Peptidomimetic macrocycles with improved properties

1. A method of screening for an improved alpha-helical polypeptide with an increased in vivo half-life comprising:

a. providing a parent alpha-helical polypeptide;

b. installing at least one cross-link in the parent alpha-helical polypeptide, thereby resulting in a cross-linked polypeptide of Formula (I):

wherein:

each A, C, D, and E is independently a natural or non-natural amino acid;

each B is independently a natural or non-natural amino acid, amino acid analog,

 [—NH-L 3 -CO—], [—NH-L 3 -SO 2 —], or [—NH-L 3 -]; each R 1 and R 2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or an additional cross-link L, unsubstituted or substituted with halo-;

each R 3 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ;

L is alkyl, alkenyl or alkynyl;

each L 3 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R 4 —K—R 4 -] n , each being optionally substituted with R 5 ;

each R 4 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is independently O, S, SO, SO 2 , CO, CO 2 , or CONR 3 ;

each R 5 is independently halogen, alkyl, —OR 6 , —N(R 6 ) 2 , —SR 6 , —SOR 6 , —SO 2 R 6 , —CO 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent;

each R 6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;

each R 7 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;

each R 8 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;

each of v and w is independently an integer from 1-1000;

each of x, y, and z is independently an integer from 0-10;

u is an integer of value 1 or more;

n is an integer from 1-5; wherein at least one-crosslink connects two a-carbon atoms; and

c. determining an apparent affinity (K d *) of the cross-linked polypeptide;

d. determining the in vivo half-life of the cross-linked polypeptide relative to the parent alpha-helical polypeptide; and

e. selecting the cross-linked polypeptide as an improved alpha-helical polypeptide, if the K d * of the cross-linked polypeptide is in the range of 1 to 70 micromolar and the cross-linked polypeptide has an increased in vivo half-life relative to a corresponding polypeptide lacking said at least one cross-link.

2. The method of claim 1 , wherein at least one of R 1 and R 2 is alkyl.

3. The method of claim 1 , wherein at least one of R 1 and R 2 is methyl.

4. The method of claim 1 , wherein u is 2.

5. The method of claim 1 , wherein one crosslink is formed of consecutive carbon-carbon bonds.

6. The method of claim 1 , wherein one crosslink contains at least 8 consecutive bonds.

7. The method of claim 1 , wherein one crosslink contains 9 consecutive bonds.

8. The method of claim 1 , wherein one crosslink contains 12 consecutive bonds.

9. The method of claim 1 , wherein one crosslink comprises at least 7 carbon atoms.

10. The method of claim 1 , wherein one crosslink comprises at least 10 carbon atoms.

11. The method of claim 1 , wherein the crosslinked polypeptide comprises an α-helical domain of a BCL-2 family member.

12. The method of claim 1 , wherein the crosslinked polypeptide comprises a BH3 domain.

13. The method of claim 1 , wherein the crosslinked polypeptide has a K d * of 1 to 10 micromolar.

14. The method of claim 1 , wherein the crosslinked polypeptide has a K d * in the range 10-70 micromolar.

15. The method of claim 1 , wherein the cross-linked polypeptide is selected such that the % helicity of the crosslinked polypeptide is 25% or greater at room temperature under aqueous conditions.

16. The method of claim 1 , wherein the cross-linked polypeptide is selected such that the % helicity of the crosslinked polypeptide is 50% or greater at room temperature under aqueous conditions.

17. The method of claim 1 , wherein the cross-linked polypeptide is selected such that the % helicity of the crosslinked polypeptide is 75% or greater at room temperature under aqueous conditions.

18. The method of claim 1 , wherein the in vivo half-life of said polypeptide is determined after intravenous administration.