Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.

Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370536 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6882 (Electronic) Linking ISSN: 00032700 NLM ISO Abbreviation: Anal Chem Subsets: MEDLINE

Original Publication: Washington, American Chemical Society.

Adaptor Proteins, Signal Transducing/*metabolism ; Apoptosis Regulatory Proteins/*metabolism ; Drug Evaluation, Preclinical/*methods ; Electrophoresis, Capillary/*methods ; HSP70 Heat-Shock Proteins/*metabolism; Artifacts ; Fluorescent Dyes/chemistry ; HSP70 Heat-Shock Proteins/chemistry ; Humans ; Protein Binding/drug effects ; Small Molecule Libraries/pharmacology

Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods.

Annu Rev Biochem. 2009;78:959-91. (PMID: 19298183)
Electrophoresis. 2003 Jan;24(1-2):101-8. (PMID: 12652579)
Cell. 2005 Sep 23;122(6):957-68. (PMID: 16169070)
ACS Chem Biol. 2012 Aug 17;7(8):1311-20. (PMID: 22725693)
Curr Protoc Cytom. 2010 Jan;Chapter 13:Unit 13.11.1-15. (PMID: 20069525)
Anal Chem. 1991 Jun 1;63(11):1126-32. (PMID: 1883070)
Anal Chem. 2008 Jul 1;80(13):5225-31. (PMID: 18465881)
J Biol Chem. 2013 Mar 8;288(10):6980-90. (PMID: 23341456)
Expert Rev Mol Med. 2012 Jul 26;14:e16. (PMID: 22831787)
J Am Chem Soc. 2008 Jan 9;130(1):34-5. (PMID: 18072778)
Chem Biol Drug Des. 2013 Jan;81(1):22-32. (PMID: 23253128)
J Biomol Screen. 1999;4(2):67-73. (PMID: 10838414)
Anal Chem. 1994 Jan 1;66(1):9-15. (PMID: 8116876)
Nature. 2007 Dec 13;450(7172):1001-9. (PMID: 18075579)
J Biol Chem. 2009 Apr 3;284(14):9176-83. (PMID: 19179333)
Curr Pharm Des. 2013;19(3):404-17. (PMID: 22920901)
Anal Chem. 2007 Feb 15;79(4):1690-5. (PMID: 17297974)
J Chromatogr A. 2004 Jun 25;1040(2):273-82. (PMID: 15230534)
Anal Chem. 1998 Nov 15;70(22):4761-70. (PMID: 9844572)
Electrophoresis. 2003 May;24(9):1375-82. (PMID: 12731022)
Anal Chem. 2005 Jan 15;77(2):564-72. (PMID: 15649054)
Curr Opin Chem Biol. 2009 Jun;13(3):284-90. (PMID: 19553156)
J Biol Chem. 2010 Jul 9;285(28):21282-91. (PMID: 20439464)
Biochemistry. 2011 Apr 5;50(13):2394-402. (PMID: 21332192)
Cell. 2011 Mar 18;144(6):986-98. (PMID: 21414488)
J Clin Endocrinol Metab. 2007 Mar;92(3):1159-63. (PMID: 17164298)
FEBS Lett. 2009 Jan 22;583(2):401-6. (PMID: 19111544)
J Biomol Screen. 2010 Dec;15(10):1211-9. (PMID: 20926844)
J Chromatogr B Biomed Appl. 1996 Aug 9;683(1):77-84. (PMID: 8876441)
J Biol Chem. 2004 Dec 31;279(53):56053-60. (PMID: 15509580)
Anal Biochem. 2012 Jun 15;425(2):104-12. (PMID: 22446499)
Mol Pharmacol. 2007 Jan;71(1):169-75. (PMID: 17012620)
Anal Chem. 2008 May 1;80(9):3105-11. (PMID: 18366190)
Electrophoresis. 1997 Jan;18(1):112-7. (PMID: 9059831)
J Biol Chem. 1999 Jan 8;274(2):781-6. (PMID: 9873016)
Am J Pathol. 2011 Jun;178(6):2504-12. (PMID: 21561597)
J Chromatogr A. 1994 Oct 7;680(2):395-403. (PMID: 7981823)
Anal Biochem. 1995 May 1;227(1):186-94. (PMID: 7668380)
Chem Rev. 1999 Oct 13;99(10):3081-132. (PMID: 11749511)
Chem Biol. 2011 Feb 25;18(2):210-21. (PMID: 21338918)
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2256-61. (PMID: 9482872)
J Chromatogr A. 1994 Oct 7;680(2):405-12. (PMID: 7526936)
Anal Chem. 1998 Nov 1;70(21):4540-5. (PMID: 9823713)
Electrophoresis. 2006 Jan;27(1):44-59. (PMID: 16315182)
Curr Opin Chem Biol. 2010 Jun;14(3):315-24. (PMID: 20417149)
Biochemistry. 1994 Mar 8;33(9):2561-7. (PMID: 8117717)
Nature. 2005 Oct 20;437(7062):1173-8. (PMID: 16189514)
Electrophoresis. 2006 May;27(10):1886-94. (PMID: 16703627)
Arch Biochem Biophys. 2011 Sep 1;513(1):1-9. (PMID: 21767525)
Br J Pharmacol. 2009 Nov;158(5):1405-12. (PMID: 19681889)
Anal Chem. 2010 Nov 15;82(22):9261-7. (PMID: 20949899)
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Feb 25;768(1):199-210. (PMID: 11939553)

GM102236 United States GM NIGMS NIH HHS; R01 NS059690 United States NS NINDS NIH HHS; NS095690 United States NS NINDS NIH HHS; R01 GM102236 United States GM NIGMS NIH HHS; F32 NS095690 United States NS NINDS NIH HHS

0 (Adaptor Proteins, Signal Transducing)
0 (Apoptosis Regulatory Proteins)
0 (BAG3 protein, human)
0 (Fluorescent Dyes)
0 (HSP70 Heat-Shock Proteins)
0 (Small Molecule Libraries)

Date Created: 20130925 Date Completed: 20140819 Latest Revision: 20211021

20221213

PMC3845662

10.1021/ac4023082

24060167