key: cord-0719250-dnqbae3e
authors: Lagoumintzis, George; Chasapis, Christos T.; Alexandris, Nikolaos; Kouretas, Dimitrios; Tzartos, Socrates; Eliopoulos, Elias; Farsalinos, Konstantinos; Poulas, Konstantinos
title: Nicotinic Cholinergic System and COVID-19: In Silico Identification of Interactions Βetween α7 Nicotinic Acetylcholine Receptor and the Cryptic Epitopes of SARS-Co-V and SARS-CoV-2 Spike Glycoproteins
date: 2021-01-24
journal: Food Chem Toxicol
DOI: 10.1016/j.fct.2021.112009
sha: 60fb30c4f3c914f55efff10535f3545410d2c12b
doc_id: 719250
cord_uid: dnqbae3e

SARS-CoV-2 is the coronavirus that originated in Wuhan in December 2019 and has spread globally. Studies have shown that smokers are less likely to be diagnosed with or be hospitalized for COVID-19 but, once hospitalized, have higher odds for an adverse outcome. We have previously presented the potential interaction between SARS-CoV-2 Spike glycoprotein and nicotinic acetylcholine receptors (nAChRs), due to a "toxin-like" epitope on the Spike glycoprotein, with homology to a sequence of a snake venom toxin. This epitope coincides with the well-described cryptic epitope for the human anti-SARS-CoV antibody CR3022. In this study, we present the molecular complexes of both SARS-CoV and SARS-CoV-2 Spike glycoproteins, at their open or closed conformations, with the model of the human α7 nAChR. We found that all studied protein complexes' interface involves a large part of the "toxin-like" sequences of SARS-CoV and SARS-CoV-2 Spike glycoproteins and toxin binding site of human α7 nAChR. Our findings provide further support to the hypothesis about the protective role of nicotine and other cholinergic agonists. The potential therapeutic role of CR3022 and other similar monoclonal antibodies with increased affinity for SARS-CoV-2 Spike glycoprotein against the clinical effects originating from the dysregulated cholinergic pathway should be further explored.

While the global pandemic of Corona Virus Disease 2019 (COVID-19), a disease caused by the acute respiratory coronavirus 2 syndrome (SARS-CoV-2), progresses, it is crucial to reveal the pathophysiology and the risk and possibly protective factors associated with disease progression and severity in order to provide effective therapy [8] . Published metaanalyses reporting an unusually low pooled prevalence of smoking among hospitalized COVID-19 patients compared with population smoking rates [9] [10] [11] bolstered the debate about the connection between smoking and COVID-19. Other studies report that smokers For the first time in April 2020, we hypothesized that the nicotinic cholinergic system (NCS) might be involved in the pathophysiology of severe COVID-19 and recently built on this hypothesis [9, 20] . In light of the above considerations, these reports raise the possibility that nicotine -through its action on the NCS-may protect patients against the development of severe COVID-19 that would require hospitalization. Such results should, however, be viewed cautiously before being experimentally verified.

Nicotinic acetylcholine receptors (nAChRs) are membrane proteins, consisting of five spanning-membrane subunits arranged around a central pore. They are divided into the muscle and the neuronal type. Muscle AChRs are located in the skeletal muscles, where they facilitate neuromuscular communication. Neuronal AChRs are found primarily in both the peripheral nervous system (PNS) and the central nervous system (CNS), but also in non-neuronal tissues. Several different nAChRs, consisting of a specific combination of subunits, facilitate discrete cellular physiological functions. Generally, expression of the neuronal form of nAChR differs in several cells of the nervous system. However, neuronal nAChRs are commonly expressed in peripheral ganglia and some brain regions, and in nonexcitable cells, such as keratinocytes, epithelial cells, and immune cells (i.e., B cells, T cells, and macrophages). Among the various nAChR subtypes, the α7 receptor, which is widely distributed and overexpressed in the hippocampus, is the most important mediator of the anti-inflammatory properties of the cholinergic system due to its association with humoral and intrinsic immunity [21, 22] .

The NCS is an essential pathway that regulates inflammatory response. Its effects on macrophages and other immune cells are mainly controlled by the vagus nerve and by α7 nicotinic acetylcholine receptors (nAChRs) [23] . This so-called "cholinergic antiinflammatory pathway" has been found to be beneficial in animal models in the prevention J o u r n a l P r e -p r o o f of inflammatory conditions such as sepsis and Acute Respiratory Distress Syndrome (ARDS) [24] . Thus, NCS dysregulation may be a potential cause of the uncontrolled inflammatory response in COVID-19. More clinical manifestations of COVID-19, such as anosmia and thromboembolic complications, could also be explained through its action [21] .

The plasma of recovered patients containing neutralizing Abs was a relatively safe and effective treatment choice during the first SARS-CoV and MERS-CoV outbreaks to decrease viral load and minimize mortality in extreme cases [25, 26] J o u r n a l P r e -p r o o f

We compared amino acid sequences between SARS-CoV and SARS-CoV-2 Spike glycoproteins and snake venom neurotoxins. Sequence retrieval of the protein sequences of both virus-related Spike proteins and "three-finger" neurotoxins from various species was 

The following 3D structures were downloaded from the Protein Data Bank (PDB): 1) PDB id: 6LZG: SARS-CoV-2 Spike glycoprotein (S1) 3D structure in complex with the human angiotensin-converting enzyme 2 (hACE2), 2) PDB id: 6M18: hACE2 (1R41, 1R42) cryo-EM determined complex of spike protein S-ACE2-B0AT1 neutral amino acid transporter, 3) PDB id: 6NB7: structure of a neutralizing to SARS-CoV mAb cross-reacting with complexed ACE2-S protein of SARS-CoV-2, 4) PDB id: 4UY2: ECD of the nAChR α9 subunit complexed with α-bungarotoxin., 5) PDB id:6W41: SARS-CoV-2 RBD complexed with CR3022, and 6) PDB id: 3SQ9: ligand-binding domain (LBD) of a chimera pentameric α7 nAChR.

The protein structure prediction of the ECD of human α7 nAChR was performed using ROSETTA software [58], applying an automated multi-step and multi-template homology J o u r n a l P r e -p r o o f

We scrutinized the amino acid conservation between SARS-CoV-2 and other SARS-related coronavirus sequences in the Spike protein using the simple local alignment search tool BLASTP. The multiple amino acid sequence alignment of the Spike glycoprotein RBD of all SARS-related coronaviruses is shown in Figure 2 . Phylogenetically, SARS-CoV-2 is closely linked to SARS-CoV, which triggered the human outbreak of 2003. As we can see in Figure 2A , the RBD epitope is highly conserved between all SARS-related coronaviruses, including the SARS-CoV-2, suggesting a typical evolutionary pattern.

Remarkably, the aa 375-395 RBD fragments containing the neurotoxin-like residues of the nAChRs-interacting snakes are among the most conserved regions of almost all CoVs related to SARS. Exceptions are human SARS-CoV OC43, SARS-CoV HKU1, and MERS-CoV, which have a somewhat different composition of RBD amino acids that indicate a distinction from the previously described strains of the viruses, particularly in the aa 375-395 region (aa similarity <50%) ( Figure 2B ). Figure 3A presents the sequence alignment of SARS-CoV and SARS-CoV-2 S1 glycoproteins (A7J8L4, P0DTC2) with Neurotoxin homolog NL1 (Q9DEQ3). We found a double "recombination" within the same S-protein sequence (aa 375-390), which is homologous in the Neurotoxin homolog NL1 sequence, part of the "three-finger" interacting motif of the toxins. This peptide fragment (aa 375-390) is part of the SARS CoV-2 Spikes RBD (aa 333-527) and is located adjacent to the ACE2 Receptor Binding Motif (RBM), through which the Spike glycoprotein recognizes the ACE2 receptor on the host's cell surface. Quite notably, this peptide is the main part of the epitope for the CR3022 antibody, as described before [35, 49] . The main interacting amino acids between J o u r n a l P r e -p r o o f the RBD of SARS-CoV and mAb CR3022, as described in the crystal structure of CR3022 and SARS-CoV-2 Spike glycoprotein [35] , are shown in Figure 3B . Molecular models of mAb CR3022 interacting with SARS-CoV are presented in Figure 3C .

This "toxin-like" fragment on SARS-CoV (aa 362-377) and SARS-CoV-2 (aa 375-390) RBD, containing an amphipathic sequence of alternating polar and hydrophobic amino acid residues with selectively charged amino acids in a conserved order, lies on the spike protein surface and is not buried in the domain core. In ball and stick representation, the toxin-like sequence and its location in the protein surface are illustrated in Figure 4 . Neighboring the ACE2 binding motif, this entity may interact with the human α7 nAChRs like neurotoxins.

We have previously identified the interaction between the SARS-CoV-2 S1 glycoprotein (aa 381-386) and the α9 subunit of nAChR ECD (aa 189-192), a region that forms the core of the nAChR "toxin-binding site". The interaction between the two proteins is caused by the complementarity of the hydrogen bonds and shape [52] . The interaction model is very similar to the interaction between α9 nAChR and both α-bungarotoxin and the homologous neurotoxin NL1 (snake venom toxins inhibiting nAChRs). Similar interacting surfaces were observed between the SARS-CoV and SARS-CoV-2 S1 and the LBD of the pentameric α7 nAChR chimera.

Herein, the HADDOCK models show that all studied protein complexes' interface involves most of the toxin-like sequences within SARS-CoV S proteins and toxin binding sites of human α7 nAChR. The binding affinity (ΔG, expressed in kcal mol -1 ), the dissociation constant (K d at 25 ℃, expressed in Molar), electrostatic energy (expressed in kcal mol -1 ), J o u r n a l P r e -p r o o f and the buried surface area (expressed in Å 2 ) for all the modeled protein complexes are presented in Table 1 . The dissociation constant of all SARS-α7 nAChR complexes is found to be in the nM range, comparable with experimental supported Kds of well-known enzymatic interacting partners that produce stable protein complexes (i.e., E2-E3 pairs in ubiquitination pathway [62]). promote viral invasion and cell entry, leading to increased susceptibility and severity of COVID-19 [69] . However, some studies have strongly questioned that notion. Evidence from in vitro SARS-CoV studies indicates that viral replication causes ACE2 downregulation, resulting in adverse effects due to unregulated angiotensin II accumulation and activity [70] . A similar mechanism may be implicated in SARS-CoV-2 infection [71] .

Besides, many case-series of patients in the United States, Europe, and Asia reported a lack of association between the use of ACE-inhibitors (and angiotensin receptor blockers) and COVID-19 diagnosis and adverse outcome [71, 72] . In one study, ACE-inhibitors were associated with a lower risk of in-hospital death [73] . Also, ACE2 deficiency has been observed with age, diabetes mellitus, and heart disease, which tend to be risk factors for severe COVID-19 [74] [75] [76] . In contrast, children and young women have higher levels of ACE2 than older people, yet they are generally experiencing milder disease symptoms [77] .

So, it is possible that ACE2 up-regulation may protect against extreme COVID-19 in these cases [78] . According to our hypothesis, it seems that SARS-CoV-2 Spike glycoprotein has a "toxinlike" sequence in its RBD that could bind to the nAChRs alpha subunit's toxin-binding domain. This binding may produce various adverse effects by dysregulating the NCS, which involves mostly α7 nAChRs. One of the consequences may be the disruption of the anti-inflammatory cholinergic pathway, leading to cytokine storm and inability to return to homeostasis for the immune response. Cholinergic dysfunction could explain several clinical manifestations of the disease [70] . While a positive association between smoking and adverse outcomes among hospitalized COVID-19 patients has been observed, this does not necessarily reject our hypothesis. Smokers experience abrupt nicotine cessation once hospitalized (except in the unlikely scenario that they receive nicotine replacement therapies). This will result in the rapid elimination of plasma nicotine levels within a few :

Bat coronavirus BM48-31/BGR/2008

Bat coronavirus BM48-31/BGR/2008

J o u r n a l P r e -p r o o f Table 1 . Haddock parameters of SARS-CoV S1 and SARS-CoV-2 S1 with ECD of human α7 nAChR pentamer.

SARS-CoV-2 S1 

Patients: Implications for COVID-19

Smoking Upregulates Angiotensin-Converting Enzyme-2 Receptor: A Potential Adhesion Site for Novel Coronavirus SARS-CoV-2 (Covid-19)

Tobacco Smoking Increases the Lung Gene Expression of ACE2, the Receptor of SARS-CoV-2

Smoking Upregulates Angiotensin-Converting Enzyme-2 Receptor: A Potential Adhesion Site for Novel Coronavirus SARS-CoV-2 (Covid-19)

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

Aldosterone System Inhibitors in Patients with Covid-19

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Renin-Angiotensin-Aldosterone System Inhibitors and Risk of Covid-19

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COVID-19, Diabetes Mellitus and ACE2: The conundrum

Loss of angiotensin-converting enzyme 2 accelerates maladaptive left ventricular remodeling in response to myocardial infarction

COVID-19 infection and the predictive ACE2 soluble levels: the favourable protection of children and women. Front. Pediatr

Nicotine exerts an anti-inflammatory effect in a murine model of acute lung injury

The authors are grateful to the "National Research Infrastructures on Integrated Structural Biology, Drug Screening Efforts and Drug Target Functional Characterization (INSPIRED)" for personnel's financial support.