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.

Design Principles for Fragment Libraries: Maximizing the Value of Learnings from Pharma Fragment-Based Drug Discovery (FBDD) Programs for Use in Academia

“…35 The highest hit rate was found for biochemical screening (17%) followed by NMR (11%) and thermal shift assay (8%). It is important to note, however, that the authors neither The different assays were performed under similar conditions including the buffers.…”

Strukturbasierte Entwicklung einer Affinitätssonde für Sirtuin 2

“…Dies schränkt den Einsatz von biochemischen Aktivitätsassays,d ie auf spektroskopischen Methoden wie Fluoreszenz-oder UV/Vis-Spektroskopie beruhen, deutlich ein. [24] Biophysikalische Methoden wie die Biolayer-Interferometrie sind bezüglich Assayinterferenzen weniger stçranfällig und daher für fragmentbasiertes Screening besser geeignet. [25] Um die Eignung unserer SirReal-Sonde in Kombination mit der Biolayer-Interferometrie für fragmentbasierte Screeningansätze zu prüfen, testeten wir Fragmente von SirReal2 (2) [26] zu identifizieren und genomweite Bindungsprofile für Sirt2 durch Chem-seq [27] zu erstellen.…”

Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective

“…26–28 The use of MST is increasingly being reported in fragment-based drug discovery, 23,24,30–33 including kinases such as p38α 30 and MEK1, 31 and the bromodomain BRD9. 32 In both the MEK1 study by Sanofi in collaboration with NanoTemper and the BRD9 study by Boehringer Ingelheim, MST was used to confirm the binding of fragments identified by SPR and DSF, as well as to identify additional hits not detected by the other techniques.…”