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.
Native mass spectrometry coupled to ion mobility (IM-MS)
has become
an important tool for the investigation of protein structure and dynamics
upon ligand binding. Additionally, collisional activation or collision
induced unfolding (CIU) can further probe conformational changes induced
by ligand binding; however, larger scale screens have not been implemented
due to limitations associated with throughput and sample introduction.
In this work we explore the high-throughput capabilities of CIU fingerprinting.
Fingerprint collection times were reduced 10-fold over traditional
data collections through the use of improved smoothing and interpolation
algorithms. Fast-CIU was then coupled to a droplet sample introduction
approach using 40 nL droplet sample volumes and 2 s dwell times at
each collision voltage. This workflow, which increased throughput
by ∼16-fold over conventional nanospray CIU methods, was applied
to a 96-compound screen against Sirtuin-5, a protein target of clinical
interest. Over 20 novel Sirtuin-5 binders were identified, and it
was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5
gas-phase conformations. This work demonstrates that droplet-CIU can
be implemented as a high-throughput biophysical characterization approach.
Future work will focus on improving the throughput of this workflow
and on automating data acquisition and analysis.
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