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This study presents a BRET biosensor that measures how anticancer drugs cooperatively engage PRMT5 complexes in cells, revealing how cellular metabolites such as SAM and MTA enhance drug action and enable precision therapies for MTAP-deleted tumors.

This Perspective highlights advances in bioluminescence resonance energy transfer technologies for measuring intracellular drug–target engagement, expanding their use to analyze kinetics, permeability and complex mechanisms in chemical biology.

Drug molecules operate through physical interaction with specific cellular targets, and understanding this interaction is important for mechanisms and the potential therapeutic effect of drug candidates. Here, the authors show that bioluminescence resonance energy transfer can be used to monitor the intracellular engagement of a drug with its target.

Cellular signaling processes often involve trafficking of receptors and other proteins between subcellular compartments. Here the authors demonstrate a method based on the concept of Enhanced bystander Bioluminescence Resonance Energy Transfer (EbBRET) that allows efficient real time monitoring of endocytosis and trafficking.

Bioluminescence resonance energy transfer, from the substrate of a luciferase fused with a G protein–coupled receptor to a fluorescent dye covalently linked to a receptor ligand, allows the profiling of ligand affinity and binding kinetics.

The use of NMR spectroscopy and development of a cellular BRET KRAS engagement assay revealed that noncovalent ligands can access the switch-II pocket of KRAS hotspot mutants.

This report describes a nanobody targeting glycine receptor mGlyR that inhibits its ability to regulate G protein signaling and produces anti-depressant effects in mice providing an immunotherapy approach to potentially treat depression.

G protein-coupled receptor di/oligomers can diversify signaling and play roles in disease. Here authors stabilized D2 dopamine receptor di/oligomers that may selectively favor complexes with the adaptor protein β-arrestin2 to bias receptor activity.

An ‘intracrine’ signaling mechanism is proposed whereby a G-protein-coupled receptor (free fatty acid receptor 4) senses locally released fatty acids on intracellular membranes associated with lipid droplets to efficiently regulate lipolysis in adipocytes.

Signaling pathway selectivity downstream of GPCRs is not fully understood. Here, authors perform functional analysis of Frizzled mutants to uncover state-stabilizing residues or ‘micro-switches’ mediating selectivity towards Disheveled over G proteins.

The authors reveal that the C-terminal tail anchors of BCL-2 family proteins control BH3 binding and cell survival by allosteric regulation at the mitochondrial membrane, involving BAX recruitment and complex disruption upon BH3 mimetic antagonism.

Based on a BRET readout, dopamine D2 receptor agonist NPA is more potent at activating Gα_i when the D2 receptor forms a heteromer with the related D1 receptor than if it forms D2 receptor homomers, suggesting that GPCR heteromerization can result in functional selectivity.

While Ras is a promising target for cancer therapy, development of inhibitors targeting Ras signaling has proven challenging. Here, the authors report the discovery of Rasarfin, a small molecule from a phenotypic screen on G protein-coupled receptor (GPCR) endocytosis that acts as a dual Ras and ARF6 inhibitor.

Coupling selectivity was found to be partly lost with G proteins that bypass conformations that exist before GDP release, suggesting that selectivity is closely linked to the process of nucleotide release.

Here the authors show that MRAP2 tunes the melanocortin-4 receptor by boosting its G-protein signaling while limiting β-arrestin2 interaction and destabilizing receptor oligomers, shaping appetite regulation.

GPCR kinases (GRKs) regulate GPCR interactions and thus functions. Here, the authors report a comprehensive panel of GRK knockout cells, used to assess the GRK-specific β-arrestin recruitment. Selective engagement of GRKs induces distinct GPCR–β-arrestin complexes.

Beta-arrestins play central roles in the mechanisms regulating GPCR signalling and trafficking. Here the authors identify a selective inhibitor of the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP-2, which they use to dissect the role of the β-arrestin/β2-adaptin interaction in GPCR signalling.

BRET probes that monitor activation of multiple G protein isoforms reveal that angiotensin II and a biased agonist of the angiotensin II type 1A receptor stabilize distinct receptor conformations associated with different signaling outputs.

Annunziato, Quan and Donckele et al. identify G3BP2 (Ras–GAP SH3 domain-binding protein 2) as a molecular glue-induced neosubstrate of the CRL4^CRBN E3 ubiquitin ligase. The CRBN–glue neosurface uses a molecular surface mimicry mechanism to recruit and degrade G3BP2 in a compound-dependent manner.

The combination of engineered probes and spectral phasor analysis overcomes long-standing challenges associated with bioluminescence detection at the microscale, enabling multiplexed, real-time imaging of cellular features without the need for excitation light.

Here, the authors identify human Gαi protein as an intracellular pH sensor. The Gαi subunit undergoes a disorder to order transition as pH is increased over the intracellular pH range, which modulates agonist mediated Gβγ dissociation from the Gαi-Gβγ complex.

Woo et al. report new autophagy inhibitors identified through a high-throughput chemical screening using a BRET-based assay and an ATG8 synthetic sensor, that function as safe and effective fungicides against broad fungal pathogens.

There remains a demand for optogenetic tools that provide modularity, ease of integration, and minimal interference with protein function. Here, authors introduce PhoBITs as compact and modular optogenetic tools for reversible control, demonstrating a range of applications including in vivo optogenetic tumor suppression.

The single nucleotide polymorphism C115Y in the IL-23 receptor is associated with autoinflammatory diseases. Here the authors demonstrate that this mutation prevents the binding of a fluorescent cyclic peptide and IL-23 to the IL-23 receptor.

A series of intramolecular fluorescent FlAsH BRET reporters is used to monitor conformational changes in β-arrestin2 following activation of seven G-protein-coupled receptors (GPCRs), showing that different GPCRs produce distinct β-arrestin2 conformational signatures that correlate with the stability of the receptor–arrestin complex and the role of β-arrestin2 in activating or dampening downstream signalling events, which explains how different GPCRs can use a common effector for different purposes.

G protein-coupled receptors (GPCRs) are integral membrane proteins and the largest class of drug targets in the human genome. Here, Baidya et al. show that a synthetic antibody can be used to modulate GPCR trafficking and signaling in live cells.

Li et al. identify dynorphin as an endogenous ligand for orphan receptor GPR139 introducing it as a non-canonical member of the opioid receptor family that triggers excitatory signaling to balance the inhibitory effects of opioids.

Among the adhesion receptor class of GPCRs, which are understudied, the adhesion receptor ADGRL3 can be activated by its own tethered agonist and couples to G protein G12/13 and somewhat to Gq.

Chemokine receptors are G protein-coupled receptors (GPCRs) involved in immune responses and characterized by ligand promiscuity Here, the authors characterize signaling through chemokine receptors CXCR4 and CXCR7, with insights into intrinsic-bias encoded in the CXCR4-CXCR7 system.

Using molecular dynamics simulations and functional assays, authors track the structural changes in heterotrimeric G proteins in response to receptor coupling that lead to the ejection of GDP, the rate-limiting step during G-protein activation.

Activated GPCRs signal through multiple pathways. Ligands that signal through a single pathway are highly valued. The authors demonstrate that tethering ligands to receptors via conjugation with binding nanobodies enables pathway-specific signaling.

Autism spectrum disorders (ASD) are characterized by social impairments, communication deficits and repetitive stereotyped behaviours. Here, the authors show that de novo missense mutations, but not inherited missense mutations, in TBR1disrupt the protein function and contribute to ASD aetiology.

D1-like dopamine receptors are coupled to Golf proteins in the dorsal striatum but Gs in cortical and other areas. Here, the authors demonstrate selective agonism of Gs-coupled versus Golf-coupled D1 receptors.

Murray et al. identified and characterized a small-molecule inhibitor of human COQ8A, which belongs to the UbiB protein family and is essential for coenzyme Q biosynthesis.

RAF kinases are essential for RAS protein signalling but how RAS binding regulates dimerization and activation of RAF has remained unclear. Here, the authors report cryoEM structures that provide mechanistic insights into the RAS-mediated monomer-to-dimer transition of full-length BRAF.

Class F receptors are therapeutic targets in human disease and understanding their structural changes during receptor activation may provide important pharmacological insight. Here, the authors combine computational and experimental methods to identify a molecular switch in TM6/7 of Class F receptors that mediates receptor activation.

GPR179, an orphan class C GPCR crucial for visual signaling, is expressed at dendritic tips in the retina. Here, authors present cryo-EM structure of GPR179, revealing its dimerization mode and curvature.

Molecular glues have great potential for drug discovery if they can be systematically discovered. Konstantinidou, et al describe a scaffold-hopping approach using multicomponent reaction chemistry to design molecular glues that induce 14-3- 3σ/ERα formation in cells.

IL-37 is a member of the IL-1 family with profound anti-inflammatory functions. Nold and colleagues identify the receptor for IL-37 and clarify the molecular nature of the anti-inflammatory pathway induced by this cytokine.

Pertussis toxin is used extensively for perturbing Gαi/o pathways in the study of physiology and disease, but an equivalent inhibitor of Gαq signalling is not currently available to the research community. Here the authors characterize FR900359 as a specific Gq inhibitor and demonstrate its utility to dissect GPCR signalling and its potential to inhibit melanoma cells.

Many neurotransmitters act on receptors coupled to GTP-binding G proteins. Here authors report the structure and activity of a mutant that locks the nucleotide-free and receptor-bound state of the G protein, leading to a rare neurological disorder.

Agonists of the glucagon-like peptide-1 receptor are used to treat diabetes and obesity. Here, Wright et al. investigate the subcellular location of the receptor’s signaling events and uncover associations between signaling profiles and adverse drug reactions.