Combination of MS Binding Assays and affinity selection mass spectrometry for screening of structurally homogenous libraries as exemplified for a focused oxime library addressing the neuronal GABA transporter 1
Abstract
This study presents an efficient screening approach based on combination of mass spectrometry (MS) based binding assays (MS Binding Assays) and affinity selection mass spectrometry (ASMS) customized for screening of structurally homogeneous libraries sharing a common mass spectrometric fragmentation pattern. After reaction of a nipecotic acid derivative possessing a hydroxylamine functionality with aldehydes, the resulting oxime library was screened accordingly toward the GABA transporter subtype 1 (GAT1), a drug target for several neurological disorders. After assessing sublibraries' activities for inhibition of reporter ligand binding, hits in active ones were directly identified. This could be achieved by recording mass transitions for the reporter ligand as well as those predicted for the library components in a single LC-MS/MS run with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Identification of hits with a predefined affinity could be reliably accomplished by calculation of IC 50-values from specific binding concentrations of library constituents and reporter ligand. Application of this strategy revealed six hits, from which two of them were resynthesized for further biological evaluation. Thereby, the best one displayed a pK i of 7.38 in MS Binding Assays and a pIC 50 of 6.82 in [3 H]GABA uptake assays for GAT1.
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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 [81]
Gabriel 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 [81]
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- Authors
- Gabriel, J., Höfner, G., & Wanner, K. T.
- Affiliations
- Ludwig-Maximilians-Universität München (DE)
- Corresponding author
- Wanner, Klaus T.
- Venue
- European Journal of Medicinal Chemistry
- Publisher
- Elsevier BV
- Published
- 2020-11
- Volume
- 206
- Pages
- 112598
- ISSN
- 0223-5234 (ISSN-L)
- OpenAlex
- W3045935278
- Cited by
- 3
- References
- 42
- Citations by year
- 2025: 1 · 2024: 2
- FWCI
- 0.00
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- European Journal of Medicinal Chemistry (publishedVersion)
- PubMed (publishedVersion)
- 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 › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
- Keywords
- GABA transporter 1, affinity selection-mass spectrometry, high-throughput screening, GABA uptake inhibition, hit identification, multiple reaction monitoring
- MeSH terms
- Drug Evaluation, Preclinical, Humans, Ligands, Neurons, Oximes, Protein Binding, Mass Spectrometry, GABA Plasma Membrane Transport Proteins
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