Enhancing the solubility of SARS-COV-2 inhibitors to increase clinical potential

Abstract

Described herein are soluble HRC-based Hpopeptide inhibitors of coronaviruses identified using structure-guided design to incorporate charged or polar residues at specific sites in the peptide to enhance aqueous solubility. Also described are methods of treating a coronavirus infection using soluble HRC-based hpopeptide inhibitors.

Description

Attorney Docket 44010.227WO-PCT / / CU25029 ENHANCING THE SOLUBILITY OF SARS-COV-2 INHIBITORS TO INCREASE CLINICAL POTENTIAL CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 683,596, filed August 15, 2024, titled “ENHANCING THE SOLUBILITY OF SARS-COV-2 INHIBITORS TO INCREASE CLINICAL POTENTIAL”. The entire contents of the above- identified applications are hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under AI160961, AI160953, GM056414, GM122263, and AI176876 awarded by the National Institutes of Health. The government has certain rights in the invention INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

[0003] The official copy of the sequence listing is submitted electronically in an .xml file format having the file name “44010.227SeqList.xml” created on August 14, 2025, and having a size of 14,897 bytes, and is filed concurrently with the specification. The Sequence Listing is part of the specification and is herein incorporated by reference in its entirety. TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to soluble HRC-based lipopeptide inhibitors of SARS-CoV-2 infection. BACKGROUND

[0005] Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the causative agent of the 2019 coronavirus disease (COVID-19), now appears to be established in the human population. Rapid emergence of new strains and declining interest in vaccination and other preventative measures make this pathogen an ongoing threat, especially for the elderly and those with impaired immune systems. A prophylactic antiviral strategy that maintains efficacy as SARS-CoV-2 evolves would represent a significant contribution to public health.Attorney Docket 44010.227WO-PCT / / CU25029

[0006] Infection by SARS-CoV-2 is initiated by fusion of the viral and host cell membranes, a process mediated by the SARS-CoV-2 spike (S) glycoprotein.1, 2S is a homotrimer, with each monomer consisting of S1 and S2 subunits. A receptor-binding domain (RBD) within the S1 subunit promotes cell surface attachment and activation. Receptor- mediated activation triggers a series of structural transitions that leads to formation of a stable six-helix bundle (6HB) assembly between N- and C-terminal heptad repeat (HRN and HRC) domains within the S2 subunit, ultimately driving membrane fusion and infection.

[0007] Applicants have previously reported lipopeptides that potently inhibit fusion mediated by SARS-CoV-2 S and block infection by SARS-CoV-2 S in cell monolayers (in vitro), human airway tissues (ex vivo), and animal models (in vivo).3, 4These molecules contain a peptide segment corresponding to the HRC domain of SARS-CoV-2 S. The lipopeptides are believed to engage the trimeric HRN domain assembly of SARS-CoV-2 S, thereby disrupting the structural rearrangements of S that drive membrane fusion. Analogous HRC-based lipopeptides prevent infection by several other viruses (HIV, measles, Nipah, parainfluenza, influenza), and can be administered via the airway.3-26Treatment is effective for some of these viruses even several days after exposure.8

[0008] These lipopeptides bear an appended cholesterol moiety that anchors them in the host cell membrane, thus concentrating the lipopeptides at the site of action and enhancing antiviral efficacy.7-10, 18-20These precedents suggest that HRC-based lipopeptides could be effective prophylactic agents for protection from COVID.

[0009] The first lipopeptide inhibitor Applicants described, designated HRC-L-PEG4-Chol here (Figure 1), was based on a 36-residue segment of the native SARS-CoV2 S HRC domain (Wuhan strain)3. This inhibitor was efficacious in cell-based assays3, and pegylated and / or dimerized derivatives of this lipopeptide were highly effective at preventing SARS-CoV2 transmission in vivo.4The 36-mer peptide and lipopeptide derivatives exhibited poor aqueous solubility and a strong propensity for gelation that hindered synthesis, purification, characterization, and evaluation. These studies revealed that prospects for clinical application of this approach would be improved if Applicants could enhance the solubility of the precursor HRC peptide. Here Applicants describe the path to increasing aqueous solubility by modifications of the HRC-derived segment, which resulted in new lipopeptide inhibitors of SARS-CoV-2 infection. Low nasal cavity volume allows for a maximum volume of only <500 μL for intranasal delivery in humans; thus, efforts to enhance lipopeptide solubility in early-Attorney Docket 44010.227WO-PCT / / CU25029 stage studies should enhance long-term prospects for clinical development.47–53Here, Applicants describe a rational approach to increasing aqueous solubility of the HRC-derived peptide that resulted in new lipopeptide inhibitors of SARS-CoV-2 infection.

[0010] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention. SUMMARY

[0011] In one aspect, the present invention provides for a synthetic peptide comprising a C-terminal heptad repeat region (HRC) of SARS-CoV-2 S protein, wherein one or more residues of the HRC of SARS-CoV-2 S protein (SEQ ID NO: 1) are substituted with corresponding residues having charged or polar side chains at solvent-facing positions of the synthetic peptide when in a six-helix bundle assembly with the SARS-CoV-2 N-terminal heptad repeat region (HRN), thereby improving solubility of the synthetic peptide while maintaining side chains for stabilizing the six-helix bundle assembly of a viral HRN trimer. In certain embodiments, wherein the synthetic peptide comprises a substitution with at least one negatively charged amino acid and at least one positively charged amino acid. In certain embodiments, the C-terminal HRC comprises a sequence of: DISQINASVVNIEYEIKKLEEVAKKLEESLIDLQEL (SEQ ID NO: 2); or SIDQINATFVDIEYEIKKLEEVAKKLEESYIDLKEL (SEQ ID NO: 3). In certain embodiments, the synthetic peptide further comprises a linker segment at the C-terminus of the peptide. In certain embodiments, the linker segment comprises a Gly-Ser-Gly-Cys linker segment. In certain embodiments, the synthetic peptide further comprises a cholesterol moiety conjugated to the linker segment at the Cys residue. In certain embodiments, the cholesterol moiety is conjugated to the linker segment by a polyethylene glycol linker. In certain embodiments, the polyethylene glycol linker is PEG4, PEG8, PEG11, PEG24, or PEG28. In certain embodiments, the synthetic peptide further comprises an N-terminally extended SARS- CoV-2 HRC peptide corresponding to S residues 1162-1203, wherein the peptide includes PDVDLG (SEQ ID NO: 6) at the N-terminus. In certain embodiments, the C-terminal HRC comprises a sequence of PDVDLGDISQINASVVNIEYEIKKLEEVAKKLEESLIDLQEL (SEQ ID NO: 4).

[0012] In another aspect, the present invention provides for a lipoprotein comprising: the HRC of any of embodiment herein; a cholesterol unit; and at least one polyethylene glycolAttorney Docket 44010.227WO-PCT / / CU25029 linker, wherein: the cholesterol unit and the at least one polyethylene glycol linker are attached to form a polyethylene glycol-cholesterol unit, and the polyethylene glycol-cholesterol unit is linked to the C-terminus of the synthetic peptide. In certain embodiments, the at least one polyethylene glycol linker is PEG4, PEG8, PEG11, PEG24, or PEG28. In certain embodiments, the lipoprotein further comprises a linker segment at the C-terminus of the lipoprotein. In certain embodiments, the linker segment comprises a Gly-Ser-Gly-Cys linker segment. In certain embodiments, the lipoprotein structure comprises HRC-linker segment-PEG- cholesterol.

[0013] In another aspect, the present invention provides for a method of inhibiting SARS- CoV-2 or MERS infection comprising administering to a subject in need thereof the synthetic peptide or lipoprotein according to any embodiment herein. In certain embodiments, the synthetic peptide or lipoprotein are administered by inhalation, parenterally, intravenously, intramuscularly, or subcutaneously.

[0014] In another aspect, the present invention provides for structure-guided method of improving solubility of a peptide capable of disrupting a six-helix bundle assembly of a viral N-terminal heptad repeat region (HRN) and C-terminal heptad repeat region (HRC) of SARS- CoV-2 S protein, comprising: selecting at least one amino acid within a 3-D structure of the peptide in complex with a six-helix bundle assembly, wherein the amino acid is not critical for stabilizing the six-helix bundle assembly with a viral HRN trimer, wherein the amino acid has at least one side chain facing a solvent in the 3-D structure; engineering the peptide to substitute the at least one amino acid with a negatively charged amino acid or a positively charged amino acid to improve solubility of the peptide; testing the engineered peptide with the at least one substituted amino acid for stabilizing the six-helix bundle assembly with a viral HRN trimer; and selecting an optimal combination of amino acid substitutions which preserves the stabilization while enhancing the aqueous solubility of the peptide. In certain embodiments, the substitutions include one or more substitutions made to the HRC of SARS-CoV-2 S protein selected from the substitutions in SEQ ID NO:2 or SEQ ID NO: 3. In certain embodiments, the negatively charged amino acid comprises aspartate (Asp) or glutamate (Glu) and the positively charged amino acid comprises arginine (Arg) or lysine (Lys).

[0015] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.Attorney Docket 44010.227WO-PCT / / CU25029 BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0018] FIG.1A-FIG.1G – (FIG.1A) Simplified schematic diagram of SARS-Co-V-2 S. The N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), N- terminal heptad repeat (HRN, periwinkle), C-terminal heptad repeat (HRC, salmon), transmembrane (TM), and cytoplasmic tail (CP) domains are depicted. (FIG. 1B) Sequences of the native HRC, EK1, and designed variants, QE and SK. Residue replacements in SK and QE are shown in red. (FIG.1C) Definition of peptides referred to in text and the structure of cholesterol-conjugated HRC. (FIG.1D) Analysis of the six-helix bundle assembly formed by HRN (periwinkle) and HRC (salmon). (FIG. 1E-G) Analysis of side chain orientation within the α-helical segments of the HRC (FIG.1E, salmon), QE (FIG.1F, yellow), and SK (FIG.1G, green) heptad repeat sequences.

[0019] FIG. 2 – Solubility of HRC-L, QE-L, and SK-L peptides as measured by UV absorbance at 280 (QE-L and SK-L) and 205 nm (HRC-L).

[0020] FIG. 3A-FIG. 3F – Circular dichroism studies of SARS-CoV-2 HRN + HRC hybrid co-assemblies. Full spectrum scan at 25oC of (FIG. 3A) peptide mixtures and (FIG. 3B) individual peptides. (FIG.3C) Temperature dependent denaturation of HRN alone. (FIG. 3D-E) temperature-dependent denaturation of co-assemblies formed between mixtures of either (FIG. 3D) HRN or (FIG.3E) truncated HRN peptides (tHRN) with HRC, QE, and SK. 50 μM total peptide concentration in 10 mM phosphate buffer, pH 7.4. (FIG.3F) Table of Tmfor the indicated peptides.

[0021] FIG.4A-FIG.4H – X-ray crystal structure of QE bound to the HRN domain of SARS-CoV-2 S. (FIG. 4A) 6HB co-assembly formed between SARS-CoV- 2 HRN (periwinkle) and HRC (salmon) (PDB 6LXT). (FIG. 4B) 6HB co-assembly formed between SARS-CoV-2 HRN (periwinkle) and QE (yellow) (PDB 6X45). (FIG. 4C) Alternate view ofAttorney Docket 44010.227WO-PCT / / CU25029 PDB 6LXT. (FIG. 4D) Alternate view of PDB 6X45 with changed residues shown in bright red. (FIG.4E) Overlay of SARS-CoV-2 HRC (salmon) and QE (yellow). (FIG.4F-G) Overlays of 6HB co-assemblies formed between SARS-CoV-2 HRN with either QE (yellow) or SK (green; AlphaFold2). (FIG.4H) Overlays of 6HB co-assemblies formed between SARS-CoV- 2 HRN with either QE (yellow) or SK (green; AlphaFold2) with changed sidechains illustrated. Legend shown in bottom right.

[0022] FIG. 5A-FIG. 5B – Inhibition of SARS-CoV-2 spike (S)-mediated cell-cell fusion. Fusion inhibitory activity of different peptides against SARS-CoV-2 S variants D614G (FIG. 5A) and BA.5 (FIG. 5B). The percent inhibition is depicted for four SARS-CoV-2 inhibitory peptides at increasing concentrations. The percent fusion inhibition was calculated as the ratio of luminescence in the presence of peptide at a specific concentration (X) to the luminescence in the absence of inhibitor. Percent inhibition = 100 x [1 - (luminescence at X) / (luminescence in absence of peptide)]. Data in (FIG.5A) and (FIG.5B) are mean ± standard error of the mean (SEM) from three distinct experiments with a four parameter variable curve.

[0023] FIG. 6A-FIG. 6B – Inhibition of SARS-CoV-2 and MERS viral infection by the designed peptides. Plaque reduction assay with SARS-CoV-2 virus (FIG.6A) or MERS virus (FIG. 6B). The QE and SK lipopeptides were assessed alongside the native HRC lipopeptide; this dataset included the native HRC published in2and reproduced here.

[0024] FIG. 7 – Technical issues encountered during synthesis, purification, and analysis of HRC and HRC-L peptides. During cleavage from solid support in 95% trifluoracetic acid (TFA), HRC precipitates out of solution. During dissolution of crude peptide in common HPLC solvents, such as DMSO, MeOH, and acetonitrile, HRC undergoes irreversible gel formation or precipitation. Once pure, HRC precipitates and / or gels in several experimental aqueous conditions, such as when sonicated, agitated, or slightly heated.

[0025] FIG. 8 – Solubility assessment of peptides. Solubility of HRC, QE, and SK peptides in 50 mM phosphate buffer (pH 7.4) as measured by UV absorbance at 280 (QE and SK) and 205 nm (HRC) over time. ε205(HRC-L) = 118,820 M-1cm-1and ε205(HRC) = 101,450 M-1cm-1.

[0026] FIG. 9 –. Circular dichroism studies of SARS-CoV-2 tHRN + EK1 co- assembly. Temperature-dependent denaturation of co-assembly formed between a 1:1 mixture of tHRN and EK1.10 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. Tm= 38 ± 0.3 °CAttorney Docket 44010.227WO-PCT / / CU25029 EK1: Ac- S L D Q I N V T F L D L E Y E M K K L E E A I K K L E E S Y I D L K E L - NH2(SEQ ID NO: 5)

[0027] FIG.10 – Peptide toxicity. To determine the toxicity of the new lipopeptides, a 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed in parallel with the fusion inhibition studies. Toxicity of the lipopeptides was minimal for each lipopeptide tested (<20% at 100 nM). No toxicity was observed for any of the lipopeptides at the respective IC90s. For SK, no toxicity was observed up to 10 µM peptide concentration, while all other lipopeptides exhibited ~20% toxicity at this concentration.

[0028] FIG. 11A-FIG. 11D – Inhibition of SARS-CoV-2 spike (S)-mediated cell-cell fusion with oxidized peptides. Fusion inhibitory activity of oxidized (-ox) and non-oxidized peptides against SARS-CoV-2 S variant BA.5. The non-oxidized peptides have a thioether linkage at the C-terminal Cys side chain, while the oxidized peptides contain a sulfoxide. The percent inhibition is depicted for QE (FIG.11A), SK (FIG.11B), Native HRC (FIG.11C), and QE-ext (FIG.11D) at increasing concentrations. The percent fusion inhibition was calculated as the ratio of luminescence in the presence of peptide at a specific concentration (X) to the luminescence in the absence of inhibitor. Percent inhibition = 100 x [1 - (luminescence at X) / (luminescence in absence of peptide)]. Data in (FIG. 11A) and (FIG. 11B) are mean ± standard error of the mean (SEM) from three distinct experiments with a four-parameter variable curve.

[0029] FIG.12A-FIG.12C – Circular dichroism studies of SARS-CoV-2 HRN + HRC hybrid co-assemblies. Full CD scans from temperature-dependent denaturation of co- assemblies formed by 1:1 mixtures of HRN with (FIG. 12A) HRC, (FIG. 12B) QE or (FIG. 12C) SK. 50 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. Legends indicate temperature in °C.

[0030] FIG. 13A-FIG. 13C – Circular dichroism studies of SARS-CoV-2 tHRN + HRC hybrid co-assemblies. Full CD scans from temperature-dependent denaturation of co- assemblies formed in 1:1 mixtures of tHRN with (FIG. 13A) HRC, (FIG. 13B) QE or (FIG. 13C) SK. 50 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. Legends indicate temperature in °C.

[0031] FIG.14 – Circular dichroism studies of SARS-CoV-2 HRN + the Ile12-->Ala variant of the native HRC. Temperature-dependent denaturation of co-assemblies formed inAttorney Docket 44010.227WO-PCT / / CU25029 1:1 mixtures of HRN with QE or QE-A12. QE-A12 Tm= 74 °C. 50 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. QE: Ac –D I S Q I N A S V V N I E Y E I K K L E E V A K K L E E S L I D L Q E L - NH2(SEQ ID NO: 2) QE-A12: Ac –D I S Q I N A S V V N A E Y E I K K L E E V A K K L E E S L I D L Q E L - NH2(SEQ ID NO: 11)

[0032] FIG. 15 – Circular dichroism studies of SARS-CoV-2 HRN + truncated variants of QE. Temperature-dependent denaturation of co-assemblies formed in 1:1 mixtures of HRN with QE, QE-25, or QE-19. QE-25 Tm= 53 °C; QE-19 Tm= 50 °C. 50 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. QE-25 (fragment of QE): Ac – I E Y E I K K L E E V A K K L E E S L I D L Q E L - NH2(SEQ ID NO: 12) QE-19 (fragment of QE): Ac – I E Y E I K K L E E V A K K L E E S L - NH2(SEQ ID NO: 13)

[0033] FIG. 16 – Circular dichroism studies of SARS-CoV-2 HRN + QE-EXT. Temperature-dependent denaturation of co-assemblies formed in 1:1 mixtures of HRN with QE or QE-EXT.50 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. QE1-EXT: Ac – P D V D L G - D I S Q I N A S V V N I E Y E I K K L E E V A K K L E E S L I D L Q E L - NH2(SEQ ID NO: 4)

[0034] FIG.17 – Circular dichroism studies of SARS-CoV-2 HRN peptides. Full CD spectrum scans at 25 °C of HRN or tHRN. 25 µM total peptide concentration in 10 mM phosphate buffer, pH 7.4. HRN: Ac – T Q N V L Y E N Q K L I A N Q F N S A I G K I Q D S L S S T A S A L G K L Q D V V N Q N A Q A L N T L V K Q L - NH2(SEQ ID NO: 14) tHRN: Ac –L I A N Q F N S A I G K I Q D S L S S T A S A L G K L Q D V V N Q N A Q A L N T L V K Q - NH2(SEQ ID NO: 15)

[0035] FIG. 18 – Oxidation of cysteine thioether to the corresponding sulfoxide, which occurs spontaneously during chemical ligation of the cholesterol moiety. The sulfur atom of a sulfoxide is a stereogenic center; therefore, the sulfoxide form is presumably a mixture of two stereoisomers that differ in terms of the configuration at the sulfur center.

[0036] FIG.19A-FIG.19C – Solubility Studies. UPLC analysis of QE solubility in PBS after equilibration times of (FIG.19A) 1 hour, (FIG.19B) 1 day, and (FIG.19C) 2 weeks. AfterAttorney Docket 44010.227WO-PCT / / CU25029 one day, a second peak appeared corresponding to disulfide formation between cysteine residues at the C-termini of QE (FIG. 19B). Tris(2-carboxyethyl)phosphine (TCEP) addition after 2 weeks eliminated the second peak, as a result of disulfide reduction (FIG. 19C). No peptide degradation was observed after 2 weeks of incubation in PBS at room temperature. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm.

[0037] FIG.20A-FIG.20C – Solubility Studies. UPLC analysis of SK solubility in PBS after equilibration times of (FIG.20A) 1 hour, (FIG.20B) 1 day, and (FIG.20C) 2 weeks. After one day, a second peak appeared corresponding to disulfide formation between cysteine residues at the C-termini of SK (FIG.20B). TCEP addition after 2 weeks eliminated the second peak, as a result of disulfide reduction (FIG.20C). No peptide degradation was observed after 2 weeks of incubation in PBS at room temperature. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm.

[0038] FIG.21 – MALDI-TOF and UPLC analysis of QE. Purity = 99.3%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 4184.2; Observed [M+H]+: 4184.1

[0039] FIG.22A-FIG.22B – MSMS data for QE. (FIG.22A) MS and MSMS spectra of QE. (FIG. 22B) Fragmentation data for QE. Most b and y’ ions were observed during fragmentation, which strongly supports the existence of the proposed peptide sequence. Analysis was done in GPMAW 10.

[0040] FIG. 23 – MALDI-TOF and UPLC analysis of QE-L. Purity = 98.4%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. QE-L: Ac –D I S Q I N A S V V N I E Y E I K K L E E V A K K L E E S L I D L Q E L GSGSGC- NH2(SEQ ID NO: 6) Calculated monoisotopic [M+H]+: 4632.4; Observed [M+H]+: 4632.9

[0041] FIG.24 – MALDI-TOF and UPLC analysis of SK. Purity = 99.8%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 4297.3; Observed [M+H]+: 4297.5Attorney Docket 44010.227WO-PCT / / CU25029

[0042] FIG.25A-FIG.25B – MSMS data for SK. (FIG.25A) MS and MSMS spectra of SK. (FIG. 25B) Fragmentation data for SK. Most b and y’ ions were observed during fragmentation, which strongly supports the existence of the proposed peptide sequence. Analysis was done in GPMAW 10.

[0043] FIG. 26 – MALDI-TOF and UPLC analysis of SK-L. Purity = 99.8%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. SK-L: Ac-SIDQINATFVDIEYEIKKLEEVAKKLEESYIDLKEL-GSGSGC-NH2(SEQ ID NO: 7) Calculated monoisotopic [M+H]+: 4745.4; Observed [M+H]+: 4745.4

[0044] FIG. 27 – MALDI-TOF and UPLC analysis of HRC. Purity = 97.6%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity BEH C4, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 4048.1; Observed [M+H]+: 4049.6

[0045] FIG. 28 – MALDI-TOF and UPLC analysis of HRC-L. Purity = 95.4%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity BEH C4, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. HRC-L: Ac- D I S G I N A S V V N I Q K E I D R L N E V A K N L N E S L I D L Q E L GSGSGC- NH2(SEQ ID NO: 8) Calculated monoisotopic [M+H]+: 4496.3; Observed [M+H]+: 4496.8

[0046] FIG. 29 – MALDI-TOF and UPLC analysis of HRN-55. Purity = 98.1%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. HRN-55: Ac – T Q N V L Y E N Q K L I A N Q F N S A I G K I Q D S L S S T A S A L G K L Q D V V N Q N A Q A L N T L V K Q L - NH2(SEQ ID NO: 9) Calculated monoisotopic [M+H]+: 5983.2; Observed [M+H]+: 5983.0

[0047] FIG.30 – MALDI-TOF and UPLC analysis of HRN-44. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. HRN-44: Ac –L I A N Q F N S A I G K I Q D S L S S T A S A L G K L Q D V V N Q N A Q A L N T L V K Q - NH2(SEQ ID NO: 10) Calculated monoisotopic [M+H]+: 4652.5; Observed [M+H]+: 4652.6Attorney Docket 44010.227WO-PCT / / CU25029

[0048] FIG. 31 – MALDI-TOF and UPLC analysis of QE-A12. Purity = 98.9%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 4142.2; Observed [M+H]+: 4142.5

[0049] FIG. 32 – MALDI-TOF and UPLC analysis of QE-25. Purity = 98.5%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 3043.7; Observed [M+H]+: 3043.4

[0050] FIG. 33 – MALDI-TOF and UPLC analysis of QE-19. Purity = 99.2%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 2332.3; Observed [M+H]+: 2332.0

[0051] FIG. 34 – MALDI-TOF and UPLC analysis of QE-ext. Purity = 96.9%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. QE-EXT (N-terminally extended QE): Ac – PDVDLG - D I S Q I N A S V V N I E Y E I K K L E E V A K K L E E S L I D L Q E L - NH2(SEQ ID NO: 4) Calculated monoisotopic [M+H]+: 4780.5; Observed [M+H]+: 4780.3

[0052] FIG. 35 – MALDI-TOF and UPLC analysis of EK1. Purity = 95.0%. UPLC gradient = 10-90% MeCN / H2O over 10 minutes (0.3 mL / min; column = Waters Acquity CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm). Detection = 220 nm. Calculated monoisotopic [M+H]+: 4371.3; Observed [M+H]+: 4371.7

[0053] The figures herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

[0054] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green andAttorney Docket 44010.227WO-PCT / / CU25029 Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y.1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0055] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0056] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0057] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0058] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0059] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, femaleAttorney Docket 44010.227WO-PCT / / CU25029 ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0060] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0061] Amino acids may be referred to by either the three letter symbols or by the one- letter symbols recommended by the IUPAC, the IUAPC letter code are as follows: G = Glycine; A = Alanine; L = Leucine; M = Methionine; F = Phenylalanine; W = Tryptophan; K = Lysine; Q = Glutamine; E = Glutamic Acid; S = Serine; P = Proline; V = Valine; I = Isoleucine; C = Cysteine; Y = Tyrosine; H = Histidine; R = Arginine; N = Asparagine; D = Aspartic Acid; T = Threonine.

[0062] Amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids may be grouped together based on the properties of their side chains alone. Conservative substitutions may be made, for example according to Table A below which describes a generally accepted grouping of amino acids. Table A.Attorney Docket 44010.227WO-PCT / / CU25029

[0063] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0064] Reference is made to US Patent Application publication US20230159597A1. Reference is also made to Kuhn AJ, Outlaw VK, Marcink TC, Yu Z, Mears MC, Cajimat MN, Kreitler DF, Cleven PR, Mook JC, Bente DA, Porotto M, Gellman SH, Moscona A. Enhancing the solubility of SARS-CoV-2 inhibitors to increase future prospects for clinical development. J Virol. 2025 Mar 18;99(3):e0215924. doi: 10.1128 / jvi.02159-24. Epub 2025 Feb 4. PMID: 39902960; PMCID: PMC11915835.

[0065] All publications, published patent documents, and patent applications cited herein, including the Supplemental Materials associated with the cited publications, are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.Attorney Docket 44010.227WO-PCT / / CU25029 OVERVIEW

[0066] Embodiments disclosed herein provide soluble HRC-based lipopeptide inhibitors of SARS-CoV-2 infection. SARS-CoV-2 poses an ongoing threat to human health as variants continue to emerge. Several effective vaccines are available, but only approximately 22% of Americans received the updated 2023 vaccine. Public hesitancy towards vaccines and common occurrence of “breakthrough” infections (i.e., infections of vaccinated individuals) highlight the need for alternative methods to reduce viral transmission. SARS-CoV-2 enters cells by fusing its envelope with the target cell membrane in a process mediated by the viral spike protein, S. The S protein operates via a Class I fusion mechanism in which fusion between the viral envelope and host cell membrane is mediated by structural rearrangements of the S trimer. Applicants previously reported lipopeptides derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibit fusion by SARS-CoV-2, both in vitro and in vivo. These lipopeptides bear an attached cholesterol unit to anchor them in the membrane. Here, to improve potential clinical utility, Applicants employed structure-guided design to incorporate charged residues at specific sites in the peptide to enhance aqueous solubility. This effort resulted in three new, potent lipopeptide inhibitors. SYNTHETIC PEPTIDES

[0067] In example embodiments, synthetic peptides corresponding to the HRC of SARS- CoV-2 are modified to improve solubility of the synthetic peptide while maintaining side chains for stabilizing the six-helix bundle assembly with a viral HRN trimer. In example embodiments, the HRC of SARS-CoV-2 (SEQ ID NO: 1) is modified. As used herein, “Heptad repeat region” (HR) refers to an α-helical domain characterized by a repeating pattern of seven amino acids (denoted a–g), wherein positions a and d are typically occupied by hydrophobic residues, enabling the formation of a coiled-coil structure. The C-terminal heptad repeat region (HRC) of the SARS-CoV-2 S protein is responsible for forming the outer layer of the six-helix bundle when bound to the HRN trimer (Xia et al., 2020). The N-terminal heptad repeat region (HRN) refers to the N-terminal α-helical domain of the S2 subunit, forming a central trimeric coiled coil in the post fusion six-helix bundle structure. HRC Modifications

[0068] In example embodiments, solvent facing residues are modified. In example embodiments, a solvent facing residue is modified to a charged residue. In example embodiments, more than 1 solvent facing residue is modified to a charged residue. In exampleAttorney Docket 44010.227WO-PCT / / CU25029 embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 residues are modified to increase solubility. In preferred embodiments, at least 8 residues are modified. As used herein, “solvent-facing positions” in the HRC are amino acid side chains oriented outward toward the aqueous environment when the HRC is bound to the HRN trimer in a six-helix bundle. These positions are generally found at b, c, e, f, and g positions of the heptad repeat (see, e.g., FIG. 1E). As used herein, “charged side chains” refers to amino acid residues whose side chains bear a net positive or negative charge at physiological pH (pH ~7.4), including but not limited to lysine (positive), arginine (positive), histidine (partial positive), aspartic acid (negative), and glutamic acid (negative). In the context of the present invention, glutamine (neutral polar) may also be used as a hydrophilic substitution to enhance solubility while preserving hydrogen bonding capability.

[0069] In example embodiments, solvent facing residues are modified with residues possessing charged or hydrophilic side chains without disrupting the hydrophobic interface necessary for HRN binding. In example embodiments, at least one residue is modified at each of b, c, e, and f positions of the heptad repeat. In example embodiments, at least two residues are modified at each of c and e positions of the heptad repeat. In example embodiments, each of a and d positions of the heptad repeat are not modified or modified with a hydrophobic conservative residue.

[0070] In example embodiments, solvent-facing positions are determined by mapping the HRC onto the HRN-HRC coiled-coil crystal structure and analyzing solvent-accessible surface area (SASA) values for each residue.

[0071] In example embodiments, the synthetic peptide is the sequence as set forth in SEQ ID NO: 2 (QE). In example embodiments, the synthetic peptide is the sequence as set forth in SEQ ID NO: 3 (SK). In example embodiments, the synthetic peptide is the sequence as set forth in SEQ ID NO: 4 (QE-EXT) In example embodiments, the synthetic peptides include conservative substitutions. LIPOPROTEINS

[0072] In example embodiments, the synthetic peptides are part of a lipoprotein. As used herein, “lipoprotein” refers to a conjugate comprising a peptide covalently linked to a lipid moiety, optionally through a spacer such as PEG, which can enhance membrane association. The lipoprotein can include any lipid capable of anchoring the lipoprotein in the membrane (e.g., cholesterol, tocopherol, or palmitate). In preferred embodiments, the lipid is a cholesterolAttorney Docket 44010.227WO-PCT / / CU25029 moiety. As used herein, “cholesterol moiety” or “cholesterol unit” refers to a sterol-based hydrophobic group, derived from cholesterol or analogs thereof, which can insert into lipid bilayers and facilitate peptide localization to cellular or viral membranes. In example embodiment, the synthetic peptide includes a linker segment that is covalently linked to the lipid moiety, optionally through a spacer. In example embodiments, the lipoprotein structure comprises HRC-linker segment-PEG-cholesterol. Although cholesterol is preferred, other lipid moieties such as palmitic acid, stearic acid, or tocopherol derivatives may be substituted in certain embodiments. Linkers

[0073] In example embodiments, a linker segment is included in the synthetic peptide to separate the HRC sequence from the sequence conjugated to the lipid molecule. In example embodiments, the linker segment is a peptide sequence covalently attached to the C-terminal or N-terminal end of the HRC peptide to provide flexibility, spacing, and / or reactive functionality for conjugation. In preferred embodiments, the linker segment is at the C- terminus of the synthetic peptide. In example embodiments, the linker segment is a Gly-Ser linker. Peptide linkers consisting of repeats of glycine and serine residues are commonly chosen to introduce flexible and hydrophilic spacers between protein domains. In example embodiments, the linker segment is a Gly-Ser-Gly-Cys sequence, providing a thiol group on the cysteine residue for conjugation to lipids. Alternative linker designs are also contemplated. Suitable amino acid linkers include Gly-Gly-Cys (GGC), Gly-Ser-Cys (GSC), Gly-Gly-Gly- Cys (GGGC), and other glycine-rich sequences providing flexibility and terminal cysteine functionality.

[0074] In example embodiments, the lipid moiety is directly linked to the synthetic peptide by reacting with the terminal cysteine residue of the linker segment. For example, the terminal cysteine residue of the linker segment is reacted with a maleimide-activated cholesterol derivative to form a stable thioether bond. Other thiol-reactive cholesterol derivatives, such as iodoacetamide–cholesterol or bromoacetamide–cholesterol, may also be employed. Polyethylene glycol linkers

[0075] In example embodiments, the cholesterol moiety is conjugated to the linker segment by a polyethylene glycol linker. As used herein, “polyethylene glycol linker” (PEG) refers to an oligomer or polymer of ethylene glycol repeating units (–O–CH₂–CH₂–)^ of defined length, wherein n corresponds to the total number of ethylene glycol units. In certain embodiments,Attorney Docket 44010.227WO-PCT / / CU25029 PEG linkers have defined lengths corresponding to PEG4, PEG8, PEG11, PEG24, or PEG28, where the number indicates the number of ethylene glycol subunits. In preferred embodiments, the cholesterol moiety is attached to the peptide via a polyethylene glycol (PEG) spacer of defined length. Not being bound by a theory, PEG spacers improve peptide solubility, reduce aggregation, and allow the lipid anchor to insert into membranes without sterically hindering HRN binding. THERAPEUTIC METHODS

[0076] In example embodiments, the lipoproteins of the present invention are used for the inhibition of coronavirus entry into host cells. In humans, several coronaviruses have been identified that range in severity, from causing common colds to more severe, life-threatening illnesses. Viral entry for both SARS-CoV-2 and MERS-CoV requires formation of a six-helix bundle between the HRN and HRC domains of the spike (S) glycoprotein. Not being bound by a theory, the modified HRC peptides described herein bind to the HRN trimer and prevent this six-helix bundle assembly, thereby blocking membrane fusion and viral genome delivery into the host cytoplasm. In example embodiments, the lipoproteins are used to treat SARS-CoV-2, MERS-CoV, SARS-CoV, or other coronaviruses with similar HRN HRC structures. In example embodiments, the invention provides methods of treating, preventing, or reducing the severity of infection in a subject in need thereof. In example embodiments, prophylactic administration may be initiated prior to high-risk exposure (e.g., healthcare workers, contacts of confirmed cases) and continued during the exposure period. Therapeutic administration may begin as soon as possible following symptom onset or positive diagnostic test.

[0077] In example embodiments, the lipoprotein can be administered by any pharmaceutically acceptable route. In example embodiments, the lipoprotein is administered by inhalation, parenterally, intravenously, intramuscularly, or subcutaneously. In example embodiments, effective doses range from 0.5 mg / kg to 5 mg / kg per day.

[0078] In example embodiments, the lipoproteins are administered in a pharmaceutical composition. A “pharmaceutical composition” refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells or to a subject. The term “pharmaceutically acceptable” as used throughout this specification is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.Attorney Docket 44010.227WO-PCT / / CU25029

[0079] Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically- acceptable carrier. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms. As used herein, “carrier” or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, aromatizers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilizers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active components is well known in the art. Such materials should be non-toxic and should not interfere with the activity of the cells or active components. The precise nature of the carrier or excipient or other material will depend on the route of administration.

[0080] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm.203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-Attorney Docket 44010.227WO-PCT / / CU25029 some emerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol.52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0081] The medicaments of the invention are prepared in a manner known to those skilled in the art, for example, by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, 20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.

[0082] Administration can be systemic or local. In addition, it may be advantageous to administer the composition into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may also be desirable to administer the agent locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant. Combination therapy

[0083] In example embodiments, the lipoproteins of the present invention are used in combination with antivirals, monoclonal antibodies, or anti-inflammatory agents (e.g., corticosteroids). Non-limiting antivirals include paxlovid (nirmatrelvir / ritonavir), remdesivir, and molnupiravir.

[0084] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.Attorney Docket 44010.227WO-PCT / / CU25029 EXAMPLES Example 1 – Results Peptide Design

[0085] Applicants aimed to improve upon previous SARS-CoV-2 peptide design3, 4by identifying analogues of the 36-residue SARS-CoV-2 C-terminal heptad repeat region (Fig. 1A-B) that retained the ability to form a six-helix bundle assembly with the SARS-CoV-2 HRN, but displayed higher aqueous solubility relative to native HRC peptide, which is designated HRC below. The design strategy led to two 36-mer sequences, one designated QE and the other SK. The ability of an HRC peptide to form a stable six-helix bundle (Fig. 1D) often correlates with the ability of a lipopeptide derivative to inhibit viral infection8, 10. Applicants have previously shown that cholesterol attachment enhances the antiviral efficacy of peptide-derived fusion inhibitors relative to peptides lacking the lipid appendage7, 8, 18, 20, 23. Other peptide-based approaches have been described.27

[0086] Applicants conjugated with a cholesterol moiety by extending the C-termini with a Gly-Ser-Gly- Ser-Gly-Cys linker segment10, and the cysteine side chain was used to attach a tetra-ethylene glycol– cholesterol unit (PEG4–Chol; Fig. 1C). The design strategy was based on combining features of the native SARS-CoV-2 HRC, which is very potent but has limited solubility, and a peptide derived from the human coronavirus OC43 HRC domain designated EK1 (Fig.1B), which was reported by Xia et al28-30. EK1 has lower antiviral potency toward SARS-CoV-2 relative to the native SARS-CoV-2 HRC,3but EK1 displays high solubility28-30. Multiple changes to the OC43 HRC sequence were made to generate EK1 with the goal of improving solubility while maintaining side chains thought to be critical for stabilizing a six- helix bundle assembly with a viral HRN trimer.

[0087] Xia et al. used the structure of the MERS HRC-HRN six-helix bundle in the EK1 design process,30since the available evidence suggests that coronavirus HRC-HRN assemblies are generally similar to one another,31-34and the OC43 six-helix bundle structure has not been determined. Residue changes that generated EK1 from the OC43 HRC sequence were located at sites expected to project side chains from the surface of the six-helix bundle into the surrounding solvent; native side chains expected to form close contacts with the HRN trimer were preserved. Most of these changes were located in the central portion of the OC43 HRC, which is expected to adopt an α-helical conformation. For all coronaviruses, the N- and C- terminal portions of each HRC component adopt extended conformations in the six-helixAttorney Docket 44010.227WO-PCT / / CU25029 bundle assembly.29, 31-34The EK1 design featured non-native lysine and glutamic acid residues in positions that would support formation of solvent-facing salt bridges in the helical conformation. These charged side chains were expected to enhance peptide solubility relative to the native OC43 HRC sequence.30

[0088] EK1 was reported to inhibit SARS-CoV-2 infection in cell-based assays with a potency comparable to that of the native SARS-CoV-2 HRC peptide28. Subsequently, it was reported that a cholesterol-bearing derivative of EK1, designated EK1C4, was substantially more potent against SARS-CoV-2 infection than EK1 itself29, which is consistent with prior findings that lipopeptides display enhanced antiviral potency relative to unmodified HRC peptides3, 10. However, Applicants found that an EK1-derived lipopeptide was 10- to 100-fold less potent than the native HRC-derived lipopeptide for inhibition of SARS-CoV-2 infection.3

[0089] Applicants reasoned that the poor performance of the EK1-derived lipopeptide as an inhibitor of SARS-CoV-2 infection results from the multiple differences between EK1 and the native SARS-CoV-2 HRC at sites that form intimate contacts with the HRN trimer, which lies at the core of the six-helix bundle assembly. Crystallographic data indicate that fourteen of the SARS-CoV-2 HRC residues contribute side chains to the HRC-HRN interface in the native six-helix bundle;29seven of these interfacial residues differ between the SARS-CoV-2 HRC domain and EK1. Applicants therefore hypothesized that the antiviral potency of the native SARS-CoV-2 HRC peptide could be maintained while solubility was improved by combining the solubility-promoting residues of EK1 with the native interfacial residues.

[0090] Applicants tested their design hypothesis with two peptides, one designated QE and the other SK. Relative to the native SARS-CoV-2 peptide, HRC, QE contains eight residue substitutions derived from EK1 (Fig. 1B, F). SK contains an additional eight residue substitutions derived from EK1 (Fig. 1B, F). In both cases, all substitutions are expected to occur at solvent-facing positions of the HRC-derived peptide in a six-helix bundle assembly with the SARS-CoV-2 HRN. In both QE and SK, all of the native SARS-CoV-2 residues that contact the HRN in the six-helix bundle are preserved. Aqueous solubility of modified peptides

[0091] The published SARS-CoV-2 HRC peptide has proved exceptionally difficult to produce and reliably work with due to technical difficulties ranging from precipitation to gel formation (Fig.7). Applicants used the grand average of hydropathy (GRAVY) score to predict the relative aqueous solubilities of the peptides QE, SK and HRC. GRAVY scores areAttorney Docket 44010.227WO-PCT / / CU25029 calculated by summing the individual hydropathy scores for each residue, then dividing by the total number of residues in the peptide or protein sequence.35The GRAVY scores, -0.197 for QE, -0.386 for SK and -0.178 for HRC, suggested that the designs would display the desired improvement in solubility.

[0092] For experimental assessment of aqueous solubility, Applicants used the peptides HRC-L, QE-L, and SK-L. Each of these peptides contains a C-terminal Gly-Ser-Gly-Ser-Gly- Cys extension, relative to HRC, QE or SK (Fig. 1C). This “linker” extension provides the attachment site for the cholesterol unit, as described above. UV absorbance was used to determine peptide concentration (Fig.2). QE-L contains a single residue with an aromatic side chain (Tyr), and SKL contains two such residues (both Tyr). For these peptides, concentration was calculated based on absorbance at 280 nm and a well-established extinction coefficient.36HRC-L does not contain any aromatic side chain, and in this case concentration was calculated based on absorbance at 205 nm following reported protocols.37QE-L and SK-L were ~2-fold more soluble than HRC-L; this difference was maintained over seven days. When solubility was tested for analogous peptides lacking the GSGSGC linker, i.e., for QE, SK and HRC (Fig. 1C), solubility was substantially reduced in each case (Fig.8), indicating the importance of the linker segment for peptide solubility. In addition, QE, SK, and their derivatives were substantially less prone to gelation relative to HRC and its derivatives (Fig.7). QE and SK form stable assemblies with the native SARS-CoV-2 HRN

[0093] Applicants used circular dichroism (CD) data to ask whether the SARS-CoV-2 HRC-derived peptides co-assemble with SARS-CoV-2 HRN-derived peptides in solution to form six-helix bundles similar to those found in the post-fusion state of SARS-CoV-2 S. Applicants have shown that the stability of the HRN-HRC assembly can correlate with antiviral efficacy of HRC-derived peptides from other viruses that employ a Class I fusion mechanism to enter target cells8, 14, 23, 38. The CD studies involved 1:1 pairings of HRC, QE or SK with a 55-residue peptide derived from the native SARS-CoV-2 HRN. Although Applicants use the term “six-helix bundle” to describe the resulting assemblies, as is standard in this field, Applicants acknowledge that for coronaviruses the HRC components are only partially helical in the assembled state.29, 31-34

[0094] CD data in Figure 3 are presented in terms of mean residue ellipticity, which means that the data are normalized for the number of residues. This approach enables comparisons among peptides with different numbers of residues (e.g., HRC vs. HRN) (Fig.3A and Fig.17)Attorney Docket 44010.227WO-PCT / / CU25029 shows data for individual peptides. The HRN peptide displays a strong α-helix signature, with prominent minima at 208 and 222 nm. These data suggest that the HRN peptide spontaneously forms a helical assembly, which is presumed to be the three-helix assembly that lies at the core of the HRC+HRN six-helix bundle. In contrast, the CD data for HRC, QE and SK indicate that each of these peptides is largely unfolded in the absence of the HRN peptide.

[0095] CD data for a 1:1 mixture of HRC, QE or SK with the HRN peptide (Fig. 3B; 25 μM each peptide) display an α-helix signature in each case, which Applicants interpret to indicate that a six-helix bundle assembly forms in each case. This interpretation is consistent with the fact that HRC cocrystallizes with the HRN peptide to form a six-helix bundle;29Applicants report below that a very similar six-helix bundle is observed in the co-crystal structure involving QE and the native HRN peptide. For each of the 1:1 mixtures, the minima at 208 and 222 nm are less intense than for the HRN peptide alone. This observation presumably reflects that fact that many of the HRC residues occur in extended rather than helical conformations in the six-helix bundle assembly.29, 30, 32-34

[0096] Variable-temperature CD (VT-CD) measurements were used to assess the stability of the helix-bundle assemblies; in these measurements, the CD signal at 220 nm was monitored (Fig.3C-E). VT-CD data for the HRN peptide alone (Fig.3C) display a sigmoidal transition, which indicates that the helical state present at lower temperatures is cooperatively disrupted upon heating. The midpoint of such transitions is commonly designated the “melting temperature” (Tm). In this case, Tm= 50oC.

[0097] For each 1:1 combination, involving the HRN peptide and HRC, QE or SK, the helical assembly appears to be stable over much of the accessible temperature range, with disruption detectable only above ~70oC (Fig.3D). The fully disrupted state does not appear to be reached for any of these pairs at the highest temperature (95oC), because there is no plateau in the signal. These data suggest that all three forms of the six-helix bundle are quite stable. Fully disrupted states have been achieved in related VT-CD studies reported by others, but in these cases the HRN and HRC sequences were truncated or otherwise modified relative to the peptides Applicants used.29, 39-41Applicants tested their interpretation of the data in Fig.3D by examining a second set of 1:1 mixtures containing HRC, QE or SK (Fig.3E). In these cases, the 55-residue native HRN peptide was replaced with a truncated variant (44 residues) from which the 1 N-terminal residue and 10 C-terminal residues had been removed (designated tHRN).29Based on analysis of the HRC+HRN crystal structure, Applicants predicted that thisAttorney Docket 44010.227WO-PCT / / CU25029 truncation would destabilize the six-helix bundle. Indeed, all three mixtures with tHRN displayed sigmoidal transitions (Fig. 3E), implying complete disruption of helix-bundle assemblies over the accessible temperature range. These data suggested that the assemblies formed by HRN or QE with tHRN were similar in stability, and that the assembly formed by SK was slightly more stable. This VT-CD comparison was extended to EK1 (Fig.3F, and Fig. 9); this 1:1 mixture with tHRN displayed a thermal transition at a substantially lower temperature range relative to the three shown in Fig.3E, as indicated in Fig.3F and Fig.9. Structural mimicry of the native SARS-CoV-2 HRC

[0098] QE and the native HRN peptide were co-crystallized. The resulting structure (PDB 6X45; 2.20 Å resolution; Fig. 4A-D) revealed a six-helix bundle nearly identical to the assembly formed by HRC and the native HRN peptide (PDB 6LXT Fig. 4A)29, with a root mean square deviation (RMSD) of 0.68 Å for the Cαatoms. Three copies of the HRN form a parallel trimer around which three copies of QE are arrayed (Fig. 4B). The central α-helical portion of each QE peptide, spanning residues I1181 to L1199 (S protein numbering), displays a bend similar to that in the native assembly. The residues altered in QE relative to HRC (highlighted in red in Fig.4D) are exposed to the solvent, as expected. Non-helical segments are observed at both ends of QE, spanning residues N1159-N1180 and I1200-1205L. An overlay of HRC and QE (Fig. 4E) highlights the conformational similarity of these two peptides. In the new crystal structure, all three copies of QE in the helix-bundle assembly are crystallographically non-equivalent, which matches the non-equivalence of the three HRC molecules in the previously reported structure. QE vs. HRC comparisons involving different versions of these molecules gave RMSD values of 0.5 to 1.0 Å for the Cαatoms. The relatively high R-free value of 0.278 is consistent with other coil–coil crystal structures14, 23, 29.

[0099] Extensive efforts to co-crystallize SK and the native HRN peptide were unsuccessful. Applicants therefore turned to AlphaFold 2 (ColabFOLD V1.5.5)42to predict the structure of this assembly (Fig. 4F-H). The predicted structure was nearly indistinguishable from the six-helix bundle formed by QE and the HRN peptide (Fig.4D) with an RMSD of 0.36 Å for the Cαatoms. Inhibition of fusion mediated by SARS-CoV- 2 S

[0100] Applicants compared the efficacy of the lipopeptide derivatives of HRC, QE and SK for inhibiting fusion mediated by SARS-CoV-2 S using a cell-cell fusion assay. The lipopeptides were generated from derivatives of HRC, QE and SK bearing a Gly-Ser- Gly-Ser-Attorney Docket 44010.227WO-PCT / / CU25029 Gly-Cys “linker” segment at the C-terminus, and the cysteine side chain was used as an attachment point for the tetra-ethylene glycol–cholesterol (PEG4–Chol) appendage (Fig.1C). The resulting lipopeptides are designated HRC-L-PEG4-Chol, QE-L-PEG4-Chol, and SK-L- PEG4- Chol. In the assay, cells expressing human angiotensin-converting enzyme 2 (hACE2), the receptor for SARS-CoV-2 S, and the N-terminal portion of β-galactosidase (β-gal) were mixed with cells expressing either the D614G or BA.5 SARS-CoV-2 S protein and the C- terminal portion of β-gal. When fusion mediated by S occurs, the two portions of β-gal combine to generate a catalytically active species, and fusion is detected via the luminescence that results from substrate processing by the reconstituted β-gal.

[0101] HRC-L-PEG4-Chol, QE-L-PEG4-Chol and SK-LPEG4-Chol inhibited D614G S- mediated fusion comparably, with 50% inhibitory concentrations (IC50) of 33 nM, 25 nM, and 48 nM respectively (Table 1). All three peptides were significantly more effective toward SARS-CoV- 2 BA.5 relative to SARS-CoV-2 D614G (Table 1). For the BA.5 variant, HRC- L-PEG4-Chol was moderately but significantly more potent than either QE-L-PEG4-Chol or SK-L-PEG4-Chol. Each of the lipopeptides displayed little or no toxicity in a cell-based assay (Fig.10).

[0102] The final step in the synthesis of the lipopeptide derivatives involves attachment of the PEG4–Chol unit to the Cys side chain via nucleophilic displacement. The desired product contains a thioether at the attachment site (Fig.1C); however, the reaction process inevitably generates a significant amount of the sulfoxide derivative, which apparently results from spontaneous oxidation of the thioether in air. Each sulfoxide lipopeptide by-product could be readily separated from the corresponding thioether lipopeptide via HPLC. It seems possible that the sulfoxide form would be generated in the lung if a lipopeptide were administered via inhalation. Applicants therefore considered whether the sulfoxide form of a lipopeptide (presumably a pair of diastereomers at sulfur) differs in antiviral activity relative to the thioether form. Thioether vs. sulfoxide comparisons revealed no impact of this oxidation on fusion-inhibitory activity in any of the three cases (Fig.11). This result is promising from the perspective of in vivo applications.

[0103] It was recently reported that an N-terminally extended SARS-CoV-2 HRC peptide, corresponding to S residues 1162-1203, was significantly more potent at inhibiting SARS- CoV-2 fusion in a cell-cell fusion assay and a VSV-SARS2-CoV chimera infection assay relative to the HRC peptide Applicants previously employed (S residues 1168-1203)43. ThisAttorney Docket 44010.227WO-PCT / / CU25029 report motivated Applicants to assess the fusion inhibitory activity of the lipopeptide derived from the N-terminally extended version of QE, i.e., QE-EXT-L-PEG4-Chol. The fusion inhibitory efficacies of QE-EXT-L-PEG4-Chol and QE-L-PEG4-Chol were statistically indistinguishable vs. D614G (Fig. 5A) or BA.5 (Fig. 5B) (Table 1). The control lipopeptide corresponding to the parainfluenza 3(HPIV3) HRC was shown in Reference (4) and (1) to be ineffective vs SARS-Cov-2. In contrast to the observation that extension of the native SARS- CoV-2 HRC peptide increased fusion inhibitor activity43, Applicants did not observe an analogous effect for the QE HRC peptide. (Fig.16). Table 1. IC50and IC90from inhibition of SARS-CoV-2 spike (S)-mediated cell–cell fusion.Inhibition of SARS-CoV-2 and MERS infection by the designed peptides

[0104] Applicants compared the efficacy of the lipopeptides HRC-L-PEG4-Chol, QE-L- PEG4-Chol and SK-L-PEG4-Chol at inhibiting entry of SARS-CoV-2 into Vero E6 cells using a plaque reduction neutralization test (PRNT). HRC-L-PEG4-Chol (IC506.3 nM; IC9015 nM; Table 2), QE-L-PEG4-Chol (IC505.9 nM; IC9011 nM), and SK-L-PEG4-Chol (IC507.7 nM; IC9014 nM) were similar in potency at inhibiting entry (Fig.6A). Applicants tested these three lipopeptide also for inhibition of MERS entry. Applicants have previously shown that a lipopeptide derived from the SARS-CoV-2 HRC is effective at inhibiting MERS3. Fig. 6B shows that HRC-L-PEG4-Chol and QE-L-PEG4-Chol had similar efficacy against MERS, while SK-L-PEG4-Chol was modestly less potent. The control lipopeptide HPIV3 HRC published in Reference (4) was shown here in parallel to be relatively ineffective vs SARS- CoV-2 (50% inhibition not obtained) and MERS (IC50~70nM). Table 2. IC50 and IC90 from live virus plaque reduction neutralization test.Attorney Docket 44010.227WO-PCT / / CU25029 Discussion

[0105] This work focused on improving the solubility of peptides used to generate inhibitors of SARS-CoV-2 infection. As discussed above, lipopeptide inhibitors of viral entry could represent a useful contribution in the struggle to protect vulnerable populations from COVID-19. The effort was motivated by the importance of peptide solubility in manufacturing and delivery44. This strategy has been successful with HPIV3 fusion inhibitors23, and the first step toward this goal was to identify variants of the native HRC sequence with improved solubility.

[0106] Applicants employed a design strategy inspired by the antiviral peptide EK1, which was derived from the HRC domain of the human coronavirus OC43.30Xia et al. generated EK1 by modifying the native OC43 sequence at positions that are not expected to engage the HRN domain in the six-helix bundle assemble of the post-fusion S state. These modifications enhanced the complement of polar / charged side chains in EK1 relative to the native OC43 HRC, which was expected to increase solubility. However, the native OC43 HRC residues that are predicted to contact the HRN core in the six-helix bundle assembly differ at many positions from the corresponding residues in the SARS-CoV-2 HRC domain. These differences presumably explain why Applicants found a lipopeptide derived from EK1 to be substantially less potent at inhibiting fusion mediated by SARS-CoV-2 S relative to the analogous lipopeptide derived from the native SARS-CoV-2 HRC (i.e., HRC-L-PEG4-Chol).3

[0107] The design strategy generated two novel analogues of the native SARS-CoV-2 HRC domain, QE and SK. Both contain the native SARS-CoV-2 residues at all positions that contact the trimeric HRN core in the six-helix bundle assembly (PDB 6LXT).29However, QE contains 8 substitutions at solvent-exposed positions relative to HRC, and SK contains 16 substitutions. When evaluated with derivatives containing a short C-terminal “linker” segment, both of the designed sequences display significantly enhanced solubility relative to the native HRC. Despite the considerable number of sequence changes in QE and SK relative to HRC, these three peptides form assemblies of comparable stability with the native HRN, presumably six-helix bundles, according to VT-CD data. Applicants solved a QE+HRN co-crystal structure that reveals an assembly very similar to that reported for the native HRC+HRN pair.29

[0108] Two assay formats were employed to evaluate the functional consequences of the new designs, one involving cell-cell fusion mediated by SARS-CoV-2 S, and the other involving viral infection of Vero E6 cells. These studies employed lipopeptides derived fromAttorney Docket 44010.227WO-PCT / / CU25029 QE, SK or HRC. The fusion assays were conducted with either of two S variants; with D614G, the three lipopeptides were indistinguishable, while with BA.5, the HRC lipopeptide displayed a modest advantage relative to the QE or SK lipopeptide. The three lipopeptides were indistinguishable in their ability to inhibit Vero E6 cell infection (Wuhan strain). The HRC and QE lipopeptides were comparably potent inhibitors of MERS infection as well, while the SK lipopeptide was moderately less potent against this virus. Overall, these results show that the native SARS-CoV-2 HRC domain can be substantially modified to enhance solubility while retaining potent antiviral activity.

[0109] The final step in the lipopeptide synthesis method creates a thioether linkage between the peptide and the cholesterol unit, and this process invariably generates the corresponding sulfoxide as a by-product. This unintended oxidation seems likely to occur in vivo as well.45It is therefore significant that sulfoxide forms of all three lipopeptides, derived from HRC, QE or SK, match the fusion-inhibitory potencies of the thioether analogues. Applicants used the QE system to evaluate another type of modification in which the HRC domain is lengthened by six residues at the C-terminus. Such an extension might lead to additional favorable contacts in the six-helix bundle assembly and therefore enhance antiviral potency; however, in distinction to a recent report that extension of the native HRC segment resulted in dramatically improved antiviral potency43Applicants observed no change in fusion- inhibitory activity for the extended QE lipopeptide.

[0110] The QE sequence introduced here appears to represent a favorable candidate for further development intended to deliver a prophylactic agent that could protect vulnerable patient populations from COVID-19. Future studies will assess the efficacy of this candidate in vivo for preventing and treating SARS-CoV-2. The next step will be to incorporate backbone modifications into the QE sequence in an effort to inhibit degradation by proteases in vivo. The combination of enhanced solubility and potent antiviral efficacy that extends beyond SARS- CoV-2 suggests that QE could lead to a significant public health advance. Methods

[0111] Peptide synthesis. All peptides were produced by standard Fmoc-based solid-phase synthetic methods using a Liberty Blue microwave-assisted peptide synthesizer. The cholesterol moiety was attached to peptides as previously described,3, 7, 8via a chemoselective reaction between the thiol group of the C-terminal cysteine residue and a bromoacetyl derivative of cholesterol.Attorney Docket 44010.227WO-PCT / / CU25029

[0112] Instrumentation. Solid-phase peptide synthesis was performed on a CEM Liberty Blue 1.0 microwave-assisted peptide synthesizer. Preparative HPLC was performed on an Agilent 1260 Infinity II Instrument. Peptide purity measurements were performed on a Waters Acquity H-Class UPLC. Mass spectra were obtained on a Bruker microflex LRF MALDI- TOF-MS. MSMS data were collected on a Bruker Impact II. Circular dichroism experiments were performed on a JASCO J-1500 CD spectrometer.

[0113] General Procedures for Synthesis and Purification. Peptides were prepared on Rink amide resin using microwave-assisted solid-phase peptide synthesis (MA-SPPS) procedures via a Liberty Blue 1.0. Resin was purchased from Millipore–Sigma. Fmoc-amino acids and coupling reagents were purchased from Chem-Impex International. Protected Fmoc-α-amino acids included: Asp(t-Bu ester), Glu(t-Bu ester), His(trityl), Lys(Boc), Asn(trityl), Gln(trityl), Arg(Pbf), Ser(t-Bu ether), Thr(t-Bu ether), Trp(Boc), and Tyr(t-Bu ether). Rink amide resin was pre-swelled with 1:1 DCM:DMF. Coupling reactions were performed using solutions comprised of 4 equivalents Fmoc-amino acid, 4 equivalents of OxymaPure and 8 equivalents of DIC in biotechnology-grade dimethylformamide (DMF) at a final concentration of 200 mM Fmoc-amino acid. Coupling reactions were carried out by microwave-assisted synthesis using a 2 min hold at 90 ºC. Deprotection was conducted by addition of 20% (v / v) piperidine in biotechnology-grade DMF. The deprotection reactions were carried out by microwave-assisted synthesis using a 2 min ramp to 80 ºC followed by a 2 min hold at 80 ºC. Each peptide was cleaved with 8.5:1:0.25:0.25 TFA / TIPS / ethanedithiol / phenol and precipitated by addition of cold diethyl ether

[0114] Preparative HPLC was performed using an Agilent 1250 Infinity II Prep HPLC equipped with a Waters XSelect CSH Prep C18 column (5 µm OBD, 19x250mm). Peptide purity measurements were performed on a Waters Acquity H-Class UPLC equipped with equipped with an Acquity UPLC CSH C18 (130Å, 1.7 µm, 2.1 x 100 mm) or an Acquity UPLC BEH C4 (300Å, 1.7 µm, 2.1 x 100 mm) column. Mass spectra were obtained on a Bruker microflex LRF-MALDI-TOF-MS. MS-MS data were collected on a Bruker Impact II. MSMS data were analyzed using Bruker Compass DataAnalysis 4.3 and GPMAW 10.

[0115] Synthesis of Lipopeptides. The cholesterol conjugation was achieved using displacement of an α-bromoamide. In a nitrogen-purged vial, BrAcNH-PEG4-Chol reagent (1.5 equiv.) was dissolved in degassed DMSO. In a separate nitrogen-purged vial outfitted with a stir bar, Ac-peptide-GSGSGC-NH2 (1.0 equiv.) was dissolved in degassed DMSO. TheAttorney Docket 44010.227WO-PCT / / CU25029 BrAcNH-PEG4-Chol solution was added to the peptide solution dropwise via syringe, followed by addition of N,N-diisopropylethylamine (DIEA). The reaction mixture was stirred for 90 minutes. Tris(2-carboxyethyl)phosphine (2.0 equiv.) was added, and the mixture was stirred for an additional 15 minutes. The resulting peptide-cholesterol conjugate was purified by reverse-phase HPLC on an Agilent Prep HPLC using a gradient of 55-75% acetonitrile+0.1% trifluoroacetic acid in water+0.1% trifluoroacetic acid over 30 minutes at a flow rate of 18 mL / min.

[0116] B-Gal complementation cell-cell fusion assay. To evaluate cell-cell fusion, Applicants modified a fusion assay relying on alpha complementation of B-galactosidase (B- gal).8For this assay, cells co-expressing the hACE2 receptor and the omega peptide of B-gal are mixed with cells bearing SARS-Cov-2 glycoprotein S and the alpha peptide of B-gal. The fusion of cells allows for complementation of the alpha and omega peptides of B-gal, and this process continues until fusion is terminated by lysing the cells. The substrate (The Tropix Galacto-Star chemiluminescent reporter assay system, Applied Biosystem) is introduced, and luminescence is quantified on a Cytation 5 (BioTek) or a Tecan M1000. All data was plotted in GraphPad Prism. A nonlinear regression model was applied to the data, yielding a fitted curve.

[0117] In vitro cytotoxicity assessment. An MTT (3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide) assay was used to determine the cytotoxicity of lipopeptides HRC-L-PEG4-Chol, QE-LPEG4- Chol or SK-L-PEG4-Chol toward HEK 293T cells. The toxicity observed for each lipopeptide was <20% at 5 μM.

[0118] Peptide concentration determination. QE-L contains a single residue with an aromatic side chain (Tyr), and SK-L contains two Tyr residues. For these peptides, concentration was calculated based on absorbance at 280 nm and a well-established extinction coefficient (ε280= 1,490 M-1cm-1for QE; 2,980 M-1cm-1for SK).36The HRC-L does not contain an aromatic side chain, and concentration was calculated based on absorbance at 205 nm from the peptide backbone (ε205= 118,820 M-1cm-1).37It should be noted that absorbance at 205 nm could not be used for measurements with QE-L or SK-L because there is signal contributionAttorney Docket 44010.227WO-PCT / / CU25029 from the UV absorbance of the Tyr side chain.37Solubility over time was measured in phosphate-buffered saline (PBS) at room temperature by adapting the saturation shake-flask method46. A 12.5 mg / mL mixture of HRC-L, QE-L or SK-L in PBS was placed in an Eppendorf tube, vortexed, sonicated, and centrifuged. The tube was allowed to stand at room temperature for 1 hr, and then a large portion of the supernatant was transferred to another tube. The absorbance at the appropriate wavelength (280 nm for QE-L or SK-L, or 205 nm for HRC-L) was measured at this point, and again at 18 hr, 72 hr 144, and 168 hr after sample preparation. At each time point, the sample was centrifuged before the absorbance measurement. UPLC analysis demonstrated that all three peptides remained intact during the solubility study.

[0119] GRAVY. GRAVY scores are calculated by summing the individual hydropathy scores for each residue, then dividing by the total number of residues in the peptide or protein sequence.35

[0120] Structure determination.

[0121] X-ray Crystallography

[0122] Crystallization conditions. Lyophilized powders of HRN and QE peptides were dissolved in an aqueous solution of 1% w / v b-D-octylglucoside to final respective concentrations of 4 mg / mL and 3 mg / mL. Peptide crystals were grown with hanging drop vapor diffusion, in which drops comprised of 1 uL peptide solution combined with 1 uL of well solution (0.1 M Tris pH 7.8, 26% w / v PEG3350, 0.3 M MgCl2) were equilibrated against 150 uL of well solution in a VDXm plate at 20°C. Crystals were cryo-protected by sequential soaking for 2-3 minutes each in cryo-solution A (0.1 M Tris pH 7.8, 37% w / v PEG3350, 0.3 M MgCl2) and cryo-solution B (0.1 M Tris pH 7.8, 48% w / v PEG3350, 0.3 M MgCl2) followed by vitrification in liquid nitrogen.

[0123] X-ray Data Collection. X-ray diffraction data were collected from a single crystal at the AMX beamline (17-ID-1) at NSLS-II at Brookhaven National Laboratory, Upton, NY.

[0124] X-ray Data Processing, Structure Solution, and Refinement. Diffraction data were indexed and integrated with XDS (1), then scaled and merged with XDS / aimless as implemented in the autoPROC software package (2). Initial phases were obtained with molecular replacement via phenix.phaser using the corresponding residues in the HRN / HRC hairpin dimer from 6LXT (3-4). Refinement of XYZ, ADP, and TLS parameters was performed with phenix.refine in space group P21212 combined with iterative real space refinement in Coot (3,5).Attorney Docket 44010.227WO-PCT / / CU25029 Table 3. SARS-CoV-2 HRN + SARS2-QE

[0125] Circular dichroism. All circular dichroism (CD) experiments were performed on a JASCO J-1500 CD spectrometer. Samples were prepared in 1 mm quartz cuvettes with 10 mM phosphate buffer, pH 7.4. Wavelength scans were collected from 190 to 260 nm with a 1 nm bandwidth, 4 second averaging time, and scanning speed of 50 nm / min. Individual peptides were prepared at 25 μM, and peptide combinations were prepared at 50 μM (25 μM each component). For variable temperature experiments, ellipticity was measured at 222 nm asAttorney Docket 44010.227WO-PCT / / CU25029 temperature was raised from 5oC to 95oC in 5oC increments with a 5-minute equilibration time at each new temperature and a 5-second averaging time for each measurement. All experiments were repeated in triplicate. Data are presented as mean + / - standard error.

[0126] Cells. Human embryonic kidney (HEK) 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and antibiotics in 5% CO2. Vero E6 cells (ATCC CRL-1586) were grown in minimum essential medium with Earle’s salts (EMEM; Gibco) supplemented with 6% FBS and antibiotics in 5% CO2.

[0127] Plasmids. The cDNAs coding for hACE2 fused to the fluorescent protein Venus, SARS-CoV-2 S D614G and SARS-CoV-2 S BA.5, (codon optimized for mammalian expression) were cloned in a modified version of the pCAGGS (with puromycin resistance for selection).

[0128] Viral titration and plaque reduction neutralization assay. As performed previously,3titers of virus stocks were determined by plaque assay in Vero E6 cells grown in six-well tissue culture plates. Virus stocks were serially diluted 10-fold in PBS, and 0.2 ml of each dilution was inoculated into quadruplicate wells and allowed to adsorb at 37oC for 1 h with rocking every 15 min. Monolayers were rinsed with Dulbecco’s phosphate-buffered saline (DPBS; Corning) and then overlaid with a semisolid medium containing MEM, 5% FBS, antibiotics, and ME agarose (0.6%). Cultures were incubated at 37oC for 3 days and overlaid with DPBS containing neutral red (3.33 g / liter; Thermo Fisher Scientific) as a stain (10%), and plaques were counted after 4 to 5 h.

[0129] Peptides were tested for inhibitory activity against SARS-CoV-2 and MERS-CoV by plaque reduction neutralization assay. Peptides were serially diluted in molecular biology grade water (1,000 nM or 500 nM through 0.5 nM), each peptide dose was mixed with an equal volume of virus containing 500 particle-forming units (PFU) / ml in MEM, and the peptide / virus mixtures were incubated at 37oC for 1 h. Each peptide dose / virus mixture was inoculated into triplicate wells of Vero E6 cells in six-well plates (0.2 ml per well) and allowed to adsorb at 37oC for 1 h with rocking every 15 min. Monolayers were rinsed with DPBS prior to the addition of medium overlay containing MEM, 5% FBS, antibiotics, and ME agarose (0.6%). Cultures were incubated at 37oC for 2 (SARS-CoV-2) to 3 (MERS CoV) days and overlaid with medium containing neutral red as a stain, and plaques were counted after 4 to 5 h. Virus controls were mixed with sterile water instead of peptide.Attorney Docket 44010.227WO-PCT / / CU25029

[0130] Statistical Analysis. Inhibitory concentrations 50% and 90% (IC50and IC90respectively) in fusion assays were calculated with a log variable slope dose verses response curve. Data are mean ± standard error of the mean (SEM) from three distinct experiments with a four-parameter variable curve. All line graphs were compared by 2-way ANOVA repeated measures. All statistics were performed with GraphPad Prism V9. References 1. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-3. Epub 2020 / 02 / 23. doi: 10.1126 / science.abb2507. PubMed PMID: 32075877; PMCID: PMC7164637. 2. Bosch BJ, van der Zee R, de Haan CA, Rottier PJ. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol. 2003;77(16):8801-11. Epub 2003 / 07 / 30. doi: 10.1128 / jvi.77.16.8801- 8811.2003. PubMed PMID: 12885899; PMCID: PMC167208. 3. Outlaw VK, Bovier FT, Mears MC, Cajimat MN, Zhu Y, Lin MJ, Addetia A, Lieberman NAP, Peddu V, Xie X, Shi PY, Greninger AL, Gellman SH, Bente DA, Moscona A, Porotto M. Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain. mBio. 2020;11(5). Epub 2020 / 10 / 22. doi: 10.1128 / mBio.01935-20. PubMed PMID: 33082259; PMCID: PMC7587434. 4. de Vries RD, Schmitz KS, Bovier FT, Predella C, Khao J, Noack D, Haagmans BL, Herfst S, Stearns KN, Drew-Bear J, Biswas S, Rockx B, McGill G, Dorrello NV, Gellman SH, Alabi CA, de Swart RL, Moscona A, Porotto M. Intranasal fusion inhibitory lipopeptide prevents directcontact SARS-CoV-2 transmission in ferrets. Science. 2021;371(6536):1379- 82. Epub 2021 / 02 / 19. doi: 10.1126 / science.abf4896. PubMed PMID: 33597220; PMCID: PMC8011693. 5. Porotto M, Doctor L, Carta P, Fornabaio M, Greengard O, Kellogg GE, Moscona A. Inhibition of hendra virus fusion. J Virol. 2006;80(19):9837-49. doi: 10.1128 / JVI.00736-06. PubMed PMID: 16973588; PMCID: PMC1617219. 6. Lee KK, Pessi A, Gui L, Santoprete A, Talekar A, Moscona A, Porotto M. Capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide: a newAttorney Docket 44010.227WO-PCT / / CU25029 antiviral strategy for influenza virus. J Biol Chem. 2011;286(49):42141-9. Epub 2011 / 10 / 14. doi: 10.1074 / jbc.M111.254243. PubMed PMID: 21994935; PMCID: PMC3234914. 7. Porotto M, Yokoyama C, Palermo LM, Mungall B, Aljofan M, Cortese R, Pessi A, Moscona A. Viral entry inhibitors targeted to the membrane site of action. J Virol. 2010. PubMed PMID: 20357085. 8. Porotto M, Rockx B, Yokoyama CC, Talekar A, Devito I, Palermo LM, Liu J, Cortese R, Lu M, Feldmann H, Pessi A, Moscona A. Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry. 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[0131] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

Attorney Docket 44010.227WO-PCT / / CU25029 CLAIMS What is claimed is:

1. A synthetic peptide comprising a C-terminal heptad repeat region (HRC) of SARS- CoV-2 S protein, wherein one or more residues of the HRC of SARS-CoV-2 S protein (SEQ ID NO: 1) are substituted with corresponding residues having charged or polar side chains at solvent-facing positions of the synthetic peptide when in a six-helix bundle assembly with the SARS-CoV-2 N-terminal heptad repeat region (HRN), thereby improving solubility of the synthetic peptide while maintaining side chains for stabilizing the six-helix bundle assembly of a viral HRN trimer.

2. The synthetic peptide of claim 1, wherein the synthetic peptide comprises a substitution with at least one negatively charged amino acid and at least one positively charged amino acid.

3. The synthetic peptide of claim 1 or 2, wherein the C-terminal HRC comprises a sequence of: DISQINASVVNIEYEIKKLEEVAKKLEESLIDLQEL (SEQ ID NO: 2); or SIDQINATFVDIEYEIKKLEEVAKKLEESYIDLKEL (SEQ ID NO: 3).

4. The synthetic peptide of any of claims 1 to 3, further comprising a linker segment at the C-terminus of the peptide.

5. The synthetic peptide of claim 4, wherein the linker segment comprises a Gly-Ser-Gly- Cys linker segment.

6. The synthetic peptide of claim 5, further comprising a cholesterol moiety conjugated to the linker segment at the Cys residue.

7. The synthetic peptide of claim 6, wherein the cholesterol moiety is conjugated to the linker segment by a polyethylene glycol linker.

8. The synthetic peptide of claim 7, wherein the polyethylene glycol linker is PEG4, PEG8, PEG11, PEG24, or PEG28.Attorney Docket 44010.227WO-PCT / / CU25029 9. The synthetic peptide of any of claims 1 to 8, further comprising an N-terminally extended SARS-CoV-2 HRC peptide corresponding to S residues 1162-1203, wherein the peptide includes PDVDLG (SEQ ID NO: 6) at the N-terminus.

10. The synthetic peptide of claim 9, wherein the C-terminal HRC comprises a sequence of PDVDLGDISQINASVVNIEYEIKKLEEVAKKLEESLIDLQEL (SEQ ID NO: 4).

11. A lipoprotein comprising: the HRC of any of claims 1-10; a cholesterol unit; and at least one polyethylene glycol linker, wherein: the cholesterol unit and the at least one polyethylene glycol linker are attached to form a polyethylene glycol-cholesterol unit, and the polyethylene glycol-cholesterol unit is linked to the C-terminus of the synthetic peptide.

12. The lipoprotein of claim 11, wherein the at least one polyethylene glycol linker is PEG4, PEG8, PEG11, PEG24, or PEG28.

13. The lipoprotein of claim 11 or 12, further comprising a linker segment at the C-terminus of the lipoprotein.

14. The lipoprotein of claim 13, wherein the linker segment comprises a Gly-Ser-Gly-Cys linker segment.

15. The lipoprotein of claim 14, wherein the lipoprotein structure comprises HRC-linker segment-PEG-cholesterol.

16. A method of inhibiting SARS-CoV-2 or MERS infection comprising administering to a subject in need thereof the synthetic peptide or lipoprotein according to any of claims 1-15.

17. The method of claim 16, wherein the synthetic peptide or lipoprotein are administered by inhalation, parenterally, intravenously, intramuscularly, or subcutaneously.Attorney Docket 44010.227WO-PCT / / CU25029 18. A structure-guided method of improving solubility of a peptide capable of disrupting a six-helix bundle assembly of a viral N-terminal heptad repeat region (HRN) and C-terminal heptad repeat region (HRC) of SARS-CoV-2 S protein, comprising: selecting at least one amino acid within a 3-D structure of the peptide in complex with a six-helix bundle assembly, wherein the amino acid is not critical for stabilizing the six-helix bundle assembly with a viral HRN trimer, wherein the amino acid has at least one side chain facing a solvent in the 3-D structure; engineering the peptide to substitute the at least one amino acid with a negatively charged amino acid or a positively charged amino acid to improve solubility of the peptide; testing the engineered peptide with the at least one substituted amino acid for stabilizing the six-helix bundle assembly with a viral HRN trimer; and selecting an optimal combination of amino acid substitutions which preserves the stabilization while enhancing the aqueous solubility of the peptide.

19. The structure-guided method of claim 18, wherein the substitutions include one or more substitutions made to the HRC of SARS-CoV-2 S protein selected from the substitutions in SEQ ID NO:2 or SEQ ID NO:

3.

20. The structure-guided method of claim 18 or 19, wherein the negatively charged amino acid comprises aspartate (Asp) or glutamate (Glu) and the positively charged amino acid comprises arginine (Arg) or lysine (Lys).

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