Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power.

Bioluminescence emitted from a luciferase-catalyzed oxidation of luciferin has been broadly utilized to report on biological events, predominantly through relative changes in the light output. Recent advances in protein engineering and synthetic chemistry have yielded bioluminescent systems with markedly improved brightness and bioavailability. These developments have enabled not only the detection of biological events at far lower expression levels but also new opportunities utilizing bioluminescence to power photochemistry in cells.

Luminogenic HiBiT Peptide-Based NanoBRET Ligand Binding Assays for Melatonin Receptors.

The two human melatonin receptors MT and MT, which belong to the G protein-coupled receptor (GPCR) family, are important drug targets with approved indications for circadian rhythm- and sleep-related disorders and major depression. Currently, most of the pharmacological studies were performed using [H]melatonin and 2-[I]iodomelatonin (2-[I]-MLT) radioligands. Recently, NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a sensitive, nonradioactive alternative for quantifying GPCR ligand engagement on the surface of living cells in equilibrium and real time.

An Integrated Approach toward NanoBRET Tracers for Analysis of GPCR Ligand Engagement.

Gaining insight into the pharmacology of ligand engagement with G-protein coupled receptors (GPCRs) under biologically relevant conditions is vital to both drug discovery and basic research. NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a robust and sensitive method to quantify ligand engagement with specific GPCRs genetically fused to NanoLuc luciferase or the luminogenic HiBiT peptide. However, development of fluorescent tracers is often challenging and remains the principal bottleneck for this approach.

Equilibrium and Kinetic Measurements of Ligand Binding to HiBiT-tagged GPCRs on the Surface of Living Cells.

G-protein coupled receptors (GPCRs) remain at the forefront of drug discovery efforts. Detailed assessment of features contributing to GPCR ligand engagement in a physiologically relevant environment is imperative to the development of new therapeutics with improved efficacy. Traditionally, binding properties such as affinity and kinetics were obtained using biochemical radioligand binding assays.

Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag.

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions.

The luminescent HiBiT peptide enables selective quantitation of G protein-coupled receptor ligand engagement and internalization in living cells.

G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization.

Utilizing a Simple Method for Stoichiometric Protein Labeling to Quantify Antibody Blockade.

Ligand binding assays routinely employ fluorescently-labeled protein ligands to quantify the extent of binding. These ligands are commonly generated through chemical modification of accessible lysine residues, which often results in heterogeneous populations exhibiting variable binding properties. This could be remedied by quantitative, site-specific labeling.

Complex Formation between VEGFR2 and the β-Adrenoceptor.

Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and angiogenesis via its receptor VEGFR2. A common tumor associated with elevated VEGFR2 signaling is infantile hemangioma that is caused by a rapid proliferation of vascular endothelial cells. The current first-line treatment for infantile hemangioma is the β-adrenoceptor antagonist, propranolol, although its mechanism of action is not understood.

Quantitative, Real-Time Measurements of Intracellular Target Engagement Using Energy Transfer.

Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement.

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGFa, VEGFb, and VEGFa. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.

For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells.

Target Identification Using Cell Permeable and Cleavable Chloroalkane Derivatized Small Molecules.

An important aspect for gaining functional insight into the activity of small molecules revealed through phenotypic screening is the identification of their interacting proteins. Yet, isolating and validating these interacting proteins remains difficult. Here, we present a new approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules to isolate their respective protein targets.

Real-time analysis of the binding of fluorescent VEGFa to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGFa-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGFa-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET).

Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag.

The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells.

Target engagement and drug residence time can be observed in living cells with BRET.

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase.

Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag.

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets.

Purification of Recombinant Proteins from Cultured Mammalian Cells by HaloTag Technology.

Cultured mammalian cells provide an environment ideal for producing functional recombinant mammalian proteins. However, low expression levels of recombinant proteins present a challenge for their detection and purification. This unit will focus on HaloTag, a protein fusion tag designed to bind selectively and covalently to a chloroalkane ligand that may be attached to a variety of functional groups, allowing both protein detection and immobilization.

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction.

HaloTag-based purification of functional human kinases from mammalian cells.

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification.

HaloTag7: a genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification.

Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment.

HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces.