| 主权项 |
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-L3-CO—], [—NH-L3-SO2—], or [—NH-L3-]; each R1 and R2 are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or an additional cross-link L, unsubstituted or substituted with halo-; each R3 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5; L is alkyl, alkenyl or alkynyl;
each L3 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R4—K—R4-]n, each being optionally substituted with R5;each R4 is independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;each K is independently O, S, SO, SO2, CO, CO2, or CONR3;each R5 is independently halogen, alkyl, —OR6, —N(R6)2, —SR6, —SOR6, —SO2R6, —CO2R6, a fluorescent moiety, a radioisotope or a therapeutic agent;each R6 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;each R7 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with a D residue;each R8 is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, 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 (Kd*) 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 Kd* 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. |
