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SARS-CoV-2 infection and ACE2 inhibition

2021; Lippincott Williams & Wilkins; Volume: 39; Issue: 8 Linguagem: Inglês

10.1097/hjh.0000000000002859

1473-5598

Fabio Angeli, Gianpaolo Reboldi, Paolo Verdecchia,

Computational Drug Discovery Methods

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread across the world and it is still a major public health concern [1]. SARS-CoV-2 was found to share similarities with the SARS-CoV (sharing about 80% genome sequence identity [2]), which caused the SARS epidemic in 2002 [3]. The new coronavirus SARS-CoV-2 is perhaps less deadly but far more contagious than SARS-CoV with higher proportion of symptomatic patients requiring hospital admission [4]. Exploring what makes SARS-CoV-2 different from SARS-CoV [4], it is worth mentioning that both SARS-CoV-2 and SARS-CoV use the same receptor [angiotensin-converting enzyme 2 (ACE2)] to initiate the virus entry into human cells. Nonetheless, despite similarities, there are differences in the structure and dynamics of the interactions of SARS-CoV-2 and SARS-CoV with ACE2. Of note, robust experimental data suggest that SARS-CoV-2 exhibits a considerably higher binding affinity to human ACE2 compared with SARS-CoV [5–7]. Such difference might explain the higher transmission rate of SARS-CoV-2. Shang et al. [7] determined the crystal structure of the receptor-binding domain (RBD) of the Spike protein (a 1273 amino acid long protein which belongs to the viral envelope and protrudes outwards with a 'corona' like appearance) of SARS-CoV-2 in complex with ACE2. In comparison with the SARS-CoV RBD, they showed that an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation [7]. Furthermore, some residue changes in the SARS-CoV-2 RBD seem to stabilize two virus-binding hotspots at the RBD–ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity [7]. Similar results were also obtained by other dynamics simulations performed to better understand the structural stability and interfacial interactions between the receptor binding domain of the Spike protein of SARS-CoV-2 and SARS-CoV bound to ACE2 [6]. A key to tackle the pandemic is to explain the receptor recognition mechanisms of the virus. More specifically, the mechanisms of virus entry into cells mediated by the binding to ACE2 are fundamental to understand the viral invasion of host cells. It is currently hold that the entry of SARS-CoV-2 into cells is mediated by the efficient binding of the Spike protein to the ACE2 receptors [8,9]. The ACE2 is a homolog to ACE with 40% structural identity [10] and ACE2 receptors are almost ubiquitous. Notably, ACE2 receptors are expressed in the heart (endothelium of coronary arteries, fibroblasts, myocites, epicardial adipocites), vessels (vascular endothelial and smooth cells), gut (intestinal epithelial cells), lung (macrophages, tracheal and bronchial epithelial cells, type 2 pneumocytes), kidney (luminal surface of tubular epithelial cells), testis and brain [11]. The ACE2 receptor is a transmembrane type I glycoprotein (mono-carboxypeptidase) composed by 805 amino acids, which uses a single extracellular catalytic domain to cleave an amino acid from angiotensin I to form angiotensin1–9 and to remove an amino acid from angiotensin II to form angiotensin1–7[12]. Of note, the catalytic active site of ACE2 appears to be distant from the SARS binding site [13]. Schematically, the viral entry process consists of three main steps (Fig. 1) [8,14]. In the first step (Fig. 1a), the N-terminal portion of the viral protein unit S1 binds to a pocket of the ACE2 receptor. The second step (Fig. 1b) is the protein cleavage between the S1 and S2 units, which is operated by the receptor transmembrane protease serine 2 (TMPRSS2), which is structurally contiguous to ACE2 receptor. Finally, after the cleavage of the viral protein by TMPRSS2, the viral S2 unit undergoes a conformational rearrangement driving the fusion between the viral and cellular membrane, with subsequent entry of the virus into cell, release of its content, replication and infection of other cells (Fig. 1c) [8,15,16].FIGURE 1: Steps of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry process (see text for details). The main step after the invasion of SARS-CoV-2 is binding to membranal angiotensin-converting enzyme 2 receptor (ACE2). ACE2, angiotensin-converting enzyme 2; TMPRSS2, receptor transmembrane protease serine 2.Although vaccines to prevent SARS-CoV-2 infection are considered the most promising approach for curbing the pandemic, there is an ongoing search for therapeutics to build an armamentarium finalized to block the transition from infection to severe forms of coronavirus disease 2019 (COVID-19). Among the different therapeutic strategies (including the prevention of the viral RNA synthesis and replication, the restoration of the host's innate immunity and the modulation of the host's specific receptors or enzymes), the blockade of SARS-CoV-2 from binding to human cell receptors is the object of both in-silico and in-vivo experiments [17,18]. Given the uncertainty about the role of the inhibition of the binding of SARS-CoV-2 RBD to ACE2 [5,19–21], results of the analysis by Nami et al. are welcome [22]. Briefly, Nami et al. [22] evaluated the effect of ACE2 inhibition on the SARS-CoV-2 Spike protein binding to ACE2 by docking the Spike proteins to ACE structures. They used a selective, cell-permeable, and highly potent ACE2 inhibitor (MLN-4760). Dynamics of the complexes, ligand-protein and protein-protein interactions have been studied by molecular dynamics simulation [22]. First, Nami et al. [22] evaluated the difference between the Spike protein RBDs of SARS-CoV-2 and SARS-CoV in terms of structural morphology. Previous reports showed an overall 90% genomic similarity of SARS-CoV-2 to SARS-CoV [5–7]. However, the documented 25.6% difference between the structure of the RBD of the SARS-CoVs is relatively significant [22]. As aforementioned, previous reports demonstrated that the SARS-CoV-2 Spike protein binds to ACE2 with a higher affinity than SARS-CoV Spike protein [5–7]. In this context, Nami et al. [22] highlighted that both the Spike protein RBDs of SARS-CoV-2 and SARS-CoV bind to the same region of ACE2. However, they confirmed the evidence that SARS-CoV-2 Spike protein RBD binds to ACE2 with higher affinity and form stronger interaction with ACE2 compared to SARS-CoV [22]. Analysis of the binding affinity of the interactions between Spike protein RBDs of SARS-CoV-2 and ACE2 showed a ΔG and a dissociation constant (Kd) of the interactions equal to -12.5 kcal/mol and 7.2e-10 molar, respectively [22]. Conversely, the SARSCoV RBD/ACE2 complex had a ΔG of -10.9 kcal/mol and a Kd of 1.1e-8 mol/l [22]. More importantly, Nami et al. [22] showed that MLN-4760 binding to ACE2 had no major effect on the binding of the RBD to ACE2. The Spike protein binds to inhibited ACE2 (closed/inactive conformation) with higher affinity than the native ACE2 with open/active conformation [22]. Moreover, simulation analyses show that the Spike protein binding to the ACE2 inhibited by MLN-4760 changes its closed/inhibited conformation to open/active conformation [22]. This suggests that binding the SARS-CoV-2 Spike protein to the ACE2 inhibited by MLN-4760 abrogates the inhibitory effect of MLN-4760 and probably rescues the enzymatic activity of ACE2 by removing MLN-4760 from the ACE2 enzymatic active site and promoting the viral entry process (Fig. 2, upper).FIGURE 2: Different potential mechanisms of action of ACE2 inhibitors. The upper part depicts the inhibition of ACE2 by MLN-4760 as documented by Nami et al. [22]. In the middle part, a compound with insurmountable and complete inhibition of ACE2 blocks the binding of the viral Spike protein to the pocket of the ACE2 receptor exerting inactivation of ACE2 enzymatic activities (with subsequent enhancement of the ACE-Angiotensin II-AT1 receptor axis and angiotensin1–7 deficiency). In the lower part, the ideal ACE2 inhibition modulates SARS-CoV-2 binding to ACE2 without inactivation of this enzyme to avoid angiotensin1–7 deficiency. A1,7, angiotensin1,7; ACE2, angiotensin-converting enzyme 2; AII, angiotensin II.Taken together, the findings of the simulation study by Nami et al. [22] provide insights on the interactions between the viral glycoproteins and their host receptors (namely ACE2). Keeping in mind that SARS-CoV-2 uses ACE2 as a Trojan horse to invade target cells (Fig. 1), at least three different mechanisms of action of ACE2 inhibitors may be postulated (Fig. 2). Obviously, ACE2 inhibitors with properties similar to MLN-4760 (i.e. promoting the affinity of viral Spike proteins with ACE2) do not have any potential utility (Fig. 2, upper). Conversely, compounds with insurmountable inhibition of ACE2, blocking or attenuating the binding of the viral Spike protein to the pocket of the ACE2 receptor, have the potential to prevent viral internalization into ACE2-expressing cells. However, pharmacological inhibition of ACE2 may exert enzymatic activities with (Fig. 2, middle) or without (Fig. 2, lower) inactivation of ACE2. The real challenge in the field of ACE inhibition is to modulate SARS-CoV-2 binding to ACE2 without blocking the crucial protective properties of this enzyme. Indeed, it has been suggested that the loss of ACE2 receptor activity as a consequence of the viral elimination and downregulation processes leads to less angiotensin II inactivation and less generation of antiotensin1–7[15,23,24]. The imbalance between angiotensin II overactivity and angiotensin1–7 deficiency reduces the activation of the Mas receptor and endothelial nitric oxide synthase [25], triggers inflammation, thrombosis and other adverse reactions, finally worsening COVID-19 [15,23,24]. In other words, the ideal ACE2 inhibition should preserve the catalytic activities of ACE2 (Fig. 2, lower). Conversely, the use of inhibitors causing loss of enzymatic properties of ACE2 should be ideally combined with angiotensin1,7 replacement therapy or, alternatively, with ACE2 activators (Fig. 2, middle). In conclusion, the blockade of ACE2 may be a clever way to eradicate the disadvantageous contribution of ACE2 as a viral entry route. Nonetheless, mechanisms and significance in clinical practice of this therapeutic intervention remain to be fully elucidated and deserve further evaluation. The main target of ACE2 inhibition or modulation is to modify the enzyme configuration to attenuate its binding affinity to the SARS-CoV-2. Although an approach targeted to modulate the molecular interaction of SARS-CoV-2 binding to the ACE2 receptor seems attractive, it should not come at the expense of lessening the beneficial effects of ACE2 [24,26]. Indeed, the potential attenuation of the catalytic properties of ACE2 due to pharmacological manipulation may require either replacement therapy or ACE2 activators to guarantee adequate circulating levels of Angiotensin1,7[27]. ACKNOWLEDGEMENTS Conflicts of interest None of the authors of this study has financial or other reasons that could lead to a conflict of interest.