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

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. One question, multiple answers: Fragment‐based lead discovery depends on structural information about target–fragment complexes and, therefore, on reliable screening methods used to select a subset of small molecules that are analyzed by X‐ray crystallography. A comprehensive screening campaign on a single protease target, however, revealed a disappointingly low overlap between the results of the six applied assays with no single common hit.