Integrating Functional Response and Target Binding for Mechanism-Centered Drug Screening by High-Mass MALDI-MS

Early stage drug discovery is limited by the disjunction of function and binding assays, creating an information gap that leads to the high failure rate in hit advancement. This limitation is particularly pronounced for protein−protein interactions, whose large and shallow interfaces make it difficult to distinguish hits mechanistically. To address this, we developed a cross-linking matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) platform that integrates biochemical functional response and target binding in a single assay, thereby generating a multidimensional pharmacological profile. Using the SARS-CoV-2 RBD−ACE2 interaction and a set of 17 drug candidates for a proof-of-concept study, the platform revealed a clear difference between two inhibitors that appeared indistinguishable in conventional functional assays: one showed stronger affinity and preferential ACE2 binding, while the other showed weaker and less specific binding. These mechanistic differences were consistent with the results of a cellular antiviral assay, in which only the high-affinity inhibitor improved cell viability. This work presents a mechanism-centered, rapid screening strategy that provides early multiparameter insight, enables rational selection of high-quality leads for challenging drug targets, and is compatible with high-throughput formats.