Method for identifying modulators of G3BP activity

Patent metadata

Field	Value
Publication number	US-12372526-B2
Application number	US-202117541806-A
Country	US
Kind code	B2
Filing date	Dec 3, 2021
Priority date	Jun 26, 2019
Publication date	Jul 29, 2025
Grant date	Jul 29, 2025

Abstract

A method of identifying a lead or candidate compound that modulates the activity of GTPase-Activating Protein SH3 Domain-Binding Proteins (G3BP) is provided, which includes determining whether a compound modulates the interaction between the N-terminal Nuclear Transport Factor 2-like (NTF2L) domain of G3BP and FGDF peptide of ubiquitin specific protease 10 (USP10) or non-structural protein 3 (nsP3).

First claim

What is claimed is:

1. A kit for identifying a lead or candidate compound that modulates the activity of GTPase-Activating Protein SH3 Domain-Binding Proteins (G3BP) comprising:
(i) a N-terminal Nuclear Transport Factor 2-like (NTF2L) domain of G3BP,
(ii) a peptide containing a FGDF motif, and
(iii) a tag-specific antibody conjugated to a metal liganding moiety,
wherein at least on one of (i) or (ii) is conjugated to a tag that binds the tag-specific antibody conjugated to the metal liganding moiety.

2. The kit of claim 1 , wherein the NTF2L domain has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3.

3. The kit of claim 1 , wherein the peptide containing a FGDF motif has the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

4. The kit of claim 1 , wherein the NTF2L domain of G3BP is conjugated to the tag.

5. The kit of claim 4 , wherein the tag comprises V5, myc, His 6 (SEQ ID NO: 6), glutathione-S-transferase, the Fc portion of human IgG, or maltose binding protein.

6. The kit of claim 4 , wherein the peptide containing a FGDF motif is conjugated to a fluorescent acceptor moiety.

7. The kit of claim 6 , wherein the fluorescent acceptor moiety comprises fluorescein, rhodamine, GFP, GFP derivatives, FITC, 5-carboxyfluorescein (5-FAM), 6-FAM, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, or 7-amino-4-methylcoumarin-3-acetic acid.

8. The kit of claim 1 , wherein the metal liganding moiety comprises lanthanide metal ions.

9. The kit of claim 6 , wherein the kit further comprises an unlabeled control peptide containing a FGDF motif.

Assignees

• St Jude Childrens Res Hospital

Inventors

• Taylor J Paul
• Yang Peiguo
• Lin Wenwei
• Chen Taosheng

Classifications

• G01N2333/914

  Hydrolases (3) · CPC title

• G01N2500/02

  Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) · CPC title

• G01N2021/6441

  with two or more labels · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/6445

  Measuring fluorescence polarisation · CPC title