Development of high-throughput mass spectrometry modalities to enhance drug discovery in immunology

Abstract

Mass spectrometry (MS) is a powerful analytical technique, enabling unbiased and comprehensive analysis of analytes without requiring potentially disadvantageous chemical modifications that are used in label-based methods traditionally relied on for instance in high throughput (HT) screening. In this thesis, I will showcase the advantages of different MS approaches which provide increased throughput suitable for early drug discovery. I will apply these approaches to the immunology field and establish workflow performance by comparison to established techniques. First, I developed a matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS based biochemical drug discovery assay to identify inhibitors of ERAP1, an antigen modulator and therapeutic target for immuno-oncology and auto-immune diseases. With equal performance, this workflow increased throughput, enabled miniaturisation, and reduced costs compared to an established MS workflow. I addressed the demand to improve the translation of early drug candidates into the clinic by developing an innovative MALDI-TOF MS-based phenotypic screening assay, utilising human induced pluripotent stem cell-derived macrophages to study cell activation. The assay consistently distinguished between resting and pro-inflammatory macrophages, and successfully identified polarisation-specific “biomarkers”. A screen of 87 broad mechanism of action compounds demonstrated assay robustness. Screening of 86 inflammation-focused compounds identified 21 hits which correlated with an established label-based cytokine secretion assay. Complementary proteomics analysis provided insights into hit compounds mechanism of action and determined undesirable off target effects. In summary, I established two novel HT capable screening applications that leverage MALDI TOF MS. For both, biochemical and cellular applications, I demonstrated their power to support early drug discovery in immunology. Additionally, I showed the utility of proteomics for early drug discovery by functionally grouping compounds at the molecular level based on on-target and off-target pathway engagement. Further adoption of these MS techniques promises to enhance hit compound selection efficacy in early drug discovery.