WO2021195009A1 - Peptide antagonists of ace2-binding proteins - Google Patents

Abstract

Provided are ACE2 peptides having modifications in the spike-interacting region, compositions comprising the ACE2 peptides, and methods of inhibiting binding of an ACE2- binding protein, such as a coronavirus spike (S) protein, using the ACE2 peptides.

Description

PEPTIDE ANTAGONISTS OF ACE2-BINDING PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority ofU.S. Provisional Patent Application No. 62/993,660, filed on March 23, 2020.

BACKGROUND

[0002] 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 SI 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 SI 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 S 1 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). Entry of a virus into a target (host) cell is the first step in the viral life cycle.

[0003] 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

[0004] 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 are intended to fall within the scope of the present invention. [0005] The invention provides ACE2 variant peptides comprising a modified portion of the ACE2 spike-interacting region. One embodiment is a modified ACE2 peptide comprising a variant of the amino acid sequence

STIEEQAKTFLDKFNHEAEDLFYQSSLAS (SEQ ID NO: 3). In certain embodiments, the variant is modified at one or more positions of SEQ ID NO: 3 as follows: T9 is substituted with L; F10 is substituted with A; LI 1 is substituted with A or Q; D12 is substituted with E or L; K13 is substituted with L; F14 is substituted with A or Q; N15 is substituted with A; H16 is substituted with A; D20 is substituted with E; L21 is substituted with A or K; F22 is substituted with A or Q; Q24 is substituted with E or K; S25 is substituted with A; S26 is substituted with A. The modified ACE2 peptide can additionally comprise a modification wherein between 1 and 8 consecutive amino acids of SEQ ID NO: 3 are truncated from the N-terminus, beginning at SI, or wherein between 1 and 12 consecutive amino acids of SEQ ID NO: 3 are truncated from the C-terminus, beginning at S29.

[0006] In one embodiment, the modified ACE2 peptide comprises an amino acid sequence selected from the group consisting of: QAKTFQDKQNHEAEDKQYQSSL (SEQ ID NO: 4); QAKTFQDKQNHEAEDAQYQSSL (SEQ ID NO: 5); QAKTFLEKFNHEAEELFYQSSL (SEQ ID NO: 6); QAKLFLLLFNHEAEDLFYQSSL (SEQ ID NO: 7); and QAKLFQLLQNHEAEEKQYQSSL (SEQ ID NO; 8); QAKTFLDKANHEAEDLFYQSSL (SEQ ID NO: 9); QAKTFLDKFAHEAEDLFYQSSL (SEQ ID NO: 10); QAKTFLDKFNHEAEDLAYQSSL (SEQ ID NO: 11); QAKTFLDKFNHEAEDLFYQASL (SEQ ID NO: 12); QAKTFLDKFNHEAEDLFYQSAL (SEQ ID NO: 13); QAKTFLDKFNHEAEDLFYQAAL (SEQ ID NO: 14); QAKTFLDKFNAEAEDLFYQSSL (SEQ ID NO: 15);

Q AKTFLDKFNHEAEDLF YK SSL (SEQ ID NO: 16); QAKTFLDKFNHEAEDAFYQSSL (SEQ ID NO: 17); QAKTFLDKFNHEAEDLAYQSSL (SEQ ID NO: 18);

Q AKTFLDKFNHEAED AAY Q S SL (SEQ ID NO: 19); QAKTALDKFNHEAEDLFYQSSL (SEQ ID NO: 20); QAKTFADKFNHEAEDLFYQSSL (SEQ ID NO: 21); and Q AKTFLDKFNHEAEDLF YES SL (SEQ ID NO: 22). Optionally, the modified ACE2 peptide comprises a glutamic acid residue N-terminal to Q1 of the foregoing sequences.

[0007] Also included are retro-inverso modified ACE2 peptides comprising D-amino acids in a reversed amino acid sequence relative to an amino acid sequence disclosed herein. In one embodiment, the modified ACE2 peptide comprises a D-amino acid sequence selected from the group consisting of: LSSQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 23); LSSQYQKDEAEHNQKDQFTKAQ (SEQ ID NO: 24); LSSQYQADEAEHNQKDQFTKAQ (SEQ ID NO: 25); LSSQYFLEEAEHNFKELFTKAQ (SEQ ID NO: 26); LSSQYFLDEAEHNFLLLFLKAQ (SEQ ID NO: 27); LSSQYQKEEAEHNQLLQFLKAQ (SEQ ID NO: 28); LSSQYFLDEAEHNAKDLFTKAQ (SEQ ID NO: 29); LSSQYFLDEAEHAFKDLFTKAQ (SEQ ID NO: 30); LSSQYALDEAEHNFKDLFTKAQ (SEQ ID NO: 31);

L S AQ YFLDEAEHNFKDLFTK AQ (SEQ ID NO: 32); LASQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 33); LAAQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 34); LSSQYFLDEAEANFKDLFTKAQ (SEQ ID NO: 35); LSSKYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 36); LSSQYALDEAEHNFKDLFTKAQ (SEQ ID NO: 37); LSSQYFADEAEHNFKDLFTKAQ (SEQ ID NO: 38); LSSQYAADEAEHNFKDLFTKAQ (SEQ ID NO: 39); LSSQYFLDEAEHNFKDLATKAQ (SEQ ID NO: 40); and LSSQYFLDEAEHNFKDAFTKAQ (SEQ ID NO: 41);

LSSEYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 42). Optionally, the modified ACE2 peptide comprises a D-glutamic acid residue C-terminal to Q22 of the foregoing sequences.

[0008] In some embodiments, the modified ACE2 peptide comprises an N-terminal acetyl group and/or a C-terminal amide group.

[0009] In one aspect, modified ACE2 peptides of the invention are for use in inhibiting binding of an ACE2-binding protein to ACE2. In one embodiment, the ACE2 protein is a coronavirus spike (S) protein, for example, a SARS-CoV-2 S protein. [0010] Further aspects of the invention provide a composition comprising a modified ACE2 peptide of the invention, for example, a pharmaceutical composition; a kit comprising a modified ACE2 peptide of the invention; and a nucleic acid molecule encoding a modified ACE2 peptide of the invention.

[0011] The invention additionally provides methods of inhibiting binding of an ACE2-binding protein to a spike-interacting region of ACE2, the methods comprising contacting the ACE2-binding protein with a modified ACE2 peptide of the invention. In one embodiment, the ACE-2 binding protein is a coronavirus spike (S) protein. In a particular embodiment, the coronavirus is SARS-CoV-2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows the amino acid sequence of human angiotensin-converting enzyme 2 (ACE2) (SEQ ID NO: 1). Contact residues that participate in SARS-Cov-2 spike (S) protein binding are shown in bold type and underlined.

[0013] FIG. 2 shows the amino acid sequence of SARS-Cov-2 S protein (SEQ ID NO: 2). Contact residues that participate in ACE2 binding are shown in bold type and underlined.

DETAILED DESCRIPTION OF THE INVENTION

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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

[0018] 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.

[0019] 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.

[0020] 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).

[0021] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are included.

[0022] Units, prefixes, and symbols are denoted in their Systeme 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.

[0023] 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.

[0024] 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.) 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 el al. 2009), or disulfide bridges (Wang etal. 2006). Bridges or staples can be spaced, for example, 3, 4, 7, or 8 amino acids apart.

[0025] 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.

[0026] 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 a-center chirality of the amino acid subunits) to help maintain side-chain topology similar to that of the original L-amino acid peptide.

[0027] 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. ah, 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, ΙII: basic, IV: hydrophobic, V: aromatic, bulky amino acids.

Table I

[0028] In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; VI: neutral or hydrophobic, VII: acidic, VIII: basic, LX: polar, X: aromatic.

Table II

[0029] 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 Bulks et al. , Proc. Natl. Acad. Sci. U.S.A. 94:412-417 (1997)).

[0030] 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.

[0031] 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 etal., 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 etal, 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 NW Sgapdna. 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., SIAMJ. Applied Math., 48:1073 (1988).

[0032] 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.

[0033] An “isolated” molecule is one that is in a form not found in nature, including those which have been purified.

[0034] 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).

[0035] “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 (KD). 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.

[0036] 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, KD or Kd, Kon, K_orr) are made with standardized solutions of binding partners and a standardized buffer, as known in the art.

[0037] 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.”

[0038] An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.

[0039] 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.

[0040] 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 ACE2 Peptides and Compositions

[0041] Angiotensin-converting enzyme 2 (ACE2) is an 805 amino acid eukaryotic metallopeptidase that catalyzes the conversion of angiotensin I to angiotensin II (Douglas et al. 2004). ACE2 is a transmembrane protein found on the surface of cell types that include pulmonary and intestinal epithelial cells and vascular endothelial cells. The extracellular portion of ACE2 contains a zinc-binding catalytic region comprised of two sub-domains, which form an active-site cleft (Towler et al. 2004). The full amino acid sequence of wild-type human ACE2 is set forth as GenBank Accession No. AAT45083.1 and in FIG 1. Contact residues that participate in SARS-Cov-2 spike (S) protein binding are indicated in FIG. 1 and are referred to herein as the “spike-interacting region.”

[0042] The SARS-CoV-2 spike (S) glycoprotein is a 1273 amino acid protein of probable bat origin (Zhou et al. 2020). S protein facilitates entry of the virus into host cells by binding to ACE2 on the surface of a target host cell (Hoffman et al. 2020; Xia et al. 2020; Zhou 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. 2. Contact residues that participate in ACE2 binding are indicated in FIG. 2 and are referred to herein as the “ACE2-interacting region.”

[0043] An X-ray crystal structure of a complex comprising residues 336-516 of the SARS-CoV-2 S protein receptor binding domain (RBD) bound to residues 19-615 of the N-terminal peptidase domain of ACE2 has recently been solved (Lan et al. 2020). The contact residues at the RBD-ACE2 interface were identified (Lan et al. 2020) and are shown in FIG. 1 and FIG. 2.

[0044] Modified ACE2 peptides of the invention are variants of a region corresponding to residues 19-47 of ACE2, STIEEQ AKTFLDKFNHEAEDLF Y Q S SL AS (SEQ ID NO: 3), which region is critical for binding by coronavirus S protein. Accordingly, the invention provides modified ACE2 peptides having one or more amino acid modifications relative to SEQ ID NO: 3. The modified ACE2 peptide preferably comprises a peptide corresponding to at least positions 6-17 of SEQ ID NO: 3, or to at least positions 6-20 of SEQ ID NO: 3, or to at least positions 6-24 of SEQ ID NO: 3, or to at least positions 6-27 of SEQ ID NO: 3, or to at least positions 9-20 of SEQ ID NO: 3, or to at least positions 9-24 of SEQ ID NO: 3, or to at least positions 9-27 of SEQ ID NO: 3, or to at least positions 16-27 of SEQ ID NO: 3, and comprising at least one addition, deletion, or substitution relative to SEQ ID NO: 3.

[0045] The modified ACE2 peptide can have or comprise, for example, an amino acid sequence shown in Table 1. Substitutions in SEQ ID NO:3 are shown in underlined bold type.

[0047] Variants of these sequences are also included in the scope of the invention, modified ACE2 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.

[0048] Modified ACE2 peptides of the invention are capable of interfering with or inhibiting binding of ACE2 -binding proteins, such as coronavirus S protein, to a spike- interacting region of ACE2. For example, the modified ACE2 peptides of the invention are capable of binding to S protein and competing with S protein binding to native ACE2. Binding can be assessed by any of several assays known in the art.

[0049] Accordingly, the invention also includes methods of screening candidate molecules for the ability to bind to a spike-interacting region of ACE2. One method of identifying an ACE2-binding protein comprises contacting a candidate molecule with a modified ACE2 peptide of the invention and determining whether the candidate molecule binds to the modified ACE2 peptide.

[0050] Modified ACE2 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.

[0051] Modified ACE2 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.

[0052] The modified ACE2 peptides can have a modified N-terminus and/or a modified C-terminus. For example, modified ACE2 peptides can optionally include an N-terminal acetyl group and/or a C-terminal amide group.

[0053] Modified ACE2 peptides of the invention can optionally be cyclic. For example, modified ACE2 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. [0054] Modified ACE2 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 ACE2 peptides of the invention can optionally include one or more labels.

[0055] In certain aspects, the invention provides a composition, e.g., a pharmaceutical composition, comprising a modified ACE2 peptide of the invention, optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0056] Also within the scope of the invention are kits comprising the modified ACE2 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.

[0057] The invention includes methods of inhibiting binding of an ACE2-binding protein to a spike-interacting region of ACE2 by contacting an ACE2-binding protein with the modified ACE2 peptide. Modified ACE2 peptides of the invention can be contacted with ACE2-binding proteins by methods known in the art. The method of introduction chosen will depend, for example, on the intended application. For example, the modified ACE2 peptides can be introduced directly into a physiological environment or fluid medium comprising ACE2-binding proteins, such as a coronavirus particle, which can be a SARS- CoV-2 particle.

[0058] In some instances, DNA or RNA encoding the modified ACE2 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

[0059] Modified ACE2 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.

[0060] Accordingly, also provided are nucleic acid molecules encoding modified ACE2 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 modified ACE2 peptide and selection of those codons that are favored in the host cell in which the recombinant modified ACE2 peptide will be produced. Standard methods can be applied to synthesize a nucleic acid molecule encoding a modified ACE2 peptide of interest.

[0061] Once prepared, the nucleic acid encoding a particular modified ACE2 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 modified ACE2 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.

[0062] 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 pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, such as Ml 3, and filamentous single- stranded DNA phages.

[0063] 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

Douglas GC, et al. The Novel Angiotensin-Converting Enzyme (ACE) Homolog, ACE2, Is Selectively Expressed by Adult Ley dig Cells of the Testis. Endocrinol. 145:4703-4711 (2004).

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).

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.

Li F, et al. Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor. Science 309:1864- 1868 (2005).

Le Chevalier Isaad A, et al. Side chain-to-side chain cyclization by click reaction. J. Pept. Sci. 15:451-454 (2009).

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).

Schafineister 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. 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

1. A modified ACE2 peptide comprising a D-amino acid sequence selected from the group consisting of: i. LSSQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 23); ii. LSSQYQKDEAEHNQKDQFTKAQ (SEQ ID NO: 24); iii. LSSQYQADEAEHNQKDQFTKAQ (SEQ ID NO: 25);

IV. LSSQYFLEEAEHNFKELFTKAQ (SEQ ID NO: 26); v. LSSQYFLDEAEHNFLLLFLKAQ (SEQ ID NO: 27); vi. LSSQYQKEEAEHNQLLQFLKAQ (SEQ ID NO: 28); vii. LSSQYFLDEAEHNAKDLFTKAQ (SEQ ID NO: 29); viii. LSSQYFLDEAEHAFKDLFTKAQ (SEQ ID NO: 30);

IX. LSSQYALDEAEHNFKDLFTKAQ (SEQ ID NO: 31); x. LSAQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 32); xi. LASQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 33); xii. LAAQYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 34); xiii. LSSQYFLDEAEANFKDLFTKAQ (SEQ ID NO: 35);

XIV. LSSKYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 36); xv. LSSQYALDEAEHNFKDLFTKAQ (SEQ ID NO: 37); xvi. LSSQYFADEAEHNFKDLFTKAQ (SEQ ID NO: 38); xvii. LSSQYAADEAEHNFKDLFTKAQ (SEQ ID NO: 39); xviii. LSSQYFLDEAEHNFKDLATKAQ (SEQ ID NO: 40); xix. L S S Q YFLDE AEHNFKD AFTK AQ (SEQ ID NO: 41); and xx. LSSEYFLDEAEHNFKDLFTKAQ (SEQ ID NO: 42).

2. The modified ACE2 peptide according to claim 1, comprising a D-glutamic acid residue C-terminal to Q22. 3. A modified ACE2 peptide comprising a variant of the amino acid sequence STIEEQ AKTFLDKFNHEAEDLF Y Q S SL AS (SEQ ID NO: 3), wherein the variant is modified at one or more positions of SEQ ID NO: 3 as follows:

1. T9 is substituted with L;

11. F10 is substituted with A;

111. LI 1 is substituted with A or Q;

IV. D12 is substituted with E or L;

V. K13 is substituted with L; vi. F14 is substituted with A or Q;

Vll. N15 is substituted with A;

Vlll. HI 6 is substituted with A;

IX. D20 is substituted with E;

X. L21 is substituted with A or K;

XI. F22 is substituted with A or Q;

XU. Q24 is substituted with E or K;

Xlll. S25 is substituted with A;

XIV. S26 is substituted with A.

4. The modified ACE2 peptide according to claim 3, wherein between 1 and 8 consecutive amino acids of SEQ ID NO: 3 are truncated from the N-terminus or wherein between 1 and 12 consecutive amino acids of SEQ ID NO: 3 are truncated from the C-terminus.

5. The modified ACE2 peptide according to claim 3, wherein the peptide comprises D-amino acids in a reversed amino acid sequence relative to the amino acid sequence of any preceding claim.

6. A modified ACE2 peptide comprising an amino acid sequence selected from the group consisting of: i. QAKTFQDKQNHEAEDKQYQSSL (SEQ ID NO: 4); ii. QAKTFQDKQNHEAEDAQYQSSL (SEQ ID NO: 5); iii. Q AKTFLEKFNHE AEELF Y Q S SL (SEQ ID NO: 6); iv. QAKLFLLLFNHEAEDLFYQS SL (SEQ ID NO: 7); v. QAKLFQLLQNHEAEEKQYQSSL (SEQ ID NO; 8); vi. QAKTFLDKANHEAEDLFYQSSL (SEQ ID NO: 9); vii. QAKTFLDKFAHEAEDLFYQSSL (SEQ ID NO: 10); viii. QAKTFLDKFNHEAEDLAYQSSL (SEQ ID NO: 11); ix. QAKTFLDKFNHEAEDLFYQASL (SEQ ID NO: 12); x. Q AKTFLDKFNHEAEDLF YQ SAL (SEQ ID NO: 13); xi. Q AKTFLDKFNHEAEDLF YQ AAL (SEQ ID NO: 14); xii. Q AKTFLDKFNAEAEDLF YQ S SL (SEQ ID NO: 15); xiii. QAKTFLDKFNHEAEDLF YKS SL (SEQ ID NO: 16); xiv. Q AKTFLDKFNHEAED AF Y Q S SL (SEQ ID NO: 17); xv. QAKTFLDKFNHEAEDLAYQSSL (SEQ ID NO: 18); xvi. Q AKTFLDKFNHEAED AAYQSSL (SEQ ID NO: 19); xvii. QAKTALDKFNHEAEDLFYQSSL (SEQ ID NO: 20); xviii. Q AKTF ADKFNHEAEDLF Y Q S SL (SEQ ID NO: 21); and xix. QAKTFLDKFNHEAEDLF YES SL (SEQ ID NO: 22).

7. The modified ACE2 peptide according to claim 6, comprising a glutamic acid residue N-terminal to Ql.

8. The modified ACE2 peptide according to any preceding claim, wherein the peptide comprises an N-terminal acetyl group and/or a C -terminal amide group.

9. A composition comprising the modified ACE2 peptide according to any preceding claim.

10. The composition according to claim 9, which is a pharmaceutical composition.

11. A kit comprising the modified ACE2 peptide according to any one of claims 1 to

8.

12. A nucleic acid molecule encoding the modified ACE2 peptide according to any one of claims 1 to 8.

13. A method of inhibiting binding of an ACE2-binding protein to a spike-interacting region of ACE2, the method comprising contacting an ACE2-binding protein with the modified ACE2 peptide according to claim 1.

14. The method according to claim 13, wherein the ACE2-binding protein is a coronavirus spike (S) protein.

15. The method according to claim 14, wherein the coronavirus is SARS-CoV-2.

16. A modified ACE2 peptide according to any one of claims 1 to 8 for use in inhibiting binding of an ACE2-binding protein to ACE2.

17. The modified ACE2 peptide according to claim 16, wherein the ACE2-binding protein is a coronavirus spike (S) protein.

18. The modified ACE2 peptide according to claim 17, wherein the coronavirus is

SARS-CoV-2.