Update: Target engagement assessment of drug-like and pan-assay interfering compounds using Affinity Selection Mass Spectrometry

Affinity Selection Mass Spectrometry whitepaper update

Pivot Park Screening Centre (PPSC) has adopted Affinity Selection Mass Spectrometry (ASMS) workflow into its daily high-throughput screening (HTS) operations. ASMS 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 complexes from unbound compounds by in-plate size exclusion chromatography (SEC), followed by establishing bound ligand identity using MS. In contrast to other conventional biophysical assays (like SPR, MST, and TSA), ASMS does not require mobilization or extensive assay development, it is label-free  , and is HTS-amenable, allowing the analysis of large compound libraries.

Thrombin, a well-studied protease, was used to establish and validate an ASMS-based screening pipeline. To this end, we setup a fluorogenic biochemical assay, a label-free MALDI-TOF MS functional biochemical assay, and a biophysical ASMS binding assay to screen our in-house ‘Robustness  set’ compound collection against thrombin. The Robustness set collection comprises  various classes of compounds with assay interfering properties and non-drug-like mode-of-action such as autofluorescence, aggregation, chelation, chemical reactivity, and redox activity, as well as a chemically diverse drug-like subset for which no obvious interferences are expected. In the primary ASMS assay, five compounds were identified as potential binders to thrombin, of which only two were identified as inhibitors of the functional enzymatic activity. We examined the specificity of the five binders by comparing their binding to bovine γ-globulin (BGG) using a counter-screen  ASMS assay setup. This resulted in confirming target engagement of two compounds with inhibitory activity in both biochemical enzymatic setups and identifying one compound that selectively binds to thrombin, albeit without functional activity.

The results from testing the Robustness set compound collection show that the ASMS compliments hit discovery and triaging workflows by enabling early assessment of target engagement. The throughput for ASMS screening of single compound libraries is around 5,000 samples per day and about 250,000 per day for pooled compound libraries.


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