Abstract

Correct identification of targets and mechanism of action (MoA) are bottlenecks and keys in drug development. For phenotype-based approaches, our chemical proteomics methods can perform MS-based and proteome-wide analyses of cell culture and / or lysates samples treated by any molecule, deciphering how any small molecule or treatment works in biological systems, also with multiplexed comparisons of analogues and integrated orthogonal approaches in one assay. Our full proteome profiling analyses with unbiased, MS-based proteomics at the throughput, sustainability, depth, confidence and sensitivity needed in drug development overcome limitations of the affinity-based approaches, with no need of modified ligands. Our recent PISA assay identifies targets and early MoA proteins as solubility-shifted proteins in a 30-60 min treatment, avoiding relying only on late cell responses, and overcoming limitations of using thermal proteome profiling (TPP). PISA can multiplex in one assay more analogs at various concentrations (up to five treatments plus control, each in triplicate), dramatically minimizes costs, analysis time, missing values. PISA increases throughput (10–100-fold higher than TPP), number of replicates, proteome depth (~10,000 protein IDs), and avoids potentially incorrect “protein melting” interpretations, also with miniaturized sample amounts (e.g., primary cells and iPSCs). PISA allows integrating orthogonal methods in one assay: PISA-Express combines PISA with global protein amount changes for cell proteome regulation at later times; PISA-REX adds to the above the reduction-oxidation changes; PISA-SIESTA enables high throughput identification of substrates of enzymes; ResT-PISA assay measures drug residence time on targets and off-targets, thus enabling more accurate prediction of drug in vivo efficacy and toxicity. For target-based approaches we provide H/D exchange MS (HDX-MS) to map the binding site and conformational changes upon ligand binding.