Fusion lipopeptides for inhibiting coronavirus

Abstract

A fusion peptide comprising a coronavirus HR2-derived peptide fused to an ACE2-derived peptide or a lipopeptide comprising the fusion protein and cholesterol is a broad spectrum inhibitor of a coronavirus.

Description

[0001] Cross-references to related applications This application claims priority to PCT International Application PCT / CN2023 / 087911, filed on April 12, 2023, which is incorporated herein by reference in its entirety.

[0002] Invention Field This invention relates to polypeptide drugs, and more specifically to lipopeptides for inhibiting coronaviruses. Background of the Invention The ongoing COVID-19 pandemic has resulted in over 6 million deaths. While many approved vaccines and therapies are effective in reducing hospitalizations and deaths from SARS-CoV-2 infection, different variants of concern for SARS-CoV-2, particularly the Omicron variant, have been shown to evade immunity on a broad scale, significantly impairing the efficacy of vaccines and therapies (1–7). Therefore, the need for developing highly effective, broad-spectrum antiviral drugs that can inhibit current and emerging SARS-CoV-2 variants remains high.

[0004] Similar to other coronaviruses, SARS-CoV-2 infection requires fusion between the viral envelope and the cell membrane, a process mediated by the viral spike (S) glycoprotein. The SARS-CoV-2 S protein consists of S1 and S2 subunits and enters the cell via a type I enveloped viral fusion mechanism (8-11). The S1 subunit binds to the cell receptor angiotensin-converting enzyme 2 (ACE2) via its receptor-binding domain (RBD), triggering a conformational change in the S2 subunit. This results in the formation of a six-hexagonal bundle (6-HB) between the heptapeptide repeat 1 (HR1) and heptapeptide repeat 2 (HR2) domains, driving the fusion of the target cell and viral membrane (10-13). Because the HR1 and HR2 domains are highly conserved among different coronaviruses, peptides derived from these two domains are considered promising antiviral agents (10, 11, 14). Indeed, peptides derived from both the HR1 and HR2 domains have been shown to inhibit viral infection, with IC50... 50 The values ​​are in the micromolar range (15-18). Several HR2 helical conformation stabilization strategies, such as chemical stapling and the introduction of salt bridges, have been reported to improve the activity of these peptide inhibitors, IC50. 50 The values ​​ranged from low micromolar to high nanomolar (14, 19). Cholesterol modification of HR2-derived peptides is another effective method, which can significantly enhance inhibitory activity and IC50. 50 The values ​​typically reach low nanomolar concentrations (20-22).

[0005] In the past, peptides derived from the ACE2 protein have been explored for use as antiviral agents. However, due to the low spike protein binding affinity of ACE2-derived peptides, these peptides have had limited success in inhibiting SARS-CoV-2 infection (23-25).

[0006] Invention Summary In one aspect, the present invention provides a fusion polypeptide comprising a coronavirus HR2-derived peptide fused to an ACE2-derived peptide.

[0007] In some implementations, the ACE2-derived peptides are derived from the ACE2 region that interacts with the spike protein of the coronavirus.

[0008] In some implementations, the ACE2-derived peptides are derived from the α-helix 1 region of ACE2.

[0009] In some embodiments, the ACE2-derived peptide contains the amino acid sequence of IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFR (SEQ ID NO: 6) or a functional variant thereof.

[0010] In some implementations, the HR2-derived peptide of the coronavirus competitively interacts with the HR1 domain to block the fusion of the coronavirus with the host cell.

[0011] In some implementations, the coronavirus HR2-derived peptide is derived from the spike protein of SARS-CoV, SARS-CoV-2, or MERS-CoV.

[0012] In some implementations, the coronavirus HR2-derived peptide contains the amino acid sequence of DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO: 7) or a functional variant thereof.

[0013] In some implementations, a peptide derived from coronavirus HR2 is fused to a peptide derived from ACE2 via a linker.

[0014] In some implementations, the linker is a flexible peptide linker containing at least 25 amino acids.

[0015] In some implementations, the connector is composed of glycine and serine.

[0016] In some implementations, the connector includes (GGGGS)5, (GGGGS)6, (GGGGS)7, (GGGGS)8, (GGGGS)9, or (GGGGS) 10 The amino acid sequence.

[0017] In some implementations, the coronavirus HR2-derived peptide is located at the C-terminus of the ACE2-derived peptide.

[0018] In another aspect, the present invention provides a lipopeptide comprising a fusion protein and a lipid according to any one of claims 1-12.

[0019] The present invention also provides a lipopeptide comprising a peptide and lipid derived from coronavirus HR2, wherein the coronavirus HR2-derived peptide comprises the amino acid sequence DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO: 7).

[0020] In some implementations, lipids are attached to the C-terminus of the fusion protein.

[0021] In some implementations, the lipids are cholesterol, tocopherol, and palmitate.

[0022] In some implementations, lipids are attached to the fusion protein via spacers.

[0023] In some implementations, the spacers are PEG-based spacers.

[0024] In some implementations, the spacer comprises PEG4 or AEEA-AEEA.

[0025] In some implementations, the spacer is selected from the following formula: and .

[0026] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned fusion protein or the aforementioned lipopeptide, and a pharmaceutically acceptable excipient.

[0027] In some implementations, the pharmaceutical composition is a dosage form suitable for intranasal or intravenous administration.

[0028] In another aspect, the present invention provides a method for preventing or treating coronavirus infection in a subject in need, the method comprising administering an effective amount of the above-described pharmaceutical composition to the subject.

[0029] In some implementations, the pharmaceutical composition is administered intranasally or intravenously.

[0030] In some implementations, the coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV, or a variant thereof.

[0031] In some implementations, the coronavirus is an Omicron variant.

[0032] In another aspect, the present invention provides the use of the above-described fusion protein or lipopeptide in the preparation of a medicament for the prevention or treatment of coronavirus infection in subjects in need.

[0033] In some implementations, the coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV, or a variant thereof.

[0034] In some implementations, the coronavirus is an Omicron variant.

[0035] In another aspect, the present invention provides the above-described pharmaceutical composition for the prevention or treatment of coronavirus infection in subjects in need.

[0036] In some implementations, the pharmaceutical composition is administered intranasally or intravenously.

[0037] In some implementations, the coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV, or a variant thereof.

[0038] In some implementations, the coronavirus is an Omicron variant. Brief description of the attached diagram Figure 1 Design and characterization of A1L35HR2m, HR2mL35A1, A1L5HR2m, and A1HR2m. (A) Design of the peptides A1L35HR2m, HR2mL35A1, A1L5HR2m, and A1HR2m, and sequences of the A1 and HR2m peptides. A1L35HR2m, HR2mL35A1, A1L5HR2m, and A1HR2m were characterized by SDS-PAGE (B) and LC-MS (C).

[0040] Figure 2Conjugation of ACE2-derived peptide A1 to the N-terminus of HR2m peptide significantly enhanced its inhibitory activity against SARS-CoV-2 infection. (A) Inhibitory activity of A1L35HR2m, HR2mL35A1, A1L5HR2m, and A1HR2m against SARS-CoV-2 pseudovirus infection in Caco-2 cells. (B) Inhibitory activity of A1L35HR2m, HR2m, A1, and a mixture of A1 / HR2m against SARS-CoV-2 pseudovirus infection in Caco-2 cells. (C) Inhibition of SARS-CoV-2 D614GS-mediated cell fusion in Caco-2 cells by A1L35HR2m, HR2m, and A1. (D) Inhibition of SARS-CoV-2 D614GS-mediated cell fusion in Calu-3 cells by A1L35HR2m, HR2m, and A1. Each sample was tested three times, and data are expressed as mean ± SEM. (E) The binding affinity of A1L35HR2m to HR1 was determined by BLI. (F) The binding affinity of HR2m to HR1 was determined by BLI. The fitted curves were analyzed using ForteBio Data Analysis 12.0 software.

[0041] Figure 3 A1L35HR2m-Chol broadly and effectively inhibits pseudovirus infection. (A) Chemical structure of A1L35HR2m-Chol. Inhibitory activity of A1L35HR2m-Chol, HR2m-Chol, A1L35HR2m, and HR2m in pseudovirus infection assays against SARS-CoV-2 D614G (B), SARS-CoV-2 Alpha variant (C), SARS-CoV-2 Beta variant (D), SARS-CoV-2 Delta variant (E), SARS-CoV-2 Omicron variant (F), SARS-CoV (G), MERS-CoV (H), and VSV-G (I). Each sample was tested three times, and data are expressed as mean ± SEM.

[0042] Figure 4A1L35HR2m-Chol effectively inhibited real SARS-CoV-2 Omicron in vitro and in vivo. In micro-neutralization assays, A1L35HR2m and A1L35HR2m-Chol inhibited real Omicron BA.2.12.1 (A) and BA.5 (B). In viral load reduction assays, A1L35HR2m and A1L35HR2m-Chol inhibited real Omicron BA.2.12.1 (C) and BA.5 (D). Viral load was quantified by RT-qPCR. (E) Dilution factor of mouse plasma samples treated intranasally with A1L35HR2m (5 mg / kg) or A1L35HR2m-Chol (5 mg / kg) that achieved 50% inhibition of SARS-CoV-2 D614G pseudovirus. (F) Estimated concentrations of functionally active A1L35HR2m and A1L35HR2m-Chol in plasma samples. (G) Schematic diagram of the prevention and treatment experimental design. On day 0, K18-hACE transgenic mice were challenged with Omicron BA.5 (10000 PFU). In the prevention study, A1L35HR2m-Chol (2 mg / kg) was administered intranasally on days 2 and 1 before infection. In the treatment study, A1L35HR2m-Chol (2 mg / kg) was administered intranasally on days 1 and 2 post-infection. Tissue samples were collected on designated dates following viral infection or drug treatment. In the prevention experiment, nasal turbinates and lungs were collected on day 2 post-infection for viral load (H) and infectious viral titer (I). In the treatment experiment, nasal turbinates and lungs were collected on day 3 post-infection for viral load (J) and infectious viral titer (K). (A) to (F), each sample was tested three times, and data are expressed as mean ± SEM. (H) to (K), PBS, n=5; A1L35HR2m-Chol, n=5. The dashed lines in (I) and (J) represent the limits of detection (LOD). An unpaired two-tailed t-test was performed.

[0043] Figure 5 Characterization of HR2m-Chol. HR2m-Chol was characterized by RP-HPLC (A) and characterizing electrospray ionization mass spectrometry (ESI-MS) (B).

[0044] Figure 6 Characterization of A1 peptide. The A1 peptide was characterized by analytical HPLC (A) and ESI-MS (B).

[0045] Figure 7 Characterization of HR2m peptide. The HR2m peptide was characterized by analytical HPLC (A) and ESI-MS (B).

[0046] Figure 8 Characterization of the HR1 peptide. The sequence of the HR1 peptide and its characterization by LC-MS.

[0047] Figure 9 Characterization of A1L35HR2m-Chol. A1L35HR2m-Chol was characterized by LC-MS.

[0048] Figure 10 Inhibitory activity of A1L35HR2m-Chol. (A) Inhibitory activity of A1L35HR2m-Chol against SARS-CoV-2 pseudovirus infection. (B) Inhibitory activity of A1L35HR2m-Chol against cell fusion between 293T / EGFP / S and Caco-2 or Calu-3 cells.

[0049] Figure 11 Cytotoxicity of A1L35HR2m-Chol to Caco-2 and Calu-3 cells. The effects of A1L35HR2m-Chol (A) and A1L35HR2m (B) on the viability of Caco-2 and Calu-3 cells were evaluated using CCK-8 assay.

[0050] Figure 12 Dynamic light scattering (DLS) analysis of A1L35HR2m-Chol. The particle size distribution of A1L35HR2m-Chol was measured by DLS. Invention Details Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples herein are for illustrative purposes only and are not intended to be limiting.

[0052] When used with numerical variables, the terms “about” and “approximately” typically refer to the value of the variable as well as all values ​​of the variable within a range of measurements or experimental errors (e.g., a 95% confidence interval of the mean) or within a wider range of specified values ​​(e.g., ±10%).

[0053] As used in this article, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0054] The term "comprise" and its variations, such as "comprises" and "comprising," as well as "contain," "containing," "have," "include," and "including," indicate that the stated step or element is included, but do not exclude other steps or elements. "Constitutes of" means that any unspecified step or element is excluded. "Substantially constitutes of" means that steps or elements that do not substantially affect the essential and novel features of the claimed invention are not excluded. The term "comprise" and its variations also include cases where "consist of" and "substantially constitutes" are used.

[0055] When a range of values ​​is provided, it should be understood that the upper and lower limits, as well as each smaller range between the upper (or lower) limit and any intermediate value, or between any two intermediate values ​​within that range, should be considered specifically disclosed. Any intermediate range within the range of values, as well as all individual values, may be excluded from the range of values.

[0056] The term “and / or” refers to any one, several or all of the elements connected by the term.

[0057] Unless otherwise stated, nucleic acids are written from left to right in the direction from 5' to 3'; amino acid sequences are written from left to right in the direction from N-terminus to C-terminus.

[0058] The terms “associate,” “interact,” “conjugate,” “link,” “connect,” “couple,” “bond,” or similar terms refer to one element (e.g., a compound) being directly connected to another element (e.g., another compound), or indirectly connected to another element (e.g., another compound) through one or more intermediate parts, in a covalent or non-covalent manner. Examples of non-covalent association include electrostatic forces, hydrogen bonds, hydrophobic effects, van der Waals forces, etc., such as the interaction between a protein and its ligand, the interaction between an aptamer and its adaptor protein, or the interaction between an antigen and an antibody.

[0059] The terms “first” and “second” are used only to distinguish elements or steps and do not imply a specific order of priority or positional relationship. It should be understood that when referring to a “second” element or step, it is intended to distinguish it literally from other elements or steps, and does not necessarily imply a corresponding “first” element or step.

[0060] Furthermore, it should be understood that although the specification may present the methods and / or processes of the present invention as having a specific sequence of steps, the method or process should not be limited to the specific sequence of steps described herein, as long as it does not depend on such a sequence. Those skilled in the art will understand that other sequences of steps are also possible. Therefore, the specific order of steps listed in the specification should not be construed as a limitation of the claims.

[0061] This invention provides a fusion polypeptide comprising a coronavirus HR2-derived peptide fused to an ACE2-derived peptide.

[0062] ACE2-derived peptides are peptides derived from ACE2. ACE2-derived peptides may contain, or consist of, a coronavirus spike protein (S protein) binding fragment of ACE2 or a functional variant thereof. In some embodiments, ACE2-derived peptides are capable of interacting with (or binding to) the coronavirus S protein, particularly the RBD of the coronavirus S protein. In some embodiments, ACE2-derived peptides are capable of competitively inhibiting the interaction between the coronavirus S protein and the host's ACE2. In some embodiments, ACE2-derived peptides can block coronavirus entry into host cells.

[0063] In some embodiments, ACE2 may be a mammalian ACE2, such as human ACE2 (hACE2) or any homolog or ortholog thereof, such as ACE2 derived from mice, rats, hamsters, pigs, primates, or cattle. In some embodiments, ACE2 may be wild-type. For therapeutic or preventative purposes, preferably, the ACE2 is derived from the same organism as the organism to be treated to minimize the risk of immunogenic reactions; for example, a peptide derived from human ACE2 may be used to treat human subjects. However, it should be understood that a different ACE2 from the organism to be treated may also be used; for example, a fusion polypeptide containing peptides derived from ACE2 from a different organism may be used to treat humans, mice, rats, hamsters, pigs, primates, or cattle.

[0064] The coronavirus spike protein-binding fragment of ACE2 is an ACE2 fragment that can interact with the coronavirus S protein (especially the RBD of the coronavirus S protein).

[0065] In some implementations, the length of the ACE2-derived peptide can be 20-40 amino acids, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0066] ACE2 is known to interact primarily with the RBD of the S protein via the α-helix 1 region located at the N-terminus of ACE2. Therefore, in some embodiments, the coronavirus spike protein-binding fragment of ACE2 may contain, or be composed of, the α-helix 1 region of ACE2. In some implementations, the coronavirus spike protein binding fragment of ACE2 may contain one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus of the α-helix 1 region of ACE2 and / or contain one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the α-helix 1 region of ACE2.

[0067] In some implementations, the coronavirus spike protein binding fragment of ACE2 may include the sequence of amino acids 21 to 45 of hACE2 (i.e., the sequence IEEQAKTFLDKFNHEAEDLFYQSSL (SEQ ID NO: 17), or composed of, or containing one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus of the amino acid sequence from position 22 to position 44 of ACE2 and / or containing one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the amino acid sequence from position 21 to position 45 of ACE2.

[0068] It should be understood that, in addition to the coronavirus spike protein binding fragment of ACE2 containing the natural sequence of the ACE2 fragment, functional variants of the coronavirus spike protein binding fragment of ACE2 can also be used as peptides derived from ACE2 in this invention.

[0069] Functional variants of the ACE2 coronavirus spike protein binding fragment can be peptides comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid insertions, substitutions, and / or deletions compared to the amino acid sequence of the ACE2 coronavirus spike protein binding fragment, or peptides formed by inserting, substituting, and / or deleting one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids into the amino acid sequence of the ACE2 coronavirus spike protein binding fragment. Substitutions can be conserved or non-conserved. Functional variants of the ACE2 coronavirus spike protein binding fragment can also be peptides having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ACE2 coronavirus spike protein binding fragment. Functional variants of the ACE2 coronavirus spike protein binding fragment can interact with the coronavirus spike protein in a manner similar to the interaction between ACE2 and the coronavirus S protein.

[0070] In some implementations, the ACE2-derived peptide can be a peptide mimic of the α-helix 1 region of ACE2. The peptide mimic of the α-helix 1 region can form an α-helix structure and be able to interact with the coronavirus S protein (which has a binding affinity for the spike RBD of SARS-CoV-2).

[0071] Numerous publications describe the crystal structures of several coronavirus spike receptor-binding domains (RBDs) that bind to hACE2, the regions where ACE2 interacts with the coronavirus spike protein, key interacting amino acid residues, and various ACE2 α-helix 1 region peptide mimics, such as P. Karoyan et al., Human ACE2 peptide-mimics block SARS-CoV-2 pulmonary cell infection. Commun. Biol. 4, 197 (2021); DP Han, A. Penn-Nicholson, MW Cho, Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor. Virology 350, 15-25 (2006); SK Panda, PS Sen Gupta, S. Biswal, AK Ray, MK Rana, ACE-2-Derived Biomimetic Peptides for the Inhibition of Spike Protein of SARS-CoV-2. J. Proteome Res. 20, 1296-1303. (2021); MV Sidorova et al., Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor andTheir Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain, DoklBiochem Biophys. 507, 1, 237-241 (2022); Lan, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2receptor.Nature581, 215–220 (2020); Fang Li et al., Structure of SARS-Coronavirus Spike Receptor-Binding Domain Complexed with Receptor, Science, 309, 5742, 1864-1868; Abdul Basit et al., Designing Short Peptides to Block the Interaction of SARS-CoV-2 and Human ACE2 for COVID-19 Therapeutics, Front. Pharmacol., 12, 1-8, (2021); Sajjan Rajpoot et al., A Novel Therapeutic Peptide Blocks SARS-CoV-2 Spike Protein Binding with Host Cell ACE2 Receptor, Drugs in R&D, 21, pages 273–283 (2021); These are incorporated herein by reference in their full text. Those skilled in the art will recognize that these ACE2 α-helix region 1 peptide mimics can also be used in this invention, and will understand that those skilled in the art can further obtain various ACE2 coronavirus spike protein (S protein) binding fragments or functional variants thereof, or various ACE2 α-helix region 1 peptide mimics, in accordance with the teachings of these documents.

[0072] In some embodiments, the ACE2-derived peptide comprises, or is composed of, the amino acid sequence IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFR (SEQ ID NO: 6). In some embodiments, the ACE2-derived peptide may comprise, or be composed of, an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid insertions, substitutions, and / or deletions compared to SEQ ID NO: 6. Substitutions may be conservative or non-conservative. In some embodiments, the ACE2-derived peptide may comprise, or be composed of, a peptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0073] HR2 is one of the two heptapeptide repeat domains (HR1, also known as HRN, and HR2, also known as HRC) of the coronavirus S protein. During the binding of the coronavirus S protein to the receptor ACE2, HR2 and HR1 associate to form a 6-helix bundle (6-HB), thereby promoting the fusion of the viral envelope with the target host cell membrane. Receptor binding triggers a conformational change in the S protein (Hoffman M, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and IsBlocked by a Clinically Proven Protease Inhibitor. Cell 181:1-10 (2020); Xia S, et al. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1domain in spike protein. Cell. Mol. Immunol. (2020)).

[0074] The HR2-derived peptide is derived from the HR2 domain of the coronavirus S protein and can interact with the HR1 domain of the coronavirus to be inhibited. The HR2-derived peptide can competitively inhibit the formation of the 6-HB structure of both the HR1 and HR2 domains of the coronavirus to be inhibited. In some embodiments, the HR2-derived peptide can block the fusion of the coronavirus with and entry into host cells.

[0075] In some embodiments, the HR2-derived peptide is derived from the S protein of SARS-CoV, SARS-CoV-2, or MERS-CoV (including variants of SARS-CoV, SARS-CoV-2, or MERS-CoV, as described below). The S protein can be the wild-type coronavirus S protein or the S protein of naturally occurring variants of these viruses. For therapeutic or preventative purposes, preferably, the HR2-derived peptide is derived from the S protein of the same species as the coronavirus to be inhibited to improve specificity; for example, a peptide derived from the HR2 domain of SARS-CoV-2 has been used to inhibit SARS-CoV-2 infection. However, it should be understood that HR2-derived peptides from the S protein of a different species than the coronavirus to be inhibited can also be used; for example, fusion peptides containing HR2-derived peptides from the S protein of different coronavirus species can be used to inhibit SARS-CoV, SARS-CoV-2, or MERS-CoV infection.

[0076] In some implementations, the length of the HR2-derived peptide can be 20-60 amino acids, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0077] HR2-derived peptides may contain or be composed of fragments of the coronavirus S protein, wherein the fragments of the coronavirus S protein may contain or be composed of the HR2 domain of the coronavirus S protein or fragments thereof.

[0078] Fragments of the coronavirus S protein may contain one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus of the HR2 domain of the coronavirus S protein, and / or contain one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the HR2 domain of the coronavirus S protein.

[0079] In some embodiments, the fragment of the coronavirus S protein may comprise the amino acid sequence from position 1145 to position 1190 of the SARS-CoV-2 S protein (i.e., DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO: 7) or consisting of the sequence of the SARS-CoV-2 S protein, or containing one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the N-terminus of the amino acid sequence of the SARS-CoV-2 S protein from position 1145 to position 1190 and / or containing one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the amino acid sequence of the SARS-CoV-2 S protein from position 1145 to position 1190 and / or containing one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 amino acids) at the C-terminus of the amino acid sequence of the SARS-CoV-2 S protein.

[0080] It should be understood that, in addition to the natural sequence of the coronavirus S protein fragment, functional variants of the coronavirus S protein fragment can also be used as HR2-derived peptides in this invention.

[0081] Functional variants of coronavirus S protein fragments can be peptides containing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid insertions, substitutions, and / or deletions compared to the amino acid sequence of a coronavirus S protein fragment containing an HR2 domain or an HR2 domain fragment, or peptides formed by inserting, substituting, and / or deleting one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids into the amino acid sequence of a coronavirus S protein fragment containing an HR2 domain or an HR2 domain fragment. Substitutions can be conserved or non-conserved. Functional variants of the ACE2 coronavirus spike protein binding fragment can also be peptides having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a coronavirus S protein fragment containing an HR2 domain or an HR2 domain fragment.

[0082] In some embodiments, the HR2-derived peptide may contain, or consist of, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid insertions, substitutions, and / or deletions compared to SEQ ID NO: 7. Substitutions may be conserved or non-conserved. In some embodiments, the HR2-derived peptide may contain, or consist of, a peptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0083] Those skilled in the art will recognize that various HR2-derived peptides, such as those described in the publications mentioned above or known in the prior art, can also be used in this invention.

[0084] Several HR2-derived peptides (HR2Ps) from organisms such as SARS-CoV, SARS-CoV-2, or MERS-CoV have been described as inhibitors of coronavirus infection by blocking the fusion of coronaviruses with host cells and their entry into host cells. See, for example, US 7,151,163; S. Xia et al., Middle East respiratory syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike protein. Virus Res. 194, 200-210 (2014); Xia S, et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci. Adv. 5:eaav4580 (2019); S. Xia et al., Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol. Immunol. 17, 765-767 (2020); S. Liu et al., Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenicmechanism and identification of fusion inhibitors. Lancet 363, 938-947(2004); Lu L, et al. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat. Commun.5:3067 (2014); S. Xia etal., Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highlypotent pan-coronavirus fusion inhibitor targeting its spike protein thatharbors a high capacity to mediate membrane fusion. Cell research 30, 343-355(2020); Y. Zhu, D. Yu, H. Yan, H. Chong, Y. He, Design of Potent MembraneFusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with HighFusogenic Activity. J. Virol. 94, e00635-20 (2020); Berend Jan Bosch et al.,Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibitionusing spike protein heptad repeat-derived peptides, Proc Natl Acad Sci U S A,101, 22, 8455-60, (2004); Kailu Yang et al., Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpinintermediate of the spike protein, Proc Natl Acad Sci U S A, 119, 40,e2210990119, (2022); Yanhui Xu et al.The descriptions of the heptad repeat regions, HR1 and HR2, and the design of a fusion core structure model of the spike protein from severe acute respiratory syndrome (SARS) coronavirus, in *Biochemistry*, 9, 43, 44, 14064-71 (2004), are incorporated herein by reference in their entirety. Those skilled in the art will recognize that these HR2Ps can also be used in this invention.

[0085] In some implementations, the coronavirus HR2-derived peptide may be located at the C-terminus of the ACE2-derived peptide.

[0086] In some implementations, a peptide derived from coronavirus HR2 is fused to a peptide derived from ACE2 via a linker.

[0087] Suitable linkers include straight-chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. Linkers used herein can also be covalent bonds (carbon-carbon or carbon-heteroatom bonds). In some embodiments, linkers are used to separate the two peptides to which they are linked by sufficient distance to ensure that each peptide retains its desired functional properties.

[0088] In some embodiments, the length of the peptide linker can be from about 25 to 100 amino acids. The length of the peptide linker can be, for example, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids. The length of the linker can be, for example, at least 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 amino acids. In other embodiments, the length of the peptide linker is, for example, at most 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids. Any range including any combination of these endpoints is also contemplated.

[0089] In some embodiments, a flexible peptide linker is used (i.e., comprising amino acids with high degrees of freedom, such as glycine, alanine, and / or serine). In some embodiments, the peptide linker comprises one, two, or more of the amino acids Gly, Asn, and Ser. In some embodiments, about 80% or 90% or more of the amino acids in the linker sequence are glycine, alanine, and / or serine residues, particularly glycine and serine residues. In some embodiments, the linker may consist of glycine and serine residues. In some embodiments, the linker may comprise a GGGGS sequence or a GGGGS sequence repeated n times in tandem, where n can be in the range of about 5 to 10, for example, 5, 6, 7, 8, 9, or 10. In some implementations, for example, (GGGGS)5 (SEQ ID NO: 12), (GGGGS)6 (SEQ ID NO: 13), (GGGGS)7 (SEQ ID NO: 9), (GGGGS)8 (SEQ ID NO: 14), (GGGGS)9 (SEQ ID NO: 15) or (GGGGS) may be used herein. 10 The connector (SEQ ID NO: 16).

[0090] Peptides derived from ACE2, peptides derived from HR2, and fusion polypeptides can be produced by recombinant DNA technology or solid-phase synthesis, as is known to those skilled in the art.

[0091] The present invention also provides a lipopeptide, which is a fusion of lipids and polypeptides. The lipopeptide may comprise the aforementioned fused polypeptide, with lipids attached to the C-terminus of the fused polypeptide (lipid tag).

[0092] The lipopeptide may also comprise an HR2-derived peptide with a lipid attached to its C-terminus. The HR2-derived peptide preferably comprises, or consists of, the amino acid sequence from positions 1145 to 1190 of the SARS-CoV-2 S protein (i.e., the sequence of DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO:7)), or either, or contains one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids) at the N-terminus of the amino acid sequence from positions 1145 to 1190 of the SARS-CoV-2 S protein and / or [other amino acids]. The C-terminus of the amino acid sequence from position 1145 to 1190 of the S protein contains one or more additional amino acids (e.g., 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids). In some embodiments, the HR2-derived peptide preferably contains, or is composed of, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid insertions, substitutions, and / or deletions compared to SEQ ID NO: 7. Substitutions can be conservative or non-conservative. In some embodiments, the HR2-derived peptide may comprise, or be composed of, a peptide having, or being composed of, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0093] Lipids attached to fusion peptides or HR2-derived peptides can enhance anti-coronavirus activity and prolong in vivo, in vitro, or ex vivo half-life. Lipids can be cholesterol, tocopherol, or palmitate esters.

[0094] Methods for attaching lipid molecules to peptides are well known in the art. For example, amino acid residues containing reactive groups (e.g., thiols, amines, or carboxyl groups) can be added to the end of a fusion peptide, and lipid molecules can be attached to the end of the fusion peptide by forming amide bonds, ester bonds, or thioether bonds with the reactive groups.

[0095] In some implementations, cysteine ​​or lysine may be introduced into the C-terminus of HR2-derived peptides to provide a reactive group for attaching lipid molecules, particularly cholesterol molecules.

[0096] In some embodiments, lipid molecules are attached to the fusion peptide via spacers. In some embodiments, the spacers are flexible or rigid. In some embodiments, the spacers are PEG-based spacers containing PEG, modified PEG, or PEG derivatives. In some embodiments, the spacers contain 4-24 PEG units (PEG4-PEG24), for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 PEG units. The term "PEG unit" refers to a single polyethylene glycol unit, i.e., -(CH2CH2O)-. Advantageously, the spacers according to the invention are flexible, non-immunogenic, not easily cleaved by proteolytic enzymes, and enhance the solubility of the peptide in aqueous media. In some implementations, the spacer contains four PEG units, which may be referred to as PEG4.

[0097] In some embodiments, the spacer comprises one or more short polyethylene glycol molecules (“mini-PEG”, also known as short PEG subunits), such as one or more AEEA, AEEEA, AEEP, AEEEP, or any combination thereof. In some embodiments, the spacer may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (i.e., 1-10) short PEG subunits. In some embodiments, the spacer comprises AEEA-AEEA.

[0098] The term AEEA stands for -NH(CH2CH2O)2CH2CO- or NH2(CH2CH2O)2CH2CO-, which is derived from the N-terminal or C-terminal group of [2-(2-aminoethoxy)-ethoxy]-acetic acid (also known as 8-amino-3,6-dioxanoic acid).

[0099] The term AEEEA stands for -NH(CH2CH2O)3CH2CO- or NH2(CH2CH2O)3CH2CO-, which is derived from the N-terminal or C-terminal group of {2-[2-(2-aminoethoxy)-ethoxy]-ethoxy]-acetic acid (also known as 11-amino-3,6,9-trioxaundecanoic acid).

[0100] The term AEEP stands for -NH(CH2CH2O)2CH2CH2CO- or NH2(CH2CH2O)2CH2CH2CO-, which is derived from the N-terminal or C-terminal group of [3-(2-aminoethoxy)-ethoxy]-propionic acid (also known as 9-amino-4,7-dioxanonanoic acid).

[0101] The term AEEEP stands for -NH(CH2CH2O)3CH2CH2CO- or NH2(CH2CH2O)3CH2CH2CO-, which is derived from the N-terminal or C-terminal group of [3-[2-(2-aminoethoxy)-ethoxy]-ethoxy]-propionic acid (also known as 12-amino-4,7,10-trioxododecanoic acid).

[0102] In some embodiments, the spacer comprises Lys-PEG4 or Cys-PEG4. In some embodiments, the spacer comprises Lys-AEEA-AEEA or Cys-AEEA-AEEA. In some embodiments, the spacer is selected from the following formula: ,and .

[0103] The fusion peptides and lipopeptides of this invention possess antiviral activity, particularly inhibitory activity against coronaviruses. These fusion peptides and lipopeptides can prevent or treat coronavirus infection in subjects by inhibiting spike protein-mediated virus-cell fusion and coronavirus replication. Furthermore, these fusion peptides and lipopeptides can inhibit viral transmission.

[0104] The term "coronavirus" includes naturally occurring (e.g., wild-type) coronaviruses; naturally occurring coronavirus variants; and laboratory-generated coronavirus variants, including variants generated through selection, variants generated through chemical modification, and genetically modified variants (e.g., coronaviruses modified in the laboratory using recombinant DNA methods). Coronaviruses described herein include, for example, SARS-CoV-2, SARS-CoV, or MARS-CoV, including their wild-type strains or variants. In some embodiments, the term coronavirus includes variants classified by the World Health Organization (WHO) as "variants of interest (VOI)" or "variants of concern (VOC)." The spike glycoprotein of variant coronaviruses can mutate, which can alter viral characteristics such as the severity of infection or transmissibility. WHO-classified variants of coronaviruses of concern include alpha, beta, gamma, delta, and omicron, while WHO-classified variants of interest include lambda and mu. Each variant can be defined by the mutations in its spike glycoprotein compared to the wild-type spike glycoprotein. For example, the Omicron variant of SARS-CoV-2 (also known as B.1.1.529) contains mutations selected from the following: A67V, Δ69-70, T95I, G142D / Δ143-145, Δ211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, or any combination thereof. Furthermore, these variants can have further lineages and encompass a group of related viruses. For example, the Omicron variant has BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, and BA.4 / BA.5 lineages. In some embodiments, the coronaviruses discussed herein include Omicron.

[0105] In some embodiments, the fusion peptides and lipopeptides of the present invention possess broad-spectrum anti-coronavirus activity. In some embodiments, the fusion peptides and lipopeptides of the present invention can inhibit infection by multiple coronavirus strains, such as any one, any two, or all of SARS-CoV-2, SARS-CoV, and MARS-CoV. The fusion peptides and lipopeptides of the present invention can inhibit infection by multiple variants of coronaviruses (e.g., multiple variants of SARS-CoV-2, multiple variants of SARS-CoV, and / or multiple variants of MARS-CoV, including Omicron BA.2.12.1 and BA.5).

[0106] The fusion polypeptides and lipopeptides of the present invention can be formulated into pharmaceutical compositions.

[0107] Pharmaceutical compositions may contain pharmaceutically acceptable carriers.

[0108] As used herein, the term “pharmaceutically acceptable carrier” means any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; water, such as pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions; and, at the pharmacist's discretion, other non-toxic and compatible lubricants, such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings and aromas, preservatives, and antioxidants. Other suitable pharmaceutically acceptable excipients are described in “Remington’s Pharmaceutical Sciences,” Mack Pub. Co., New Jersey, 1991, which is incorporated herein by reference.

[0109] The pharmaceutical compositions described herein may be administered to subjects in dosage units containing conventional, non-toxic, pharmaceutically acceptable carriers, adjuvants, and solvents, as needed.

[0110] The formulation methods are well known in the art, for example, as disclosed in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions used in this disclosure may be in the form of sterile, pyrogen-free liquid solutions or suspensions, coated capsules or lipid particles, lyophilized powders, or other forms known in the art.

[0111] The pharmaceutical compositions described herein can be formulated for delivery as liquids, aerosols, or inhalable dry powders. Peptides can be delivered via nasal or inhalation. Liquid aerosol formulations can be largely atomized into particle sizes suitable for delivery to the respiratory tract.

[0112] The pharmaceutical composition can be used as a preventative agent (e.g., a vaccine) or a therapeutic agent.

[0113] Vaccines may further contain one or more adjuvants.

[0114] The pharmaceutical compositions of the present invention can be administered to a subject via injection (e.g., subcutaneous, intraperitoneal, intravenous, intra-arterial, intramuscular, or intravenous), inhalation, oral administration, intranasal administration, or any other parenteral, mucosal, or transdermal route (e.g., rectal or vaginal).

[0115] In some embodiments, the pharmaceutical composition of the present invention can be administered via a mucosal route. Suitable routes of mucosal administration include oral, intranasal, gastric, pulmonary, intestinal, rectal, ocular, and vaginal routes. The pharmaceutical composition is suitable for mucosal administration. For example, in the case of oral administration, it can be in the form of tablets or capsules, optionally enteric-coated, liquid, etc. In the case of intranasal administration, it can be in the form of nasal spray, nasal drops, gel, or powder.

[0116] For preventative purposes, the pharmaceutical composition of the present invention may be administered to a subject 0-7 days prior to the expected date of coronavirus infection (e.g., 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or combinations thereof), once or multiple times, e.g., once daily, once every two days, or once every three days. For therapeutic purposes, the pharmaceutical composition of the present invention may be administered to a subject 0-7 days after coronavirus infection (e.g., within 6 hours, 12 hours, 24 hours, 48 ​​hours, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days), and may be administered once or multiple times, e.g., once daily, once every two days, or once every three days.

[0117] Effective amounts of the pharmaceutical compositions of the present invention discussed herein may include amounts effective in preventing (or preventing) coronavirus infection and / or effective in treating coronavirus infection. Effective amounts for treatment generally include any amount sufficient to detectably inhibit or reduce the replication or spread of coronavirus, or to alleviate or eliminate symptoms in a subject infected with coronavirus. Effective amounts for prevention include any amount sufficient to prevent or reduce the occurrence of coronavirus infection and one or more symptoms caused by coronavirus infection. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. However, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific peptide used, age, weight, general health condition, sex, diet, time of administration, route of administration, excretion rate, drug concomitant use, and the severity of the specific disease being treated. For a given situation, the effective amount for treatment or prevention can be readily determined through routine experiments and is within the skill and judgment of an average clinician.

[0118] It should be understood that the total daily dosage of the pharmaceutical composition of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. For any particular subject, the specific effective dose level for treatment or prevention will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific peptide used; the specific composition used; the patient's age, weight, general health status, sex, and diet; the timing, route of administration, and excretion rate of the specific peptide used; the duration of treatment; drugs used in combination with or concurrently with the specific peptide used; and similar factors well known in the medical field.

[0119] For example, for therapeutic or preventative purposes, the appropriate dose range for intravenous administration is approximately 0.01–50 mg of the active ingredient per kilogram of body weight, which can be administered in single-dose or multiple-dose regimens. The appropriate dose range for intranasal administration is typically approximately 0.01 mg / kg body weight to 100 mg / kg body weight (e.g., 1 mg / kg body weight to 10 mg / kg body weight), which can be administered in single-dose or multiple-dose regimens.

[0120] definition: Peptide: The terms "peptide" and "polypeptide" are used interchangeably. The term "peptide" refers to an amino acid polymer containing two or more amino acids (usually at least 3, preferably at least 20, more preferably at least 30, e.g., at least 50 amino acids). The term "peptide sequence" is also used interchangeably with the term "amino acid sequence." ACE2: The term "ACE2" refers to the host cell receptor angiotensin-converting enzyme 2.

[0121] Originating from: As used herein, the term “originating from” or “derived from” generally indicates that a sequence can be separated from, is associated with, is based on, or is homologous to a reference sequence. Therefore, sequences “derived from” a reference sequence include sequences identical to the reference sequence (i.e., full-length sequences exhibiting 100% sequence identity with the reference sequence), as well as variants, fragments, and derivatives of the reference sequence. This definition applies to both amino acid sequences and nucleic acid sequences.

[0122] Host: As used herein, the term “host” refers to a cell or organism (including animals, such as mammals, particularly humans) that has been infected with or is at risk of being infected with a coronavirus. In some implementations, the host is a subject or patient.

[0123] Orthologue and Homologue: The terms “orthologue” (also referred to herein as “ortholog”) and “homologue” (also referred to herein as “homolog”) are well known in the art. With further guidance, as used herein, a “homologue” of a protein is a protein of the same species that performs the same or similar function as the protein to which it is a homologue. Homologous proteins may, but do not have to, be structurally related, or may only be structurally partially related. As used herein, an “orthologue” of a protein is a protein of a different species that performs the same or similar function as the protein to which it is a homologue. Orthologous proteins may, but do not have to, be structurally related, or may only be structurally partially related.

[0124] Fragment: As used herein, the term "fragment" generally refers to a peptide consisting of a continuous subsequence of the full-length amino acid sequence of a reference (or "parent") protein, which, in terms of its amino acid sequence, is truncated at the N-terminus, C-terminus, and / or intra-sequence compared to the amino acid sequence of the reference protein. In other words, a "fragment" is generally a shorter portion of the full-length amino acid sequence.

[0125] Variant: In the context of peptides, the term "variant" refers to a peptide that contains an amino acid sequence that differs from the reference (or "parental") amino acid sequence of a reference (or "parental") protein or peptide at at least one amino acid residue. A "variant" peptide may contain at least one amino acid mutation, substitution, insertion, and / or deletion in its amino acid sequence compared to its reference sequence. Substitutions may be selected from conserved or non-conserved substitutions.

[0126] Functional variants: In the context of peptides, the term "functional variant" refers to a variant that has the same or substantially the same function as the parent peptide. In the context of HR2-derived peptides, function can refer to the ability to interact with the HR1 domain of the coronavirus S protein, or competitively inhibit the formation of the 6-HB structure or block the fusion of the coronavirus with and entry into host cells. In the context of ACE2-derived peptides, function can refer to the ability to interact with the coronavirus S protein (particularly its RBD), or competitively inhibit the interaction of the coronavirus S protein with host ACE2, or block the entry of the coronavirus into host cells.

[0127] Peptide mimics: As used herein, the term "peptide mimic" refers to a molecule that mimics the structural and / or functional characteristics of a peptide. Peptide mimics can be peptides or non-peptides. Functional variants of peptides are considered peptide mimics.

[0128] Conservative Substitution: As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue in a peptide by another amino acid residue having a similar side chain without substantially affecting the inhibitory function of the peptide variant. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. Conservative substitution can include mutual substitution between amino acids within the same group. Preferred groups of conserved amino acid substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0129] Sequence identity: As used herein, the term “sequence identity” refers to the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when they are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Sequence alignment and the calculation of sequence identity percentages can be performed using suitable computer programs known in the art. Such programs include, but are not limited to, BLAST, ALIGN, ClustalW, EMBOSS Needle, etc. An example of a local alignment program is BLAST (Basic Local Alignment Search Tool), which is available from the website of the National Center for Biotechnology Information (NCBI), currently at http: / / www.ncbi.nlm.nih.gov / / , and was originally described in Altschul et al. (1990) J. Mol. Biol. 215; 403-410. Examples of global alignment programs (optimized full-length sequence alignment) include the EMBOSS Needle and EMBOSS Stretcher programs based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), "A general method applicable to the search for similarities in the amino acid sequence of two proteins", Journal of Molecular Biology 48 (3): 443-53), both of which are available at http: / / www.ebi.ac.uk / Tools / psa / .

[0130] Connector: The term "connector" refers to the molecular part that connects two parts of a composition.

[0131] Peptide linker: The term “peptide linker” refers to a peptide or polypeptide that contains two or more amino acid residues linked together by peptide bonds.

[0132] Treatment: The term "treatment" refers to the reduction or improvement of the severity of at least one symptom or indication of coronavirus infection. This term also includes suppressing disease progression or worsening of coronavirus infection. It also includes a positive prognosis for coronavirus infection, i.e., the subject may be free of infection or have a reduced or no viral titer after administration of a therapeutic agent (such as the therapeutic agent of this invention).

[0133] Prevention: The term "prevent, preventing, prevention" refers to suppressing or reducing the occurrence of coronavirus infection or any symptoms or signs of coronavirus infection. This term includes preventing the spread of infection to individuals exposed to the virus or at risk of coronavirus infection.

[0134] Subject: The terms “subject” and “patient” are used interchangeably herein. As used herein, the term “subject” means any organism to which the active agent of the compositions of the present invention may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates such as chimpanzees and other ape and monkey species, and humans). Subjects can be mammals, particularly humans, including males or females, and include newborns, infants, adolescents, teenagers, adults, or the elderly, and further include various races and ethnicities. In some instances, a subject refers to an individual requiring diagnosis, treatment, or prevention of coronavirus infection, who may have been infected with coronavirus or is at risk of infection with coronavirus.

[0135] Coronavirus: The term “coronavirus” or “CoV” refers to any virus in the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV and SARS-CoV.

[0136] Coronavirus infection: As used in this article, the term “coronavirus infection” or “CoV infection” refers to the invasion and replication of coronaviruses. This term includes respiratory coronavirus infections, which typically occur in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms (such as diarrhea), organ failure (kidney failure and renal dysfunction), septic shock, and in severe cases, even death.

[0137] Example The following examples are for illustrative purposes only and should not be construed as limiting the invention in any way. The following examples and detailed descriptions are for illustrative purposes only and not for limitation.

[0138] Example 1 The continuous emergence of concerning variants (VOCs) of SARS-CoV-2 poses a significant challenge to vaccines and antiviral therapies, as they broadly evade immunity. To address the issue of viral mutation escape, we introduced the ACE2-derived peptide A1L35HR2m into the N-terminus of the viral HR2-derived peptide HR2m via a longer, flexible linker, generating a broad-spectrum anti-SARS-CoV-2 fusion peptide, A1L35HR2m, which exhibited significantly enhanced antiviral activity. Further cholesterol modification of the C-terminus of A1L35HR2m greatly enhanced its inhibitory activity against SARS-CoV-2, SARS-CoV-2 VOCs, SARS-CoV, and MERS-CoV pseudoviruses, with an IC50 value of [missing value]. 50 Values ​​ranged from 0.16 nM to 5.53 nM. A1L35HR2m-Chol also effectively inhibited spike protein-mediated cell-cell fusion and the replication of real Omicron BA.2.12.1 and BA.5. Importantly, A1L35HR2m-Chol was found to have a very long half-life in vivo (t). 1 / 2 =81.8 h). Intranasal administration of A1L35HR2m-Chol to K18-hACE2 transgenic mice effectively inhibited Omicron BA.5 infection, both prophylactically and therapeutically.

[0139] result Conjugating ACE2-derived peptides to the N-terminus of HR2-derived peptides significantly enhanced inhibitory activity. We introduced the ACE2-derived peptide A1 to the N-terminus of the HR2m peptide via a relatively long flexible linker (GGGGS)7, generating A1-(GGGGS)7-HR2m (A1L35HR2m). Figure 1 A). We also constructed three control fusion peptides (A1L5HR2m and A1HR2m) by swapping the positions of the A1 peptide and HR2m peptide (HR2mL35A1) or shortening the linker length. Figure 1 A). All four fusion peptides were prepared via recombinant expression, and the purity of the resulting peptides was characterized using SDS-PAGE and liquid chromatography-mass spectrometry (LC-MS). Figure 6 B and Figure 6 C).

[0140] Next, we evaluated the inhibitory activity of A1L35HR2m, HR2mL35A1, A1L5HR2m, and A1LHR2m using a SARS-CoV-2 pseudovirus infection assay. Figure 2 As shown in Figure A, A1L35HR2m exhibits highly effective and strong inhibitory effects against SARS-CoV-2 infection, with an IC50 concentration of [missing information]. 50The value was 27 nM, which is comparable to many cholesterol-modified HR2-derived peptides. HR2mL35A1, A1L5HR2m, and A1HR2m all showed significantly reduced antiviral activity. We then compared A1L35HR2m, A1 (… Figure 6 ), HR2m ( Figure 7 The study also examined the inhibitory activity of the A1 / HR2m mixture against SARS-CoV-2 pseudovirus infection. Again, A1L35HR2m exhibited the highest activity among these tested peptides, with an IC50 value of [missing value]. 50 A value of 21 nM indicates that the potency of A1L35HR2m is at least 80 times that of other peptides. Figure 2 B). These results collectively confirm that adding ACE2-derived peptide A1 to the N-terminus of the HR2m peptide, which has a long flexible linker, can indeed significantly enhance anti-SARS-CoV-2 activity.

[0141] A1L35HR2m is more effective than HR2m peptide in inhibiting SARS-CoV-2 D614G S protein-mediated cell-cell fusion. Next, we explored the A1L35HR2m virus inhibition mechanism using an S protein-mediated cell-cell fusion assay, as previously described (26). We found that A1L35HR2m effectively inhibited SARS-CoV-2 D614G S protein-mediated cell fusion between 293T / EGFP / S and Caco-2 cells, with an IC50 concentration of 1.5%. 50 The value is 7.02 nM, which is HR2m (IC). 50 The potency is approximately 62 times that of (=436 nM). Figure 2 C). We also found that A1L35HR2m has inhibitory activity against cell-cell fusion mediated by D614G S protein between 293T / EGFP / S and Calu-3 cells (IC50). 50 =9.54 nM) is much higher than HR2m (IC 50 =745 nM) and A1 peptide (IC 50 >50μM) Figure 2 D). These results suggest that the enhanced anti-SARS-CoV-2 activity of A1L35HR2m may be due to its enhanced ability to inhibit the fusion of SARS-CoV-2 with target cells.

[0142] Numerous studies have shown that peptides derived from SARS-CoV-2 HR2 can competitively interact with the HR1 domain to block viral fusion with the target cell membrane (12, 21, 27). To investigate whether the introduction of the A1 peptide into A1L35HR2m alters the original function of the HR2m peptide, we performed biolayer interference (BLI) experiments to characterize the binding affinity of the HR1 peptide. Figure 8 We found that A1L35HR2m has a high affinity (K) for HR1. D =35.3 nM) is the original HR2m peptide (K D =116 nM) 3 times ( Figure 7 E and Figure 7 F). This indicates that the addition of peptide A1 to A1L35HR2m does indeed increase HR1 binding affinity, which may help improve viral inhibitory activity.

[0143] A1L35HR2m-Chol exhibits broad and potent inhibitory activity against various SARS-CoV-2 variants of interest, including SARS-CoV and MERS-CoV.

[0144] Previous reports have shown that lipid modification of HR2-derived peptides can significantly enhance antiviral activity (20-22, 27, 28). Considering that the antiviral mechanism of A1L35HR2m is similar to that of HR2-derived peptides, we performed site-specific modification of the C-terminus of A1L35HR2m with cholesterol to generate A1L35HR2m-Chol (…). Figure 8 (A and Figure 84). We confirmed that A1L35HR2m-Chol exhibits extremely high inhibitory activity against SARS-CoV-2, IC50. 50 The value reached 134 pM ( Figure 10 A), indicating that its efficacy is A1L35HR2m (IC). 50 It is approximately 200 times more potent than (27 nM), and is one of the most effective peptide inhibitors reported to date. A1L35HR2m-Chol can also effectively block D614G S protein-mediated cell-cell fusion between 293T / EGFP / S and Caco-2 or Calu-3 cells, with an IC50 concentration of approximately 27 nM. 50 The values ​​are 0.17 and 0.31 nM respectively. Figure 10 B). To eliminate the influence of cytotoxicity on inhibitory activity, Caco-2 or Calu-3 cells were treated with gradient concentrations of A1L35HR2m-Chol and A1L35HR2m for 48 hours, and their viability was evaluated using Cell Counting Kit-8 (CCK-8). A1L35HR2m-Chol and A1L35HR2m did not show significant cytotoxicity against Caco-2 or Calu-3 cells at concentrations up to 2.5 μM and 10 μM, respectively, and their IC50 inhibitory effects on SARS-CoV-2 were [values ​​missing]. 50 The value is more than 18,656 times and 57 times ( Figure 12 A and Figure 12 (B) This indicates that the inhibitory activity of the inhibitor cannot be attributed to its cytotoxicity.

[0145] We further evaluated the antiviral activity of A1L35HR2m-Chol and A1L35HR2m against pseudo-SARS-CoV-2 D614G, SARS-CoV-2 VOC, SARS-CoV, and MERS-CoV. We found that A1L35HR2m-Chol was effective against SARS-CoV-2 D614G, SARS-CoV-2 VOC, and SARS-CoV (ICV14G, SARS-CoV-2 VOC, and MERS-CoV). 50 Values ​​from 160 pM to 550 pM showed better performance than A1L35HR2m (IC). 50 Stronger inhibitory activity (values ​​from 17 nM to 49 nM) Figure 8 B to Figure 8 G). We also found that A1L35HR2m-Chol effectively inhibited MERS-CoV (IC). 50 =5.53 nM)( Figure 3 H), while A1L35HR2m showed only minimal activity against MERS-CoV pseudovirus. Neither A1L35HR2m-Chol nor A1L35HR2m showed inhibitory activity against the negative control VSV-G, which was consistent with expectations. Figure 3 I). For comparison, we also tested the antiviral activity of HR2m and HR2m-Chol against pseudotyped SARS-CoV-2 D614G, SARS-CoV-2VOC, SARS-CoV, and MERS-CoV. Figure 8 B to Figure 8 G). We found that HR2m has weak inhibitory activity, IC50. 50 Values ​​ranged from 2.2 μM to 0.59 μM. HR2m-Chol exhibited fairly good inhibitory activity, with an IC50 value of [missing value]. 50 The values ​​ranged from 10.5 nM to 2.2 nM, but the activity was significantly lower than that of A1L35HR2m-Chol.

[0146] A1L35HR2m-Chol effectively inhibited infection of real SARS-CoV-2 Omicron in vitro. We then evaluated the antiviral efficacy of A1L35HR2m-Chol against real Omicron BA.2.12.1 and BA.5 strains. First, we performed a micro-neutralization assay on VeroE6-TMPRSS2 cells. The results showed that A1L35HR2m could inhibit the replication of BA.2.12.1 and BA.5 strains, with an IC50 concentration of [missing value]. 50 The values ​​were 507 nM and 878 nM, respectively, while A1L35HR2m-Chol effectively inhibited the replication of BA.2.12.1 and BA.5, with an IC50 value of 507 nM and 878 nM, respectively. 50 The values ​​are 22.0 nM and 23.7 nM respectively. Figure 9 A and Figure 9 B). We also performed viral load reduction assays for Omicron BA.2.12.1 and BA.5 in VeroE6-TMPRSS2 cells. Inhibitory activity was analyzed by quantifying viral yield in cell culture supernatant using RT-qPCR. We consistently found that A1L35HR2m-Chol significantly inhibited real Omicron BA.2.12.1 and BA.5 infection (BA.2.12.1 IC50 was significantly higher than that of BA.2.12.1). 50 =5.9 nM; BA.5 IC 50 =10.4 nM) is superior to A1L35HR2m (BA.2.12.1 IC) in terms of performance. 50 =109 nM; IC BA.5 50 =236 nM) significantly more effective ( Figure 9 C and Figure 9 D).

[0147] A1L35HR2m-Chol exhibited durable in vitro anti-SARS-CoV-2 activity and effectively inhibited real SARS-CoV-2 Omicron BA.5 infection in hACE2 transgenic mice. Based on encouraging in vitro results, we wanted to know whether A1L35HR2m-Chol could inhibit SARS-CoV-2 in vivo. Previous studies have shown that lipid HIV-1 fusion inhibitors can enhance in vitro anti-HIV-1 activity and improve in vivo stability (29, 30). Here, we used an in vitro inhibition assay to investigate whether A1L35HR2m-Chol also exhibited enhanced in vitro anti-SARS-CoV-2 activity and a prolonged in vivo half-life. A1L35HR2m-Chol (5 mg / kg) or A1L35HR2m (5 mg / kg) was administered intranasally (nasal drops) to mice, and plasma samples were diluted at each time point to determine their inhibitory activity against SARS-CoV-2 pseudovirus infection. The peptide concentration was then estimated based on the dilution factor that achieved 50% inhibition of pseudovirus infection. Figure 9 E and Figure 9 F). Plasma samples from mice treated with A1L35HR2m-Chol showed peak in vitro inhibitory activity at 8 hours post-administration, corresponding to peak peptide concentrations at a mean dilution of 10284-fold, inhibiting 50% of SARS-CoV-2 pseudovirus infection. A1L35HR2m-Chol appeared to maintain high plasma concentrations even after 336 hours. In contrast, A1L35HR2m showed significantly lower peak in vitro inhibitory activity (corresponding to peak peptide concentrations) and was rapidly cleared at 8 hours post-administration. Figure 9 E and Figure 9Based on the estimated concentrations, we calculated the pharmacokinetic parameters of A1L35HR2m-Chol and A1L35HR2m using PKSolver software. The plasma half-life of A1L35HR2m-Chol was approximately 81.8 hours, which was significantly longer than that of A1L35HR2m (t). 1 / 2 =2.6 hours) 30 times longer ( Figure 4 F and Table 1). The area under the average concentration-time curve (AUC 0-t) of A1L35HR2m-Chol is ,Compare The effect was approximately 58 times greater (Table 1). These results indicate that C-terminal conjugation of cholesterol to the A1L35HR2m significantly prolongs the plasma half-life. Similar phenomena have been observed with other fusion peptide inhibitors (29, 30).

[0148] Next, we used the established K18-hACE2 transgenic mouse model to evaluate the preventive and therapeutic efficacy of A1L35HR2m-Chol. Figure 4 G)(31). In the prevention study, mice were administered A1L35HR2m-Chol (2 mg / kg) intranasally twice, 48 hours and 24 hours before viral infection. On day 0, each mouse was challenged intranasally with Omicron BA.5 (10000 PFU). Two days post-infection (2 dpi), mice were sacrificed for viral RNA and infectious virus titer analysis. We found that the viral RNA copy number in lung homogenates from mice treated with A1L35HR2m-Chol decreased by >2log10, P<0.01. Figure 4 The viral RNA copy number in the nasal turbinates and H) decreased by >1 log10, P<0.0001. Figure 4 The viral load (H) in mice treated with A1L35HR2m-Chol was significantly lower than that in the control group. We also found that the infectious viral titer in mice treated with A1L35HR2m-Chol was significantly lower than that in the control group (reduction >2 log10, P < 0.01). Figure 4 I). Notably, almost no viral titers were detected in the nasal turbinates of mice treated with A1L35HR2m-Chol, suggesting that A1L35HR2m-Chol has the potential to inhibit viral transmission.

[0149] For the treatment study, all mice were first challenged with Omicron BA.5 (10000 PFU) on day 0, followed by intranasal delivery (nasal drops) of A1L35HR2m-Chol (2 mg / kg) at 1 dpi and 2 dpi. Mice were sacrificed at 3 dpi for viral RNA and infectious virus titer analysis. We found that the nasal turbinate virus RNA copy number was significantly lower in mice receiving A1L35HR2m-Chol compared to the control group (P<0.05), while there was no significant difference in viral RNA copy number in lung homogenate between the two groups. Figure 4 J). Importantly, A1L35HR2m-Chol completely suppressed infectious viral titers in lung homogenates, and no detectable live SARS-CoV-2 particles were found. Figure 4 In summary, A1L35HR2m-Chol demonstrated strong protective effects against SARS-CoV-2 infection in both preventative and therapeutic settings.

[0150] discuss As SARS-CoV-2 continues to spread globally, new variants are emerging rapidly, posing a significant challenge to existing vaccines and therapies. Membrane fusion mechanisms between the virus and target cells are highly conserved across different coronaviruses, and the formation of a six-helix bundle between the HR1 and HR2 domains plays a crucial role in driving membrane fusion (12, 15). Because the sequences of HR1 and HR2 are highly conserved across different coronaviruses, peptides derived from HR1 and HR2 are considered promising antiviral agents that could potentially overcome the problem of viral mutation escape (14, 21).

[0151] To explore novel HR2 peptide engineering strategies that could generate antiviral drugs with enhanced inhibitory activity, we introduced the short ACE2-derived peptide A1 into the HR2-derived peptide HR2m to construct A1L35HR2m. We found that while the ACE2-derived peptide A1 was ineffective in inhibiting pseudovirus infection and the HR2m peptide exhibited only minimal inhibitory activity, A1L35HR2m displayed high pseudovirus inhibitory activity, suggesting a synergistic effect when the A1 and HR2m peptides are combined. Our binding studies indicate that the A1 peptide does indeed enhance the binding affinity of the HR2m peptide to the HR1 peptide, thereby contributing to improved inhibitory activity. The A1 peptide may also weakly interact with the viral spike protein, thereby enriching the local concentration of A1L35HR2m around the spike protein, ultimately enhancing its inhibitory activity. However, this explanation may be difficult to prove because neither the A1 peptide nor A1L35HR2m showed significant binding to the spike protein in the biolayer interference binding experiments.

[0152] Further modification of A1L35HR2m with cholesterol at the C-terminus significantly enhanced the inhibitory activity of A1L35HR2m-Chol against different SARS-CoV-2 VOCs and SARS-CoV pseudoviruses, with an IC50 value of [missing value]. 50 The value reached 0.16-0.55 nM. A1L35HR2m-Chol can also effectively inhibit MERS-CoV pseudovirus infection, IC50... 50 The value was 5.5 nM. A1L35HR2m-Chol was found to effectively inhibit the replication of real Omicron BA.2.12.1 and BA.5, further demonstrating its broad coverage against different coronaviruses. Cholesterol modification may facilitate the binding of A1L35HR2m-Chol to the cell surface or viral envelope. In either case, this would reduce the dimensionality of the A1L35HR2m-Chol peptide diffusion process, thereby accelerating its binding to the viral HR1 domain and resulting in enhanced inhibitory activity. If A1L35HR2m-Chol binds to cells, the 3D diffusion in solution will be converted to 2D diffusion on the membrane to search for the viral HR1 domain during fusion. If A1L35HR2m-Chol binds to the viral envelope, the 3D search in solution will be converted to a search for the viral HR1 domain inside the virus. Cholesterol can also directly enhance HR1 binding affinity, which can directly contribute to improved antiviral inhibitory activity (20).

[0153] Notably, we found that A1L35HR2m-Chol exhibited unusually sustained in vitro anti-SARS-CoV-2 activity, with an in vivo half-life extended to 81.8 hours, which is extraordinary for peptide inhibitors. We believe that the extended in vivo half-life is mainly due to cholesterol modification. Cholesterol has been shown to bind to serum albumin, leading to a prolonged cycle time of the bound peptide (32). Cholesterol modification can also cause A1L35HR2m-Chol to form micelles in solution, resulting in slower clearance. In fact, dynamic light scattering experiments showed that A1L35HR2m-Chol forms micelles (165 nm in diameter) in aqueous solution (32). Figure 12 Cholesterol modification may also facilitate the adhesion of A1L35HR2m-Chol to endothelial cells and the reticuloendothelial system via physical absorption followed by slow release, which may also prolong the in vivo half-life of A1L35HR2m-Chol. The increased half-life in fresh pigs may significantly contribute to the in vivo antiviral activity of A1L35HR2m-Chol. In fact, we found that intranasal administration of A1L35HR2m-Chol effectively inhibited Omicron BA.5 infection and replication in hACE2 transgenic mouse models, both prophylactically and therapeutically.

[0154] In summary, we have established a novel method to significantly enhance the antiviral activity of the HR2 peptide. The engineered A1L35HR2m-Chol effectively inhibits different SARS-CoV-2 VOCs, SARS-CoV, and MERS-CoV. We believe that A1L35HR2m-Chol is a promising antiviral molecule that will retain inhibitory activity against future SARS-CoV-2 variants, and therefore warrants further translational research.

[0155] Materials and methods chemicals All chemicals were purchased from commercial suppliers and were ready for use without further purification. Fmoc-L-Gly-OH, Fmoc-L-Leu-OH, Fmoc-L-Ile-OH, Fmoc-L-Val-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Lys(Alloc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Glu(OtBu Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Phe-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-His(Boc)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Pro-OH, and Fmoc-L-Met-OH were all purchased from GL Biochem. Fmoc-Rink amide resin was purchased from Tjhecheng. Bromoacetic acid, [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid, N,N'-diisopropylcarbodiimide (DIC), ethyl cyanoglyoxylate-2-oxime (Oxyma), N,N-dimethylformamide (DMF), dichloromethane (DCM), diethyl ether, trifluoroacetic acid, triisopropylsilane, 4-methylpiperidine, and HPLC-grade acetonitrile were purchased from Energy Chemical.

[0156] peptides Peptides were synthesized via solid-phase peptide synthesis (SPPS) on a Liberty Blue automated microwave peptide synthesizer (CEM Corporation, North Carolina, USA). The resin was swollen in DMF for 5 minutes before use. The resin and Fmoc groups assembling the amino acids were removed by treatment with 20% 4-methylpiperidine in DMF at 40 °C. Amino acid coupling was performed at 80 °C with 5 equivalents of Fmoc-amino acid, 5 equivalents of Oxyma, and 5 equivalents of DIC. The coupling reaction of Fmoc-His(Trt)-OH was carried out at 50 °C for 10 minutes to prevent side reactions at high temperatures. After stepwise SPPS completion, the resin was thoroughly washed with DCM and dried under vacuum. The peptides were then cleaved from the resin and treated with 2% (v / v) water and 2.5% (v / v) triisopropylsilane in pure trifluoroacetic acid (TFA) at room temperature for 2.5 hours to deprotect the side chains. The resulting peptide-containing solution was precipitated and washed three times with cold diethyl ether. The resulting solid was dissolved in 50% H₂O:50% acetonitrile containing 0.1% TFA and then lyophilized. The crude peptide was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to a purity greater than 95%, as determined by RP-HPLC and characterized by electrospray ionization mass spectrometry (ESI-MS). The peptide was dissolved using dimethyl sulfoxide (DMSO) or PBS.

[0157] Preparation of HR2m-Chol (HR2m-Lys-AEEA-AEEA-Chol) HR2m-chol main chain sequence D1VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQK1(SEQ ID NO: 11) D1:Boc-L-Asp(OtBu)-OH K1: Fmoc-L-Lys(Alloc)-OH

[0158] The main-chain peptide was synthesized via solid-phase peptide synthesis (SPPS) on a Liberty Blue automated microwave peptide synthesizer (CEM Corporation, North Carolina, USA). The resin was swollen in DMF for 5 minutes before use. The resin and Fmoc groups for assembling amino acids were removed by treatment with 20% 4-methylpiperidine in DMF at 40°C. Amino acid coupling was then performed at 80°C for 4 minutes with 5 equivalents of Fmoc-amino acids, 5 equivalents of Oxyma, and 5 equivalents of DIC. After stepwise SPPS completion, the resin was washed with DCM. Alloc groups were then removed by treatment three times with tetrakis(triphenylphosphine)palladium for 20 minutes each at room temperature. Alloc protection group removal conditions: 11 mg of tetrakis(triphenylphosphine)palladium was dissolved in 6 mL of DCM, followed by the addition of 246 µL of phenylsilane to obtain the final reaction buffer. After alloc protection group removal, the PEG linker and cholesterol were sequentially coupled to the side chain of K1 via standard solid-phase peptide synthesis (SPPS) on an automated microwave peptide synthesizer (Biotage). After SPPS, the resin was thoroughly washed with DCM and dried under vacuum. The peptide was then cleaved from the resin and treated with 2% (v / v) water and 2.5% (v / v) triisopropylsilane in pure trifluoroacetic acid (TFA) at room temperature for 2.5 hours to remove side chain protection. The resulting peptide-containing solution was precipitated and washed three times with cold diethyl ether. The resulting solid was dissolved in 50% H₂O:50% acetonitrile containing 0.1% TFA and lyophilized. The crude peptide was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) to a purity greater than 95%, determined by RP-HPLC and characterized by electrospray ionization mass spectrometry (ESI-MS).

[0159] plasmids and cells The envelope expression plasmids pcDNA3.1-SARS-CoV-S and pcDNA3.1-SARS-CoV-2-S, as well as the luciferase expression plasmid pNL4-3.Luc.RE, were kindly provided by Dr. Shibo Jiang and Dr. Lu Lu of Shanghai Medical College, Fudan University. The SARS-CoV-2 S mutant envelope expression plasmid was generated using overlap PCR. Cell lines (including 293T, Caco-2, and Calu-3) were also kindly provided by Dr. Shibo Jiang and Dr. Lu Lu and were cultured in DMEM containing 10% fetal bovine serum (FBS).

[0160] Expression and purification of fusion peptides The genes encoding A1L35HR2m, HR2mL35A1, A1L5HR2m, and A1HR2m were synthesized by Genewiz (Suzhou, China) and cloned into the pET28a vector. The plasmids pET28a-A1L35HR2m, pET28a-HR2mL35A1, pET28a-A1L5HR2m, and pET28a-A1HR2m were transformed into *E. coli* BL21 (DE3). Cells were shaken in LB medium at 37°C until the OD600 reached 0.8. Protein expression was induced for 6 hours at 37°C with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside. Cells were then harvested, sonicated in PBS containing 1% Triton, and centrifuged at 12000 × g for 10 min. The supernatant was loaded onto Ni-NTA beads (Smart-Lifesciences, Cat. SA004100) and washed with 10 mM imidazole in PBS. Protein was then eluted with elution buffer (250 mM imidazole in PBS). Peak fractions were collected and concentrated using 3 kDa ultrafiltration tubes (Millipore, Germany). Protein analysis was then performed by SDS-PAGE and LC-MS.

[0161] Preparation of A1L35HR2m-Chol protein His-TEV-A1L35HR2m-Cys protein was expressed in *E. coli* BL21 and purified using NTA-Ni beads. The purified His-TEV-A1L35HR2m-Cys protein was digested overnight with TEV protease in 50 mM Tris-HCl (pH=7.4) containing 1 mM DTT at 4 °C. The A1L35HR2m-Cys protein was then purified again by semi-preparative RP-HPLC and lyophilized to obtain a white powder. Bromoacetyl-AEEA-AEEA-chol was synthesized on H-Rink-Amid-ChemMatrix resin, purified by semi-preparative RP-HPLC, and lyophilized to obtain a light yellow powder. In an acetonitrile / PBS (70 / 30: v / v) (pH 8.0) mixed buffer, A1L35HR2m-Chol (A1L35HR2m-Cys-AEEA-AEEA-Chol, with the structure shown below) was synthesized via a nucleophilic substitution reaction between the terminal thiol group on the peptide cysteine ​​residue and the bromoacetyl-AEEA-AEEA-chol molecule. Figure 3 The protein (shown in the image) was purified by semi-preparative RP-HPLC and lyophilized to obtain a white powder. The identity of the A1L35HR2m-Chol protein was verified by ESI-MS. Figure 9 ).

[0162] Inhibiting pseudovirus infection SARS-CoV-2 and its variants were generated as previously described (20). In short, 293T cells were co-transfected with a plasmid encoding the S protein of SARS-CoV-2 or its variants and an HIV-1 genome plasmid expressing luciferase using Vigofect reagent (Vigorous Biotech, Beijing, China). The supernatant was collected 48 hours after transfection, centrifuged at 3000×g for 10 minutes, and stored at -80°C for single-round infection. Caco-2 cells (10) were infected with a SARS-CoV-2 pseudovirus mixed with an inhibitor. 4 The antiviral activity of the protein was determined using cells / well. Twelve hours post-infection, the supernatant was replaced with fresh DMEM containing 10% FBS, and the cells were cultured at 37°C for another 48 hours. Cells were lysed, and luciferase activity was measured using a luciferase kit (Promega, Madison, WI, USA) on a microplate reader (Thermo, Variokan LUX). All best-fit inhibition curves were plotted using GraphPad Prism.

[0163] Binding affinity was determined using biolayer interferometry (BLI). According to the manufacturer's instructions, the HR1 peptide was biotinylated using EZ-Link NHS-PEG12-Biotin (Thermo Fisher Scientific) at a theoretical 1:3 molar ratio. Unreacted biotin was removed by ultrafiltration using an Amicon column (3 kDa MWCO, Millipore). For kinetic analysis, HR1 was captured on a streptavidin biosensor. The biotinylated HR1 was diluted to 5 µg / mL in dilution buffer (PBS containing 0.02% Tween 20). The background signal was measured using a reference sensor loaded with HR1 but without inhibitor binding, and this background signal was subtracted from the corresponding inhibitor-bound sensor. Curve fitting was performed using ForteBio data analysis software. The results were obtained by analyzing all samples related to R... 2 The average k is determined by averaging the theoretically fitted and matched combination curves with a value of 0.96. on k off and K D value.

[0164] Inhibition of SARS-CoV-2 D614GS-mediated cell-cell fusion The establishment and detection of cell-cell fusion assays were similar to those for HIV-1 Env-mediated cell-cell fusion assays (33). Briefly, 293T cells were transfected with plasmid pcDNA3.1 encoding either EGFP or SARS-CoV-2 D614G S protein (293T / EGFP / S protein) or EGFP (293T / EGFP) and used as effector cells. Calu-3 or Caco-2 cells were used as target cells. For SARS-CoV-2 D614G S-mediated cell-cell fusion assays, target cells were incubated overnight at 37°C, followed by the addition of 293T / EGFP / S cells with or without different concentrations of the test inhibitor. After 5 hours of co-culturing at 37°C, three fields of view were randomly selected from each well, and the number of fused and non-fused cells was counted under an inverted fluorescence microscope (ZEISS, Germany).

[0165] Cell viability assay According to the manufacturer's instructions, the potential cytotoxicity of the peptide to Calu-3 and Caco-2 cells was measured using the Cell Counting Kit-8 (CCK-8, Dojindo). In short, 100 μl of the peptide at the indicated concentration was added to an average volume of cells (103) in a 96-well plate. 4 Cells were incubated in wells at 37°C for 2 days. Cell viability was evaluated using a CCK-8 assay kit. After a further 2-hour incubation at 37°C with the CCK-8 reagent, absorbance at 450 nm was measured using an ELISA reader, and the percentage of cytotoxicity was calculated.

[0166] In vitro inhibition and plasma half-life of fusion peptides As previously described (29, 33), the in vitro anti-SARS-CoV-2 D614G activity of A1L35HR2m or A1L35HR2m-Chol in plasma samples was detected. Briefly, BALB / c mice were intranasally administered 5 mg / kg of A1L35HR2m or A1L35HR2m-Chol. Serum was collected from these mice before injection (0 h) and at 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 120 h, 168 h, 240 h, and 336 h after injection, and its inhibitory activity against SARS-CoV-2 D614G infection was tested as described above. The serum dilution factor (DF-IC) at which 50% inhibition was achieved was calculated. 50 ) DF-IC based on computation 50 The values ​​were used to estimate the peptide concentration in the plasma sample, and the plasma half-life and other pharmacokinetic parameters were calculated using PKSolver software, as previously described (33).

[0167] Trace neutralization assay As previously described (31, 34), the inhibitory activity of A1L35HR2m-Chol against real Omicron BA.2.12.1 and BA.5 infections was determined. In short, VeroE6 cells overexpressing TMPRSS2 were infected one day prior to infection at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates. Serially diluted peptides were pre-incubated for 1 hour at 37°C with 2500 plaque-forming units (PFU) of SARS-CoV-2 OmicronBA.2.12.1 or BA.5. The virus-peptide mixture was then seeded into the cells and incubated at 37°C in a 5% CO2 incubator for 36 hours. After incubation, the supernatant was removed, and cells were fixed with 10% formaldehyde solution. Following fixation, cells were permeabilized with 0.2% Triton X-100 in PBS and stained with rabbit anti-SARS-CoV-2 N protein at room temperature for 1 hour, followed by staining with a secondary antibody (LifeTechnology) cross-linked with goat anti-rabbit Alexa Fluor 488 conjugated IgG (H+L). Signals were quantified using a Sapphire biomolecular imaging system (Azure Biosystems), and images were analyzed using ImageJ software. IC50 was plotted in GraphPad Prism by fitting a nonlinear regression curve. 50 value.

[0168] Viral load reduction assay As previously described (35), viral load reduction was assessed in VeroE6-TMPRSS2 cells by RT-qPCR. Cell culture supernatant or animal tissue homogenate was lysed in RLT buffer, and RNA was extracted using the RNeasy mini kit (Qiagen). Viral load was quantified using the one-step TB green RT-PCR kit (Takara) according to the protocol of the LightCycle 480 real-time PCR system (Roche). Primers used for quantification targeted the SARS-CoV-2 RdRp gene: forward sequence: 5'-CGCATACAGTCTTRCAGGCT-3'; reverse sequence: 5'-GTGTGATGTTGAWATGACATGGTC-3'.

[0169] Efficacy of A1L35HR2m-Chol in the prevention and treatment of Omicron BA.5 infection in K18-hACE2 transgenic mice The K18-hACE transgenic mice were obtained from the Centre for Comparative Medicine at the University of Hong Kong. All experimental protocols were approved by the Animal Ethics Committee of the University of Hong Kong and conducted in accordance with the standard operating procedures for biosafety level 3 animal facilities.

[0170] Peptide prophylaxis experiments were conducted in a K18-hACE transgenic mouse model. In short, 6- to 8-week-old male mice (n=5) were anesthetized and intranasally treated with either lipopeptide A1L35HR2m-Chol or a solvent once daily for 2 days before and 1 day after infection. On day 0, mice were intranasally inoculated with 10,000 PFU of Omicron BA.5. Mice were sacrificed 2 days post-infection. Nasal turbinates and lungs were collected for qPCR and infection titer analysis.

[0171] For the treatment experiment, 6- to 8-week-old male K18-hACE2 mice (n=5) were challenged intranasally with 10,000 PFU of Omicron BA.5. On days 1 and 2 post-infection, mice were intranasally treated with either A1L35HR2m-Chol or a solvent. Mice were sacrificed on day 3 post-infection, and the nasal turbinates and lungs were harvested and homogenized. Viral load and infection titer in the tissues were determined as previously described (36).

[0172] Table 1. Pharmacokinetic parameters of A1L35HR2m (n=3) and A1L35HR2m-Chol (n=3) in mice based on in vitro virus inhibition assays.

[0173]

[0174] Sequences used in the examples A1L35HR2m, amino acid sequence (SEQ ID NO: 1) MG IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFRGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ LEHHHHHH A1L35HR2m, nucleic acid sequence (SEQ ID NO: 2) ATGGGCATTGAAGAACAAGCGAAAACGTTTCTGGATAAATTTAATCATGAAGCGGAAGATCTGTTTTATCAGAGCAGCTTAGGCGGTAGCAAAGGCGATTTTCGCGGCGGTGGCGGCAGTGGTGGCGGTGGCAGCGGCGGTGGCGGCAGTGGCGGTGGTGGCAGCGGCGGTGGCGGCAGTGGCGGTGGCGGTAGCGGTGGCGGTGGCAGTGATGTGGATCTGGGCGATATTAGCGGCATTAACGCGAGCGTGGTGAACATTCAGAAAGAAATTGATCGCCTGAACGAAGTGGCGAAAAACCTGAACGAAAGCCTGATTGATCTGCAAGAACTGGGCAAATATGAACAGCTCGAGcaccaccaccaccaccactga His-TEV-A1L35HR2m-Cys, Amino Acid Sequence (SEQ ID NO: 3) MGHHHHHHHHGSENLYFQG IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFRGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ C His-TEV-A1L35HR2m-Cys, Nucleic Acid Sequence (SEQ ID NO: 4) ATGGGCCATCATCATCATCATCACCATCACGGCTCTGAAAACTTATATTTTCAGGGCATTGAAGAACAAGCGAAAACCTTTCTGGATAAATTTAACCATGAAGCGGAAGATCTGTTTTATCAGAGCAGCTTAGGCGGTAGCAAAGGCGATTTTCGCGGCGGTGGCGGCAGTGGTGGCGGTGGCAGCGGCGGTGGCGGCAGTGGCGGTGGTGGCAGCGGCGGTGGCGGCAGTGGCGGTGGCGGTAGCGGTGGCGGTGGCAGTGATGTGGATCTGGGCGATATTAGCGGCATTAACGCGAGCGTGGTGAACATTCAGAAAGAAATTGATCGCCTGAACGAAGTGGCGAAAAACCTGAACGAAAGCCTGATTGATCTGCAAGAACTGGGCAAATATGAACAGtgctga A1L35HR2m-Chol (A1L35HR2m-Cys-AEEA-AEEA-Chol, structure as follows) Figure 3 (as shown), amino acid sequence (SEQ ID NO: 5) G IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFRGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ C(-AEEA-AEEA-Chol) A1, amino acid sequence (SEQ ID NO: 6): IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFR HR2m, amino acid sequence (SEQ ID NO: 7): DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ HR2m-Lys-AEEA-AEEA-Chol (HR2m-Chol, structure see Materials and Methods), amino acid sequence (SEQ ID NO: 8): DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQK1(AEEA-AEEA-Chol) Connector L35 (SEQ ID NO: 9) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS Connector L5 (SEQ ID NO: 10) GGGGS References 1. 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Claims

1. A fusion peptide comprising a coronavirus HR2-derived peptide fused to an ACE2-derived peptide.

2. The fusion polypeptide of claim 1, wherein the ACE2-derived peptide is derived from the ACE2 region that interacts with the spike protein of the coronavirus.

3. The fusion polypeptide according to claim 1 or 2, wherein the ACE2-derived peptide originates from the α-helix 1 region of ACE2.

4. The fusion polypeptide according to any one of claims 1-3, wherein the ACE2-derived peptide comprises the amino acid sequence of IEEQAKTFLDKFNHEAEDLFYQSSLGGSKGDFR (SEQ ID NO: 6) or a functional variant thereof.

5. The fusion polypeptide according to any one of claims 1-4, wherein the coronavirus HR2-derived peptide competitively interacts with the HR1 domain to block the fusion of the coronavirus with the host cell.

6. The fusion polypeptide according to any one of claims 1-5, wherein the coronavirus HR2-derived peptide is derived from the spike protein of SARS-CoV, SARS-CoV-2, or MERS-CoV.

7. The fusion polypeptide according to any one of claims 1-6, wherein the coronavirus HR2-derived peptide comprises the amino acid sequence of DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO: 7) or a functional variant thereof.

8. The fusion polypeptide according to any one of claims 1-7, wherein the coronavirus HR2-derived peptide is fused to the ACE2-derived peptide via a linker.

9. The fusion polypeptide of claim 8, wherein the linker is a flexible peptide linker comprising at least 25 amino acids.

10. The fusion polypeptide according to claim 8 or 9, wherein the linker is composed of glycine and serine.

11. The fusion polypeptide according to any one of claims 8-10, wherein the linker comprises (GGGGS)5, (GGGGS)6, (GGGGS)7, (GGGGS)8, (GGGGS)9, or (GGGGS) 10 The amino acid sequence.

12. The fusion polypeptide according to any one of claims 1-11, wherein the coronavirus HR2-derived peptide is located at the C-terminus of the ACE2-derived peptide.

13. A lipopeptide comprising a fusion protein and a lipid according to any one of claims 1-12.

14. A lipopeptide comprising a peptide and lipid derived from coronavirus HR2, wherein the coronavirus HR2-derived peptide comprises the amino acid sequence DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ (SEQ ID NO: 7).

15. The lipopeptide of claim 13 or 14, wherein the lipid is attached to the C-terminus of the fusion protein.

16. The lipopeptide according to any one of claims 13-15, wherein the lipid is cholesterol, tocopherol, or palmitate.

17. The lipopeptide according to any one of claims 13-15, wherein the lipid is attached to the fusion protein via a spacer.

18. The lipopeptide of claim 17, wherein the spacer is a PEG-based spacer.

19. The lipopeptide of claim 17 or 18, wherein the spacer comprises PEG4 or AEEA-AEEA.

20. The lipopeptide according to any one of claims 17-19, wherein the spacer is selected from the following formula: and 。 21. A pharmaceutical composition comprising a fusion protein of any one of claims 1-12 or a lipopeptide of any one of claims 13-20, and a pharmaceutically acceptable excipient.

22. The pharmaceutical composition according to claim 21, wherein it is a dosage form suitable for intranasal or intravenous administration.

23. A method for preventing or treating coronavirus infection in a subject in need, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 21 or 22.

24. The method of claim 23, wherein the pharmaceutical composition is administered intranasally or intravenously.

25. The method according to claim 23 or 24, wherein the coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV, or a variant thereof.

26. The method of claim 23 or 24, wherein the coronavirus is an Omicron variant.

27. Use of the fusion protein of any one of claims 1-12 or the lipopeptide of any one of claims 13-20 in the preparation of a medicament for the prevention or treatment of coronavirus infection in a subject in need.

28. The use according to claim 27, wherein the coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV or a variant thereof.

29. The use according to claim 27 or 28, wherein the coronavirus is an Omicron variant.

30. The pharmaceutical composition according to claim 22 or 23, for the prevention or treatment of coronavirus infection in a subject in need.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition is administered intranasally or intravenously.

32. The pharmaceutical composition according to claim 30 or 31, wherein the coronavirus is SARS-CoV, SARS-CoV-2, MERS-CoV or a variant thereof.

33. The pharmaceutical composition according to any one of claims 30-32, wherein the coronavirus is an Omicron variant.

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