Peptides having reduced toxicity that stimulate cholesterol efflux
The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.
1. An isolated polypeptide having cholesterol efflux activity, the polypeptide comprising an amphipathic α-helix that has a non-polar surface and a polar surface, wherein the polar surface comprises charged and uncharged amino acid residues at the lipid-water interface and, wherein the α-helix amino acid sequence:
has at least 60% identity to SEQ ID NO:1;
has an acidic amino acid residue at positions 1, 7, 8, 15, 18, and 19 as determined with reference to SEQ ID NO:1;
has an aliphatic amino acid independently selected from V, L, or I, or an uncharged amino acid analog thereof, at positions 10, 13, 16, and 20, as determined with reference to SEQ ID NO:1; wherein the uncharged amino acid analog has an aliphatic side chain and has a hydrophobicity of V or greater than V; and
has an uncharged polar amino acid residue at at least two of positions 3, 14, and 23 as determined with reference to SEQ ID NO:1.
2. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence is 24 amino acids in length and further comprises a position 25 and 26, wherein position 25 is K or N and position 26 is S or Y.
3. The isolated polypeptide of claim 1 , wherein the amino acid sequence has citrulline or an uncharged analog of citrulline that maintains a salt-bridge configuration in the α-helix at at least one of positions 3, 14, or 23.
4. The isolated polypeptide of claim 1 , wherein the amino acid sequence has a citrulline or an uncharged analog of citrulline that maintains a salt-bridge configuration in the α-helix at two of the positions 3, 14, or 23.
5. The isolated polypeptide of claim 4 , wherein the α-helix amino acid sequence has an R or K at the third position.
6. The isolated polypeptide of claim 4 , wherein the two positions that have a citrulline or uncharged analog of citrulline are positions 3 and 14.
7. The isolated polypeptide of claim 6 , wherein the α-helix amino acid sequence has R or K at the third position.
8. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence has Q, N, L, V, I, or A at position 3, 14, or 23.
9. The isolated polypeptide of claim 8 , wherein the α-helix amino acid sequence has Q or N at position 3, 14, or 23.
10. The isolated polypeptide of claim 1 , wherein the amino acid residue at at least two of the three positions 3, 14, and 23 is independently selected from Q or N.
11. The isolated polypeptide of claim 10 , wherein the amino acid residue at the third position is R, K, or citrulline or an uncharged analog of citrulline that maintains a salt bridge configuration in the α-helix.
12. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence comprises an aliphatic amino acid at at least one of positions 2, 6, 9, 17, and 24 as determined with reference to SEQ ID NO:1.
13. The isolated polypeptide of claim 12 , wherein the aliphatic amino acid at at least one of positions 2, 6, 9, 17, and 24 is independently selected from L, V, A, or I.
14. The isolated polypeptide of claim 13 , where the aliphatic amino acid at at least one of positions 2, 6, 9, 17, and 24 is L.
15. The isolated polypeptide of claim 1 , wherein the aliphatic amino acid at at least two, or at at least three, of positions 10, 13, 16, or 20 is independently selected from V, L, or I.
16. The isolated polypeptide of claim 15 , wherein the aliphatic amino acid at at least two, or at at least three, of positions 10, 13, 16, or 20 is independently selected from L or I.
17. The isolated polypeptide of claim 16 , wherein the α-helix amino acid sequence comprises L at at least two, or at at least three, of positions 10, 13, 16, or 20.
18. The isolated polypeptide of claim 16 , wherein the α-helix amino acid sequence comprises I at at least two, or at at least three, of positions 10, 13, 16, or 20.
19. The isolated polypeptide of claim 1 , wherein the aliphatic amino acid at each of positions 10, 13, 16, and 20 is independently selected from V, L, or I.
20. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence comprises L at each of position 6, 10, 13, 16, and 20; or comprises I at each of position 6, 10, 13, 16, and 20.
21. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence has at least 70% identity to SEQ ID NO:1.
22. The isolated polypeptide of claim 1 , wherein the polypeptide further comprises a protecting group.
23. The isolated polypeptide of claim 1 , wherein all enantiomeric amino acids are “D” amino acids; or the enantiomeric amino acids are a mixture of “L” amino acids and “D” amino acids.
24. A retro-inverso or a retro-enantio analog of a peptide of claim 1 .
25. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable carrier.
26. A composition comprising a polypeptide of claim 1 complexed with a lipid.
27. A kit for treating a symptom of atherosclerosis, said kit comprising a container containing a polypeptide of claim 1 .
28. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence has citrulline at at least two of the positions 3, 14, or 23; and an R or K at the third position.
29. The isolated polypeptide of claim 28 , wherein the α-helix amino acid sequence has citrulline at positions 3 and 14 and R or K at position 23.
30. The isolated polypeptide of claim 29 , wherein the aliphatic amino acid at at least two, or at least three, of positions 10, 13, 16, and 20 is independently selected from V, L, or I.
31. The isolated polypeptide of claim 29 , wherein the α-helix amino acid sequence has an aliphatic amino acid residue at positions 2, 6, 21 and 24.
32. The isolated polypeptide of claim 29 , wherein the aliphatic amino acid at each of positions 10, 13, 16, and 20 is independently selected from L, V, or I.
33. The isolated polypeptide of claim 32 , wherein the α-helix amino acid sequence comprises L at each of positions 6, 10, 13, 16, and 20.
34. The isolated polypeptide of claim 32 , wherein the α-helix amino acid sequence comprises I at each of positions 6, 10, 13, 16, and 20.
35. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence comprises SEQ ID NO:1.
36. The isolated polypeptide of claim 1 , wherein the α-helix amino acid sequence comprises SEQ ID NO:1 in which I is substituted for L at positions 10, 13, 16, and 20.
37. An isolated polypeptide having cholesterol efflux activity, wherein the polypeptide comprises an amphipathic α-helix that has a non-polar surface and a polar surface, wherein the α-helix amino acid sequence has at least 60% identity to SEQ ID NO:1, comprises an acidic amino acid residue at positions 1, 7, 8, 15, 18, and 19 as determined with reference to SEQ ID NO:1; comprises at least one chemical staple at positions 3, 14, and 23 and at least two of positions 3, 14, and 23 are polar uncharged amino acids as determined with reference to SEQ ID NO:1; and comprises an aliphatic amino acid independently selected from V, L, or I, or an uncharged amino acid analog thereof, at positions 10, 13, 16, and 20, as determined with reference to SEQ ID NO:1; wherein the uncharged amino acid analog has an aliphatic side chain and has a hydrophobicity of V or greater than V.
38. The isolated polypeptide of claim 37 , wherein the α-helix amino acid sequence comprises an aliphatic amino acid at positions 2, 6, 21, and 24, as determined with reference to SEQ ID NO:1.
39. The isolated polypeptide of claim 37 , wherein the aliphatic amino acid at each of positions 10, 13, 16, or 20 is independently selected from L or I.