Too weak for biophysics: Prioritising and progressing fragment hits from X-ray crystallographic screening.

Thesis

In the last decade, X-ray crystallography has matured to a powerful primary screening technique for fragment hit identification in structure-based drug discovery. Unmatched in its sensitivity, it provides valuable knowledge of precise fragment binding location in a protein-fragment complex. The comparison to ¹⁹F NMR fragment campaign conducted in this work emphasises the strength of crystallographic screening by revealing additional binding sites and interactions for the breast cancer target NUDT5.

The high sensitivity of X-ray crystallography allows to identify even weakly binding fragment hits, which are often undetectable by commonly used biophysical methods. The lack of orthogonal tools with sufficient detection limits to quantify their interactions impedes prioritisation of fragment hits for elaboration. For this purpose, the use of both NMR-based techniques as well as concentration-dependent crystallographic soaking are explored. While further investigation of relaxation-based T_1rho NMR for rapid ranking of weak binders is necessary, the crystallographic approach demonstrates a correlation between the lowest detectable soaking concentration (LDSC) and fragment binding affinity. A low-concentration counter-soak is proposed as a simple solution to triage fragment hits according to their binding strengths, streamlining their selection for further elaboration.

Weak fragment hits can be informative for lead discovery: they can reveal unknown binding sites and can be progressed into more potent compounds. For the target NUDT5, crystallographic screening identified a novel, previously unreported binding pocket. The elaboration efforts utilising SAR by catalogue approach and crystallographic soaking as readout highlight the value of crystallography for the progression of weak binders in absence of other assays.

Authors: Dubianok, Y

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: crystallographic soaking; NUDT5; fragment-based drug dicovery; crystallographic fragment screening; fragment elaboration
• Subjects: protein crystallography; structural biology; drug discovery