Viral fusion inhibitors

Abstract

The present invention relates to viral fusion inhibitors, as well as use of these viral fusion inhibitors in the treatment or prevention of infectious disease or infection.

Description

25856 VIRAL FUSION INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No.63 / 617,452 filed January 04, 2024, the entire contents of which are incorporated by referenceherein. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (25856-WO-PCT-SL.xml; Size:1,326,732 bytes; and Date of Creation: November 27, 2024) are herein incorporated by referencein their entirety. BACKGROUND OF THE INVENTION

[0003] In the process of infection, severe acute respiratory syndrome coronavirus (SARS-CoV)enters host cells by a process in which fusion occurs between the viral membrane and the host cell membrane. The fusion process for SARS-CoV occurs at either the cell surface membrane or at the endosomal membrane. The fusion process is mediated by the envelope S glycoprotein that is a type I transmembrane glycoprotein. The envelope glycoprotein of class I viruses plays an important role in the fusion process for all human coronaviruses similarly to many other viruses including human immunodeficiency virus (HIV), Influenza, Ebola, Marburg, etc. In these viruses, one of the two enveloped glycoproteins mediates the fusion process after a conformational transition is triggered upon receptor binding. In the coronaviruses, the key player in the infectivity process is the S protein consisting of two sub-units, S1 and S2. The S1 subunit binds the cellular receptor through its receptor-binding domain (RBD), followed byconformational changes in the S2 subunit, which allows the fusion peptide to insert into the hosttarget cell membrane. The heptad repeat 1 (HR1) region in the S2 subunit forms a homo-trimeric assembly, which exposes three conserved hydrophobic grooves on the surface that bind heptad repeat 2 (HR2). A pre hairpin intermediate of this six-helix bundle (6-HB) core structure is formed during the fusion process and helps bring the viral and cellular membranes into close proximity for viral fusion and entry.SUMMARY

[0004] The present disclosure is based upon the discovery that fusion between a viralmembrane and a host cell membrane can be reduced by a compound that interrupts interaction ofenvelope glycoproteins (e.g., interaction between HR1 and HR2).

[0005] Among other things, the present disclosure provides viral fusion inhibitors comprising apeptide moiety, a linker moiety, and a lipid moiety. In some embodiments, viral fusion inhibitors of the present disclosure provide pan-coronavirus activities.

[0006] In embodiments of the present invention, a peptide moiety of a viral fusion inhibitor isbased on in general the HR2 region of SARS-CoV-2. In some embodiments, a certain portion of the HR2 region, when incorporated in a viral fusion inhibitor, may in fact provide more effective antiviral activities and / or pan-coronavirus activities. The present disclosure is based, in part, on the insight that one or more mutations or substitutions in a peptide from the HR2 region can enhance antiviral potency.

[0007] Additionally or alternatively, the present disclosure describes certain features (e.g.,conjugation chemistry between a peptide moiety and a linker moiety, conjugation chemistry between a linker moiety and a lipid moiety, and the length or physical properties of a linker moiety) of the viral fusion inhibitors, which may play an important role in antiviral potency and / or pan-coronavirus activity.

[0008] In one aspect, provided are inhibitors of viral fusion comprising (i) a peptide moiety, (ii)a linker moiety, and (iii) a lipid moiety, wherein the linker moiety connects the C-terminal part ofthe peptide moiety to the lipid moiety, wherein the peptide moiety comprises at least tencontiguous amino acids of the heptad repeat 2 (HR2) domain of severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2), and wherein the N-terminal part of the peptide moietyis PDVD as set forth in SEQ ID NO. 418. In some embodiments, the peptide moiety comprises apeptide as set forth in SEQ ID NO:1, X1is P, and X2is D.

[0009] In one aspect, provided are inhibitors of viral fusion comprising (i) a peptide moiety, (ii)a linker moiety, and a lipid moiety, wherein the linker moiety connects the C-terminal part of thepeptide moiety to the lipid moiety, wherein the peptide moiety comprises at least ten contiguousamino acids of the heptad repeat 2 (HR2) domain of severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2), and wherein the N-terminal part of the peptide moiety is PEVD asset forth in SEQ ID NO. 419. In some embodiments, the peptide moiety comprises a peptide asset forth in SEQ ID NO:1, X1 is P, and X2 is E.

[0010] In one aspect, provided are inhibitors of viral fusion comprising (i) a peptide moiety, (ii)a linker moiety; and (iii) a lipid moiety, wherein the linker moiety connects the C-terminal part ofthe peptide moiety to the lipid moiety, wherein the peptide moiety comprises at least tencontiguous amino acids of the heptad repeat 2 (HR2) domain of severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2), and wherein the N-terminal part of the peptide moietyis (2R,4S)Prot4OacOH-DVD. In some embodiments, the peptide moiety comprises a peptide asset forth in SEQ ID NO:1, X1 is (2R,4S)Prot4OacOH, and X2 is D.

[0011] In some embodiments, the peptide moiety comprises at least one amino acid that isdifferent from the HR2 domain of SARS-CoV-2.

[0012] In some embodiments, the peptide moiety comprises at least one amino acid that is notnatural.

[0013] In some embodiments, the peptide moiety further comprises C at the C-terminal part ofthe peptide moiety. In some embodiments, the peptide moiety further comprises CGSG as setforth in SEQ ID NO.421 at the C-terminal part of the peptide moiety. In some embodiments, the peptide moiety further comprises GGGSGGGGSG as set forth in SEQ ID NO.422 at the C-terminal part of the peptide moiety. In some embodiments, the peptide moiety further comprisesGSGGGSGG as set forth in SEQ ID NO. 423 at the C-terminal part of the peptide moiety. Insome embodiments, the peptide moiety further comprises KYEQYIG as set forth in SEQ ID NO. 424 at the C-terminal part of the peptide moiety.

[0014] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOs: 2-39. In some embodiments, the peptide moiety is selectedfrom the group consisting of peptides as set forth in SEQ ID NOs: 3-39. In some embodiments,the peptide moiety comprise a peptide as set forth in SEQ ID NO: 40. In some embodiments, thepeptide moiety is selected from the group consisting of peptides as set forth in SEQ ID NOs: 279,287, 288, 291, 292, 296, 300, 303, 308-376, 395-398, and 407-413. In some embodiments, thepeptide moiety is selected from the group consisting of peptides as set forth in SEQ ID NOs: 309,389, 399, or 401-404. In some embodiments, the peptide moiety is selected from the groupconsisting of peptides as set forth in SEQ ID NOs: 377, 378, 405, 406, and 414-416.

[0015] In some embodiments, the lipid moiety comprises cholesterol, tocopherol, or palmitate.In some embodiments, the lipid moiety comprises cholesterol.

[0016] In some embodiments, the linker moiety is attached to the lipid moiety via an etherbond, an ester bond, a carbamate bond, or a triazole bond. In some embodiments, the linker25856moiety is attached to the lipid moiety via an ether bond. In some embodiments, the linker moietyis attached to the lipid moiety via an triazole bond. In some embodiments, the linker moietycomprises an amino acid. In some embodiments, the linker moiety is selected from the groupconsisting of compounds listed in Table 2. In some embodiments, the linker moiety comprisesPEG12-C(propyl)-NH2, Ttds2-C(propyl)-NH2, Ttds3-C(propyl)-NH2, Ttds4-C(propyl)-NH2, Ttds2-Dab(triazole-propyl)-NH2, Ttds3-Dab(triazole-propyl)-NH2, Ttds4-Dab(triazole-propyl)-NH2, Ttds2-Orn(triazole-propyl)-NH2, or Ttds3-Orn(triazole-propyl)-NH2.

[0017] In some embodiments, the inhibitor is selected from the group consisting of compoundslisted in Table 3A.

[0018] In some embodiments, the N-terminal part of the peptide moiety is acetylated.

[0019] In some embodiments, the inhibitor further comprises a half-life extension moiety. Insome embodiments, the half-life extension moiety comprises 4-(p-iodophenyl)butyric acid(4IPhBut). In some embodiments, the the half-life extension moiety is attached to the N-terminalpart of the peptide moiety. In some embodiments, the half-life extension moiety is selected fromthe group consisting of 4IPhBut-eK-Succinyl, 4IPhBut-eK-Succinyl-AEEA, 4IPhBut-eK-Succinyl-(AEEA)2, 4IPhBut-eK-Succinyl-(AEEA)3, and 4IPhBut-eK-Succinyl-GSGSG (SEQID NO: 425).

[0020] In some embodiments, the peptide moiety has a Lys, D-Lys or Orn mutation, and thehalf-life moiety is attached to the mutation.

[0021] In some embodiments, the peptide moiety has Lys in position 1168.

[0022] In some embodiments, the peptide moiety comprise a peptide as set forth in SEQ IDNO: 407.

[0023] In some embodiments, the half-life extension moiety is selected from the groupconsisting of 4IPhBut-eK-Succinyl, 4IPhBut-eK-Succinyl-AEEA, 4IPhBut-eK-Succinyl- (AEEA)2, and 4IPhBut-eK-Succinyl-(AEEA)3.

[0024] In one aspect, provided are compositions comprising the inhibitor, or a pharmaceuticallyacceptable carrier or diluent.

[0025] In one aspect, provided are inhibitors for use in a method of treatment or prevention of asubject. In some embodiments, the treatment or prevention is for infection by a virus selectedfrom the group consisting of SARS-CoV-1, SARS-CoV-2, MERS, and HIV-I. In someembodiments, the inhibitor is capable of treating and / or preventing two or more types of thevirus. In some embodiments, the virus is SARS-CoV-2 or MERS.

[0026] In one aspect, provided are methods of treating or preventing infectious disease in asubject, comprising administering to the subject an effective amount of a composition comprisingthe inhibitor. In some embodiments, the infectious disease is caused by a virus selected from thegroup consisting of SARS-CoV-1, SARS-CoV-2, MERS, and HIV-I. In some embodiments, thecomposition is administered intranasally, intratracheally, subcutaneously or intranasally. In someembodiments, the composition is administered as nasal drops, nasal powder, or a spray.

[0027] In one aspect, provided are uses of the inhibitor for the manufacture of a medicament forthe treatment or prevention of infection or infectious disease. In some embodiments, theinfectious disease is caused by a virus selected from the group consisting of SARS-CoV-1, SARS-CoV-2, MERS, and HIV-I. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows a structure of an exemplary viral fusion inhibitor comprising a peptidemoiety, a linker moiety, and a lipid moiety.

[0029] Figure 2 shows an exemplary modification in a linker moiety of the viral fusioninhibitor in Figure 1 and its resulting structure of another exemplary viral fusion inhibitor. n may be 1, 2, 3, or 4.

[0030] Figure 3 shows a structure of another exemplary viral fusion inhibitor comprising atriazole bond.

[0031] Figures 4A and 4B depict structures of certain amino acids or reagents used for mutationof the peptide moiety.

[0032] Figure 5 shows a structure of another exemplary viral fusion inhibitor using Glu orhGlu-based conjugation. X may be 2 or 3, and Y may be 1 or 4.

[0033] Figure 6 shows structures of another exemplary viral fusion inhibitor using Orn-basedconjugation.

[0034] Figure 7 shows a structure of another exemplary viral fusion inhibitor comprisingcarbamate.

[0035] Figures 8, 8-1 and 8-2 depict an exemplary synthesis process for viral fusion inhibitorscomprising Glu-C3-O-cholesterol. Figures disclose SEQ ID NO: 682.

[0036] Figures 9, 9-1 and 9-2 depict an exemplary synthesis process for viral fusion inhibitorscomprising hGlu-C3-O-cholesterol. Figures disclose SEQ ID NO: 683.

[0037] Figures 10, 10-1 and 10-2 depict an exemplary synthesis process for viral fusioninhibitors comprising Glu-C6-O-cholesterol. Figures disclose SEQ ID NO: 684.

[0038] Figures 10, 11-1 and 11-2 depict an exemplary synthesis process for viral fusioninhibitors comprising hGlu-C6-O-cholesterol. Figures discloses SEQ ID NO: 685.

[0039] Figures 12, 12-1 and 12-2 depict an exemplary synthesis process for viral fusioninhibitors comprising Orn-C3-O-cholesterol. Figures disclose SEQ ID NO: 686.

[0040] Figures 11 and 13-1 depict an exemplary synthesis process for viral fusion inhibitorscomprising Lys-carbamate-cholesterol. Figures disclose SEQ ID NO: 687.

[0041] Figures 14, 14-1, 14-2 and 14-3 depict an exemplary synthesis process for viral fusioninhibitors comprising PEG11- carbamate-cholesterol.

[0042] Figure 12 shows a structure of a viral fusion inhibitor comprising lysine of which sidechain is functionalized.

[0043] Figures 13 and 16-1 depict an exemplary synthesis process for viral fusion inhibitorscomprising amide conjugation points. Figures disclose SEQ ID NO: 682.

[0044] Figures 17 and 17-1 depict another exemplary synthesis process for viral fusioninhibitors comprising amide conjugation points. Figures disclose SEQ ID NO: 688.

[0045] Figures 14-21 show pharmacokinetics of certain viral fusion inhibitors after intravenousand subcutaneous dosing on mouse.

[0046] Figures 22-24 show mouse antiviral efficacy at various subcutaneous doses withPepSP1141.

[0047] Figures 25-26 show mouse antiviral efficacy at various intravenous doses withPepSP1141.

[0048] Figures 27-28 show hamster antiviral efficacy at various subcutaneous doses withPepSP1545.

[0049] Figure 29A depicts a structure of 4-(p-iodophenyl)butyric acid (4IPhBut), also known asAlbutag.

[0050] Figure 29B depicts a structure of succinyl-eK(4IPhBut).

[0051] Figure 30 shows Albutag derivatization at position 1168 or the N-terminus. Figurediscloses SEQ ID NOS 689 and 425, respectively, in order of appearance.

[0052] Figure 31 shows a structure of Compound X. Figure discloses SEQ ID NOS 690-691,respectively, in order of appearance.

[0053] Figure 32 shows a structure of Compound Y. Figure discloses SEQ ID NOS 692-694,respectively, in order of appearance.

[0054] Figure 33 depicts concentrations of compounds in mouse plasma after a 15 mg / kgsubcutaneous dosing.

[0055] Figure 34 demonstrates levels of compounds in mouse lung tissue after the 15 mg / kgsubcutaneous dosing, detected by LC-MS / MS.

[0056] Figure 35 shows ex vivo analysis of mouse lung tissue by a pseudoneutralizationbioassay after the 15 mg / kg subcutaneous dosing. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0057] Certain technical and scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure relates.

[0058] “About” when used to modify a numerically defined parameter means that theparameter is within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of the stated numerical value or range for that parameter; where appropriate, the stated parameter may be rounded to the nearest whole number.

[0059] As used herein, including the appended claims, the singular forms of words such as “a,”“an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

[0060] The terms “administration” or “administer” refers to the act of injecting or otherwisephysically delivering a substance as it exists outside the body into a patient, such as by oral,mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any othermethods of physical delivery described herein or known in the art.

[0061] The term “agent” refers to a compound or entity of any chemical class including, forexample, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. As will be clear from context, in some embodiments, an agent can be or comprise a cell or organism, or a fraction, extract, or component thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and / or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that are man-made in that it isdesigned, engineered, and / or produced through action of the hand of man and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized include small molecules, antibodies, antibody fragments, aptamers, siRNAs, shRNAs, DNA / RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, peptide nucleic acids, small molecules, etc. In some embodiments, an agent is or comprises a polymer. In some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an agent comprises a therapeutic, diagnostic and / or drug.

[0062] As used herein, the term “amino acid” refers to any compound and / or substance that canbe incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid;in some embodiments, an amino acid is an L-amino acid. In some embodiments, an amino acid,including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structuralmodification as compared to the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared to the general structure.

[0063] As used herein, the term “N-terminal group” is a group that is covalently bonded to theN-terminus amino group and may in some cases mimic an amino acid side chain. Suitable N- terminal capping groups include acyl groups, for example, CH3CO—, CH3CH2CO—, CH3CH2CH2CO—, CH3(CH2)3CO—, CH3(CH2)4CO—, CH3(CH2)5CO—, CH3(CH2)6CO—, CH3(CH2)7CO—, CH3(CH2)8CO—, CH3(CH2)9CO—, CH3(CH2)10CO—, CH3(CH2)11CO—,CH3—(CH2)4(cis-CH2 2)CH2(cis- 2)(CH2)7—CO—, CH3(CH2)4(cis-2(cis- 2(cis- —(CH2)4—CO—, CH3(CH2)12—CO— andCH3(CH2)14—CO—.

[0064] The term “C-terminal capping group” is a group that is covalently bonded to the C-terminal carboxy group or transforms the C-terminal carboxy group into an amide group. In some embodiments, the C-terminal capping group assists in stabilization of helical structure, forexample, by participating in hydrogen bonds. Suitable C-terminal capping groups include amineand substituted amines. Examples of amines include, but are not limited to, —NH2, —NH(alkyl)such as —NH(methyl), —NH(ethyl), —NH(propyl), and —N(alkyl)2 such as —N(methyl)2, —N(ethyl)2 and —N(methyl)(ethyl), especially —NH2 and —NH(alkyl).

[0065] As used herein, the terms “at least one” item or “one or more” item each include a singleitem selected from the list as well as mixtures of two or more items selected from the list.

[0066] The term “pan-coronavirus activity” or “pan-coronavirus potency” refers to anti-viralactivity against two or more different types of coronavirus (e.g., alphacoronavirus, betacoronavirus (e.g., embecovirus, sarbecovirus, merbecovirus, nobecovirus, hibecovirus), gammacoronavirus, deltacoronavirus). In some embodiments, pan-coronavirus activity or pan- coronavirus potency refers to anti-viral activity against two or more of SARS-CoV-1 (i.e., the virus behind the 2002–2003 severe acute respiratory syndrome outbreak), mutant forms that the SARS-CoV-1 could adopt, SARS-CoV-2, mutant forms that the SARS-CoV-2 could adopt, and / or major lineages of coronavirus (e.g., sarbecoviruses that is the subgenus including all the SARS-like viruses, betacoronaviruses that is the larger branch of the family tree that counts the pathogen responsible for Middle East respiratory syndrome (MERS), some seasonal coronaviruses that cause the common cold).

[0067] The term “peptide” or “polypeptide” refers to a string of at least two (e.g., at least three)amino acids linked together by peptide bonds. In some embodiments, a polypeptide comprises naturally-occurring amino acids; alternatively or additionally, in some embodiments, a polypeptide comprises one or more non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain and / or amino acid analogs as are known in the art may alternatively be employed). In some embodiments, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.

[0068] The term “lactam bridge” refers to the formation of an amide bond —NH— —that closes a ring to form a cyclic structure.

[0069] The term “subject” (alternatively “patient”) as used herein refers to a mammal that hasbeen the object of treatment, observation, or experiment. The mammal may be male or female. The mammal may be one or more selected from the group consisting of humans, bovine (e.g., cows), porcine (e.g., pigs), ovine (e.g., sheep), capra (e.g., goats), equine (e.g., horses), canine25856 (e.g., domestic dogs), feline (e.g., house cats), lagomorph (e.g., rabbits), rodent (e.g., rats or mice), Procyon lotor (e.g., raccoons). In particular embodiments, the subject is human.

[0070] The term “subject in need thereof” as used herein refers to a subject diagnosed with orsuspected of having cancer or an infectious disease as defined herein.

[0071] The therapeutic agents and compositions provided by the present disclosure can beadministered via any suitable enteral route or parenteral route of administration. The term“enteral route” of administration refers to the administration via any part of the gastrointestinaltract. Examples of enteral routes include oral, mucosal, buccal, and rectal route, or intragastric route. “Parenteral route” of administration refers to a route of administration other than enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumor, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal, subcutaneous, or topical administration. The therapeutic agents and compositions of the disclosure can be administered using any suitable method, such as by oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The suitable route and method of administration may vary depending on a number of factors such as the specific therapeutic agent being used, the rate of absorption desired, specific formulation or dosage form used, type or severity of the disorder being treated, the specific site of action, and conditions of the patient, and can be readily selected by a person skilled in the art.

[0072] As used herein, the term “treat” or “treating” means to administer a therapeuticcomposition comprising a viral fusion inhibitor to a subject or patient having one or more disease symptoms as provided herein. Typically, the agents are administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of the agents that is effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapeutic combination to elicit a desired response in the subject. Whether adisease symptom has been alleviated can be assessed by any clinical measurement typically usedby physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.25856

[0073] “Prevent” refers to causing a disease or symptom or manifestation of a disease not tooccur for at least a period of time in at least some individuals.

[0074] The term “pharmaceutically acceptable carrier” refers to any inactive substance that issuitable for use in a formulation for the delivery of a therapeutic agent. A carrier may be an anti- adherent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial, or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifying agent, buffer, and the like. Examples of suitable pharmaceutically acceptable carriersinclude water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and thelike), dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0075] The terms “treatment regimen,” “dosing protocol,” and “dosing regimen” are usedinterchangeably to refer to the dose and timing of administration of each therapeutic agent in a combination therapy of the disclosure.

[0076] As used herein, the term “effective amount” refers to an amount of a viral fusioninhibitor that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of an infection or a disease. An effective amount further refers to that amount of the viral fusion inhibitor sufficient to result in at least partial amelioration of symptoms. An effective amount of a therapeutic may result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in asubjective measure in cases where subjective measures are used to assess disease severity.Toxicity and therapeutic efficacy of the viral fusion inhibitor of the invention, administered aloneor in combination with another therapeutic agent, can be determined by any number of systemsor means. For example, the toxicity and therapeutic efficacy of the viral fusion inhibitor or compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD50 / ED50). The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of25856 circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

[0077] It is understood that wherever embodiments are described herein with the language“comprising,” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided.

[0078] “Consists essentially of,” and variations such as “consist essentially of” or “consistingessentially of,” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition.

[0079] Unless expressly stated to the contrary, all ranges cited herein are inclusive; i.e., therange includes the values for the upper and lower limits of the range as well as all values in between. As an example, temperature ranges, percentages, ranges of equivalents, and the like described herein include the upper and lower limits of the range and any value in the continuum there between. Numerical values provided herein, and the use of the term “about”, may includevariations of ± 1%, ± 2%, ±3%, ± 4%, ± 5%, ± 10%, ± 15%, and ± 20% and their numericalequivalents. All ranges also are intended to include all included sub-ranges, although not necessarily explicitly set forth. For example, a range of 3 to 7 days is intended to include 3, 4, 5, 6, and 7 days. In addition, the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination.

[0080] Where aspects or embodiments of the disclosure are described in terms of a Markushgroup or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in the claims.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers25856 but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0082] Exemplary methods and materials are described herein, although methods and materialssimilar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0083] The term “moiety” refers to a specific segment or functional group of a molecule.Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

[0084] The term “peptide moiety” refers to a segment of a viral fusion inhibitor, whichcomprise a peptide. In some embodiments, the C-terminal part of a peptide moiety is connected to a linker moiety.

[0085] The term “linker moiety” refers to a segment of a viral fusion inhibitor, which ispositioned between a peptide moiety and a lipid moiety. In some embodiments, the linker moiety connects a C-terminal part of the peptide moiety and the lipid moiety.

[0086] The term “lipid moiety” refers to a segment of a viral fusion inhibitor, which comprise alipid. In some embodiments, a lipid moiety is connected to a linker moiety.

[0087] The term “viral fusion inhibitor” refers to an agent whose presence, level, or degreecorrelates with decreased level or activity of viral fusion that occurs between a viral membrane and a host cell membrane. In some embodiments, the viral fusion inhibitor comprises a peptide moiety, a linker moiety, and a lipid moiety. Viral Fusion inhibitors

[0088] In some embodiments, a viral fusion inhibitor comprises a peptide moiety, a linkermoiety, and a lipid moiety. In some embodiments, the linker moiety connects the peptide moiety and the lipid moiety. In some embodiments, the linker moiety is connected to the C-terminal part of the peptide moiety.

[0089] In some embodiments, a peptide moiety is based on the HR2 domain of SARS-CoV-2.In some embodiments, the peptide moiety comprises at least five contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least six contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide25856 moiety comprises at least seven contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least eight contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least nine contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least ten contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least eleven contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least twelve contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least thirteen contiguous amino acids of the HR2 domain of SARS- CoV-2. In some embodiments, the peptide moiety comprises at least fourteen contiguous amino acids of the HR2 domain of SARS-CoV-2. In some embodiments, the peptide moiety comprises at least fifteen contiguous amino acids of the HR2 domain of SARS-CoV-2.

[0090] In some embodiments, a peptide moiety is based on the HR2 domain of SARS-CoV-1.In some embodiments, the peptide moiety comprises at least five contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least six contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least seven contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least eight contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least nine contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least ten contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least eleven contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least twelve contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least thirteen contiguous amino acids of the HR2 domain of SARS- CoV-1. In some embodiments, the peptide moiety comprises at least fourteen contiguous amino acids of the HR2 domain of SARS-CoV-1. In some embodiments, the peptide moiety comprises at least fifteen contiguous amino acids of the HR2 domain of SARS-CoV-1.

[0091] In some embodiments, the peptide moiety comprises the amino sequence spanning theHR2 region of SARS-CoV-2 in 1168-1203, 1162-1203, 1147-1203, 1156-1203, 1159-1203, or1168-1203, all of which are listed in Table 1. In some embodiments, the peptide moietycomprises the amino sequence spanning the HR2 region of SARS-CoV-2 in 1168-1203. In some25856 embodiments, the present disclosure recognizes that a viral fusion inhibitor comprising a certain portion of the HR2 domain provide improved antiviral activities.

[0092] In some embodiments, the peptide moiety based on the HR2 domain of SARS-CoV-2comprises one or more substitution or mutations, so that the peptide moiety is not identical to a portion of the HR2 domain. In some embodiments, the peptide moiety comprises hydrophobic, natural, and / or non-natural amino acids. In some embodiments, one or more amino acids corresponding to the HR2 region at 1162 (P), 1163 (D), 1164 (V), 1165 (D), 1166 (L), 1169 (I), 1170 (S), 1172 (I), 1173 (N), 1174 (A), 1175 (S), 1176 (V), 1177 (V), 1178 (N), 1181 (K), 1183 (I), 1186 (L), 1187 (N), 1189 (V), 1197 (L), 1198 (I), 1199 (D), or 1200 (L) are substitutedand / or mutated. Certain amino acids or reagents used for mutation are shown in Figures 4A and4B.

[0093] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides listed in Table 1.

[0094] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 1. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 2-39. In some embodiments, the peptide moiety is selectedfrom the group consisting of peptides as set forth in SEQ ID NOS: 3-39. In some embodiments,the peptide moiety comprises a peptide as set forth in SEQ ID NO: 2. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 3. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 4. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 5. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 6. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 7. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 8. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 9. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 10. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 11. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 12. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 13. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 14. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 15. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 16. In some embodiments, the25856 peptide moiety comprises a peptide as set forth in SEQ ID NO: 17. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 18. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 19. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 20. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 21. In some embodiments, thepeptide moiety comprises a peptide as set forth in SEQ ID NO: 22. In some embodiments, thepeptide moiety comprises a peptide as set forth in SEQ ID NO: 23. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 24. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 25. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 26. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 27. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 28. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 29. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 30. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 31. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 32. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 33. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 34. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 35. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 36. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 37. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 38. In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ ID NO: 39.

[0095] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 40. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 41-78. In some embodiments, the peptide moiety isselected from the group consisting of peptides as set forth in SEQ ID NOS: 42-78.

[0096] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 79. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 80-117. In some embodiments, the peptide moiety isselected from the group consisting of peptides as set forth in SEQ ID NOS: 81-117.25856

[0097] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 118. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 119-156. In some embodiments, the peptide moiety isselected from the group consisting of peptides as set forth in SEQ ID NOS: 120-156.

[0098] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 157. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 158-195. In some embodiments, the peptide moiety isselected from the group consisting of peptides as set forth in SEQ ID NOS: 159-195.

[0099] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 196. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 197-234. In some embodiments, the peptide moiety isselected from the group consisting of peptides as set forth in SEQ ID NOS: 198-234.

[0100] In some embodiments, the peptide moiety comprises a peptide as set forth in SEQ IDNO: 235. In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 236-273. In some embodiments, the peptide moiety isselected from the group consisting of peptides as set forth in SEQ ID NOS: 237-273.

[0101] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 274-350.

[0102] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOS: 351-394.

[0103] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in 368-416.

[0104] In some embodiments, the N-terminal part of the peptide moiety is PDVD as set forth inSEQ ID NO. 418. In some embodiments, the N-terminal part of the peptide moiety is PEVD asset forth in SEQ ID NO. 419. In some embodiments, the N-terminal part of the peptide moiety is(2R,4S)Prot4OacOH-DVD.

[0105] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOs: 310, 390, 400, or 402-405.

[0106] In some embodiments, the peptide moiety is selected from the group consisting ofpeptides as set forth in SEQ ID NOs: 378, 379, 406, 407, and 415-417.25856 Table 1. Exemplary peptide moiety sequences SEQ ID NO: SEQUENCEX1X2VDX3GX4X5SGINX6X7X8X9X10X11QX12EIDX13LX14EX15AKNLX16ESX17IX18X19X20ELG Bn,25856 SEQ ID NO: SEQUENCE23 PDVDFGSLSGINASLAbuF3DLQKEIDSLNEVAKNLNESYIDLQELGBn,25856 SEQ ID NO: SEQUENCE46 PDVDLGDISGINASVVDIQKEIDRLNEVAKNLNESLIDtBuAQEL25856 SEQ ID NO: SEQUENCEX6 = A, or AbuX7 = S T or BhV Bn,25856 SEQ ID NO: SEQUENCE107 PDVDFGSLSGINASLVDLQKEIDSLNEVAKNLNESYIDLKELGCBn,25856 SEQ ID NO: SEQUENCE130 PDVDLGDISGINAbuSVVDIQaMeKEIDRLNEVAKNLNESLIDLQELGSGSGGG25856 SEQ ID NO: SEQUENCEX15 = V, or tBuGX16= N or EBn,SGSEQ ID NO: SEQUENCE191 PDVDLGDtBuGSGINAbuSVVDIQaMeKEIDRLNEVAKNLNESYIELQELGGGGSGGGGSGGG Bn,SEQ ID NO: SEQUENCE214 PDVDLGDISGINATVVDIQaMeKEIDRLNEtBuGAKNLNESLIDtBuAQELGGSGGGSGGBn,25856 SEQ ID NO: SEQUENCE237 PDVDAbuF3GDISGINAbuSVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIG25856 SEQ ID NO: SEQUENCE275 PDChgDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG25856 SEQ ID NO: SEQUENCE313 PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIELQELG25856 SEQ ID NO: SEQUENCE351 PDVDLGDtBuGSGINAbuBhVVDIQaMeKEIDRLNEVAKNLNES(Phe-4-CO2H)IDLQELG25856 SEQ ID NO: SEQUENCE400 PDVDLGDtBuGSGINAbuBhVVVDIQAibEIDRLNEVAKNLNESYIDLQELG, y p y p . nsome embodiments, the linker moiety comprises one or more hydrophilic units. In some embodiments, the linker moiety comprises one or more amphiphilic units.

[0108] In some embodiments, the linker moiety comprises polyethylene glycol (PEG2)n=1-4,PEG4, PEG8, PEG12, or PEG24. In some embodiments, the linker moiety comprises PEG12. In some embodiments, the linker moiety comprises PEG24. In some embodiments, the linker moiety comprises 1-Amino-15-oxo-4,7,10-trioxa-14-azaoctadecan-18-oic acid (Ttds), Ttds2, Ttds3, or Ttds4. In some embodiments, the linker moiety comprises Ttds2. In some embodiments, the linker moiety comprises Ttds3. In some embodiments, the linker moiety comprises Ttds4. In some embodiments, the linker moiety comprises 11-amino undecanoic acid (11-Aun).

[0109] In some embodiments, the linker moiety comprises an amino acid. In someembodiments, the amino acid is selected from the group consisting of Cys, Glu (E), hGlu (hE), Orn, Lys (K), 2,4-Diaminobutyric Acid (Dab).

[0110] In some embodiments, the amino acid in the linker moiety is not directly connected tothe C-terminal part of the peptide moiety. For example, one or more of PEG2, PEG4, PEG8, PEG12, or PEG24, Ttds, Ttds2, Ttds3, Ttds4, and 11-Aun are positioned between the peptide moiety and the amino acid in the linker moiety.25856

[0111] In some embodiments, the linker moiety is selected from the group consisting ofstructures listed in Table 2. In some embodiments, the linker moiety comprises PEG12-C(propyl)-NH2, Ttds2-C(propyl)-NH2, Ttds3-C(propyl)-NH2, Ttds4-C(propyl)-NH2, Ttds2-Dab(triazole-propyl)-NH2, Ttds3-Dab(triazole-propyl)-NH2, Ttds4-Dab(triazole-propyl)-NH2, Ttds2- Orn(triazole-propyl)-NH2, or Ttds3-Orn(triazole-propyl)-NH2. Table 2. Exemplary Linker Moiety Structures ID StructureL1 PEG12-C(propyl)-NH2L2 PEG2-C( ro l)-NH225856 ID StructureL44 PEG24-K(triazole-propyl)-NH2L45 PEG12-Dab(triazole-propyl)-NH225856ID Structure25856 ID Structure[, p y g p g fcholesterol, tocopherol, and palmitate. In some embodiments, the lipid moiety comprises cholesterol.

[0113] In some embodiments, the linker moiety and the lipid moiety is connected via an etherbond, an ester bond, a carbamate bond, or a triazole bond. In some embodiments, the linkermoiety and the lipid moiety is connected via an ether bond. In some embodiments, the linkermoiety and the lipid moiety is connected via an ester bond. In some embodiments, the linkermoiety and the lipid moiety is connected via a carbamate bond. In some embodiments, the linkermoiety and the lipid moiety is connected via a triazole bond.

[0114] In some embodiments, the attachment of the lipid moiety via an ether bond may provideimproved metabolic stability compared to an ester bond. In some embodiments, the metabolic stability is measured by a remaining time in mouse plasma.

[0115] In some embodiments, the lipid moiety is connected to a side chain of the amino acid inthe linker moiety.

[0116] In some embodiments, the viral fusion inhibitor comprise a peptide moiety listed inTable 1, a linker moiety listed in Table 2, and a lipid moiety selected from the group consisting of cholesterol, tocopherol, and palmitate. In some embodiments, the viral fusion inhibitorcomprises a compound listed in Table 3A. In some embodiments, the viral fusion inhibitorcomprises a compound listed in Table 3B.25856

[0117] In some embodiments, antiviral activity of the viral fusion inhibitors is characterized bya pseudo-type neutralization assay. In some embodiments, when a pseudo-type neutralization assay is performed similarly to Example 1, EC50 of the viral fusion inhibitors is about sub nanomolar range, about 1 digit nanomolar range, or about 2 digits nanomolar range.

[0118] In some embodiments, the viral fusion inhibitors of the present disclosure exhibit pan-coronavirus activity or pan-coronavirus potency. In some embodiments, pan-coronavirus activity or pan-coronavirus potency is characterized by a pseudo-type neutralization assay. In some embodiments, when a pseudo-type neutralization assay is performed similarly to Example 1, EC50 of the viral fusion inhibitors exhibiting pan-coronavirus activity or pan-coronavirus potency is about sub nanomolar range, about 1 digit nanomolar range, or about 2 digits nanomolar range at least for two or more different types of coronavirus.Table 3A. Exemplary Inhibitor CompoundsID SEQUENCE & Structure SEQ IDNOA PDVDLGDISGINASVVNI KEIDRLNEVAKNLNESLIDL ELG PEG C l O25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVDIQKEIDRLNEtBuGAKNLNESLIDLQELG-PEG12-C(propyl-O- 4 825856 ID SEQUENCE & Structure SEQ IDNOAc- PDVDFGSLSGINASLVDLQKEIDSLNEVAKNLNESYIDLSELG-PEG12-C(propyl-O-4 725856 ID SEQUENCE & Structure SEQ IDNOAc-SFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG- P P1 1 47725856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESYIDLQELG-PEG12-C(propyl-O- P P12 4725856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINATVVDIQKEIDRLNEVAKNLNESLIDtBuAQELG-PEG12-C(propyl-O- P P14 1725856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuBhVVAbuF3DIQaMeKEIDRLNEtBuGAKNLNESYIDLQELG- P P14 725856 ID SEQUENCE & Structure SEQ IDNOCompound Ac-PDVDFGSLSGINASLVDLQKEIDSLNEVAKNLNESYIDLQELG-PEG12-hE(propyl-O- 525856 ID SEQUENCE & Structure SEQ IDNOCompound Ac-PDVDLGDtBuGSGINAbuBhVVDIQaMeKEIDRLNEVAKNLNEShYIDLQELG-PEG12- 7525856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG- nd25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuBhvVVDIQaMeKEIDRLNEVAKNLNESYIDLQELG-25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuBhVVVDIQAibEIDRLNEVAKNLNESYIDLQELG-PEG12- P P242 1025856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuBhVVVDIQaMeKEIDRLNEVAKNLNESYIDLQELG(Sar)10- P P227 1125856 ID SEQUENCE & Structure SEQ IDNO4IPhBut-eK-Succinyl-GSGSG-Table 4B. Additional Exemplary Inhibitor Compounds ID SEQUENCE & Structure SEQ IDNO25856 ID SEQUENCE & Structure SEQ IDNOAc-PDChgDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-PEG12-C(propyl-O- 4625856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEChgAKNLNESLIDLQELG-PEG12-C(propyl-O- 6

[0119] Peptides are an attractive class of molecules that combine the beneficial properties ofsmall molecule drugs with the therapeutic properties of proteins. However, a main limitation of peptides as drug candidates is their short half-life in circulation, mainly due to proteolytic degradation and / or rapid renal clearance. To address this limitation, a variety of half-life extension approaches have been developed over the past decades. Peptide derivatization with ligands that non-covalently bind to albumin, which is the most abundant serum protein with a circulating half-life of 19 days in humans, is one of the most successful strategies for generating long-lasting biotherapeutics with enhanced pharmacokinetic properties and improved efficacy in the clinic.

[0120] Among the albumin-binder molecules developed in recent years, Dumelin et al.(Angew. Chem. Int. Ed.2008, 47, 3196 –3201) used a large DNA-encoded chemical library, toidentify a small organic albumin binder molecule, 4-(p-iodophenyl)butyric acid (4IPhBut), alsoknown as Albutag (see Figure 29A).

[0121] In some embodiments, the inhibitor comprises a half-life extension moiety. In someembodiments, the half-life extension moiety comprises 4-(p-iodophenyl)butyric acid (4IPhBut).In some embodiments, the carboxylate group of 4IPhBut can be linked to amino groups on theside chains of various amino acids.

[0122] In some embodiments, the half-life extension moiety is attached to the N-terminal partof the peptide moiety. In some embodiments, the half-life extension moiety is selected from thegroup consisting of 4IPhBut-eK-Succinyl, 4IPhBut-eK-Succinyl-AEEA, 4IPhBut-eK-Succinyl-25856(AEEA)2, 4IPhBut-eK-Succinyl-(AEEA)3, and 4IPhBut-eK-Succinyl-GSGSG (SEQ ID NO:425). In some embodiments, the half-life extension moiety comprises 4IPhBut-eK-Succinyl. Insome embodiments, the half-life extension moiety comprises 4IPhBut-eK-Succinyl-AEEA. Insome embodiments, the half-life extension moiety comprises 4IPhBut-eK-Succinyl-(AEEA)2. Insome embodiments, the half-life extension moiety comprises 4IPhBut-eK-Succinyl-(AEEA)3. Insome embodiments, the half-life extension moiety comprises 4IPhBut-eK-Succinyl-GSGSG (SEQ ID NO: 425).

[0123] The present disclosure encompasses the insight that Lys, D-Lys or Orn in a peptidemoiety can be used to attach a half-life extension moiety. In some embodiments, the peptidemoiety comprises a mutation to which the half-life moiety is attached. In some embodiments, oneor more amino acids of the peptide moiety are substituted to Lys, D-Lys or Orn. In someembodiments, an amino acid of the peptide moiety is substituted to Lys. In some embodiments,the peptide moiety has Lys in position 1168. In some embodiments, the peptide moiety comprisea peptide as set forth in SEQ ID NO: 408. In some embodiments, the half-life extension moiety isselected from the group consisting of 4IPhBut-eK-Succinyl, 4IPhBut-eK-Succinyl-AEEA,4IPhBut-eK-Succinyl-(AEEA)2, and 4IPhBut-eK-Succinyl-(AEEA)3. In some embodiments, thehalf-life extension moiety comprises 4IPhBut-eK-Succinyl. In some embodiments, the half-lifeextension moiety comprises 4IPhBut-eK-Succinyl-AEEA. In some embodiments, the half-lifeextension moiety comprises 4IPhBut-eK-Succinyl-(AEEA)2. In some embodiments, the half-lifeextension moiety comprises 4IPhBut-eK-Succinyl-(AEEA)3. Dosing and Administration

[0124] Further provided herein are dosing regimens and routes of administration for treating orpreventing an infection using a viral fusion inhibitor.

[0125] The viral fusion inhibitor disclosed herein may be administered by doses administeredintravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. In certain embodiments, the doses are administered intravenously. In certain embodiments, the doses are administered subcutaneously. In certain embodiments, the doses are administered orally. In certain embodiments, the doses are nasally.

[0126] In some embodiments, a provided viral fusion inhibitor is administered according to adosing regimen sufficient to achieve a reduction in the degree and / or prevalence of symptoms of infection of a specified percentage of a population of patients to which the viral fusion inhibitor25856 is administered. In some embodiments, the specified percentage of population of patients towhich the viral fusion inhibitor was administered is at least about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 31 %, about 32%. about 33%. about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41 %, about 42%, about 43%,about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51 %,about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81 %, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0127] To give but a few illustrative examples, in some embodiments, administration of at leastone provided composition comprising a viral fusion inhibitor according to a dosing regimen issufficient to achieve a reduction in the degree and / or prevalence of infection of at least about 20% in at least about 50% of the population of patients to which the composition was administered. In some embodiments, administration of at least one composition according to adosing regimen is sufficient to achieve a reduction in the degree and / or prevalence of infection ofat least about 30% in at least about 50% of the population of patients to which the composition was administered.

[0128] In some embodiments, at least one provided pharmaceutical composition comprising aviral fusion inhibitor is administered according to a dosing regimen sufficient to achieve a delayin the onset of symptoms of infection. In some embodiments, at least one provided composition is administered according to a dosing regimen sufficient to prevent the onset of one or more symptoms of infection.

[0129] In some embodiments, a provided dosing regimen comprises or consists of a singledose. In some embodiments, a provided dosing regimen comprises or consists of multiple doses,separated from one another by intervals of time that may or may not vary. In some embodiments,a provided dosing regimen comprises or consists of dosing once every 20 years, once every 10 years, once every 5 years, once every 4 years, once every 3 years, once every 2 years, once per year, twice per year, 3 times per year, 4 times per year, 5 times per year, 6 times per year, 7 times per year, 8 times per year, 9 times per year, 10 times per year, 11 times per year, once per month,25856 twice per month, three times per month, once per week, twice per week, three times per week, 4 times per week, 5 times per week, 6 times per week, daily, twice daily, 3 times daily, 4 timesdaily, 5 times daily, 6 times daily, 7 times daily, 8 times daily, 9 times daily, 10 times daily, 11times daily, 12 times daily, or hourly.

[0130] In some embodiments, a provided dosing regimen comprises or consists of an initialdose with one or more booster doses. In some embodiments, one or more booster doses areadministered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 1 month, 2 months,6 months, 1 year, 2 years, 5 years, 10 years, or longer than 10 years after the initial dose.

[0131] In some embodiments, an initial dose comprises a series of doses administered over aperiod of time. For example, in some embodiments, an initial dose comprises a series of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more doses administered at regular intervals, e.g., intervals that are close in time to one another, such as 5 minute intervals, 10 minute intervals, 15 minute intervals, 20 minute intervals, 25 minute intervals, 30 minute intervals, 45 minute intervals, hourly intervals, every 2 hours, etc.

[0132] In some embodiments, a provided dosing regimen comprises or consists ofadministration of multiple doses over the course of the subject's entire lifespan. In some embodiments, a provided dosing regimen comprises administration of multiple doses over the course of several years (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 years). In some embodiments, a provided dosing regimen comprises or consists ofmultiple doses over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.Pharmaceutical Compositions

[0133] In yet another aspect, provided herein are pharmaceutical compositions comprising thetherapeutic agents disclosed herein (e.g., a viral fusion inhibitor).

[0134] In certain embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier.

[0135] The pharmaceutical compositions comprising a viral fusion inhibitor, can be preparedfor storage by mixing the antibodies or compounds having the desired degree of purity withoptionally physiologically acceptable carriers, excipients, or stabilizers (see,e.g., Remington,Remington’s Pharmaceutical Sciences (18th ed. 1980)) in the form of aqueous solutions orlyophilized or other dried forms.

[0136] The pharmaceutically acceptable carriers, excipients, or stabilizers are non-toxic to thecell or mammalian being exposed thereto at the dosage and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include buffers, such as phosphate, citrate, acetate, andother organic acids; antioxidants, such as ascorbic acid; low molecular weight (e.g., fewer thanabout 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM. Thepharmaceutically acceptable carriers can also refer to a diluent, adjuvate (e.g., Freund’s adjuvate(complete or incomplete)), excipient, or vehicle. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier whena composition (e.g., a pharmaceutical composition) is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water,ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Kits

[0137] In still another aspect, provided herein are kits comprising the therapeutic agentsdisclosed herein (e.g., a viral fusion inhibitor) or pharmaceutical compositions thereof, packaged into suitable packaging material. A kit optionally includes a label or packaging insert that includea description of the components or instructions for use in vitro, in vivo, or ex vivo, of thecomponents therein.

[0138] In certain embodiments, the kit further comprises instructions for administering to ahuman patient the viral fusion inhibitor. In one embodiment, the kit comprises: (a) one or moredosages of a viral fusion inhibitor; and (b) instructions for administering to a human patient the viral fusion inhibitor.

[0139] In some embodiments, the kit comprises means for separately retaining the components,such as a container, divided bottle, or divided foil packet. A kit of this disclosure can be used for administration of different dosage forms, for example, oral and parenteral, for administration of the separate compositions at different dosage intervals, or for titration of the separate compositions against one another. General Methods

[0140] Standard methods in molecular biology are described in, for example, Sambrook,Fritsch and Maniatis (1982 & 19892ndEdition, 20013rdEdition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Springand Wu (1993) Recombinant DNA, Vol.217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol.1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol.4).

[0141] Methods for protein purification including immunoprecipitation, chromatography,electrophoresis, centrifugation, and crystallization are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol.1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols inProtein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol.3, John Wiley and Sons, Inc., NY, pp.16.0.5-16.22.17; and Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp.45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp.384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol.1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY; and Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol.4, John Wiley, Inc., New York).

[0142] Methods for flow cytometry, including fluorescence activated cell sorting (FACS), areavailable (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nded.; Wiley-Liss, Hoboken, NJ; and Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primersand probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (see,e.g., Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; and Sigma- Aldrich (2003) Catalogue, St. Louis, MO).

[0143] Analytical methods suitable for evaluating the product stability include size exclusionchromatography (SEC), dynamic light scattering test (DLS), differential scanning calorimetery(DSC), iso-asp quantification, potency, UV at 340 nm, UV spectroscopy, and Fourier-transforminfrared spectroscopy (FTIR). SEC (see, e.g., J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); and J. Pharm. Bio. Anal., 14:1133- 1140 (1986)) measures percent monomer in the product and gives information of the amount of soluble aggregates. DSC (see, e.g., Pharm. Res., 15:200 (1998); and Pharm. Res., 9:109 (1982)) gives information of protein denaturation temperature and glass transition temperature. DLS (see, e.g., American Lab., November (1991)) measures mean diffusion coefficient, and gives information of the amount of soluble and insoluble aggregates. UV at 340 nm measures scattered light intensity at 340 nm and gives information about the amounts of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278 nm and gives information of protein concentration. FTIR (see, e.g., Eur. J. Pharm. Biopharm., 45:231 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); and J. Pharm. Scien., 87:1069 (1998)) measures IR spectrum in the amide one region, and gives information of protein secondary structure.

[0144] The iso-asp content in the samples is measured using the Isoquant IsoaspartateDetection System (Promega). The kit uses the enzyme Protein Isoaspartyl Methyltransferase (PIMT) to specifically detect the presence of isoaspartic acid residues in a target protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to isoaspartic acid at the alpha.-carboxyl position, generating S-adenosyl-L-homocysteine (SAH) in the process. This is arelatively small molecule, and can usually be isolated and quantitated by reverse phase HPLC using the SAH HPLC standards provided in the kit.

[0145] All publications mentioned herein are incorporated by reference for the purpose ofdescribing and disclosing methodologies and materials that might be used in connection with the present invention.

[0146] Having described different embodiments of the invention herein with reference to theaccompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims. EXAMPLES Example 1: Experimental methods

[0147] This Example explains experimental methods used for synthesizing and / orcharacterizing inhibitor compounds comprising a peptide moiety, a linker moiety and a lipid moiety. General synthesis procedure

[0148] The synthesis of peptides was performed by standard fluorenylmethoxycarbonylprotecting group (Fmoc) stepwise solid phase peptide synthesis (SPPS) on a Liberty BlueTMmicrowave synthesizer (CEM Corporation, Matthews, NC). The assembly was performed using a Rink amide AM Resin polystyrene + 1% divinylbenzene (DVB) Chem-Impex (0.33 mmol / g, 100-200 mesh) on a 100 μmol scale.

[0149] For compounds comprising a PEG12 unit, at the beginning of the assembly, the C-terminal Fmoc-Cys(Trt)-OH (CAS No.103213-32-7) required for conjugation was manuallyacylated followed by acylation of the linker Fmoc-PEG12-OH (CAS No. 1952360-91-6) usingstandard methods (3 equivalents (Eq) Fmoc-protected residue, 3 Eq 1-hydroxy-7- azabenzotriazole (HOAt), 6 Eq diisopropylcarbodiimide (DIC) in dimethylformamide (DMF), 1.5-4 hours).

[0150] For inhibitors incorporating 1-Amino-15-oxo-4,7,10-trioxa-14-azaoctadecan-18-oic acid(Ttds) units prior to the assembly, the C-terminal Fmoc-Cys(Trt)-OH was manually acylated25856followed by acylation of the linker Fmoc-Ttds-OH (CAS No. 172089-14-4) from 1 up to 4 Ttdsunits using standard methods.

[0151] For inhibitors incorporating 11-amino undecanoic acid (11-Aun), PEG2, PEG4, or PEG8linkers, before the assembly, the C-terminal Fmoc-Cys(Trt)-OH was manually acylated followed by acylation of the linkers Fmoc-11Aun-OH (CAS No.88574-07-6), and / or Fmoc-PEG4-OH (CAS No.557756-85-1), or Fmoc-PEG8-OH (CAS No.868594-52-9), or Fmoc-PEG2-OH (CAS No.166108-71-0) using standard methods (3 Eq of the corresponding Fmoc-residue, 3 Eq of HOAt, 6 Eq of DIC in DMF, 1.5 hours).

[0152] For PepSP1510, at the beginning of the assembly, the C-terminal Fmoc-Lys(N3)-OH(CAS No.159610-89-6) required for conjugation was manually acylated followed by acylationof the linker Fmoc-PEG12-OH (CAS No. 1952360-91-6) using standard methods (3 Eq Fmoc-protected residue, 3 Eq HOAt, 6 Eq DIC in DMF, 1.5-4 h).

[0153] Amino acids were used with standard side chain protecting groups unless otherwisenoted. Aspartic acid was coupled as Fmoc-Asp(OMpe)-OH (CAS No.180675-08-5) to minimize aspartimide formation.

[0154] Each amino acid (0.4 M in DMF) was acylated with a 5-fold excess using 10 Eq of DIC(1 M) and 5 Eq of OxymaPure® (1 M) in DMF as activators. Fmoc deprotection was performedwith 20% piperidine (v / v) in DMF for 1 minute under microwave (MW) irradiation (75°C for 15 seconds, then 90°C for 50 seconds). After deprotection, the resin was washed three times with DMF. Single and double couplings were performed under microwave irradiation at 90°C for 2 minutes (75°C for 15 seconds, 90°C for 110 seconds), then drained and washed 2 times. Double acylation reactions were performed for all Fmoc-Arg(Pbf)-OH (CAS No. No.154445-77-9) andfor the residues between positions 1172 (Ile) –1179 (Ile) and 1185 (Arg) – 1189 (Val). At the endof the sequence assembly, the resin was acetylated using 10 Eq of acetic anhydride in DMF. The peptides were cleaved using a solution of 82.5% trifluoroacetic acid (TFA), 5% H2O, 5%thioanisole, 2.5% ethanedithiol (EDT), 5% phenol for 1.5 hours at room temperature (RT) andthen precipitated with cold tert-butyl methyl ether. After centrifugation, the peptide pellets were washed with diethyl ether, dried, dissolved in 0.1% formic acid in H2O / CH3CN (1:1), and lyophilized.25856 Analytical characterization

[0155] Crude and purified peptides were analyzed by ultra-high performance liquidchromatography (UPLC) with ultraviolet (UV) and mass spectrometry detection (UPLC-UV- MS). Analyses were performed on a Waters Acquity UPLC system equipped with an analytical WatersTMBEH300 C4 (2.1x100 mm, 1.7μm, at 45°C) column. Detection was performed by UV absorbance at 214 nm wavelength. Mass analysis was performed on a WatersTMsingle quadrupole (SQ) detector with electrospray ionization in positive ion detection mode and the scan range of the mass-to-charge ratio was 400-1800. Analyses were performed using linear gradient of binary mixtures of H2O containing 0.1%TFA (A) and acetonitrile containing 0.1%TFA (B). The linear gradients of B used were 35%.

[0156] 35%B (1 minute), 35%B-55%B (5 minutes), 55%B-80%B (0.1 minute); and 40%B-40%B (1 minute), 40%B-60%B (5 minutes), 60%B-90%B (0.1 minute) at 0.4 milliliter per minute (mL / min) and 45°C.

[0157] Reversed-phase high-performance liquid chromatography (HPLC) of crude peptideswas performed with a preparative HPLC WatersTMsystem using C8 (Dr Maisch, ReproSil acetonitrile in water, 0.1% TFA (30%B-45%B in 25 minutes; 35%B-50%B in 25 minutes;40%B- min). Fractions containing the desired productwere combined and lyophilized. The characterization was performed on a WatersTM AcquityBEH300 C42.1x100 mm, 1.7μm column using the linear gradient of acetonitrile in water with the following conditions: 35%B-35%B (1 minute), 35%B-55%B (5 minutes), 55%B-80%B (0.1 minute); 40%B-40%B (1 minute), 40%B-60%B (5 minutes), 60%B-90%B (0.1 minutes); eluents of A= H2O + 0.1% TFA and B= CH3CN + 0.1%TFA; flow rate of 0.4 mL / min; wavelength of 214 nm; temperature of 45°C; and MS of Waters Acquity ESI+, single quadrupole. General protocol for the synthesis of compounds comprising cholesterol via ether bond

[0158] In order to yield the cholesterol ether analogs, the corresponding purified peptideprecursor (C-terminus cysteine free) and 1Eq of Br-acetamide-propyl-O-cholesterol were dissolved together in H2O + 0.05% w / v 2-hydroxypropyl-b-cyclodextrine / tetrahydrofuran (THF) 6:4 (final peptide concentration 20 mg / mL).1% v / v diisopropylethylamine (DIEA) was25856 added to reach pH 8-9. After 30 minutes, 0.1Eq of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (solubilized in H2O + 0.05% w / v cyclodextrin) was added. The reaction was stirred at room temperature for an additional 30 minutes. The reaction was monitored by UPLC analysis on a WatersTMAcquity BEH300 C4 column (2.1x100 mm, 1.7 μm) with the following conditions: a gradient of 30%B-30%B (1 minute), 30%B-90%B (5 minutes), 90%B-90%B (0.5 minute); eluents of A= H2O + 0.1% TFA; B= CH3CN + 0.1%TFA; flow rate of 0.4 mL / min; wavelength of 214 nm; temperature of 45°C; and MS of WatersTMAcquity ESI+, single quadrupole.

[0159] After 1 hour, the reaction was quenched with TFA, diluted with dimethyl sulfoxide(DMSO), loaded on a Reversed-phase HPLC using a Delta Pak C4200x25 mm 300A 15 μm column and purified using the linear gradients of 40%B-55%B in 20 minutes or 45%B-60%B in20 minutes; eluents of A= H2O + 0.1% TFA and B= CH3CN + 0.1%TFA; flow rate of 50mL / min; wavelength of 214 nm. Fractions containing the desired product were combined and lyophilized. The analytical characterization was performed on a WatersTMAcquity BEH300 C4 2.1x100 mm, 1.7 μm column with the following conditions: a gradient of 45%B-45%B (1min), 45%B-65%B (5min), 65%B-90%B (0.1min); or 50%B-50%B (1 minute), 50%B-70%B (5 minutes), 70%B-90%B (0.1 minute); or 55%B-55%B (1 minute), 55%B-75%B (5 minutes), 75%B-90%B (0.1 minute); eluents of A= H2O + 0.1% TFA and B= CH3CN + 0.1%TFA; flow rate of 0.4 mL / min; wavelength of 214 nm; temperature of 45°C; and MS of WatersTMAcquity ESI+, single quadrupole.General protocol for the synthesis of compounds comprising cholesterol via triazole bond

[0160] In order to yield the cholesterol triazole compounds, the corresponding purified peptideprecursor (e.g., C-terminus Dab(N3) or Lys(N3) or Orn(N3)) was dissolved in DMSO and 1.6 Eq of pentyn-O-cholesterol (dissolved in THF), 3 Eq of CuSO4(CAS 7758-99-8, dissolved in H2O), and 5Eq of (+)-Sodium L-ascorbate (CAS 134-03-2, dissolved in H2O) were added. The final peptide concentration was 20 mg / mL. The final solvent ratio was H2O 4.5% in DMSO / THF 1:1. The reaction was monitored by UPLC analysis on a WatersTMAcquity BEH300 C4 column (2.1x100 mm, 1.7 μm) with the following conditions: a gradient of 30%B-30%B (1 minute), 30%B-90%B (5 minutes), 90%B-90%B (0.5 minute); eluents of A= H2O + 0.1% TFA; B= CH3CN + 0.1%TFA; flow rate of 0.4 mL / min; wavelength of 214 nm; temperature of 45 °C; and MS of WatersTMAcquity ESI+, single quadrupole.25856

[0161] After 40 minutes, the reaction was completed and it was quenched with TFA, dilutedwith DMSO, loaded on a Reversed-phase HPLC using a Delta Pak C4200x25mm 300A 15 mcolumn and purified using the linear gradients: 40%B-55%B in 20 min (eluents: A= H2O + 0.1% TFA; B= CH3 product were combined and lyophilized. The analytical characterization was performed on a BEH300 C4 Acquity Waters 2.1x100 mm, 1.7μm column with a gradient of 45%B-45%B (1min), 45%B-65%B (5min), 65%B-90%B (0.1min); or 50%B-50%B (1min), 50%B-70%B(5min), 70%B-90%B (0.1min); eluents: A= H2O + 0.1% TFA; B= CH3CN + 0.1%TFA; flow: 0.4Synthesis of compounds comprising Glu-C3-O-Cholesterol

[0162] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) as described above. The sequence assembly was performed using a ProTideTMrink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yieldpeptidyl resin 1 in Figures 8, 8-1 and 8-2. Fmoc-amino acids were used as described above,except for the C-terminal two amino acids, Fmoc-Glu(Oall)-OH (CAS No.133464-46-7) required for conjugation and Fmoc-PEG12-OH.

[0163] The above peptidyl resin was submitted to allyl deprotection as described above. Thedesired product formation was assessed by micro-cleavage (TFA:TIS:H2O = 95:2.5:2.5) followedby UPLC-MS analysis to confirm peptidyl resin 2 (see Figures 8, 8-1 and 8-2) was yielded.

[0164] The above peptidyl resin 2 (0.1 mmol) reacted with Compound A (see Figures 8, 8-1and 8-2, 2 Eq), along with PyAOP (CAS No.156311-83-0, 1.5 Eq), HOAt (CAS No.39968-33- 7, 1.5 Eq), dissolved in DMF.2M DIEA in N-methylpyrrolidone (NMP) was added to make thepH of the solution basic. The reaction was performed under microwave irradiation for 10 minutesat 65°C. Then, the pH was adjusted to make it basic and the reaction was kept at roomtemperature overnight. The peptidyl resin 3 (see Figures 8, 8-1 and 8-2) was yielded.

[0165] Peptidyl resin 3 was cleaved as described above.Synthesis of compounds comprising hGlu-C3-O-Cholesterol

[0166] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) on a Liberty Blue microwave synthesizer (CEM Corporation, Matthews, NC) as described above. The sequence assembly was performed using a ProTideTM25856 rink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yield peptidyl resin7 (see Figures 9, 9-1 and 9-2). Fmoc-amino acids were used as described above, except for the C-terminal two amino acids, Fmoc-hGlu(Oall)-OH (CAS No.133464-45-6) and Fmoc-PEG12-OH were used.

[0167] Peptidyl resin 7 was submitted to allyl deprotection as described above. The desiredproduct formation was assessed by microcleavage (TFA:TIS:H2O = 95:2.5:2.5) followed byUPLC-MS analysis to confirm peptidyl resin 8 (see Figures 9, 9-1 and 9-2) was yielded.

[0168] For the conjugation of cholesterol on the resin, peptidyl resin 8 (0.1 mmol) reacted withCompound B (see Figures 9, 9-1 and 9-2, 4 Eq), along with HATU (CAS No. 148893-10-1) (4Eq), dissolved in DMF.2M DIEA in NMP was added to make pH of solution basic. The reaction was performed under microwave irradiation for 8 minutes at 50°C. The pH of solution was adjusted to make it basic, and the reaction was kept overnight. The peptidyl resin 9 (see Figures 9, 9-1 and 9-2) was yielded. Peptidyl resin was cleaved as described above. Synthesis of compounds comprising Glu-C6-O-Cholesterol

[0169] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) on a Liberty Blue microwave synthesizer (CEM Corporation, Matthews, NC) as described above. Assembly of the sequences was performed using a ProTideTMrink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yieldpeptidyl resin 4 (see Figures 10, 10-1 and 10-2). Fmoc-amino acids were used as describedabove, except for the C-terminal two amino acids, Fmoc-Glu(Oall)-OH required for conjugation and Fmoc-PEG12-OH.

[0170] The above peptidyl resin 4 was submitted to allyl deprotection as described above. Thedesired product formation was assessed by microcleavage (TFA:TIS:H2O = 95:2.5:2.5) followedby UPLC-MS analysis to confirm peptidyl resin 5 (see Figures 10, 10-1 and 10-2) was yielded.

[0171] For conjugation of Cholesterol on the peptidyl resin 5, the above peptidyl resin 5 (0.05mmol) reacted with Compound C (see Figures 10, 10-1 and 10-2, 4 eq), along with HATU (4 eq),dissolved in DMF.2M DIEA in NMP was added to make the pH of the solution basic. The reaction was performed at room temperature overnight. The resin was drained. Peptidyl resin 6(see Figures 10, 10-1 and 10-2) was yielded. Peptidyl resin 6 was cleaved as described above25856 Synthesis of compounds comprising hGlu-C6-O-Cholesterol

[0172] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) on a Liberty Blue microwave synthesizer (CEM Corporation, Matthews, NC) as described above. The sequence assembly was performed using a ProTideTMrink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yield peptidyl resin10 (see Figures 15, 11-1 and 11-2). Fmoc-amino acids were used as described in GeneralProtocol above, except for the C-terminal two amino acids, Fmoc-hGlu(Oall)-OH required for conjugation and Fmoc-PEG12-OH.

[0173] The above peptidyl resin 10 was submitted to allyl deprotection as described above. Thedesired product formation was assessed by microcleavage (TFA:TIS:H2O = 95:2.5:2.5) followedby UPLC-MS analysis to confirm peptidyl resin 11 (see Figures 16, 11-1 and 11-2) was yielded.

[0174] For conjugation of cholesterol on the resin, the above peptidyl resin 11 (0.1 mmol)reacted with Compound D (see Figures 17, 11-1 and 11-2, 4 Eq), along with PyAOP (1.5 Eq),HOAt (1.5 Eq), dissolved in DMF.2M DIEA in NMP was added to make the pH of the solution basic. The reaction was performed under microwave irradiation at 50°C for 10 minutes. Then, the pH of the solution was adjusted to basic, and the reaction was kept overnight. Peptidyl resin 12(see Figures 18, 11-1 and 11-2) was yielded. Peptidyl resin 12 was cleaved and purified asdescribed above. Synthesis of compounds comprising Orn-C3-O-Cholesterol

[0175] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) on a Liberty Blue microwave synthesizer (CEM Corporation, Matthews, NC) as described above. The sequence assembly was performed using a ProTideTMrink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yield peptidyl resin13 (see Figures 12, 12-1 and 12-2). Fmoc-amino acids were used as described above, except forthe C-terminal two amino acids, Fmoc-Orn(Alloc)-OH (CAS No. 147290-11-7) required forconjugation and Fmoc-PEG12-OH.

[0176] The above peptidyl resin 13 was submitted to Alloc deprotection as described above.The desired product formation was assessed by microcleavage (TFA:TIS:H2O = 95:2.5:2.5)followed by UPLC-MS analysis to confirm peptidyl resin 14 (see Figures 12, 12-1 and 12-2) wasyielded.25856

[0177] For the Conjugation of Cholesterol on the resin, the above peptidyl resin 14 (0.1 mmol)reacted with Compound E (see Figures 12, 12-1 and 12-2, 5 eq), along with PyAOP (5 eq), HOAt(5 eq), dissolved in DMF.2M DIEA in NMP was added to make the pH of the solution basic. The reaction was performed under microwave irradiation at 50°C for 10 minutes. Then, the pH ofthe solution was adjusted to make it basic, and the reaction was kept overnight. The peptidylresin 15 was yielded. Peptidyl resin 15 (see Figures 12, 12-1 and 12-2) was cleaved and purifiedas described above. Synthesis of compounds comprising PEG11-Carbamate-Cholesterol

[0178] The peptidyl resin 18 in Figures 19 and 13-1 (0.1 mmol) reacted with cholesterolchloroformate (CAS 7144-08-3) (10 eq), dissolved in DMF.2M DIEA in NMP was added to make the pH of the solution basic. The reaction was performed at room temperature for 1 hour,then drained, and repeated once more. The peptidyl resin 20 in Figures 20 and 13-1 was yielded.Peptidyl resin 20 was cleaved and purified. Synthesis of compounds comprising PEG11-Carbamate-Cholesterol

[0179] A pre-stirred solution of amino-PEG11-amine (424 mg, 0.779 mmol) and Et3N (0.031mL, 0.223 mmol) in CH2Cl2(1 mL) was added to a stirred solution of cholesteryl chloroformate (100 mg, 0.223 mmol) in CH2Cl2 (1 mL) dropwise over 30 minutes at ambient temperature and the resulting reaction mixture was stirred overnight. Upon completion of the reaction, crude reactions contents were subjected to normal phase column chromatography (24G Gold RediSepcolumn; 0% to 20% MeOH / CH2Cl2) to provide title compound (3R,8R,9R,10S,13S,14R,17S)-10,13-dimethyl-17-((S)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl (35-amino-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)carbamate (Fa, see Figures 14, 14-1, 14-2 and 14-3).

[0180] To a stirred solution of Compound Fa (60 mg, 0.063 mmol) in CH2Cl2 (1 mL) was add2-bromoacetic anhydride (19.55 mg, 0.075 mmol), and DIEA (0.044 mL, 0.251 mmol) at ambient temperature. The resulting reaction mixture was stirred for 1h at the same temperature. Upon completion of the reaction, crude reaction contents were subjected to normal phase column chromatography (12g RediSep Gold®; 0-25% MeOH / CH2Cl2) to provide (3R,8R,9R,10S,13S,14R,17S)-10,13-dimethyl-17-((S)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl (1-bromo-25856 2-oxo-6,9,12,15,18,21,24,27,30,33,36-undecaoxa-3-azaoctatriacontan-38-yl)carbamate(Compound F, see Figures 14, 14-1, 14-2 and 14-3).

[0181] To a stirred solution of peptide (34.3 mg, 7.15 μmol) in dry DMSO (1.5 mL) was addedpre-made solution of Compound F (10 mg, 5.95 μmol) in THF (100 μL). DIEA (21.80 μL, 0.125 mmol) was added and the resulting reaction was run at room temperature for 48 hours. Upon completion of the rection, crude contents were subjected to HPLC purification to provideCompound G (see Figures 14, 14-1, 14-2 and 14-3).Synthesis of compounds comprising amide conjugation points

[0182] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) as described above. The sequence assembly was performed using a ProTideTMrink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yieldpeptidyl resin 21 in Figures 21 and 16-1. Fmoc-amino acids were used as described above,except for the C-terminal two amino acids, Fmoc-Glu(OAll)-OH (CAS 133464-46-7), or Fmoc- homoGlu(OAll) (CAS 133464-45-6), or Fmoc-Bis homoGlu(OAll) (NB-hughesgr-5067973- 0022), along with Fmoc-PEG12-OH (CAS 1952360-91-6).

[0183] The above peptidyl resin 21 was submitted to allyl deprotection. The desired productformation was assessed by micro-cleavage (TFA:TIS:H2O = 95:2.5:2.5) followed by UPLC-MSanalysis to confirm peptidyl resin 22 (see Figures 22 and 16-1) was yielded.

[0184] The above peptidyl resin 22 (0.1 mmol) reacted with the appropriate amine (4 eq), alongwith PyAOP (4 eq) (CAS 156311-83-0), HOAt (4) (CAS 39968-33-7), dissolved in DMF.2M DIEA in NMP was added to make the pH of the solution basic. The reaction was performed at40 °C overnight. Peptidyl resin 23 (see Figures 23 and 16-1) was yielded.

[0185] Peptidyl resin 23 was cleaved using a solution comprising 75% TFA, 10% TFE, 10%DCM, 2.5% TIS, and 2.5% water for 40 minutes. The crude peptide was lyophilized immediately. HPLC purification was performed.

[0186] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) as described above. The sequence assembly was performed using a ProTideTMrink amide resin (CEM, 0.19 mmol / g, 100-200 mesh) on a 0.1 mmol scale to yieldpeptidyl resin 24 in Figures 17 and 17-1. Fmoc-amino acids were used as described above,except for the C-terminal two amino acids, Fmoc-Orn(1-(4,4-dimethyl-2,6-dioxocyclohex-1-25856 ylidene)-3-methylbutyl, iVDde)-OH (CAS 1198321-33-3) or Fmoc-Lys(iVDde) (CAS 204777- 78-6); and Fmoc-PEG12-OH (CAS 1952360-91-6).

[0187] The above peptidyl resin 24 was submitted to iVDde deprotection with 5% hydrazine inDMF for 10 minutes, then repeated 2 times. The resin was washed thoroughly. The desired product formation was assessed by microcleavage (TFA:TIS:H2O = 95:2.5:2.5) followed byUPLC-MS analysis to confirm peptidyl resin 25 (see Figures 17 and 17-1) was yielded.

[0188] The above peptidyl resin 25 (0.1 mmol) reacted with Compound H1 (5 Eq) orCompound H2 (5 Eq), along with PyAOP (5 Eq) (CAS 156311-83-0), HOAt (5 Eq) (CAS 39968-33-7), dissolved in DMF.2M DIEA in NMP was added to make the pH of the solutionbasic. The reaction was performed at 40 °C, overnight. Peptidyl resin 26 (see Figures 17 and 17-1) was yielded. Peptidyl resin 26 was cleaved and purified as described above.Synthesis of compounds comprising lysin sidechain-based linker

[0189] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) as described above. The sequence assembly was performed using a ProTideTMrink amide resin.4 Eq of Fmoc-amino acids (0.2 M in DMF), 4 Eq of HATU (0.4 M in DMF) and 8 Eq of 4-methylmorpholine (NMM) (0.8 M in DMF) were used. The reaction was performed at room temperature for 60 minutes. All Arg, beta-branched amino acids, residues coupled to beta-branched amino acids and the final ten amino acids of the peptide were double coupled to ensure quantitative incorporation of the incoming residue. The Fmoc group wasremoved with 20% pyrrolidine with 0.1 M OxymaPure® for 10 minutes and the N-terminus wasacetylated with 10% acetic anhydride in DMF. After the completion of the linear synthesis, the IvDde group was selectively removed by the treatment of the peptidyl resin with 5% hydrazine / DMF, 3 times for 20 minutes. Additional residues were then incorporated as needed, followed by acylation with the cholesterol acid derivative (2 Eq of Fmoc-amino acids, 2 Eq of HATU, 4 Eq of N, N-Diisopropylethylamine (DIPEA)) for 3 hours at room temperature. The peptide was cleaved from the resin using 10 mL of cleavage cocktail (95% TFA, 2.5% TIS, 2.5% H2O). The cleavage solution containing the crude peptide was then precipitated with 1:1 hexane / diethyl ether, incubated on ice for 10 minutes and then centrifuged (30 minutes, 5000 RPM). The ether was carefully decanted. The pellet was dissolved in H2O / acetonitrile (ACN) andlyophilized. A sample of crude material was analyzed by UPLC-MS. The crude peptide was thendissolved in a minimal amount of DMF and purified by Gilson C8 HPLC (gradient 25%-85%25856 H2O / ACN + 0.1% TFA over 30 minutes). The desired fractions were pooled and lyophilized to give a white solid.

[0190] The synthesis of the peptide was performed by standard Fmoc stepwise solid phasepeptide synthesis (SPPS) as described above. The sequence assembly was performed using aProTideTM rink amide resin. 5 Eq of Fmoc-amino acid (0.2 M in DMF), 10 Eq of DIC (1 M inDMF) and 5 Eq of OxymaPure®(1 M in DMF) for 90 °C for 4 minutes. All Arg, beta-branched amino acids, residues coupled to beta-branched amino acids and the final ten amino acids of the peptide were double coupled to ensure quantitative incorporation of the incoming residue. The Fmoc group was removed with 20% pyrrolidine with 0.1 M OxymaPure®for 2 minutes and the N-terminus was acetylated with 10% acetic anhydride in DMF. After the completion of the linear synthesis, the IvDde group was selectively removed by the treatment of the peptidyl resin with 5% hydrazine / DMF, 3 times for 20 mins. Additional residues were then incorporated as needed,followed by acylation with the cholesterol acid derivative (2 Eq of Fmoc-amino acids, 10 EqDIC, 5 Eq OxymaPure®for 4 minutes at 90 °C). The peptide was cleaved from the resin using 10 mL of cleavage cocktail (95% TFA, 2.5% TIS, 2.5% H2O). The cleavage solution containing the crude peptide was then precipitated with 1:1 hexane / diethyl ether, incubated on ice for 10 min and then centrifuged (30 minutes, 5000 RPM). The ether was carefully decanted. The pellet was dissolved in H2O / ACN and lyophilized (a sample of crude material was analyzed by UPLC-MS). The crude peptide was then dissolved in a minimal amount of DMF and purified by Gilson C8 HPLC (gradient 25%-85% H2O / ACN + 0.1% TFA over 30 min). The desired fractions were pooled and lyophilized. Pseudovirus assay methods •Generation of SARS-CoV pseudoviruses

[0191] SARS-CoV-2 (multiple strains), SARS-CoV-1 and MERS pseudoviral particles werecomposed of a replication-incompetent lentivirus pseudotyped with SARS-CoV Spike (S) protein. The pseudoviral platform was constructed using two plasmids: pNL4-3-GFP-dENV that encodes the lentiviral packaging proteins and a green fluorescent protein (GFP) reporter; and pV1Jns that expresses a SARS-CoV S protein. The pseudovirus stock production begun with transfection of both plasmids into 293T Lenti-X cells, followed by supernatant collection, clarification, and concentration. A panel of S protein plasmids was constructed for each SARS- CoV strain. Each plasmid was used to generate a small scale pseudovirus batch. These small25856 batches were screened to identify the optimal construct for each strain, from which large scale working stocks of pseudovirus were generated. •Assay cell lines

[0192] A549-ACE2-TMPRSS2 stable overexpression cells (GenScript Biotech Corporation,Piscataway, NJ) were permissive to SARS-CoV-2 and SARS-CoV-1 infection via direct fusionwith the cell membrane. Overexpressed angiotensin-converting enzyme 2 (ACE2) receptor was engaged with CoV S protein receptor binding domain (RBD), exposing the S2 subunit for cleavage by transmembrane protease, serine 2 (TMPRSS2); which then caused a S protein conformational transition and pseudoviral envelope fusion with the cell membrane.

[0193] HeLa-ACE2 stable overexpression cells (GenScript Biotech Corporation, Piscataway,NJ) were permissive to SARS-CoV2 and SARS-CoV1 infection via endosomal entry. Overexpressed ACE2 receptor was engaged with CoV S protein RBD. This complex was internalized via clathrin-mediated endocytosis. The S2 subunit was cleaved by cathepsin L in the late endolysosome, which then caused a S protein conformational transition and pseudoviral envelope fusion with the vesicular membrane.

[0194] Huh-7 cells (Japanese Collection of Research Bioresources Cell Bank) were permissiveto MERS infection via endosomal entry. Dipeptidyl-peptidase 4 (DPP4) receptor was engaged with MERS S protein RBD. This complex was internalized via clathrin-mediated endocytosis. The S2 subunit was cleaved by cathepsin L in the late endolysosome, which then caused a S protein conformational transition and pseudoviral envelope fusion with the vesicular membrane. •Pseudoviral entry inhibition assay

[0195] The pseudoviral entry inhibition assay assesses the functional capacity of a compound toinhibit pseudoviral fusion either directly with the cell membrane, or with the vesicular membrane in the late endolysosome. Test compound stocks were serially diluted in a 10-point, 3-fold titration in DMSO, then dispensed to a 384-well tissue culture plate, using an Echo®acoustic liquid handler (Labcyte Inc., San Jose, CA). Along with 31 samples plus a reference, DMSO alone was plated for a negative control to represent minimal effect (Emin), and for a positive control either.10 μL DMSO per well (sufficient volume to result in 100% cell death) or a sufficient concentration of a potent compound was plated to inhibit 100% of pseudoviral fusion and represent maximal effect (Emax). The total volume of DMSO added per well was 0.5% of25856 the final volume of media to be added, which was well below a level where DMSO toxicity occurs.

[0196] Pseudoviruses were diluted in assay media consisting of Dulbecco’s minimum essentialmedium (DMEM) with glutamax and high glucose, plus 2% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin. Dilution factors were determined for each pseudovirus and host cell line pair to result in 400-600 objects counted per well, so that a single, green fluorescent protein (GFP)-expressing cell can be resulted from infection by a pseudovirus. Host cell lines were dissociated from a tissue culture flask using 0.25% Trypsin- ethylenediaminetetraacetic acid (EDTA), and then diluted in an assay media to have 250K cells / well for A549 and Huh-7 cell lines, and 125k cells / well for HeLa.

[0197] To each assay plate containing diluted compound samples and controls, 20 μL of dilutedpseudovirus was added. Then the plates were incubated at 37°C, 5% CO2, and 80% relative humidity (RH). After one hour incubation, 20 μL diluted cell suspension was added to each plate using the liquid dispenser. The plates were returned to the incubator for 72 hours. Following the incubation, total green fluorescent object counts per well were read using Acumen®EX3 microplate cytometer. A 4P curves was fit to these data for each sample peptide titration, and pseudoviral inhibition potency of each peptide was reported as the half-maximal effective concentration (EC50).

[0198] A version of the pseudoviral assay was modified so that inhibition could be measured insamples of ex vivo tissues or bronchoalveolar lavage fluids (BALF) from animals to which dosesof compounds had been administered. The modifications included pre-plating the cells to 384- well tissue culture plates 24 hours prior to assay setup. The plates of tissue homogenate or lavage fluid samples were then serially diluted in an assay media, followed by addition of diluted pseudovirus and one hour incubation. After the incubation, the culture media was removed from the pre-plated cells. The diluted samples and pseudovirus were transferred to the plate of cells. The plates were analyzed as above.

[0199] The modified version of the assay from the previous paragraph was used with a standardcurve of known peptide concentrations. The assay was spiked into tissue homogenate or BALF from untreated animals to interpolate unknown peptide concentrations in samples from treated animals. The accuracy of this quantitative modification to the assay was verified against LC- MS / MS results from the same samples. Further testing revealed that this “pseudoquant” assay was more sensitive for low concentrations than the LC-MS / MS method.25856 •Cytotoxicity assay

[0200] After the pseudoviral inhibition assay plates have been read for total infected cells / wellvia GFP object count, a second assay was performed to determine whether toxicity of the peptidesamples might generate a false positive result by negatively impacting cell viability. Thiscytotoxicity was assessed using the Cell Titer Glo 2.0 (CTG) assay kit (Promega Corporation, Madison, WI) which measured adenosine triphosphate (ATP) as a surrogate for viable cells.

[0201] After thawing the CTG 2.0 reagent and equilibrating both the reagent and pseudovirusinhibitor assay plates to room temperature, a liquid dispenser was used to add a volume of CTG reagent equal to 50% of the volume of cell culture media in the assay plates. The plates were incubated for 9 minutes at room temperature, and agitated on a plate shaker for 1 minute. Total luminescence of each plate well was measured. Live virus assay: SARS -CoV2 microneutralization assay (MNA) experimental protocol

[0202] All cell culture work and sample handling was performed under sterile conditions in aBiosafety Cabinet under Biosafety Level (BSL)-2 conditions. Vero E6 TMPRSS2 cells were maintained in Growth Media containing 10% FBS. Cells were split 2 times per week at confluency. All live virus work was performed in a BSL-3 facility in accordance with current BSL3 SOPs. •Plate Cells For Assay

[0203] Vero E6 TMRPSS2 cells from flasks with 0.25% Trypsin (Gibco; Cat. No. 25200056)were harvested and resuspend in a growth media (DMEM + GlutaMax (Gibco, Cat. No.10569) containing 100 μ / mL Penicillin-Streptomycin (Gibco; Cat. No.15140-122), 1x Minimum Essential Medium (MEM) non-essential amino acids (Gibco; Cat. No. AAJ15694AE) and 10% heat inactivated FBS (Gibco, Cat. No. A38400). Cells were counted and diluted in growth media to have 625,000 cells / mL. The diluted cells were added to assay plates (Perkin Elmer, Cell Carrier 96, Cat. No.6055300), and incubated overnight at 37 °C 5% CO2. •Prepare Virus / Peptide Dilutions

[0204] Virus-inactivation samples (1:4) in Infection Medium (Growth Media containing only2% FBS) were serially diluted in a 96-well dilution plate (Corning, Cat. No.3585). Eight25856 dilutions were prepared for each sample at 2x the final test concentration and sufficient volume of each diluted sample was prepared for 2 replicate test wells (60 μL). •Live Virus work:

[0205] Viruses were thawed and diluted to 4,000 to 8,000 plaque-forming units (pfu) / mL ininfection medium. Each 25 μL contained approximately 100 to 200 pfu. Virus titers and optimal infection time for each SARS-CoV2 variant were determined previously on the assay cell line. The diluted virus (60 μL / well) was added to the sample (60 μL / well) in the dilution plates. For virus control wells, the diluted virus was added to the infection medium in the dilution plate. Thesample / virus mixtures were incubated in diluti 2 incubator.

[0206] The cell / assay plates were removed from the incubator and media from cells wasremoved using a multi-channel electronic pipette.

[0207] The diluted sample / virus mixture (50 μL / well) was transferred to each well of the pre-seeded assay plates in duplicate. For uninfected cell control wells, 50 μL of infection medium was added. The plates were incubated for 1 hour in a 35°C, 5% CO2 incubator.

[0208] Methylcellulose overlay (100 μL / well) was added to all wells of the plates.Methylcellulose overlay was prepared by adding 3.75 g of Methylcellulose (Sigma; Cat. No. M0512) to a 1L glass bottle. The solution comprising DMEM and GlutaMax (480 mL) was then added to the bottle. The bottle was stirred until the methylcellulose has dissolved completely. All plates were incubated at 35°C, 5% CO2incubator for 24 to 48 hours. •Plate Fixation and Blocking

[0209] The inactivated live virus and cells were fixed with formaldehyde. The methylcelluloseoverlay was first removed. The wells were washed once with PBS (100 μL / well). The cells arethen fixed by adding fixation solution (PBS + 3.7% Formaldehyde (PolySciences Inc., Cat. No.04018), 100 μl / well) and incubating for 15 minutes at room temperature. The fixation solution was removed using a plate washer (BioTek, Model EL406). The cells was permeabilized with two 5-minute incubations with a permeabilization buffer (PBS + 0.1% Triton X-100 (Sigma Cat. No. T-9284), 100 μL / well).

[0210] After the second permeabilization, the plates were washed 4 times with 150 μL / well ofwash buffer (PBST; PBS + 0.05% Tween-20 (Sigma Aldrich; Cat. No. P7949)) and blocked25856 overnight at 4 °C with 200 μl / well of a blocking buffer (PBST + 1% BSA Fraction V (Gibco, Cat. No.15260). •Immunostaining and Plate Reading

[0211] SARS-CoV-2 plaques were stained using two primary antibodies for optimalvisualization of variants. Primary antibodies (Spike RBD Antibody, Rabbit PAb (Sino Biologics, Cat. No.40592-T62); and SARS-CoV2 Nucleocapsid Ab, Rabbit Mab (Invitrogen, Cat. No. MA5-36284)) were diluted in the blocking buffer at the ratio of 1:1000. The diluted primary antibodies (100 μL / well) were added to the wells of the assay plates. The assay plates wereincubated for 1 hour at room temperature. Then the plates were washed three times with 150μL / well of PBST on the plate washer.

[0212] A secondary antibody (anti-rabbit IgG AlexaFluor 488 (Invitrogen, Cat. No. A11008))was diluted in the blocking buffer at the ratio of 1:200 and protected from light until ready to be used. The diluted secondary antibody (100 μL / well) was added to the wells of the assay plates. The assay plates were incubated at room temperature for 1 hour. The plates were washed three times with PBST as described above. For optimal imaging, after the final wash, the plates were inverted onto a paper towel. The bottom of the plates were wiped with a damp Kim wipe to remove any smudges from the glass, and then dried with a clean Kim wipe. PBS (200 μL / well) was added to the plates. The plaques were counted. •Calculations and Neutralizing Titer (NT50) Determination

[0213] After the plates have been read, the Viral Control and Cell Control wells were reviewedto ensure average viral counts were within an acceptable range (e.g. plaques; Cell control < 5 plaques).

[0214] 50% neutralizing titer (NT50) is defined as the dilution level at which a 50% reductionin virus is observed relative to the virus control. The NT50 values were determined by 4- parameter curve fit with GraphPad Prism software v.8.1.1 which plots the log transformed sample concentration (x-axis) by the percent neutralization (y-axis). Percent neutralization was calculated by the following equation:

[0215] % Neutralization = (1 – ((sample plaque count – average cell control plaque count) / (average virus control plaque count – average cell control plaque count)) *10025856

[0216] The highest sample dilution tested may not have wells which reach 100% neutralization.Because of this limitation, constraints are placed on the non-linear regression curves (Top = 100%, Bottom = 0%). Example 2: Viral fusion inhibitors comprising a peptide moiety, a linker moiety, and a lipid moiety

[0217] This example shows exemplary inhibitor compounds comprising a peptide moiety, alinker moiety, and a lipid moiety.

[0218] PepSP987 and PepSP988 (as shown in Table 4) comprise peptide moieties havingsequences corresponding to peptides of the HR2 region of SARS-CoV-2 in the different ranges, e.g., 1168-1203 and 1162-1203, respectively. Each of PepSP1095, PepSP990, PepSP991, and PepSP1408 (as shown in Table 4) comprises a peptide moiety having an amino acid sequence corresponding to the HR2 region in the range of 1162-1203 where an extra Cysteine residue was added at the C-terminus. PepSP1095 is the corresponding acetamidated compound in which the C-terminal cysteine was reacted with bromo-acetamide reagent. For PepSP990, PepSP991, and PepSP1408, the thiol group of the cysteine was used for conjugation with bromo cholesterol reagents having a PEG4or a PEG12or a PEG24in the linker moieties to yield the final compounds, respectively. Table 5. Exemplary viral fusion inhibitors ID SEQUENCE & Structure SEQID NO25856Table 6. Pseudo-type neutralization assay results of compounds in Table 4 against SARS-CoV-1,SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela CoV-1 A549 CoV-1 Hela MERS Pseudoneut EC50 - Pseudoneut EC50 - Pseudoneut EC50 – Pseudoneut EC50 – Pseudoneut EC50 in on )1)1)=4)=2)=4)[ ] s s own n a e , e ncorpora on o s x a ona am no ac s a e - erm nus(e.g., PepSP988), yields an almost 2-fold more potent compound compared to PepSP987 against SARS-CoV-2, highlighting the importance of the N-terminal region for maintaining the antiviral activity in the low nanomolar range. Regarding MERS, the two compounds were not active.

[0220] Compared to PepSP987 and PepSP988, the incorporation of an extra cysteine residue atthe C-terminus for conjugation with Bromo cholesterol reagents embedding PEG4 (e.g., PepSP990), PEG12 (e.g., PepSP991), or PEG24 (e.g., PepSP1408) in the linker moieties gives rise to a boost in antiviral activity. These compounds show the 1 or 2 digits nanomolar potency against SARS-CoV-2 and 2 or 3 digits nanomolar potency against MERS.

[0221] The acetamidated compound (e.g., PepSP1095) showed a 3 digits nanomolar potencyagainst SARS-CoV-2 direct fusion route and is not active against SARS-CoV-2 endosomal fusion route and MERS. The presence of the cholesterol group may be crucial to keep the activity in the low nanomolar range and against the SARS-CoV-2 and MERS endosomal fusion route.

[0222] The incorporation of a flexible linker moiety comprising glycine and serine residues(i.e., GSGSG (SEQ ID NO: 675)) and a cysteine residue at the C-terminus for conjugation with25856 Bromo cholesterol (PEG4-COO-cholesterol or a PEG12-COO-cholesterol), resulted in an enhancement of the antiviral activity against SARS-CoV-2, being these inhibitors active in the 1 or 2 digits nanomolar or sub-nanomolar range (PepSP1032 and PepSP1053, respectively). Withrespect to MERS, the incorporation of a flexible linker moiety at the C-terminus did not lead to afurther enhancement of the antiviral activity (PepSP1032 and PepSP1053). Example 3: Modifications in the linker moiety and / or conjugation between the linker moiety and the lipid moiety

[0223] This example shows exemplary modifications of the linker moiety and / or the lipidmoiety conjugation.

[0224] PepSP1093 is an acetamidated control, PepSP1094 is a cholesterol conjugate by an esterbond, and PepSP1141 as shown in Figure 1 is a cholesterol conjugate by an ether bond. In addition, PEG12 of these inhibitors is located between two amino acids, i.e., Glycine at position 1203 and cysteine required for conjugation with a Bromo acetyl-COO-cholesterol (PepSP1094).Table 7. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNOA PDVDLGDISGINASVVNI KEIDRLNEVAKNLNESLIDL ELG PEG C CH CONH-1,SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela CoV-1 A549 CoV-1 Hela MERS P d n t EC50 - P d n t EC50 - P d n t EC50 – P d n t EC50 – in on 1)4)25856

[0225] In comparison to the PepSP991, the incorporation of PEG12 between the two aminoacids and away from the lipid moiety is beneficial for the antiviral activity, resulting in compounds with activity in the sub and 1 digit nanomolar range against SARS-CoV-2 and SARS-CoV-1, and low nanomolar range against MERS (e.g., PepSP1094 and PepSP1141). In addition, the presence of the cholesterol group is advantageous for maintaining activity in the low nanomolar range, since the acetamidated control peptide PepSP1093 exhibited an activity ofabout 71.2 ± 6.78 nM (n=2) against SARS-CoV-2 direct fusion route and is not active againstSARS-CoV-2 endosomal fusion route and MERS.

[0226] Comparing the metabolic stability of PepSP991 and PepSP1141, it was found thatPepSP1141 showed complete (100%) stability at 6 hours incubation in mouse plasma. ForPepSP991, only 46% peptide was detected at 6 hours in mouse plasma (mainly loss of the cholesterol moiety). Example 4: Elongation or truncation in the N-terminal and C-terminal regions of the peptide moiety

[0227] This example shows exemplary inhibitors that have elongation or truncation in the N-terminal and C-terminal regions of the peptide moiety.

[0228] The peptide moiety was elongated at the N-terminus, spanning the HR2 region inthe different ranges, e.g., 1147-1203 (PepSP1460, PepSP1518, see Table 8), 1156-1203 (PepSP1442, see Table 8), and 1159-1203 (PepSP1441, see Table 8). Alternatively, the peptidemoiety was shortened at the N-terminus, spanning the HR2 region in 1168-1203 (PepSP1407, seeTable 8). Alternatively, the peptide moiety was truncated at the C-terminus, spanning the HR2region in 1162-1197 (PepSP1461, see Table 8).Table 9. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856 ID SEQUENCE & Structure SEQ IDNOAc-DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-PEG12-C(propyl-O-Cholesterol)- Pe SP1407 475V-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh7[ ] ompare w ep , ep , runca e a e - erm nus y on y res ues,showed a nearly 4-fold decrease in activity. Extension to the N-terminal region 1162-1168 may be important for improving antiviral activity in both the endosomal and direct route against SARS-CoV-2 (Table 9). On the other hand, PepSP1141 and PepSP1407 showed similar activity against MERS. Similar conclusions can be drawn for the peptide PepSP1461, truncated at the C- -helix present in the HR2 domain ends. Compared to PepSP1141, the truncation of the C-terminal region is detrimental for the antiviral activity being this compound almost 5-fold and 3-fold less active against SARS-CoV-2 and MERS, respectively. For compounds in which the peptide was elongated at the N-terminus beyond Pro at position 1162 by three (PepSP1441) and six (PepSP1442) additional residues, the results showed that the compounds are slightly less potent against SARS-CoV-2 but more potent against MERS (only endosomal route) than PepSP1141.

[0230] Regarding PepSP1460, elongated at the N-terminus by 15 additional residues up toposition 1147 and without the cholesterol moiety, the pseudo neutralization assay results revealedthat it is almost 2-fold more active than PepSP988 but is not active against MERS. However, for25856 PepSP1518, although no improvement in antiviral activity against SARS-CoV-2 was observed compared with PepSP1141, the compound was nearly 2-fold more active in the endosomal fusion pathway against MERS.

[0231] In conclusion, the HR2 optimal sequence lies within residues 1162-1203 of the HR2domain. Example 5: Length and / or physical properties of the linker moiety

[0232] This example demonstrates the effect of the length and / or the physical properties of thelinker moiety.

[0233] The oligoethylene glycol unit (e.g., PEG12) in the linker moiety was replaced with oneor more Ttds units as shown in Figure 2. The use of the Ttds unit could allow for easier synthesis of the compounds and less expensive manufacturing.

[0234] Also, the PEG12 in the linker moiety was replaced with (a) a hydrophobic unit byinserting 1 or 2 units of 11-Aun (e.g., PepSP1432 and PepSP1433); (b) an amphiphilic unit by inserting a unit of 11-Aun and a unit of PEG4 (e.g., PepSP1431); or (c) a hydrophilic unit byinserting two units of PEG4 or one unit of PEG8 (e.g., PepSP1453, PepSP1463).

[0235] Additionally, the PEG12 in the linker moiety was replaced with shorter PEG2 and PEG4units (PepSP1897 and PepSP1898) or by a longer PEG24unit (PepSP1899).

[0236] An extra flexible spacer of glycine and serine residues (i.e., GSGS (SEQ ID NO: 678))was added before Glycine 1203 in combination with PEG2, PEG4, or PEG12 linkers (e.g., PepSP1736, PepSP1737, PepSP1738). Table 11. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856 Ac-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-11-Aun-11-Aun- PepSP1432 480 C(propyl-O-Cholesterol)-NH2a e . seu o-ype eu a a o assay esu s o co pou s a e aga s - oV-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G in Pseudoneut EC50 - SARS2 D614G in25856 PepSP1737 <0.512 nM (n=1) 11.7 ± 1.884 nM (n=2) Not availablePepSP1738 <0.512 nM (n=1) 10.1 ± 3.771 nM (n=2) Not available1Ttds (PepSP1292) resulted in a less potent compound against SARS-CoV-2, whereas similar antiviral potency was observed against MERS, as shown in Table 11. The incorporation of 2, 3, and 4 units of Ttds (PepSP1293, PepSP1294, PepSP1295, respectively) yielded similar or slightly less potent compounds against SARS-CoV-2 and MERS with respect to PepSP1141. Itappears that the best linker length is 3 or 4 units of Ttds. Moreover, in all cases tested, thecompounds are less potent in the endosomal fusion route against SARS-CoV-2 suggesting that the amide linkages within the multiple Ttds linkers can be detrimental for the endosomal fusion route.

[0238] The pseudo-neutralization assay results revealed that the incorporation of shorter (i.e., 2units of PEG4 (PepSP1453) or one unit of PEG8 (PepSP1463)) or amphiphilic (i.e., PEG4-11-Aun (PepSP1431)) linker moieties may not be beneficial for the antiviral activity yielding compounds slightly less potent against SARS-CoV-2 but slightly more potent against MERS endosomal fusion route.

[0239] The incorporation of hydrophobic linker moieties (i.e., 1 or 2 units of 11-Aun,PepSP1432 and PepSP1433, respectively) appears to be detrimental for the antiviral activity. These compounds are active in the 2 or 3 digit nanomolar range against SARS-CoV-2 and inactive (PepSP1432) or slightly active (PepSP1433) against MERS.

[0240] The substitution of PEG12 with PEG2 (PepSP1897) and PEG4 (PepSP1898) spacersresulted in less potent analogs against CoV-2 and MERS. On the other hand, the incorporation of PEG24 (PepSP1899) yielded a similar potent analog against SARS-CoV-2, but slightly more potent against MERS endosomal fusion route than PepSP1141.

[0241] Furthermore, when the flexible GSGS spacer (SEQ ID NO:678) was combined withPEG2(PepSP1736), PEG4(PepSP1737) or PEG12(PepSP1738) in the linker moieties, the compounds showed similar or slightly lower potency against the direct SARS-CoV-2 pathway and less active against the endosomal SARS-CoV-2 pathway. Example 6: Mutations in the peptide moiety

[0242] This example shows exemplary mutations in the peptide moiety.25856

[0243] One or more amino acids in the peptide moiety were mutated with hydrophobic, natural,and / or non-natural amino acids. For example, one or more amino acids corresponding to the HR2 region at positions selected from the group consisting of: 1162 (P), 1163 (D), 1164 (V), 1165 (D), 1166 (L), 1169 (I), 1170 (S), 1172 (I), 1173 (N), 1174 (A), 1175 (S), 1176 (V), 1177 (V), 1178 (N), 1181 (K), 1183 (I), 1186 (L), 1187 (N), 1189 (V), 1197 (L), 1198 (I), 1199(D), and1200 (L) were mutated. Without wishing to be bound by any particular theory, as these positionsmay make contact into the HR1 trimeric coiled coil, mutations in the positions may increase binding interactions and therefore antiviral potency.

[0244] To improve the quality of the synthesis of the compounds, the aspartic acid residues inpositions 1163, 1165, 1168, 1184, and 1199 were mutated with glutamic acid residues to prevent side reactions that might occur during the synthetic process (e.g., PepSP1351, PepSP1352, PepSP1353, PepSP1354, and PepSP1355, Table 12).

[0245] The structures listed in Table 12 include one mutation.Table 13. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNOA -PCh VDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-PEG -C( r l-O-25856 Ac-PDVDLGDISGIChgASVVNIQKEIDRLNEVAKNLNESLIDLQELG-PEG12-C(propyl-O- PepSP1363 654 Cholesterol)-NH2Ac-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLFDLQELG-PEG12-C(propyl-O- PepSP1443 668 Cholesterol)-NH2a e . seu o-ype eu a a o assay esu s o co pou s a e aga s - oV-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GPepSP1361 1.38 ± 0.2682 nM (n=2) 3.34 ± 2.477 nM (n=2) 23.8 ± 5.092 nM (n=4)PepSP1508 4.13 ± 0.6057 nM (n=2) 8.89 ± 4.226 nM (n=4) 6.09 ± 2.647 nM (n=2)

[0246] The insertion of tBuG in position 1169 (PepSP1325), Abu in position 1174(PepSP1297), and AbuF3 in position 1177 (PepSP1333) of the HR2 gave rise to compounds with similar antiviral potency against SARS-CoV-2 and almost 2 or 3-fold higher potency against MERS than PepSP1141. The introduction of Asp in position 1178 and tBuG in position 1189 (PepSP1357 and PepSP1358) yielded constructs with sub-nanomolar and 1-digit nanomolar potency in the direct and endosomal fusion route against SARS-CoV-2 and similar or nearly 1.5- fold enhanced potency against MERS endosomal fusion route, respectively.25856

[0247] Additionally, the pseudo neutralization assay results exhibit that the insertion of Leu inposition 1177 (PepSP1299) and Tyr in position 1197 (PepSP1328) resulted in an improvement in the antiviral activity against MERS, being these compounds active at an EC50 around 9.79 ± 1.621 nM (n=2) and 18.3 ± 0.7866 nM (n=2), respectively. The insertion of Thr in position 1175 (PepSP1361) mutation shows 1-digit nanomolar potency in both the direct and endosomal pathways against SARS-CoV-2.

[0248] Likewise, the insertion of BhV in position 1175 (PepSP1508) resulted in a compoundwith low nanomolar potency in the direct and endosomal pathways against SARS-CoV-2 and 1- digit nanomolar potency against the endosomal fusion route of MERS (EC50: 6.09 ± 2.647 nM (n=2)).

[0249] The series of compounds in which aspartic acid residues were replaced with glutamicacid residues yielded compounds with similar or slightly lower potency against SARS-CoV-2, but slightly higher potency against MERS, compared with PepSP1141. PepSP1355 incorporating Glu at position 1199 showed 1-digit nanomolar potency against SARS-CoV-2 and low nanomolar potency against MERS endosomal route (EC50: 16.8 ± 7.987 nM (n=2)).

[0250] Based on the pseudo-neutralization assay results (Table 13), two or more amino acids inthe peptide moiety of PepSP1141 were mutated. The corresponding peptide structures are listed in Table 14.Table 15. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINASVVNIQKEIDRLNEVAKNLNESLIDYQELG-PEG12-C(propyl-O- Pe SP1425 50825856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINATVVDIQKEIDRLNEVAKNLNESLIELQELG-PEG12-C(propyl-O- Pe SP1505 51825856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuTVVDIQaMeKEIDRLNEtBuGAKNLNESYIELQELG-PEG12- Pe SP1543 53325856Table 16. Pseudo-type neutralization assay results of compounds in Table 13 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh725856 CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh7

[0251] Five compounds with multiple mutations exhibit high antiviral potency against SARS-CoV-2 and MERS, such as PepSP1439 (tBuG insertion at position 1169, Abu insertion at position 1174, Tyr insertion at position 1197), PepSP1464 (AbuF3 insertion at position 1177, Asp insertion at position 1178, aMeK insertion at position 1181), PepSP1465 (Abu insertion at position 1174, Asp insertion at position 1178, aMeK insertion at position 1181), PepSP1469 (tBuG insertion at position 1169, Abu insertion at position 1174, AbuF3 insertion at position 1177, Asp insertion at position 1178, aMeK insertion at position 1181, Tyr insertion at position 1197), and PepSP1545 (tBuG insertion at position 1169, Abu insertion at position 1174, BhV insertion at position 1175, Asp insertion at position 1178, aMeK insertion at position 1181, Tyr25856 insertion at position 1197). All these compounds showed sub-nanomolar potency (direct fusion route) and low nanomolar potency (endosomal fusion route) against SARS-CoV-2 and excellent potency against MERS (1-digit nanomolar activity in the direct and endosomal pathways for most of them). Only specific combinations of the mutations resulted in improved antiviral activity.

[0252] The aspartic acid residue at position 1199 has been replaced with a glutamic acidresidue (e.g., PepSP1657, PepSP1658, PepSP1660, PepSP1661, PepSP1662). This replacement can help to prevent side reactions that could occur during the synthetic process (i.e., aspartimide formation), thereby improving the quality of the synthesis of the compounds.

[0253] Compared to the PepSP1545, the substitution of an aMeK with an aMeY in position1181 as in PepSP2310, PepSP2311, resulted in similar potent analogs against SARS-CoV-2 and MERS endosomal pathway. In addition, N-terminal of Pro at position 1162 was mutated with a 4-carboxymethoxy substituted proline, such as (2R,4S)-4-(carboxymethoxy)pyrrolidine-2-carboxylic acid ((2R,4S)Prot4OH) in PepSP2431 resulting in the compound with sub or 1-digitnanomolar potency against SARS-CoV-2 and MERS.

[0254] PepSP1545 were further modified to (i) further strengthen the binding interactionbetween HR1 and HR2 by incorporating new single point mutations, (ii) improve the manufacturing process (quality of the synthesis) and the stability of the analogs in formulation,(iii) find alternative chemo-selective chemistries for cholesterol conjugation to substitute thethioether bond and make the compound more stable in a formulation (e.g., by removing oxidativeliability due to the sulfur atom of the cysteine) or (iv) modulate the chemical and physicalproperties of the linker to replace the PEG12 with a more affordable moiety for manufacturingpurposes. The modified compounds are listed in Table 16.Table 17. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856Table 18. Pseudo-type neutralization assay results of compounds in Table 16 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh7, ,PepSP2422, PepSP2423, respectively) yielded similar potent compounds against CoV-2 and MERS than PepSP1545. The feasibility of replacing Asn in position 1194 with a Glu or Aspresidue allows for (i) enhanced binding interactions with HR1 and (ii) improved chemicalstability of the sequence when in the formulation (e.g., due to avoidance of Asn deamidation).

[0256] The exchange of Asp in positions 1163 and 1199 with Glu (i.e., PepSP2437) led toPepSP1545-like antiviral activity against CoV-2 and MERS. In addition to the strong antiviralpotency, it helps to prevent side reactions that could occur during the synthetic assembly (e.g., aspartimide formation), thus improving the quality of the synthesis of the compounds.

[0257] The insertion of Aib in position 1181 (i.e., PepSP2452) maintained the antiviral potencyof the compound in the low nanomolar range and similar to PepSP1545. This result pointed outhowever, its side chain had low influence on the antiviral activity. Therefore, the aMeLys1181replacement with Aib, as a much cheaper aMeAA, can be beneficial for improving the manufacturing process of the analog. Example 7: Modifications in the linker moiety in combination with mutations in the peptide moiety

[0258] This example shows exemplary modifications in the linker moiety in combination withmutations in the peptide moiety. PEG12in the linker moiety was replaced with the following: 1) two or three units of Ttds (PepSP1793 and PepSP1794);25856 2) a flexible but more hydrophobic GS-like units: (GGGS)2 (SEQ ID NO: 680) inPepSP2174 and (GGSG)2G-PEG2 (SEQ ID NO: 681) in PepSP2175;3) a hydrophilic poly-Sarcosine polypeptoid, i.e., (Sar)10-PEG2 in PepSP2254; and / or4) 7 residues from the HR2 of SARS-CoV-2 C-terminal sequence with or withoutPEG2 or PEG4 units (PepSP2253, PepSP2176, PepSP2177).Table 19. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuBhVVVDIQaMeKEIDRLNEVAKNLNESYIDLQELG-(Ttds)2-Table 20. Pseudo-type neutralization assay results of compounds in Table 18 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pd t EC50 SARS2 D614G P d t EC50 SARS2 D614G25856 PepSP2253 3.73 ± 0.6327 nM (n=2) 2.27 ± 0.5393 nM (n=2) 11.5 ± 3.686 nM (n=2)PepSP2176 1.75 ± 0.1589 nM (n=2) 3.17 ± 0.6103 nM (n=2) 2.75 ± 0.1662 nM (n=2)4,respectively), the GS-like units (PepSP2174, PepSP2175), and poly-Sarcosine polypeptoid unit (PepSP2254) yielded compounds with similar antiviral potency against SARS-CoV-2 and MERS to PepSP1545. Replacing PEG12 with more affordable units for manufacturing purposes may be possible.

[0260] Concerning the feasibility of substituting the PEG12 with 7 residues from the HR2 C-terminal sequence of SARS-CoV-2, whether or not combined with PEG2or PEG4units, the pseudo-neutralization assay results (Table 19) revealed that these mutations may not be beneficial for the antiviral activity, resulting in compounds with similar or slightly less potency against SARS-CoV-2 and MERS. Example 8: Alternative chemistry for conjugation between the linker moiety and the lipid moiety

[0261] This example shows additional exemplary modifications in the linker moiety.

[0262] Various compounds were made using amino acids other than Cys. The amino acids usedas conjugation points are Glu or homoGlu, with either 3 or 6 carbon spacers between thesidechain amide and the cholesterol moiety. The compounds are generically represented by thestructure shown in Figure 5. Also, several compounds using Orn as a conjugation point was also prepared, as detailed in Figure 6. The corresponding structures are listed in Table 20.Table 21. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-PEG12-E(propyl-O- C d 9 554Table 22. Pseudo-type neutralization assay results of compounds in Table 20 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela MERS25856 Compound 8 3.66 ± 1.033 nM (n=2) 7.42 ± 0.8277 nM (n=2) 8.1 ± 0.5331 nM (n=2)Compound 9 2.56 ± 0.3647 nM (n=2) 25.6 ± 8.794 nM (n=2) 58.5 ± 0.8242 nM (n=2), edgood pan-corona activity, but somewhat decrease versus similar Cys containing conjugates. Thebest compounds in this series were Compound 57 and Compound 18.

[0264] Additionally, the thioether bond was substituted with a triazole bond to performconjugation between the linker moiety and the lipid moiety via a click chemistry reaction (see Figure 3).

[0265] The C-terminal cystine required for the conjugation was replaced with a Lys(N3) orDab(N3) residues (i.e., (S)-2-(Fmoc-amino)-6-azido-hexanoic acid and (S)-2-(Fmoc-amino)-4- azido-butanoic acid), which reacts with a pentyn-O-cholesterol reagent yielding PepSP2172, PepSP2173, PepSP2424, and PepSP2430. The corresponding compound structures are listed in Table 22.Table 23. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856Table 24. Pseudo-type neutralization assay results of compounds in Table 22 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh7resulted in compounds nearly 2-fold less potent against SARS-CoV-2 (direct route), with similar activity against SARS-CoV-2 endosomal route, but with similar (e.g., PepSP2172) or sub- nanomolar activity (e.g., PepSP2173) against MERS endosomal route than PepSP1545. The replacement of the thioether bond by a triazole bond may provide more stability in formulation and improve its shelf-life compared to PepSP1545.

[0267] Regarding the compounds comprising 2 or 3 units of Ttds combined with the cholesterolconjugation via a triazole bond (i.e., PepSP2424, and PepSP2430), the results of the pseudo- neutralization assay revealed the achievement of constructs with excellent antiviral potency against CoV-2 and MERS similar to that of PepSP1545.

[0268] Additionally, a series of compounds was prepared that contain carbamate-basedlinkages to the cholesterol instead of the ether linkages. The various carbamate structuresprepared are shown in Figure 7 and the corresponding peptide sequences are detailed in Table 24.Table 25. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856Table 26. Pseudo-type neutralization assay results of compounds in Table 24 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela Pseudoneut EC50 - SARS2MERS Pseudoneut EC50 - SARS2 D614G in, -corona potency that was somewhat less than that seen with similar ether linked constructs. The most potent compound in this series was Compound 22, which contained a direct linkage between the PEG unit and the cholesterol. Example 9: Alternative chemistry for conjugation between the peptide moiety and the linker moiety

[0270] This example shows additional exemplary alternative chemistry for conjugating the lipidmoiety.

[0271] A series of compounds was prepared, which comprises a Lys appended to the C-terminus of the peptide moiety with the linker moiety attached via the epsilon (side chain) nitrogen of the Lys amino acid. A generic structure representing these varied compounds is shown in Figure 15. Specific structures and data are shown in Table 26.Table 27. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG-25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELG- nd25856 ID SEQUENCE & Structure SEQ IDNOAc-PDVDLGDtBuGSGINAbuBhvVVDIQaMeKEIDRLNEVAKNLNESYIDLQELG-Table 28. Pseudo-type neutralization assay results of compounds in Table 26 against SARS-CoV-2, and MERS CoV-2 A549 CoV-2 Hela Pseudoneut EC50 - SARS2MERS25856 Compound 54 1.86 ± 0.2418 nM (n=2) 5.15 ± 1.922 nM (n=2) 8.2 ± 1.185 nM (n=2)Compound 55 4.54 ± 0.04412 nM (n=2) 21.1 ± 2.248 nM (n=2) 24.4 ± 2.53 nM (n=2)

[0272] PepSP2430 (as shown in Table 28) was synthesized by replacing the thioether bond andPEG12 of PepSP1545 with the triazole bond and Ttds moieties. The replacement of the thioetherbond by a triazole bond may show more stability in formulation and improve its shelf-life.Additionally, Ttds moieties were beneficial for manufacturing purposes, as they are moreaffordable than PEG moieties. Table 29. Exemplary viral fusion inhibitorsSEQ ID ID SEQUENCE & StructureNO. yp y p g V-2, and MERS. CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614G

[0273] New sequences in Table 30 were prepared to combine newly identified point mutationsand linkers with the triazole chemistry for cholesterol conjugation. Table 31. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNO25856 Ac-PDVDLGDtBuGSGINAbuBhVVVDIQaMeKEIDRLNEVAKNLNESYIDLQELG-Ttds3- PepSP2442 609 Orn(triazole-propyl-O-Cholesterol)-NH2oV-, and S. CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh7chain of different length was performed. For this purpose, the C-terminal Dab(N3) required for the conjugation was replaced with a Lys(N3) or Orn(N3) residues (i.e., (S)-2-(Fmoc-amino)-6- azido-hexanoic acid and (S)-2-(Fmoc-amino)-5-azido-pentanoic acid), and combined with 2 or 3 Ttds moieties, resulting in the compounds: PepSP2439, PepSP2440, PepSP2441, and PepSP2442.

[0275] As shown in Table 33, the combination of the C-terminal Lys(N3) or Orn(N3) with 2 or3 Ttds units yielded slightly less active analogs against CoV-2 and MERS than PepSP1545 andPepSP2430.

[0276] In addition, spacer, such as the (Sar)10-PEG2 and (GGSG)2G-PEG2 (SEQ ID NO: 681)(i.e., PepSP2527, PepSP2528, respectively) with the triazole bond for the cholesterol conjugationwere incorporated. However, the pseudo-neutralization results revealed that the compounds wereslightly less active against CoV-2 and MERS.

[0277] Additional mutations were introduced to PepSP2430. In these new compounds, the(Ttds)3spacer and triazole bond for cholesterol conjugation were combined.25856

[0278] In particular Pro in position 1162 was replaced by a (2R,4S)Prot4OAcOH. Asp inposition 1163 and Asp in position 1199 were exchanged with Glu residues. Asn in position 1187and Asn in position 1194 were substituted with Glu. aMeK in position 1181 was replaced with anAib residue.Table 33. Exemplary viral fusion inhibitorsID SEQUENCE & Structure SEQ IDNOAc-PEVDLGDtBuGSGINAbuBhVVVDIQaMeKEIDRLEEVAKNLNESYIELQELG-Ttds3- 5. oV-2, and MERS. CoV-2 A549 CoV-2 Hela MERS25856 PepSP2608 1.31 ± 0.1615 nM (n=2) 3.64 ± 0.1182 nM (n=2) 4.97 ± 0.088 nM (n=2)PepSP2609 1.24 ± 0.203 nM (n=2) 2.89 ± 0.6473 nM (n=2) 4.86 ± 0.009 nM (n=2)glutamate residues combined with the mutation of the Asn in position 1187 or Asn in position1194 for Glu as well as the aMeK in position 1181 to Aib mutation led to combo mutant peptidesexhibited excellent antiviral potency in the 1-digit and sub-nanomolar range against CoV-2 andMERS (e.g., PepSP2531, PepSP2784, PepSP2608, PepSP2609, PepSP2708, PepSP2785).

[0280] The substitution of the Pro in position 1162 with a 4-carboxymethoxy substitutedproline, such as (2R,4S)-4-(carboxymethoxy)pyrrolidine-2-carboxylic acid ((2R,4S)Prot4AcOH)(e.g., PepSP2529, PepSP2530, PepSP2783) revealed the achievement of constructs withoutstanding antiviral potency against CoV-2 and MERS.

[0281] The below shows additional exemplary inhibitor compounds and their Pseudo-typeneutralization assay results. Table 35. Exemplary viral fusion inhibitorsSEQ ID ID SEQUENCE & StructureNO25856 SEQ ID ID SEQUENCE & StructureNO25856 SEQ ID ID SEQUENCE & StructureNO. oV-2, and MERS. CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID P d t EC50 i Hh725856 CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID Pseudoneut EC50 in Huh7

[0282] This example shows exemplary half-life extension of viral fusion inhibitors.

[0283] To increase the half-life, PepSP2430 was functionalized with Albutag using a Succinyl-eK(4IPhBut) building block. Albutag was introduced at position 1168 and / or at the N-terminus.Two different spacers were also tested to identify the optimal linker for PK extension: differentnumber of AEEA units, moving from 0 to 3; and a flexible stretch of GSGSG residues (SEQ IDNO: 675). The structures of the compounds are listed in Table 36.Table 37. Exemplary viral fusion inhibitorsSEQ ID ID SEQUENCE & StructureNO25856 SEQ ID ID SEQUENCE & StructureNOoV-2, and MERS. CoV-2 A549 CoV-2 Hela MERS Pseudoneut EC50 - SARS2 D614G Pseudoneut EC50 - SARS2 D614GID P d t EC50 i H h7

[0284] As shown in the Table above, the incorporation of the Albutag derivatization either atthe N-terminus (e.g., PepSP2737, PepSP2738, PepSP2739, PepSP2736) or at position 1168 (e.g.,PepSP2749, PepSP2750, PepSP2751) with the AEEAn (n=0,1,3) or GSGSG spacer (SEQ ID NO:675) is tolerated and has low impact on the antiviral activity. Indeed, the resulting analogsexhibited antiviral potency in the sub- and 1-digit nanomolar range against CoV-2 and MERS,similarly to PepSP2430. Example 12: Live Virus Assay

[0285] This example shows exemplary live virus assays against several inhibitors.

[0286] Some inhibitors (see Table 38) were tested in a number of varied live virus assaysagainst several strains of CoV2 including WA1 (WT CoV2) and BA.5 (current Omicron variant). Asmall subset of inhibitors was tested against WA1, BA.2, BA2.12.1, BA.4, and BA.5, and the25856 results are summarized in Table 38. Broader testing was done against WA1 (WT CoV2) and BA.5 (current Omicron variant), and that data is summarized in Table 39. This assay is highly reflective of the real-world behavior of these molecules in the presence of actual infective virus. Table 39. Live virus assay results CoV2 WA1 CoV2 BA.2 CoV2 BA.2.12.1 CoV2 BA.4 CoV2 BA.5 ID (NT50, pM) (NT50, pM) (NT50, pM) (NT50, pM) (NT50, pM)ID CoV2 WA1 (NT50, nM) CoV2 BA.5 (NT50, nM)056 0036

[0287] In general, the compounds that were highly potent in the pseudoneutralization screeningassay are as potent or more potent in these live virus assays. Potency against the WA1 WT virus shows enhanced potency in most cases against the Omicron variant(s).Example 13: Plasma stability and In vivo experiments with selected compounds25856 Pharmacokinetics after IV and SC dosing

[0288] Selected compounds were evaluated for pharmacokinetics after intravenous andsubcutaneous dosing to mice. The pharmacokinetics of selected compounds were evaluated in mice or hamsters. For each study, the individual test compounds was administered to subjects in via a preselected route of administration which included intranasal (IN), intratracheal (IT),intravenous (IV), or subcutaneous (SC). Plasma, liver, lung, and / or bronchoalveolar lavage fluid(BALF) were collected up to 30 hours following dose administration and samples were submittedfor bioanalysis to determine analyte concentrations.

[0289] The metabolic stability of test compounds was assessed in fresh plasma, from mice at afinal concentration of 10 μM. The tissue homogenates were prepared at a 3:1 ratio of tissue:phosphate buffered saline. Reactions were initiated by the addition of the test compounds into the matrix and incubated in a 5% CO2environment at 37°C with serial sampling out to 4 hours. The Samples were quenched by the addition of an organic solvent and then analyzed by LC-HRMS using calcitonin as an internal standard. Peak area ratios of the test compound to the internal standard at each time point were expressed as a percent of the peak area at the zero- minute time point which represented the 100% value.

[0290] The results are summarized in Table 40 and in Figures 18-21.Table 41. Plasma and lung levels of selected compounds after IV and SC dosing in mice Compound # In vitro mouse In vivo mouse In vivo mouse In vivo mouselasma stabilit terminal T1 / 2 (h) clearance stead state volume

[0291] Selected compounds were evaluated for antiviral efficacy after intranasal andsubcutaneous dosing to mice and / or hamsters. ^Mouse antiviral efficacy at various SC doses with PepSP1141

[0292] 8-9 week-old K18-humanized ACE2 mice (male and female) were nasally inoculatedwith SARS CoV-2 virus strain USA-WA1 / 2020 (P4, Catalog#: NR-53873.9.2x105median tissue25856culture infectious dose (TCID50) / ml (Vero E6), Lot#:70039812); Virus titer 4.6x104TCID50 / animal) on day zero. The test compound was dosed via SC administration on day zero at 2 hours before viral inoculation and 6 hours after viral inoculation, and subsequently twice daily at 8 hour intervals for days 1,2, and 3 of the experiment. Subcutaneous dosing was performed as follows: 1. Attach a 20-27 gauge needle to a syringe and fill the syringe with the material to beinjected. Remove air bubbles from the syringe. (A maximum volume of up to 3 mL may be injected). 2. Site for injection: nape of the neck, around hip.3. Manually restrain the animal.4. Elevate skin to produce a tent.5. Insert needle through the loose skin, bevel up, being careful not to penetratemusculature or the opposite side of the tent. 6. Pull back gently on plunger. If blood enters syringe, a blood vessel has beenpenetrated. Remove needle and change injection site. 7. Inject material slowly. You should notice a moveable swelling form under skin.8. Withdraw needle.9. Put the needle / syringe assembly into a red Sharps container. Do not recap the syringe.Treatment groups were as follows: Group 1, vehicle control, (15% 2- hydroxypropyl- betacyclodextrin in 50 mMPhosphate buffer) 5 mL / kg, SC, twice a day (BID) Group 2, 10 mL / kg (5 ml / kg), SC, BID Group 3, 25 mL / kg (5 ml / kg), SC, BID Group 4, 50 mL / kg (5 ml / kg), SC, BID Group 5, 50 mL / kg (5 ml / kg), SC, once a day (QD) Group 6, Human convalescent IgG, 15 mg / kg IM, day 0

[0293] Viral load in lung was determined on day 4 and the results are shown in Figures 22-24.PepSP1141 demonstrated statistically significant reductions in viral load and viral RNA in this experiment. ^Mouse antiviral efficacy at various IN doses with PepSP114125856

[0294] 8-9 week-old K18-humanized ACE2 mice (male and female) were nasally inoculatedwith SARS CoV-2 virus strain USA-WA1 / 2020 (P4, Catalog#: NR-53873.9.2X105TCID50 / ml(Vero E6), Lot#:70039812); Virus titer 4.6x104 TCID50 / animal) on day zero. The test compoundwas dosed intranasally on day 0 at 2 hours before infection, and once a day on days 1-3. Intranasal doing was performed as follows: 1. Mice anaesthetized by isoflurane.2. Set the positive-displacement pipette to the volume of material to be delivered. Loadthe tip with the inoculum (0.05 mL maximum volume). 3. Hold the mouse upright at an approximate 30-degree angle.4. Place the tip to the nares of the mouse. Slowly release the inoculum dropwise onto thenares making sure the entire drop is inhaled. 5. Place mouse on its back in a clean cage and observe until the mouse is up and movingaround the cage. Treatment groups were as follows: Group 1, vehicle control, (15% 2- hydroxypropyl- betacyclodextrin in 50 mMPhosphate buffer) 50 μL (25 μL per nare), IN, QDGroup 2, 1 mg / kg (50 μL, 25 μL per nare), IN, QD Group 3, 3 mg / kg (50 μL, 25 μL per nare), IN, QD Group 4, 10 mg / kg (50 μL, 25 μL per nare), IN, QD Group 5, 50 mg / kg (5 mL / kg), SQ, BID (control based on above SC data) Group 6, Human convalescent IgG, 15 mg / kg IM, day 0

[0295] Viral load in lung was determined on day 4 and the results are shown in Figures 25-26.PepSP1141 demonstrated dose dependent, statistically significant reductions in viral load and viral RNA in this experiment. ^Hamster antiviral efficacy at various SC doses with PepSP1545 with PepSP1141 as SCcontrol

[0296] Male syrian hamsters were nasally inoculated with SARS CoV-2 virus strain USA-WA1 / 2020 (P4, Catalog#: NR-53873. 9.2x105 TCID50 / ml (Vero E6), Lot#:70039812); Virus titer4.6x104TCID50 / animal) on day zero. The test compound was dosed SC on day zero at 2 hours before viral inoculation and 6 hours after viral inoculation, and subsequently BID at 8 hours intervals for days 1,2, and 3 of the experiment. Subcutaneous dosing was performed as follows:25856 1. Attach a 20-27 gauge needle to a syringe and fill the syringe with the material to beinjected. Remove air bubbles from the syringe. (A maximum volume of up to 3ml can be injected). 2. Site for injection: nape of the neck, around hip.3. Manually restrain the animal.4. Elevate skin to produce a tent.5. Insert needle through the loose skin, bevel up, being careful not to penetratemusculature or the opposite side of the tent. 6. Pull back gently on plunger. If blood enters syringe, a blood vessel has beenpenetrated. Remove needle and change injection site. 7. Inject material slowly. You should notice a moveable swelling form under skin.8. Withdraw needle.9. Put the needle / syringe assembly into a red Sharps container. Do not recap the syringe.Treatment groups were as follows: Group 1, vehicle control, (15% 2- hydroxypropyl- betacyclodextrin in 50 mMPhosphate buffer) 5 mL / kg, SC, BID Group 2, PepSP1545, 25 mg / kg (5 mL / kg), SC, BID Group 3, PepSP1545, 10 mg / kg (5 mL / kg), SC, BID Group 4, PepSP1545, 2.5 mg / kg (5 mL / kg), SC, BID Group 5, PepSP1141, 25 mg / kg (5 mL / kg), SC, BID in vehicle 1

[0297] Viral load in lung was determined on day 4 and the results are shown in Figures 27 and28. PepSP1545 demonstrated an approximately 10 times improved in vivo effect on viral loadand viral RNA levels in mouse lung versus PepSP1141 in this experiment, with the 2.5 mg / kg dose of PepSP1545 showing equal potency to the 25 mg / kg dose of PepSP1141.

[0298] In general, the compounds tested demonstrated substantial and statistically significanteffects on lowering viral load and viral RNA in hamsters and mice, demonstrating great antiviral efficacy in both species. Example 14

[0299] This example demonstrates comparative data against existing compounds, such asCompound X (as shown in Figure 31) and Compound Y (as shown in Figure 32).25856

[0300] Compound X and Compound Y were publicly disclosed in a manuscript published indeVries, R.D., et al.:Science 2021, 371, 1379. The manuscript demonstrated that aftersubcutaneous dosing to ferrets, no detectable levels of Compound X or Compound Y were found in lung tissue. It was necessary to administer Compound X or Compound Y directly intranasally in order to observe any level of them in lung tissue.

[0301] The pharmacokinetics of PepSP1141, Compound X, and Compound Y were evaluatedafter dosing to mice, similarly to Example 13 using LC-MS / MS detection. The levels ofcompounds in mouse lung after the same subcutaneous dosing regimen were analyzed.

[0302] At a subcutaneous dose of 15 mg / kg, PepSP1411 exhibited slightly better plasma levelsat 2 hours, 4 hours, and 8 hours compared to Compound X as shown in Figure 33. However, at24 hours, PepSP1411 showed approximately 10 times higher plasma levels than Compound X.Compound Y was not detectable at any of the time points.

[0303] A number of the animals were sacrificed and their lung exposures were determinedusing two methods of detection: LC-MS / MS, and ex vivo analysis of lung tissue using apseudoneutralization bioassay. Both methods showed that the levels of PepSP1141 in the lungtissue were consistently higher than those demonstrated by Compound X. The levels ofCompound Y were undetectable in the lung tissue.

[0304] Furthermore, in the testing described in Example 1, PepSP1141 and the compoundsdisclosed herein exhibited greater potency and a wider range of activity against the coronavirus compared to Compound X and Compound Y.

Claims

25856 WHAT IS CLAIMED IS:

1. An inhibitor of viral fusion comprising:(i) a peptide moiety; (ii) a linker moiety; and (iii) a lipid moiety, wherein the linker moiety connects the C-terminal part of the peptide moiety to the lipid moiety, wherein the peptide moiety comprises at least ten contiguous amino acids of the heptad repeat 2 (HR2) domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), and wherein the N-terminal part of the peptide moiety is PDVD as set forth in SEQ ID NO. 418.

2. The inhibitor of claim 1, wherein the peptide moiety comprises a peptide as set forth inSEQ ID NO:1, X1 is P, and X2 is D.

3. An inhibitor of viral fusion comprising:(i) a peptide moiety; (ii) a linker moiety; and (iii) a lipid moiety, wherein the linker moiety connects the C-terminal part of the peptide moiety to the lipid moiety, wherein the peptide moiety comprises at least ten contiguous amino acids of the heptad repeat 2 (HR2) domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), and wherein the N-terminal part of the peptide moiety is PEVD as set forth in SEQ ID NO. 419.

4. The inhibitor of claim 3, wherein the peptide moiety comprises a peptide as set forth inSEQ ID NO:1, X1is P, and X2is E.258565. An inhibitor of viral fusion comprising:(i) a peptide moiety; (ii) a linker moiety; and (iii) a lipid moiety, wherein the linker moiety connects the C-terminal part of the peptide moiety to the lipid moiety, wherein the peptide moiety comprises at least ten contiguous amino acids of the heptad repeat 2 (HR2) domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), and wherein the N-terminal part of the peptide moiety is (2R,4S)Prot4OacOH-DVD.

6. The inhibitor of claim 5, wherein the peptide moiety comprises a peptide as set forth inSEQ ID NO:1, X1 is (2R,4S)Prot4OacOH, and X2 is D.

7. The inhibitor of any one of claims 1-6, wherein the peptide moiety comprises at least oneamino acid that is different from the HR2 domain of SARS-CoV-2.

8. The inhibitor of claim 7, wherein the peptide moiety comprises at least one amino acidthat is not natural.

9. The inhibitor of any one of claims 1-8, where the peptide moiety further comprises C atthe C-terminal part of the peptide moiety.

10. The inhibitor of any one of claims 1-8, where the peptide moiety further comprises CGSGas set forth in SEQ ID NO. 421 at the C-terminal part of the peptide moiety.

11. The inhibitor of any one of claims 1-8, where the peptide moiety further comprisesGGGSGGGGSG as set forth in SEQ ID NO. 422 at the C-terminal part of the peptidemoiety.

12. The inhibitor of any one of claims 1-8, where the peptide moiety further comprisesGSGGGSGG as set forth in SEQ ID NO. 423 at the C-terminal part of the peptide moiety.2585613. The inhibitor of any one of claims 1-8, where the peptide moiety further comprisesKYEQYIG as set forth in SEQ ID NO. 424 at the C-terminal part of the peptide moiety.

14. The inhibitor of claim 1, wherein the peptide moiety is selected from the group consistingof peptides as set forth in SEQ ID NOs: 2-39.

15. The inhibitor of claim 14, wherein the peptide moiety is selected from the groupconsisting of peptides as set forth in SEQ ID NOs: 3-39.

16. The inhibitor of claim 1, wherein the peptide moiety comprise a peptide as set forth inSEQ ID NO: 40.

17. The inhibitor of claim 2, wherein the peptide moiety is selected from the group consistingof peptides as set forth in SEQ ID NOs: 279, 287, 288, 291, 292, 296, 300, 303, 308-376,395-398, and 407-413.

18. The inhibitor of claim 4, wherein the peptide moiety is selected from the group consistingof peptides as set forth in SEQ ID NOs: 309, 389, 399, or 401-404.

19. The inhibitor of claim 6, wherein the peptide moiety is selected from the group consistingof peptides as set forth in SEQ ID NOs: 377, 378, 405, 406, and 414-416.

20. The inhibitor of any one of claims 1-19, wherein the lipid moiety comprises cholesterol,tocopherol, or palmitate.

21. The inhibitor of claim 20, wherein the lipid moiety comprises cholesterol.

22. The inhibitor of any one of claims 1-21, wherein the linker moiety is attached to the lipidmoiety via an ether bond, an ester bond, a carbamate bond, or a triazole bond.2585623. The inhibitor of claim 22, wherein the linker moiety is attached to the lipid moiety via anether bond.

24. The inhibitor of claim 22, wherein the linker moiety is attached to the lipid moiety via antriazole bond.

25. The inhibitor of any one of claims 1-24, wherein the linker moiety comprises an aminoacid.

26. The inhibitor of any one of claims 1-25, wherein the linker moiety is selected from thegroup consisting of compounds listed in Table 2.

27. The inhibitor of claim 26, wherein the linker moiety comprises PEG12-C(propyl)-NH2,Ttds2-C(propyl)-NH2, Ttds3-C(propyl)-NH2, Ttds4-C(propyl)-NH2, Ttds2-Dab(triazole-propyl)-NH2, Ttds3-Dab(triazole-propyl)-NH2, Ttds4-Dab(triazole-propyl)-NH2, Ttds2- Orn(triazole-propyl)-NH2, or Ttds3-Orn(triazole-propyl)-NH2.

28. The inhibitor of any one of claims 1, 5 or 8, wherein the inhibitor is selected from thegroup consisting of compounds listed in Table 3A.

29. The inhibitor of any one of claims 1-28, wherein the N-terminal part of the peptidemoiety is acetylated.

30. The inhibitor of any one of claims 1-29, further comprising a half-life extension moiety.

31. The inhibitor of claim 30, wherein the half-life extension moiety comprises 4-(p-iodophenyl)butyric acid (4IPhBut).

32. The inhibitor of any one of claims 30-31, wherein the half-life extension moiety isattached to the N-terminal part of the peptide moiety.2585633. The inhibitor of claim 32, wherein the half-life extension moiety is selected from thegroup consisting of 4IPhBut-eK-Succinyl, 4IPhBut-eK-Succinyl-AEEA, 4IPhBut-eK- Succinyl-(AEEA)2, 4IPhBut-eK-Succinyl-(AEEA)3, and 4IPhBut-eK-Succinyl-GSGSG(SEQ ID NO: 425).

34. The inhibitor of any one of claims 30-31, wherein the peptide moiety has a Lys, D-Lys orOrn mutation, and the half-life moiety is attached to the mutation.

35. The inhibitor of claim 34, wherein the peptide moiety has Lys in position 1168.

36. The inhibitor of claim 35, wherein the peptide moiety comprise a peptide as set forth inSEQ ID NO: 407.

37. The inhibitor of any one of claims 34-36, wherein the half-life extension moiety isselected from the group consisting of 4IPhBut-eK-Succinyl, 4IPhBut-eK-Succinyl- AEEA, 4IPhBut-eK-Succinyl-(AEEA)2, and 4IPhBut-eK-Succinyl-(AEEA)3.

38. A composition comprising the inhibitor of any preceding claim, or a pharmaceuticallyacceptable carrier or diluent.

39. An inhibitor of any one of claims 1 to 37, for use in a method of treatment or preventionof a subject.

40. The inhibitor of claim 39, wherein the treatment or prevention is for infection by a virusselected from the group consisting of SARS-CoV-1, SARS-CoV-2, MERS, and HIV-I.

41. The inhibitor of claim 40, wherein the inhibitor is capable of treating and / or preventingtwo or more types of the virus.

42. The inhibitor of claim 41, wherein the virus is SARS-CoV-2 or MERS.2585643. A method of treating or preventing infectious disease in a subject, comprisingadministering to the subject an effective amount of a composition comprising the inhibitor of any one of claims 1 to 37.

44. The method of claim 43, wherein the infectious disease is caused by a virus selected fromthe group consisting of SARS-CoV-1, SARS-CoV-2, MERS, and HIV-I.

45. The method of any one of claims 43-44, wherein the composition is administeredintranasally, intratracheally, subcutaneously or intranasally.

46. The method of any one of claims 43-44, wherein the composition is administered as nasaldrops, nasal powder, or a spray.

47. Use of the inhibitor of any one of claims 1 to 37 for the manufacture of a medicament forthe treatment or prevention of infection or infectious disease.

48. The use of claim 47, wherein the infectious disease is caused by a virus selected from thegroup consisting of SARS-CoV-1, SARS-CoV-2, MERS, and HIV-I.

Images