Abstract

High-throughput screening (HTS) outputs routinely contain false
positive hits caused by samples acting via undesirable mechanisms of action.
Pursuit of these ’hits’ can result in wasted time and resource, making their
early identification highly desirable. Redox cycling compounds (RCCs) produce
reactive oxygen species, such as H₂O₂, which can damage
protein function and falsely appear as hits. The high throughput assays available
to identify RCCs in are indirect, have limited sensitivity or are prone to
flagging false positives. The direct measurement of tris(2-carboxyethyl)phosphine
(TCEP) oxidation was demonstrated to be a sensitive and accurate measure of
redox cycling. However, the current NMR based detection method is not
compatible with the magnitude required for triage of an HTS campaign (Tarnowski et al. 2018). The development of Acoustic Mist Ionisation MS (AMI-MS) has made it possible
to rapidly measure oxidation of TCEP at large scale. AMI-MS uses acoustic energy to fire nanolitre
volumes of ionised sample directly from microtitre plates into the mass
spectrometer, allowing for multiple ion species to be measured rapidly and
simultaneously. Utilising this technique we are now able to accurately flag RCCs
that catalyse the oxidation of TCEP, in a high throughput manner (2 samples
per second). Applying this assay to HTS outputs revealed that 80% of hits from an
Open Innovation project and 18% of hits from a dehyrdogenase target were
capable of redox cycling. Incorporating this triaging assay into our standard
HTS screening cascade has facilitated the delivery of high-quality data that
facilitates faster prioritisation of robust lead series.