Abstract
The primary objective of early drug development is to identify hits and leads for a target of interest. To achieve this aim, rapid, and reliable screening techniques for a huge number of compounds are needed. Mass spectrometry based binding assays (MS Binding Assays) represent a well-established technique for library screening based on competitive binding experiments revealing active sublibraries due to reduced binding of a reporter ligand and following hit identification for active libraries by deconvolution in further competitive binding experiments. In the present study, we combined the concepts of MS Binding Assays and affinity selection mass spectrometry (ASMS) to improve the efficiency of the hit identification step. In that case, only a single competitive binding experiment is performed that is in the first step analyzed for reduced binding of the reporter ligand and-only if a sublibrary is active-additionally for specific binding of individual library components. Subsequently, affinities of identified hits as well as activities of reduced sublibraries (i.e., all sublibrary components without hit) are assessed in additional competitive binding experiments. We exemplified this screening concept for the identification of ligands addressing the most widespread GABA transporter subtype in the brain (GAT1) studying in the beginning a library composed of 128 and further on a library of 1,280 well-characterized GAT1 inhibitors, drug substances, and pharmacological tool compounds. Determination of sublibraries' activities was done by quantification of bound NO711 as reporter ligand and hit identification for the active ones achieved in a further LC-ESI-MS/MS run in the multiple reaction monitoring mode enabling detection of all sublibrary components followed by hit verification and investigation of reduced sublibraries in further competitive binding experiments. In this way, we could demonstrate that all GAT1 inhibitors reducing reporter ligand binding below 50% at a concentration of 1 μM are detected reliably without generation of false positive or false negative hits. As the described strategy is apart from its reliability also highly efficient, it can be assumed to become a valuable tool in early drug research, especially for membrane integrated drug targets that are often posing problems in established screening techniques.