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

ultra-High Throughput Screening using Mass Spectrometry at Pivot Park Screening Centre

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 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 or extensive assay development, and is HTS-amenable, allowing the analysis of large compound libraries.

Thrombin, a well-studied protease was used to establish an ASMS-based screening pipeline. To this end, we setup a label-based and a label-free functional biochemical, as well as a biophysical ASMS binding assay to screen the ‘Robustness Set’ compound collection against thrombin. The Robustness set collection is comprised of various classes of compounds with assay interfering properties and non-druglike mode-of-action such as autofluorescence, aggregation, chelation, chemical reactivity, and redox activity, as well as a focused chemically diverse drug-like subset for which no obvious interferences are expected. In the primary ASMS assay, a total 14 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 14 binders by comparing their binding to bovine γ-globulin (BGG) using a confirmation ASMS setup. This resulted in confirming target engagement of two compounds with inhibitory activity in the biochemical enzymatic setup and identifying one compound that selectively binds to the target, yet didn’t inhibit the
functional activity of thrombin.

The results from testing robustness set compound collection show that the ASMS setup, coupled with unspecific binders counter-screen confirmation setup is complementary to the biochemical workflows for early assessment of target engagement. Although the throughput is around 5,000 samples/wells per day, compound pooling can drastically increase the throughput of ASMS-based screening for use in primary screening of large compound libraries.

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