Schake, P. | Search bioRxiv

bioRxiv2025

The structural basis of drugs targeting protein-protein interactions uncovered with the protein-ligand interaction profiler PLIP

Promiscuity of drugs and targets plays an important role in drug-target prediction, ranging from the explanation of side effects to their exploitation in drug repositioning. A specific form of promiscuity concerns drugs, which interfere with protein-protein interactions. With the rising importance of such drugs in drug discovery and with the large-scale availability of structural data, the question arises on the structural basis of this form of promiscuity and the commonalities of the underlying protein-ligand (PLI) and protein-protein interactions (PPI). To this end, we employ the protein-ligand interaction profiler, PLIP, to characterize the PLI and PPI of MDM2/p53, Bcl-2/BAX, XIAP/Casp9, CCR5/gp120, and BRD/H4. We show that the wealth of existing complexes in PDB, of predicted protein structures, and of molecular docking results gives deep insights into the design principles for drugs targeting protein interactions. Drugs targeting protein interaction interfaces are a promising avenue in drug discovery. Understanding drug and target promiscuity at a structural level will pave the way to deliver on this promise.

bioinformatics↗

bioRxiv2023

Farewell to single-well: An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Single-molecule FRET (smFRET) has become a widely used tool for probing the structure, dynamics, and functional mechanisms of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Investigations by smFRET often require sampling of a large parameter space, for example, by varying one or more constituent molecular components in ten or more steps to reliably extract distances, kinetic rates, and other quantitative parameters. Confocal smFRET measurements, for example, which are amongst the widely used smFRET assays, are typically performed in a single-well format and measurements are conducted in a manual manner, making sampling of many experimental parameters laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality into a confocal microscope to allow for continuous and automated smFRET measurements. We show that the multiwell plate assay is on par with conventional single-well smFRET measurements in terms of accuracy and precision yet enables probing tens to hundreds of conditions in a fully automized manner. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, and competitive and cooperative protein-DNA interactions, revealing new insights that would be hard if not impossible to achieve with conventional single-well format measurements. The higher sampling density afforded by the multiwell plate format increases the accuracy of data analysis by at least 10-fold. We further showcase that the assay provides access to smFRET-based screening of drug-protein interactions. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software. Taken together, the automated multiwell plate assay developed here opens up new possibilities to acquire high-content smFRET datasets for in-depth single-molecule analysis of biomolecular conformations, interactions, and dynamics.

biophysics↗