Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions

Abstract

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 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 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to 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.

Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

“…4 We found adsorption of Bag3 to be a persistent problem, even at pH 10, so that no discernible complex peaks were observed in samples containing Hsp70-488 and Bag3 when using free solution CE (Figure S-1A). Bag3 adsorption is likely a result of it being intrinsically disordered which is common with PPI targets.…”

“…In APCE, an equilibrated mixture of binding partners is electrophoresed to allow for detection of non-covalent interactions. 1–3 APCE has been used to investigate many biomolecular interactions such as protein-protein 4–7 , antibody-antigen 8–13 , protein-DNA 10,14–17 , protein-peptide 10,18 and protein-aptamer 19–21 . Typically, one binding partner is fluorescently labeled enabling sensitive detection by laser induced fluorescence (LIF).…”

“…Strategies to minimize protein-wall interactions include capillary derivatization, 4,15,16,19 extreme pH, 22,23 surfactant additives 24 and high ionic strength buffers. 25,26 Techniques to minimize protein adsorption to the capillary are often not compatible with maintaining non-covalent protein interactions or require optimization for each protein binding partner of interest.…”

Stabilizing the Hsp70-Tau Complex Promotes Turnover in Models of Tauopathy

“…Procedure is adapted from previously reported methods (Rauch et al, 2013). Biotinylated Hsc70 was immobilized on polystyrene streptavidin coated beads (Spherotech), incubated with Alexa-Fluor 488 labeled NEF (50 nM) and increasing amounts of compound in buffer A (25 mM HEPES, 5 mM MgCl 2 , 10 mM KCl, 0.03% Tween-20 pH 7.5).…”

Capillary Electrophoresis for Quantitative Studies of Biomolecular Interactions

“…53 The Bag3-Hsp70 pair is known to stabilize a number of key oncogenes that makes this complex a promising anticancer target. The NECEEM method yielded K d = 23 nM for the Hsp70-Bag3 complex.…”