COMPOUNDS THAT INTERACT WITH THE RAS SUPERFAMILY FOR THE TREATMENT OF CANCERS, INFLAMMATORY DISEASES, RASOPATHIES, AND FIBROTIC DISEASE
Provided herein are methods and compositions for treating cancers, inflammatory diseases, rasopathies, and fibrotic disease involving aberrant Ras superfamily signaling through the binding of compounds to the GTP binding domain of Ras superfamily proteins including, in certain cases, K-Ras and mutants thereof, and a novel method for assaying such compositions.
1 . An assay for identifying a compound as a modulator of Ras activity, wherein the assay comprises:
a) contacting a compound with a Ras protein;
b) incubating a cyanine-labeled GTP with the compound and the Ras protein; and
c) measuring the amount of the cyanine-labeled GTP bound to the Ras protein.
2 . The assay of claim 1 , wherein the assay is a cell-free assay.
3 . The assay of claim 1 , wherein the Ras protein is DIRASI; DIRAS2; DIRAS3; ERAS; GEM; HRAS; KRAS; MRAS; NKIRAS1; NKIRAS2; NRAS; RALA; RALB; RAPIA; RAPIB; RAP2A; RAP2B; RAP2C; RASDI; RASD2; RASLIOA; RASL10B; RASLI IA; RASLI IB; RASL12; REMI; REM2; RERG; RERGL; RRAD; RRAS; or RRAS2.
4 . The assay of claim 1 , wherein the Ras protein is HRAS; KRAS; or NRAS, or a mutant thereof.
5 . The assay of claim 1 , wherein the Ras protein is immobilized.
6 . The assay of claim 1 , wherein the compound competitively inhibits GTP binding to the Ras GTP binding domain.
7 . The assay of claim 1 , wherein the compound binds to the Ras protein GTP binding domain with greater than 25% inhibition at 20 uM.
8 . The assay of claim 1 , wherein the compound has a binding affinity (K d ) to the Ras protein GTP binding domain of less than 10 uM.
9 . The assay of claim 1 , wherein the compound inhibits the Ras activity and has an IC 50 value of less than 10 uM.
10 . The assay of claim 1 , wherein the compound inhibits binding of the cyanine-labeled GTP with an IC 50 value of less than 10 uM
11 . The assay of claim 1 , wherein the cyanine-labeled GTP is a Cy3- or a Cy5-labeled GTP.
12 . A Ras modulating compound, wherein the Ras modulating compound:
a) is predicted to bind to the GTP binding site of wild-type Ras protein using in silico molecular modeling software; and
b) inhibits GTP binding to wild-type Ras protein in the assay of claim 1 .
13 . The Ras modulating compound of claim 12 , wherein the Ras modulating compound competitively inhibits GTP binding to the wild-type Ras protein GTP binding domain.
14 . The Ras modulating compound of claim 12 , wherein the in silico molecular modeling software is Molecular Operating Environment software (MOE).
15 . A Ras modulating compound, wherein the Ras modulating compound:
a) is predicted to bind to the GTP binding site of a Ras protein using molecular modeling software; and
b) competitively inhibits GTP binding to the Ras protein in the assay of claim 1 .
16 . The Ras modulating compound of claim 15 , wherein the compound binds to the Ras protein GTP binding domain with greater than 25% inhibition at 20 uM.
17 . The Ras modulating compound of claim 15 , wherein the Ras modulating compound inhibits activation of the MAPK signaling pathway.
18 . The Ras modulating compound of claim 15 , wherein the Ras modulating compound inhibits phosphorylation of MAPK in a cell-based assay.
19 . The Ras modulating compound of claim 15 , wherein the Ras modulating compound inhibits cellular proliferation.
20 . The Ras modulating compound of claim 15 , wherein the Ras modulating compound has a molecular weight of less than 2000 Daltons.
21 . The Ras modulating compound of claim 15 , wherein the in silico molecular modeling software is Molecular Operating Environment software (MOE).
22 . A complex between a wild-type Ras protein and a compound, wherein the compound:
a) binds in the GTP binding site of the wild-type Ras protein; and
b) is capable of interacting with one or more amino acid residues at a position of 11, 12, 14, 15, 16, 17, 18, 28, 29, 30, 31, 32, 33, 34, 35, 36, 60, 61, 117, 119, 120, 145, 146, or 147, or with Mg202, in the wild-type Ras GTP binding domain.
23 . The complex of claim 22 , wherein the wild-type Ras protein is wild-type KRas.
24 . The complex of claim 22 , wherein the compound is predicted to interact with the one or more amino acid residues using Molecular Operating Environment software.
25 . A complex between a Ras protein and a compound, wherein the compound:
a) competitively binds in the GTP binding site of the Ras protein; and
b) is capable of interacting with one or more amino acid residues at a position of 11, 12, 14, 15, 16, 17, 18, 28, 29, 30, 31, 32, 33, 34, 35, 36, 60, 61, 117, 119, 120, 145, 146, or 147, or with Mg202, in the Ras GTP binding domain.
26 . The complex of claim 25 , wherein the Ras protein is wild-type HRAS, KRAS, or NRAS, or a mutant thereof.
27 . The complex of claim 25 , wherein the compound competitively inhibits GTP binding to the GTP binding site of the Ras protein.
28 . The complex of claim 25 , wherein the compound competitively inhibits GTP binding to the Ras protein in the assay of claim 1 .
29 . The complex of claim 25 , wherein the compound is the Ras modulating compound of claim 12 .
30 . The complex of claim 25 , wherein the compound is the Ras modulating compound of claim 15 .