Harnessing the Sars-cov-2 Spike Protein Binding Anity to Ace2 Receptor Through Narcotinic Compounds Combined With Adjuvants: an in Silico Insight

Protein products of SARS-CoV-2 spike (S) coding gene sequence, were all analyzed and compared to other SARS-CoV S proteins to elucidate structural similarities of spike proteins. A homology modeling of SARS-CoV-2 S protein was obtained and used in molecular docking studies to nd binding anities of spike protein for angiotensin-converting enzyme 2 (ACE2). The two most important binding sites of S protein, namely, RBD and CTD, critically responsible for binding interactions, were identied. Finally, binding anity of RBD and CTD domains of S protein with narcotic analgesics are studied. Moreover, interactions of ACE2 receptor- S protein with narcotic compounds when mixed with small molecule adjuvants to improve the immune response and increase the ecacy of potential vaccines, were taken into consideration. In-silico results suggest that the combination of narcotine hemiacetal with mannide monooleate shows a stronger binding anity with CTD, while carprofen-muramyl dipeptide and squalene have stronger binding anities for the RBD portion of S protein. Thus, a suitable combination of these narcotic is proposed to yield potent site-blocking ecacy for ACE2 receptor against SARS-CoV-2 spike proteins.


Introduction
Recently emerged novel coronavirus (2019-nCoV) originated in Wuhan, China [1] is currently the most signi cant ongoing threat to human health worldwide [2,3]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic pneumonia coronavirus after SARS-CoV-1 and MERS-CoV (middle east respiratory syndrome-related coronavirus) to cause a full-blown pandemic [4,5]. According to the World Health Organization (WHO), by 17 February 2021, over 109 million infection cases globally and almost 2,413,000 deaths have been reported [6]. To inhibiting SARS-CoV-2, there are potentially effective strategies such as the design of an effective vaccine and the discovery of a potential drug that capable target active sites of SASR-CoV-2 [7][8][9][10][11][12]. Meanwhile, how the virus enters a host cell has a signi cant effect to develop a novel drug and/or repurpose existing drugs.
Angiotensin-converting enzyme 2 (ACE2) contributes to viral cell entry through the interaction with S protein [13][14][15]. Finding a compound that can block the formation of the complex between the SARS-CoV-2 S protein receptor binding domain (RBD) and the ACE2 receptor or disrupt in RBD-ACE2 complex has been followed as a reasonable strategy to come up with a rational drug discovery for COVID-19 [16]. Since the development or discovery of a novel drug is often costly and might take several years [17], repurposing a drug for timely release to the market signi cantly lowers the cost and help ght the pandemic in its most aggressive phases. Although several efforts are underway, there is so far no promising antiviral agent for SARS-CoV-2 in nal quarter of 2020. Therefore, we still need to review existing drugs in order to discover their antiviral properties [18].
Narcotic compounds are bioactive natural alkaloids mostly with plant origins which act as stimulants and inhibitors in the biological system of mammals including humans. Although use of high-dose narcotic analgesics such as morphine and fentanyl have been observed to cause immune suppression, they are safe, economic, and effective for the management of severe cancer pain when used by medical advice and with precautions [19][20][21]. Recent studies suggest that narcotic compounds such as fentanyl and lidocaine can minimize aerosolization risk of coughing during extubation [22][23][24][25][26][27]. In China, a preliminary analysis in a case study report, examined data from 5 case series of hospitalized COVID-19 patients, and calculated a smoking prevalence of 10.2 % (95 % CI: 8.7-11.8 %) while the estimated expected prevalence was 31.3 % (95 % CI: 8.7-11.8 %) [28]. The analysis was further expanded well into the second pandemic wave, by examining 13 Chinese studies and 5960 hospitalized COVID-19 patients, with a pooled smoking prevalence of 6.5 % (95 % CI: 4.9-8.2 %) [29]. This suggests a hypothesis that nicotine may be protective against severe COVID-19, which is biologically plausible and should be further investigated in clinical trials on nicotine as a drug candidate [30]. These clinical trials should test the effects of the smoking and nicotine on the risk of being infected with COVID-19 (NCT04429815). We recently reported combinations, i.e., nicotine and caffeine, for blocking ACE2 receptor against SARS-CoV-2 [31]. In this paper, we investigated for the rst time a hypothesis about the potential bene ts of narcotic analgesics using in delivery systems as an adjuvant (i.e., Montanide ISA 720, muramyl dipeptide, squalene and Adjuvant Systems 01 (AS01)) to improve the immune response and increase the e cacy of vaccines. We hypothesized that there could be a potential S protein-ACE2 site-blocking activity through forming a complex between narcotic compounds with small molecule adjuvants.

Methods
The interactions of narcotic compounds with the SARS-CoV-2 S protein-ACE2 compared to SARS-CoV S protein with ACE2 complex were investigated. The chemical structure backbones of narcotic compounds resemble some anti-COVID-19 drug candidates. Therefore, we decided to investigate the possible interactions and potential blocking activity of these narcotic compounds with the CTD/RBD-ACE2. Such interaction sites are the epitopes of S protein and the corresponding ACE2 receptor responsible for COVID-19 cell binding mechanisms. This blocking behavior is intensi ed when the compounds are mixed with adjuvants such as Montanide ISA 720, muramyl dipeptide, squalene and AS01. Our focus will be directed toward the potency of narcotic compounds acting as COVID-19 antiviral drugs by blocking the ACE2 receptor mechanism.

Selected Narcotic Compounds
Several narcotic compounds have been introduced in treatment of human immunode ciency virus (HIV), SARS-CoV and COVID-19. Accordingly, we've selected twenty narcotic analgesics as presented in Figure 1. The interaction of narcotic analgesics with the RBD/CTD-ACE2 receptor when combined with these adjuvants were investigated to study their inhibition mechanism as well as their e cacy as potential drug candidates.

Selected small molecule adjuvants
Available vaccines can induce weak immunogenicity and often do not conduce an effective immune response [32]. An underexplored approach for maximising the e cacy, e ciency and enduring effect of vaccines is to discover, develop and optimize e cacious adjuvants [33][34][35]. Adjuvants can be used by enhancing antigen presentation to antigen-speci c immune cells in order to both improve immunogenicity and conduce long-term protection against pathogens [35]. Adjuvants can range in their chemical structure from proteins, complex natural products, oligonucleotides, drug-like small molecules to certain delivery systems, such as those based on liposomes, which possess intrinsic adjuvant activity [34]. Here four selected adjuvants are discussed, see Figure 2.

Montanide ISA 720
Oil adjuvants, based on non-mineral oil such as Squalene which is a natural product with animal or plant origin, has been also used in human clinical trials [36]. Recently water in oil adjuvant based on squalene like Montanide ISA 720 that contains a mannide monooleate emulsi er [37], are tested in various clinical trial representing more than 500 patients and 1500 injections [36]. Many studies with oil-based adjuvant like Montanide ISA 720 reported the use of these adjuvants for immunotherapy such as HIV [38][39][40] treatment and even for prophylactic vaccine where no relevant treatment exists like malaria [41][42][43].

Muramyl Dipeptide
It is found that adjuvants based on peptidoglycan constituents, known as muramyl dipeptide (MDP) can be used to modulate the immune response [44]. It was reported that MDP and their derivatives confer their adjuvanticity effect by activating the NF-κB pathway through the NOD2 receptor [45]. In an in silico study on mices rapid identi cation of SARS-CoV derived antigenic peptides was observed due to recognition mechanisms by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). It was suggested that muramyl dipeptide can conduce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+antigen presenting cells, and it was administered as an adjuvant [46]. It is also proposed that a synthetic vaccine might be designed based on T-epitopes as haptens (for cell response and immune system memory), molecular adjuvant (e.g., muramyl dipeptide), and possibly excitatory or anti-inhibitory peptides for SARS-CoV-2 [47].

Homology modeling of SARS-CoV-2 S protein
All genomic sequences of SARS-CoV-2 S protein (YP_009724390.1) was obtained from National Center for Biotechnology Information (NCBI) nucleotide database. The nucleotide sequences were aligned with whole database using BLASTn to search for homology viral genomes. Accession numbers of 50 sequences in GenBank are listed as follows: QKY78084.  Figure S1.
To nd possible sites of positive or negative selection Adaptive Evolution Server was used (http://www.datamonkey.org/). Statistically signi cant positive or negative selection was based on p value < 0.05 [48].
Recently, the two crystal structures of the complex between SARS-CoV-2 S protein and ACE2 receptor (e.g., 6LZG, 6VW1) are resolved by X-ray diffraction and cryoelectron microscopy (cryo-EM). According to reported structures for ACE2 and SARS-CoV-2 complex, there are two active sites in SARS-CoV-2 S protein, interacting with ACE2. The SARS-CoV-2 RBD and CTD structures as active sites of the SARS-CoV-2 S protein were selected in this study to further investigate the binding mechanism to ACE2 receptor. The nucleotide sequence editing was conducted using Bioedit program v7.0.5 [49], and sequence has been aligned using ClustaIW [50]. The evolutionary history was inferred using the Neighbor-Joining method in molecular evolutionary genetics analysis version X (MEGA-X) [51] software package. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test was determined by 500 replicates. Phylogenetic tree was generated with Jones-Taylor-Thornton (JTT) evolutionary model.
Protein homology modelling has been attempted using the website Swiss-Model [52]. Each model was then individually superimposed over the template and root mean square deviation (RMSD) was estimated in Å using SWISS-PdBViewer 4.1.0 [53]. Three dimensional structures have been analyzed and displayed using PyMOL [54].

Molecular docking
It is suggested that a fast and economical tool (such as molecular docking) can be combined with molecular dynamic simulations (high precision but time consuming) to e ciently conduct more reliable and highly precise calculations in protein-ligand complexes. Moreover, for the fast screening of large libraries, docking can be e ciently utilized to explore the conformations of the protein receptors, optimize the structures of the nal complexes, and nally to calculate accurate energies [55].
For targeting the interface of the S protein (in SARS-CoV-2) and the ACE2 receptor by a blocking agent, we evaluated the blocking potential of narcotic compounds, and their combined forms with adjuvants. All the narcotic molecules (www.drugbank.ca/) and adjuvants were energy minimized using Steepest descent algorithm employed in Avogadro. Reported crystal structures in Protein Data Bank (PDB) for ACE2 (PDB: 1R42), SARS-CoV (PDB: 2AJF) and SARS-CoV-2 CTD/RBD encoded were used. All water molecules, ligands and ions were removed in these crystal structures and nally hydrogen atoms were enhanced to better serve the purpose of this study. Binding a nity estimation for the interactions between the S protein RBD/CTD-ACE2 and narcotic compounds mixed with adjuvants mentioned were all performed via molecular docking.
We used the AutoDock v4.2 package for the docking studies [56,57]. Also, the charges of the molecules were applied. We selected a 60 × 60 × 60 Å grid box, and the distance between two grid points was set at 1.0 Å centering on the structures. In this paper, the rigid structure of the proteins was considered, so that, in this state, the drug is assumed to be xed in shape. By using the Lamarckian genetic algorithm (LGA) [58], we performed molecular docking. In molecular docking through genetic algorithms (GA) [59], the particular arrangement of a ligand and a protein can be de ned by a set of values describing the translation, orientation, and conformation of the ligand with respect to the protein. All thigh pro les were produced under the following conditions: an initial population of 150 randomly placed individuals and a maximum number of 2.5 × 106 energy evaluations, a maximum number of 27,000 generations, a mutation rate of 0.02, a crossover rate of 0.80, and an elitism value of 1. Finally, results were clustered and analyzed considering binding energies and main interacting residues in each complex by bioinformatics module of ICM 3.7.3 modeling software on an Intel i7 4960 processor (MolSoft LLC, San Diego, CA, USA) [60].

Results
Here, we present our in silico results for calculated binding a nities of narcotic compounds mixed with adjuvants to target proteins such as RBD-ACE2 and CTD-ACE2. Also, we highlight ligands of narcotic compounds mixed with adjuvants that we believe may be targeting the binding between S protein and ACE2, and thus are of special interest for experimental evaluation. A structural representation of the interaction between ACE2/SARS-CoV-2-CTD and SARS-CoV RBD with narcotine hemiacetal binding to mannide monooleate is shown in Figure 3.
Any small molecule bound to S protein at this time may interfere the re-folding of S protein, therefore inhibits the viral infection process. Furthermore, small molecule that can target any part of S protein may be a good starting point to design PROTAC based therapy [61]. For mannide monooleate, we found SARS-CoV-2 CTD-ACE2+ narcotine hemiacetal and codeine could be helpful for viral infection treatment, whereas fentanyl is the best option for SARS-CoV RBD-ACE2, as illustrated in Figure 4. Based on our results for strongest binding a nity, initial repurposing may be better suited to carprofen mixed with muramyl dipeptide for both of RBD-ACE2 of SARS-CoV-2 and SARS-CoV. Also, the most e cient compounds are narcotine hemiacetal with SARS-CoV-2 RBD-ACE2 complex, whereas noscapine has the most e cient compounds in SARS-CoV RBD-ACE2. Figure 5 reveals that, in case of the squalene, compounds such as RBD-ACE2+carprofen and noscapine, also, CTD-ACE2+ noscapine could be helpful for viral infection treatment of SARS-CoV-2. But acetohexamide and fentanyl have the strongest binding a nity to SARS-CoV RBD-ACE2. For AS01, we found only one compound, hydrocodone, cannot effectively target the binding between RBD/CTD and ACE2 for both of SARS-CoV-2 and SARS-CoV. The compounds identi ed here could potentially target the binding site between Spike RBD/CTD and ACE2. As suggested by the result, authors believe that these compounds are inhibitors of ACE2 driven virus infections.
The binding energy is due to the energy contributions of all different amino acids and residues around the cavity of target protein on interaction site with the screened molecules. Energy contributions of these residues are due to different interactions like hydrogen bonding, van der Waals, electrostatic interactions, π-π stacking, etc. [62]. As the binding of the S protein to ACE2 is undesirable, it is preferable to diminish the ligand-interface interactions that may bridge, and therefore stabilize, the interaction between the S protein and the ACE2 receptor. The detail of RBD/CTD-ACE2 interface binding to narcotics compounds, i.e., narcotine hemiacetal, codeine, carprofen and noscapine mixed with adjuvants (mannide monooleate, muramyl dipeptide and squalene) were evaluated (Figures 6-8 and S2-S4).
As shown in Figures 6-8 the binding of adjuvants in the active pocket of RBD/CTD-ACE2 were compared to SARS-CoV RBD-ACE2. Also, the binding between S protein and ACE2 with narcotics compounds were compared to SARS-CoV RBD-ACE2 as represented in Figures S2-S4. It seems that variations in the binding free energies occur due to the difference in the hydrophobic interactions and hydrogen bonding formation between RBD/CTD-ACE2′s amino acid residues with narcotics compounds and adjuvants.

Conclusions
In summary, the binding of two crucial active sites of the Spike protein (i.e., RBD and CTD) when complexed to ACE2 receptor was theoretically evaluated using in-silico docking studies. Based on bioinformatics analysis two homology structures of 50 SARS-CoV-2 S proteins were built and were setup for high throughput protein-protein docking studies with ACE2 structures.
The results of the protein-protein docking revealed SARS-CoV-2 S protein (i.e., RBD and CTD) have strong binding a nity toward ACE2. However, this interaction is weaker than that of SARS-CoV S protein.
Possible mutations in speci c loops of S protein, RBD/CTD, might promote binding interaction with the ACE2 receptors. The binding a nity of RBD/CTD when complexed to ACE2 receptor were studied in the presence of accessible natural bioactive alkaloids, i.e., narcotic compounds, via molecular docking. Combination of narcotic compounds with small molecule adjuvants-including mannide monooleate, muramyl dipeptide, squalene and AS01 were found as potential complimentary agents of the ACE2 receptor. The results of the molecular docking revealed a promising binding tendency in narcotic compounds with the ACE2 receptor; to an extent where consequent blocking of ACE2 receptor against SARS-CoV-2 can be optimally achieved.
Combinations of narcotine hemiacetal and mannide monooleate in blocking CTD-ACE2 as well as the combination of carprofen with muramyl dipeptide and squalene in blocking RBD-ACE2 were both shown to be e cient. In conclusion, our results suggest that narcotine hemiacetal, carprofen, codeine and noscapine compounds are capable of interacting with the S protein and ACE2 interface and thus interfere with their binding mechanism through blocking active sites. Our results might have signi cant applications in considering potential drug candidates in therapeutic treatment of SARS-CoV-2 infection. Declarations