Stabilized polypeptides as regulators of RAB GTPase function

Patent metadata

Field	Value
Publication number	US-9487562-B2
Application number	US-201214126642-A
Country	US
Kind code	B2
Filing date	Jun 15, 2012
Priority date	Jun 17, 2011
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

The present invention provides inventive polypeptides comprising a C terminal RAB binding domain (RabBD) of RAB family interacting proteins (FIPs) stabilized by peptide stapling, and pharmaceutical compositions thereof. Also provided are methods for modulating RAB function comprising contacting an inventive stapled polypeptide with a RAB protein, and methods of treatment associated with modulation of RAB activity. The present invention also provides methods of making the inventive stapled polypeptides by ring closing metathesis of unstapled polypeptide precursors.

First claim

The invention claimed is:

1. An unstapled polypeptide of Formula (I):
R f -[X AA ] s -[X 1-26 ]-[X AA ] t -R e   (I)

or a pharmaceutically acceptable salt thereof;
wherein:
each instance of X AA is a natural amino acid or an unnatural amino acid;
s is 0 or an integer between 1 and 100, inclusive;
t is 0 or an interger between 1 and 100, inclusive;
R f is an N-terminal group selected from the group consisting of hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; acyl; a resin; an amino protecting group; and a label optionally joined by a linker, wherein the linker is selected from the group consisting of optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; and acylene;
R e is a C-terminal group selected from the group consisting of hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; —OR E , —N(R E ) 2 , or —SR E , wherein each instance of R E is, independently, hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; acyl; a resin; a protecting group; or two R E groups taken together form an optionally substituted heterocyclic or optionally substituted heteroaryl ring;
-[X 1-26 ]- is an unstapled amino acid sequence of the formula:

-[X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -

X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 ]-

wherein:
X 1 is an amino acid selected from the group consisting of Q, H, and I;
X 2 is an amino acid selected from the group consisting of V, I, and N;
X 3 is an amino acid selected from the group consisting of R and F;
X 4 is an amino acid selected from the group consisting of E and R;
X 5 is amino acid L;
X 6 is an amino acid selected from the group consisting of E, Q, and R;
X 7 is an amino acid selected from the group consisting of D, Q, and an amino acid of formula (i);
X 8 is an amino acid selected from the group consisting of Y and an amino acid of formula (i);
X 9 is an amino acid selected from the group consisting of I and M;
X 10 is amino acid D;
X 11 is an amino acid selected from the group consisting of N, R, K, and an amino acid of formula (i) or (ii);
X 12 is an amino acid selected from the group consisting of L, I, and an amino acid of formula (i) or (ii);
X 13 is an amino acid selected from the group consisting of L and I;
X 14 is an amino acid selected from the group consisting of V and L;
X 15 is an amino acid selected from the group consisting of R, A, and an amino acid of formula (i) or (ii);
X 16 is an amino acid selected from the group consisting of V and I;
X 17 is an amino acid selected from the group consisting of M and L;
X 18 is an amino acid selected from the group consisting of E and D;
X 19 is an amino acid selected from the group consisting of E, T, H and an amino acid of formula (i);
X 20 is an amino acid selected from the group consisting of T and N;
X 21 is amino acid P;
X 22 is an amino acid selected from the group consisting of N and S;
X 23 is amino acid I;
X 24 is amino acid L;
X 25 is an amino acid selected from the group consisting of R and E;
X 26 is an amino acid selected from the group consisting of I and V;

provided that the amino acid sequence comprises at least two independent occurrences of an amino acid of formula (i) or (ii);
wherein the amino acid of formula (i) is:

and the amino acid of Formula (ii) is:

wherein:
each instance of K, L 1 , and L 2 , is, independently, optionally substituted alkylene; optionally substituted heteroalkylene; optionally substituted arylene; or optionally substituted heteroarylene;
each instance of R a1 and R a2 is, independently, hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; acyl; or an amino protecting group; and
each instance of R b is, independently, hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; or optionally substituted heteroaryl.

2. A stapled polypeptide of the Formula (II):
R f -[X AA ] s -[X 1-26 ]-[X AA ] t -R e   (II)

or a pharmaceutically acceptable salt thereof;
wherein:
each instance of X AA is a natural amino acid or unnatural amino acid;
s is 0 or an integer between 1 and 100, inclusive;
t is 0 or an integer between 1 and 100, inclusive;
R f is an N-terminal group selected from the group consisting of hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; acyl; a resin; an amino protecting group; and a label optionally joined by a linker, wherein the linker is selected from the group consisting of optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; and acylene;
R e is a C-terminal group selected from the group consisting of hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; —OR E , —N(R E ) 2 , or —SR E , wherein each instance of R E is, independently, hydrogen; optionally substituted aliphatic; optionally substituted heteroaliphatic; optionally substituted aryl; optionally substituted heteroaryl; acyl; a resin; a protecting group; or two R E groups taken together form an optionally substituted heterocyclic or optionally substituted heteroaryl ring; and
-[X 1-26 ]- is a stapled amino sequence of the Formula:

-[X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -

X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 -X 25 -X 26 ]-

wherein:
X 1 is an amino acid selected from the group consisting of Q, H, and I;
X 2 is an amino acid selected from the group consisting of V, I, and N;
X 3 is an amino acid selected from the group consisting of R and F;
X 4 is an amino acid selected from the group consisting of E and R;
X 5 is amino acid L;
X 6 is an amino acid selected from the group consisting of E, Q and R;
X 7 is an amino acid selected from the group consisting of D and Q, or X 7 and X 11 are stapled amino acids of For

Assignees

• Moellering Raymond E
• Verdine Gregory L
• Harvard College

Inventors

• Moellering Raymond E
• Verdine Gregory L

Classifications

• C07K14/001 Primary

  by chemical synthesis · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K14/4702 Primary

  Regulators; Modulating activity · CPC title