Application of high-throughput affinity-selection mass spectrometry for screening of chemical compound libraries in lead discovery

2007 ◽  
Vol 2 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Hartmut Zehender ◽  
Lorenz M Mayr
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Chemical Compound ◽  
Lead Discovery ◽  
Affinity Selection ◽  
Compound Libraries

Solution-Based Indirect Affinity Selection Mass Spectrometry—A General Tool For High-Throughput Screening Of Pharmaceutical Compound Libraries

2014 ◽  
Vol 86 (15) ◽  
pp. 7413-7420 ◽  
Author(s):  
Thomas N. O’Connell ◽  
Jason Ramsay ◽  
Steven F. Rieth ◽  
Michael J. Shapiro ◽  
Justin G. Stroh
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Affinity Selection ◽  
Pharmaceutical Compound ◽  
Compound Libraries ◽  
General Tool

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

2021 ◽  
pp. 247255522110232
Author(s):  
Michael D. Scholle ◽  
Doug McLaughlin ◽  
Zachary A. Gurard-Levin
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Rhinovirus ◽  
Binding Potential ◽  
Affinity Selection ◽  
Response Curves ◽  
Desorption Ionization ◽  
Self Assembled

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

scholarly journals Discovery and Characterization of Orthosteric and Allosteric Muscarinic M2 Acetylcholine Receptor Ligands by Affinity Selection-Mass Spectrometry

2005 ◽  
Vol 11 (2) ◽  
pp. 194-207 ◽  
Author(s):  
Charles E. Whitehurst ◽  
Naim Nazef ◽  
D. Allen Annis ◽  
Yongmin Hou ◽  
Denise M. Murphy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Acetylcholine Receptor ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Size Exclusion ◽  
Affinity Selection ◽  
Binding Properties ◽  
Small Molecule Ligands

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

scholarly journals Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry

2011 ◽  
Vol 17 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Sue E. Hutchinson ◽  
Melanie V. Leveridge ◽  
Michelle L. Heathcote ◽  
Peter Francis ◽  
Laura Williams ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
High Throughput ◽  
Sampling Rate ◽  
Peptide Substrate ◽  
Lead Discovery ◽  
Assay Sensitivity ◽  
Histone Lysine ◽  
Histone Lysine Demethylases ◽  
Mass Spectrometry Assay

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe2+, O2, and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z′ values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein–Ligand Interactions at High Throughput

2020 ◽  
Vol 26 (1) ◽  
pp. 44-57
Author(s):  
Roman P. Simon ◽  
Martin Winter ◽  
Carola Kleiner ◽  
Lucie Wehrle ◽  
Michael Karnath ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
High Throughput ◽  
Binding Assay ◽  
Size Exclusion ◽  
Affinity Selection ◽  
Maldi Tof ◽  
Protein Ligand Interactions ◽  
Small Molecule Ligands ◽  
Ligand Interactions

Demonstration of in vitro target engagement for small-molecule ligands by measuring binding to a molecular target is an established approach in early drug discovery and a pivotal step in high-throughput screening (HTS)-based compound triaging. We describe the setup, evaluation, and application of a ligand binding assay platform combining automated affinity selection (AS)-based sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The platform enables mass spectrometry (MS)-based HTS for small-molecule target interactions from single-compound incubation mixtures and is embedded into a regular assay automation environment. Efficient separation of target–ligand complexes is achieved by in-plate size exclusion chromatography (SEC), and small-molecule ligands are subsequently identified by MALDI-TOF analysis. In contrast to alternative HTS-capable binding assay formats, MALDI-TOF AS-MS is capable of identifying orthosteric and allosteric ligands, as shown for the model system protein tyrosine phosphatase 1B (PTP1B), irrespective of protein function. Furthermore, determining relative binding affinities (RBAs) enabled ligand ranking in accordance with functional inhibition and reference data for PTP1B and a number of diverse protein targets. Finally, we present a validation screen of more than 23,000 compounds within 24 h, demonstrating the general applicability of the platform for the HTS-compatible assessment of protein–ligand interactions.

scholarly journals A Chemoinformatics Analysis of Hit Lists Obtained from High-Throughput Affinity-Selection Screening

2005 ◽  
Vol 11 (2) ◽  
pp. 123-130 ◽  
Author(s):  
Nathan Brown ◽  
Hartmut Zehender ◽  
Kamal Azzaoui ◽  
Ansgar Schuffenhauer ◽  
Lorenz M. Mayr ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Size Exclusion ◽  
Label Free ◽  
Affinity Selection ◽  
High Performing ◽  
Exclusion Chromatography ◽  
Screening Platform ◽  
Screening Technology

The high-throughput affinity-selection screening platform SpeedScreen was recently reported by the Novartis Institutes for BioMedical Research as a homogeneous, label-free screening technology with mass-spectrometry readout. SpeedScreen relies on the screening of compound mixtures with various target proteins and uses fast size-exclusion chromatography to separate target-bound from unbound substances. After disintegration of the target-binder complex, the binder molecules are identified by their molecular masses using liquid chromatography/mass spectrometry. The authors report an analysis of the molecular properties of hits obtained with SpeedScreen on 26 targets screened within the past few years at Novartis using this technology. Affinity-based SpeedScreen is a robust high-throughput screening technology that does not accumulate frequent hitters or potential covalent binders. The hits are representative of the most commonly identified scaffold classes observed for known drugs. Validated SpeedScreen hits tend to be enriched on more lipophilic and larger-molecular-weight compounds compared to the whole library. The potential for a reduced SpeedScreen screening set to be used in case only limited protein quantities are available is evaluated. Such a reduced compound set should also maximize the coverage of the high-performing regions of the chemical property and class spaces; chemoinformatics methods including genetic algorithms and divisive K-means clustering are used for this aim.

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