Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits: A Case Study

Fragment-based lead discovery (FBLD) has become a pillar in chug development. Typical, applications, of this Method,comprise at least two, biophysical screens as, prefilter and a follow-up crystallographic experiment on a subset of fragments. structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment bound structures. We therefore Set out to screen 361 fragments for binding to endothiapepsin,,a representative of the challenging group of aspartic-proteases, employing Six screening techniques and Crystallography in parallel. Crystallography resulted in the very high number Of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening:cascade, building on the results from two or more screening methods, would have failed to -predict at least 73%;of these bits. We thus conclude that, at least in the present case, the frequently applied biophysical preScreening filters deteriorate, the number of possible X-ray hits,while only the immediate use of crystallography enables exhaustive: retrieval Of a-maximum offragment structures, which represent a:rich:source guiding 'hit-to-lead-to-drug evolution.