Annotation of the results obtained in 2023
The work plan of the second year of the project was aimed at a detailed characterization of the interaction of the receptor-binding domain (RBD) of the SARS-Cov2 virus of the China/Wuhan strain with various subtypes of the nicotinic acetylcholine receptor (nAChR), including binding parameters and action on the functional activity of the receptor, supported by computer modeling, to identify the interface of interaction between RBD and nAChR. The obtained data were to become the basis for clarifying the localization of RBD sites responsible for interaction with nAChR, which was planned to be confirmed by mutagenesis of RBD and synthesis of its fragments capable of recognizing the receptor. If key RBD sites interacting with nAChR were identified, these data could be used to search for compounds that block such interactions and create antiviral drugs based on them. During the second stage of the project, 3 batches of RBD S-protein of the SARS-Cov-2 virus strain China/Wuhan were expressed in Leishmania tarentolae cells for a statistically reliable assessment of its binding and functional activity against nAChR. The gene design assumed the expression into the extracellular space of a recombinant glycosylated protein containing a histidine tag for purification. RBD purification from the culture medium was carried out by two stages of chromatography. As a result of each expression, from 1.8 to 2.5 mg of purified recombinant product was obtained with purity of 92-95%. The average yields of RBD were 1.4-1.8 mg/l of culture. The measured mass of products (ESI-MS) of all three expressions was 23708.0 ± 0.5 Da. Samples of all batches of RBD showed the ability to interact with muscle type α1β1γδ nAChR of the Torpedo californica ray and human α7 neuronal nAChR in a competitive radioligand test. The calculated affinity values (IC50) for three samples with respect to the muscle type receptor were 5.5 ± 0.3, 10.0 ± 1.4 and 13.9 ± 2.7 µM, and with respect to α7 human neuronal nAChR - 6.4 ± 1.3, 8.8 ± 1.6 and about 15 µM. RBD samples also showed the ability to block the functional activity of distinct nAChR subtypes expressed in Xenopus laevis oocytes in electrophysiological tests. Similar inhibitory activity was detected with respect to α7 and α9α10 subtypes of nAChR, but not α4β2. At the same time, the effectiveness of the action, as in radioligand analysis tests, was different for different RBD samples. Thus, at a concentration of 10 µM, three different RBDs inhibited the current through the α7 receptor by 10, 20 and 0%, and through the α9α10 receptor by 20, 30 and 50%, respectively. In order to understand the reason for the noticeable differences in binding and functional activity of different RBD batches, more thorough structural studies of one of the new RBD samples were carried out, including chromatography-mass spectrometric analysis of its tryptic fragments and identification of the glycosylation site with clarification of the glycan structure. Analysis of the tryptic fragments of the recombinant product confirmed its compliance with the RBD structure (complete overlap with MS2 fragmentation of more than 86% of the entire domain structure was revealed). The complete structure of the recombinant RBD was also established - in addition to the corresponding sequence C336-P527 of the domain itself, the structure contains a glycan of the composition (Hex)3(HexNAc)2 at the N343 residue, as well as the additional 3 amino acids SLD at its N-terminus and the sequence GTHHHHHH at the C-terminus. In order to identify the interface of interaction between RBD and nAChR, their complexes were modeled for α1β1γδ and α9α10 receptors. These models suggested the important role of some amino acid residues of RBD for interaction with nAChRs. In the case of a muscle-type receptor, those in RBD may be Lys378, Arg403, Asp405, Arg408, Gln409, Pro412, Gly413, Gln414, Thr415, Gly416, Lys417, Asp420, Gly446, Tyr449, Tyr453, Leu455, Phe456, Glu484, Phe486, Asn487, Tyr489, Phe490, Gln493, Ser494, Tyr495, Gly496, Phe497, Gln498, Thr500, Asn501, Gly502, Tyr505. Interestingly, for α9α10 nAChR, computer modeling gives a completely different location of RBD on the receptor. Accordingly, the important for recognizing residues are Trp21, Arg23, Arg25, Tyr64, Pro94, Asp96, Arg122, Phe124, Arg125, Lys126, Ser127, Asn128, Leu129, Lys130, Pro131, Phe132, Glu133, Arg134, Asp135, Ile136, Ser137, Thr138, Glu139, Ile140, Tyr141, Gln142, Thr146, Pro147, Asn149, Gly150, Val151, Pro159, Glu184, Leu185, Leu186, His187. According to the obtained computer models of the RBD complex with muscle-type nAChR, the amino acid residues of the receptor recognizing domain are located in three regions that were selected to produce 3 synthetic fragments - Peptide 1 – F400-K424, Peptide 2 - N440-F464 and Peptide 3 – C480-Y505. These three fragments were obtained by solid-phase peptide synthesis in amounts of 1.5 mg, 1.7 mg and 1.8 mg, respectively, with a purity of 92 to 95%, and their structures were confirmed by ESI mass spectrometry. However, none of the synthesized Peptides showed any binding and functional inhibitory activity up to a concentration of 10 µM with respect to the muscle-type and α7 receptor subtypes in radioligand tests, and with respect to α9α10 nAChR in electrophysiology. Also, none of the synthesized Peptides at a concentration of 10 µM showed in the SPR tests the ability to bind to the angiotensin converting enzyme 2 (ACE2) immobilized on the chip. The obtained result supports the version that RBD peptide fragments with a length of 25-26 residues probably do not retain their secondary structure, which leads to a loss of their ability to recognize not only the auxiliary target, nAChR, but also the main target, ACE2. Since the possibility of identifying the key amino acid residues of RBD responsible for its interaction with nAChR through the synthesis of peptide fragments of the domain as a method proved unsuccessful, this problem was tried to be solved through RBD mutagenesis. However, computer simulations have identified more than 30 potential amino acid residues that can interact with nAChR. The scope (time and cost) of this project does not allow for the sequential mutagenesis of all predicted amino acid residues in the domain. As an alternative, it was decided to obtain and investigate the cholinergic properties of the RBD S-protein of the SARS-Cov-2 virus of the Omicron strain, which differs from that of the China/Wuhan strain by 15 substitutions. The constructed models of the complexes of muscle-type and α9α10 nAChP with RBD from the Omicron strain suggest the preferred binding of this domain variant to a muscle-type receptor, but not to a α9a10. Therefore, a genetic construct was obtained for the expression of RBD S-protein of the SARS-Cov-2 virus of the Omicron strain (B 1.1.529) in Leishmania tarentolae cells according to the method developed on the China/Wuhan strain. However, for reasons that have not yet been clarified, the culture transformed by this construction turns out to be unviable, which requires further selection of conditions or modification of the transformation protocol.

 

Publications

---

Annotation of the results obtained in 2022
This project "Nicotinic cholinoreceptor as a possible target for SARS-CoV-2 virus recognition" arose a year ago as a reaction to theoretical data that appeared in the scientific literature on the possible participation of nicotinic acetylcholine receptors (nAChR) in interaction with the coronavirus envelope protein, in addition to the main target of its action ‑ angiotensin-converting enzyme 2 (ACE2). Confirmation or refutation of this hypothesis would make a significant contribution to understanding the full picture of the molecular mechanism of infection with SARS-CoV‑2 virus of host cells and spread throughout the organism and experimental verification of it seemed not too difficult. It included, at the first stage of this project, computer modeling of the complexes of the extracellular domain (ECD) of the SARS-Cov-2 virus of the China/Wuhan strain with nAChR with the identification of the domains in the structure of the S-protein - likely participants in the recognition of the receptor, obtaining recombinant forms of both the whole ECD and its domains and their characterization by various available methods (radioligand analysis, electrophysiology, surface plasmon resonance) of the interaction of the obtained S-protein fragments with different nAChR subtypes, as well as their water-soluble structural and functional homologues – acetylcholine-binding proteins (AChBP), which are a remarkable object for crystallization, including in complex with S-protein domains. The main efforts of the first year of this project were focused at obtaining recombinant forms of ECD and its shortened fragment – receptor-binding protein (RBD), which is the area of ACE2 recognition, which we performed in the LEXSY expression system, specially designed to work in Leishmania tarentolae cells. This system, quite simple and cheap, makes it possible to obtain a functionally active protein in the cell culture medium of correct folding and glycosylation close to natural, as well as protein complexes in a heteromeric form. This LEXSY expression system was successfully tested and optimized during the project to obtain AChBP from Lymnaea stagnalis, making it possible to obtain 8 mg of highly active protein in homopentameric form with a purity of at least 95% and a yield of 1.2 mg of product per liter of culture. However, in the case of obtaining ECD of the S-protein of the SARS-Cov-2 virus with the use of this proven technique, we were faced with the problem of extremely low yields of the target product, which even the modification of the ECD gene recommended by the literature did not help to significantly increase. At the same time, its shortened fragment – RBD – avoided problems with expression in the LEXSY system, giving 1.5-1.7 mg of the product per liter of culture, which eventually allowed to produce about 10 mg of this domain with a purity of at least 95%. Interestingly, in 2022, computer modeling data began to appear in the literature on the possible interaction of RBD with some subtypes of nAChR, which were also confirmed by our calculations of the models of the structures of ECD and RBD complexes with the muscle-type nAChR that was planned for the first year of the project. Thus, the recombinant RBD of the S-protein of the SARS-Cov-2 virus obtained by us during the reporting period became the main object for studying its "cholinergic" properties. The binding ability of the domain was tested by the method of competitive radioligand analysis for its effectiveness in displacing radioactively labeled α-bungarotoxin from α1β1γδ muscle-type nAChR from Torpedo californica, the human neuronal α7 receptor, as well as AChBPs. As a result, it was experimentally established for the first time that RBD quite effectively inhibits the specific binding of the radioligand to muscle-type and neuronal α7 nAChR subtypes (IC50 values were 6.6 ± 0.4 µM and 3.0 ± 0.1 µM, respectively), but does not displace labeled α-bungarotoxin from AChBPs. Electrophysiological studies initiated in the first year of the project on the effect of RBD on the functional activity of some nAChR subtypes showed preliminary inhibitory activity of the domain on the activation of the α7 receptor induced by acetylcholine. The method of surface plasmon resonance and the preparation of a radioactive form of RBD for the possible evaluation of the parameters of its direct interaction with nAChRs or AChBPs (Kd) were also undertaken in the first year of the project, but encountered certain difficulties and will be continued at the next stage. At the same time, the main efforts are planned to focus on localization in the RBD of the nACHR recognition site and identification of key amino acid residues that determine their interaction. This is planned to be done by mutagenesis methods in RBD, based on the model of the structure of the RBD complex with muscle-tupe nAChR from Torpedo californica obtained by us. Summing up in one phrase the main result of the work in the first year of this project, we can say that for the first time we experimentally confirmed a sufficiently effective interaction of the RBD of the SARS-Cov-2 virus S protein with the muscle and α7 neuronal subtypes of the nicotinic cholinoreceptor.

 

Publications

---