Endothelial nitric oxide synthase antagonists and uses thereof for inhibiting oxygen toxicity
View Patent ↗Compositions and methods for inhibiting the interaction between eNOS and β-actin are provided for use in inhibiting or reducing lung injury from oxygen toxicity. One embodiment provides a synthetic or recombinant polypeptide having the β-actin binding domain of eNOS, wherein the polypeptide inhibits or reduces eNOS activity in lung endothelial cells.
1. An isolated polypeptide 6 to 30 residues in length comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:1 (LGLRWYAL), wherein the polypeptide is operably linked to a cell penetrating peptide, wherein the polypeptide binds β-actin.
2. The isolated polypeptide of claim 1 , wherein the polypeptide binds to β-actin in vivo.
3. The isolated polypeptide of claim 1 , wherein the polypeptide comprises a conservative amino acid substitution in SEQ ID NO:1.
4. The isolated polypeptide of claim 3 , wherein the polypeptide comprises at least 6 consecutive amino acids of SEQ ID NO:1.
5. The isolated polypeptide of claim 1 , wherein the cell penetrating peptide is TAT.
6. The isolated polypeptide of claim 5 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO:2 (RKKRRQRRRALGLRWYAL).
7. The isolated polypeptide of claim 1 operably linked to a lung-homing peptide.
8. The isolated polypeptide of claim 7 , wherein the lung-homing peptide is the tripeptide motif gly-phe-glu (GFE).
9. A synthetic or recombinant polypeptide comprising the β-actin binding domain of eNOS, wherein the polypeptide inhibits eNOS activity in lung endothelial cells, and wherein the polypeptide consists of SEQ ID NO:2.
10. A pharmaceutical composition comprising an effective amount of the polypeptide of any one of claims 1 - 4 , and 5 - 9 to inhibit or reduce eNOS binding to β-actin in vivo and a pharmaceutically acceptable excipient.
11. A method of inhibiting eNOS association with β-actin in a cell, comprising contacting the cell with a polypeptide 6 to 30 amino acids in length comprising the amino acid sequence SEQ ID NO:1.
12. The method of claim 11 , wherein the polypeptide comprises a cell penetrating peptide.
13. The method of claim 12 , wherein the cell penetrating peptide is TAT.
14. The method of claim 13 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO:2.
15. The method of claim 12 , wherein the polypeptide comprises a lung-homing peptide.
16. The method of claim 15 , wherein the lung-homing peptide is the tripeptide motif gly-phe-glu (GFE).
17. The method of any one of claims 11 to 16 , wherein the polypeptide inhibits or reduces damage in the cell from hyperoxia.
18. A method of inhibiting peroxynitrite formation in a cell, comprising contacting the cell with a polypeptide comprising the β-actin binding domain of eNOS, wherein the polypeptide inhibits eNOS activity in the cell.
19. The method of claim 18 , wherein the β-actin binding domain of eNOS comprises the amino acid sequence SEQ ID NO:1.
20. The method of claim 18 , wherein the polypeptide comprises a cell penetrating peptide.
21. The method of claim 20 , wherein the cell penetrating peptide is TAT.
22. The method of claim 21 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO:2.
23. The method of claim 20 , wherein the polypeptide comprises a lung-homing peptide.
24. The method of claim 23 , wherein the lung-homing peptide is the tripeptide motif gly-phe-glu (GFE).
25. A method of inhibiting, reducing or attenuating lung damage by hyperoxia in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising the β-actin binding domain of eNOS, wherein the purified polypeptide does not comprise full-length eNOS.
26. The method of claim 25 , wherein the β-actin binding domain of eNOS comprises the amino acid sequence SEQ ID NO:1 (LGLRWYAL), and wherein the polypeptide further comprises a cell penetrating peptide.
27. The method of claim 26 , wherein the cell penetrating peptide is TAT.
28. The method of claim 27 , wherein the polypeptide comprises the amino acid sequence SEQ ID NO:2.
29. The method of claim 27 , wherein the polypeptide comprises a lung-homing peptide.
30. The method of claim 29 , wherein the lung-homing peptide is the tripeptide motif gly-phe-glu (GFE).
31. An isolated fragment of eNOS consisting of SEQ ID NO:1.
32. An isolated β actin-binding polypeptide consisting of SEQ ID NO:1.