Lytic peptide prodrugs
View Patent ↗A cytotoxin can be rendered non-toxic by charge neutralizing the amino acids salient to pore assembly and/or sterically inhibiting formation of the peptide's active conformation. In the presence of specific proteases, the inactive peptide or procytotoxin can be activated to assemble into its lytic conformation and selectively destroy a target cell.
1. A procytotoxin comprising at least one lysine residue bound via a peptide bond to at least one amino acid via the ε-amino group of said lysine residue and further comprising a cytotoxic peptide bound to an inactivator via a peptide bond, wherein said peptide is a pore-forming cytolytic peptide that comprises an amphipathic alpha-helical structure, and wherein said peptide bond is susceptible to cleavage by PSMA.
2. The procytotoxin of claim 1 , further comprising a targeting molecule.
3. The procytotoxin of claim 2 , wherein said targeting molecule is selected from the group consisting of a molecule that targets the neo-vasculature and an antibody.
4. The procytotoxin of claim 3 , wherein said targeting molecule is an RGD targeting sequence.
5. The procytotoxin of claim 1 , wherein said cytolytic peptide is selected from the group consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar , brevinin- 1 E, brevinin-2E, barbatolysin, cytolysin of Enterococcus faecalis , delta hemolysin, diphtheria toxin, E1 Tor cytolysin of Vibrio cholerae , equinatoxin, enterotoxin of Aeromonas hydrophila , esculentin, granulysin, haemolysin of Vibrio parahaemolyticus , intermedilysin of Streptococcus intermedius , the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomycetemcomitans , magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perform perfringolysin O, theta-toxin, of Clostridium perfringens , phallolysin, phallotoxin, streptolysin, D,L-α-amino acid cyclic peptides.
6. The procytotoxin of claim 1 , wherein said cytolytic peptide is a melittin.
7. The procytotoxin of claim 6 , wherein said cytolytic peptide comprises the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro](residues 1-32 of SEQ ID NOS 1 or 2).
8. The procytotoxin of claim 7 , further comprising a targeting molecule.
9. A pharmaceutical composition, comprising one or more procytotoxins of claim 7 and a pharmaceutically suitable carrier or excipient.
10. The procytotoxin of claim 6 , wherein said cytolytic peptide comprises the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(or Tyr)-Tyr-Ser-Gly(or Ser)] (residues 1-32 of SEQ ID NOS 3 or 4).
11. The procytotoxin of claim 10 , further comprising a targeting molecule.
12. A pharmaceutical composition, comprising one or more procytotoxins of claim 10 and a pharmaceutically suitable carrier or excipient.
13. A pharmaceutical composition, comprising one or more procytotoxins of claim 1 and a pharmaceutically suitable carrier or excipient.
14. The procytotoxin of claim 1 , wherein said inactivator is selected from the group consisting of a microbead, an amino acid, a peptide, phage and a phage filament.
15. A method for selectively destroying a target cell, comprising contacting a target cell expressing PSMA with a procytotoxin, which comprises a cytotoxic peptide bound via a peptide bond to an inactivator, wherein said peptide is a pore-forming cytolytic peptide that comprises an amphipathic alpha-helical structure, wherein said procytotoxin further comprises at least one lysine residue bound via a peptide bond to at least one amino acid via the ε-amino group of said lysine residue, and wherein said peptide bond is susceptible to cleavage by PSMA.
16. The method of claim 15 , wherein said procytotoxin further comprises a targeting molecule.
17. The method of claim 16 , wherein said targeting molecule is selected from the group consisting of a molecule that targets the neo-vasculature and an antibody.
18. The method of claim 17 , wherein said targeting molecule is an RGD targeting sequence.
19. The method of claim 15 , wherein said cytolytic peptide is selected from the group consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar , brevinin- 1E, brevinin-2E, barbatolysin, cytolysin of Enterococcus faecalis , delta hemolysin, diphtheria toxin, E1 Tor cytolysin of Vibrio cholerae , equinatoxin, enterotoxin of Aeromonas hydrophila , esculentin, granulysin, haemolysin of Vibrio parahaemolyticus , intermedilysin of Streptococcus intermedius , the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomycetemcomitans, magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perforin, perfringolysin O, theta-toxin, of Clostridium perfringens , phallolysin, phallotoxin, streptolysin, D,L- α-amino acid cyclic peptides.
20. The method of claim 15 , wherein said cytolytic peptide is a melittin.
21. The method of claim 20 , wherein said cytolytic peptide comprises the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro] (residues 1-32 of SEQ ID Nos 1 or 2).
22. The method of claim 21 , further comprising a targeting molecule.
23. The method of claim 20 , wherein said cytolytic peptide comprises the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(or Tyr)-Tyr-Ser-Gly(or Ser)] (residues 1-32 of SEQ ID Nos 3 or 4).
24. The method of claim 23 , further comprising a targeting molecule.
25. The method of claim 15 wherein the target cell is a prostate cancer cell.
26. The method of claim 15 , wherein said inactivator is selected from the group consisting of a microbead, an amino acid, a peptide, phage and a phage filament.
27. A method of making a Procytotoxin, comprising modifying a cytotoxic peptide to include an inactivator, wherein said cytoxic peptide is a pore-forming cytolytic peptide that comprises an amphipathic alpha-helical structure; wherein said procytotoxin further comprises at least one lysine residue bound via a peptide bond to at least one amino acid via the ε-amino group of said lysine residue, and wherein the inactivator is cleaved from the procytotoxin by PSMA.
28. The method of claim 27 , wherein said procytotoxin further comprises a targeting molecule.
29. The method of claim 28 , wherein said targeting molecule is selected from the group consisting of a molecule that targets the neo-vasculature and an antibody.
30. The method of claim 29 , wherein said targeting molecule is an RGD targeting sequence.
31. The method of claim 27 , wherein said cytolytic peptide is selected from the group consisting of Ae I, cytolysin of sea anemone, aerolysin, amatoxin, amoebapore, amoebapore homolog from Entamoeba dispar , brevinin-1E, brevinin-2E, barbatolysin, cytolysin of Enterococcus faecalis , delta hemolysin, diphtheria toxin, El Tor cytolysin of Vibrio cholerae , equinatoxin, enterotoxin of Aeromonas hydrophila , esculentin, granulysin, haemolysin of Vibrio parahaemolyticus , intermedilysin of Streptococcus intermedius , the lentivirus lytic peptide, leukotoxin of Actinobacillus actinomycetemcomitans , magainin, melittin, membrane-associated lymphotoxin, Met-enkephalin, neokyotorphin, neokyotorphin fragment 1, neokyotorphin fragment 2, neokyotorphin fragment 3, neokyotorphin fragment 4, NK-lysin, paradaxin, perform perfringolysin O, theta-toxin, of Clostridium perfringens , phallolysin, phallotoxin, streptolysin, D,L-α-amino acid cyclic peptides.
32. The method of claim 27 , wherein said cytolytic peptide is a melittin.
33. The method of claim 27 , wherein said pore-forming cytolytic peptide comprises the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Gly-Ala-Ile-Gly-Gln-Pro](residues 1-32 of SEQ IDNOS 1 or 2).
34. The method of claim 33 , further comprising adding a targeting molecule to said procytotoxin.
35. The method of claim 34 wherein said targeting molecule is selected from the group consisting of a molecule that targets neovascular and an antibody.
36. The method of claim 32 , wherein said cytolytic peptide comprises the following structure: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-[Gln-Ser-Ser-Phe(or Tyr)-Tyr-Ser-Gly (or Ser)] (residues 1-32 of SEQ ID NOS 3 or 4).
37. The method of claim 27 , wherein said inactivator is selected from the group consisting of a microbead, an amino acid, a peptide, phage and a phage filament.
38. A method of treating a cancer patient comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 13 to a patient having prostate cancer.