Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs)

Abstract

Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

High-throughput matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry–based deubiquitylating enzyme assay for drug discovery

“…This allowed the miniaturization and scalability of MALDI-TOF MS-based assays to 384 and 1,536 well formats. To date, MALDI-TOF MS-based assays have been established for a large variety of enzymes-including E3 ligases 62 , kinases 63,64 , phosphatases 65,66 , β-secretases (BACE1) 67 , histone demethylases and acetylcholinesterases 68 and cyclic GMP-AMP synthases 69 -and have been applied to the analysis of N-glycans [70][71][72] . The success of MALDI-TOF MS within HTS campaigns relies on several factors.…”

The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway

“…To address these issues, we have developed a sensitive and fast assay to quantify in vitro E2/E3 enzyme activity using MALDI-TOF MS. It builds on our DUB MALDI-TOF assay ( Ritorto et al., 2014 ), which has enabled us to screen successfully for a number of selective DUB inhibitors ( Kategaya et al., 2017 , Magiera et al., 2017 , Weisberg et al., 2017 ), and adds to the increasing number of drug discovery assays utilizing label-free high-throughput MALDI-TOF MS. Apart from E2/E3 enzymes and DUBs ( Ritorto et al., 2014 ), high-throughput MALDI-TOF MS has now successfully been used for drug discovery screening of protein kinases ( Heap et al., 2017b ), protein phosphatases ( Winter et al., 2018 ), histone demethylases, and acetylcholinesterases ( Haslam et al., 2016 ), as well as histone lysine methyltransferases ( Guitot et al., 2014 ).…”

The MALDI TOF E2/E3 ligase assay as an universal tool for drug discovery in the ubiquitin pathway

“…To address these issues, we have developed a sensitive and fast assay to quantify in vitro E2/E3 enzyme activity using MALDI TOF MS. It builds on our DUB MALDI TOF assay 32 , which has enabled us to screen successfully for a number of selective DUB 295 inhibitors 56, 57, 58 , and adds to the increasing number of drug discovery assays utilising labelfree high-throughput MALDI TOF MS. Apart from E2/E3 enzymes and DUBs 32 , highthroughput MALDI TOF MS has now successfully been used for drug discovery screening of protein kinases 59 , histone demethylases and acetylcholinesterases 60 as well as histone lysine methyltransferases 61 . 300 Unlike other current assays, all these label-free MALDI TOF MS methods use unmodified substrates, such as mono-ubiquitin.…”