Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.
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.
Department of Biological Chemistry, ‡Department of Chemistry, and the§ Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.
Jennifer N
JN
Rauch
Jing
J
Nie
Tonia J
TJ
Buchholz
Jason E
JE
Gestwicki
Robert T
RT
Kennedy
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D016477:Artifacts; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C414431:BAG3 protein, human; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D054852:Small Molecule Libraries