An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics.

Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is 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 to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein-DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.

An interaction-based drug discovery screen explains known SARS-CoV-2 inhibitors and predicts new compound scaffolds.

The recent outbreak of the COVID-19 pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has shown the necessity for fast and broad drug discovery methods to enable us to react quickly to novel and highly infectious diseases. A well-known SARS-CoV-2 target is the viral main 3-chymotrypsin-like cysteine protease (Mpro), known to control coronavirus replication, which is essential for the viral life cycle. Here, we applied an interaction-based drug repositioning algorithm on all protein-compound complexes available in the protein database (PDB) to identify Mpro inhibitors and potential novel compound scaffolds against SARS-CoV-2. The screen revealed a heterogeneous set of 692 potential Mpro inhibitors containing known ones such as Dasatinib, Amodiaquine, and Flavin mononucleotide, as well as so far untested chemical scaffolds. In a follow-up evaluation, we used publicly available data published almost two years after the screen to validate our results. In total, we are able to validate 17% of the top 100 predictions with publicly available data and can furthermore show that predicted compounds do cover scaffolds that are yet not associated with Mpro. Finally, we detected a potentially important binding pattern consisting of 3 hydrogen bonds with hydrogen donors of an oxyanion hole within the active side of Mpro. Overall, these results give hope that we will be better prepared for future pandemics and that drug development will become more efficient in the upcoming years.