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NanoBRET Implementation in HTS For Drug Discovery

Part # PS273

Abstract

Gediminas Vidugiris1, Jacqui Méndez11, Danette L. Daniels1, Cristopher Cowan1 and Michael Forbush2
1Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711, USA; 2EDC BioSystems 49090 Milmont Dr. Fremont, CA 94538

Identifying small molecule modulators, inhibitors or activators, of protein:protein interactions (PPI) remains challenging, largely due to the difficulty of developing robust and high-throughput screening tools. Bioluminescent resonance energy transfer (BRET) has been used to monitor real-time protein:protein interactions in cells but current approaches suffer from limited sensitivity and narrow dynamic range.

Here we present a new BRET method, termed NanoBRET, based on a small and extremely bright NanoLuc luciferase coupled to a HaloTag - long-wavelength fluorophore. This highly effective energy donor-acceptor combination boosts BRET performance and the higher sensitivity facilitates application of the method in high density plates and high throughput screening (HTS). Application of acoustic dispensing instruments simplifies assay assembly in HTS formats by allowing dispensing of stock reagents without pre-dilutions. We present examples of inhibitor screening for bromodomain/histone and p53/Mdm2 transcriptional protein interactions; as well pathway activators for the EFGR/GRB2 membrane interaction and cRaf/bRaf signaling pathway.

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