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

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


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.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartic Acid Endopeptidases / antagonists & inhibitors
  • Aspartic Acid Endopeptidases / metabolism
  • Biochemistry / methods*
  • Biophysics / methods*
  • Drug Discovery / methods
  • High-Throughput Screening Assays / methods*
  • Magnetic Resonance Spectroscopy
  • Protease Inhibitors / chemistry
  • Protease Inhibitors / pharmacology
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology
  • Spectrometry, Mass, Electrospray Ionization / methods


  • Protease Inhibitors
  • Small Molecule Libraries
  • Aspartic Acid Endopeptidases
  • Endothia aspartic proteinase