Introduction
Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery.
Areas Covered
This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described.
Expert opinion
Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.
Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then the complex separated from free protein allowing direct determination of bound to free ratios. Although possessing many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain non-covalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS-complexes. The method gives good quantitative results, e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.
Tools for measuring affinities and stoichiometries of protein-protein complexes are valuable for elucidating the role of protein-protein interactions (PPIs) in governing cell functions and screening for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and include stoichiometries from dimers to high order oligomers. Also, most techniques require large amounts of protein which can hamper research for difficult to obtain proteins. Protein cross-linking capillary electrophoresis (PXCE) has the potential to directly measure PPIs and even resolve multiple PPIs while consuming attomole quantities. Previously PXCE has only been used for high affinity, 1 : 1 complexes; here we expand the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. Use of glutaraldehyde as the cross-linking agent was key to advancing the method because of its rapid reaction kinetics. A 10 s reaction time was found to be sufficient for cross-linking and quantification of seven different PPIs with Kd values ranging from low μM to low nM including heat shock protein 70 (Hsp70) interacting with heat shock organizing protein (3.8 ± 0.7 μM) and bcl2 associated anthanogene (26 ± 6 nM). Non-specific cross-linking of protein aggregates was found to be minimal at protein concentrations <20 μM as assessed by size exclusion chromatography. PXCE was sensitive enough to measure changes in PPI affinity induced by the protein nucleotide state or point mutations in the protein-binding site. Further, several interactions could be resolved in a single run, including Hsp70 monomer, homodimer and Hsp70 complexed the with c-terminus of Hsp70 interacting protein (CHIP). Finally, the throughput of PXCE was increased to 1 min per sample suggesting potential for utility in screening.
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