Author response: How to measure and evaluate binding affinities

Jarmoskaite, Inga; AlSadhan, Ishraq; Vaidyanathan, Pavanapuresan P.; Herschlag, Daniel

doi:10.7554/elife.57264.sa2

Cited by 2 publications

(2 citation statements)

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“…Three databases-FireProtDB (Stourac et al, 2021), ProtaBank (Wang et al, 2018a(Wang et al, , 2018b, and ProTherm (Gromiha et al, 1999)-have been created to collect, organize, and validate stability data from the literature. This growing collection of data also underscores the importance of establishing best practices for performing and reporting stability measurements, as has been pioneered for other types of complex data from diverse sources (Conesa et al, 2016;Jarmoskaite et al, 2020). High-throughput techniques provide high-resolution estimates of relative energetic differences between constructs in a single experiment (e.g., DDG fold ) but typically do not provide absolute estimates (e.g., DG fold ).…”

Section: Combining Data Across Platformsmentioning

confidence: 99%

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

et al. 2021

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Section: Combining Data Across Platformsmentioning

confidence: 99%

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

et al. 2021

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“…For ligand displacement assays, the tracer is canonically used at or around its KD,app. In contrast, in protein quantification experiments, the "titration regime" is desired where the tracer concentration is much greater than the KD,app to maximize occupancy 19,20 . In our preliminary experiments, dBET6, a potent BRD4 degrader, was chosen as a positive control due to its well-established activity 21 .…”

Section: Quantifying Brd4 Levels In Response To Degrader Treatmentmentioning

confidence: 99%

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

Payne

Maksoud

Tannous

et al. 2021

Preprint

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We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras; PROTACs), capable of both accurately quantifying protein levels in whole cell lysates in less than 1 h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small molecule inhibitors and PROTACs in high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation

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Author response: How to measure and evaluate binding affinities

Cited by 2 publications

References 0 publications

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

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