Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery

Abstract

In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.drug design | hot spots | protein-ligand interactions

Le criblage de fragments

“…En général, le criblage de fragments est réalisé avec une première technique, puis confirmé par une seconde approche. D'autres techniques ont aussi été utilisées, comme la spectrométrie de masse en conditions douces [12], la chromatographie à faible affinité [13], la mesure de la température de dénaturation des protéines, ou encore la calorimétrie [14]. La calorimétrie permet d'obtenir des données cruciales pour développer des molécules spécifiques, en favorisant les interactions dues à l'enthalpie, via des interactions polaires telles que liaison hydrogène ou attraction électrostatique.…”

The Impact of Binding Thermodynamics on Medicinal Chemistry Optimizations

“…The limitations of ITC measurements, namely throughput and the amount of protein required have been significantly alleviated by recent technological developments [65,35] and this is likely to increase the role of thermodynamic measurements both in hit identification and in optimization. This trend is expected to apply also for fragment based drug discovery since recent results have clearly demonstrated the feasibility of applying ITC in this domain [32,34,35].…”

“…Owing to their low affinity, compounds have to be screened at high concentration that requires high solubility. In line with their small size and polarity that allows fragments to form few, good quality polar interactions without significant contribution from apolar desolvation fragment hits typically bind with favorable enthalpy [17,30,31,32,33,34,35] ( Figure 5). The few compounds in Figure 5 that bind with unfavorable enthalpy are all anionic species with highly unfavourable desolvation profile.…”

Structural Annotation of the Mycobacterium tuberculosis Proteome

“…Several other examples are featured in the recent literature, such as the optimization of inhibitors of protein kinases such as protein kinase G by AX20017 (98). Fragment-based design has also been used to guide derivatization of a lead series of β-lactamase inhibitors against M. tuberculosis β-lactamase (BlaC) (99) and has been used successfully in the screening of fragment libraries against pantothenate synthetase to detect candidates that can be further modified to generate potent inhibitors (100).…”