Title: Illuminating biophysics with BRET: from thermodynamics of target engagement to cooperative pharmacology in living cells
Abstract:
Drug action is predicated on engagement of cellular targets. However, characterizing target engagement and mode-of-action in a physiologically relevant context remains a central challenge in drug discovery. The cellular milieu profoundly shapes target conformation, complex assembly, and the thermodynamics of engagement — yet most biophysical methods are incompatible with intracellular analysis. Our lab has developed bioluminescence resonance energy transfer (BRET)-based methods designed to quantitatively resolve target engagement directly in living cells, bridging biophysics and cellular pharmacology.
The foundation of this platform is NanoBRET target engagement (TE), which enables direct, quantitative measurement of small molecule binding to intracellular targets in live cells via competitive displacement of cell-permeable, fluorescent probes. Beyond binary occupancy, NanoBRET TE enables kinetic and thermodynamic dissection of ligand-target interactions in a cellular context — redefining selectivity assessment through residence time and thermodynamic profiling that cannot be recapitulated in biochemical formats. This approach has been applied broadly across the kinome and beyond, revealing how the live-cell environment reshapes ligand selectivity in ways invisible to in vitro assays. However, NanoBRET TE requires a priori knowledge of the binding site to design a displacement tracer — motivating the need for a complementary, tracer-independent approach capable of reaching the broader proteome.
To address this, we developed BRETSA, a broadly applicable BRET-based thermal shift method for detecting ligand-protein interactions in intact cells without prior knowledge of binding site or mechanism. In BRETSA, cell-permeable denaturation-sensitive dyes decorate proteins and generate energy transfer with a luciferase-tagged target upon thermal challenge. Ligand interactions manifest as a dose-dependent change in the thermal stability profile of the target, and because cooperativity is encoded in that stability landscape, BRETSA is uniquely positioned to resolve induced-proximity mechanisms at defined biomolecular interfaces. BRETSA has been applied broadly across the proteome — spanning transcription factors, structural proteins, E3 ligases, and their complexes — including targets traditionally considered intractable to cellular biophysical analysis. By measuring intracellular α values directly in cells, BRETSA quantifies the thermodynamic cooperativity of ternary complex assembly at defined protein-protein interfaces — including molecular glues and tricomplex inhibitors targeting the GTP-bound state of KRAS. Together, NanoBRET TE and BRETSA constitute a unified biophysical platform for dissecting the full spectrum of ligand-target interactions in living cells — from classical binary engagement to the cooperative pharmacology driving the next generation of induced proximity therapeutics.
Bio:
Matthew Robers is an Associate Research Director at Promega Corporation. Matthew received his initial post-graduate training at University of Wisconsin-Madison, studying iron-sulfur cluster enzymes in bacteria. Matthew conducted his PhD thesis at Goethe University in Frankfurt, studying target engagement at biomolecular complexes with Dr. Stefan Knapp. Matthew received his PhD in 2024, while simultaneously leading a team of chemists and chemical biologists in his industry role.
In industry, Matthew first focused on technology development and cellular pathway analysis at Life Technologies (Invitrogen). Since joining Promega in 2010, Matthew has developed novel biophysical technologies to assess intracellular target engagement, residence time, and drug cooperativity. Matthew’s most noteworthy contributions include the creation of the NanoBRET Target Engagement platform, and bioluminescent methods to quantify protein thermal shift in cells. He has authored over 10 issued patents involving novel luciferase reporters, structural complementation, energy transfer, and target engagement.
Matthew’s team actively collaborates with biopharma and academic labs worldwide, including various groups within the Structural Genomics Consortium. In 2023, Matthew was awarded the title of Distinguished Scientist at Promega, owing to his patent and publication record in the field of chemical biology.
Complete bibliography
https://www.ncbi.nlm.nih.gov/myncbi/matthew.robers.1/bibliography/public/
Keywords:
Chemical Biology
Target Engagement
Thermal shift
NanoBRET
Cooperativity
Host: Prof. Helen Blackwell