) profiles that could mitigate the hyperinflammation and oxidative stress caused by SARS-CoV-2 infection, thereby preventing disease progression and reducing the severity of cases and the number of hospitalizations. In addition, considering COVID-19 as a procoagulant disease, the effects of AT1-inverse agonists on the coagulation cascade could prevent thrombotic events and help to counteract the proinflammatory influence of cytokines. At this stage, it must be emphasized that current evidence suggests that AT1-inverse agonists also downregulate apoptotic protein expression or apoptotic pathway activation, reducing cellular death, and multiorgan failure. This hypothesis should be given serious consideration as the excessive inflammatory response commonly reported in severe cases of COVID-19 has been associated with a worsening of the respiratory condition. In addition, SARS-CoV-2 is capable of producing important in vitro cytopathic effects in human lung cell lines without significant cell death, even in the presence of a high concentration of viral particles, and is not proving to be a significant inducer of apoptosis in this condition (Chu et al., 2020) . This suggests that modulation of host systems, including RAS, during SARS-CoV-2 infection may play an important role in the pathogenesis of the disease, and be responsible for the intense focal lysis of type 2 pneumocytes.

The RAS system has a possible central role in relation to COVID-19 because ACE2 is the main receptor for SARS-CoV-2, as well as for SARS-CoV. Although it is a marker of susceptibility to these coronavirus subtypes, its expression decreases markedly after coronavirus infection, which can generate excess Ang II in the tissue microenvironment with its pro-inflammatory, pro-thrombotic and proapoptotic effects, mainly produced by activating the AT1R. Interestingly, the previous use of exogenous Ang II (Giapreza®) in sepsis therapy showed complications similar to those found in severe cases of COVID-19, in addition to the intense vasoconstrictor phenomena that may explain the rapid change in clinical condition and poor clinical outcome, and its use as a vasopressor should be expressly avoided in patients with COVID-19 (Speth, 2020) .

Currently, to the best of our knowledge, there are nine registered studies assessing the use of ARBs in treating patients with COVID-19, bringing promising results to this approach. As COVID-19 has a biphasic pattern, the effectiveness of therapy with ARBs could be related to the "timing" of infection, as well as patient risk factors, previous use of ARBs -even without important tachyphylaxis, and other class and molecular effects of a specific ARB. It is important to emphasize that at present the effect of ARB drugs on COVID-19 is a controversial subject in the literature, and several authors have shown that this kind of drug has a dual phase, with possible antagonistic effects (Dworakowska and Grossman, 2020; Mehta et al., 2020) . Controlled studies are necessary to establish whether these drugs are effective in the treatment of COVID-19, and, if they are, what is the appropriate time to prescribe them.

project. We dedicate this article to all the doctors and frontline health workers and other staff for their dedication in the fight against COVID-19.

No financial or otherwise, are declared by the authors.

No conflicts of interest or otherwise, are declared by the authors.

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Diabetes and COVID-19

Biased signaling of the angiotensin II type 1 receptor can be mediated through distinct mechanisms

Differential renal effects of candesartan at high and ultra-high doses in diabetic micepotential role of the ACE2/AT2R/Mas axis

Predictors of Intensive Care Unit admission in patients with coronavirus disease 2019 (COVID-19)

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology. Semin Immunopathol

Involvement of angiotensin II type 1 receptor and calcium channel in vascular remodeling and endothelial dysfunction in rats with pressure overload

Valsartan regulates TGF-β/Smads and TGF-β/p38 pathways through lncRNA CHRF to improve doxorubicin-induced heart failure

an angiotensin II receptor blocker, attenuates Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1β in murine macrophages

Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: An observational study. The Lancet Microbe

Modena Covid-19 Working Group (MoCo19)#. SARS-CoV-2, the virus that causes COVID-19: Cytometry and the new challenge for global health

Valsartan: A review of its use in patients with heart failure and/or left ventricular systolic dysfunction after myocardial infarction

Comparison of the effect of valsartan and lisinopril on autonomic nervous system activity in chronic heart failure

Acute kidney injury and special considerations during renal replacement therapy in children with coronavirus disease-19: Perspective from the critical care nephrology section of the European Society of Paediatric and Neonatal Intensive Care

Renin-angiotensin-aldosterone system inhibitors and COVID-19 infection or hospitalization: A cohort study

Renin-angiotensin system inhibitors in management of hypertension during the COVID-19 pandemic

Ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study

Double-blind comparison of eprosartan and enalapril on cough and blood pressure in unselected hypertensive patients. Eprosartan Study Group

Delayed protective effect of telmisartan on lung ischemia/reperfusion injury in valve replacement operations

Severe acute respiratory syndrome coronavirus 2 lethality did not change over time in two Italian provinces

Relationship between decrease in ambulatory blood pressure and heart rate variability due to the effects of taking olmesartan medoxomil

Effect of telmisartan on QT interval variability and autonomic control in hypertensive patients with left ventricular hypertrophy

Expression profiling meta-analysis of ACE2 and TMPRSS2, the putative anti-inflammatory receptor and priming protease of SARS-CoV-2 in human cells, and identification of putative modulators

Angiotensin II receptor blockers attenuate lipopolysaccharide-induced memory impairment by modulation of NF-κB-mediated BDNF/CREB expression and apoptosis in spontaneously hypertensive rats

The involvement of renin-angiotensin system in lipopolysaccharide-induced behavioral changes, neuroinflammation, and disturbed insulin signaling

Beneficial effects of losartan or telmisartan on the local hepatic renin-angiotensin system to counter obesity in an experimental model

Identifying critically ill patients at risk of death from coronavirus disease

Neuroprotective potential of azilsartan against cerebral ischemic injury: Possible involvement of mitochondrial mechanisms

Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics

The effects of known cardioprotective drugs on proinflammatory cytokine secretion from human coronary artery endothelial cells

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Covid-19, ACE2, and the kidney

Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS

Candesartan and arterial baroreflex sensitivity and sympathetic nerve activity in patients with mild heart failure

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Age and multimorbidity predict death among COVID-19 patients: Results of the SARS-RAS study of the Italian Society of Hypertension. Hypertension

Effects of amlodipine and valsartan on vascular damage and ambulatory blood pressure in untreated hypertensive patients

Candesartan improves impaired endothelial function in the human coronary artery

Angiotensin-converting enzyme 2 protects from severe acute lung failure

Sacubitril and valsartan protect from experimental myocardial infarction by ameliorating oxidative damage in Wistar rats

Coronavirus 2019 disease (COVID-19), systemic inflammation, and cardiovascular disease

Inhibition of Fas-associated apoptosis in granulation tissue cells accompanies attenuation of postinfarction left ventricular remodeling by olmesartan

ACE2 of the heart: From angiotensin I to angiotensin

Candesartan ameliorates arsenic-induced hypertensive vascular remodeling by regularizing angiotensin II and TGF-beta signaling in rats

Suppressive effects of type I angiotensin receptor antagonists, candesartan and irbesartan on allergic asthma

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury

Effects of azilsartan compared to other angiotensin receptor blockers on left ventricular hypertrophy and the sympathetic nervous system in hemodialysis patients

Olmesartan inhibits angiotensin II-Induced migration of vascular smooth muscle cells through Src and mitogen-activated protein kinase pathways

Effects of eprosartan on mitochondrial membrane potential and H2O2 levels in leucocytes in hypertension

Modulation of AT-1R/MAPK cascade by an olmesartan treatment attenuates diabetic nephropathy in streptozotocin-induced diabetic mice

The effect of enalapril and telmisartan on clinical and biochemical indices of sympathetic activity in hypertensive patients

Valsartan attenuates intimal hyperplasia in balloon-injured rat aortic arteries through modulating the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor axis

The G-protein coupled receptor family: actors with many faces

Losartan improves measures of activity, inflammation, and oxidative stress in older mice

Candesartan ameliorates acute myocardial infarction in rats through inducible nitric oxide synthase, nuclear factor-κB, monocyte chemoattractant protein-1, activator protein-1 and restoration of heat shock protein 72

an angiotensin II type 1 receptor blocker, attenuates tert-butyl hydroperoxide-induced endothelial cell injury through inhibition of mitochondrial dysfunction and anti-inflammatory activity

Cardiovascular medications in angiogenesis--how to avoid the sting in the tail

Ligand binding determinants for angiotensin II type 1 receptor from computer simulations

An AT1-receptor antagonist and an angiotensin-converting enzyme inhibitor protect against hypoxia-induced apoptosis in human aortic endothelial cells through upregulation of endothelial cell nitric oxide synthase activity

Azilsartan, an angiotensin II type 1 receptor blocker, restores endothelial function by reducing vascular inflammation and by increasing the phosphorylation ratio Ser1177/Thr497 of endothelial nitric oxide synthase in diabetic mice

Acute respiratory distress syndrome

Nitric oxide-dependent antiplatelet action of AT1-receptor antagonists in a pulmonary thromboembolism in mice

COVID-19 -A theory of autoimmunity to ACE-2

COVID-19: Consider cytokine storm syndromes and immunosuppression

Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor

Olmesartan reduces arterial stiffness and serum adipocyte fatty acid-binding protein in hypertensive patients

Long-term treatment with valsartan improved cyclic variation of the myocardial integral backscatter signal and diastolic dysfunction in hypertensive patients: the echocardiographic assessment

Ahmed MM, Al-Rejaie SS. LCZ696 mitigates diabeticinduced nephropathy through inhibiting oxidative stress, NF-κB mediated inflammation and glomerulosclerosis in rats

Valsartan attenuates bleomycin-induced pulmonary fibrosis by inhibition of NF-κB expression and regulation of Th1/Th2 cytokines

Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats

Hemodynamic and cardiac effects of chronic eprosartan and moxonidine therapy in stroke-prone spontaneously hypertensive rats

New angiotensin II type 1 receptor blocker, azilsartan, attenuates cardiac remodeling after myocardial infarction

Paracrine regulation of the renal microcirculation

Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19

Tetrazole and carboxylate groups of angiotensin receptor antagonists bind to the same subsite by different mechanisms

AT1-R is involved in the development of long-lasting, region-dependent and oxidative stress-independent astrocyte morphological alterations induced by Ketamine

Possible role of angiotensin-converting enzyme 2 and activation of angiotensin II type 2 receptor by angiotensin-(1-7) in improvement of vascular remodeling by angiotensin II type 1 receptor blockade

In vitro antagonistic properties of a new angiotensin type 1 receptor blocker, azilsartan, in receptor binding and function studies

Telmisartan improves vascular function independently of metabolic and antihypertensive effects in hypertensive subjects with impaired glucose tolerance

Effects of AT1 receptor antagonism with candesartan on endothelial function in patients with hypertension and coronary artery disease

Evidence for angiotensin mediation of the late histopathological effects of pulmonary fat embolism: Protection by losartan in a rat model

Azilsartan: Current evidence and perspectives in management of hyperension

C-reactive protein (CRP)-lowering agents

Multivalent ligand-receptor interactions elicit inverse agonist activity of AT(1) receptor blockers against stretch-induced AT(1) receptor activation. Hypertens Res

The efficacy and tolerability of azilsartan in mice with left ventricular pressure overload or acute myocardial infarction

Protective effect of a direct renin inhibitor in acute murine model of cardiotoxicity and nephrotoxicity

Impact of the therapy by renin-angiotensin system targeting antihypertensive agents perindopril versus telmisartan on prothrombotic state in essential hypertension

Telmisartan ameliorates dextran sodium sulfate-induced colitis in rats by modulating NF-κB signalling in the context of PPARγ agonistic activity

Olmesartan ameliorates chemically-induced ulcerative colitis in rats via modulating NFκB and Nrf-2/HO-1 signaling crosstalk

but not candesartan improves left ventricular diastolic function in patients with hypertension and heart failure

Effect of valsartan on cardiac senescence and apoptosis in a rat model of cardiotoxicity

Impact of COVID-19 outbreak on regional STEMI care in Germany

Modulation of carbon tetrachloride-induced hepatic oxidative stress, injury and fibrosis by olmesartan and omega-3

Impact of olmesartan medoxomil on amiodarone-induced pulmonary toxicity in rats: Focus on transforming growth factor-ß1

2019 novel coronavirus of pneumonia in Wuhan, China: Emerging attack and management strategies

Renoprotective effects of olmesartan medoxomil on diabetic nephropathy in streptozotocin-induced diabetes in rats

Effects of aminoguanidine, a potent nitric oxide synthase inhibitor, on myocardial and organ structure in a rat model of hemorrhagic shock

Angiotensin II administration to COVID-19 patients is not advisable

Biotransformation of losartan to its active carboxylic acid metabolite in human liver microsomes. Role of cytochrome P4502C and 3A subfamily members

Cardioprotective effects of telmisartan against heart failure in rats induced by experimental autoimmune myocarditis through the modulation of angiotensin-converting enzyme-2/angiotensin 1-7/mas receptor axis

Telmisartan modulates mitochondrial function in vascular smooth muscle cells

Structurefunction basis of attenuated inverse agonism of angiotensin II type 1 receptor blockers for active-state angiotensin II type 1 receptor

Current topics in angiotensin II type 1 receptor research: Focus on inverse agonism, receptor dimerization and biased agonism

The non-biphenyl-tetrazole angiotensin AT1 receptor antagonist eprosartan is a unique and robust inverse agonist of the active state of the AT1 receptor

Laboratory diagnosis of COVID-19: Current issues and challenges

Olmesartan inhibits cardiac hypertrophy in mice overexpressing renin independently of blood pressure

Mas axis and NADPH oxidase expression

MolDock: A new technique for high-accuracy molecular docking

Angiotensin II receptor antagonists: An emerging new class of cardiovascular therapeutics

COVID-19 and heart failure: From infection to inflammation and angiotensin II stimulation. Searching for evidence from a new disease

The protective effects of compound 21 and valsartan in isoproterenol-induced myocardial injury in rats

Correlation between renin-angiotensin system and severe acute respiratory syndrome Coronavirus 2 infection: What do we know?

Dual pathway for angiotensin II formation in human internal mammary arteries

Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan

Losartan suppresses the inflammatory response in collagen-induced arthritis by inhibiting the MAPK and NF-κB pathways in B and T cells

Telmisartan protects against high glucose/high lipidinduced apoptosis and insulin secretion by reducing the oxidative and ER stress

Preventing a covid-19 pandemic

Coronavirus disease 2019 (COVID-19): Situation report. Geneva: WHO, 2020

Principles and applications of halogen bonding in medicinal chemistry and chemical biology

Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7) or an angiotensin II receptor antagonist

Nonpeptide angiotensin II receptor antagonist: pharmacokinetics and pharmacodynamics in rats of EXP3174, an active metabolite of losartan

Valsartan decreases platelet activity and arterial thrombotic events in elderly patients with hypertension

Losartan promotes myocardial apoptosis after acute myocardial infarction in rats through inhibiting Ang II-induced JAK/STAT pathway

Structural basis for the recognition of SARS-CoV-2 by fulllength human ACE2

Role of blood pressure reduction in prevention of cardiac and vascular hypertrophy

AT1 receptor blocker added to ACE inhibitor provides benefits at advanced stage of hypertensive diastolic heart failure

Candesartan inhibits inflammation through an angiotensin II type 1 receptor independent way in human embryonic kidney epithelial cells

Preliminary investigation of human serum albumin-V beta inhibition on toxic shock syndrome induced by staphylococcus enterotoxin B in vitro and in vivo

Structural basis for ligand recognition and functional selectivity at angiotensin receptor

Association of inpatient use of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers with mortality among patients with hypertension hospitalized with COVID-19

Telmisartan attenuates kidney apoptosis and autophagy-related protein expression levels in an intermittent hypoxia mouse model

The authors would like to thank FAPI-TEC-SE, CAPES, CNPq and EpiSERGIPE