A Library Screening Strategy Combining the Concepts of MS Binding Assays and Affinity Selection Mass Spectrometry

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.

Solving the Competitive Binding Equilibria between Many Ligands: Application to High-Throughput Screening and Affinity Optimization

“…The main limitation, aside from requiring a suitable known ligand, is that it does not inform which ligand (or ligands) in a given pool is the strongest binder, for which additional experiments are needed (see below). A similar approach has recently been reported by Gabriel et al, where they set a 50% reduction in reporter binding as the cutoff, to identify pools containing binders to GAT1, a membrane-bound target . HAMS (high-affinity MS screening) allows the identification ligands with “very high affinity” from a pool.…”

“…Finding the optimal threshold for hit identification and, if the number of hits is relatively high, how to rank the hits for further follow-up remain a critical challenge for HTS methods. In MS-based HTS, the relative signal intensity for different compounds is not simply proportional to their relative concentrations because the ionization efficiency of every molecule is different and generally unknown. , Therefore, it is not possible to produce a ranking from a single AS–MS experiment, though a separate negative control experiment (e.g., with an unrelated target protein) may be used to eliminate some false positives. , A summary of current AS–MS approaches is presented in Table .…”

Development of a High-Throughput Affinity Mass Spectrometry (AMS) Platform Using Laser Diode Thermal Desorption Ionization Coupled to Mass Spectrometry (LDTD-MS)

“…AMS is a true label-free platform where direct detection of an analyte in the presence of a target protein is possible. The appeal of AMS technology has led to multiple reports of the implementation of AMS platforms in industry or in academia, 35,49,50 either as an orthogonal screening approach or as an orthogonal assay to confirm hits identified by activity-based screens. At Amgen, we have employed Agilent’s RF-MS technology for small-molecule AMS.…”

Affinity selection–mass spectrometry for the discovery of pharmacologically active compounds from combinatorial libraries and natural products

“…As an example of a target not accessible using conventional HTS approaches, Gabriel et al 41 used a variation of PUF AS‐MS to screen for ligands to membrane‐bound GABA transporter GAT1, which is a therapeutic target for a variety of disorders including neuropathic pain and depression. PUF AS‐MS has even been applied to the screening of cell organelles like mitochondria for ligands 42 .…”

“…33 Anticancer targets have included retinoid X receptor (RXR)-α, 34 quinone reductase-2, 22 and urokinasetype plasminogen activator. 35 Other PUF AS-MS assays have been reported for targets such as estrogen receptor (ER)-α and ER-β, 13,36,37 human serum albumin, 38 thrombin, 39 and α-glucosidase, 15,24,25 For a review of recent applications of PUF AS-MS to natural product screening, see Han et al 40 As an example of a target not accessible using conventional HTS approaches, Gabriel et al 41 used a variation of PUF AS-MS to screen for ligands to membrane-bound GABA transporter GAT1, which is a therapeutic target for a variety of disorders including neuropathic pain and depression. PUF AS-MS has even been applied to the screening of cell organelles like mitochondria for ligands.…”