Search

US-20260125426-A1 - CYCLIC CELL-PENETRATING PEPTIDES AND USES THEREOF

US20260125426A1US 20260125426 A1US20260125426 A1US 20260125426A1US-20260125426-A1

Abstract

Disclosed are peptides comprising: a cell-penetrating peptide domain of from about 7 to about 25 amino acids in length; wherein the cell-penetrating peptide domain comprises any combination of at least two arginines and at least two amino acids having a hydrophobic side chain selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted, and wherein the cell penetrating peptide domain comprises at least three thiol containing residues and which are separated from one another by at least one amino acids.

Inventors

  • Dehua Pei
  • Jeremy RITCHEY

Assignees

  • OHIO STATE INNOVATION FOUNDATION

Dates

Publication Date
20260507
Application Date
20230731

Claims (20)

  1. 1 . A peptide, comprising: a cell-penetrating peptide domain of from about 7 to about 25 amino acids in length; wherein the cell-penetrating peptide domain comprises any combination of at least two arginines or arginine analogs and at least two amino acids having a hydrophobic side chain selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted, and wherein the cell penetrating peptide domain comprises at least three thiol containing residues and which are separated from one another by at least one amino acid.
  2. 2 . The peptide of claim 1 , wherein the cell penetrating peptide domain is from about 8 to about to about 14 amino acids amino acids in length.
  3. 3 . The peptide of claim 1 , wherein one or more of the thiol containing residues are cysteine or cysteine analog.
  4. 4 . The peptide of claim 1 , wherein one or more of the amino acids in the cell penetrating peptide domain are non-natural amino acids.
  5. 5 . The peptide of claim 1 , wherein one or more of the amino acids in the cell penetrating peptide domain are D-amino acids.
  6. 6 . The peptide of claim 1 , wherein at least one of the hydrophobic amino acids has an aryl side chain.
  7. 7 . The peptide of claim 1 , further comprising bismuth.
  8. 8 . The peptide of claim 7 , wherein the cell penetrating domain is represented by the following structure: wherein, X 1 and X 2 are amino acids; n and m are independently selected from 1 to 20; j and k are independently selected from 1 to 4; p is selected from 0 to 10; Y 1a and Y 1b are independently selected from H, OH, when Y 1b is not OH or NR 1 Y 1 , NR 1 Y 1 , when Y 1b is not OH or NR 1 Y 1 , C 1 -C 6 alkyl, arylC 0 -C 6 alkyl, heteroarylC 0 -C 6 alkyl where aryl and heteroaryl are optionally substituted; Y 1 is H, a protecting group, a counterion, a cargo moiety joined by an optional linker, or a third peptide domain with an optional linker moiety; R 1 , R 2 , and R 3 are independently selected from H, C 1 -C 6 alkyl, arylC 1 -C 6 alkyl, heteroarylC 1 -C 6 alkyl where aryl and heteroaryl are optionally substituted; Y 2 is OH, NH 2 , a protecting group of the carboxylate, a counterion of the carboxylate, a cargo moiety joined by an optional linker, or a third peptide domain with an optional linker moiety; wherein, the methylene group in (CH 2 )p, (CH 2 )k or (CH 2 )j is optionally substituted with C 1 -C 6 alkyl, or linked to R 1 , R 2 or R 3 to form a ring; and wherein at least two arginines or arginine-analogs and at least two amino acids having a hydrophobic side chain selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted, are present among (X 1 )n and (X 2 )m.
  9. 9 . The peptide of claim 8 , wherein the at least two arginines or arginine-analogs are in (X 1 )n or (X 2 )m.
  10. 10 . The peptide of claim 8 , wherein the at least two arginines or arginine-analogs are distributed among (X 1 )n and (X 2 )m.
  11. 11 . The peptide of claim 8 , wherein the at least two amino acids having hydrophobic side chains are in (X 1 )n or (X 2 )m.
  12. 12 . The peptide of claim 8 , wherein the at least two amino acids having hydrophobic side chains are distributed among (X 1 )n and (X 2 )m.
  13. 13 . The peptide of claim 8 , wherein the at least two or three amino acids having hydrophobic side chains selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted are distributed among the two domains.
  14. 14 . The peptide of claim 8 , wherein the peptide domains comprise at least three, at least four, at least five, at least six, or at least seven arginines.
  15. 15 . The peptide of claim 8 , wherein the peptide domains comprise four arginines.
  16. 16 . The peptide of claim 8 , wherein either (X 1 )n or (X 2 )m comprises at least two or at least three adjacent amino acids having hydrophobic side chains selected from an aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted, and the other (X 1 )n or (X 2 )m comprises at least three, at least four, at least five, at least six, or at least seven adjacent arginines or arginine analogs.
  17. 17 . The peptide of claim 8 , wherein either (X 1 )n or (X 2 )m comprises two adjacent amino acids having hydrophobic side chains selected from fused aryl, fused heteroaryl, or non-aromatic polycyclic cycloalkyl radicals.
  18. 18 . The peptide of claim 8 , wherein the arginines or arginine-analogs are adjacent to one another or distributed throughout the CPP.
  19. 19 . The peptide of claim 8 , wherein Y 1a , Y 1b , Y 1 , or Y 2 comprises a cargo moiety.
  20. 20 . A peptide having SEQ ID NO. 12-85.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application Nos. 63/393,649, filed Jul. 29, 2022, 63/415,415, filed Oct. 12, 2022, and 63/463,953, filed May 4, 2023, which are each incorporated by reference herein in their entireties. ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT This invention was made with government support under grant/contract number GM122459 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Cell-penetrating peptides (CPPs) are short peptides (usually 5-30 aa) that are capable of entering the eukaryotic cell without causing significant damage to the cell membrane. As such, CPPs have potential applications in delivering membrane-impermeable cargoes (e.g., peptides, proteins, nucleic acids, and nanoparticles) into the interior of mammalian cells to act as novel therapeutics Since the initial discovery of Tat (Vivès, E. et al. (1997) Truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-16017) and Penetratin (Derossi, D. et al. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444-10450), nearly 2000 different CPPs have been reported. The first-generation CPPs are linear peptides, which generally have low cytosolic entry efficiencies and are also susceptible to proteolytic degradation in vivo. These shortcomings prompted us and other researchers to develop cyclic peptides as second-generation CPPs (Lättig-Tünnemann. G., et al. (2011) Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides. Nat. Commun. 2, 453-459: Mandal, D., et al. (2011) Cell-penetrating homochiral cyclic peptides as nuclear-targeting molecular transporters. Angew. Chem. Int. Ed. 50 (41), 9633-9637; Qian, Z. et al. (2013) Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs. ACS Chem. Biol. 8 (2). 423-431). Cyclic CPPs, as exemplified by cyclo(phe-Nal-Arg-arg-Arg-arg-Gln) (CPP9, where phe is D-phenylalanine, arg is D-arginine, and Nal is L-naphthylalanine) and cyclo(Phe-phe-Nal-Arg-arg-Arg-arg-Gln) (CPP12) (Qian, Z. et al. (2016) Discovery and mechanism of highly efficient cyclic cell-penetrating peptides. Biochemistry 55, 2601-2612), are highly resistant to proteolytic degradation and have vastly improved cytosolic entry efficiencies, compared to their linear counterparts. The latter properties in turn facilitated the mechanistic studies of how CPPs enter the eukaryotic cell. It is now established that CPPs bind to the proteoglycans and/or phospholipids on the cell membrane and are taken into the early endosome by endocytic/pinocytic mechanisms (Dougherty, P. G. et al. (2019) Understanding cell penetration of cyclic peptides. Chem. Rev. 119, 10241-10287). As the endosomes mature and become progressively more acidic, the CPPs bind to the endosomal membrane with increased affinity and cluster the phospholipids into CPP-enriched lipid domains (Qian. Id.: Sahni, A. et al. (2020) Cell-penetrating peptides escape the endosome by inducing vesicle budding and collapse. ACS Chem. Biol. 15, 2485-2492). The lipid domains bud out as small, unstable vesicles, which subsequently collapse, releasing their luminal contents into the cytosol (Id.). At high concentrations, CPPs can also induce the vesicle budding-and-collapse (VBC) mechanism at the plasma membrane, leading to direct translocation of the CPPs across the plasma membrane. Cyclization of CPPs increases their membrane-binding affinity and therefore the cell entry efficiency (Qian. Id.). Cyclic CPPs have been used to deliver a variety of drug modalities into the mammalian cell in vitro and in vivo, including peptides (Qian, Z., et al. (2014) Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery. Biochemistry 53, 4034-4046: Dougherty, P. G., et al. (2020) A peptidyl inhibitor that blocks calcineurin-NFAT interaction and prevents acute lung injury. J. Med. Chem. 63 (21), 12853-12872: Dougherty. P. G., et al. (2019) Enhancing the cell permeability of stapled peptides with a cyclic cell-penetrating peptide. J. Med. Chem. 62 (22), 10098-10107: Salim, H., et al. (2020) Development of a cell-permeable cyclic peptidyl inhibitor against the Keap1-Nrf2 interaction. J. Org. Chem. 85(3), 1416-1424), proteins (Nischan, N., et al. (2015) Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. Angew. Chem. Int. Ed. 54(6), 1950-1953; Herce, H. D., et al. (2017) Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nature Chem. 9, 762-771; Schneider. A. F. L., et al. (2019) Targeted subcellular protein delivery using cleavable cyclic cell-penetrating pep