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ChemMedChem. 2015 Sep;10(9):1511-21. doi: 10.1002/cmdc.201500267. Epub 2015 Aug 10.

One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists.

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Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).
Proteros Biostructures, Bunsenstr. 7a, 82152 Martinsried (Germany).
NovAliX, BioParc, Blvd. Sébastien Brant BP 30170, 67405 Illkirch (France).
Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Straße 65, 88397 Biberach/Riß (Germany).
NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich (Germany).
Department of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35032 Marburg (Germany).


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.


comparative analysis; endothiapepsin; fragment-based drug discovery; inhibitors; screening methods

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