Preliminary Exploration Of MERS - CoV Fusion Inhibitors With N - Peptide Bonded

Since 2012, a new type of coronavirus zoonotic virus called Middle East Respiratory Syndrome Corona Virus(MERS-Co V) have been outbreak in the Middle East, Asia, Europe and South Korea, causing more than 1,600 people infected. The mortality rate is as high as36%, but there is no specific treatment. Therefore, the development of a specific drugs used against the highly pathogenic and highly contagious of MERS-Co V, become a top priority.MERS-Co V belongs to the class I fusion enveloped virus. The virus uses its Class I Fusion protein as the mediating molecule, which is a necessary step during the virus infection.In this process, the transmembrane subunit of the fusion protein undergoes intramolecular folding. Its N-terminal repeat(NHR) and C-terminal repeat(CHR) interact into structure of6-helix-bundle(6-HB). Adding the peptide derived from NHR or CHR into the process can prevent the formation of 6-HB, which will inhibit the process of virus-cell fusion. Virus stopped out of the host cell will be removed by the body’s immune defense. Previous studies have indicated that HR2P-6S, a MERS-Co V CHR deriving peptide, has a certain fusion inhibitory activity against MERS and potential druggability. NHR is an important structure in the process of 6-HB formation. The interaction between NHR and CHR makes them target each other. NHR derivatives(N-peptides) can combine with CHR competitively to interfere the formation of 6-HB, which can inhibit the viral fusion. Therefore, the development of MERS-Co V N-peptide fusion inhibitor can furtherly demonstrate the mechanism of MERS-Co V fusion protein, and might develop a fusion inhibitors with new mechanism and target.In the current MERS-Co V N-peptide inhibitors development, there are several problems.Unlike CHR, natural NHR has a complicated hydrophobic environment. The hydrophobic residues on the N-peptide surface leads the water solubility poor; from the perspective of 1st structure, hydrophilic and hydrophobic residues of N-peptide sequence appear alternately. The characteristic of this structure is more in line with the structure of beta folding or hydrogels.These two points make it difficult to reproduce the N-peptide helical trimer(N-trimer) in vitro,which is the activity structure. Thus N-peptide fusion activity is poor. Further, N-peptides under physiological conditions is easily integrate into hydrogel or irreversible precipitation,makes the experimental operation difficult. The problems above are ubiquitous in all kinds of enveloped virus N-peptide fusion inhibitor development process. Therefore, this study is in the purpose of improving the biophysical properties of MERS-Co V N-peptide to enable the reproducible of N-trimer advanced structure in vitro, and the stability of active conformation in order to simulate natural structure of MERS-Co V N-trimer molecular, conduce the further development of N-peptide fusion inhibitors.Based on our previous research, we investigated two methods here to study MERS-Co V N-peptide fusion inhibitors. The first method is to add an auxiliary sequence to N-peptide,then investigate inter-chain isopeptide bonds to stabilize the N-trimer. The auxiliary sequence can induce N-peptides assemble into triple helix. IZm is an artificially designed polypeptide sequence, which is rich in hydrophilic residues. Chimeric peptide sequence can improve the water solubility by weakening the tendency of aggregation; amino acid in a, d site of IZm is isoleucine, and b, f sites, e, g’ site is glutamic acid and lysine, which can induce the formation of α-helix, and strengthen the self-assembility into trimer. On this basis, we used an acyl-transfer-reaction under physiological conditions, to form isopeptide bond between adjacent N-peptide, which between lysine residue and glutamic acid residue in e, g ’site, in order to build an auxiliary sequence modified N-trimer stabled by isopeptide bonds.Second method is to use the triple-helix forming strategy and the isopeptide bonds forming strategy to stabilize the structure of N-trimer. Research shows that in the α-helix,strengthen the interaction between i/i+3 and i/i+4 site can enhance the helicity; introduced isoleucine(Ile) in a and d sites can enhance the tendency of self-assemble into trimers;glutamic acid-lysine ion bridge in e, g’ site between the adjacent helical chain can further stabilize the trimer structure. So we introduced glutamate(Glu) and lysine(Lys) that can form ion-bridge in b, c, f site of the N-peptide, in order to improve the helicity and water solubility;introduced isoleucine in a and d sites, to help the formation of N-trimer; and on these basis,we introduced the inter-chain isopeptide bonds in e, g’ site instead of ionic bond, forming the natural N-trimer molecules stabilized by isopeptide bond.We used benzyl thiol ester formed with carboxyl functional group in glutamic acid residue to active lysine, make the amino group nucleophilic attack on the carbonyl carbon to form isopeptide bonds. We synthesized with orthogonal protection strategy and complete the thioester modification in the resin. Classic Fmoc solid phase synthesis, Rink Amide resin,synthesized with allylmethyl protecting Glu. Peptide capped by N-terminal acetylation,dimedone and [Pd]0 to remove the allylmethyl protecting group. Synthesized the exposing carboxyl group to benzyl thiol ester catalysed by EDC/HBTU. Cleaved to remove resin and other protective groups with TFA, followed by purified and freeze-dried. The purified peptide was dissolved in 10 m M PBS in p H=7.2 solution containing 10% acetonitrile. Acetonitrile provides a state of natural membrane lipid environment, make the peptide to form the correct structure; moiety ionic conditions make thioester activate the alkaline in acyl transfer reaction.Covalent reaction was monitored by reversed phase high performance liquid chromatography(RP-HPLC), the purified products is confirmed by matrix-assisted laser desorption / ionization time of flight mass spectrometry instrument(MALDI-TOF-MS) or electrospray ionization mass spectrometry(ESI-MS). Secondary structure is determined by Circular dichroism(CD), and cell to cell fusion experiment was used to evaluation the N peptide inhibiting activity.Result of RP-HPLC and MALDI-TOF-MS showed that we have successfully constructed MERS-Co V NHR deriving N-trimer molecular stabled by isopeptide bond. Due to the chemical environment changes before and after covalent cross-linking, chromatography retention time of stabled N-trimer is shorter than monomer. This phenomenon matches our preliminary studies. CD results showed that the 2nd structure of auxiliary modified N peptide have improved compared with natural N peptide HR1 P.In summary, we have cross-linked 11 covalent sequences based on the synthesized polypeptide. Isopeptide bond was formed under physiological conditions to construct MERS-Co V covalent stabled N-trimer model. Although this model can provide a reference for constructing Middle East respiratory syndrome syndrome coronavirus spike protein transmembrane subunit N-terminal helix trimer in vitro, but as fusion inhibitor, an IC50 value of above 25μM in cell-cell fusion model did not show the expected activity, which needs further study and improvement. Also, on the basis of our research group previous work, we have further verified the general applicability of this isopeptide bond formation method, this method can provide a reference for the construction of other helix trimer model.