A Library Screening Strategy Combining the Concepts of MS Binding Assays and Affinity Selection Mass Spectrometry
Open accessAbstract
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.
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The experimental set-up combining denaturation, LC-MS, and removal of nonspecific binders facilitated the determination of new potential binders.
“By using denaturation of target to liberate ligands in both experiments, determining the compound composition by LC-MS, and subsequently removing nonspecific binders from the total binding experiment, this experimental set-up facilitated the determination of new potential binders.”
From Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products (2024) · cites this paper as [80]
The Introduction (page unavailable) says bound ligands were liberated from the target, analyzed by LC-ESI-MS, and identified as hits by comparing total with non-specific binding. Materials and Methods (MS Binding Assay; page unavailable) describes elution with methanol, but our copy does not say that target denaturation was the means of liberation. (LLM, conf=0.82)
Experimental — accuracy not yet measuredGabriel et al. combined MS binding assays with affinity-selection MS for an activity-based drug discovery screen.
“These classical MS methods are most commonly used for the investigation of enzyme kinetics; however, Gabriel et al. recently combined MS binding assays with affinity-selection MS for an activity-based drug discovery screen.80,81”
From Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products (2024) · cites this paper as [80]
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- Authors
- Gabriel, J., Höfner, G., & Wanner, K. T.
- Affiliations
- Ludwig-Maximilians-Universität München (DE)
- Corresponding author
- Wanner, Klaus T.
- Venue
- Frontiers in Chemistry
- Publisher
- Frontiers Media SA
- Published
- 2019-10-04
- Volume
- 7
- Pages
- 665-665
- ISSN
- 2296-2646 (ISSN-L)
- PMC
- PMC6787468
- OpenAlex
- W2978958386
- Cited by
- 20
- References
- 52
- Citations by year
- 2025: 4 · 2024: 2 · 2023: 4 · 2022: 2 · 2021: 3 · 2020: 5
- FWCI
- 1.00
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Chemistry (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Open access LMU (Ludwid Maxmilian's Universitat Munchen) (submittedVersion, cc-by)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- affinity selection-mass spectrometry, compound library screening, hit identification, competitive binding, GABA transporter 1, early drug development
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