Measuring Protein–Protein Interactions in Cells using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) Assay

Szewczyk, Magdalena M.; Owens, Dominic; Baršytė-Lovejoy, Dalia

doi:10.1007/978-1-0716-3397-7_10

Cited by 2 publications

(2 citation statements)

References 27 publications

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“…In this assay, the target protein TEAD1 is fused to a nanoLuc tag and YAP is tagged with a Halo-tag, which covalently binds to a chloroalkane fluorophore that generates a measurable signal only when the two proteins are in close proximity. 50 Compounds HC-258 and AF-857 (28) at 10 μM did not diminish the nanoBRET signal and thus did not disrupt the YAP−TEAD interaction in cells (Figure S1). To determine the binding of HC-258, we next performed cocrystallization experiments with hTEAD2 217−447 .…”

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“…We then used a nanoluciferase (nanoLuc) bioluminescence resonance energy transfer assay (nanoBRET) to characterize the YAP–TEAD interaction disruption in cells. In this assay, the target protein TEAD1 is fused to a nanoLuc tag and YAP is tagged with a Halo-tag, which covalently binds to a chloroalkane fluorophore that generates a measurable signal only when the two proteins are in close proximity . Compounds HC-258 and AF-857 ( 28 ) at 10 μM did not diminish the nanoBRET signal and thus did not disrupt the YAP–TEAD interaction in cells (Figure S1).…”

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Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration

Fnaiche,

Chan,

Paquin

et al. 2023

ACS Med. Chem. Lett.

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confidence: 99%

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confidence: 99%

Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration

Fnaiche,

Chan,

Paquin

et al. 2023

ACS Med. Chem. Lett.

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A resource to enable chemical biology and drug discovery of WDR Proteins

Ackloo,

Li,

Szewczyk

et al. 2024

Preprint

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Protein class-focused drug discovery has a long and successful history in pharmaceutical research, yet most members of druggable protein families remain unliganded, often for practical reasons. Here we combined experiment and computation to enable discovery of ligands for WD40 repeat (WDR) proteins, one of the largest human protein families. This resource includes expression clones, purification protocols, and a comprehensive assessment of the druggability for hundreds of WDR proteins. We solved 21 high resolution crystal structures, and have made available a suite of biophysical, biochemical, and cellular assays to facilitate the discovery and characterization of small molecule ligands. To this end, we use the resource in a hit-finding pilot involving DNA-encoded library (DEL) selection followed by machine learning (ML). This led to the discovery of first-in-class, drug-like ligands for 9 of 20 targets. This result demonstrates the broad ligandability of WDRs. This extensive resource of reagents and knowledge will enable further discovery of chemical tools and potential therapeutics for this important class of proteins.

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Measuring Protein–Protein Interactions in Cells using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) Assay

Cited by 2 publications

References 27 publications

Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration

Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration

A resource to enable chemical biology and drug discovery of WDR Proteins

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