Peptidomimetic macrocycles
Biologically active crosslinked polypeptides with improved properties relative to their corresponding precursor polypeptides, having good cell penetration properties and reduced binding to human proteins; and methods of identifying and making such improved polypeptides.
1. A method of preparing a polypeptide with optimized cellular efficacy in human whole blood, the method comprising:
(a) providing a parent polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the parent polypeptide, and wherein the parent polypeptide penetrates cell membranes by an energy-dependent process and binds to an intracellular target;
(b) producing a modified polypeptide having the same sequence as the parent polypeptide sequence except that at least one acidic side chain in the modified polypeptide is replaced with a neutral side chain; wherein the acidic side chain in the parent polypeptide is adjacent to a large hydrophobic side chain and is not essential for target binding, and wherein the modified polypeptide comprises a cross-linker connecting a first amino acid and a second amino acid of the modified polypeptide;
(c) measuring an in vitro efficacy of the modified polypeptide in a whole cell assay wherein activity of the modified polypeptide is mediated by binding of the modified polypeptide to the intracellular target, in the presence and absence of human serum;
(d) calculating an apparent affinity (K d *) of the modified polypeptide to human serum proteins and an EC 50 of the modified polypeptide; and
(e) selecting the modified polypeptide as an optimized polypeptide if the modified polypeptide has a higher K d * and an equal or lower EC 50 than the parent polypeptide.
2. The method of claim 1 , wherein the selected polypeptide has a K d * of less than 70 micromolar.
3. The method of claim 1 , wherein the selected polypeptide has a K d * of about 1-10 micromolar.
4. The method of claim 1 , wherein the selected polypeptide possesses an estimated free fraction in human blood of about 0.1-50%, wherein the estimated free fraction is defined by the equation
FreeFraction
=
K
d
*
K
d
*
+
[
HSA
]
total
and [HSA] total is 700 micromolar.
5. The method of claim 1 , wherein the selected polypeptide possesses an estimated free fraction in human blood of about 0.5-10%.
6. The method of claim 1 , wherein the energy-dependent process is endocytosis.
7. The method of claim 1 , wherein at least one of the first and the second amino acids is an α,α-disubstituted amino acid.
8. The method of claim 1 , wherein the first amino acid and the second amino acid are α,α-disubstituted.
9. The method of claim 1 , wherein the selected polypeptide comprises a helix.
10. The method of claim 1 , wherein the selected polypeptide comprises an alpha-helix.
11. The method of claim 1 , wherein the first amino acid and the second amino acid are separated by three amino acids.
12. The method of claim 1 , wherein the cross-linker comprises from 6 to 14 consecutive bonds.
13. The method of claim 1 , wherein the cross-linker comprises from 8 to 12 consecutive bonds.
14. The method of claim 1 , wherein the selected polypeptide comprises a ring of from about 18 atoms to 26 atoms.
15. The method of claim 1 , wherein the first amino acid and the second amino acid are separated by six amino acids.
16. The method of claim 1 , wherein the cross-linker comprises from 8 to 16 consecutive bonds.
17. The method of claim 1 , wherein the cross-linker comprises from 10 to 13 consecutive bonds.
18. The method of claim 1 , wherein the selected polypeptide comprises a ring of from about 29 atoms to 37 atoms.
19. The method of claim 10 , wherein the cross-linker spans from 1 turn to 5 turns of the alpha-helix.
20. The method of claim 10 , wherein the cross-linker spans 1 turn of the alpha helix.
21. The method of claim 10 , wherein the cross-linker spans 2 turns of the alpha helix.
22. The method of claim 10 , wherein the length of the cross-linker is about 5 Å to about 9 Å per turn of the alpha-helix.
23. The method of claim 1 , wherein the selected polypeptide carries a net positive charge at pH 7.4.
24. The method of claim 1 , wherein the selected polypeptide comprises a halogen, an alkyl group, a fluorescent moiety, an affinity label, a targeting moiety, or a radioisotope.
25. The method of claim 1 , wherein the selected polypeptide provides a therapeutic effect.
26. The method of claim 1 , wherein the selected polypeptide possesses an apparent affinity to human serum proteins of about 1 micromolar or weaker.
27. The method of claim 1 , wherein the selected polypeptide possesses an apparent affinity to human serum proteins of about 3 micromolar or weaker.
28. The method of claim 1 , wherein the selected polypeptide possesses an apparent affinity to human serum proteins of about 10 micromolar or weaker.
29. The method of claim 1 , wherein the selected polypeptide possesses an improved ability to penetrate a cell membrane by an energy-dependent process relative to a corresponding uncrosslinked polypeptide.
30. The method of claim 1 , wherein the acidic and the large hydrophobic side chains are not essential to binding the target and are replaced with neutral and less hydrophobic side chains, respectively.
31. The method of claim 1 , wherein K d * is defined by the equation
E
C
50
′
=
E
C
50
+
P
(
n
1
+
K
d
*
E
C
50
)
where n is 1, and EC 50 is an in vitro efficacy measured in a whole cell assay in the absence of any human serum, and EC′ 50 is an in vitro efficacy measured in a whole cell assay in N % human serum, wherein P equals (N/100)×(700) micromolar.