Abstract
Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.
Highlights
IntroductionA unique feature of NMR is its robust capability to detect weak intermolecular interactions
NMR spectroscopy is a highly versatile screening method for drug discovery [1,2,3]
Whether a hit/lead compound is capable of dissociating a particular protein–protein interaction (PPI) is determined by the strength of its binding to one of the two protein components of the protein–protein interactions (PPIs)
Summary
A unique feature of NMR is its robust capability to detect weak intermolecular interactions. This feature makes NMR ideal for fragment-based screening in which the binary binding between low-affinity fragments and target proteins is studied [1,2,3,6,7,8]. This type of NMR “binary screening” does not provide information about whether a compound can inhibit or dissociate protein–protein interactions.
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