Target engagement is the cornerstone for validating hits identified in HTS campaigns. Pivot Park Screening Centre can rapidly profile target engagement and residence time through kinetic cellular read-outs as well as biophysical assays such as Affinity Selection Mass Spectrometry (ASMS), Thermal Shift Assay (TSA) and Surface Plasmon Resonance (SPR), providing a more refined hit validation.
Hit compounds worth pursuing
A major challenge in evaluating hits is to select hit compounds worth pursuing as a chemical scaffold during drug discovery. The success of HTS campaigns depends on post-screen hit-triaging , because of undesired compound-assay interference leading to false-positive data. These could be categorized into technical false-positives (e.g. quenchers, auto-fluorescence, inhibitors of secondary enzymatic reactions) and hits with undesired modes-of-action (chelators, aggregators, redox, etc.). Thus, a comprehensive hit triaging cascade of assays including counter-screening, deselection, orthogonal, selectivity as well as target engagement biophysical assays such as TSA, MST, ITC, SPR are required.
Follow-up assays are either dependent on fluorescent labels and therefore minimally de-risk the identified hits or require large amounts of reagents and are generally low in throughput and laborious. The hit confirmation phase of a conventional drug discovery project requires ~1.5 years. The above-mentioned secondary assays tailored for the project requirements also drive about 85% of the cost of hit confirmation.
We have recently adopted Affinity Selection Mass Spectrometry (ASMS) workflow into our daily high-throughput screening (HTS) operations. ASMS basically is a biophysical method to study protein-ligand interactions using mass spectrometry (MS) in microtiter plates. High-throughput ASMS is based on separation of protein-ligand bound complexes from unbound compounds achieved by in-plate size exclusion chromatography (SEC), followed by identifying the ligands using MS. In contrast to other conventional biophysical assays, ASMS is label-free, does not require mobilization, and is HTS-amenable, allowing the analysis of large compound libraries. Currently, the methodology is used for early target engagement assessment post uHTS campaigns. Pivot Park Screening Centre actively develops the technology towards both compound pooling and biophysical primary screening applications.
Our experience in biophysical assay development and expertise in interpreting the data from hit biophysical characterisation ensures direct interaction of hits with target proteins and assists with chemotype prioritisation.
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