Discovering ERAP1 Inhibitors with MALDI-TOF MS

The dysregulation of endoplasmic reticulum aminopeptidase 1 (ERAP1) protein has been linked to cancer and both autoinflammatory and autoimmune diseases – making it a conspicuous therapeutic target for drug discovery. Existing high-throughput screening campaigns – such as fluorescence and chemiluminescence methods – are used to target specific proteins in a sea of interfering compounds. Unfortunately, these labed-based methods are susceptible to artifacts, and can often result in perturbed biology. 

A group of UK researchers decided to tackle these challenges by developing a high-throughput MALDI-TOF mass spectrometry (MS) assay to screen for small molecule inhibitors of ERAP1 (1). They based their assay on the existing in vitro RapidFire MS (RF MS) assay for greater reproducibility and robustness for the platform. The label-free nature of the assay was particularly valuable in the case of ERAP1, considering enzyme activity is mediated by the substrate’s structural features. 

The team discovered that the previous RF MS peptides – YTAFTIPSI substrate trimmed into the product TAFTIPSI – had low ionization efficiency and assay stability. Therefore, arginine residues were added into the peptide sequence to increase the limit and linearity of detection. The assay conditions were optimized and then validated with established ERAP1 inhibitors that targeted the active side of the enzyme.

Using their novel MALDI-TOF MS process, the researchers tested a set of 699 ERAP1 binders that had been previously identified through an affinity selection mass spectrometry screen. They first tested the compounds at a single concentration, before moving onto a dose-response format. The team compared the collection of compounds found using the new setup with the existing RF MS setup, noting that both platforms delivered similar results; however, the authors state that their MALDI-TOF MS setup saw additional benefits, such as shorter sample cycle times, lower reagent consumption, and a lower tight-binding limit – making it an effective candidate for ERAP1 screening.