title: One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists.
abstract: Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Johannes
authors.initials: J
authors.lastname: Schiebel
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Nedyalka
authors.initials: N
authors.lastname: Radeva
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Helene
authors.initials: H
authors.lastname: Köster
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Alexander
authors.initials: A
authors.lastname: Metz
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Timo
authors.initials: T
authors.lastname: Krotzky
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Maren
authors.initials: M
authors.lastname: Kuhnert
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Wibke E
authors.initials: WE
authors.lastname: Diederich
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
authors.forename: Andreas
authors.initials: A
authors.lastname: Heine
authors.affiliation: Proteros Biostructures, Bunsenstr. 7a, 82152 Martinsried (Germany).
authors.forename: Lars
authors.initials: L
authors.lastname: Neumann
authors.affiliation: NovAliX, BioParc, Blvd. Sébastien Brant BP 30170, 67405 Illkirch (France).
authors.forename: Cedric
authors.initials: C
authors.lastname: Atmanene
authors.affiliation: NovAliX, BioParc, Blvd. Sébastien Brant BP 30170, 67405 Illkirch (France).
authors.forename: Dominique
authors.initials: D
authors.lastname: Roecklin
authors.affiliation: NovAliX, BioParc, Blvd. Sébastien Brant BP 30170, 67405 Illkirch (France).
authors.forename: Valérie
authors.initials: V
authors.lastname: Vivat-Hannah
authors.affiliation: NovAliX, BioParc, Blvd. Sébastien Brant BP 30170, 67405 Illkirch (France).
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Renaud
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65, 88397 Biberach/Riß (Germany).
authors.forename: Robert
authors.initials: R
authors.lastname: Meinecke
authors.affiliation: NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich (Germany).
authors.forename: Nina
authors.initials: N
authors.lastname: Schlinck
authors.affiliation: NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich (Germany).
authors.forename: Astrid
authors.initials: A
authors.lastname: Sitte
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65, 88397 Biberach/Riß (Germany).
authors.forename: Franziska
authors.initials: F
authors.lastname: Popp
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65, 88397 Biberach/Riß (Germany).
authors.forename: Markus
authors.initials: M
authors.lastname: Zeeb
authors.affiliation: Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany). klebe@mailer.uni-marburg.de.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Klebe
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D001703:Biophysics / Q000379:methods*; D055808:Drug Discovery / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D011480:Protease Inhibitors; D054852:Small Molecule Libraries; D016282:Aspartic Acid Endopeptidases; C042466:Endothia aspartic proteinase
keywords: comparative analysis; endothiapepsin; fragment-based drug discovery; inhibitors; screening methods