Drug discovery researchers face a fundamental constraint in their work to develop safe, effective therapeutics: the vast majority of the human proteome remains inaccessible to conventional small molecule approaches. Proteins without defined binding pockets, those lacking known chemical probes, and protein targets that fail to translate from biochemical assays into cellular models have long been considered out of reach of standard drug discovery screening tools. As Dixit et al. describe, developing biochemical or cellular assays for all genome-encoded targets “is not scalable and likely impossible as most proteins have ill-defined or unknown activity” — these are what the authors call “the dark undruggable expanses” of the proteome [1].
That gap is now narrowing. Promega Corporation recently launched the TarSeer™ BRETSA™ Target Engagement System, a live-cell target engagement platform designed to bring previously challenging targets within reach of early-stage drug discovery.
The Problem: A Translation Gap in Early Discovery
Drug discovery teams regularly encounter a frustrating disconnect. A compound may show strong binding activity in a biochemical assay, only to fail when tested in a cellular environment. Without target-specific cellular assays, which generally aren’t available for poorly characterized proteins, researchers face difficult choices when deciding which compounds to advance through the drug development pipeline.
This challenge is especially acute for proteins in the so-called “dark proteome”: targets that lack defined binding pockets, have no established chemical probes, or belong to understudied protein families. Computational estimates suggest that only roughly 20% of the human proteome (approximately 6,300 proteins) meets the criteria for conventional drug discovery based on sequence and structural similarity to known targets [2]. For the remainder, standard target engagement methods cannot identify hits that will warrant further investment in downstream studies.
The Approach: Thermal Stability in Live Cells as a Signal
BRETSA — Bioluminescence Resonance Energy Transfer Shift Assay — takes a different approach. Rather than requiring a known probe or binding pocket, the platform measures changes in a target protein’s thermal stability when a ligand binds to it. This phenomenon, known as ligand-induced thermal stabilization, is a reliable indicator of direct compound-protein interaction.
Critically, BRETSA detects these changes in intact, living cells. This means researchers get a direct readout of target engagement in a physiologically relevant context — not in a test tube or cell lysate, but rather, inside a living cell. This matters because, as Vasta et al. have demonstrated with related BRET-based methods, potency results from biochemical assays of purified protein “have often failed to correlate with inhibition” of downstream pathway activity, underscoring the value of live-cell measurements [3].
BRETSA is unique from other cellular thermal shift methods because it depends on target protein denaturation, rather than the downstream effect of aggregation of the target protein. This eliminates the need for separation and transfer steps used in aggregation-based methods.
The platform builds on the established principles that use bioluminescence resonance energy transfer to study proximity-based interactions, such as between a target protein and a small molecule. [4]. While NanoBRET® Target Engagement has been applied to many target classes, it requires a suitable molecule that can be made into a fluorescent tracer. BRETSA eliminates this need: it is designed for targets where no such tracer exists, effectively extending the toolbox to a new class of proteins.
BRETSA Capabilities: Sensitivity, Broad Target Coverage and Scalable Workflows
Three capabilities make BRETSA particularly significant for drug discovery programs. First, enhanced sensitivity: the platform can detect and rank-order weak compound-protein interactions that would be missed or difficult to quantify with earlier methods. This is especially valuable in early drug discovery, where early chemical matter is often sub-optimal and binding signals are faint.
Second, broad applicability: BRETSA has been validated across more than 20 target classes and multiple cellular compartments. This breadth means the platform can be deployed across a wide range of programs. The reagents to develop and perform the assay are supplied, eliminating the need to source and/or develop separate target-specific detection methods.
Third, a scalable workflow: the addition-only format is compatible with both 96- and 384-well plate configurations, allowing teams to seamlessly accommodate the various stages of drug development.
Why This Matters Now: New Starting Points for Drug Discovery
The human proteome contains an estimated 20,000 proteins, yet only a fraction have been successfully targeted by approved drugs [2]. The remainder — including many proteins implicated in cancer, neurodegeneration, and other diseases — have resisted conventional drug discovery approaches, in part because the cellular biology tools to study them simply weren’t available [1].
BRETSA doesn’t solve every challenge in targeting difficult proteins. But it provides an accessible cellular method to begin interrogating these targets, accelerating researcher projects and enabling decisions about which compounds to pursue.
As Matt Robers, Associate Director of R&D at Promega, noted at the technology’s debut at SLAS 2026: “This platform will give drug discovery researchers new starting points to go after a huge fraction of previously intractable therapeutic targets within the human proteome.”
Learn more about the BRETSA platform and view example data. Or watch the video below to get an overview of the BRETSA technology.
Literature Cited
- Dixit A, Barhoosh H, Paegel BM. (2023) Translating the Genome into Drugs. Acc Chem Res. 56(4):489–499. https://doi.org/10.1021/acs.accounts.2c00791
- Plewczynski D, Rychlewski L. (2008) Meta-basic estimates the size of druggable human genome. J Mol Model. 15(6):695–699. https://doi.org/10.1007/s00894-008-0353-5
- Vasta JD et al. 2024 A Method to Conditionally Measure Target Engagement at Intracellular RAS and RAF Complexes. Methods Mol Biol. 2797:287–297. https://doi.org/10.1007/978-1-0716-3822-4_21
- Nieman AN et al. (2023) NanoBRET™ Live-Cell Kinase Selectivity Profiling Adapted for High-Throughput Screening. Methods Mol Biol. 2706:97–124. https://doi.org/10.1007/978-1-0716-3397-7_8
Further Reading
- Expand the Druggable Proteome with Intracellular Target Engagement. The Scientist
- Promega launches cellular target engagement platform – Drug Discovery World (DDW) Drug Discovery World (DDW)
- SLAS Highlights: AI Labs, Small-Molecule SPR, Protein Interaction Assays, and Paper Labware Genetic Engineering and Biotechnology News (GEN)
- Promega launches live-cell platform to expand the druggable proteome Drug Target Review
- Key Analytical Advances from SLAS 2026 Separation Science
This article was written with the assistance of an AI platform.
Michele Arduengo, PhD
