At the end of 2019, in Wuhan (China), the emergence of a new coronavirus [severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)] was reported, which is classified as belonging to the Beta-coronavirus genus and possessing as genetic material a single strip positive RNA, being capable of resulting in disease [coronavirus disease 19 (COVID-19)] that can evolve to severe acute respiratory syndrome. Although it is known that this disease mainly affects the respiratory system, it was identified that it can manifest clinic signs and symptoms related to other organs, such as nausea, vomiting, abdominal pain and diarrhea. With regard to the liver, alterations in lesion markers including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total serum bilirubin were reported, indicating hepatic injury[1,2]. Between 14%-53% of patients with COVID-19 present with abnormalities in transaminase levels[3,4] which is associated with severity of the disease, and abnormal transaminase levels can indicate a higher chance of poor prognosis and Intensive Care Unit requirement[5-7]. The associated causes of liver injury, beyond direct viral impact, include the use of drugs during treatment, hypoxia due to pulmonary symptoms, previous hepatic lesions and co-morbidities. Some of the drugs used can cause hepatotoxicity, liver injury and dysregulation, as shown for hydroxychloroquine, Azithromycin and Remdesivir, respectively[8,9]. One of the SARS-CoV-2 presentations on this system can be acute non-icteric hepatitis before the most common symptoms (fever and respiratory symptoms) and those already related to the liver, nausea, diarrhea and abdominal pain. The viral cellular entrance and its dissemination is on a vast spectrum of the organism’s systems, for example the liver-related, is a consequence of the expression of a common cellular receptor [angiotensin-converting enzyme 2 (ACE2)]. However, for effective cellular intrusion the participation of other human proteins such as Type 2 Serine Protease (TMPRSS2) and Furin (Convertase Proprotein of the Subtilisin Type)[1,2,11-14]. Therefore, concerning the entrance mechanisms, it has been identified that the viral Spike glycoprotein possesses affinity and binds to the ACE2 (responsible for the adhesion stage), as it is structurally divided into two subunits: S1 (N-terminus, that connects to the receptor) and S2 (C-terminus, that takes part in the penetration process)[2,12,13]. Between both units, S1 and S2, there is a cleavage site for Furin, that triggers the activation and conformational change of viral Spike glycoprotein after it completes this action[2,13,14]. Following the initial activation process, further cleavage between the S1/S2 and S2’ sites is essential for viral entrance. The TMPRSS2 protein performs this activity on the Spike glycoprotein of SARS-CoV-2, which allows fusion between the viral and cellular membranes, entry of the viral genetic material and the development of infection[1,11,12]. Following viral entrance, during the course of infection, modulation of the signaling pathway Akt/mTOR, that regulates apoptosis, cell survival, transcription and translation, occurs which also occurs during infection by other viruses[15-17]. This signaling possibly increases factors of viral translation while blocking mechanisms of cellular death, generating greater pathogenicity. Based on this, recent studies have indicated the possibility of using already existing drugs that interfere with this pathway for the treatment of COVID-19, including MK-2206, an Akt inhibitor.
Despite these facts, studies directly investigating the interaction between SARS-CoV-2 and liver cells, specifically the entrance mechanisms, the biochemical cascades and methods for possible infection inhibition still require further investigation. Therefore, the present study aims to associate the hepatic alterations triggered by SARS-CoV-2 with the activation and/or inhibition of transduction pathways of cellular signals in the viral infection process. In addition, possible intervention in the signaling pathways with inhibitors was analyzed to suggest potential treatments for SARS-CoV-2 infection.
Liver is the main organ involved in drug metabolism and xenobiotic detoxification; therefore, its proper functioning is essential for the effectiveness of pharmacological treatments. As altered liver function is reported in up to half of patients with COVID-19, it is important to clearly understand the possible mechanisms involved in liver injury in order to optimize the treatment outcome of this disease.
Moderate microvesicular steatosis and mild inflammation in the lobular and portal area are pathological findings in liver tissue in patients with COVID-19; thus, this may contribute to the incidence of elevated levels of hepatic transaminases reported in this disease. In the early stage of COVID-19, infected individuals have positive SARS-CoV-2 RNA in fecal and blood samples, and present gastrointestinal symptoms such as diarrhea, abdominal pain, nausea, and vomiting suggesting that SARS-CoV-2 could infect liver cells.
In SARS-CoV-2 infection, it is known that the viral Spike glycoprotein interacts with the ACE2 present in humans, leading to entry of the virus. However, for its entry to occur, there is a need for activation of the glycoprotein by host cell proteases which occurs between the S1/S2 subunits of Spike generating a conformational change in the S2 subunit and allowing the interaction of SARS-CoV-2 with ACE2, completing virus entry.
As SARS-CoV-2 interacts with the ACE2 receptor of host cells to invade them; thus, cells that have this receptor are susceptible to infection. The level of ACE2 expression is low in hepatocytes (2.6%), but in bile duct cells (cholangiocytes) this expression is high (59.7%). Therefore, SARS-CoV-2 does not necessarily directly infect liver cells, but causes bile duct dysfunction that plays an important role in liver regeneration and immune response.
In fact, according to Xu et al (2020), no direct cytopathic effect of SARS-CoV-2 on the liver was found in pathological autopsy findings. On the other hand, the findings of Pirola and Sookoian support the possibility that SARS-CoV-2 may cause direct liver injury by the viral cytopathic effect. These authors showed that the three host cell proteins-ACE2, Furin and TMPRSS2, responsible for viral infection are expressed in liver tissue. Although ACE2 has low expression in hepatocytes compared with cholangiocytes, TMPRSS2 and Furin are expressed more in hepatocytes.
The Spike glycoprotein of SARS-CoV-2 facilitates viral entry into host cells; the surface unit S1 binds to the cellular receptor-ACE2, while the transmembrane unit S2 facilitates fusion of both viral and cellular membrane. Membrane fusion depends on S protein cleavage by host cell proteases at the S1/S2 and the S2′ sites, and among these proteases, TMPRSS2 and Furin play major roles in proteolytic activation of a broad range of viruses including SARS-CoV-2. After infection, the signaling pathways of the host cell are affected to promote viral replication. Activation of the Akt/mTOR signaling pathway and through a cascade of events, mTORC1 and Akt activate host transcription and translation of specific genes. The activation of Akt/mTOR signaling during SARS-CoV-2 infection could be to sustain protein synthesis by increased access to translation components.
An analysis of the summary of the SARS-CoV-2 infection process indicates that host cell proteases and signaling cascade proteins could be a potential target for therapeutic interventions for COVID-19 symptoms, including gastrointestinal symptoms due to liver damage.
This study analyzed the interactions of SARS-CoV-2 with molecules which are necessary for the success of infection and are expressed in the liver using bioinformatics tools and in silico enzymatic inhibition tests to try to associate such interactions with the gastrointestinal findings and liver injuries in COVID-19. It was shown that the interaction affinity of some proteins present in the human body with their respective inhibitors that can act in their pathways and prevent the development of infection. The target proteins were the Furin enzyme, involved in cell invasion, and mTORC1 and Akt enzymes belonging to the signaling pathway.
To verify the interaction between the mTORC1/CC-223, Akt/MK-2206 and Furin/naphthofluorescein complexes at the molecular level, molecular docking was used. Thus, if there is an interaction between proteins and inhibitors, simulation helps us to understand the dynamics that occur in silico.
The interaction affinity calculated by AutoDock Vina for the mTORC1/CC-223 complex was -7.7 kcal/moL, for the Akt/MK-2206 complex it was -8.8, and for the complex formed by Furin/naphthofluorescein it was -9.8 kcal/moL. These values are considered significant since values lower than -6.0 kcal/moL already constitute stable interactions in silico analysis.
The complex with the highest interaction affinity was that formed by Furin and its inhibitor. This significantly low affinity energy value indicates a more stable complex, in other words, indicates that the inhibitor will have higher biological activity. Unlike other coronaviruses, SARS-CoV-2 has a potentially critical insertion of a Furin cleavage site upstream of the S1 cleavage site in the Spike glycoprotein reducing its dependence on host cell proteases for infection. The high affinity between ACE2 and the Spike glycoprotein cleaved by Furin allows SARS-CoV-2 to maintain its efficient entry into cells while preventing the action of the immune system which can contribute to the widespread infection capacity of the virus[14,37]. As ACE2 is present in type 2 alveolar cells, the gastrointestinal tract and the liver, these tissues would be more affected by COVID-19; therefore, inhibiting the action of Furin would prevent infection of these tissues and consequently the associated symptoms.
Vankadari in his study on Furin analyzed not only its structure but also how it would bind at S1/S2 subunits of the Spike glycoprotein. Thus, it was suggested that Furin binds to these subunits through the equatorial region present in the Spike glycoprotein which creates a 970 Å interface between the participants.
The Furin enzyme is required in various normal functions of the body. Prolonged Furin blocking can therefore generate side effects or damage. In this context, Furin's involvement in the viral invasion process could reduce the effectiveness of the action of this enzyme in normal physiological processes triggering pathological processes. In fact, studies suggest that Furin plays an important role in homeostasis and disease; thus, it is possible that liver cell lesions would be reduced and AST and ALT levels would be normal. On the other hand, brief Furin inhibition can be well tolerated and has therapeutic benefit; therefore, Naphthofluorescein has promising potential for treatment through this route given its high affinity for Furin in silico.
Another way of studying the symptoms resulting from liver injury triggered by SARS-CoV-2 infection is to analyze the signaling pathway affected by the virus. In fact, some studies point to the dysregulation in the Akt/mTOR signaling cascade, which could be a potential target in COVID-19 treatment.
It was observed in this study that the Akt/MK-2206 complex remains with 12 bonds, added to three Van der Waals interactions and all five rings of the MK-2206 inhibitor are bound to protein, suggesting stability of the complex, and the protein Akt would be unable to proceed with his cascade. Shi et al in his findings on the inhibition of esophageal cancer growth through the PI3K/AKT/mTOR pathway, showed that MK-2206 would be a potential allosteric inhibitor of Akt, by decreasing cell proliferation, inducing cell cycle arrest and increasing apoptosis of cancer cells. Furthermore, Appelberg et al observed that the Akt/mTOR/HIF-1 pathways participate in COVID-19 infection, in particular, Akt/mTOR are activated at the beginning of infection. It has also been shown that MK-2206 caused a decay in viral transcription in SARS-CoV-2 infected cells and supernatants by interacting with Akt. Based on these data and on the results obtained with molecular docking, in which significant affinity and stability of the bonds were observed, it is possible that the MK-2206 inhibitor may help to contain the development of COVID-19 by interacting with Akt, in a way that prevents continuity of the cascade triggered by this protein.
It is possible that in the mTORC1/CC-223 complex significant chemical bonds are established along the CC-223 structure involving three of four molecule rings. The same was observed for Van der Waals' interactions. The sum of all binding and interactions between mTORC1 and CC-223 contribute to the formation of a complex with significant affinity energy which contributes to its stability. Mortensen et al showed that CC-223 as an inhibitor has high affinity for mTOR, and the pathway to which it belongs was unfeasible. It has been described that mTOR is relevant in cell growth, proliferation, survival and metabolism; in particular, mTORC-1 plays a role in protein synthesis and cell development. In view of this, CC-223 has been reported to cause inhibition of mTORC-1 in vivo upon administration of this compound in tumor-bearing mice, suppressing continuation of the cascade. Added to this, in another study, Mortensen et al also analyzed the interaction of compounds with the PI3K/AKT/mTOR pathway, thus, selectivity of the CC-223 inhibitor for the mTORC-1 protein was highlighted, as the former has high affinity for the latter and may inhibit it. Furthermore, in line with the results obtained from molecular docking, it was found that binding of the mTORC-1 and CC-223 complex has a high affinity and stability, therefore, considering the potential of this inhibitor for blockade of the mTOR pathway, it may be used to prevent the spread of SARS-CoV-2 infection and, consequently, restrict disease spread. In this way, as the liver is the main regulatory organ of metabolism and the mTOR/Akt signaling pathway plays a key role in cellular metabolism, changes in this pathway are significantly reflected in the liver; thus, preventing changes in this pathway by the virus would help to slow down the symptoms.