WO2021207517A2

WO2021207517A2 - Modified hr2 peptide inhibitors of coronavirus fusion

Info

Publication number: WO2021207517A2
Application number: PCT/US2021/026416
Authority: WO
Inventors: Gene Merutka
Original assignee: Sapience Therapeutics, Inc.
Priority date: 2020-04-10
Filing date: 2021-04-08
Publication date: 2021-10-14
Also published as: WO2021207517A3

Abstract

Provided are modified HR2 peptides that are capable of competing with the native HR2 region of a viral spike (S) protein for binding to the HR1 region, thereby inhibiting formation of the 6-HB fusion complex and fusion with a target host cell, compositions comprising the modified HR2 peptides, and methods of inhibiting viral fusion using the HR2 peptides.

Description

MODIFIED HR2 PEPTIDE INHIBITORS OF CORONAVIRUS FUSION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of U.S. Provisional Patent Application No.63/008,469, filed on April 10, 2020. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 8, 2021, is named Sapience_011_WO1_SL.txt and is 19,097 bytes in size. BACKGROUND [0003] Coronaviruses are enveloped particles with a positive-sense single-stranded RNA genome. The membrane (M), envelope (E), and spike (S) structural proteins are anchored in the lipid bilayer of the envelope (Lai et al.1997). The S protein mediates 1) attachment of the virus to the surface of a target cell via its S1 subunit, and 2) fusion with the target cell via its S2 subunit (Li et al.2005; Han et al.2006; Hoffman et al.2020; Lan et al.2020; Xia et al.2020). In particular, a receptor binding domain within the S1 subunit binds to angiotensin-converting enzyme 2 (ACE2) on the surface of a target cell (Li et al.2005; Han et al.2006; Lan et al.2020; Xia et al.2020). ACE2 binding by S1 triggers a conformational change in the S2 subunit, in which two heptad-repeat domains (HR1 and HR2) associate to form a 6-helix bundle (6-HB) (Hoffman et al.2020). The formation of the 6-HB brings the viral envelope into proximity with the cell membrane, facilitating fusion of the viral and target cell membranes (Xia et al.2020). Membrane fusion is the mechanism by which enveloped viruses enter target host cells. Entry of a virus into a host cell is the first step in the viral life cycle. Accordingly, peptides that disrupt formation and/or stability of the 6-HB can inhibit fusion of viral particles with target host cells (Hardy et al.2004; Ashkenazi et al.2011; Xia et al.2019a; Xia et al. 2019b). [0004] Respiratory diseases such as the common cold, Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), are caused by coronavirus infection. A novel coronavirus, referred to as SARS-CoV-2 or 2019-nCoV, emerged in late 2019 and resulted in a global pandemic of coronavirus disease 19 (COVID-19) by March 2020 (Hoffman et al.2020; Lan et al.2020). To date, no effective treatments have been identified for COVID-19. SUMMARY OF THE INVENTION [0005] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations of embodiments are intended to fall within the scope of the present invention. [0006] The invention provides HR2 variant peptides comprising a modified portion of the HR2 region of a coronavirus spike (S) protein, which peptides can inhibit viral fusion by interfering with formation and/or stability of the 6-HB. One embodiment is a modified HR2 peptide (SEQ ID NO: 7) comprising a variant of the amino acid sequence DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 2). In certain embodiments, the variant is modified at one or more positions of SEQ ID NO: 2 as follows: D1 is substituted with S; I2 is substituted with F, W, M, L, or A; S3 is substituted with H, T, or D; G4 is substituted with T, A, Q, Y, or F; I5 is substituted with M, L, or A; A7 is substituted with T, L, or V; S8 is substituted with N or T; V9 is substituted with F or L; V10 is substituted with I, M, or L; N11 is substituted with S or D; I12 is substituted with L, M, W, or A; Q13 is substituted with Y, F, T, or A; K14 is substituted with F, R, or Q; I16 is substituted with L, M, V, W, or F; D17 is substituted with N, L, A, Q, or E; R18 is substituted with K, L, S, T, Q, N, or O; L19 is substituted with M, W, F, or Y; N20 is substituted with Q, S, D, E, or A; E21 is substituted with D or Q; V22 is substituted with A, L, R, M, or W; A23 is substituted with L or V; N25 is substituted with S, A, K, G, or Q; N27 is substituted with D or A; S29 is substituted with A or N; L30 is substituted with F, A, or W; I31 is substituted with M, A, W, F, or L; D32 is substituted with S, E, or N; Q34 is substituted with R; L36 is substituted with V, W, F, or Y. [0007] The modified HR2 peptide can additionally comprise a modification wherein between 1 and 11 consecutive amino acids of SEQ ID NO: 2 are truncated from the N- terminus, beginning at D1 and proceeding in the C-terminal direction, or wherein between 1 and 10 consecutive amino acids of SEQ ID NO: 2 are truncated from the C-terminus, beginning at L36 and proceeding in the N-terminal direction. In one embodiment, between 1 and 11 consecutive amino acids of SEQ ID NO: 2 are truncated from the N- terminus, beginning at D1, and between 1 and 10 consecutive amino acids of SEQ ID NO: 2 are truncated from the C-terminus, beginning at L36. [0008] Optionally, the modified HR2 peptide comprises between 1 and 3 additional amino acids at the N-terminus and/or the C-terminus, relative to SEQ ID NO: 2. In one embodiment, the N-terminal amino acids are selected from the group consisting of: (i) G; (ii) LG; (iii) FG; (iv) DLG; (v) DFG; (vi) NLG; and (vii) NFG. In one embodiment, the C-terminal amino acids are selected from the group consisting of: (i) G; (ii) GK; (iii) GN; (iv) GR; (v) GKY; (vi) GNY; (vii) GRY; (viii) GKF; (ix) GNF; (x) GRF; (xi) GKW; (xii) GNW; and (xiii) GRW. [0009] The invention also provides HR2 variant peptides comprising a retro inverso sequence derived from the HR2 region of a coronavirus spike (S) protein. One embodiment is a modified HR2 peptide (SEQ ID NO: 8) comprising a variant of the D- amino acid sequence LEQLDILSENLNKAVENLRDIEKQINVVSANIGSID (SEQ ID NO: 6), wherein the variant is modified at one or more positions of SEQ ID NO: 6 as follows: L1 is substituted with V, W, F, or Y; Q3 is substituted with R; D5 is substituted with S, E, or N; I6 is substituted with M, A, W, F, or L; L7 is substituted with F, A, or W; N10 is substituted with D or A; N12 is substituted with S, A, K, G, or Q; A14 is substituted with L or V; V15 is substituted with A, L, R, M, or W; E16 is substituted with D or Q; N17 is substituted with Q, S, D, E, or A; L18 is substituted with M, W, F, or Y; R19 is substituted with K, L, S, T, Q, N, or O; D20 is substituted with N, L, A, Q, or E; I21 is substituted with L, M, V, W, or F; K23 is substituted with F, R, or Q; Q24 is substituted with Y, F, T, or A; I25 is substituted with L, M, W, or A; N26 is substituted with S or D; V27 is substituted with I, M, or L; V28 is substituted with F or L; S29 is substituted with N or T; A30 is substituted with T, L, or V; I32 is substituted with M, L, or A; G33 is substituted with T, A, Q, Y, or F; S34 is substituted with H, T, or D; I35 is substituted with F, W, M, L, or A; D36 is substituted with S. [0010] The modified HR2 peptide can additionally comprise a modification wherein between 1 and 10 consecutive D-amino acids of SEQ ID NO: 6 are truncated from the N- terminus, beginning at L1 and proceeding in the C-terminal direction, and/or wherein between 1 and 11 consecutive D-amino acids of SEQ ID NO: 6 are truncated from the C- terminus, beginning at D36 and proceeding in the N-terminal direction. [0011] Optionally, the modified HR2 peptide comprises between 1 and 3 additional D-amino acids at the N-terminus and/or the C-terminus, relative to SEQ ID NO: 6. In one embodiment, the N-terminal D-amino acids are selected from the group consisting of: (i) G; (ii) KG; (iii) NG; (iv) RG; (v) YKG; (vi) YNG; (vii) YRG; (viii) FKG; (ix) FNG; (x) FRG; (xi) WKG; (xii) WNG; and (xiii) WRG. In one embodiment, the C-terminal amino acids are selected from the group consisting of: (i) G; (ii) GL; (iii) GF; (iv) GLD; (v) GFD; (vi) GLN; and (vii) GFN. [0012] In some embodiments, the peptide comprises a lipid. In some embodiments, the modified HR2 peptide comprises an N-terminal acetyl group and/or a C-terminal amide group. [0013] Further aspects of the invention provide a composition comprising a modified HR2 peptide of the invention, for example, a pharmaceutical composition; a kit comprising a modified HR2 peptide of the invention; and a nucleic acid molecule encoding a modified HR2 peptide of the invention. [0014] The invention additionally provides a method of inhibiting interaction between an HR1 region and an HR2 region of a coronavirus spike (S) protein, the method comprising contacting the coronavirus S protein with a modified HR2 peptide of the invention. The invention further provides a method of inhibiting fusion of a coronavirus with a target cell, the method comprising contacting the coronavirus with a modified HR2 peptide of the invention. In one embodiment, the coronavirus is SARS-CoV-2. [0015] The invention provides the use of a modified HR2 peptide of the invention for inhibiting interaction between an HR1 region and an HR2 region of a coronavirus S protein. Also provided is the use of a modified HR2 peptide of the invention are for inhibiting fusion of a coronavirus with a target cell. In one embodiment, the coronavirus is SARS-CoV-2. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG.1 shows the amino acid sequence of SARS-Cov-2 S protein (SEQ ID NO: 1). The HR1 region is shown in bold, italicized type. The HR2 region is shown in bold type and underlined. DETAILED DESCRIPTION OF THE INVENTION [0017] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. [0018] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, 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 is related. [0019] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [0020] All of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art. I. Definitions [0021] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [0022] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably. [0023] Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). [0024] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are included. [0025] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10. [0026] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Except when preceded with a “D” or in lower case, the amino acid is an L-amino acid. Groups or strings of amino acid abbreviations are used to represent peptides. Except where specifically indicated, peptides are indicated with the N-terminus of the left and the sequence is written from the N-terminus to the C-terminus. [0027] Polypeptides, peptides, and proteins can encompass natural or synthetic modifications, for example, disulfide bonds, lactam bridges, glycosylation, lipidation, acetylation, acylation, amidation, phosphorylation, or other manipulation or modification, such as conjugation with a labeling component or addition of a protecting group. Also included are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, amino-isobutyric acid (Aib); unnatural amino acids, such as naphthylalanine (Nal), etc.); non-proteinogenic amino acids, such as ornithine (O); and polypeptides comprising or consisting of D-amino acids; as well as other modifications known in the art. Polypeptides can be in one or multiple salt forms. Preferred salt forms include acetate, chloride or trifluoroacetate. In certain embodiments, the polypeptides can occur as single chains, covalent dimers, or non-covalent associated chains. Polypeptides can also be in cyclic form. Cyclic polypeptides can be prepared, for example, by bridging free amino and free carboxyl groups. Formation of the cyclic compounds can be achieved by treatment with a dehydrating agent, with suitable protection if needed. The open chain (linear form) to cyclic form reaction can involve intramolecular-cyclization. Cyclic polypeptides can also be prepared by other methods known in the art, for example, using one or more lactam bridges, hydrogen bond surrogates (Patgiri et al.2008), hydrocarbon staples (Schafmeister et al.2000), triazole staples (Le Chevalier Isaad et al. 2009), or disulfide bridges (Wang et al.2006). Bridges or staples can be spaced, for example, 3, 4, 7, or 8 amino acids apart. [0028] The term “variant” refers to a polypeptide having one or more amino acid substitutions, deletions, and/or insertions compared to a reference sequence. Deletions and insertions can be internal and/or at one or more termini. Substitution can include the replacement of one or more amino acids with a similar or homologous amino acid(s) or a dissimilar amino acid(s). For example, some variants include alanine substitutions at one or more amino acid positions. Other substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein. Some variants include non-conservative substitutions that change the charge or polarity of the amino acid. Substitution can be with either the L- or the D-form of an amino acid. [0029] A “retro inverso” polypeptide has a reversed amino acid sequence, relative to a native L-amino acid sequence, and is made up of D-amino acids (inverting the α-center chirality of the amino acid subunits) to help maintain side-chain topology similar to that of the original L-amino acid peptide. [0030] The term “conservative substitution” as used herein denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. For further information concerning phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et. al., Science 247:1306- 1310 (1990). In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; I: neutral and/or hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids. Table I

[0031] In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic. Table II

[0032] Methods of identifying conservative nucleotide and amino acid substitutions which do not affect protein function are well-known in the art (see, e.g., Brummell et al., Biochem.32 :1180-1187 (1993); Kobayashi et al., Protein Eng.12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. U.S.A.94:412-417 (1997)). [0033] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. [0034] One such non-limiting example of a sequence alignment algorithm is described in Karlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)), can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used. Other resources for calculating identity include methods described in Computational Molecular Biology (Lesk ed., 1988); Biocomputing: Informatics and Genome Projects (Smith ed., 1993); Computer Analysis of Sequence Data, Part 1 (Griffin and Griffin eds., 1994); Sequence Analysis in Molecular Biology (G. von Heinje, 1987); Sequence Analysis Primer (Gribskov et al. eds., 1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988). [0035] A “polynucleotide,” as used herein can include one or more “nucleic acids,” “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA. [0036] An “isolated” molecule is one that is in a form not found in nature, including those which have been purified. [0037] A “label” is a detectable compound that can be conjugated directly or indirectly to a molecule, so as to generate a “labeled” molecule. The label can be detectable on its own (e.g., radioisotope labels or fluorescent labels), or can be indirectly detected, for example, by catalyzing chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label) or by other means of indirect detection (e.g., biotinylation). [0038] “Binding affinity” generally refers to the strength of the sum total of non- covalent interactions between a single binding site of a molecule and its binding partner (e.g., a receptor and its ligand, an antibody and its antigen, two monomers that form a dimer, etc.). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_D). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity binding partners generally bind slowly and tend to dissociate readily, whereas high-affinity binding partners generally bind faster and tend to remain bound longer. [0039] The affinity or avidity of a molecule for its binding partner can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme- linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BIACORE™ or OCTET® analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. (See, e.g., Berzofsky et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992)). The measured affinity of a particular binding pair interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other binding parameters (e.g., K_D or Kd, K_on, K_off) are made with standardized solutions of binding partners and a standardized buffer, as known in the art. [0040] An “active agent” is an ingredient that is intended to furnish biological activity. The active agent can be in association with one or more other ingredients. An active agent that is a peptide can also be referred to as an “active peptide.” [0041] An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose. [0042] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. polyol or amino acid), a preservative (e.g. sodium benzoate), and/or other conventional solubilizing or dispersing agents. [0043] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule. II. Modified HR2 Fusion Inhibitor Peptides and Compositions [0044] The SARS-CoV-2 spike (S) glycoprotein is a 1273 amino acid protein of probable bat origin (Zhou et al.2020). The S1 subunit of S protein facilitates entry of the virus into host cells by binding to ACE2 on the surface of a target host cell, which acts as a receptor for the virion (Hoffman et al.2020; Xia et al.2020; Zhou et al.2020). Receptor binding triggers a conformational change in the S protein, in which its helical heptad-repeat domains (HR1 and HR2) associate to form a 6-helix bundle (6-HB), which facilitates fusion of the viral envelope and target host cell membrane (Hoffman et al. 2020; Xia et al.2020). The full amino acid sequence of SARS-CoV-2 S protein is set forth as GenBank Accession No. QHR63250.2 and in FIG.1; the HR1 and HR2 sequences are also indicated in FIG.1. [0045] HR2P is a 36-amino acid peptide based on a portion of the HR2 sequence from coronaviruses SARS-CoV and SARS-CoV-2 (Xia et al.2019b). HR2P can competitively inhibit 6-HB formation and coronavirus fusion with and entry into host cells (Xia et al.2019b). The amino acid sequence of HR2P corresponds to amino acids 1168-1203 of the wild-type SARS-CoV-2 spike (S) protein: DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 2). Other fusion inhibitor peptides based on coronavirus HR2 sequences include: SARS-CoV: VVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK (SEQ ID NO: 3) (US 7,151,163) MERS HR2P: SLTQINTTLLDLTYEMLSLQQVVAKALNESYIDLKEL (SEQ ID NO: 4) (Lu et al.2014); EK1: SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL (SEQ ID NO: 5) (Xia et al.2019b). [0046] The present invention provides modified HR2 peptides, which are variants of the wild-type S protein HR2 sequence that interacts with the S protein HR1 region to form the 6-HB structure required for viral fusion. In particular, the invention provides modified HR2 peptides having one or more amino acid modifications relative to SEQ ID NO: 2. The modified HR2 peptide can have or comprise, for example, an amino acid sequence wherein each amino acid at positions 1-36 is independently selected from those shown in Table 1 (SEQ ID NO: 7), with the proviso that the modified HR2 peptide is not a wild-type coronavirus S protein sequence and/or is not any of SEQ ID NO: 2-5.

ⁿ _e ⁱ _p ^a _S _- ₃ ¹ -

[0047] Modified HR2 peptides can include deletions or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids from the N-terminus and/or deletions or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids from the C-terminus of SEQ ID NO: 2. [0048] For example, the modified HR2 peptide can comprise a peptide corresponding to at least positions 12-26 of SEQ ID NO: 2, or to at least positions 12-33 of SEQ ID NO: 2,or to at least positions 12-36 of SEQ ID NO: 2, or to at least positions 4-26 of SEQ ID NO: 2, or to at least positions 4-32 of SEQ ID NO: 2, or to at least positions 4-33 of SEQ ID NO: 2, or to at least positions 2-19 of SEQ ID NO: 2, or to at least positions 2-26 of SEQ ID NO: 2, or to at least positions 2-32 of SEQ ID NO: 2, or to at least positions 2-33 of SEQ ID NO: 2, and comprising at least one addition, deletion, or substitution relative to SEQ ID NO: 2. [0049] The modified HR2 peptide can comprise, for example, additional amino acids at the N-terminus, wherein position -1 is G, wherein position -2 is L or F, wherein position -3 is D or N, relative to SEQ ID NO: 2. The modified HR2 peptide can comprise, for example, additional amino acids at the C-terminus, wherein position 37 is G, wherein position 38 is K, N, or R, wherein position 39 is Y, F, or W, relative to SEQ ID NO: 2. [0050] Retro inverso forms of all modified HR2 peptides described herein are also included. In particular, the invention provides modified HR2 peptides having one or more amino acid modifications relative to the D-amino acid sequence LEQLDILSENLNKAVENLRDIEKQINVVSANIGSID (SEQ ID NO: 6), which is the retro inverso sequence of SEQ ID NO: 2. The modified HR2 peptide can have or comprise, for example, a D-amino acid sequence wherein each D-amino acid at positions 1-36 is independently selected from those shown in Table 2 (SEQ ID NO: 8). In some embodiments, the modified HR2 peptide is not the retro inverso sequence of a wild-type coronavirus S protein sequence and/or is not the retro inverso sequence of any of SEQ ID NO: 2-5. ₁ ⁰ _. ^A ^e _P _c ⁿ _e ⁱ _p ^a _S O -₅ ¹ -

[0051] Modified HR2 peptides can include deletions or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the N-terminus and/or deletions or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the C-terminus of SEQ ID NO: 6. [0052] For example, the modified HR2 peptide can comprise a peptide corresponding to at least positions 1-25 of SEQ ID NO: 6, or to at least positions 4-25 of SEQ ID NO: 6, or to at least positions 11-25 of SEQ ID NO: 6, or to at least positions 4-33 of SEQ ID NO: 6, or to at least positions 5-33 of SEQ ID NO: 6, or to at least positions 11-33 of SEQ ID NO: 6, or to at least positions 4-35 of SEQ ID NO: 6, or to at least positions 5-35 of SEQ ID NO: 6, or to at least positions 11-35 of SEQ ID NO: 6, or to at least positions 18-35 of SEQ ID NO: 6, and comprising at least one addition, deletion, or substitution relative to SEQ ID NO: 6. [0053] The modified HR2 peptide can comprise, for example, additional D-amino acids at the N-terminus, wherein position -1 is G, wherein position -2 is K, N, or R, wherein position -3 is Y, F, or W, relative to SEQ ID NO: 6. The modified HR2 peptide can comprise, for example, additional amino acids at the C-terminus, wherein position 37 is G, wherein position 38 is L or F, wherein position 39 is D or N, relative to SEQ ID NO: 6. [0054] Variants of these sequences are also included in the scope of the invention. Modified HR2 peptides of the invention can have at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to those sequences disclosed herein. [0055] Modified HR2 peptides of the invention are preferably 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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, or 50 amino acids in length, including ranges having any of those lengths as endpoints, for example, 13-35 amino acids. [0056] Modified HR2 peptides of the invention are capable of interfering with or inhibiting association between the coronavirus S protein HR1 and HR2 domains. Peptides of the invention are capable of disrupting formation and/or decreasing stability of the 6-HB. In particular, the peptides of the invention are capable of interacting with the HR1 domain and competing with the native HR2 domain, thus preventing formation of the 6-HB and inhibiting fusion between the viral envelope and target host cell membrane. Interaction between the modified HR2 peptide and HR1 and inhibition of viral fusion can be assessed by any of several assays known in the art. [0057] Modified HR2 peptides of the invention can comprise amino acids of mixed chirality, such that one or more amino acids in the peptide are in the L form and one or more amino acids are in the D form. [0058] The modified HR2 peptides can have a modified N-terminus and/or a modified C- terminus. For example, the peptide can comprise an N-terminal and/or C-terminal hydrophobic group, such as a linear or cyclic C₂-C₁₈ aliphatic or aromatic hydrocarbon, a naphthyl group, a phenyl group, an octanyl group, a valeryl group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or a 9-fluorenylmethoxy-carbonyl group. In some embodiments, the peptide can comprise an N-terminal acetyl group and/or a C-terminal amide group. [0059] Modified HR2 peptides can comprise a lipid. For example, the peptide can be conjugated to a fatty acid, a glycerolipid, a glycerophospholipid, a prenol, a sphingolipid, or a sterol. In some embodiments, the fatty acid is a C4-C24 fatty acid, preferably a C16 fatty acid. In one embodiment, the fatty acid is palmitic acid. In some embodiments, the sterol is cholesterol or tocopherol. [0060] In some embodiments, the lipid is covalently attached to the modified HR2 peptide directly or via a linker known in the art. Exemplary linkers include, but are not limited to, an amino acid or peptide linker, a substituted alkyl, a substituted cycloalkyl, polyethylene glycol, and derivatives thereof. The lipid can be linked to the N-terminus or the C-terminus of the peptide, or via a residue side chain. [0061] Modified HR2 peptides of the invention can optionally be cyclic. For example, peptides of the invention can include one or more lactam bridges. A lactam bridge is preferably, but not necessarily, created between side chains spaced four amino acid residues apart (BxxxB). Lactam bridges can be formed, for example, between the side chains of Asp or Glu and Lys. Amino acid substitutions can be made at the site of the lactam bridge to facilitate the linkage. [0062] Modified HR2 peptides of the invention can optionally include one or more epitope and/or affinity tags, such as for purification or detection. Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, and the like. Modified HR2 peptides of the invention can optionally include one or more labels. [0063] In certain aspects, the invention provides a composition, e.g., a pharmaceutical composition, comprising a modified HR2 peptide of the invention, optionally further comprising one or more carriers, diluents, excipients, or other additives. [0064] Also within the scope of the invention are kits comprising the modified HR2 peptides and compositions as provided herein and, optionally, instructions for use. The kit can further contain at least one additional reagent, and/or one or more additional active agent. Kits typically include a label indicating the intended use of the contents of the kit. In this context, the term “label” includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit. [0065] The invention includes methods of inhibiting interaction of the HR1 and HR2 regions of a spike (S) protein, for example, a coronavirus S protein, by contacting the S protein with a modified HR2 peptide of the invention. The S protein can be contacted with the modified HR2 peptide by methods known in the art. The method of introduction chosen will depend, for example, on the intended application. For example, the modified HR2 peptide can be introduced directly into a physiological environment or fluid medium comprising S protein, such as a viral particle, which can be a coronavirus particle, for example, a SARS-CoV-2 particle. [0066] In some instances, DNA or RNA encoding the modified HR2 peptide can be delivered to and expressed in a cell. In this embodiment, the DNA or RNA can comprise a sequence encoding a signal peptide for extracellular secretion. Delivery of the DNA or RNA can be accomplished via any suitable vector, depending on the application. Examples of vectors include plasmid, cosmid, phage, bacterial, yeast, and viral vectors prepared, for example, from retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses, and envelope-pseudotyped viruses. Vectors can be introduced into cells, for example, using nanoparticles, hydrodynamic delivery, electroporation, sonoporation, calcium phosphate precipitation, or cationic polymers such as DEAE-dextran. Vectors can be complexed with lipids, such as encapsulated in liposomes, or associated with cationic condensing agents. III. Methods of Preparation [0067] Modified HR2 peptides of the invention can be chemically synthesized, for example, using solid-phase peptide synthesis or solution-method peptide synthesis, or can be expressed using recombinant methods. Synthesis or expression may occur as fragments of the peptide which are subsequently combined either chemically or enzymatically. [0068] Accordingly, also provided are nucleic acid molecules encoding modified HR2 peptides of the invention. Such nucleic acids can be constructed by chemical synthesis using an oligonucleotide synthesizer. Nucleic acid molecules of the invention can be designed based on the amino acid sequence of the desired peptide and selection of those codons that are favored in the host cell in which the recombinant peptide will be produced. Standard methods can be applied to synthesize a nucleic acid molecule encoding a modified HR2 peptide of interest. [0069] Once prepared, the nucleic acid encoding a particular modified HR2 peptide can be inserted into an expression vector and operably linked to an expression control sequence appropriate for expression of the peptide in a desired host. In order to obtain high expression levels of the peptide, the nucleic acid can be operably linked to or associated with transcriptional and translational expression control sequences that are functional in the chosen expression host. [0070] A wide variety of expression host/vector combinations can be employed to anyone known in the art. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13, and filamentous single- stranded DNA phages. [0071] Suitable host cells include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells can be established or cell lines of mammalian origin, examples of which include Pichia pastoris, 293 cells, COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and BHK cells. Cell-free translation systems can also be employed. REFERENCES Ashkenazi A, et al. Multifaceted action of Fuzeon as virus-cell membrane fusion inhibitor. Biochim. Biophys. Acta 1808:2352-2358 (2011). Douglas GC, et al. The Novel Angiotensin-Converting Enzyme (ACE) Homolog, ACE2, Is Selectively Expressed by Adult Leydig Cells of the Testis. Endocrinol.145:4703-4711 (2004). Erickson JW, et al. Antiviral Agents for the Treatment, Control and Prevention of Infections by Coronaviruses. U.S. Patent No.7,151,163 (2006). Han DP, et al. Identification of critical determinant on ACE2 for SARS-CoV entry and development of a potent entry inhibitor. Virol.350:15-25 (2006). Hardy H, et al. Enfuvirtide, a New Fusion Inhibitor for Therapy of Human Immunodeficiency Virus Infection. Pharmacother.24:198-211 (2004). Hoffman M, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181:1-10 (2020). Lai MMC, et al. The Molecular Biology of Coronaviruses. Adv. Virus Res.48:1-100 (1997). Lan J, et al. Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor. bioRxiv (2020); doi.org/10.1101/2020.02.19.956235. Le Chevalier Isaad A, et al. Side chain-to-side chain cyclization by click reaction. J. Pept. Sci. 15:451-454 (2009). Li F, et al. Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor. Science 309:1864-1868 (2005). Lu L, et al. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat. Commun.5:3067 (2014). Patgiri A, et al. A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha helical conformation. Acc. Chem. Res.41:1289-1300 (2008). Schafmeister CE, et al. An All-Hydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides. J. Am. Chem. Soc.122:5891-5892 (2000). Towler P, et al. ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis. J. Biol. Chem.279:17996-18007 (2004). Wang X-Y, et al. Synthesis of small cyclic peptides containing the disulfide bond. ARKIVOC xi:148-154 (2006). Xia S, et al. Potent MERS-CoV Fusion Inhibitory Peptides Identified from HR2 Domain in Spike Protein of Bat Coronavirus HKU4. Viruses 11:56 (2019a). Xia S, et al. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike. Sci. Adv.5:eaav4580 (2019b). Xia S, et al. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell. Mol. Immunol. (2020); doi.org/10.1038/s41423-020-0374-2. Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270-273 and Supplementary Materials/Extended data (2020). *** The present invention is further described by the following claims.

Claims

CLAIMS 1. A modified HR2 peptide (SEQ ID NO: 8) comprising a variant of the D-amino acid sequence LEQLDILSENLNKAVENLRDIEKQINVVSANIGSID (SEQ ID NO: 6), wherein the variant is modified at one or more positions of SEQ ID NO: 6 as follows: i. L1 is substituted with V, W, F, or Y; ii. Q3 is substituted with R; iii. D5 is substituted with S, E, or N; iv. I6 is substituted with M, A, W, F, or L; v. L7 is substituted with F, A, or W; vi. S8 is substituted with A or N; vii. N10 is substituted with D or A; viii. N12 is substituted with S, A, K, G, or Q; ix. A14 is substituted with L or V; x. V15 is substituted with A, L, R, M, or W; xi. E16 is substituted with D or Q; xii. N17 is substituted with Q, S, D, E, or A; xiii. L18 is substituted with M, W, F, or Y; xiv. R19 is substituted with K, L, S, T, Q, N, or O; xv. D20 is substituted with N, L, A, Q, or E; xvi. I21 is substituted with L, M, V, W, or F; xvii. K23 is substituted with F, R, or Q; xviii. Q24 is substituted with Y, F, T, or A; xix. I25 is substituted with L, M, W, or A; xx. N26 is substituted with S or D; xxi. V27 is substituted with I, M, or L; xxii. V28 is substituted with F or L; xxiii. S29 is substituted with N or T; xxiv. A30 is substituted with T, L, or V; xxv. I32 is substituted with M, L, or A; xxvi. G33 is substituted with T, A, Q, Y, or F; xxvii. S34 is substituted with H, T, or D; xxviii. I35 is substituted with F, W, M, L, or A; xxix. D36 is substituted with S.

2. The modified HR2 peptide according to claim 1, wherein between 1 and 10 consecutive D-amino acids of SEQ ID NO: 6 are truncated from the N-terminus.

3. The modified HR2 peptide according to claim 1, wherein between 1 and 11 consecutive D-amino acids of SEQ ID NO: 6 are truncated from the C-terminus.

4. The modified HR2 peptide according to claim 1, wherein between 1 and 10 consecutive D-amino acids of SEQ ID NO: 6 are truncated from the N-terminus, and wherein between 1 and 11 consecutive D-amino acids of SEQ ID NO: 6 are truncated from the C- terminus.

5. The modified HR2 peptide according to claim 1, comprising between 1 and 3 additional N-terminal D-amino acids.

6. The modified HR2 peptide according to claim 5, wherein the N-terminal D-amino acids are selected from the group consisting of: (i) G; (ii) KG; (iii) NG; (iv) RG; (v) YKG; (vi) YNG; (vii) YRG; (viii) FKG; (ix) FNG; (x) FRG; (xi) WKG; (xii) WNG; and (xiii) WRG.

7. The modified HR2 peptide according to claim 1, comprising between 1 and 3 additional C-terminal amino acids.

8. The modified HR2 peptide according to claim 7, wherein the C-terminal amino acids are selected from the group consisting of: (i) G; (ii) GL; (iii) GF; (iv) GLD; (v) GFD; (vi) GLN; and (vii) GFN.

9. A modified HR2 peptide (SEQ ID NO: 7) comprising a variant of the amino acid sequence DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 2), wherein the variant is modified at one or more positions of SEQ ID NO: 2 as follows: i. D1 is substituted with S; ii. I2 is substituted with F, W, M, L, or A; iii. S3 is substituted with H, T, or D; iv. G4 is substituted with T, A, Q, Y, or F; v. I5 is substituted with M, L, or A; vi. A7 is substituted with T, L, or V; vii. S8 is substituted with N or T; viii. V9 is substituted with F or L; ix. V10 is substituted with I, M, or L; x. N11 is substituted with S or D; xi. I12 is substituted with L, M, W, or A; xii. Q13 is substituted with Y, F, T, or A; xiii. K14 is substituted with F, R, or Q; xiv. I16 is substituted with L, M, V, W, or F; xv. D17 is substituted with N, L, A, Q, or E; xvi. R18 is substituted with K, L, S, T, Q, N, or O; xvii. L19 is substituted with M, W, F, or Y; xviii. N20 is substituted with Q, S, D, E, or A; xix. E21 is substituted with D or Q; xx. V22 is substituted with A, L, R, M, or W; xxi. A23 is substituted with L or V; xxii. N25 is substituted with S, A, K, G, or Q; xxiii. N27 is substituted with D or A; xxiv. S29 is substituted with A or N; xxv. L30 is substituted with F, A, or W; xxvi. I31 is substituted with M, A, W, F, or L; xxvii. D32 is substituted with S, E, or N; xxviii. Q34 is substituted with R; xxix. L36 is substituted with V, W, F, or Y.

10. The modified HR2 peptide according to claim 9, wherein between 1 and 11 consecutive amino acids of SEQ ID NO: 2 are truncated from the N-terminus.

11. The modified HR2 peptide according to claim 9, wherein between 1 and 10 consecutive amino acids of SEQ ID NO: 2 are truncated from the C-terminus.

12. The modified HR2 peptide according to claim 9, wherein between 1 and 11 consecutive amino acids of SEQ ID NO: 2 are truncated from the N-terminus and wherein between 1 and 10 consecutive amino acids of SEQ ID NO: 2 are truncated from the C-terminus.

13. The modified HR2 peptide according to claim 9, comprising between 1 and 3 additional N-terminal amino acids.

14. The modified HR2 peptide according to claim 13, wherein the N-terminal amino acids are selected from the group consisting of: (i) G; (ii) LG; (iii) FG; (iv) DLG; (v) DFG; (vi) NLG; and (vii) NFG.

15. The modified HR2 peptide according to claim 9, comprising between 1 and 3 additional C-terminal amino acids.

16. The modified HR2 peptide according to claim 15, wherein the C-terminal amino acids are selected from the group consisting of: (i) G; (ii) GK; (iii) GN; (iv) GR; (v) GKY; (vi) GNY; (vii) GRY; (viii) GKF; (ix) GNF; (x) GRF; (xi) GKW; (xii) GNW; and (xiii) GRW.

17. The modified HR2 peptide according to any preceding claim, wherein the peptide comprises a lipid.

18. The modified HR2 peptide according to any preceding claim, wherein the peptide comprises an N-terminal acetyl group and/or a C-terminal amide group.

19. A composition comprising the modified HR2 peptide according to any preceding claim.

20. The composition according to claim 19, which is a pharmaceutical composition.

21. A kit comprising the modified HR2 peptide according to any one of claims 1 to 18.

22. A nucleic acid molecule encoding the modified HR2 peptide according to any one of claims 1 to 18.

23. A method of inhibiting interaction between an HR1 region and an HR2 region of a coronavirus spike (S) protein, the method comprising contacting the coronavirus S protein with a modified HR2 peptide according to claim 1 or claim 9, wherein the modified HR2 peptide competes with the HR2 region for binding to the HR1 region.

24. A method of inhibiting fusion of a coronavirus with a target cell, the method comprising contacting the coronavirus with a modified HR2 peptide according to claim 1 or claim 9, wherein the modified HR2 peptide inhibits fusion of the coronavirus with the target cell.

25. The method according to claim 23 or 24, which is in in vitro method.

26. Use of a modified HR2 peptide according to any one of claims 1 to 18 for inhibiting interaction between an HR1 region and an HR2 region of a coronavirus spike (S) protein.

27. Use of a modified HR2 peptide according to any one of claims 1 to 18 for inhibiting fusion of a coronavirus with a target cell.

28. The method according to any one of claims 23 to 25, or the use according to claim 26 or 27, wherein the coronavirus is SARS-CoV-2.