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EP-4741414-A1 - PEPTIDE HAVING CELL MEMBRANE PERMEABILITY AND SCREENING METHOD THEREFOR

EP4741414A1EP 4741414 A1EP4741414 A1EP 4741414A1EP-4741414-A1

Abstract

Provided is a peptide or a salt thereof having plasma membrane-penetrating activity, the peptide comprising an amino acid sequence X that satisfies the following conditions (1) to (4): (1) the amino acid sequence X includes at least one amino acid residue selected from R, K, and H, and the total number of these three amino acid residues is 1 to 7; (2) the amino acid sequence X includes at least one amino acid residue selected from Y, W, and F, and the total number of these three amino acid residues is 1 to 5; (3) the amino acid sequence X includes at least one amino acid residue selected from L, V, and M, and the total number of these three amino acid residues is 1 to 4; and (4) the amino acid sequence X has 6 to 15 amino acid residues. Also provided is a method for screening a peptide having plasma membrane-penetrating activity, comprising the steps of: (A) preparing a cDNA display library comprising cDNA display molecules; (B) contacting cells with the cDNA display library and incubating; and (C) recovering the cDNA display molecules from intracellular contents of the cells or from a permeate through the cells.

Inventors

  • HARADA MITSUNORI
  • KITAMURA KOICHIRO

Assignees

  • Mescue-Janusys Inc.

Dates

Publication Date
20260513
Application Date
20240701

Claims (14)

  1. A peptide or a salt thereof having plasma membrane-penetrating activity, comprising an amino acid sequence X that satisfies the following conditions (1) to (4): (1) the amino acid sequence X includes at least one amino acid residue selected from R, K, and H, and the total number of these three amino acid residues is 1 to 7; (2) the amino acid sequence X includes at least one amino acid residue selected from Y, W, and F, and the total number of these three amino acid residues is 1 to 5; (3) the amino acid sequence X includes at least one amino acid residue selected from L, V, and M, and the total number of these three amino acid residues is 1 to 4; and (4) the amino acid sequence X has 6 to 15 amino acid residues.
  2. The peptide or the salt thereof according to claim 1, wherein the amino acid sequence X is linear, and within the amino acid sequence X, the number of consecutive occurrences of D, F, L, Q, R, S, V, and Y is 2 or less for each such residue, and the number of consecutive occurrences of the other amino acid residues is 1, or wherein the amino acid sequence X forms a cyclic structure, and within the amino acid sequence X, the number of consecutive occurrences of F, L, and Y is 2 or less for each such residue, the number of consecutive occurrences of R is 3 or less, and the number of consecutive occurrences of the other amino acid residues is 1.
  3. The peptide or the salt thereof according to claim 1, wherein the amino acid sequence X is linear, and a proportion of the total number of eight amino acid residues E, M, A, L, Q, K, R, and H to the total number of amino acid residues constituting the amino acid sequence X is 25% to 60%, or wherein the amino acid sequence X forms a cyclic structure via a disulfide bond between two cysteine residues, and a proportion of the total number of eight amino acid residues E, M, A, L, Q, K, R, and H to the total number of amino acid residues constituting the amino acid sequence X excluding the two cysteine residues is 25% to 60%.
  4. The peptide or the salt thereof according to claim 1, which exhibits cell-type-selective plasma membrane-penetrating activity.
  5. The peptide or the salt thereof according to claim 1, which exhibits selective plasma membrane-penetrating activity toward at least one selected from the group consisting of macrophage cells, cancer cells, neutrophils, mast cells, T cells, B cells, gastrointestinal epithelial cells, the blood-brain barrier, and skin tissue.
  6. The peptide or the salt thereof according to claim 1, wherein the number of amino acid residues is 6 to 30.
  7. The peptide or the salt thereof according to claim 1, wherein the peptide or the salt thereof has the GRAVY value of from -2.6 to 1.9, and is linear.
  8. The peptide or the salt thereof according to claim 1, comprising an amino acid sequence represented by any one of SEQ ID NOs: 1-40 or a homologous sequence thereof, wherein the homologous sequence comprises 1 to 5 amino acid residue substitutions, insertions, or deletions in the amino acid sequence represented by any one of SEQ ID NOs: 1-40 and has a sequence identity of 60% or more to the amino acid sequence.
  9. A drug-peptide conjugate, comprising a drug and the peptide or the salt thereof according to claim 1, the peptide or the salt thereof being bonded to the drug.
  10. The drug-peptide conjugate according to claim 9, wherein the drug is a protein or a nucleic acid.
  11. Use of the peptide or the salt thereof of claim 1 for drug delivery.
  12. A method for screening a peptide having plasma membrane-penetrating activity, comprising the steps of: (A) preparing a cDNA display library comprising cDNA display molecules; (B) contacting cells with the cDNA display library and incubating the resulting mixture; and (C) recovering the cDNA display molecules from intracellular contents of the cells or from a permeate through the cells.
  13. The screening method according to claim 12, further comprising preparing a sub-cDNA display library using the recovered cDNA display molecules.
  14. The screening method according to claim 12, wherein a selection comprising the steps (A), (B), and (C) in this order is repeated twice or more.

Description

Technical Field The present invention relates to peptides having plasma membrane-penetrating activity and methods for screening such peptides. Background Art In recent years, a molecularly targeted agent that acts on a specific protein or gene serving as a target has been attracting attention, and hence a protein and a gene present in cells are each one of attractive potential drug targets. As a method of delivering a drug into a cell, there has been known a method including bonding a peptide having plasma membrane-penetrating activity (hereinafter also referred to as "cell-penetrating peptide") to impart the cell-penetrating activity to a drug or a carrier thereof (e.g., Patent Literature 1). Citation List Patent Literature [PTL 1] JP 2021-531274 A Summary of Invention Technical Problem The number of cell-penetrating peptides, which exhibit high intracellular delivery efficiency and hence have been actually applied to clinical uses, is small, and hence there has been a demand for a novel cell-penetrating peptide. In addition, while existing cell-penetrating peptides are introduced into a wide variety of cell types; however, they are generally considered to lack cell selectivity. Accordingly, a cell-type-selective cell-penetrating peptide has been desired for alleviating a side effect. In view of the foregoing, a primary object of the present invention is to provide a novel peptide exhibiting high plasma membrane-penetrating activity, and another object thereof is to provide a novel cell-penetrating peptide that is cell-type-selective. Solution to Problem [1] According to one aspect of the present invention, there is provided a peptide or a salt thereof having plasma membrane-penetrating activity, including an amino acid sequence X that satisfies the following conditions (1) to (4): (1) the amino acid sequence X includes at least one amino acid residue selected from R, K, and H, and the total number of these three amino acid residues is 1 to 7; (2) the amino acid sequence X includes at least one amino acid residue selected from Y, W, and F, and the total number of these three amino acid residues is 1 to 5; (3) the amino acid sequence X includes at least one amino acid residue selected from L, V, and M, and the total number of these three amino acid residues is 1 to 4; and (4) the amino acid sequence X has 6 to 15 amino acid residues.[2] In the peptide or the salt thereof according to the above-mentioned item [1], the amino acid sequence X may be linear, and within the amino acid sequence X, the number of consecutive occurrences of D, F, L, Q, R, S, V, and Y may be 2 or less for each such residue, and the number of consecutive occurrences of the other amino acid residues may be 1; or the amino acid sequence X may form a cyclic structure, and within the amino acid sequence X, the number of consecutive occurrences of F, L, and Y may be 2 or less for each such residue, the number of consecutive occurrences of R may be 3 or less, and the number of consecutive occurrences of the other amino acid residues may be 1.[3] In the peptide or the salt thereof according to the above-mentioned item [1] or [2], the amino acid sequence X may be linear, and a proportion of the total number of eight amino acid residues E, M, A, L, Q, K, R, and H to the total number of amino acid residues constituting the amino acid sequence X may be 25% to 60%; or the amino acid sequence X may form a cyclic structure via a disulfide bond between two cysteine residues, and a proportion of the total number of eight amino acid residues E, M, A, L, Q, K, R, and H to the total number of amino acid residues constituting the amino acid sequence X excluding the two cysteine residues may be 25% to 60%.[4] The peptide or the salt thereof according to any one of the above-mentioned items [1] to [3] may exhibit cell-type-selective plasma membrane-penetrating activity.[5] The peptide or the salt thereof according to any one of the above-mentioned items [1] to [4] may exhibit selective plasma membrane-penetrating activity toward at least one selected from the group consisting of macrophage cells, cancer cells, neutrophils, mast cells, T cells, B cells, gastrointestinal epithelial cells, the blood-brain barrier, and skin tissue.[6] In the peptide or the salt thereof according to any one of the above-mentioned items [1] to [5], the number of amino acid residues may be 6 to 30.[7] The peptide or the salt thereof according to any one of the above-mentioned items [1] to [6] may have the GRAVY value of from -2.6 to 1.9, and may be linear.[8] The peptide or the salt thereof according to any one of the above-mentioned items [1] to [7] may include an amino acid sequence represented by any one of SEQ ID NOs: 1-40 or a homologous sequence thereof, wherein the homologous sequence may include 1 to 5 amino acid residue substitutions, insertions, or deletions in the amino acid sequence represented by any one of SEQ ID NOs: 1-40 and may have a sequence identity of 60% or m