Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the disclosure relates to a chimera comprising a molecule having the structure of any one of the molecules depicted in Fig. 7 or Fig. 14A.
• this disclosure features a pharmaceutical composition
• a pharmaceutical composition comprising a peptide or a chimera disclosed herein and a pharmaceutically acceptable carrier.
• the pharmaceutical composition is formulated for oral, intravenous, topical, buccal, rectal, parenteral, intraperitoneal, intradermal, subcutaneous, intramuscular, transdermal, intranasal, pulmonary, or intratracheal administration.
• this disclosure provides a method of treating or preventing a viral infection caused by a coronavirus in a subject in need thereof.
• the method comprises administering to the subject a therapeutically effective amount of a peptide, a chimera, or a pharmaceutical composition disclosed herein.
• the disclosure provides a method for blocking the replication of SARS-CoV or SARS-CoV-2 in a subject in need thereof.
• the method comprises administering to the subject a therapeutically effective amount of a peptide, a chimera, or a pharmaceutical composition disclosed herein.
• the subject is selected from a group consisting of a human, a primate, a bat, a bird, a mouse, a turkey, a cow, a pig, a cat and a dog. In one instance, the subject is a human.
• the disclosure features a stabilized (e.g., stapled, stitched) peptide comprising a peptide comprising the amino acid sequence set forth in SEQ ID NOs.: 2 or 3 with 1, 2, 3, 4, 5, or 6 amino acid substitutions, wherein at least two amino acid substitutions replace amino acids separated by three or six amino acids with non-natural amino acids, and wherein the peptide binds and inhibits Mpro.
• the amino acid substitutions replace amino acids separated by three amino acids with non-natural amino acids, the non-natural amino acids are both S5.
• the disclosure features a peptide comprising the amino acid sequence set forth in SEQ ID NOs.: 4 or 5, or a variant thereof, wherein the peptide binds NSP9. In some instances, the peptide inhibits dimerization of NSP9.
• this disclosure provides a method of treating or preventing a coronaviral infection in a subject in need thereof.
• the method involves administering to the subject a therapeutically effective amount of a peptide, a stabilized peptide, or a pharmaceutical composition described herein.
• the subject is a human subject.
• the subject is a cat, dog, horse, sheep, chicken, or cow.
• the disclosure also features a pharmaceutical composition comprising a means for inhibiting Mpro or NSP9 and a pharmaceutically acceptable carrier.
• Figure 2 middle panel shows the generation of stapled peptides with staples of various lengths spanning discrete distances along the length of a peptide helix (/, i+ 3, i, i+4, and i, i+ 7)
• Figure 3 shows various kinds of double and triple stapling strategies along with exemplary staple walks for generating stapled peptides.
• Figure 4 illustrates exemplary staple walks for generating stitched peptides.
• Figure 5 shows the various ways in which the peptide ligand component(s) of SP-PROTACs can be optimized by mutagenesis, differential staple or stitch insertion(s), and mutations of the peptide sequence by substation, deletion, addition, or derivatization.
• FIG 6 shows an exemplary ring closing metathesis (RCM) stapling reaction that can be employed to generate stapled peptides (in this case an i, i+ 7 stapled peptide).
• Figure 7 depicts the structure of two stapled peptide chimeras designed to target an essential SARS-CoV-2 protein (PLpro) and a host protein (BRD4 and/or BRD2) that binds to a SARS-CoV-2 protein: BRD4 (JQ1/SP645 SP-PROTAC, top) and PLpro (GRL0617/SP645 SP-PROTAC, bottom).
• PLpro essential SARS-CoV-2 protein
• BRD4 and/or BRD2 host protein
• Figure 8A shows the levels of a-helical stabilization of various HDM2- binding stapled p53 peptides (SAH-p53-l to 4; SEQ ID NOs.: 40-43 respectively) compared to wild type p53i4-29 (SEQ ID NO: 11).
• Figure 9B shows traces to determine complex formation as assessed by size exclusion chromatography (SEC) for various molecules and chimeras.
• SEC size exclusion chromatography
• Figure 10A are images that show that unlike HeLa cells treated with vehicle (top row), cells treated with SP-PROTAC-BRD4 exhibit relocalization of HDM2 from the cytosol (diffuse staining) to the nuclear lamina where BRD4 is experimentally anchored (bottom row).
• Figure 10B is an image of a western blot of PLpro protein and ubiquitylated- PLpro protein levels in the presence of vehicle or SP-PROTAC-PLpro as determined by an in vitro ubiquitylation assay.
• Figure 11A is an image of a western blot of BRD4 and p53 levels in SJSA-1 cells exposed to various concentrations of SP-PROTAC-BRD4. Actin antibody is used as the loading control.
• Figure 1 IB is an image of a western blot of BRD4 and p53 levels in SJSA-1 cells exposed to various concentrations of SP-PROTAC-BRD4 in the presence of the selective proteasome inhibitor, carfilzomib. Actin antibody is used as the loading control.
• Figure 12A shows the levels of BRD proteins (BRD2/3/4) and the p53 transcriptional target, HDM2, in cells treated with SP645 alone.
• Figure 12B depicts the levels of BRD proteins (BRD2/3/4) and the p53 transcriptional target, HDM2, in cells treated with JQ1 alone.
• Figure 13A shows the percentage (%) viability of SJSA-1 cells in the presence of SP645, JQ1, or SP-PROTAC-BRD4. Data represents mean ⁇ SEM., *p ⁇ 0.05 pairwise for each group.
• the viral target for degradation may be any viral protein (e.g., an essential viral protein such as a coronaviral protease or other non-structural protein (NSP)), or a host protein that facilitates viral pathogenesis (e.g., a bromodomain and extraterminal domain (BET) protein)).
• the viral protein is coronaviral papain-like protease (PLpro), or main protease (Mpro); the coronaviral NSP is NSP9 or NSP12.
• the BET protein is bromodomain 2 (BRD2), bromodomain 3 (BRD3) or bromodomain 4 (BRD4).
• the host protein is Sec61, an endoplasmic reticulum membrane protein translocon.
• This disclosure provides chimeras that act as protein degradation inducing moieties, by combining a first moiety that targets a viral protein target (e.g., binds to a viral protein (e.g., an essential viral protein) or a host protein that facilitates viral pathogenesis), with a second moiety (e.g., a ligand) that binds to a protein that is, or recruits, “a protein degrader.”
• a protein degrader e.g., recruits an enzyme or a complex that catalyzes the ubiquitination of the target, labeling in this way the target for degradation, which in turn is degraded by the proteasome.
• the “protein degrader” is an E3 ligase that ubiquitylates and thus targets the viral protein for degradation.
• the first moiety is an Mpro binder, such as Lopinavir, Ritonavir, Darunavir, ASC09, GC376, GC813, Ebselen carboxylic acid, or a peptide comprising an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
• Mpro binder such as Lopinavir, Ritonavir, Darunavir, ASC09, GC376, GC813, Ebselen carboxylic acid, or a peptide comprising an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
• the first moiety is an NSP9 binder, such as a peptide comprising an amino acid sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
• the first moiety is an NSP12 binder, such as remdesivir acid, sofosbuvir acid or analogs thereof.
• an essential viral protein that can be targeted with the chimeras of this disclosure includes any viral protein that plays a role in viral replication, and/or pathogenesis, including viral entry into a cell.
• the essential viral protein is a viral protein from an RNA virus, such as Severe acute respiratory syndrome-associated coronavirus-2 SARS-CoV), SARS-CoV2, and Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV).
• the viral protein is a NSP such as a coronaviral protease from S ARS-CoV2.
• the coronaviral protease is a PLpro or an Mpro.
• the NSP is NSP9 or NSP12.
• the essential viral protein is a viral protein from an RNA virus such as Hepatitis C Virus (HCV), Human immunodeficiency virus (HIV), Herpes simplex virus (HSV), Zika virus, and enterovirus.
• Mpro plays a major role in the autoprocessing proteolytic reactions that yield mature proteins (NSP5-16) essential to the viral life cycle (Chang, G.G. et al. Molecular Biology of the SARS-Coronavirus , 115-128 (2009)).
• NSP5-16 mature proteins
• 3CLpro 3C-like protease
• PLpro is a multifunctional cysteine protease that processes the viral polyprotein and host cell proteins required for viral replication (Baez-Santos, Y.M., et al. J Virol 88, 12511-27 (2014)). PLpro is also involved in viral mechanisms for promoting p53 degradation (Ma-Lauer, Y. et al. Proc Natl Acad Sci USA 113, E5192-201 (2016)) and evading the host immune system (deuibiquitylating and delSGylating activity (Ratia, K., et al. PLoS Pathog 10, el004113 (2014)). Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells.
• coronaviral non-structural proteins are essential proteins that can be targeted using the chimeras of this disclosure.
• These non- structural proteins include coronaviral proteases, NSP9 and NSP12.
• Coronaviral replication and transcription are driven by the 15 or 16 viral NSPs encoded in the replicase gene, any of which can be targeted by the chimeras of this disclosure.
• These NSPs are produced during co- and post-translational processing of the PPI a and PPlab replicase polyproteins (te Velthuis, Aartjan JW. et al. Nucleic acids research 38,1 (2010): 203-14).
• the first moiety of a chimera of this disclosure binds to a protein (e.g., a coronaviral protease) that is targeted for degradation.
• the first moiety can include a small molecule, a small molecule derivatized with a warhead, a peptide, a stapled peptide, a peptide or stapled peptide derivatized with a warhead, or a nucleotide analog.
• the first moiety can target a viral protein (e.g., an essential viral protein needed for viral replication or pathogenesis).
• the first moiety can target a host protein that aids in viral pathogenesis.
• the first moiety can fully or partially act as a “molecular glue” which can bind to the protein targeted for degradation but itself does not necessarily have any inhibitory or agonistic effects.
• the Mpro binders encompassed by the present disclosure include agents that directly interact with Mpro, such as Mpro inhibitors.
• the Mpro inhibitors can inhibit Mpro dimerization and/or Mpro enzymatic activity.
• Mpro inhibitors such as those described in Jin Z et al. Nature 2020 582:289-293; Zumla A et al; Nat. Rev. Drug Disc. 2016 (10):327; Li G and Clercq ED. Nature Rev. Drug Disc., Feb 2020; Ghosh A.K. et al., ChemMedChem 2020 15(11): 907-932 can be used in the present disclosure.
• the peptides can have at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 75% identity, at least or about 80%, at least 85%, at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity to those amino acids in SEQ ID NO: 2 or 3, wherein the peptides bind to Mpro.
• the peptides can include amino acid substitutions and/or deletions, whether conservative or not.
• the peptide can include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, less than 10, less than 5, less than 4, less than 3, or less than 2 amino acid substitutions, deletions, and/or additions, whether conservative or not, provided that the peptide can still bind to Mpro.
• the amino acid sequence of any of the Mpro binding peptides disclosed herein can be varied so long as the variant peptide binds to Mpro.
• the peptides differ from the peptides of SEQ ID NO: 2 or 3 in that they vary from SEQ ID NO: 2 or 3 in having 1 to 4 (e.g., 1, 2, 3, 4) amino acid substitutions.
• the positions labeled “X” can be substituted in SEQ ID NO: 2 or 3 as follows: ATXNVLXWLYXAVIXGD (SEQ ID NO: 51).
• X can be a conservative or non-conservative amino acid residue.
• each X is anon-natural amino acid with olefinic side chains (e.g., S5).
• Mpro binders that can be utilized in the chimeras described herein have the structures provided below:
• Mpro binders shown in Table 1 or Mpro-binding analogs thereof can be utilized in the chimeras of this disclosure.
• PLpro binders that can be utilized in the chimeras described herein have the structures provided below:
• NSP9 binders are agents that can directly interact with NSP9.
• An NSP9 binder can be an NSP9 inhibitor which suppresses NSP9 dimerization and/or NSP9 enzymatic activity.
• an NSP9 binder is a peptide (e.g., a recombinant or synthetically produced peptide). Such peptides can be non-cross-linked, stapled, or stitched, so long as the peptides interact with NSP9 as described herein.
• NSP9 The enzymatic activity of NSP9 relies on forming a homodimer mediated by an alpha-helical sequence: NLNRGMVLGSLAATVRLQ (SEQ ID NO: 10).
• SEQ ID NO: 10 can be used to create peptide-based dimerization binders.
• NSP9 binder peptides can include at least six (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous amino acids of SEQ ID NO: 10).
• NSP9 binder peptides can include at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions or deletions in SEQ ID NO: 10 so long as the variant peptide still binds NSP9.
• an NSP9 binder peptide includes the sequence GXXXG (SEQ ID NO: 8), where each X can be any amino acid. In some instances X is any one M, norleucine (B), V, L, A, G, or I.
• an NSP9 binder peptide of SEQ ID NO: 4 or 5 or variants thereof can be shortened by 1, 2, or 3 amino acids at each end of the sequence.
• an NSP9 binder peptide of SEQ ID NO: 4 or 5 or variants thereof can include either no staple, one staple (e.g., a staple formed between R8 and S5), or be double stapled.
• the BET binders of the present disclosure include agents that directly interact with a BET protein, such as bromodomain 2 (BRD2), BRD3 and/or BRD4.
• a BET protein binder can be a BET protein inhibitor which suppresses BET enzymatic activity.
• BET inhibitors that can be utilized in the chimeras described herein have the structures provided below:
• E3 ubiquitin ligases include human double minute 2 (HDM2), Von Hippel-Lindau (VHL), Cereblon, X- linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein (cIAP), or Constitutive photomorphogenic 1 (COP1).
• HDM2 human double minute 2
• VHL Von Hippel-Lindau
• XIAP X- linked inhibitor of apoptosis protein
• cIAP cellular inhibitor of apoptosis protein
• COP1 Constitutive photomorphogenic 1
• the second moiety of the chimera of this disclosure can be a cereblon binding moiety that is or includes a small molecule.
• small molecules that bind to cereblon include but are not limited to thalidomide, pomalidomide, lenalidomide, avadomide, analogs thereof, and pharmaceutically acceptable salts thereof.
• the cereblon binding moiety is conjugated to the N- terminus of the first protein-targeting stapled peptide. In some instances, the cereblon moiety is conjugated to the C-terminus of the first protein-targeting stapled peptide. In certain instances, the cereblon moiety is contained within a non-natural amino acid inserted in the peptide sequence between the N- and C-terminus of the first proteintargeting stapled peptide. In some instances, the small molecule (thalidomide) that binds to cereblon can be conjugated at the N-terminus of the first protein-targeting moiety (e.g., a peptide). When the small molecule (thalidomide) is conjugated at the N-terminus of a peptide, it has the structure shown below:
• the small molecule when the small molecule (thalidomide) is conjugated at the C-terminus of a first protein-targeting peptide, it has the structure shown below:
• the cereblon-binding moiety is contained within a nonnatural amino acid inserted in the peptide sequence between the N- and C-terminus of the first protein-targeting stapled peptide.
• the second moiety of the chimera of this disclosure can be a VHL binding moiety that is or comprises a small molecule.
• VHL binding moiety that is or comprises a small molecule.
• small molecules that bind to VHL include but are not limited to VH 032 and VHL-binding analogs thereof.
• VHL moiety when the VHL moiety is conjugated to an amine, the carboxylate analog pictured below is employed:
• the VHL binding moiety is conjugated to the N- terminus of the first protein-targeting stapled peptide. In some instances, the VHL moiety is conjugated to the C-terminus of the first protein-targeting stapled peptide. In certain instances, the VHL moiety is contained within a non-natural amino acid inserted in the peptide sequence between the N- and C-terminus of the first proteintargeting stapled peptide. In certain instances, the VHL binding moiety is a peptide that has the amino acid sequence of a peptide listed below, or a VHL-binding variant thereof:
• LAPYIPMDDDFQL (SEQ ID NO: 13) - E3 ligase: VHL.
• the variant can differ from SEQ ID NO: 12 or 13 at e.g., 1, 2, 3, 4, 5, or 6 amino acid positions (e,g., by substitution with another amino acid, insertion, or deletion), provided it still binds VHL.
• the second moiety of the chimera of this disclosure can be an HDM2 binding moiety that is or comprises a small molecule, a peptide or a stapled peptide or an otherwise chemically-stabilized peptide (e.g., stitched) of the transactivation domain of p53 that binds HDM2 and/or HDMX.
• small molecules that bind to HDM2 include but are not limited Nutlin-3, Nutlin-3a, Nutlin-3a derivatives such as RG7112 and RG7388 (Idasanutlin), and HDM2-binding analogs thereof.
• Nutlin-3a has the following structure:
• RG7112 has the following structure:
• RG7388 has the following structure:
• the HDM2 binding moiety is a p53 peptide or a stapled p53 peptide known in the art (e.g., a peptide comprising the transactivation domain of p53 (LSQETFSDLWKLLPEN (SEQ ID NO: 11)), a variant thereof that binds HDM2, or a stapled version thereof).
• the HDM2 binding moiety is a stabilized or stapled p53 peptide that directly binds to and recruits a complex between HDMX and the HDM2.
• a stapled p53 peptide can bind HDMX and recruit the HDMX/HDM2 complex.
• a stapled p53 peptide binds, directly or indirectly, to HDM2, or to HDMX which binds to HDM2.
• the HDM2-binding stapled peptides of the disclosure have the residues (R5), (R8), (S5), and (S8).
• R and S refers to the stereochemistry of the non-natural amino acids
• 5 and 8 refer to the number of carbon residues in the olefinic side chains of the non-natural amino acid.
• (R5) is (R)-2-(4’-pentenyl)alanine [also known as (R)-a-(4'- pentenyl)alanme]
• “(R8)” is (R)-2-(7’-octenyl)alanine [also known as (R)-a-(7'-octenyl)alanine]
• “(S5)” is (S)-2-(4’-pentenyl)alanine [also known as (S)-a- (4'- pentenyl)alanine]
• (S8)” is (S)-2-(7’-octenyl)alanine [also known as (S)-a- (7'-octenyl)alanine]
• SEQ ID NOs.: 1, 7, and 40-47 can be varied such that (R8) is replaced by (R5) and concurrently, (S5) is replaced by (S8).
• variant HDM2-binding stapled peptides are encompassed by this disclosure.
• These variants of SEQ ID NOs. 1, 7, and 40-47 retain the ability to bind to HDM2.
• a variant sequence of SEQ ID NO: 1 is SEQ ID NO: 49; and a variant sequence of SEQ ID NO: 7 is SEQ ID NO: 50
• Non-limiting examples of stapled p53 peptides that may be used in the chimeras of this disclosure are listed below:
• LTF(R8)EYWAQ#(S5)SAA (SEQ ID NO: 7) - ATSP-7041# is cyclobutylalanine
• stapled p53 peptides and other peptides that bind to HDM2 and/or HDMX and can be used in the chimeras of this disclosure include but are not limited to ALRN-6924, and the stapled or stitched p53 peptides provided in US Patent Nos.10,202,431; 9,617,309; 9,556,227; 9,527,896; 9,517,252; 9,505,804; 9,505,801; 9,408,885; 9,175,045; 9,163,330; 8,927,500; 8,889,632; 8,592,377; 8,586,707; U.S. Patent Application Publication Nos.
• variants of any one of SEQ ID NOs.: 1, 7, 40-47, and 49- 50 that still bind HDM2 and/or HDMX are encompassed by this disclosure. These variants can differ from any one of SEQ ID NOs.: 1, 7,40-47 and 49-50 at 1, 2, 3, 4, 5, or 6 amino acid positions (e.g., by substitution with a different amino acid, insertion, or deletion) wherein the variants retains its ability to bind HDM2.
• the tryptophan (“W”) residue in SEQ ID NO: 1 or 7 can be substituted by 3-(2-Naphthyl)-L-alanine).
• a variant differs from the peptide of SEQ ID NO: 1 or 7 in that it varies from SEQ ID NO: 1 or 7 in having 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) amino acid substitutions.
• the residues labeled Xi, X2, X3, X4, and X5 can be substituted in SEQ ID NO: 1 or 7 as follows: XiTF(R8)X2YX 3 AQX4(S5)X 5 AA (SEQ ID NO: 48) where Xi, X 2 , and X 5 are any amino acid (e.g., A, W, F, L, V, I, naphthylalanine, E, D, Y, cyclobutylalanine and side chain analogs thereof); X3: W, F, or 3-(2-naphthyl)-L-alanine; and X : L or a leucine mimetic (e.g., cyclobutyla
• the second moiety peptide has the amino acid sequence of one of the peptides listed below, or HDM2-binding variants thereof (variations may be made as described in the preceding paragraph), such as those described in Chong Li et al., J Mol Biol. 2010 Apr 30; 398(2): 200-213: FSDLWKLL (SEQ ID NO: 38) - p53 transactivation domain sequence;
• TSFAEYWNLLSP (SEQ ID NO: 39) - PMI duodecimal peptide inhibitor.
• LSQETFSDLWKLLPEN (SEQ ID NO: 11) which corresponds to amino acids 14-29 of full length p53. Any or all amino acids of the stapled p53 peptide except for the essential interacting amino acids (see above) can be substituted, and/or one or more of the essential interacting amino acids (see above) can be substituted with one or more conservative substitutions. See, e.g., Coffill et al Genes Dev 2016 30: 281-292 and Baek at el JACS 2012 13: 103-6. In some cases, the first, second third, fourth and/or fifth N-terminal amino acids of SEQ ID NO: 11 may be deleted.
• one, two, and/or three of the C-terminal amino acids of SEQ ID NO: 11 may be deleted.
• the first, second third, fourth and/or fifth N-terminal amino acids and one, two, and/or three of the C-terminal amino acids of SEQ ID NO: 11 may be deleted.
• Conservative substitutions suitable for inclusion in the stapled p53 peptides disclosed herein are discussed below can include substitutions in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
• the essential amino acid phenylalanine (Fl 9) in SEQ ID NO: 11 can be replaced with alanine.
• the second moiety of the chimera of this disclosure can be an XIAP binding moiety that is or comprises a small molecule.
• small molecules that bind to XIAP include but are not limited to A410099.1 or an XIAP- binding analog thereof
• the XIAP binding moiety A410099.1 has the following structure:
• the second moiety of the chimera of this disclosure can be a cIAP binding moiety that is or comprises a small molecule.
• small molecules that bind to cIAP include but are not limited to SM-1295 or SM-1280 or a cIAP -binding analog thereof.
• the cIAP binding moiety SM-1295 has the following structure:
• the second moiety of the chimera of this disclosure can be a COP1 binding moiety that is or comprises a peptide or stapled peptide.
• a peptide that binds to COP1 includes but is not limited to the Tribbles Pseudokinase 1 (Tribl) peptide with the following sequence: DQIVPEY (SEQ ID NO: 6) or variants thereof.
• the peptides can have at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 75% identity, at least or about 80%, at least 85%, at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity to those amino acids in SEQ ID NO: 6, wherein the peptides bind to COP1.
• the peptides can include amino acid substitutions and/or deletions, whether conservative or not.
• the second moiety of the chimera of this disclosure can be a peptide that binds a protein that is the substrate adaptor for an E3 ubiquitin ligase.
• the peptide binds a WD-40 protein that is the substrate adaptor for an E3 ubiquitin ligase.
• the peptide binds to the substrate recognition domain of the E3 ubiquitin ligase in a shallow groove and is tolerant of elaboration (i.e., conjugation of a stapled peptide sequence) at either the N- or C-terminus.
• Exemplary substrate adaptors for an E3 ubiquitin ligase include HDM2, and VHL.
• the second moiety peptides are based on a natural binding consensus sequence of a peptide that binds a WD40-repeat protein that is the substrate adaptor for an E3 ubiquitin ligase.
• the second moiety peptides are variants (e.g., substitution, deletion, or insertion variants) of the natural binding consensus sequence of a peptide that binds a WD40-repeat protein that is the substrate adaptor for an E3 ubiquitin ligase.
• Non-limiting examples of natural binding consensus sequence of a peptide that binds a WD40-repeat protein that is the substrate adaptor for an E3 ubiquitin ligase are provided below: f
• the motif pattern uses the following nomenclature: ‘ .’ specifies any amino acid type, ‘[X]’ specifies the allowed amino acid type(s) at that position, ‘"X’ at the beginning of the pattern specifies that the sequence starts with amino acid type X, ‘
• conserveed residue positions within the primary degron that are known to be post- translationally modified are shown in boldface.
• the second moiety peptide can be a stabilized or stapled peptide. Any other peptides known in the art can also be incorporated as a second moiety into the chimeras of this disclosure. See, e.g., Meszaros et al., Sci. Signal., 10(470):eaak9982 (2017); Guharoy et al., Nature Communications, 7:10239, doi:10.1038/ncommsl0239 (2016); U.S. Patent Nos. 9,783,575; 9,297,017; and 9,115,184, all of which are incorporated by reference herein in their entireties.
• Variants of the peptides of this disclosure include peptides with one or more (e.g., 1, 2, 3, 4, 5) amino acid substitutions; one or more deletions (e.g., 1, 2, 3); one or more insertions (e.g., 1, 2, 3); or a combination of any two or more thereof.
• the variants that interact with the relevant E3 ligase are selected.
• a second moiety peptide of this disclosure can bind its relevant E3 ligase with a binding affinity lower than 1000 nM.
• the second moiety peptide is 4 to 20 amino acids in length (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).
• the first and/or second moiety of the chimeras of this disclosure can include stabilized peptides (e.g., a stapled peptides).
• stabilized peptides e.g., a stapled peptides.
• a peptide helix is an important mediator of key protein-protein interactions that regulate many important biological processes (e.g, apoptosis); however, when such a helix is taken out of its context within a protein and prepared in isolation, it can unfold and adopt a random coil conformation, leading to a drastic reduction in biological activity and thus diminished therapeutic potential.
• structurally stabilized peptides comprise at least two modified amino acids joined by an internal (intramolecular) cross-link (or staple).
• Stabilized peptides as described herein include stapled peptides, stitched peptides, peptides containing multiple stitches, peptides containing multiple staples, or peptides containing a mix of staples and stitches, as well as peptides structurally reinforced by other chemical strategies (see. e.g., Balaram P. Cur. Opin. Struct. Biol. 1992;2:845; Kemp DS, et al., J. Am. Chem. Soc. 1996;118:4240; Omer BP, et al., J. Am. Chem. Soc.
• polypeptides can be stabilized by peptide stapling (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated by reference herein in its entirety).
• Stapled peptides reinforce the natural a-helical shape of bioactive peptides, conferring stabilized structure, protease resistance in vivo, enhanced target binding affinity, and favorable pharmacology (Walensky, L.D. & Bird, G.H. J Med Chem 57, 6275-88 (2014); Walensky, L.D. et al. Science 305, 1466-70 (2004)).
• a peptide is “stabilized” in that it maintains its native secondary structure.
• “Peptide stapling” is a term coined from a synthetic methodology wherein two olefin-containing side-chains (e.g., cross-linkable side chains) present in a polypeptide chain are covalently joined (e.g., “stapled together”) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994).
• RCM ring-closing metathesis
• peptide stapling includes the joining of two (e.g., at least one pair ol) double bond-containing side-chains, triple bond-containing side-chains, or double bond-containing and triple bond-containing side chain, which may be present in a polypeptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly “stapled” polypeptide.
• multiply stapled polypeptides refers to those polypeptides containing more than one individual staple, and may contain two, three, or more independent staples of various spacing.
• polypeptides can be stabilized by, e.g, hydrocarbon stapling.
• the stapled peptide includes at least two (e.g, 2, 3, 4, 5, 6) amino acid substitutions, wherein the substituted amino acids are separated by two, three, or six amino acids, and wherein the substituted amino acids are non-natural amino acids with olefinic side chains.
• the substituted amino acids are non-natural amino acids with olefinic side chains.
• unnatural amino acids are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N- methyl-L-threonine, N- methyl-L-leucine, 1 -amino-cyclopropanecarboxylic acid, 1- amino-2-phenyl- cyclopropanecarboxylic acid, 1 -amino-cyclobutanecarboxylic acid, 4- aminocyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3- diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4- (aminomethyl)benzoic acid,
• cross-links between XI and X4, or between XI and X5, or between XI and X8 are useful hydrocarbon stapled forms of that peptide, as are cross-links between X2 and X5, or between X2 and X6, or between X2 and X9, etc.
• the use of multiple cross-links e.g., 2, 3, 4, or more is also contemplated.
• the use of multiple cross-links is very effective at stabilizing and optimizing the peptide, especially with increasing peptide length.
• R- propenylalanine and S-pentenylalanine; or R- pentenylalanine and S-pentenylalanine are substituted for the amino acids at those positions.
• S -pentenyl alanine is substituted for the amino acids at those positions.
• S-pentenyl alanine and R-octenyl alanine are substituted for the amino acids at those positions.
• the amino acids of the peptide to be involved in the “stitch” are substituted with Bis-pentenylglycine, S- pentenylalanine, and R-octenylalanine; or Bis-pentenylglycine, S- octenylalanine, and R-octenylalanine.
• Fig. 2 shows exemplary chemical structures of non-natural amino acids that can be used to generate various crosslinked compounds.
• Fig. 2 (middle) illustrates peptides with hydrocarbon cross-links between positions i and i+3; i and i+4 and i and i+ 7 residues.
• Fi. 2 (bottom) illustrates a staple walk along a peptide sequence.
• Fig- 3 shows various peptide sequences with double and triple stapling strategies, and exemplary staple walks.
• Fig. 4 illustrates exemplary staple walks using various lengths of branched stitched moieties.
• Rs is halo, alkyl, ORe, N(Re)2, SRe, SORe, SO2R6, CO2R6, Re, a fluorescent moiety, or a radioisotope;
• K is O, S, SO, SO 2 , CO, CO2, CONRe, or
• the tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., Cs, Cs, or Cn alkyl, a Cs, Cs, or Cn alkenyl, or Cs, Cs, or Cn alkynyl).
• the tethered amino acid can be alpha disubstituted (e.g., C1-C3 or methyl).
• x is 2, 3, or 6.
• each y is independently an integer between 1 and 15, or 3 and 15.
• Ri and R2 are each independently H or Ci-Ce alkyl.
• Ri and R2 are each independently C1-C3 alkyl.
• at least one of Ri and R2 are methyl.
• Ri and R2 can both be methyl.
• R3 is alkyl (e.g., Cs alkyl) and x is 3.
• R3 is Cn alkyl and x is 6.
• R3 is alkenyl (e.g., Cs alkenyl) and x is 3.
• x is 6 and R3 is Cn alkenyl.
• Rs is [R4 — K — R4] n ; and R4 is a straight chain alkyl, alkenyl, or alkynyl.
• the disclosure features internally cross-linked (“stapled” or “stitched”) peptides, wherein the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple; the side chains of three amino acids are replaced by an internal stitch; the side chains of four amino acids are replaced by two internal staples, or the side chains of five amino acids are replaced by the combination of an internal staple and an internal stitch.
• the stapled/ stitched peptide can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.
• the stabilized peptide is a peptide of an intracellular protein that binds to a protein targeted for degradation (e.g., a coronaviral protease), a peptide that binds to E3 ubiquitin ligase or a peptide that binds to a substrate adaptor for an E3 ubiquitin ligase (e.g., a WD40-repeat protein).
• a protein targeted for degradation e.g., a coronaviral protease
• a peptide that binds to E3 ubiquitin ligase or a peptide that binds to a substrate adaptor for an E3 ubiquitin ligase e.g., a WD40-repeat protein.
• the stapled peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs.: 1- 7, 12, 13, 38-47, and 49-50.
• this disclosure features stabilized peptides that differ from the peptides disclosed above in that they vary in the location of the staple/stitch.
• this disclosure features stabilized peptides that differ from the peptides disclosed above in that they vary from the above-disclosed sequences in having 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, 7) amino acid substitutions on the non-interacting face of the alpha-helix of these peptides. In certain instances, the substitutions are conservative. In other instances, the substitutions are non-conservative. In certain embodiments, this disclosure features stabilized peptides that differ from the peptides disclosed above in that they vary from the above-disclosed sequences in having 1 to 5 (e.g., 1, 2, 3, 4, 5) amino acid substitutions on the interacting face of the alpha-helix of these peptides. In certain instances, the substitutions are conservative. Exemplary types of variations/modifications to stapled peptides are illustrated in Fig. 5.
• Hydrocarbon tethers are used in the stabilized peptides of this disclosure.
• other tethers can also be employed in the stabilized peptides of this disclosure.
• the tether can include one or more of an ether, thioether, ester, amine, or amide, or triazole moiety.
• a naturally occurring amino acid side chain can be incorporated into the tether.
• a tether can be coupled with a functional group such as the hydroxyl in serine, the thiol in cysteine, the primary amine in lysine, the acid in aspartate or glutamate, or the amide in asparagine or glutamine.
• Triazole- containing (e.g., 1, 4 triazole or 1, 5 triazole) crosslinks can be used (see, e.g, Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO 2010/060112).
• other methods of performing different types of stapling are well known in the art and can be employed (see, e.g., Lactam stapling-. Shepherd et al., J. Am. Chem.
• the length of the tether can be varied. For instance, a shorter length of tether can be used where it is desirable to provide a relatively high degree of constraint on the secondary alpha-helical structure, whereas, in some instances, it is desirable to provide less constraint on the secondary alpha-helical structure, and thus a longer tether may be desired.
• tethers spanning from amino acids i to i+3, i to i+4, and i to i l are common in order to provide a tether that is primarily on a single face of the alpha helix, the tethers can be synthesized to span any combinations of numbers of amino acids and also used in combination to install multiple tethers.
• Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized.
• the epoxide can be treated with a nucleophile, which provides additional functionality that can be used, for example, to attach a therapeutic agent.
• Such derivatization can alternatively be achieved by synthetic manipulation of the amino or carboxy -terminus of the polypeptide or via the amino acid side chain.
• Other agents can be attached to the functionalized tether, e.g., an agent that facilitates entry of the polypeptide into cells.
• alpha disubstituted amino acids are used in the polypeptide to improve the stability of the alpha helical secondary structure.
• alpha disubstituted amino acids are not required, and instances using mono-alpha substituents (e.g., in the tethered amino acids) are also envisioned.
• the stapled polypeptides can include a drug, a toxin, a derivative of polyethylene glycol; a second polypeptide; a carbohydrate, etc. Where a polymer or other agent is linked to the stapled polypeptide it can be desirable for the composition to be substantially homogeneous.
• PEG polyethelene glycol
• n 2 to 10,000 and X is H or a terminal modification, e.g., a Ci-4 alkyl; and Y is an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N- terminus) of the polypeptide. Y may also be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine).
• Other methods for linking PEG to a polypeptide, directly or indirectly, are known to those of ordinary skill in the art.
• the PEG can be linear or branched.
• Various forms of PEG including various functionalized derivatives are commercially available.
• macromolecular polymer e.g, PEG
• the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art.
• the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.
• a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Non-peptide linkers are also possible.
• lower alkyl e.g., Ci-Ce
• halogen e.g., Cl, Br
• CN NH2, phenyl, etc.
• U.S. Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates having a peptide at each of the PEG linker termini.
• the stabilized peptides can also be modified, e.g., to further facilitate cellular uptake or increase in vivo stability, in some embodiments.
• acylating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
• the stapled peptides disclosed herein have an enhanced ability to penetrate cell membranes (e.g., relative to non-stapled peptides).
• the stabilized peptides can be made by chemical synthesis methods, which are well known to the ordinarily skilled artisan. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the a-NFL protected by either t-Boc or Fmoc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431.
• SPPS solid phase peptide synthesis
• the C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule.
• This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products.
• the N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.
• peptides could be made by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols.
• a gene encoding a peptide of this invention the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed.
• a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary.
• the synthetic gene is inserted in a suitable cloning vector and transfected into a host cell.
• the peptide is then expressed under suitable conditions appropriate for the selected expression system and host.
• the peptide is purified and characterized by standard methods.
• the peptides can be made in a high-throughput, combinatorial fashion, e.g, using a high-throughput multiple channel combinatorial synthesizer available from Advanced Chemtech.
• polypeptides can be further modified by: acetylation, amidation, biotinylation, cinnamoylation, famesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation.
• peptides where an i linked to i+ 7 staple is used either: a) one S5 amino acid and one R8 is used; or b) one S8 amino acid and one R5 amino acid is used.
• R8 is synthesized using the same route, except that the starting chiral auxiliary confers the R-alkyl-stereoisomer.
• 8-iodooctene is used in place of 5-iodopentene.
• Inhibitors are synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MBHA resin (see, e.g, WO 2010/148335).
• SPPS solid-phase peptide synthesis
• Fmoc-protected a-amino acids other than the olefinic amino acids Fmoc-Ss- OH, Fmoc-Rs-OH , Fmoc-Rs-OH, Fmoc-Ss-OH and Fmoc-Rs-OH
• Rink Amide MBHA are commercially available from, e.g., Novabiochem (San Diego, CA).
• DMF Dimethylformamide
• NMP N-methyl-2-pyrrolidinone
• DIEA N,N- diisopropylethylamine
• TFA trifluoroacetic acid
• DCE 1,2-di chloroethane
• FITC fluorescein isothiocyanate
• piperidine is commercially available from, e.g., Sigma- Aldrich. Olefinic amino acid synthesis is reported in the art (Williams et al., Org. Synth., 80:31, 2003).
• the peptides are substantially free of non-stapled peptide contaminants or are isolated.
• Methods for purifying peptides include, for example, synthesizing the peptide on a solid-phase support. Following cyclization, the solid-phase support may be isolated and suspended in a solution of a solvent such as DMSO, DMSO/dichloromethane mixture, or DMSO/NMP mixture.
• a solvent such as DMSO, DMSO/dichloromethane mixture, or DMSO/NMP mixture.
• the DMSO/dichloromethane or DMSO/NMP mixture may comprise about 30%, 40%, 50% or 60% DMSO. In a specific embodiment, a 50%/50% DMSO/NMP solution is used.
• the solution may be incubated for a period of 1, 6, 12 or 24 hours, following which the resin may be washed, for example with dichloromethane or NMP. In one embodiment, the resin is washed with NMP. Shaking and bubbling an inert gas into the solution may be performed.
• the linker is an amino acid such as amino-propionic-acid, amino- butanoic-acid, amino-pentanoic-acid, or amino-hexanoic-acid.
• the linker is an oligoethylene glycol, i.e., NH2-(CH2-CH2-O)x-CH2-CH2-COOH.
• the linker is a peptide linker.
• any arbitrary single-chain peptide comprising about one to 30 residues can be used as a linker.
• the linker is 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 144, or 10 to 150 amino acids in length.
• the linker contains only glycine and/or serine residues.
• peptide linkers examples include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 16); Ser Gly Gly Gly (SEQ ID NO: 17); Gly Gly Gly Gly Ser (SEQ ID NO: 18); Ser Gly Gly Gly Gly Gly Gly
• the linker has multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the amino acid sequence of SEQ ID NO: 16 with the exception that the serine residue in each copy of the linker is replaced with another amino acid.
• the linker is a peptide linker, a chemical linker, a Glycine-Serine linker, (G4S) 3 (SEQ ID NO: 26), (G4S)s (SEQ ID NO: 27), a betaalanine (Z) linker, a beta-alanine and alanine (ZA) linker, or a polyethylene glycol linker.
• the linker comprises beta-alanine.
• the linker comprises a beta-alanine and alanine linker.
• anon- natural amino acid (e.g., S5) with olefinic side chains is inserted at positions 3 and 7 of SEQ ID NO: 2
• a non-natural amino acid with olefinic side chains is inserted at positions 11 and 15 of SEQ ID NO: 2.
• a non-natural amino acid (e.g., S5) with olefinic side chains is inserted at positions 3 and 7 and positions 11 and 15 of SEQ ID NO: 2.
• Each of these stapled peptides can further include 1, 2, 3, 4, or 5 amino acid substitutions so long as these stapled peptides retain their ability to bind and inhibit Mpro.
• the peptides can include amino acid substitutions and/or deletions, whether conservative or not.
• the peptide can include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, less than 10, less than 5, less than 4, less than 3, or less than 2 amino acid substitutions, deletions, and/or additions, whether conservative or not, provided that the peptide can still bind to Mpro and inhibit its dimerization and/or enzymatic activity.
• the amino acid sequence of any of the Mpro inhibitor peptides disclosed herein can be varied so long as the variant peptide binds to and inhibits Mpro.
• methods include selecting a subject and administering to the subject an effective amount of one or more of the chimeras of the disclosure, e.g, in or as a pharmaceutical composition, and optionally repeating administration as required for the prophylaxis or treatment of a coronaviral infection, e.g., SARS or COVID-19, and can be administered orally, intravenously, topically, buccally, rectally, parenterally, intraperitoneally, intradermally, subcutaneously, intramuscularly, transdermally, intranasally, pulmonarily, or intratracheally.
• a subject can be selected for treatment based on, e.g., determining that the subject has or is suspected of having a coronaviral infection.
• a maintenance dose of a chimera, composition or combination of this disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Subjects may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
• compositions of this disclosure can include one or more chimeras and any pharmaceutically acceptable carrier and/or vehicle.
• a pharmaceutical composition can further include one or more additional therapeutic agents in amounts effective for achieving a modulation or amelioration of disease or infection (e.g., COVID-19) or disease symptoms.
• the one or more therapeutic agents include but are not limited to a corticosteroid, hydrocortisone, methylprednisolone, dexamethasone, remdesivir, an IL-6 inhibitor, an IL-1 inhibitor, a kinase inhibitor, a complement inhibitor, ivermectin, hydroxychloroquine, favipiravir, interferon-beta, and icatibant.
• Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-
• compositions of this disclosure may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles.
• pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the chimera or its delivery form.
• parenteral as used herein includes subcutaneous, intra- cutaneous, intra-venous, intra-muscular, intra-articular, intra-arterial, intra-synovial, intra-stemal, intra-thecal, intra-lesional and intra-cranial injection or infusion techniques.
• compositions can be in the form of a solution or powder for inhalation and/or nasal administration.
• Such compositions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.
• suitable dispersing or wetting agents such as, for example, Tween 80
• suspending agents such as, for example, Tween 80
• such compositions may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
• compositions of this disclosure may also be administered in the form of suppositories for rectal administration.
• These compositions can be prepared by mixing a chimera of this disclosure with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
• suitable non-irritating excipient include, but are not limited to, e.g., cocoa butter, beeswax, and polyethylene glycols.
• association is covalent. In other embodiments, the association is non-covalent.
• Non- covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc.
• An indirect covalent interaction is when two entities are covalently connected, optionally through a linker group.
• Polymeric carriers can be a natural or a synthetic material containing one or more primary and/or secondary amino groups, azido groups, or carboxyl groups. Carriers can be water soluble. In some embodiments, the present disclosure provides methods for using any one or more of the chimeras (indicated below as 'X') disclosed herein in the following methods:
• the motif patterns in SEQ ID NOs.: 14 and 15 use the following nomenclature: ‘ specifies any amino acid type, ‘ [X] ’ specifies the allowed amino acid type(s) at that position, ‘"X’ at the beginning of the pattern specifies that the sequence starts with amino acid type X, ‘
• conserveed residue positions within the primary degron that are known to be post-translationally modified (for example, phosphorylation and proline hydroxylation) are shown in boldface.
• Xi, X 2 , and X 5 are any amino acid (e.g., A, W, F, L, V, I, naphthylalanine, E, D, Y, cyclobutylalanine and side chain analogs thereof);
• X 3 W, F, or 3-(2-naphthyl)-L-alanine; and
• X 4 L or a leucine mimetic (e.g., cyclobutylalanine).
• (R5) is (R)-2-(4’-pentenyl)alanine
• S8) is (S)-2-(7’- octenyl)alanine.
• SEQ ID NOs.: 7 and 50 # is cyclobutylalanine.
• Xi and X 2 are (S)-2-(4’-pentenyl)alanine.
• B is norleucine.
• X is any amino acid that can be inserted into the sequence and not result in a polypeptide comprising the sequence GSG, and n is 0 to 4.
• SEQ ID NOs.: 51-52 X is (S)-2-(4’-pentenyl)alanine.
• SEQ ID NOs.: 54-55 Z is beta-alanine.
• Example 1 Synthesis of SP-PROTACs to Degrade Essential Proteins of the SARS-CoV-2 Life Cycle
• SP645 was synthesized by replacing two amino acids with the non-natural amino acids S-octenyl alanine and R-pentenyl alanine flanking 6 amino acids (/, i+7 position) (see, Fig. 6).
• Fig. 7 shows the structure of SP645 (SEQ ID NO: 1). Syntheses of the requisite a,a-disubstituted amino acids was performed as described previously (Walensky, L.D. et al. Science 305, 1466-70 (2004); Schafmeister, C.E., et al. J. Am. Chem. Soc. 122, 5891-5892 (2000)).
• Solid-phase Fmoc chemistry and ruthenium-catalyzed olefin metathesis were used for peptide synthesis and staple formation, followed by appending the linker and small molecule on-resin using standard coupling chemistries.
• the prototype panel of SP-PROTACs incorporated PLpro and Mpro targeting small molecules, including reported compounds as described in Baez-Santos, et al. J. Virol., 88, 12511-27 (2014); Ghosh, A.K. et al. J. Med. Chem., 52, 5228-40 (2009); Baez-Santos, et al. Antiviral Res., 115, 21-38 (2015); and Zhang, L. et al. Science, 368, 409-412 (2020), and Gorgulla, C. et al. Nature, 580, 663-668 (2020)).
• Additional peptide and molecular modulators of key SARS-CoV-2 targets may be incorporated into SP-PROTACs using the methods described above.
• Table 5 lists the SARS-CoV-2 target, the type of the molecule, and the name/structure of the molecule that is incorporated into the SP- PROTAC.
• SP-PROTACs that incorporate PLpro, Mpro, NSP9 inhibitors and NSP12 inhibitors.
• Table 5 Examples of small molecules, peptide sequences, and nucleotide analogs that engage viral protein targets, for incorporation into SP-PROTACs
• a qualitative size-exclusion chromatography (SEC) based assay is designed to monitor formation of a ternary complex between the SP-PROTAC, HDM2, and SARS-CoV-2 protein target.
• Recombinant HDM2 and SARS-CoV-2 proteins e.g. PLpro, Mpro
• an N- terminal hexahistidine tag SEQ ID NO: 53
• a thrombin cleavage site are cloned into the pET28a vector, expressed in BL21(DE3) E.coli, and purified by affinity Ni- NTA chromatography, followed by tag cleavage and SEC as described (Ben-Nun, Y.
• SJSA-1 cells were passaged at 37°C in a humidity-controlled, CCh-equilibrated incubator in DMEM (Life Technologies, Grand Island, NY) culture medium (CM) containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin (Pen Strep).
• CM culture medium
• FBS fetal bovine serum
• Pen Strep penicillin/ streptomycin
• SP-PROTAC compounds from high-content screening were tested in natively-susceptible human-derived Huh7 cells (Mossel, E.C. et al. J Virol 79, 3846-50 (2005)) and Calu-3 cells (Tseng, C.T. et al. J Virol 79, 9470-9 (2005)) that express ACE2. Plated cells were treated for 1 h with a serial dilution of SP- PROTACs (10 pM starting dose, in triplicate) followed by a challenge with SARS- CoV-2 (SARS-CoV-2/human/USA/WA-CDC-WAl/2020; Genbank Accession MN985325.1).
• SP-PROTACs that exhibited the most potent anti-viral activity were used to treat Huh7 and Calu-3 cells (>50% infectivity) followed by monitoring for dynamic changes in protein levels.
• Treated cells were lysed in 10% SDS-containing buffer over time (e.g. 2, 4, 6, 8 hours) and lysates subjected to western analysis for p53, the SARS-CoV-2 target (PLpro, Mpro), and actin control. Lysates from treated cells that manifest on-mechanism p53 induction and target-protein-specific degradation (e.g., PLpro, Mpro, BRD4) also undergo global proteomic analyses, performed as described (Winter, G.E. et al. Science 348, 1376-81 (2015)).
• SP-PROTAC-BRD4 significantly improved on- mechanism cytotoxicity (decrease in percent viability) of SJSA-1 cells compared to SP645 and JQ1 alone.
• a high-throughput viral detection platform has been developed for SARS- CoV-2 based on previous screens against Ebolaviruses (Anantpadma, M. et al. Antimicrob Agents Chemother 60, 4471-81 (2016)).
• Vero E6 cells plated in 384-well format were treated for 1 h with a serial dilution of SP-PROTACs (50 pM starting dose), performed in triplicate, followed by a 4 h challenge with SARS-CoV-2 (SARS- CoV-2/human/USA/WA-CDC-WAl/2020; Genbank Accession MN985325.1) to achieve control infection of 10-20% cells (optimal infectivity to assess dynamic range of test compounds).
• SAH-p53-4 SEQ ID NO: 43
• SAH-p53-l-3 SEQ ID NOs.: 40-42
• wild type p53 peptide SEQ ID NO: 11
• a key feature of stapled peptides is their capacity to resist proteolysis in vivo due to shielding of the labile amide bonds by the staple itself and induced helicalfolding.
• LC-MS Liquid chromatography-mass spectrometry
• Reaction samples are composed of 5 pl SP-PROTAC in DMSO (1 mM stock) and 195 pl buffer consisting of 50 mM Tris HC1, pH 7.4.
• FP binding analyses are performed in which C -terminally FITC-derivatized SP-PROTAC (e.g. 25 nM) with a serial dilution of each protein individually (e.g. HDM2, PLpro) in binding buffer (50 mM NaCl, 20 mM HEPES pH 7.4, 5 mM DTT) is incubated.
• FP is measured at equilibrium on a Spectramax M5 Microplate Reader (Molecular Devices) and the data plotted and Kd values calculated using Prism software (Graphpad).
• An example of the application of this FP analysis is shown in Bemal F., et al., J. Am. Chem.
• Fig. 8B shows the relative binding affinity of various stapled p53 peptides (SEQ ID NOs.: 40-43) to HDM2. SAH-p53- 4 was found to bind with higher affinity to HDM2 compared with SAH-p53-l-3 or the wild type p53 peptide.
• Example 12 Anti-Viral Activity of Lead SP-PROTACs in the Humanized ACE2 Receptor Mouse Model of SARS-CoV-2 Infection
• One group receives vehicle as control. Mice are continuously monitored to record body weights and clinical signs.
• On day 4 (peak of viremia) 4 mice from each group are euthanized and viral load quantitated by qPCR from lung homogenate supernatants, prepared as described (Bao, L. et al. Nature 583, 830-833 (2020) using a tissuelyzer (Qiagen).
• Doses for the most effective treatment are refined to determine the minimum amount to protect mice. The same experimental design is used but the 3 groups receive lower doses of the treatment in 4-fold increments. Each experiment addresses sex as a biological variable with equal numbers of male and female
• mice/dose/compound and 10 mice for saline control are used, and mice are euthanized at day 10 (or earlier for adverse signs) and examined for toxicity.
• an SP- PROTAC comprised of either (1) an i, i+4 single-stapled NSP9 binding peptide (SEQ ID NO: 4) positioned at the N-terminus and linked to SP-645 (SEQ ID NO: 1), named SP-PROTAC-NSP9-1 (SEQ ID NO: 54), or (2) SP-645 (SEQ ID NO: 1) positioned a the N-terminus and linked to an i, i+4 single-stapled NSP9 binding peptide (SEQ ID NO: 4), named SP-PROTAC-NSP9-2 (SEQ ID NO: 55; Fig. 14A).
• the SP-PROTAC successfully nucleated the ternary complex between recombinant HDM2, SP-PROTAC -NSP9, and recombinant NSP9 to enable HDM2 ubiquitylation of the viral target protein, NSP9.
• In vitro ubiquitylation assays were performed using the HDM2 ubiquitin ligase kit (Boston Biochem) and the corresponding ubiquitin-shifts in viral protein molecular weight were monitored by western blot analysis.
• SP-PROTAC -NSP9-1 and -2 (10 pM) induced HDM2-mediated ubiquitylation of NSP9 in vitro.

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