Molecular target in cancer
View Patent ↗Disclosed are methods for identifying small molecule modulators of protein-RNA interactions that could potentially function as therapeutic agents in cancer treatment. Methods of identifying inhibitors of the RNA-binding functions of MSI2, as well as inhibitors identified thereof, are also disclosed.
1 . A method of identifying a modulator of protein-RNA interaction comprising: (a) contacting a fluorophore-labeled RNA-binding protein (RBP) with a fluorophore-labeled target RNA in a reaction mix, (b) detecting a first fluorescence signal generated by the interaction of the RBP and the target RNA, (c) introducing an agent into the reaction mix, (d) detecting a second fluorescence signal in the presence of the agent, wherein the agent is an inhibitor of the RBP-RNA interaction if the second fluorescence signal is decreased relative to the first fluorescence signal, and wherein the agent is an enhancer of the RBP-RNA interaction if the second fluorescence signal is increased relative to the first fluorescence signal, wherein the RBP is U1A and the fluorophore-labeled target RNA comprises AUUGCACUCCUUUUA-Biotin (SEQ ID NO: 2).
2 . The method of claim 1 , wherein the RBP is labeled by coupling to a fluorophore donor and the target RNA is labeled by coupling to a fluorophore acceptor.
3 . The method of claim 1 , wherein the RBP is labeled by coupling to a fluorophore acceptor and the target RNA is labeled by coupling to a fluorophore donor.
4 . The method of claim 1 , wherein detecting the first fluorescence signal or detecting the second fluorescence signal is carried out by a FRET-based assay.
5 . The method of claim 4 , wherein the FRET-based assay is a Homogeneous Time Resolved Fluorescence (HTRF) assay.
6 . The method of claim 1 , wherein the target RNA is biotinylated at the 5′ end or at the 3′ end.
7 . The method of claim 6 , wherein the biotinylated target RNA is coupled to streptavidin.
8 . The method of claim 7 , wherein the streptavidin is labeled with XL665 or d2.
9 . The method of claim 1 , wherein the RBP comprises a His tag.
10 . The method of claim 9 , wherein the His tagged RBP is coupled to Terbium (Tb) cryptate via an anti-His antibody attached to Terbium (Tb) cryptate.
11 . The method of claim 1 , wherein the modulator of protein-RNA interaction is selected from the group consisting of 2-{[4-(2,5-dimethyl-1H-pyrrol-1-yl)phenyl]sulfanyl}acetic acid, 4-[2-(2,5-dimethyl-1H-pyrrol-1-yl)thiophene-3-carbonyl]-3,4-dihydro-2H-1,4-benzoxazine-2-carboxamide, 5-({3-phenyl-5H,6H,7H,8H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl}methyl)-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one, 1-{3-[2-({5,7-dimethyl-[1,2,4]triazolo[4,3-a]pyrimidin-3-yl}sulfanyl) acetyl]phenyl}pyrrolidin-2-one, N-(1-acetylpiperidin-4-yl)-4,5-dimethylthiophene-2-carboxamide, and a derivative thereof.
12 . The method of claim 1 , wherein the target RNA comprises an mRNA sequence comprising SEQ ID NO: 2.