WO2023159113A1 - Peptide fusion inhibitors exhibiting pan-coronavirus inhibitory activity - Google Patents

Abstract

The invention provides compositions, kits and methods utilizing peptides provided herein for treating and/or preventing coronavirus infections.

Description

PEPTIDE FUSION INHIBITORS EXHIBITING

PAN-CORONAVIRUS INHIBITORY ACTIVITY

Priority

This patent application claims the benefit of priority to US Provisional Application Serial No. 63/310,891, filed February 16, 2022, which is incorporated by reference herein in its entirety.

Incorporation by Reference of Sequence Listing

A Sequence Listing is provided herewith as an xml file, “2310603.xml” created on February 16, 2023, and having a size of 344,978 bytes. The content of the xml file is incorporated by reference herein in its entirety.

Background of the Invention

One of the earliest steps of virus infections is the engagement of specific receptors on the host cell surface mediated by the receptor binding domain of the viral Spike protein. ’This interaction in turn activates a different part of the Spike protein to mediate virion fusion. The molecular process of fusion allows for virions bound to receptors to fuse their membranes with the plasma membrane or endosomal membrane of the cell thereby promoting pathogen entry into the host cell . For fusion to occur, the viral protein mediating fusion must undergo major conformational changes that mediate membrane fusion and infection. First, a fusion peptide comprised of hydrophobic amino acids is deployed to “harpoon” the host cell membrane. Next, the trimeric viral Spike protein mediating fusion folds back on themselves via the binding of two amphipathic heptad repeats. This process forms a six-helix bundle structure that approximates the membranes facilitating completion of the fusion process. The six-helix bundle motif is a common fusion strategy used by many viral families including die coronaviruses.

Summary

Described herein (Figures 10-14) are peptide inhibitors of six helix bundle formation that exhibit pan-coronavirus inhibition due to conservation of die heptad repeats structures in ad of the sequenced coronaviruses.

Provided herein are compositions, kits and methods utilizing the disclosed peptides that are useful for treating, inhibiting and/or preventing coronavirus infections. One aspect provides a peptide comprising the following sequence: R¹ - SIDQINASVVNIQKEIDRLNEVAKNLNESLIDLQEL - R² (SEQ ID NO: 1) or sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto, where changes can include conservative amino acid substitutions, wherein R¹ is non-amino acid end group; and wherein is R² is lipid moiety. In one aspect the lipid moiety is selected from the group consisting of cholesterol, tocopherol, pregnenolone and palmitate. In another aspect, the non- amino acid end group is selected from the group consisting of acetyl (Ac), 4-phenylbutanoic acid (PBA), 3 -phenylpropionic acid (PPA), 4-(naphthalen-2-yl)butanoic acid (NBA)and 3- (naphthalen-l-yl)propanoic acid (NPA). In one aspect, one or more of the amino acids are D- amino acids or the sequence includes one or more non-natural amino acids. In another aspect, the peptide further comprises a spacer, such as a spacer which comprises a polyethylene glycol (PEG), including PEG4 or PEG3. In one aspect, the C-terminal comprises either one of:

GSGSG-PEG4-Lys(C16)

GSGSG-Lys[PEG4-Lys-(Mai-C14)2]

ID NO: 2 and 19).

In one aspect, the C-terminus comprises GSGSG-PEG4-Lys(C16) (SEQ ID NO: 2).

One aspect provides a peptide comprising or consisting essentially of any one of the peptides in Figures 10-14 or sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto, wherein the peptide is not EK1 or EK1C4 (including or excluding N- terminus and/or C-terminus modifications, D-amino acids, non-amnio acids and/or peptide staples).

In one aspect, the peptide has the following structure PBA-

SIDQINAS VVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-PEG3 -Ly s(C 16) (CGM23 ; SEQ ID NO: 1)) or an amino acid sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto.

One embodiment provides a composition disclosed herein or a combination thereof and a pharmaceutically acceptable carrier.

One aspect provides a method to prevent or treat a coronavirus infection comprising administering to a subject in need thereof an amount of a peptide or composition disclosed herein effective to prevent or treat said coronavirus infection. Another aspect provides a method to inhibit transmission of a coronavirus to an animal cell comprising contacting said animal cells with an effective amount a peptide or composition described herein so that the coronavirus is inhibited from infecting the cell. Another aspect provides a method to inactivate a coronavirus comprising contacting the coronavirus wi t h an effective dose of a peptide or composition described herein so that the coronavirus is rendered inactive (non- infectious). In one aspect, the peptide or composition is administered by inhalation and/or subcutaneous injection. In some aspects the peptide(s) and/or composition is administered intranasally. In some aspects the peptide(s) and/or composition is administered as nasal drops or a spray. In some aspects more than one peptide is administered.

In another aspect, there is a kit comprising the peptide and/or composition described herein and/or the composition described herein and instructions for use.

Brief Description of the Drawings

Fig. I provides a summary of SARS-CoV-2 fusion from Huang, Y ., Yang, C., Xu, Xf. et al. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin 41, 1 141-1149 (2020). Note trimeric Spike engagement of ACE2 receptor with prior cleavage of S1/S2 at furin cleavage site triggers conformational extension of fusion peptide in S2 subunit of Spike to engage the host membrane. Fusion is completed by folding back of this extended structure through interactions of Heptad Repeat 1 (HR1) and Heptad Repeat 2 (HR2) that form a six-helix bundle structure within the S2 trirner. Peptide fusion inhibitors mimicking HR2 can be developed that block six helix bundle formation and prevent virion fusion and entry' of the pathogen into cells. Because the HR1 and HR2 are highly conserved within the coronavirus family, these peptide inhibitors can display pan-coronavirus activity.

Figure 1A provides a mechanism of fusion core formation and blocking effect of antiviral peptide. The S proteins are embedded in the viral membrane and are composed of the SI and S2 subunits. The SI subunit contains one receptor-binding domain (RBD). The S2 subunit mediates the virus/ceH membrane fusion and the entry of the virus. RBD of the SI subunit binds to the host ACE2 receptor when the SARS-CoV-2 contacts with the cell membrane. Furin cleaves the S protein into the SI subunit and the S2 subunit. The fusion peptide (FP) of S2 is exposed and is inserted into the target cell membrane. Three HR1 s and three HR2s combine to form the fusion core, pulling the viral membrane to fuse with the host cell membrane. The designed and computational-optimized anti-virus peptides can bind to HR I more tightly, preventing the HR1 s and HR2s from forming fusion core. (Figure l/\ is from Ling et al. Peptides. Vol.130, August 2020, 170328.) There is also an additional ‘"priming” cleavage after furin cutting mediated by TMPRSS2 (usually at the plasma membrane) or one of the lysosomal cathepsins in fusion occurs in the late endosome after endocytosis.

FIG. 2 provides results for SARS-CoV-2 syncytia assay. It is noted that the ICso for CGM23 is 2n\i. while EK1C4 is 12 nti.

FIGs. 3A-3B depicts inhibition of infection detected in VSV virions pseudotyped with SARS-CoV-2 Spike. It is noted that there was a lower ICso for CGM23 compared to EK1C4 in both the first and second assays (top and middle panel). ICso’s in Assays 1 and 2 are summarized in the provided Table (3B).

Fig. 4 depicts the results from live SARS CoV-2 viral infection assays. There was a 50-fold greater potency of CGM23 (ICso = 0.95 nM) compared to EK1C4 (ICso =53.8 nM) involving infection of Calu-6-ACE2 cells with the Wuhan strain of SARS-CoV-2.

FIG. 5 provides data from a cell viability assay. CGM23 showed less toxicity at high input concentrations than EK1C4. Both peptides (CMG23 and EK1C4) display essentially no toxicity at their ICso or IC90.

Fig. 6 depicts data from a pan-coronavirus assay. There were lower ICso values for CGM23 for inhibition of 229E Spike, SARS Spike, and MERS Spike compared to EK1C4 and markedly greater inhibition of NL63 Spike fusion by CGM23 compared EK1C4.

FIGs. 7A-7C provides in vivo peptide efficacy data.

FIGs. 8A-8N provide HPLC stability data (SEQ ID NOs: 3-10 and 11-18).

FIGs. 9A-9D demonstrate uninfected mouse lung with no spike expression, and a few macrophages naturally present in uninfected controls. They also depict an abundant expression of spike in bronchioles and accumulation of macrophages markedly above baseline in the lung of mice infected with SARS-CoV-2. The figures further demonstrate a lack of spike expression and macrophage migration above baseline in animals treated with CGM23 (a fusion inhibitor peptide). And finally, the figures depict less effective effects of EK1C4, including some spike expression and some macrophage (MAC2 marker) immigration. Macrophages engulfing SARs-CoV-2 infected cells are also depicted.

FIGs. 10-14 provide peptide inhibitors of six helix bundle formation that exhibit pancoronavirus inhibition due to conservation of the heptad repeats structures in all of the sequenced coronaviruses (SEQ ID NOs: 2 and 19-196 (FIG. 10), 2 and 197-241 (FIG. 11 ), 242-244 (FIG 12), and 245-246 (FIG 13)). FIGs. 15 and 16 demonstrate that the peptide fusion inhibitor acts effectively to prevent SARS-CoV-2 subvariant BA.5 infection; in vivo CGM23 challenge. Description

’The present invention is directed to compositions, kits and methods utilizing peptides. The compositions are useful for treating and/or preventing viral infections.

Peptides which interfere with the viral fusogenic process can be used for the prevention and treatment of viral infections. For example, provided herein are a series of peptides that inhibit fusion of, for example, S/MlS-CoV-2 to target/host cells. It is believed that these peptides bind to the HR-1 region of the S2 component of Spike preventing the natural interaction of the HR1 and HR2 regions required to fuse the virion and host cell membranes. Such peptides include those sequences in Figures 10-14, such as following sequence PBA-SIDQINASVVNIQKEIDRLNEV AKNLNESLIDLQEL-GSGSG-PEG3- Lys(C16) (CGM23; SEQ ID NO: 1) or a sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto. Definitions

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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, several embodiments with regards to methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section.

For the purposes of clarity and a concise description, features can be described herein as part, of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

References in the specification to “one embodiment", “an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. As used herein, the indefinite articles “a”, “an” and “the” should be understood to include plural reference unless the context clearly indicates otherwise.

The phrase “and/or,” as used herein, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases.

As used herein, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating a listing of items, “and/or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number of items, and, optionally, additional unlisted items. Only terms dearly indicated to the contrary, such as “only one of¹ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “'one of,” “only one of,” or “exactly one of.”

As used herein, the terms “including,” “includes,” “having,” “has,” “wdth,” or variants thereof, are intended to be inclusive similar to the term “’comprising.”

’The terms "individual," "subject," and "patient," are used interchangeably herein and refer to any subject for whom diagnosis, treatment, or therapy is desired, including a mammal or a bird. Mammals include, but are not limited to, humans, farm animals, sport animals and pets. A “subject”' is a vertebrate, such as a mammal, including a human. Mammals include, but are not limited to, humans, farm animals, sport animals and companion animals. Included in the term “animal” is dog, cat, gerbil, guinea pig, hamster, horse, rabbit, swine, mouse, monkey (e.g., ape, gorilla, chimpanzee, orangutan) rat, sheep, goat, cow and bird.

The terms “treat,” and “treating,” as used herein, shall mean decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or decrease the occurrence of pathological cells (e.g., infected cells) in an animal who is infected with the viral disorder. The treatment may be complete, e.g., the total absence of virus in a subject. The treatment may also be partial, such that the occurrence of infected ceils in a subject is less than that which would have occurred without the present invention. Treatment results in the stabilization, reduction or elimination of the infected cells, an increase in the survival of the patient or decrease of at least one sign or symptoms of the disease.

The terms “prevent,"' “preventing,” and “prevention,” as used herein, shall refer to a decrease in the occurrence of a disease, or decrease in the risk of acquiring a disease, or a decrease in the presentation of at least one sign or associated symptom of the di sease in a subject. The prevention may be complete, e.g., the total absence of disease or pathological cells in a subject. The prevention may also be partial, such that die occurrence of the disease or pathological cells in a subject is less than that which would have occurred without the present invention.

The term “inhibits” as used herein with reference to a viral infection refers to a decrease in viral transmission, decrease in virus binding to a cellular target or decrease in disease. For example, the peptides of the present invention are used to inhibit viral transmission, syncytia formation, and disease associated with the virus (e.g., coronavirus). A peptide of the invention can be screened by many assays, known in the art and described herein, to determine whether the peptide inhibits the virus (e.g., infectivity, transmission, etc.). For example, a peptide of the invention can be assayed for its ability to inhibit viral infectivity by contacting a cell culture that is incubated with the vims with a test peptide. The peptide is found to inhibit viral infectivity when viral infectivity is 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%), 20%, 10%, 5% or less in the presence of the test peptide as compared to a suitable control (population of cells not subjected to inhibitor or exposed to an inactive control peptide).

The term ‘‘inhibit transmission,” as used herein, refers to the peptide’s ability to inhibit viral infection of cells, via, for example, cell-cell fusion or free virus infection. Such infection may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving a viral structure and a cellular structure.

The term “inhibiting syncytia formation,” as used herein, refers to a peptide’s ability to inhibit or reduce the level of membrane fusion events between two cells, one of which expresses the Spike protein while the other expresses the cognate receptor.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

The terms “effective amount,” or “effective dose” refers to that amount of a peptide to produce the intended pharmacological, therapeutic or preventive result. The pharmacologically effective amount results in die amelioration of one or more symptoms of a viral disorder, or prevents the advancement of a viral disease, or causes the regression of the disease or decreases viral transmission. For example, a therapeutically effective amount preferably refers to the amount of a therapeutic peptide that decreases the rate of transmission, decreases viral load, or decreases the number of infected cells, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90%, at least 95%, or more.

The term "amine acid’’ refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D- and L-isomers of the 20 common naturally occurring amino acids found in peptides (e.g., A, R. N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letter abbreviations)) as well as the naturally occurring and non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.

A ‘‘conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Other conserved amino acid substitutions can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide.

The term “amino acid side chain” refers to a moiety attached to the a-carbon in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e g., an amino acid metabolite) or those that are made synthetically (e.g., an alpha di -substituted amino acid).

The term polypeptide or peptide encompasses two or more naturally occurring or synthetic amino acids linked by a covalent bond (e.g., an amide bond). Peptides as described herein include full length proteins (e g., fully processed proteins) as well as shorter amino acids sequences (e.g., fragments of naturally occurring proteins or synthetic peptide fragments). As used herein, the terms “identity” or “percent identity,” refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two peptides. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two peptides is occupied by serine, then they are identical at that position. The identity between two sequences is a direct function of the number of matching or identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length), of the positions in two peptide sequences are identical, then the two sequences are 50% identical; if 90% of the positions, e g., 9 of 10 are matched, the two sequences share 90% sequence identity. The identity between two sequences is a direct function of the number of matching or identical positions. Thus, if a portion of the reference sequence is deleted in a particular peptide, that deleted section is not counted for purposes of calculating sequence identity. Identity is often measured using sequence analysis software e.g., BLASTN or BLAST? (available at the world wide web site (“www”) of the National Center for Biotechnology Information of the National Institutes of Health of the U.S. government, in the “Blast” directors- (“/BLAS T/”). The default parameters for comparing two sequences (e.g., “Blasf’-ing two sequences against each other), by BLASTN (for nucleotide sequences) are rew-ard for match=l, penalty for mismatch=-2, open gap=5, extension gap=2. When using BLAST? for protein sequences, the default, parameters are reward for match ^:::0, penalty for mismatched, open gap=l l, and extension gap=T. Additional, computer programs for determining identity are known in the art.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

The term “standard” or “control” as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound (e.g., peptide), or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or peptide on a parameter or function. Standard can also refer to an “internal standard”, such as an agent or peptide which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley- Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859- 1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981.

Any compositions, peptides or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

As used herein, the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof, are intended to be inclusive similar to the term “comprising.”

The terms “comprises,” “comprising,” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including” and the like. As used herein, “including” or “includes” or the like means including, without limitation.

Peptides/niodifications

Described herein are peptides which exhibit antiviral activity. It is believed that the peptides exhibit antiviral activity via their ability to inhibit virus-cell fusion by interfering with viral coat proteins. The peptides of the invention may include one or more modification, including N-terminal blocking agents, internal amino acid substitutions, stapling (including hydrocarbon stapling), and C-terrninal modifications with various lipids (cholesterol, tocopherol, or palmitate).

While not limited to any theory of operation, the peptides of the invention are potent inhibitors of viral infection and fusion, likely by their ability to form complexes with viral glycoproteins and interfere with the fusogenic process, e g., during the structural transition of the viral protein from the native structure to the fusogenic state. While not being bound by theory, it is believed the peptides gain access to their respective binding sites on the viral glycoprotein and exert a disruptive influence which inhibits fusion of the virus with the cell. In a first aspect, the invention is directed to peptides having 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100 identity to the sequences in Figure 10-14, including the following sequence. SIDQINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 1).

The amino acid substitutions may be of a conserved or non-conserved nature. The substitutions and/or insertions can also be one or more non-natural amino acids, such as norleucine, citrulline, 4-methyl phenylalanine, ornithine, and/or L-propargylalanine. Such substitutions/insertions can increase helicity of the peptide and/or blocking activity.

Amino acid insertions may consist of single amino acid residues or stretches of residues. The insertions may be made at the carboxy or amino terminal end of the peptides, as well as at a position internal to the peptide. Such insertions will generally range from 2 to 15 amino acids in length. It is contemplated that insertions made at either the carboxy or amino terminus of the peptide of interest may be of a broader size range, with about 2 to about 50 amino acids. One or more insertions may be introduced into the peptides described herein as long as such insertions result in peptides that exhibit anti-fusogenic or antiviral activity.

Deletions of the peptides are also within the scope of the invention. Such deletions consist of the removal of one or more amino acids from the peptide sequence, with the lower limit length of the resulting peptide sequence being 4 to 6 amino acids. Such deletions may involve a single contiguous or greater than one discrete portion of the peptide sequences. One or more deletions may be introduced into the peptides, as long as such deletions result in peptides which still exhibit anti -fusogenic or antiviral activity.

The peptide could also be stabilized with at least one staple, such as a hydrocarbon staple (hydrocarbon stapling is described in U.S. Patent Publication No. 2005/0250680, which is herein incorporated by reference in its entirety). The alpha helix heptad repeat domain is stabilized with at least one hydrocarbon staple, but could include two, three or more hydrocarbon staples. The inclusion of multiple hydrocarbon staples is particularly suited for alpha helical peptides that are 20 or more amino acids in length. In fact, the inclusion of two more hydrocarbon staples, as shown herein, may provide for exceptional structural, acid and thermal stability of the peptides, yielding bioactive peptides with strikingly enhanced pharmacologic properties in vivo.

As used herein, the term “hydrocarbon stapling (cross-link or tether refers to a process for stably cross-linking a peptide via at least two amino acids that helps to conformationally bestow the native secondary structure of that peptide. This stability increases resistance of the peptide to proteolytic cleavage and heat, and also may increase hydrophobicity. Accordingly, hydrocarbon stapled (cross-linked) peptides described herein can have improved biological activity relative to a corresponding non-hydrocarbon stapled (uncrosslinked) peptide. In some aspects, the cross-linked peptide can be used to inhibit virus entry into a cell. The cross-linked peptides described herein can be used therapeutically, e.g., to treat coronavirus infection.

The use of multiple cross-links (e.g., 2, 3, 4 or more) is also contemplated. The use of multiple cross-links is can further stabilize the peptide, especially with increasing peptide length. Thus, the invention encompasses the incorporation of more than one crosslink within the peptide sequence to either further stabilize the sequence or facilitate the structural stabilization, proteolytic resistance, acid stability, thermal stability, and biological activity enhancement of longer peptide stretches.

While hydrocarbon tethers have been described, others are also envisioned. For example, the tether can include one or more of an ether, thioether, ester, amine, or amide moiety. In some cases, a naturally occurring amino acid side chain can be incorporated into the tether. For example, a tether can be coupled with a functional group such as the hydroxyl in serine, die thiol in cysteine, the primary amine in lysine, die acid in aspartate or glutamate, or the amide in asparagine or glutamine. Accordingly, it is possible to create a tether using naturally occurring amino acids rather than using a tether that is made by coupling two non- naturady occurring amino acids. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid.

The term '■‘stable’'’ or “■stabilized,'” as used herein with reference to a peptide, refers to peptides which have been hydrocarbon-stapled to maintain their structure and/or improve protease resistance and/or improve acid stability and/or improve thermal stability.

As can be appreciated by the skilled artisan, methods of synthesizing the peptides described herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired peptides. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the peptides described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wdley and Sons (1991 ); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. The pepddes of this invention can be made by chemical synthesis methods, winch are well known to the ordinarily skilled artisan and described herein. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the alpha-NH2 protected by either t-Boc or F-moc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431, the AAPPTEC multichannel synthesizer APEX 396 or Biotage Syro II.

One manner of making of the peptides is using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, w⁷hich is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.

Longer peptides could be made by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. Furthermore, the host cell is engineered so as to be able to incorporate the nonnatural amino acids for the hydrocarbon staple. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. See Liu et al. Proc. Nat. Acad. Sci (USA), 94: 10092-10097 (1997). The peptide is purified and characterized by standard methods.

The peptides can be made in a high-throughput, combinatorial fashion, e.g., using a high-throughput polychannel combinatorial synthesizer.

The peptides can be further modified with, for example, N-terminal blocking agents, and C-terminal modifications with various lipids. For example, the N-terminus can be capped with by addition of 4-phenyl butyruc acid (4-phenylbutanoic acid; PBA), PPA, 4- (naphthalen-2-yl)butanoic acid (NBA), dimethylproline or 3-(naphthalen-l -yl)propanoic acid (NPA). Such blocking agents can improve binding and confer increased stability to the peptide.

The C-terminws of the peptides described herein can spacers, linkers and/or a lipid moiety, for example, PEG, such as PEG4 as spacer, and an optional linker, such as GSGSG, including for example, GSGSG-PEG4-Lys(C16). Other C-terminal modifications can include for example, a bifunctional linker GSGSG-Lys-PEG4-Lys-(Mal-Ci4)2 that provides for addition of two CM lipid chains. It is believed that lipidation likely positions the peptide at the membrane where the fusion process takes place providing the peptide with an increased opportunity to interfere with six helix bundle formation.

GSGSG-PEG4-Lys(C16)

GSGSG-Lys[PEG4-Lys-(Mal-C14)2]

(SEQ ID NO: 2 and 19).

/\ny of the peptides described herein can be present in a composition (e.g., pharmaceutical composition) or kit. In some embodiments of the invention, the composition or kit comprises two or more peptides.

Viruses

The peptides described herein are active against coronaviruses. Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Mild illnesses in humans include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS, and COVID-19. In cows and pigs, they cause diarrhea, while in mice they cause hepatitis and encephal omy eliti s .

Coronaviruses constitute the subfamily Orthocoronavirmae, in the family Coronaviridae, order Nidovirales, and realm Riboviria (“ICTV Taxonomy history': Orthocoronavirinae". International Committee on Taxonomy of Viruses (ICTV); Fan Y, et al. (March 2019). "Bat Coronaviruses in China". Viruses. 11 (3): 210). They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry (Cherry J, et ah (2017). Feigin and Cherry's Textbook of Pediatric Infectious Diseases. Elsevier Health Sciences, p. PT6615). The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, one of the largest among RNA viruses (Woo PC, et al, (August. 2010). “Coronavirus genomics and bioinformatics analysis". Viruses. 2 (8): 1804- 20). They have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent of the solar corona, from which their name derives (Almeida JD, et ah (November 1968). "Virology: Coronaviruses". Nature. 220 (5168): 650).

The pan-coronavirus activity of the peptide fusion inhibitors was tested against pseudotyped virions expressing the Spike from different coronaviruses. The peptides described herein demonstrated potency and broad activity against each of the five coronaviruses tested: SARS-CoV-2 (severe acme respiratory syndrome coronavirus 2; aka COVID-19), SARS-CoV-1 (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome-related coronavirus), NL63 (Human coronavirus NL63), and 229E (Human coronavirus 229E) (other known human coronaviruses, to which the peptides described herein also find use, include, Human coronavirus OC43 and Human coronavirus HKU 1). There was no evident toxicity at concentrations less than 10 micromolar.

’The peptide inhibitors described herein can be developed as an inhaled and/or subcutaneous therapeutic for treatment of early COVID-19 infection, or other coronavirus infections, prior to patients requiring hospitalization (currently, the field lacks highly active small molecule antivirals that can be deployed outside of the hospital setting).

The peptides described herein can also be used to prevent coronavirus infection. Further, because of the high conservation of the HR1 and HR2 domains in the coronavirus family, these peptides will exhibit pan-Coronavirus activity including against family members that have not yet established zoonotic infections in humans.

Thus, the peptides described herein are active against infections including SARS- CoV-2, MERS, all of the seasonal coronaviruses and new/novel coronavirus outbreaks.

Methods for evaluating the ability of a peptide to inhibit membrane fusion and/or exhibit anti-viral activity both in vitro and in vivo are provided are well known to those with ordinary skill in the art.

For example, the antiviral activity exhibited by the peptides described herein can be measured, for example, by m vitro assays, such as those described herein and known by those of ordinary skill in the art, which can test the peptides' ability to inhibit syncytia formation, or their ability to inhibit infection (Madani, N., et al., Journal of Virology, 2007. 81(2): p. 532- 538; Si, Z. H., M. Cayabyab, and J. Sodroski, Journal of' Virology, 2001. 75(9): p. 4208- 4218; Si, Z. H., et al., PNAS USA, 2004. 101(14): p. 5036-5041).

Using these assays, such parameters as the relative antiviral activity of the peptides exhibit against a given strain of virus and/or the strain specific inhibitory activity of the pepdde can be determined.

For example, peptides described herein have been tested in three primary assays including syncytia formation, fusion of SARS-CoV-2 Spike pseudotyped virions (VSV backbone) and live SAR.S-CoV-2 infection of various targets cells expressing ACE2 receptors. All peptides have been tested in cell viability assays and at concentrations less than about 10 uM, no toxicity was observed.

Pharmaceutical Compositions and Administration

As used herein, the peptides described herein, are defined to include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative or prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a peptide of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a peptide of this invention. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the peptides of this invention when such peptides are administered to a mammal or which enhance delivery of the parent peptide to a biological compartment (e.g., lymphatic system) relative to the parent species. Prodrugs include derivatives where a group which enhances aqueous solubility or active transport through the gut membrane is appended to the structure described herein.

The peptides of the invention can, for example, be administered by inhalation, injection, intravenously, sub-dennally, intraperitoneally, intramuscularly, or subcutaneously; or buccally, nasally, transmucosally, intravaginally, cervically, topically, in an ophthalmic preparation, with a dosage ranging from about 0.001 to about 100 mg/kg of body weight, including from 0.5-10 mg/kg of body weight. The methods herein contemplate administration of an effective amount of peptide or peptide composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vaty depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 1% to about 95% active peptide (w/w). Alternatively, such preparations contain from about 20% to about 80% active peptide. Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific peptide employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Upon improvement of a patient's condition or prevention of infection, a maintenance dose of a peptide described herein may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise a peptide described herein or a pharmaceutically acceptable salt thereof; an optional additional agent including for example, a steroid, and any pharmaceutically acceptable carrier, adjuvant, or vehicle.

The term "'pharmaceutically acceptable carrier or adjuvant’’ refers to a carrier or adjuvant that may be administered to a patient, together with a peptide of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient, to deliver a therapeutic amount of the peptide.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a- tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as ’Tween® or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, vcater, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wmol fat. Cyclodextrins such as alpha-, beta-, and gammacyclodextrin, may also be advantageously used to enhance delivery' of peptides described herein.

The pharmaceutical compositions of this invention may be administered enterally for example by parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The pharmaceutical compositions of this invention may contain any conventional nomtoxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated peptide or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

Examples of dosage forms include, but are not limited to: dispersions, suppositories, ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art. using suitable dispersing or wetting agents (such as, for example, ’Tween® 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The pharmaceutical compositions of the invention may be administered topically. The pharmaceutical composition will be formulated with a suitable ointment containing the active components suspended or dissolved in a earner. Carriers for topical administration of the peptides of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene poly oxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active peptide suspended or dissolved in a carrier. Topical transdermal patches and iontophoretic administration are also included in this invention.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

When the compositions of this invention comprise a combination of a peptide of the formulae described herein and one or more additional therapeutic or prophylactic agents, both the peptide and the additional agent should be present at dosage levels of between about I to 100%, including between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately, as part of a multiple dose regimen, from the peptides of this invention. Alternatively, those agents may be part of a single dosage form, mixed together with the peptides of this invention in a single composition.

Effective dosages of the peptides described herein to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity.

A therapeutically effective dose refers to that amount of the peptide sufficient to result in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such peptides can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Peptides which exhibit large therapeutic indices are particularly useful. The data obtained from these cell culture assays, and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such pepddes lies for example within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any peptide used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test peptide which achieves a half-maximal inhibition of the fusogenic event, such as a half-maximal inhibition of viral infection relative to the amount of the event in the absence of the test peptide) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography (HPLC) or mass spectrometry (MS).

Kits

The present invention also encompasses a finished packaged and labeled pharmaceutical product. This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed. The pharmaceutical product may contain, for example, a peptide of the invention in a unit dosage form in a first container, and in a second container, sterile water for injection. Alternatively, the unit dosage form may be a solid suitable for, intranasal, intravaginal, cervical ring, or topical delivery.

In a specific embodiment, the unit dosage form is suitable for intravenous, intramuscular, intranasal, intravaginal, cervical, topical, inhalation or subcutaneous delivery. Thus, the invention encompasses solutions, solids, foams, aerosol, gels, preferably sterile, suitable for each delivery route.

As with any pharmaceutical product, the packaging material and container are designed to protect the stability of the product during storage and shipment. Further, the products of the invention include instructions for use or other informational material that advise the physician, technician, or patient on how to appropriately prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures (e.g., detection and quantitation of infection), and other monitoring information.

Specifically, the invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within said packaging material, wherein said pharmaceutical agent comprises a peptide of the invention, and wherein said packaging material includes instruction means which indicate that said peptide can be used to prevent, manage, treat, and/or ameliorate one or more symptoms associated with a viral disease by administering specific doses and using specific dosing regimens as described herein.

The following examples are provided merely as illustrative of various aspects of the invention and shall not be construed to limit the invention in any way.

Examples

Example 1 - In vitro

SARS-CoV-2 Syncytia Assay

All peptides were initially adjusted to 20pM and then serially diluted 1 :5 in DMEM before testing in the syncytia assay. 293T cells constitutively expressing SARS-CoV-2 spike protein and green fluorescent protein (GFP) cells were plated in 96 well plates and mixed with the peptides. After incubation for 30 minutes at room temperature, Calu-6 cells constitutively expressing ACE2 and red fluorescent protein (RFP) cells were added to the mixtures and incubated overnight at 37 °C. The 96 well plates were then analyzed by automated microcopy (ArrayScan Instrument, Thermo Scientific) to determine the number of multinucleated large syncytia formed expressing both GFP and RFP fluorescence) for calculating the ICso values. (See Figure 2 for results; see Figures 10-14 for amino acid sequence of CG126, CG158, CG159, CG160, CG161, CG162, CGM24, CG157, CGM17, CGM20.)

SARS-CoV-2 Psuedotyped Virion Assay

Inhibition of infection detected in vesicular stomatitis virus (VSV) virions pseudotyped with SARS-CoV-2 Spike

G protein-deficient vesicular stomatitis virus (VSVAG) containing an integrated GFP reporter gene was pseudotyped with SARS-CoV-2 Spike by transfection of expression vectors for each into 293T cells. The SARS-CoV-2 Spike pseudotyped virions harvested from the supernatant of the 293 T cells was assayed for titer and then aliquots mixed for 30 minutes with peptides. The mixtures were then used to infect Calu-6 cells constitutively expressing ACE2 plated in 96 well plates and incubated overnight. Levels of infection were measured by Renilla luciferase quantification (Renilla Luciferase Assay System, Promega). Other 4 coronavirus (SARS-CoV-1, MERS, NL63, and 229E) pseudotyped virions were produced by the same method as the SARSCoV-2 pseudotyped virion and used for testing pancoronavirus activity of peptides. A scrambled peptide with EK1C4 C-terminal lipidation was used as a negative control, and EK1C4 was employed as a positive control in these studies. (See Figure 3 for results.) Live SARS CoV-2 Viral Infection Assay

Live virus experiments were performed with Calu-6 epithelial cells (ATCC HTB56) stably expressing human Angiotensin Converting Enzyme 2 (hACE2) (OriGene, RC08442) as target cells. Viral stocks were prepared using an infectious clone of SARS CoV-2, expressing a Nano-luciferase reporter, (icSARS-C0V-2-Nluc, Xie, X et al. 2020). For each experiment, 24 hours before administration of virus inoculum or virus/ peptide cocktail, 5xl0⁴ Calu-6-hACE2 cells were plated per well of a 96 well flat bottom tissue culture treated plate in 200 uLs of complete DMEM10. At the time of the experiment, test peptides were diluted in DMEM10 to 6 concentrations in triplicate as follows; 20uM, 4uM, 800nM, 160nM, 32nM and 6.4nM. A virus inoculum of 10⁴TCID50 was prepared in DMEM10 and was incubated with the peptides or media alone for 30 minutes at room temperature. Upon completion of the virus and peptide co-incubation, the Calu6 hACE2 cells were washed one time with sterile IxPBS then the peptide-virus cocktail or virus-media prep were added to the cells in triplicate. The plates were then incubated at 37°C and 5% CO2 for 24 hours. At 24 hours post infection the cells were washed once with 1 *PBS then lOOpLs of fresh DMEM10 was add per well followed by lOOpLs of Nano-Gio Luciferase substrate/buffer cocktail (Promega, N1110). The plates were incubated for 5 minutes at 37°C, and 5% CO2 then read on a GloMax Discover luminometer. Results were analyzed by Graph Pad Prism software version 9 (Graph Pad). All experiments were performed in the Gladstone ABSL3, adhering to BSL3 protocols. Results are depicted in Figure 4.

Cell Viability Assay

All peptides were adjusted to 20pM and then serially diluted 1 :5 in DMEM before testing in the cell viability assay. The culture medium with peptides was then applied on Calu-6 cells constitutively expressing ACE2 plated in 96 well plates and incubated overnight. Levels of cell viability were tested by cellular ATP measurement (Viral ToxGlo Assay, Promega). Results are depicted in Figure 5. Pan-Coronavirus assay

Testing pan-Coronavirus activity of the CGM23 peptide fusion inhibitor For testing pan-Coronavirus activity of peptides, in addition to SARS-CoV-2, four other Coronavirus (SARS-CoV-1, MERS, NL63, and 229E) pseudotyped virions were produced by the same method as the SARS-CoV-2 pseudotyped virion and aliquots mixed with peptides. Calu-6 cells constitutively expressing ACE2 were infected with SARS-CoV-1 and NL63 pseudotyped virions, VeroE6 cells were infected with MERS pseudotyped virions, and A549 cells were infected with 229E pseudotyped virions, and then levels of infection were measured by Renilla luciferase quantification.

Pseudotyped virions expressing the Spike proteins from the 229E and NL63 seasonal coronaviruses and Spike proteins from SARS-CoV-1 (SARS) and MERS-CoV (MERS) were tested in the presence of various concentrations of CGM23 or EK1C4 (as described by Lu Lu and colleagues). Target cells were engineered with the appropriate receptor for these different Spike proteins. Note lower IC50 values for CGM23 for inhibition of 229E Spike, SARS Spike, and MERS Spike compared to EK1C4 and markedly greater inhibition of NL63 Spike fusion by CGM23 compared EK1C4 (Figure 6).

Stability

Also provided is HPLC data showing that CGM23 is more stable than EK1C4 to major lung proteases (Fig. 8A-8N). For example, for neutrophil elastase - CGM23 clearly outperforms EK1C4. Note unexpected, improved performance of CGM23 (green trace in all) versus EK1C4 (red trace) against a whole variety of proteases (human neutrophil elastase, trypsin, cathepsin B, D, K and human plasma; after 0, 3, and 8 hours of exposure).

Example 2 - In vivo

To assess candidate peptide efficacy in vivo, heterozygous K18-hACE c57BL/6j mice (strain: 2B6.Cg-Tg(K18-ACE2) 2Plimn/J) were bred. At the time of the experiment, 5- to 8- week-old animals were grouped into test cohorts and transferred to the laboratory. For the infection and peptide treatment, the animals were weighed, and baseline body temperature was recorded. After weighing they were anesthetized using 150mg/kg ketamine mixed with 10 mg/kg xylazine via intra-peritoneal (I P.) injection. Anesthetized animals were treated with intranasal administration of a virus and peptide cocktail consisting of an inoculum of 100xTCID50 of SARS CoV-2 Nanoluc virus (20uL) mixed with 12.5ug of peptide (20uL), to a final volume of 40uL. On days 2 and 4 post treatment the animals were weighed, body temperatures were recorded, then they were euthanized by CO2 asphyxiation followed by cervical dislocation. Animals whose tissues were to be used for histology were anesthetized and perfused with IxPBS followed by 4% paraformaldehyde. For all other animals not to be used for histology, brain and lungs were harvested, then roughly chopped and processed for homogenates in Benchmark-prefilled zirconium bead tubes. Homogenates were assessed for infection by direct luciferase analysis. For direct luciferase assessment, lOOuL of homogenate were mixed with lOOuL of Promega Nanoluc luciferase reagent then analyzed on a Promega Gio-Max Discovery instrument. Homogenates were also tested for viral outgrowth on Calu-6- ACE2 cells. Briefly, Calu-6-ACE2 cells were plated on 12 well plates at a concentration of 2xl0⁵ cells per well. Homogenates were added to the Calu-6-ACE2 cells in a 4 step dilution series of 10¹, 10⁴, 10⁵, and 10⁶. The Homogenates were incubated on the cells for 2 to 4 hours at 37°C and 5% CO2 with frequent rocking. Homogenates were removed and normal culture media was added to the cells. The cells were incubated for 48 hours then treated with Promega Nanoluc luciferase reagent followed by analysis on a Promega Gio-Max Discovery instrument. Homogenates were additionally assessed by standard qRT-PCR for viral gene expression. (FIG. 7A).

Results

SARS-CoV-2 was used to infect human ACE2 expressing mice in the presence of a control scrambled peptide, CGM23, or EK1C4. CGM23 provided a potent antiviral effect compared to controls (scrambled peptide) and better effectiveness of compared to EK1C4. Mice treated with the scrambled peptide readily became infected displaying abundant expression of Spike protein and influx of many macrophages identified by MAC -2 staining. Note lack of these finding in animals treated with CGM23 and better effects with CGM23 than with EK1C4 (FIGs. 7B-7C).

Example 3

Data demonstrating that the peptide fusion inhibitor acts effectively to prevent SARS- CoV-2 infection and/or reinfection (one or more infections after an initial infection). In vivo CGM23 challenge (FIGs. 15 and 16).

All patents, patent applications, GenBank numbers, and published references cited herein are hereby incorporated by reference in their entirety as if they were incorporated individually. While this invention has been particularly shown and described with references to certain embodiments/aspects thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A peptide comprising the following sequence:

R¹ - X1IX3X4INASVVNIQKEIDRLNEVAKNLNESLIDLQEL - R² wherein Xi and X3 are independently S or D and X4 is G or Q, or sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto wherein R¹ comprises a non-amino acid end group; and wherein is R² comprises a lipid moiety.

2. The peptide of claim 1, wherein the lipid moiety is selected from the group consisting of cholesterol, tocopherol, pregnenolone and palmitate.

3. The peptide of claim 1 or 2, wherein the non-amino acid end group is selected from the group consisting of Ac, PBA, PPA, 4-(naphthalen-2-yl)butanoic acid (NBA)and 3- (naphthalen-l-yl)propanoic acid (NPA).

4. The peptide of any one of claims 1 to 3, wherein one or more of the amino acids are D-amino acids or the sequence includes one or more non-natural amino acids.

5. The peptide of any one of claims 1 to 4, wherein the peptide further comprises a spacer.

6. The peptide of claim 4, wherein the spacer comprises a polyethylene glycol (PEG).

7. The peptide of claim 6, wherein the spacer comprises PEG4 or PEG3.

8. The peptide of any one of claims 1-7, wherein the C-terminal comprises either one of:

GSGSG-PEG4-Lys(C16)

GSGSG-Lys[PEG4-Lys-(Mal-C14)2]

SEQ ID NO: 2 and 19).

9. The peptide of claim 8, wherein the C-terminus comprises GSGSG-PEG4-Lys(C16) (SEQ ID NO: 2).

10. A peptide comprising or consisting essentially of any one of the peptides in Figures 1 to 4 or a sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto, wherein the peptide is not EK1 or EK1C4.

11. The peptide of any one of claims 1 to 10, wherein the peptide has the following sequence PBA-SIDQINASVVNIQKEIDRLNEVAKNLNESLIDLQEL-GSGSG-PEG3- Lys(C16) (CGM23; SEQ ID NO: 1) or a sequence having at least 80%, 90%, 85%, 95% or less than 100% identity thereto.

12. The peptide of any one of claim 1 to 11, further comprising at least one peptide staple.

13. A composition comprising a peptide comprising of any one of claims 1 to 12 or a sequence having at least 80%, 90%, 85%, 95% or less than 100% identify thereto or combination thereof and a pharmaceutically acceptable carrier.

14. A method to prevent or treat a coronavirus infection comprising administering to a subject in need thereof an amount of the peptide of any one of claims 1 to 12 or the composition of claim 13 effective to prevent or treat said coronavirus infection.

15. The method of claim 14, wherein coronavirus infection is prevented.

16. The method of claim 15, wherein the infection is a reinfection.

17. A method to inhibit transmission of a coronavirus to an animal cel i comprising contacting said animal cells with an effective amount of the peptide of any one of claims 1 to 12 or the composition of claim 13 so that the coronavirus is inhibited from infecting the cell.

18. A method to inactivate a coronavirus comprising contacting the coronavirus with an effective dose of the peptide of any one of claims 1 to 12 or the composition of claim 13 so that the coronavirus is rendered inactive (non-infectious).

19. The method of any of claims 14 tol8, wherein the peptide or composition is administered by inhalation.

20. A kit comprising the peptide of any one of claims 1 to 12 and/or the composition of claim 13 and instructions for use.