Methods for making peptide conjugates and the product made therefrom

Abstract

The application provides a method to synthesize peptide conjugates via automated flow chemistry, wherein the method comprises synthesizing a covalent framework connecting to different modules, wherein the covalent framework comprises at least one diaminoaliphatic acid, preferably lysine; as well as the product made therefrom, e.g., anti¬ viral peptide conjugates.

Description

[0001] METHODS FOR MAKING PEPTIDE CONJUGATES AND THE PRODUCT MADE

[0002] THEREFROM

[0003] RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 470,257, filed on June 1, 2023. The entire teachings of the above application are incorporated herein by reference.

[0005] BACKGROUND OF THE INVENTION

[0006] Respiratory infections caused by a variety of viruses can lead to illnesses ranging from mild colds to severe pneumonia, particularly in vulnerable populations such as the elderly, infants, and immunocompromised individuals. Current medications mostly offer symptom relief, and there are few drugs that alter the course of the disease. Paramyxoviruses, a family of negative-sense RNA viruses, include several respiratory pathogens such as the human parainfluenza viruses (HPIVs), measles virus, and mumps virus, which can cause a range of respiratory illnesses from mild to severe, such as croup, pneumonia, and bronchiolitis. In addition, emerging paramyxoviruses such as Nipah virus and Hendra virus can cause severe respiratory and neurological disease in humans. Respiratory syncytial virus (RSV) is a member of the paramyxovirus family and particularly the leading cause of hospitalization in infants under the age of one year in the United States. Although most RSV infections are self-limiting, severe cases can be life-threatening, and there are currently no specific antiviral therapies available for RSV. Several approaches have been developed to prevent and treat RSV infections. For example, palivizumab, a monoclonal antibody, is used to prevent severe RSV infections in high-risk infants, and inhaled ribavirin has been used to treat severe RSV infections in immunocompromised patients. However, these treatments are limited in their effectiveness, and there is a need for improved therapies that can prevent and treat respiratory virus infections in a wider range of patients.

[0007] Furthermore, respiratory viruses can mutate rapidly, leading to the emergence of new strains that may evade existing treatments and vaccines. Thus, there is an urgent need to develop new therapeutic strategies that can keep pace with the evolving respiratory viruses, including RSV and emerging zoonotic viruses such as SARS-CoV, MERS-CoV, and SARS- CoV-2.

[0008] On the other hand, peptide conjugates are a powerful therapeutic approach in the fight against viral infections, offering specificity, stability, and efficient delivery. Solid-state synthesis is a well-established method for producing peptides through the sequential addition of protected amino acids. However, this technique may suffer from side reactions and aggregation, leading to decreased purity and longer synthesis times. To meet the challenges posed by evolving viruses and enhance accessibility, there is a need for novel methods that enable more efficient production of antiviral peptide conjugates. Such advancements will not only benefit the field of antiviral therapeutics but also broaden the possibilities for developing peptide conjugates in a more accessible manner overall.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides a novel method to synthesize peptide conjugates efficiently and precisely. It is based on the discovery that by employing automated flow chemistry to conjugate peptides, preferably derived from viral envelope proteins (such as fusion proteins or spike proteins, preferably HRC peptides), and / or other competitive ligands, to hydrophobic moieties such as lipid molecules, preferably cholesterol, new peptide conjugates can be synthesized on demand. The method offers a programable rapid response platform for the on-demand synthesis of a variety of useful peptide conjugates, e.g., potent anti-viral therapeutics. The platform includes a covalent framework that is designed to allow covalent connections to different modules (essentially a peptide and a membrane anchoring unit) via automated flow chemistry. The covalent framework comprises at least one diaminoaliphatic acid. Preferably, the covalent framework comprises a plurality of diaminoaliphatic acids, optional linkers, and / or optional spacers. Preferably, the diaminoaliphatic acid is lysine.

[0011] In some embodiments, the platform includes four modules: virus fusion peptide inhibitors, membrane anchoring units (such as lipid molecules), receptor targeting peptides, and spike binding peptides. Covalent combinations with at least two modules include a fusion peptide inhibitor conjugated to a membrane anchoring unit, resulting in an effective anti-virus compound. Modules can be connected through the covalent framework consisting of at least one lysine. Other modules consist of connections comprised of peptide linkers, polyethylene glycol (PEG) derivatives, amides, esters. These connections can be quickly made using automated flow chemistry.

[0012] The present invention also provides compounds, compositions, and methods of use thereof for treating or preventing an infection, such as compounds identified through the platform. In some embodiments, the invention provides a compound comprising one, two, three or more HRC peptides of a viral fusion protein (also called a spike protein) conjugated to a membrane anchoring moiety (such as a hydrophobic moiety) via a covalent framework that comprising at least one diaminoaliphatic acid, preferably lysine, and optionally spacers. The hydrophobic moiety can be a membrane integrating ligand, such as a sterol (e.g., cholesterol, sitosterol, and campesterol), a sphingolipid, a glycolipid, a glycerophospholipid. The HRC sequences are preferably derived from a paramyxovirus fusion protein (e.g., an RSV fusion protein, or a HPIV fusion protein), or a coronavirus spike protein. The HRC peptides of the invention inhibit viral fusion.

[0013] The invention includes compositions for the delivery of compounds of the invention, such as pulmonary or nasal delivery. The invention also provides a method of treating or preventing a viral infection, including for example a paramyxovirus infection and a SARS- CoV-2 (COVID-19) infection, in a subject in need thereof comprising administering an effective amount of a compound of the invention. The invention further provides methods of producing the compositions of the invention.

[0014] In some embodiments, anti-viral compounds that are useful for treating paramyxovirus include a compound of the invention having a formula as shown in Figure 1 A, Figure IB, Figure 1C, or Figure ID.

[0015] In some embodiments, anti-viral compounds that are useful for treating coronavirus include a compound of the invention having a formula as shown in Figure IE.

[0016] The present invention is intended to provide the efficient production of libraries of molecules. The platform and the libraries that can be produced offer the ability to interrogate variants to identify those linkers, spacers, sequences, and targeting moieties with improved antiviral activity against a variety of viruses and the variants thereof, e.g., SARS-CoV2 and its mutations, including but not limited to the alpha, beta, gamma, delta, and omicron variants.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0019] Figures 1 A through IE depict exemplary compounds of the instant invention. Figure 2 provides a schematic illustration of a system for performing peptide conjugate synthesis, according to one set of embodiments.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Compounds

[0022] The invention provides a programable rapid response platform for the on-demand synthesis of a diverse array of peptide conjugates that can hold potential for applications in prophylaxis, diagnosis, and therapy. Preferably, the peptide conjugates are therapeutic peptide conjugates, such as anti-viral peptide conjugates. The platform comprises a therapeutic peptide, a membrane anchoring moiety, and a multimeric core (B) which covalently links the peptide inhibitor to the membrane anchoring moiety. Preferably, the therapeutic peptide is a fusion peptide inhibitor. The platform optionally further comprises a spike binding peptide. The platform optionally further comprises a target peptide, such as a receptor binding domain (RBD) targeting peptide. In some embodiments, the term “compound” is used to refer to the peptide conjugates as a matter of simplicity.

[0023] Compounds of the invention can be characterized by the following General Formula: (Peptide-Linker)n-B-Membrane Anchoring Moiety wherein the Peptide is a short amino acid sequence, preferably a therapeutic peptide, the Linker is optional, B is a multimeric core which covalently links each peptide moiety to the hydrophobic moiety and comprises one or more diaminoaliphatic acids, preferably lysines (AKA, Lys or K), and optionally spacers, and n is an integer selected from 1, 2, 3 or more. The term “diaminoaliphatic acid” refers to a molecule that includes a carboxylic acid and two amine functional groups, each function group covalently linked to an aliphatic moiety. In a preferred embodiment, the therapeutic peptide is a peptide inhibitor, preferably a fusion peptide inhibitor. The term “therapeutic peptide” refers to a short amino acid sequence, consisting of 4-100 (preferably 6-80, or 9-50) amino acid residues in length, that is designed or derived from natural proteins or peptides, including wild type and variants, and has therapeutic effects.

[0024] In a preferred embodiment, the Membrane Anchoring Moiety is a membrane integrating lipid. In a preferred embodiment, the compounds are produced by chemically conjugating each moiety or a group thereof, such as an amino acid, in a sequential and continuous process. Automated flow chemistry, including the use of a solid support, is preferably used for synthesizing the compounds of the invention.

[0025] The peptide inhibitor is a short peptide molecule that can bind to a specific protein, blocking its activities, and usually consists of 4-100 amino acid residues in length. The fusion peptide inhibitor as used in this invention is preferably derived from a viral fusion protein, more preferably from C-terminus of a viral fusion protein, most preferably from C-terminus heptad repeat (HRC) region of a viral fusion protein. In some embodiments, the fusion peptide inhibitor derived from HRC region of a viral fusion protein is referred to as “HRC peptide”. The HRC region of a viral fusion protein is involved in the formation of the six- helix bundle structure that drives viral fusion. HRC peptides mimic the HRC region and can bind to the N-terminal heptad repeat (HRN) region of the fusion protein, blocking the formation of the six-helix bundle and preventing viral fusion and entry into host cells. It is noted that the acronym CHR has also been used in the literature to refer to C-terminus heptad repeat region. Therefore, “CHR peptide” or “CHR-peptide” has been used to describe the same class of peptide inhibitors as defined above, which is encompassed by “HRC peptide” as used in this application. In some embodiments, a HRC peptide is a wild type peptide derived from the HRC region of a viral fusion protein. In other embodiments, an HRC peptide is a mutant or variant peptide derived from the HRC region of a viral fusion protein, which can comprise genetic mutations that occur in nature, and / or modifications made in a laboratory setting. In some embodiments, the HRC peptide can have a length ranging from 5 to 100 amino acid residues, from 6 to 80 amino acid residues, from 8 to 60 amino acid residues, or from 10 to 50 amino acid residues. In preferred embodiments, the HRC peptide has a length of 12-40 amino acid residues. In preferred embodiments, the HRC peptide has a length of 18-39 amino acid residues. In the most preferred embodiments, the HRC peptide has a length of about 36 amino acid residues.

[0026] The optional “Linker” is defined as a bivalent moiety or group that covalently binds to a Peptide (preferably at the C or N terminus thereof) and to B.

[0027] The multimeric core B is a multivalent moiety that is designed to allow the rapid synthesis of the compounds of this invention via automated flow chemistry and can be tailored to covalently link the fusion peptide inhibitor, the membrane anchoring moiety, and other optional modules to improve anti-viral activities. B preferably comprises at least one lysine, optionally spacers, and optionally function groups such as amides, esters, and ethers. In some embodiments, B comprises 1, 2, 3, 4, 5, 6, or more lysines. In some embodiments, B comprises one lysine. In some embodiments, B comprises two lysines. In some embodiments, B comprises three lysines. In some embodiments, B comprises four lysines. In some embodiments, B comprises five lysines. In some embodiments, B comprises six lysines. When two or more lysines are present in B, the two or more lysines can be covalently linked to each other via amide bonds or via amino acid linkers such as GS linkers or polymeric linkers such as PEG linkers. In one preferable embodiment, B comprises two lysines covalently linked to each other via an amide bond. In additional preferable embodiments, B comprises one lysine. In yet additional embodiments, B comprises three lysines, covalently linked to each other via two amide bonds. In another embodiment, B comprises four lysines, covalently linked to each other via three amide bonds.

[0028] In some embodiments, B is represented by the Formula (Bl):

[0029] Formula (Bl), wherein represents covalent bonds linking to the moieties of the compound that comprise one or more fusion peptide inhibitors, a membrane anchoring moiety, optionally one or more spike binding peptide, and optionally one or more targeting peptide; m is an integer that can be 0, 1, 2, 3, 4, or more. Preferably, 4^^ represents a covalent bond to -CO- group. Preferably, m is 0, 1, or 2.

[0030] In some embodiments, the compound is represented by Formula (I):

[0031] Peptide Linker Membrane Anchoring Moiety

[0032] Peptide Linker

[0033] Formula (I),

[0034] Wherein Peptide, Linker, Membrane Anchoring Moiety, and m are as defined above, including all and preferable embodiments. In some embodiments, each Linker can be independently absent.

[0035] In some embodiments, B comprises one or more lysines, one or more spacers, and one or more additional function groups such as amides, esters, or ethers. The term “spacer”, as used herein, refers to a hydrophilic and biocompatible molecule or a chemical group that is inserted between two lysines, a lysine and a fusion peptide inhibitor, a lysine and a membrane anchoring moiety, a lysine and a spike binding peptide, or a lysine and a targeting peptide, to increase the distance between them. The spacer is used to avoid steric hindrance, reduce aggregation, improve solubility and the accessibility of the compound to the target. A common type of spacer used in the compounds is polyethylene glycol (PEG), which is a hydrophilic and biocompatible polymer that can increase the solubility and stability of the compounds in vivo. When a hydrophilic spacer PEG is present in B, a variety of PEG derivatives can be used for synthesizing the compound, such as, without limitation, amine- PEG-carboxyl acid, amine-PEG-maleimide, amine-PEG-biotin, amine-PEG-azido, azido- PEG-carboxyl acid, amine-PEG-NHS ester, maleimide-PEG-NHS ester, and biotin-PEG- NHS ester. An amine-PEG-carboxyl acid is preferably used for synthesizing the compound, preferably via automated flow chemistry, such as H2N-PEG1-40-COOH, H2N-PEG1-40- CH2COOH, or H2N-PEG1-40- CH2CH2COOH. Other hydrophilic spacers can be used, such as polyethyleneamine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl- formal) (PHF) or a carbohydrate. The length of the hydrophilic spacer can correspond to the span of the protein gap to facilitate the orientation of the HRC peptide to bind the HRN domain. B and the spacer, Linker and the spacer, or Peptide and the spacer can be joined to each other by the residue of a chemical reaction (such as an automated flow chemistry reaction).

[0036] Therefore, in some embodiments, B can be for example represented by the Formula (B2):

[0037] Formula (B2), wherein and m are as defined above, including all and preferable embodiments. p is an integer selected from 0 to 40. When a spacer PEG is present in B, in some embodiments, the compound can be therefore represented by Formula (II):

[0038] Peptide - Linker Peg - Lys - Lys — Peg - Membrane Anchoring Moiety

[0039] ^-Linker Peptide''^

[0040] Formula (II),

[0041] Wherein Peptide, Linker, Membrane Anchoring Moiety, and m are as defined above, including all and preferable embodiments.

[0042] The Membrane Anchoring Moiety is a membrane integrating lipid, such as cholesterol, a sphingolipid, sphingomyelin, a glycolipid, glycerophospholipid (such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine), ergosterol, 7- dihydrocholosterol and stigmasterol. Preferably cholesterol. Typically, B is linked directly or indirectly to a cholesterol hydroxyl group, such as 3 -OH. The Membrane Anchoring Moiety facilitates insertion of the compound of the invention into a cell membrane and can inhibit viral entry.

[0043] B can be covalently connected to a convenient position on the Membrane Anchoring Moiety. In some embodiments, connection is via a hydroxy group of the Membrane Anchoring Moiety. For example, when the Membrane Anchoring Moiety is cholesterol, B can be connected to the cholesterol by a group — C(O) — or — Ci-4 alkylene C(O) — , such as — CH2C(O)— .

[0044] In some embodiments, the Membrane Anchoring Moiety can be cholesterol. Cholesterols can include, cholesterol, 3P-amino-5-cholestene, 3P-thiol-5-cholestene, 3P- carboxymethoxyl-5-cholestene, esters of cholesterol including cholesterol hemi- succinate, salts of cholesterol including cholesterol hydrogen sulfate and cholesterol sulfate, ergosterol, esters of ergosterol including ergosterol hemi-succinate, salts of ergosterol including ergosterol hydrogen sulfate and ergosterol sulfate, lanosterol, esters of lanosterol including lanosterol hemi-succinate, salts of lanosterol including lanosterol hydrogen sulfate and lanosterol sulfate.

[0045] Examples of member-anchoring group that can be used for this invention include but are not limited to: fatty acid or long alkyl chain or 33 -cholesterylamine or 3P- cholesterylthiol or other cholesteryl analogues and derivatives, 3P-cholesterylamine type molecule enables endosome recycling of conjugate for long cell surface anchoring half-life. It can be either in monomer or dimer or trimer or oligomer format within the conjugate. The antibody binding molecule can also be either in monomer or dimer or trimer or oligomer format within the conjugate. Examples of 3P-cholesterylamine, 3P- cholesterylamine containing moiety and their derivatives that can be used for the conjugate can be found in U.S. Patent Application No. 15 / 945,741.

[0046] In some embodiments, the Membrane Anchoring Moiety can be a phospholipid. Phospholipids that can be used in this application include, without limitation, egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and phosphatidic acid (EP A); the soya counterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other phospholipids made up of ester linkages of fatty acids in the 2 and 3 of glycerol positions containing chains of 12 to 26 carbon atoms and different head groups in the I position of glycerol that include choline, glycerol, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated, and the phospholipid may be made up of fatty acids of different chain lengths and different degrees of unsaturation. In particular, the compositions of the formulations can include dipalmitoylphosphatidylcholine (DPPC), a major constituent of naturally-occurring lung surfactant. Other examples include dimyristoylphosphatidycholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatideholine (DPPQ) and dipalmitoylphosphatidylglycerol (DPPG) distearoylphosphatidylcholine (DSPQ) and distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolarnine (DOPE) and mixed phospholipids like palmitoylstearoylphosphatidyl-choline (PSPC) and palmitoylstearolphosphatidylglycerol (PSPG), and single acylated phospholipids like monool eoyl -phosphati dyl ethanol amine (MOPE) .

[0047] In some embodiments, the Membrane Anchoring Moiety can be a tocopherol. The tocopherols can include tocopherols, esters of tocopherols including tocopherol hemisuccinates, salts of tocopherols including tocopherol hydrogen sulfates and tocopherol sulfates.

[0048] In some embodiments, the Linker can have 1, 2, 3, 4, 5 or more subunits or segments. In some embodiments, the Linker comprises a subunit with one or more amino acids. The amino acids may be naturally occurring or synthetic. Thus, the Linker may comprise (Gly)n+i, (GlySerGly)n or (Gly-Pro)n where n is 1 or greater, for example, 1 to 12, 1 to 6 or 1 to 4. GlySerGly is one example of a sequence of amino acids which may form the Linker or part of the Linker.

[0049] In certain embodiments, the Linker may comprise a non-amino acid subunit. In some embodiments, examples of the non-amino acid subunit of the linker are -(OCH2CH2)m- where m is from 1 to 15, for example 2 to 10, 2 to 6 or 4. Introduction of a (poly)ethyleneglycol group assists solublity in aqueous media. In some embodiments, examples of the non-amino acid portion of the linker are -CH2C(O)- and -CH2C(O)NHCH2CH2(OCH2CH2)4C(O)-.

[0050] For example, it can be advantageous to use a Linker with 3 subunits. A first optional subunit which comprises a flexible peptide, such as -(G)m- or -(GS)mG-, where m is an integer of 1, 2, 3, 4, 5 or more, such as 2. A second subunit can be a residue of a chemical reaction (such as an automated flow chemistry reaction), such as a peptide bond, ester, or ether involving the N-terminus, C-terminus or side chain of the Peptide or first subunit. The residue can be non-cleavable, such as that formed with carbodiimide or sulfhydryl maleimide. A third optional subunit can be a hydrophilic spacer, similar or identical to the optional spacer used in the multimeric core B, such as polyethyleneglycol, polyethyleneamine, polyacetal polymer, poly(l -hydroxymethylethylene hydroxymethyl-formal) (PHF) or a carbohydrate. The hydrophilic spacers can generally be polymeric and comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more monomers. Polyethyleneglycol with 4 monomers (PEG4) is satisfactory. The length of the hydrophilic spacer can correspond to the span of the protein gap to facilitate the orientation of the HRC peptide to bind the HRN domain.

[0051] With reference to Formula (B2), when the Membrane Anchoring Moiety comprises cholesterol, in some embodiments, the compound can be represented for by Formula (III):

[0052] Formula (III), wherein peptide, linker, m, and p are as defined above, including every and preferable embodiments.

[0053] The compound of the invention can be rapidly programed to provide anti-viral effect on an evolving virus, by integrating various fusion peptide inhibitors into the compound preferably via automated flow chemistry. The target virus is preferably an enveloped virus that has viral spike proteins or viral fusion proteins, such as a coronavirus or a paramyxovirus.

[0054] In some embodiments, the compounds of the invention have anti-paramyxoviral activities. Paramyxoviruses are a family of enveloped, negative-sense RNA viruses that can cause a wide range of respiratory and systemic diseases in humans and animals. The family includes well-known viruses such as measles virus, mumps virus, and respiratory syncytial virus (RSV). The virion structure of paramyxoviruses consists of a lipid envelope containing two major glycoproteins: the attachment (HN, H, or G) and fusion (F) proteins. The attachment protein is responsible for binding to the host cell receptor, while the fusion protein is involved in fusion of the viral and cellular membranes to facilitate viral entry into the host cell. The attachment proteins interact with different cellular receptors. For example, parainfluenza virus 5 (PIV5, formerly known as SV5) HN binds sialic acid, measles H interacts with CD46 or CDwl50 / SLAM, Hendra and Nipah virus G binds to Ephrin B2, and respiratory syncytial virus (RSV) G binds heparin sulfate.

[0055] The F protein of paramyxoviruses is a type I viral fusion protein that plays a crucial role in viral entry by mediating membrane fusion between the viral envelope and host cell membrane. The F protein is synthesized as an inactive precursor (FO), which must be cleaved by host cell proteases to generate the active form of the proteins: a larger C-terminal fragment (Fl) and smaller N-terminal fragment (F2). Fl contains a hydrophobic fusion peptide at its N terminus and two hydrophobic, heptad repeat regions (HR1 and HR2). HR1 is immediately adjacent to the fusion peptide, and HR2 is proximal to the transmembrane (TM) domain, e.g., with about 250 residues separating the two heptad repeats. The F protein is highly conserved among paramyxoviruses, and it contains several functional domains, including a signal peptide, a fusion peptide, two heptad-repeat domains HR1 and HR2, a transmembrane domain, and a cytoplasmic tail.

[0056] The fusion process mediated by the F protein is triggered by the attachment of the H protein to the host cell receptor. This interaction causes a conformational change in the F protein, leading to the exposure of the fusion peptide and subsequent insertion of the peptide into the host cell membrane. The HR1 and HR2 domains of the F protein then form a six- helix bundle structure, bringing the viral and cellular membranes into close proximity and facilitating membrane fusion.

[0057] The highly conserved nature of the F protein among paramyxoviruses makes it an attractive target for the peptide inhibitors, such as HRC peptides. The HRC peptides can inhibit the F protein of paramyxoviruses by binding to the HR1 domain of the F protein, thus preventing the interaction between HR1 and HR2 domains that is necessary for the fusion process. The HR2 domain undergoes a conformational change to form a six-helix bundle with the HR1 domain during the fusion process. The HRC peptide inhibitors are designed to mimic the HR2 domain and competitively bind to the HR1 domain, preventing the formation of the six-helix bundle and inhibiting the fusion process. As a result, the virus is unable to enter the host cell and the spread of infection is inhibited.

[0058] The compound of the invention can be modulated to target a variety of paramyxoviruses, preferably by integrating a fusion peptide inhibitor for the target paramyxovirus. For example, the compound can be designed to target HPIV-3.

[0059] Human Parainfluenza Virus Type 3 (Human Respirovirus 3, HPIV-3) is a member of the paramyxovirus family and a common cause of respiratory tract infections, particularly in infants and young children. HPIV-3 is transmitted through respiratory secretions and can cause a range of symptoms, including cough, fever, runny nose, and difficulty breathing. In severe cases, it can lead to pneumonia and bronchiolitis. To the date of this application, vaccines are not currently available for HPIV-3, and treatment primarily involves supportive care.

[0060] In some embodiments, the fusion peptide inhibitor targeting HPIV-3 is a short peptide derived from the HRC region of the fusion glycoprotein of HPIV-3 (GenBank: ARV77784.1, 539 aa, see https: / / www.ncbi.nlm.nih.gOv / protein / ARV77784. l). In some embodiments, the fusion peptide inhibitor targeting HPIV-3 is an HRC peptide derived from the fusion glycoprotein of HPIV-3. In preferred embodiments, the fusion peptide inhibitor comprises an amino acid sequence that is identical to the amino acid sequence spanning residues 449V to 4841 of the HPIV-3 fusion glycoprotein in the standard N-to-C direction.

[0061] In preferred embodiments, the fusion peptide inhibitor targeting HPIV-3 comprises an amino acid sequence as shown in SEQ ID NO. 1 :

[0062] Acq-VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI (SEQ ID NO. 1), wherein q is 0 or 1. In other preferred embodiments, the fusion peptide inhibitor comprises an amino acid sequence that is a natural or non-natural variant of the amino acid sequence spanning residues 449V to 4841 of the HPIV-3 fusion glycoprotein in the standard N-to-C direction. As used herein, the term “variant” is defined as a peptide which has at least one amino acid deleted, added, or substituted in comparison with a wild type sequence, such as SEQ ID NO. 1 or other native sequence described herein. Variants preferably bind the cognate ligand of the wild type sequence. For example, a peptide wherein 1, 2, 3, 4 or 5 amino acids of SEQ ID NO. 1 are substituted can be used. Such substituted amino acids can preferably be selected from one or more corresponding amino acids identified in a different paramyxovirus strain via a sequence alignment.

[0063] In additional preferred embodiments, the fusion peptide inhibitor targeting HPIV-3 comprises an amino acid sequence selected from SEQ ID NO. 2-6:

[0064] Acq-VALDPIDISIVLNKIKSDLEESKEWIRRSNKILDSI (SEQ ID NO. 2), wherein q is 0 or 1;

[0065] Acq-VALDPIDISIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 3), wherein q is 0 or 1;

[0066] Acq-VALDPIDFSIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 4), wherein q is 0 or 1;

[0067] Acq-VALDPIDISIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 5), wherein q is 0 or 1;

[0068] Acq-VALDPIDFSIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 6), wherein q is 0 or 1.

[0069] In some embodiments, the fusion peptide inhibitor targeting HPIV-3 is an amino acid sequence of one of SEQ ID NO. 1-6.

[0070] In some embodiments, a compound of the invention has the structure as shown in Figure 1 A (also referred to as “DCOY3001”).

[0071] In some embodiments, a compound of the invention has the structure as shown in Figure IB (also referred to as “DCOY3002”).

[0072] In some embodiments, a compound of the invention has the structure as shown in Figure 1C (also referred to as “DCOY3003”).

[0073] In some embodiments, a compound of the invention has the structure as shown in Figure ID (also referred to as “DCOY3004”).

[0074] In some embodiments, in addition to the HRC peptides for paramyxoviruses, the compound optionally comprises additional peptides that target other domains of the F protein, such as the fusion peptide (FP) or the receptor binding domain (RBD). For example, one class is fusion inhibitory peptides (FIPs) that specifically target the fusion peptide (FP) of the F protein. Another class is synthetic peptides that mimic the RBD of the HN protein. For example, the compound targeting RSV can comprise HRC peptide for RSV and a RBD targeting peptide, such as 10 IF, which has been shown to block RSV infection by interfering with HN-F interactions.

[0075] In some embodiments, the compounds of the invention have anti-coronaviral activities. Coronaviruses target human cells via the spike protein binding domain attaching to the human angiotensin converting enzyme 2 (hACE2) receptor on host cells. The coronavirus spike (S) glycoprotein is a class I viral fusion protein on the outer envelope of the virion that plays a critical role in viral infection by recognizing host cell receptors and mediating fusion of the viral and cellular membranes. Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein that forms homotrimers protruding from the viral surface. S comprises two functional subunits responsible for binding to the host cell receptor (SI subunit) and fusion of the viral and cellular membranes (S2 subunit).

[0076] SI serves the function of receptor-binding and contains a signal peptide (SP) at the N terminus, an N-terminal domain (NTD), and receptor-binding domain (RBD). S2 functions in membrane fusion to facilitate cell entry, and it contains a fusion peptide (FP) domain, internal fusion peptide (IFP), two heptad-repeat domains (HR1 and HR2), transmembrane domain, and a C-terminal domain.

[0077] After binding, the spike protein is activated by the host cell transmembrane protease / serine subfamily member 2 (TMPRSS2) and consequently the virus undergoes fusion with the endosomal membrane for entry into the cell. The membrane fusion domain of the spike protein in coronaviruses is highly conserved so targeting membrane fusion may result in durable long-lasting therapeutics.

[0078] The compound comprises a fusion peptide inhibitor targeting a coronavirus, preferably a HRC peptide. The compound optionally further comprises a spike binding peptide. The compound optionally further comprises a target peptide, such as an ACE2 targeting peptides or a receptor binding domain targeting peptide.

[0079] Each peptide is independently a HRC peptide derived from a coronavirus spike protein and / or a targeting peptide, provided that there is at least one HRC peptide derived from a coronavirus spike protein. While preferred embodiments of the invention utilize native or wild-type peptides, non-natural peptides can be used as well. For example, amino acids found in one or more mutations (e.g., omicron mutations) can be combined with the native sequences of other viruses (e.g., the delta virus). The HRC peptides inhibit viral fusion, an important early step in the infection process. The wild type HRC peptide is a conserved region of the spike, or S, protein across coronaviruses. The conserved nature of the fusion regions (HRC / HRN) and mechanism of the class I enveloped viruses make it an ideal target to develop a pan-coronavirus inhibitor.

[0080] Preferred wild type HRC peptides comprise the sequence and binding fragments thereof:

[0081] Acn-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 7), wherein n is 0 or 1.

[0082] With regard to the HRC peptide in SEQ ID NO. 1, the conventional numbering of the amino acids begins with 1150 at D. 11511, 11541 and 1158V are in the hydrophobic interface pre-fusion and 1159V is exposed. These amino acids stabilize a helix. When the conformation change occurs (e.g., protease clipping to release FP), 1158V is exposed and 1159 V presents in the hydrophobic surface interacting with HRN trimer. 1155N, and 1176N are implicated in N-linked glycosylation conserved in coronavirus. The first 7 amino acids are implicated in HRN binding. The “N-Cap” region spans 1159V and 1171V. The 1171V is a conserved hydrophobe in coronaviruses and stabilizes the HRC hydro-core and is involved in the HRN interaction.

[0083] The hydrophobic core spans 11611 and 1175L and is helical pre- and post-fusion. The isoleucines, leucines and alanine are important in folding and stability of a coiled coil. The C-Cap region spans 1176N and 1185L. 1179L, 1182L and 1185L are in the hydrophobic interface pre-fusion and 11801 is exposed. These amino acids stabilize the helix. 1185Y may be implicated in hydrophobic packing between three polypeptide chains in a trimeric coiled coil. When confirmation change occurs (protease clipping to release FP), 1179L is exposed and 11801 is in the hydrophobic surface interacting with a HRN trimer. 1164E and 1184E form a salt bridge between HRC and HRN. Further, 1159V, 1160N, 1171V and 11801 have been shown to interact with HRN in crystal structures.

[0084] In certain embodiments, the peptides are selected from variants of a wild type HRC peptides comprising the sequence and binding fragments thereof:

[0085] Acq-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 7), wherein q is 0 or 1.

[0086] For example, one or both underlined isoleucines can be substituted with leucine and / or methionine, as described in the alignment provided above. The underlined alanine can be substituted by valine, leucine or isoleucine. One or both underlined leucines can be independently substituted by isoleucine, tyrosine, alanine or valine. Other conservative or nonconservative substitutions, (lysine and glutamine or aspartic acid and glutamic acid) can be selected as well, for example, as shown in the above sequence alignment. In embodiments, amino acids that are conserved amongst 2, 3, 4, 5 or more coronavirus (e.g., coronavirus isolated from bats or SARS-CoV2 mutants or variants) remain conserved in the non-natural HRC peptide.

[0087] For example, the wild type HRC sequence can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional amino acids native to the S protein at the N- and / or C termini. For example, glycine can be added to the N-terminus. Additionally, the wild type HRC peptide fragment can delete 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N and / or C termini and inhibit infection. Typically, not more than ten total amino acids are deleted in total. For example, the 10 amino acids at the C terminus can be deleted and be expected to retain inhibitory activity.

[0088] In certain embodiments, modifications to wild type sequences are desirable. For example, using one or more D amino acids can improve pharmacokinetics and the half life of the peptide. Thus, in certain embodiments, the invention includes peptides characterized by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more D-amino acids. The D-amino acids can preferably be a corresponding L-amino acid of the wild type sequence. In certain embodiments, the D-amino acid is an amino acid located at or near (e.g., within 1, 2, or 3 amino acids) a protease degradation site. In certain embodiments, the D-amino acid is a hydrophobic amino acid participating in binding with the HRN peptide and preferably at a higher affinity than the corresponding wild type sequence. Alternatively or additionally, the D-amino acid is a hydrophilic amino acid, such as lysine, aspartic acid, glutamic acid or arginine. Alternatively or additionally, the D-amino acid can be selected from the 7 amino acids at the N-terminus of SEQ ID NO: 1. Peptides that have been improved by incorporating D-amino acids are described in USSN 63 / 140,387, filed on January 22, 2021, which is incorporated by reference in its entirety.

[0089] However, swapping one or more D-amino acids for the corresponding L-amino acid can change the topology of the peptide and impact function. Therefore, a preferred nonnatural HRC peptide is a Retro-Inversion HRC peptide, or “RI HRC peptide”. Retro- Inversion HRC peptides are preferably characterized by a binding affinity of at least about 50% of the wild-type HRC peptide with its cognate ligand in a standard binding assay and decreased susceptibility to mammalian protease degradation. Retroinversion is defined as reversing a D-peptide sequence of a helical peptide or “flipping” the termini thereby restoring the presentation of the side chains to the binding ligand or target. See Kim et al, Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PBS, PNAS, February 13, 2018, 115 (7) 1505-1510, which is incorporated herein by reference in its entirety. Therefore, a non-natural peptide of the invention can include a peptide having the sequence of SEQ ID NO.: 1 wherein amino acids are D-amino acids, such as the amino acids within a region, flipping the N-terminus for a C terminus. For example, the N termini can be subjected to retroinversion as shown in SEQ ID NO. 8-14, where each D amino acid is preceded by a “d”:

[0090] Acq-dVdAdLdDdP IDISIELNKAKSDLEESKEWIRRSNQKLDSI (SEQ ID NO. 8), Acq-dVdAdLdDdP IDISIVLNKIKSDLEESKEWIRRSNKILDSI (SEQ ID NO. 9), Acq-dVdAdLdDdP IDISIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 10), Acq-dVdAdLdDdP IDFSIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 11), Acq- dVdAdLdDdP IDISIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 12), Acq- dVdAdLdDdP IDFSIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 13), and

[0091] Acq-dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO.

[0092] 14), wherein q is 0 or 1.

[0093] These examples offer a single RI region of 5 amino acids. However, as few as two amino acids can be selected (e.g., the 2 N-terminal amino acids). For example, for HRC peptides targeting a coronavirus, the RI region can span the hydrophobic core of 1160N to 1176N, or the C-cap region or a portion thereof. Alternatively, the entire peptide can be an RI peptide. Additionally, two, three or more RI regions can be included. For example, both the N-terminus and C-Cap region can be RI regions, retaining the hydrophobic core with L- amino acids.

[0094] For example, in using mirror-image phage display to screen for HRC variants, a first D-peptide can be synthesized from a HRN coronavirus peptide, or first L-peptide. The first L- peptide can be a naturally occurring L-peptide or can be a chimera of a peptide. The methods can further comprise screening for a HRC peptide, or second L-peptide, that specifically binds to the first D-peptide; then, a second D-peptide that is the mirror image of the second L-peptide can be synthesized. In one aspect of the D-peptide screening methods described herein, an N-trimer target can first be synthesized with D-amino acids, creating the mirror image of the natural L-N-trimer target. The D-N-trimer target can be used in standard peptide-based screens such as phage display, ribosome display, and / or CIS display to identify L-peptides that bind to the D-N-trimer. The identified L-peptides can then be synthesized with D-amino acids. By the law of symmetry, the resulting D-peptides bind the natural L-N- trimer and will thus target the N-trimer region of the coronavirus HRN intermediate, thereby inhibiting infection. This screening method is also described in Schumacher, et al., Identification of D-peptide ligands through mirror-image phage display, Science, 1996 Mar 29; 271(5257): 1854-7, which is hereby incorporated in its entirety by this reference.

[0095] The hotspot residues of the HRC peptide can be identified by crystal structure or NMR solution structure of the HRC peptide. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids selected from 1150D, 11511, 11541, 1155N, 1158V, 1159V, 11611, 1164E, 1171V, 1175L, 1176L, 1179L, 11801, 1182L, 1184E and / or 1185 Y, such as one or more amino acids selected from 1159V, 1160N, 1163E, 1171V, 11801, 1184E and / or 1185L of SEQ ID NO. 7 can be designated hotspot residues.

[0096] In some embodiments, the Peptide comprises a cell targeting peptide. In embodiments, the targeting peptide is selected from but not limited to an ACE2 targeting peptide or a Receptor binding domain targeting peptide. In embodiments, the targeting peptide is an ACE2 targeting peptide. In embodiments, the targeting peptide is a Receptor binding domain targeting peptide.

[0097] In some embodiments where the compound comprises only one Peptide, the peptide is HRC peptide derived from a coronavirus spike protein. In embodiments where the compound comprises 2 or more Peptides, each peptide can be a HRC peptide derived from a coronavirus spike protein. In embodiments where the compound comprises 2 or more Peptides, the Peptide can be selected from a HRC peptide derived from a coronavirus spike protein and / or a targeting peptide, provided that there is at least one HRC peptide derived from a coronavirus spike protein. Preferably, only one of the Peptides is a targeting peptide and the other peptide(s) are a HRC peptide derived from a coronavirus spike protein.

[0098] In some embodiments, a compound of the invention has the structure as shown in Figure IE (also referred to as “DCOY104”).

[0099] Automated Flow Chemistry

[0100] There is a need to be able to synthesize peptide conjugates using finely tunable and simple chemical reactions that are nontoxic to cells, and that can occur at a rapid rate and keep pace with the evolving diseases or research needs. The invention addresses this need. In broad aspect, this disclosure provides functional modules of peptide conjugate and subunits thereof that are compatible to rapid automated flow chemistry. An automated flow peptide synthesis (AFPS), as disclosed in U.S. Patent No. 10,683,325 B2 is a solid phase peptide synthesis system equipped with feedback control and can afford a high degree of control over individual coupling reactions for making peptides and / or minimize side reactions. The method of this invention provides improvements on AFPS and allows the precise and rapid synthesis of the compounds, which is therefore also referred to as “one shot synthesis”, of the compound consisting of a plurality of moieties. The method of this invention provides opportunities to rapidly and precisely synthesize a diverse array of peptide conjugates that can be used for applications in prophylaxis, diagnosis, and therapy.

[0101] In some embodiments, the synthesis of the compound can be conducted in an AFPS system that comprises a reactor that comprises peptides immobilized on a solid support. For example, as illustrated in Figure 2, reagent reservoirs (1-6) can be located upstream of and fluidically connected to a reactor (8).For example, a first reagent reservoir (1), a second reagent reservoir (2), and a third reagent reservoir (3) can be fluidically connected to the reactor via first reagent channel, second reagent channel, and third reagent channel, respectively, which connect to delivery channel that is connected to reactor. The reagent reservoirs contain at least a portion of the reagents necessary for compound synthesis. For instance, a first reagent reservoir contains amino acids, a second reagent reservoir contains an activating agent (e.g., an uronium activating agent), and a third reagent reservoir contains a reagent that comprises the Membrane Anchoring Moiety, such as a cholesterol or a derivative thereof. The system can further comprise an optional reagent reservoir that can contain a spacer reagent, such as a PEG molecule (e.g., 4, 5, or 6). The system can further comprise an optional reagent reservoir that can contain a deprotection reagent, such as piperidine or trifluoroacetic acid (e.g., 4, 5, or 6). The system can further comprise an optional reagent reservoir that can contain a base and / or optional reagent (e.g., 4, 5, or 6). The system can also comprise an optional reagent reservoir fluidically connected to the reactor that contains a solvent, such as dimethylformamide (DMF), that may be used, e.g., in a reagent removal step (e.g., 4, 5, or 6). The system can further optionally comprise reactor (7) configured to promote and / or facilitate one or more chemical reactions between certain reagents and / or reaction products thereof by, e.g., modulating the reaction kinetics and / or reaction time.

[0102] While single reservoirs have been illustrated in Figure 2 for simplicity, it should be understood that in Figure 2, where single reservoirs are illustrated, multiple reservoirs (e.g., each containing different types of amino acids, different types of activating agents, different types of deprotection agents, different types of bases, etc.) could be used in place of the single reservoir. During the amino acid addition steps, adding the amino acid (9) can result in the peptide incorporating a single additional amino acid residue (i.e., a single amino acid residue can be added to the immobilized peptides such that a given peptide includes a single additional amino acid residue after the addition step). In some such embodiments, subsequent amino acid addition steps can be used to build peptides by adding amino acid residues individually until the desired peptide has been synthesized. In some embodiments, more than one amino acid residue (e.g., in the form of a peptide) may be added to a peptide immobilized on a solid support 10 (i.e., a peptide comprising multiple amino acid residues can be added to a given immobilized peptide). Addition of peptides to immobilized peptides can be achieved through processes known to those of ordinary skill in the art (e.g., fragment condensation, chemical ligation). That is to say, during the amino acid addition step, adding an amino acid to an immobilized peptide can comprise adding a single amino acid residue to an immobilized peptide or adding a plurality of amino acid residues (e.g., as a peptide) to an immobilized peptide.

[0103] In some embodiments, the synthesis system comprises flowing fluid streams, e.g., from the reagent reservoirs through a reactor (e.g., 7, 8). For example, a fluid stream comprising a deprotection reagent may be flowed through the reactor after a coupling reaction. Fluid may exit the reactor through effluent channel connected to the reactor. The effluent channel may be fluidically connected to a detection zone (e.g., 11). In certain embodiments, the effluent channel (e.g., 12) may not comprise a separation element (e.g., size-exclusion column, affinity column) and / or may be connected to the detection zone.

[0104] In some embodiments, the detection zone (e.g., 11) may comprise one or more electromagnetic radiation detectors. The detector(s) may measure the electromagnetic absorbance and / or the electromagnetic emission of one or more fluids exiting the reactor and produce one or more signals corresponding to the fluid(s). The signal(s) may be transmitted to a unit (e.g., 13), which is in electrical communication with the detector(s). In some embodiments, the unit (e.g., 13) may be a controller that is configured to control one or more parameters of the system. In such embodiments, the controller may be operatively associated with one or more components (e.g., temperature regulator, fluid flow source) of the system and / or with one or more processors for controlling component(s) of the system. For example, the controller may be operatively associated with one or more processors for controlling flow rate, temperature, selection of reagent type, selection of reagent concentration, reaction time, selection of the ratio of reagents, the addition of an additive, or combinations thereof. Optionally, the controller may also be operatively associated with other components such as a user interface and an external communication unit (e.g., a USB), and / or other components, as described in more detail below. The user interface may be used to display the signal(s), alert the user of a problem with a certain reaction, and / or receive operation instructions from the user.

[0105] The feedback control of the system allows the rapid and precise synthesis of the compound. In some embodiments, the feedback control is conducted by detecting an electromagnetic absorbance and / or an electromagnetic emission of a fluid stream at a detection zone (e.g., 11 in Figure 2) positioned downstream of the reactor during and / or immediately after (e.g., after the stream exits the reactor, but before the next amino acid addition step) a reaction step in an amino acid addition cycle to produce a signal. Based at least in part on information derived from the signal (e.g., an intensity component and a time component derived from the signal), one or more parameter of the system may be modulated prior to and / or during a subsequent reaction (e.g., coupling reaction) in the reactor and / or prior to formation of the peptide in the reactor. The feedback can be used to modulate a parameter of the system by controlling, e.g., one or more of a pump, vacuum, valve, temperature regulator, and / or other components. In some cases, the feedback can determine problems that have occurred or are occurring in the solid phase peptide synthesis system, and the controller may send one or more signal(s) to one or more components to cause modulation of a parameter in all or portions of the system. Alternatively, when corrective action cannot be taken, the controller may send one or more signal(s) to one or more components to cause the system to shut down.

[0106] In some embodiments, detecting during a reaction step in an amino acid addition cycle may comprise detecting from the start of the reaction step to the end of the reaction step, detecting during at least a portion of the reaction step (e.g., at least about 20% of the total time for the reaction step, at least about 30% of the total time for the reaction step, at least about 40% of the total time for the reaction step, at least about 50% of the total time for the reaction step, at least about 60% of the total time for the reaction step, or at least about 75% of the total time for the reaction step and less than about 100%), and / or continuous detection during at least a portion of the two or more reaction steps (e.g., deprotection steps) and / or complete peptide synthesis.

[0107] In some embodiments, both the heating of the amino acids and the merging of the amino acids with the base and / or activating agent can be performed before and within a relatively short time of the amino acids contacting the immobilized peptides. Heating the amino acids may be performed before, during, and / or after merging the streams. The reagent used for compound synthesis comprise protecting groups, for example, on the primary amine of an amino acid, on the N-termini and / or the side chains of the peptides, on the functional group of a Linker, a subunit of the Linker, or a hydrophilic spacer. As used herein, the term “protecting group” is given its ordinary meaning in the art. Protecting groups include chemical moieties that are attached to or are configured to be attached to reactive groups (i.e., the protected groups) within a molecule (e.g., peptides) such that the protecting groups prevent or otherwise inhibit the protected groups from reacting. Protection can occur by attaching the protecting group to the molecule. In some embodiments, the side chains of the amino acid residues in the peptide can comprise protecting groups. Deprotection can occur when the protecting group is removed from the molecule, for example, by a chemical transformation which removes the protecting group. In general, any protecting group known to those of ordinary skill in the art can be used. Nonlimiting examples of protecting groups (e.g., n-terminal protecting groups) include fluorenylmethyloxycarbonyl (Fmoc), 4-methyltrityl (Mtt), tert-butyloxycarbonyl, allyloxycarbonyl (alloc), carboxybenzyl, and photolabile protecting groups. In certain embodiments, immobilized peptides comprise fluorenylmethyloxycarbonyl protecting groups. In some embodiments, immobilized peptides, B moiety, Linker, or spacer can comprise Fmoc and / or Mtt protecting groups.

[0108] As described elsewhere, an activating agent may be used to activate or complete the activation of amino acids prior to exposing the amino acids to immobilized peptides. Any suitable activating agent may be used. The activating agent comprises, in some embodiments, a carbodiimide, such as N,N'-di cyclohexylcarbodiimide (DCC), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), and the like. In certain embodiments, the activating agent comprises a uronium activating agent, such as O-(Benzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU); 2-(7-Aza-lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU); l-[(l-(Cyano-2-ethoxy-2- oxoethylideneaminooxy) dimethylaminomorpholino)] uronium hexafluorophosphate (COMU); and the like.

[0109] As described elsewhere, peptides may be immobilized on a solid support. In general, any solid support may be used with any of the addition cycles described herein. Non-limiting examples of solid support materials include polystyrene (e.g., in resin form such as microporous polystyrene resin, mesoporous polystyrene resin, macroporous polystyrene resin), glass, polysaccharides (e.g., cellulose, agarose), polyacrylamide resins, polyethylene glycol, or copolymer resins (e.g., comprising polyethylene glycol, polystyrene, etc.). The multimeric core B is designed to be compatible with the method described above, to allow the amino acids of Peptides, the optional Linker, and the Membrane Anchoring Moiety attached to it via automated flow chemistry. In some embodiments, the solid support has been preferably treated to display an amine group on the surface, and the carboxyl acid group of a lysine reacts with the amine group and thus is immobilized on the solid support, which is followed by coupling reactions with other subunits of B moiety such as a second lysine or a hydrophilic spacer, amino acids of the Peptides, Linkers, and Membrane Anchoring Moiety, wherein selective or global deprotection of function groups can be performed before a coupling reaction or between two coupling reactions.

[0110] The invention provides a method for rapidly synthesizing a peptide conjugate.

[0111] In some embodiments, the method comprises: flowing a fluid stream comprising a deprotection reagent through a reactor after a coupling reaction between an amino acid and an amino acid residue immobilized on a solid support to form a peptide fragment; detecting an electromagnetic absorbance and / or an electromagnetic emission of the fluid stream at a detection zone positioned downstream of the reactor to produce a signal; and modulating a parameter of the system prior to formation of the peptide in the reactor based at least in part on an intensity component and a time component derived from the signal, wherein the peptide comprises the peptide fragment; optionally flowing a fluid stream comprising a hydrophilic polymeric molecule through the reactor; optionally flowing a fluid stream comprising a deprotection reagent through a reactor after a coupling reaction between a peptide fragment and a hydrophilic polymeric molecule; flowing a fluid stream comprising a reagent comprising the Membrane Anchoring Moiety through the reactor. Preferably, the peptide fragment comprise a lysine residue at C-terminus. Preferably, the hydrophilic polymeric molecule is a PEG or a derivative thereof. Preferably, the Membrane Anchoring Moiety is cholesterol.

[0112] In some embodiments, the method comprises: a) binding at least one N-protected diamino acid to a solid support comprising surface amino groups to prepare an immobilized N-protected diamino acid via a coupling reaction, wherein the immobilized N-protected diamino acid is bound to the solid support via an amide bond and comprises two unbound amine groups, wherein at least one amine group is protected; b) optionally, reacting the immobilized N-protected diamino acid with at least one additional N-protected diamino acid via a coupling reaction from C to N direction to prepare an immobilized N-protected peptide, wherein the immobilized N-protected peptide has a length of 2 to 6 diamino acid residues and a C-terminus bound to the solid support; c) optionally, reacting the immobilized N-protected diamino acid of step (a) or the immobilized N-protected peptide of step (b) with a linker via a coupling reaction to prepare an immobilized conjugate framework, wherein the immobilized conjugate framework comprises at least one N-protected diamino acid and at least one linker jointed with each other at the amino group of the N-protected diamino acid via a covalent bond, wherein each linker is independently a PEG reagent that comprises a PEG2-40 chain and two reactive chain ends; d) synthesizing an amino acid sequence, preferably in an automated flow peptide synthesis system (AFPS), wherein the C-terminus of the amino acid sequence is attached to a primary amine of an immobilized moiety, wherein the immobilized moiety is selected from the immobilized N-protected diamino acid of step (a), the immobilized N- protected peptide of step (b), and the immobilized conjugate framework of step (c), via a coupling reaction; e) optionally, protecting the N-terminus of the amino acid sequence of step (d), wherein the protecting group is an acyl or acetyl; f) unprotecting the protected amine group of step (a) to form a product having a primary amine; g) optionally, reacting the primary amine of step (f) with a linker via a coupling reaction to form a product, wherein the linker is as defined in step (c); h) reacting either the primary amine of step (f) or the reactive chain end of the linker of step (g) with a functional group via a coupling reaction to form a product, wherein the functional group is a targeting moiety, a binding moiety, or a membrane integrating moiety, i) cleaving the product of step (h) from the solid support.

[0113] In some embodiments, the method comprises: a) binding at least one N-protected diamino acid, such as an N-protected lysine, which is a diamino acid substituted by a protecting group on an amine, to a solid support comprising surface amino groups to prepare an immobilized N-protected diamine comprising a primary amine; b) reacting the immobilized N-protected diamine with a diamino acid, such as lysine, or an N-protected diamino acid, such as N-protected lysine, to prepare an immobilized N-protected polyamine having one or more primary amines; c) optionally repeating step (b); d) optionally reacting each primary amine in the immobilized N-protected polyamine of step (b) or step c) with a linker moiety; e) synthesizing an amino acid sequence, preferably in an automated flow peptide synthesis system (AFPS), attached (preferably, via the C-terminus of the amino acid sequence) to the immobilized N-protected polyamine of step (b) or step (c), or more preferably, to the linker moiety of step (d); f) optionally, protecting each N-terminus of each amino acid sequence such as substituting each primary amine with an acyl, or acetyl group; g) removing each protecting group to form a product having at least one primary amine; h) optionally, reacting each primary amine with a linker moiety; i) reacting either the primary amine of the product of step (g) or the linker moiety of the product of step (h) with a functional group; j) cleaving the product of step (i) from the solid support. Preferably, the product prepared by the method has a structure: wherein n and m are independently an integer of 1 or more; wherein m+n is preferably 3 or more;

[0114] Each LI and L2 are independently the same or different linker;

[0115] Each Peptide comprises an amino acid sequence, preferably a therapeutic peptide;

[0116] Each R is a functional group; and

[0117] X is a peptide comprising two or more diamino acids.

[0118] In some embodiments, the method comprises: a) binding at least one N-protected diamino acid to a solid support comprising surface amino groups to prepare an immobilized N-protected diamine having the structure:

[0119] Wherein SS is a solid support; L is a linker and PG is a protecting group; b) reacting the immobilized N-protected diamine of formula (I) with a diamino acid to prepare an immobilized N-protected polyamine having the structure:

[0120] Wherein each L is independently a linker, preferably the same linker, such as an aliphatic linker, preferably a C4 alkylene; c) optionally reacting the immobilized N-protected polyamine of step (b) with a linker to prepare a product having the structure:

[0121] Wherein each Linker is independently a linker, such as polyethyleneglycol; and X is a reactive moiety; d) synthesizing an amino acid sequence, preferably in an automated flow peptide synthesis system (AFPS), at each reactive moiety X, e) optionally, protecting the N-terminus of the amino acid sequence;

[0122] O O wherein each Peptide is, independently, the amino acid sequence and R is, independently, H or a protecting group, such as an acyl or acetyl; f) removing Protecting Group, PG, to form a product having a primary amine with the structure: g) optionally, reacting the primary amine with a linker to form a product having the structure:

[0123] Linker-X inker-Peptide-NHR

[0124] (f)

[0125] Wherein Linker-X is as defined in step (c); h) reacting either the primary amine of formula (e) or the X of formula (f) with a functional group to form a product having the structure:

[0126] Wherein FG is a functional group, such as a targeting moiety or lipid and m is 0 or 1; i) cleaving the product of formula (g) from the solid support.

[0127] In some embodiments, the method comprises: a) synthesizing an immobilized amino acid sequence comprises the (Peptide- Linker)n, and lysine(s) and each (Peptide-Linker) is connected to a lysine directly via an amide bond, or indirectly via a polyethylene glycol (PEG) group; wherein the amino acid sequence is bound to a solid support via an amide bond between a lysine at the C-terminus of the amino acid sequence and the solid support; and wherein one -NH2 group of the lysine at the C-terminus is protected; Preferably, the amino acid sequence is represented by the protection group formula: Peptide— Linker wherein is a covalent bond between the amino acid sequence and the solid support, m is 0, 1, 2, 3, 4, or 5; each p is an integer independently selected from the range of 0-40; b) optionally, protecting (e.g., acetylating) the N-terminus of the amino acid sequence; c) deprotecting NH2 group of the lysine at the C-terminus; d) optionally, reacting the amino acid sequence with a protected PEG reagent, wherein the protected PEG reagent is covalently bound to the amino acid sequence at the C- terminus via an amide bond; for example, the protected PEG reagent is a Fmoc-NH-PEGo- CH2COOH, and O is an integer selected from 2-20; e) optionally, deprotecting the protected PEG reagent; f) reacting the amino acid sequence with a reagent comprising the Membrane Anchoring Moiety, wherein the Membrane Anchoring Moiety is covalently bound to the amino acid sequence at the C at the C-terminus via an amide bond; preferably, the Membrane Anchoring Moiety is a lipid; the reagent comprising the Membrane Anchoring Moiety is for example cholesteryl hydrogen succinate; and g) cleaving the product obtained from step F from the solid support.

[0128] In some embodiments, the lysine immobilized on the solid support of the system can be represented in the formula as shown below: Solid support Protecting group .Protecting group H wherein the protecting group can be subsequently removed, and the protected amine groups are deprotected to allow the occurrence of coupling reactions as described above. Examples of protected lysine reagents include, without limitation, Fmoc-Lys(Mmt)-OH, Fmoc- Lys(Dde)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys-OH, Fmoc- Lys(Fmoc)-OH. For example, in the method described herein, the lysine at C-terminus bound to the solid support can be preferably derived from Fmoc-Lys(Mtt)-OH. The lysine(s) that are not the lysine at C-terminus bound to the solid support can be preferably derived from Fmoc- Lys(Fmoc)-OH.

[0129] In some embodiments, the amino acids for synthesizing the Peptides, the optional Linkers and the subunits thereof, the hydrophilic spacers of B moiety, the Membrane Anchoring Moiety each are independently protected and deprotected. For example, Fmoc- NH-PEG-carboxyl acid can be used as a precursor to the hydrophilic spacer.

[0130] In some embodiments, the peptide conjugate prepared by the method is a compound as described herein. In some embodiments, the peptide conjugate prepared by the method has a structure that is selected from General Formula, Formula (I), Formula (II), and Formula (III), wherein all the variables are as defined above, including all and preferred embodiments. Nonlimiting examples of the peptide conjugates prepared by the method include those as shown in Figure 1 A-1E.

[0131] In some embodiments, the peptide conjugate prepared by the method has anti-viral effects on a SAR-Cov-2 variant. The peptide conjugate preferably comprises an amino acid sequence of SEQ ID NO: 7 or 14.

[0132] In some embodiments, the peptide conjugate prepared by the method has anti-viral effects on a paramyxovirus variant, such as HPIV3 or a variant thereof. The peptide conjugate preferably comprises an amino acid sequence of any one of SEQ ID NO: 1-6 and 8-13. Pharmaceutical Composition

[0133] The compositions of the invention comprise a compound as described herein and a pharmaceutically acceptable carrier. For example, the composition can be administered systemically or locally. The composition can be administered for oral, intravenous, intramuscular, rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, inhalation, or vaginal delivery, for example. Thus, the composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 22ndedition, 2013, ed. L. V. Allen, Pharmaceutical Press, Philadelphia, and Encyclopedia of Pharmaceutical Technology, 4.sup.th Edition, ed. J. Swarbrick, 2013, CRC Press, New York).

[0134] Compounds may be formulated in a variety of ways that are known in the art. For example, one or more compounds of the invention and any additional biologically active agent, if present, as defined herein may be formulated together or separately.

[0135] Each compound of the invention, alone or in combination with one or more active agents as described herein, can be formulated for controlled release (e.g., sustained or measured) administration, as described in U.S. Patent Application Publication Nos. 2003 / 0152637 and 2005 / 0025765, each incorporated herein by reference. For example, a compound of the invention, alone or in combination with one or more of the biologically active agents as described herein, can be incorporated into a capsule or tablet that is administered to the patient.

[0136] Controlled release formulations known in the art include specially coated pellets, polymer formulations or matrices for surgical insertion or as sustained release microparticles or nanoparticles, e.g., microspheres or microcapsules, for implantation, insertion, infusion or injection, wherein the slow release of the active medicament is brought about through sustained or controlled diffusion out of the matrix and / or selective breakdown of the coating of the preparation or selective breakdown of a polymer matrix. Other formulations or vehicles for controlled, sustained or immediate delivery of an agent to a preferred localized site in a patient include, e.g., lipid nanoparticles (LNP), suspensions, emulsions, gels, liposomes, and any other suitable art known delivery vehicle or formulation acceptable for subcutaneous or intramuscular administration. Suitable biocompatible polymers can be utilized as the controlled release material. The polymeric material may comprise biocompatible, biodegradable polymers, and, in certain preferred embodiments, is preferably a copolymer of lactic and glycolic acid. Preferred controlled release materials which are useful in the formulations of the invention include the polyanhydrides, polyesters, co-polymers of lactic acid and glycolic acid (preferably wherein the weight ratio of lactic acid to glycolic acid is no more than 4: 1 i.e., 80% or less lactic acid to 20% or more glycolic acid by weight) and polyorthoesters containing a catalyst or degradation enhancing compound, for example, containing at least 1% by weight anhydride catalyst such as maleic anhydride. Examples of polyesters include polylactic acid, polyglycolic acid and polylactic acid-polyglycolic acid copolymers. Other useful polymers include protein polymers such as collagen, gelatin, fibrin and fibrinogen and polysaccharides such as hyaluronic acid.

[0137] In additional embodiments, the controlled release material, which in effect acts as a carrier for a compound of the invention can further include a bioadhesive polymer such as pectins (polygalacturonic acid), mucopolysaccharides (hyaluronic acid, mucin) or non-toxic lectins or the polymer itself may be bioadhesive, e.g., polyanhydride or polysaccharides such as chitosan. In embodiments where the biodegradable polymer comprises a gel, one such useful polymer is a thermally gelling polymer, e.g., polyethylene oxide, polypropylene oxide (PEO-PPO) block copolymer such as PLURONIC.TM. F127 from BASF Wyandotte.

[0138] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, taste masking agents (such as hydroxypropyl methylcellulose, hydroxypropyl cellulose) and the like. One or more compounds of the invention may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, a compound of the invention is contained on the inside of the tablet, and the biologically active agent is on the outside of the tablet, such that a substantial portion of the biologically active agent is released prior to the release of the compound of the invention.

[0139] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient(s) are mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment. Formulations to the mouth may also be provided as a mouthwash, an oral spray, oral rinse solution, or oral ointment, or oral gel.

[0140] Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and / or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbon.

[0141] Liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0142] Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is from about 1 ng / ml to about 10 ug / ml, for example from about 10 ng / ml to about 1 ug / ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0143] The composition of the invention can comprise a liquid vehicle which is suitable for nasal administration. The vehicle is preferably an aqueous solution. More preferably, the vehicle is an aqueous solution which includes a viscosity enhancing agent and, optionally one or more additional excipients which, for example, improve formulation stability and / or comfort upon administration.

[0144] A variety of viscosity enhancing agents are known in the art. Viscosity enhancing agents include hydrophilic polymers, such as polysaccharides, polysaccharide derivatives, proteins and synthetic polymers. Examples include, but are not limited to, acacia, tragacanth, alginic acid, carrageenan, locust bean gum, guar gum, gelatin, hyaluronic acid, polyacrylate, polyacrylate / alkylacrylate copolymers, polyvinyl alcohol, polyvinylpyrrolidone, starch, propylene glycol alginate, maltodextrin, and cellulose ether derivatives, such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose. Where possible, salt forms of any of the foregoing are preferred. Preferred viscosity enhancing agents include hyaluronic acid, including sodium hyaluronate; carboxymethylcellulose, including sodium carboxymethylcellulose and calcium carboxymethylcellulose; methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxypropylcellulose.

[0145] The composition optionally includes one or more additional excipients which, for example, increase the ease of administration, the comfort of the subject, or the stability of the composition. Suitable additional excipients include, but are not limited to, tonicity modifiers, such as sodium chloride and dextrose; antioxidants, such as butylated hydroxyanisole; buffers, such as sodium bicarbonate, sodium citrate and sodium phosphate; preservatives, such as benzalkonium chloride, ethanol, propylene glycol, benzoyl alcohol, phenethyl alcohol, chlorobutanol or methylparaben; pH adjusters, such as hydrochloric acid, sulfuric acid and sodium hydroxide; surfactants, such as Polysorbate 80, Polysorbate 20, and polyoxyl 400 stearate; chelating agents, such as disodium EDTA; antioxidants; co-solvents, such as ethanol, PEG 400, and propylene glycol; penetration enhancers, such as oleic acid; and humectants, such as glycerin (see S. Thorat, Sch. J. App. Med. Sci. 2016, 4(8D):2976-2985; D. Marx et al., IntechOpen, DOE 10.5772 / 59468. Available from: intechopen.com / books / drug-discovery-and-development-from-molecules-to- medicine / intranasal-drug-administration-an-attractive-delivery-route-for-some-drugs).

[0146] In one embodiment, the vehicle consists of sodium hyaluronate, aloe vera, allantoin, sodium chloride, sodium bicarbonate, glycerin, propylene glycol, propylene glycol, benzalkonium chloride and USP grade purified water. A suitable vehicle is sold by NEILMED™ under the tradename NASOGEL™.

[0147] The amount of active agent in the composition can vary, for example, from about 0.5% by weight to about 25% by weight.

[0148] The pH of the formulation is tolerable in the nasal cavity and preferably at least about 8.0. Buffers that can be used in the formulation include, but are not limited to phosphate, TRIS, [tris(hydroxymethyl) methylamino] propanesulfonic acid, 2-(bis(2- hydroxyethyl)amino)acetic acid, and N-[tris(hydroxymethyl)methyl]glycine, and Alkaline Buffer (Seachem).

[0149] A pharmaceutical composition suitable for nasal or pulmonary administration comprising a water soluble solvent selected from the group consisting of propylene glycol, glycerin, polyethylene glycol, and combinations thereof. The composition can further comprise one or more of a polysaccharide gum, a non-ionic surfactant, and a preservative. An exemplary polysaccharide gum is sclerotium gum. Exemplary surfactants are poloxamers, including, but not limited, to poloxamer 188. The preservative can, for example, be benzalkonium chloride.

[0150] The composition can be a dry powder and delivered by a dry powder inhaler, suspended in a propellant or in an aqueous suspension or solution and delivered via a nebulizer.

[0151] For example, a solution or suspension of the active agent and a pulmonary excipient, such as lactose, can be spray dried to form particles having a fine particle fraction sufficient to deliver to the lungs or upper respiratory system. Alternatively, an aqueous solution or suspension can be sonicated, thereby aerosolizing the solution / suspension to a droplet size that can be inhaled, e.g., via a nebulizer.

[0152] Excipients include carbohydrates including monosaccharides, disaccharides and polysaccharides. For example, monosaccharides such as dextrose (anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol, sorbose and the like; disaccharides such as lactose, maltose, sucrose, trehalose, and the like; tri saccharides such as raffinose and the like; and other carbohydrates such as starches (hydroxy ethyl starch), cyclodextrins and maltodextrins. Other excipients suitable for use with the present invention, including amino acids, are known in the art such as those disclosed in WO 95 / 31479, WO 96 / 32096, and WO 96 / 32149. Mixtures of carbohydrates and amino acids are further held to be within the scope of the present invention. The inclusion of both inorganic (e.g., sodium chloride, etc.), organic acids and their salts (e.g., carboxylic acids and their salts such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.) and buffers is also contemplated.

[0153] The compositions may be used in the form of dry powders or in the form of stabilized dispersions comprising a non-aqueous phase. Accordingly, the dispersions or powders of the present invention may be used in conjunction with metered dose inhalers (MDIs), dry powder inhalers (DPIs), atomizers, nebulizers or liquid dose instillation (LDI) techniques to provide for effective drug delivery. With respect to inhalation therapies, those skilled in the art will appreciate that the hollow and porous microparticles of the present invention are particularly useful in DPIs. Conventional DPIs comprise powdered formulations and devices where a predetermined dose of medicament, either alone or in a blend with lactose carrier particles, is delivered as an aerosol of dry powder for inhalation.

[0154] The medicament is formulated in a way such that it readily disperses into discrete particles with a mass median aerodynamic diameters of the powders will characteristically range from about 0.5-10, preferably from about 0.5-5.0 microns MMAD.

[0155] As discussed above, the stabilized dispersions disclosed herein may also be administered to the nasal or pulmonary air passages of a patient via aerosolization, such as with a metered dose inhaler. MDIs are well known in the art and could easily be employed for administration of the claimed dispersions without undue experimentation. Breath activated MDIs, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the stabilized dispersions and present invention and, as such, are contemplated as being within the scope thereof. However, it should be emphasized that, in preferred embodiments, the stabilized dispersions may be administered with an MDI using a number of different routes including, but not limited to, topical, nasal, pulmonary or oral. Those skilled in the art will appreciate that, such routes are well known and that the dosing and administration procedures may be easily derived for the stabilized dispersions of the present invention.

[0156] Along with the aforementioned embodiments, the stabilized dispersions of the present invention may also be used in conjunction with nebulizers as disclosed in PCT WO 99 / 16420, the disclosure of which is hereby incorporated in its entirety by reference, in order to provide an aerosolized medicament that may be administered to the pulmonary air passages of a patient in need thereof. Nebulizers are well known in the art and could easily be employed for administration of the claimed dispersions without undue experimentation. Breath activated nebulizers, as well as those comprising other types of improvements which have been, or will be, developed are also compatible with the stabilized dispersions and present invention and are contemplated as being within the scope thereof.

[0157] Along with DPIs, MDIs and nebulizers, it will be appreciated that the stabilized dispersions of the present invention may be used in conjunction with liquid dose instillation or LDI techniques as disclosed in, for example, WO 99 / 16421 hereby incorporated in its entirety by reference. Liquid dose instillation involves the direct administration of a stabilized dispersion to the lung. In this regard, direct pulmonary administration of bioactive compounds is particularly effective in the treatment of disorders, especially where poor vascular circulation of diseased portions of a lung reduces the effectiveness of intravenous drug delivery. With respect to LDI the stabilized dispersions are preferably used in conjunction with partial liquid ventilation or total liquid ventilation. Moreover, the present invention may further comprise introducing a therapeutically beneficial amount of a physiologically acceptable gas (such as nitric oxide or oxygen) into the pharmaceutical microdispersion prior to, during or following administration.

[0158] Methods of Use

[0159] The invention also includes methods of using the composition of the invention for treating or preventing an infection in a subject in need thereof. The method comprises the step of administering an effective amount of the composition to the subject. The infection can be an infection of the gastrointestinal tract or upper or lower respiratory tract, including the common cold, influenza, respiratory syncytial virus infection, Severe Acute Respiratory Syndrome, Middle East Respiratory Syndrome, COVID-19 or a disease caused by another emerging zoonotic virus, such as a zoonotic coronavirus. In specific aspects, the methods of the invention treat a viral respiratory infection, such as a paramyxovirus (HPIV-3) or a SARS-CoV-2 (COVID-19) respiratory infection.

[0160] In some embodiment, the invention provides a method for treating a respiratory infection caused by a paramyxovirus or a variant thereof in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound having the formula: (Peptide-Linker)n-B-Membrane Anchoring Moiety wherein each Peptide is independently a HRC Peptide or a targeting peptide, provided that at least one peptide is a HRC Peptide, each Linker is independently a bivalent linking moiety, B is a multivalent moiety comprising one or more lysines and optionally one or more spacers, Membrane Anchoring Moiety is a lipid, and n is an integer selected from 1, 2, 3 or more. Preferably, the paramyxovirus is HPIV-3.

[0161] In some embodiments, the compound comprises a targeting peptide. The targeting peptide is a RSV fusion protein targeting peptide, a RSV attachment protein targeting peptide, a NiV / HeV attachment protein targeting peptide, or a receptor binding peptide. Or the targeting peptide is a receptor binding peptide selected from CD 169 binding peptides, CD150 binding peptides, nectin-4 binding peptides, CX3CR1 binding peptides, heparin binding peptides, chondroitin sulfate-binding peptides, dermatan sulfate-biding peptides, ephrin-B2 binding peptides, and ephrin-B3 binding peptides.

[0162] In some embodiment, the invention provides a method for treating a respiratory infection caused by a SAR-Cov-2 variant in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of a compound having the formula: (Peptide-Linker)n-B-Membrane Anchoring Moiety wherein each Peptide is independently a HRC Peptide or a targeting peptide, provided that at least one peptide is a HRC Peptide, each Linker is independently a bivalent linking moiety, B is a multivalent moiety comprising one or more lysines and optionally one or more spacers, Membrane Anchoring Moiety is a lipid, and n is an integer selected from 1, 2, 3 or more. Preferably, the SAR-Cov-2 variant comprises at least 5 mutations, wherein the at least 5 mutations are independently in the spike protein SI subunit or the S2 subunit or combinations thereof.

[0163] In embodiments, the compound comprises a targeting peptide and one or more HRC Peptide. In embodiments, the targeting peptide is an ACE2 targeting peptide or a receptor binding domain peptide. In embodiments, the Hydrophobic Moiety is cholesterol.

[0164] In some embodiments, the at least 5 mutations are independently in N-Terminal domain (NTD), the receptor binding domain (RBD), the fusion peptide (FP)domain, the heptad repeat 1 (HR1) domain, or combinations thereof. In some embodiments, the at least 5 mutations are independently selected from the mutations.

[0165] In some embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Alpha variant; at least 5 mutations from the SAR- Cov-2 Beta variant; at least 5 mutations from the SAR-Cov-2 Delta variant; or at least 5 mutations from the SAR-Cov-2 Omicron variant. In embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Alpha variant. In embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Beta variant. In embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Delta variant. In embodiments, the at least 5 mutations are independently selected from at least 5 mutations from the SAR-Cov-2 Omicron variant.

[0166] In some embodiments, the variant comprises at least 10 mutations. In additional embodiments, the variant comprises at least 15 mutations. In yet additional embodiments, the variant comprises at least 20 mutations.

[0167] In some embodiments, the SARS-CoV-2 variant comprises at least one variant selected from B.1.1.7 (Alpha), B.1.351 (Beta), P.l (Gamma), B.1.617.2 (Delta), B.1.429 / B.1.427 (Epsilon), B.1.617.1 (Kappa), B.1.525 (Eta), B.1.526 (Iota), P.3 (Theta), P.2 (Zeta), and B.1.1.529 (Omicron).

[0168] In some embodiments, the SARS-CoV-2 variant comprises at least one variant selected from A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.l lineage, P.l, P.2, P.3, and R. l.

[0169] In some embodiments, the B.l lineage comprises at least one of (including, but not limited to, B. l, B.1.1, B.l.1.7, B.l.1.7 with E484K, B.l.2, B.1.5-B.1.72, B.l.9, B. l.13, B.1.22, B.l.26, B.l.37, B.1.3-B.1.66, B. l.177, B.1.243, B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.l.526, B.l.526.1, B.l.526.2, B.1.617, B.l.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620, and B. l.621.

[0170] In some embodiments, the administration is achieved using an intranasal spray, an inhaler or a nebulizer.

[0171] In some embodiments, the compound is administered in combination with at least one other antiviral active agent or therapy.

[0172] The subject, preferably a human, can be an individual diagnosed with the infection and is either symptomatic, pre-symptomatic, or asymptomatic, or at risk for developing infection. For example, the subject can be at risk for developing the viral respiratory infection due to direct or indirect exposure or possible exposure to the virus (such as RSV, HPIV-3, SARS-CoV-2 or a mutant thereof), such as via exposure to an infected individual or a virus-contaminated fomite. The subject can be a resident of, or a visitor to, a community in which the viral respiratory infection has been identified, for example, the subject can be a family member of an infected individual or the subject can work in a health care setting caring for infected individuals. In certain embodiments, the subject at risk for infection is asymptomatic and has tested negative for presence of the virus prior to the commencement of therapy. In specific examples, the subject can be at risk for developing a condition due to exposure to the virus, such as developing COVID-19 due to exposure to a SARS-CoV-2 virus, for example, from the respiratory droplets or aerosols of an infected individual and / or contact with a contaminated fomite. In yet further aspects, the subject is suffering from a condition associated with the viral respiratory infection.

[0173] In certain embodiments of the method of the invention, the subject suffers from another disease or condition, such as chronic obstructive pulmonary disease (COPD) or ulcerative colitis, which can be exacerbated by an infection.

[0174] The composition is preferably administered to the subject before the subject is symptomatic (e.g., pre-symptomatic), or at the onset of symptoms. The composition can be administered at a variety of dosing schedules. For example, the composition can be administered one or more times and over a course of one or more days. In certain embodiments, the composition is administered one or more times per day for one to 10 days. In certain embodiments, the composition is administered one or more times per day until the subject is asymptomatic and / or testing for the virus is negative.

[0175] The composition can be administered to the nasal passages using routine methods and devices (see D. Marx et al., IntechOpen, DOI: 10.5772 / 59468. Available from: https: / / www.intechopen.com / books / drug-discovery-and-development-from-molecules-to- medicine / intranasal-drug-administration-an-attractive-delivery-route-for-some-drugs). For example, the composition can be administered to the nasal passages as drops or as an aerosol spray, for example, using an aerosol bottle or a multi-dose spray pump, which can provide a uniform metered dose. The volume per dose can be varied, but is typically from about 50 to about 150 pl. The desired volume will depend on the desired dose of the active agent and the concentration of the active agent in the composition.

[0176] Where delivery to the pulmonary system, or lungs, is desired it can be efficacious to aerosolize a low concentration solution of the active agent for an extended period, such as overnight. Combination Therapies

[0177] The compound or composition described herein can be co-administered with other active agents and therapies.

[0178] In some embodiments, the other active agent includes, but is not limited to, antibodies against a paramyxovirus such as HPIV-3. For example, the protective antibodies PI3-E12 against HPIV-3 are described in Boonyaratanakornkit et al., “Protective antibodies against human parainfluenza virus type 3 infection”, mAbs. Volume 13, 2021, Issue 1, https: / / doi.org / 10.1080 / 19420862.2021.1912884.

[0179] In some embodiments, the other active agent includes, but is not limited to, antibodies against SARS-CoV-2. Suitable antibodies are described in, for example, US 2022 / 0017604, US 2022 / 0017614; US 2021 / 0403550, US 2021 / 0395345, US 2021 / 0403537; US 2021 / 0388066, US 2021 / 0388065, US 2021 / 0347859, or US 2021 / 0309733, which are incorporated herein by reference. In some embodiments, the antibody is a monoclonal antibody such as casirivimab, imdevimab, bamlanivimab, or etesevimab. In embodiments, the antibody is a monoclonal antibody therapy such as casirivimab plus imdevimab, bamlanivimab, or bamlanivimab plus etesevimab.

[0180] The active agents and compositions of the present invention are also intended for use with general care provided patients with viral infections, including parenteral fluids (including dextrose saline and Ringer's lactate) and nutrition, antibiotic (including metronidazole and cephalosporin antibiotics, such as ceftriaxone and cefuroxime) and / or antiviral prophylaxis, fever (e.g., acetaminophen) and pain medication, antiemetic (such as metoclopramide) and / or antidiarrheal agents, vitamin and mineral supplements (including Vitamin K and zinc sulfate), anti-inflammatory agents (such as ibuprofen), pain medications, and medications for other common diseases in the patient population, such as artemether, artesunate-lumefantrine combination therapy), quinolone antibiotics, such as ciprofloxacin, macrolide antibiotics, such as azithromycin, cephalosporin antibiotics, such as ceftriaxone, or aminopenicillins, such as ampicillin), or shigellosis.

[0181] The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0182] Co-administration of a compound of the invention with one or more other active therapeutic agents generally refers to simultaneous or sequential administration of a compound of the invention and one or more other active therapeutic agents, such that therapeutically effective amounts of the compound of the invention and one or more other active therapeutic agents are both present in the body of the patient.

[0183] Co-administration includes administration of unit dosages of the compounds of the invention before or after administration of unit dosages of one or more other active therapeutic agents, for example, administration of the compounds of the invention within seconds, minutes, or hours of the administration of one or more other active therapeutic agents and / or as part of the same treatment regimen. For example, a unit dose of a compound of the invention can be administered first, followed within seconds or minutes or days by administration of a unit dose of one or more other active therapeutic agents. Alternatively, a unit dose of one or more other therapeutic agents can be administered first, followed by administration of a unit dose of a compound of the invention within seconds or minutes or days. In some cases, it may be desirable to administer a unit dose of a compound of the invention first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more other active therapeutic agents. In other cases, it may be desirable to administer a unit dose of one or more other active therapeutic agents first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a compound of the invention.

[0184] The combination therapy may provide "synergy" and "synergistic," i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.

[0185] As used herein, the words “a” and “an” are meant to include one or more unless otherwise specified. For example, the term “an agent” encompasses both a single agent and a combination of two or more agents.

[0186] The term "treating" or "treatment" as used herein covers the treatment of the disease or condition of interest (e.g., a respiratory infection) in a mammal, preferably a human, having the disease or condition of interest, and includes, for example: preventing or delaying the onset of the disease or condition from occurring in a mammal, in particular, when such mammal is at risk of developing the disease but has not yet become symptomatic and / or been diagnosed as having it; inhibiting the disease or condition, i.e., arresting its development; relieving the disease or condition, i.e., causing regression of the disease or condition; and / or stabilizing the disease or condition. Treatment includes ameliorating or lessening the severity of symptoms of the disease or condition, and / or inhibition of further progression or worsening of those symptoms. Treatment also includes shortening the time course and / or severity of a disease or condition compared to the expected or historical time course and / or severity of the disease.

[0187] As used herein the terms "preventing," means causing the clinical symptoms of a disease or condition not to develop and includes inhibiting the onset of a viral infection in a subject that may be exposed to or predisposed to the viral infection but does not yet experience or display symptoms of the infection.

[0188] An “effective amount” or a “therapeutically effective amount” of a compound or composition described herein refers to an amount of the compound that is sufficient to achieve a specific effect or result, and / or prevents or treats the disease or condition and / or the symptoms therefore, for example, alleviating, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition. The “effective amount” and “therapeutically effective amount” includes specifically an anti-viral amount of a compound of the invention (alone or in combination with another active agent) or the composition described herein.

[0189] Examples

[0190] Example 1. “All on resin” one-shot synthesis

[0191] Compounds of this application were synthesized using an automated chemistry platform, described in Hartrampf et al. “Synthesis of proteins by automated flow chemistry”. Science 2020, Vol 368, Issue 6494, 980-987. DOI: 10.1126 / science.abb2491. The synthesis of DCOY104 as an example was shown as below:

[0192]

[0193] The compound, ^ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGSGS^G-42Peg4)2-43K-44K-NH2, was synthesized using Fmoc-based solid phase peptide synthesis on an Automated Flow Peptide Synthesizer (AFPS) from C to N termini using PyAOP as an activator and DIEA as a base, where44K was Fmoc Lys (Mtt) OH,43K was Fmoc Lys (Fmoc)OH,42Peg4 was a Fmoc-NH-PEG4-CH2COOH. After taking out from AFPS, the N- terminus was acetylated and then Mtt side chain protecting group on44K was selectively deprotected, followed by coupling with Fmoc-NH-PEG4-CH2COOH. The Fmoc group was deprotected, and Peg4-amine was coupled with cholesteryl hydrogen succinate. The global deprotection was performed by subjecting resin to TFA (94%), Water (2.5%), EDT (2.5%), and TIPS (1%) for one hour. After the one-hour resin was filtered followed by washing one more time with TFA. The chilled ether was added to the combined filtrates to precipitate out the desired final product. This precipitate was purified by reverse phase-high-performance liquid chromatography to attain the desired purity. LCMS confirmed the final product. Example 2, In vitro assay

[0194] Inhibition of virus-induced cytopathic effects (CPE) and cell viability following human respiratory syncytia virus replication in Hep2 cells was measured by XTT tetrazolium dye. Cells (5 X 103cells per well) were seeded in 96-well flat-bottom tissue culture plates and allowed to adhere overnight. Following overnight incubation, diluted test compounds and virus diluted to a pre-determined titer to yield 85% to 95% cell killing at 6 days post-infection were added to the plate. Following incubation at 37 °C, 5% CO2 for six days, cell viability was measured by XTT staining. The optical density of the cell culture plate will be determined spectrophotometrically at 540 and 650 nm using Softmax Pro 4.6 software. Percent CPE reduction of the virus-infected wells and the percent cell viability of uninfected drug control wells were calculated to determine the EC50 and TC50 values using four parameter curve fit analysis. The results were shown in Table 1.

[0195] Table 1

[0196] While this invention has been particularly shown and described with references to preferred embodiments 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.

[0197] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A compound having the formula:(Peptide-Linker)n-B- Membrane Anchoring Moiety wherein each Peptide is independently a HRC Peptide or a targeting peptide, provided that at least one peptide is a HRC Peptide, each Linker is independently a bivalent linking moiety, B is a multivalent moiety comprising one or more diaminoaliphatic acids, preferably lysines, and optionally one or more spacers, Membrane Anchoring Moiety is a lipid, and n is an integer selected from 1, 2, 3 or more.

2. The compound of claim 1, wherein B comprises one, two, three, four, five, or six lysines.

3. The compound of claim 1 or 2, wherein the spacers are independently selected from polyethylene glycol (PEG) derivatives.

4. The compound of any one of the preceding claims, wherein the compound comprises a targeting peptide and one or more HRC peptides.

5. The compound of any one of the preceding claims, wherein the HRC peptide is a paramyxovirus HRC peptide.

6. The compound of any one of the preceding claims, wherein the HRC peptide is a RSV HRC peptide, a coronavirus HRC peptide, or a HPIV HRC peptide.

7. The compound of any one of the preceding claims, wherein the Membrane Anchoring Moiety is selected from cholesterol, fatty acid, long alkyl chain, 3P-cholesterylamine, 3P-cholesterylthiol, cholesteryl analogues and derivatives, 3P-cholesterylamine type molecule, and dimer, trimer or oligomer format of the above (preferably, cholesterol).

8. The compound of any one of the preceding claims, wherein the linker is AAY, EAAK, (GGGGS)3, (GGGGS)2, GGGGS, or GSGSG.

9. The compound of any one of claims 1-7, wherein the linker is a GS linker.

10. The compound of any one of the preceding claims, wherein the linker is GSGSG.

11. The compound of claim 1, wherein the compound is represented by Formula (III):Peptide — LinkerFormula (III), wherein m is 0, 1, 2, 3, 4, or 5; each p is an integer independently selected from the range of 0-40.

12. The compound of claim 10, wherein each p is an integer independently selected from the range of 2-20, and the Membrane Anchoring Moiety comprises a cholesterol.

13. The compound of any one of the preceding claims, wherein at least one peptide is a wild type HRC peptide or a variant thereof, wherein the wild type HRC peptide comprises an amino acid sequence selected from:Acq-VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI (SEQ ID NO. 1) and Acq-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 7), wherein q is 0 or 1.

14. The compound of any one of the preceding claims, wherein at least one peptide comprises an amino acid sequence selected from:Acq-VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI (SEQ ID NO. 1), Acq-VALDPIDISIVLNKIKSDLEESKEWIRRSNKILDSI (SEQ ID NO. 2), Acq-VALDPIDISIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 3), Acq-VALDPIDFSIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 4), Acq-VALDPIDISIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 5), Acq-VALDPIDFSIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 6),Acq-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 7), Acq-dVdAdLdDdP IDISIELNKAKSDLEESKEWIRRSNQKLDSI (SEQ ID NO. 8), Acq-dVdAdLdDdP IDISIVLNKIKSDLEESKEWIRRSNKILDSI (SEQ ID NO. 9), Acq-dVdAdLdDdP IDISIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 10), Acq-dVdAdLdDdP IDFSIVLNKIKSQLEESKWEIRRSNKILDSI (SEQ ID NO. 11), Acq-dVdAdLdDdP IDISIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 12),Acq-dVdAdLdDdP IDFSIVLNKIKSQLEESKEWIRRSNKILDSI (SEQ ID NO. 13), andAcq-dldGdSdldD NASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO. 14).

15. The compound of any one of claims 11-14, wherein m is 0, 1, or 2.

16. The compound of any one of claims 11-15, wherein all the peptides are the same and comprise an amino acid sequence selected from SEQ ID NO. 1-12.

17. A pharmaceutical composition comprising a compound of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. A method for treating or preventing a respiratory infection associated with a human parainfluenza virus (HPIV), in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound having the formula:(Peptide-Linker)n-B- Membrane Anchoring Moiety, wherein each Peptide is independently a HPIV HRC Peptide or a targeting peptide, provided that at least one peptide is a HPIV HRC Peptide, each Linker is independently a bivalent linking moiety, B is a multivalent moiety comprising one or more lysines, Membrane Anchoring Moiety is a lipid, and n is an integer selected from 1, 2, 3 or more.

19. The method of claim 18, wherein the HPIV is HPIV-1, HPIV-2, HPIV-3, or HPIV-4.

20. The method of claim 18 or 19, wherein the HPIV is HPIV-3.

21. The method of any one of claims 18-20, wherein the HPIC HRC peptide comprises an amino acid sequence selected from SEQ ID NO. 1-6 and 8-13.

22. The method of any one of claims 18-21, wherein the Hydrophobic Moiety is cholesterol.

23. The method of any one of claims 18-22, wherein the compound is represented by the formula:wherein the linker is GSGSG, the peptide has an amino acid sequence selected from SEQ ID NO. 1-14, and each p is independently an integer selected from 2-20.

24. The method of any one of the preceding claims, wherein the administration is achieved using an intranasal spray, an inhaler or a nebulizer.

25. A method for treating or preventing a respiratory infection associated with a SARS- Cov-2 variant, in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound having the formula: (Peptide-Linker)n-B- Membrane Anchoring Moiety, wherein each Peptide is independently a HRC Peptide of the SARS-Cov-2 variant or a targeting peptide, provided that at least one peptide is a HRC Peptide of the SARS- Cov-2 variant, each Linker is independently a bivalent linking moiety, B is a multivalent moiety comprising one or more lysines, Membrane Anchoring Moiety is a lipid, and n is an integer selected from 1, 2, 3 or more.

26. The method of claim 25, wherein the targeting peptide is an ACE2 targeting peptide or a receptor binding domain peptide.

27. The method of claim 25 or 26, wherein the HRC peptide comprises an amino acid sequence of SEQ ID NO. 7 or SEQ ID NO.

14.

28. The method of any one of claims 25-27, wherein the Hydrophobic Moiety is cholesterol.

29. The method of any one of claims 25-28, wherein the compound is represented by the formula:wherein the linker is GSGSG, the peptide has an amino acid sequence is SEQ ID NO. 7 or 14, and each p is independently an integer selected from 2-20.

30. The method of any one of claims 25-29, wherein the administration is achieved using an intranasal spray, an inhaler or a nebulizer.

31. A method of making a product wherein the method comprises the steps of: a) binding at least one N-protected diamino acid, such as an N-protected lysine, which is a diamino acid substituted by a protecting group on an amine, to a solid support comprising surface amino groups to prepare an immobilized N-protected diamine comprising a primary amine;b) reacting the immobilized N-protected diamine with a diamino acid, such as lysine, or an N-protected diamino acid, such as N-protected lysine, to prepare an immobilized N-protected polyamine having one or more primary amines; c) optionally repeating step (b); d) optionally reacting each primary amine in the immobilized N-protected polyamine of step (b) or step c) with a linker moiety; e) synthesizing an amino acid sequence, preferably in an automated flow peptide synthesis system (AFPS), attached to the immobilized N-protected polyamine of step (b) or step (c), or to the linker moiety of step (d); f) optionally, protecting each N-terminus of each amino acid sequence such as substituting each primary amine with an acyl, or acetyl group; g) removing each protecting group to form a product having at least one primary amine; h) optionally, reacting each primary amine with a linker moiety; i) reacting either the primary amine of the product of step (g) or the linker moiety of the product of step (h) with a functional group; j) cleaving the product of step (i) from the solid support.

32. The method of claim 31, wherein the product has a structure:wherein n and m are independently an integer of 1 or more; wherein m+n is preferably 3 or more; each LI and L2 are independently the same or different linker; each Peptide comprises an amino acid sequence, preferably a therapeutic peptide;each R is a functional group; andX is a peptide comprising two or more diamino acids.

33. A peptide conjugate made by the method of claim 31 or 32.

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