Mass spectrometry-based biochemical assays for enzymeinhibitor screening

Screening for inhibitors of pharmacologically-relevant enzymes is in many cases an important starting point in drug discovery. While fluorescence-based detection techniques play an important role in high-throughput screening, mass spectrometry (MS)-based assays have gained in importance in recent years. The current review focuses on the different assay formats and applications of MS-based enzyme-inhibitor screening. MS-based assays can be categorized into direct and indirect screening methods. Direct screening methods isolate and detect the enzyme-inhibitor complex or the inhibitor to prove activity against the drug target. Active compounds that form enzyme-inhibitor complexes can be analyzed directly by MS as intact complex (direct infusion), or after isolation from the unbound (non-active) compounds by capillary isoelectric focusing (CIEF), frontal affinity chromatography (FAC), size-exclusion chromatography (SEC), immobilized enzymes or ultrafiltration (UF), and dissociation. Indirect screening methods rely on detection of reporter molecules to measure enzyme-inhibitory activity of analytes. When using native substrates as reporter molecules, the assay provides a functional response rather than relying on detection of inhibitor binding alone. Indirect screening for activity is performed by continuous-flow systems (CFSs), flow-injection analysis (FIA), high-performance liquid chromatography (HPLC) or matrix-assisted laser desorption/ionization (MALDI), coupled to MS. MS-based screening methods can screen various kinds of samples (e.g., combinatorial libraries and natural-product extracts), screen large libraries, provide activity information, mass information, structural information and affinity data, and achieve high throughputs. © 2007 Elsevier Ltd. All rights reserved.