Abstract NanoLuc® luciferase (NLuc) and its complementation technologies, including the NanoBiT® LgBiT/HiBiT system, are widely applied in functional biology because of their exceptional brightness, broad dynamic range, and suitability for monitoring real-time cellular processes. Despite their versatility, NLuc-based reporters emit primarily at a single, blue-shifted wavelength (∼460 nm), restricting their use in multiplexed assays where independent spectral channels are required. To overcome this limitation, we developed NanoPrism™ luciferases, engineered NLuc variants that employ highly efficient intramolecular energy transfer to red-shift the emission. Building on BRET-based strategies, we inserted circularly permuted NLuc or LgBiT into a surface loop of HaloTag®, enabling optimal positioning to covalently bound fluorophores and achieving exceptional BRET efficiency (∼90%). By pairing these red-shifted NanoPrism™ reporters with unmodified NLuc® or NanoBiT®, we created a robust two-color bioluminescent platform with strong signal intensities and >100 nm spectral separation. This dual-channel system allows simultaneous monitoring of two proteins or cellular events, such as targeted protein degradation, signaling dynamics, or internal control measurements - in real time within a single experimental context. Citation Format: Juliano Alves, Karilyn Porter, Mike Killoran, Robin Hurst, Mark Klein, Debayan De Bakshi, Rahele Esmatpour, James J. Cali, Thomas Machleidt, Rachel F. Ohana, Hicham Zegzouti. Two-color bioluminescence analyses pairing NanoLuc® and red-shifted NanoPrism™ luciferases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 288.

Bioluminescent reporters are widely used to monitor and image biological processes. Among these, NanoLuc luciferase and its complementation variants (LgBiT/SmBiT and LgBiT/HiBiT) are commonly used due to their brightness, sensitivity, and compatibility with prolonged kinetic measurements. However, the single-channel emission of these NanoLuc-based systems (460 nm peak) limits their use in multiplexed assays. Prior efforts to shift NanoLuc's emission employed bioluminescence resonance energy transfer (BRET) to a proximal fluorescent protein or organic fluorophore. Building on this concept, we engineered high-efficiency BRET reporters, termed NanoPrism luciferases, by inserting circularly permuted NanoLuc or LgBiT into a surface loop of the self-labeling HaloTag protein. These NanoPrisms achieve a ∼90% BRET efficiency by optimally positioning NanoLuc variants near a fluorophore covalently bound to HaloTag. The binary design further supports high- and low-affinity complementation, allowing applications in HiBiT knock-in cells and tracking protein-protein interactions, respectively. Pairing red-shifted NanoPrisms with unmodified NanoLuc or its complementation variants, we created a two-color bioluminescent reporter platform featuring bright signals of similar intensity and >100 nm spectral separation, allowing quantitative, simultaneous measurement of two molecular readouts within the same sample. Here, we demonstrate the platform's utility for monitoring a degradation target alongside a control protein and for tracking two distinct events within a biological pathway, using plate-based detection and bioluminescence imaging. By enabling concurrent measurements within the same sample, the system provides insights into cellular dynamics while reducing variability and complexity associated with parallel single-channel assays.

Galanin is a neuroendocrine peptide that regulates a wide range of physiological functions, including feeding and energy homeostasis, mood and anxiety, and modulation of pain. The function of the galanin peptide is mediated through its three galanin receptors, namely, GALR1, GALR2, and GALR3, which belong to the G protein-coupled receptor family. To measure the interaction of ligands with galanin receptor 1 (GALR1) in living cells, we developed a novel HiBiT peptide-based NanoBRET ligand binding assay. We generated six bioluminescence resonance energy transfer (BRET) tracers composed of modified and truncated galanin peptide derivatives tagged with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) acceptor fluorophore. The fluorophore-tagged peptide tracers were evaluated in cells expressing GALR1 tagged with HiBiT, an 11-amino acid subunit of NanoLuc, which, upon high affinity complementation with the cell-impermeable subunit LgBiT, reconstituted a functional NanoLuc luciferase. Addition of the furimazine substrate induced BRET to the BODIPY fluorophore acceptor component of the galanin-derived peptide tracers and produced a fluorescence signal output. Using this BRET assay, we characterized the binding affinity and binding kinetics of tracers with GALR1 in both equilibrium and real time. To validate our assay, we evaluated the binding affinity and function of a panel of unmodified galanin-derived peptide ligands through the competitive displacement of bound fluorescent galanin tracers. Our data showed that the binding affinity of these galanin peptide ligands correlated well with their rank order in β-arrestin recruitment and internalization functional assays. This study demonstrates that the HiBiT peptide-based NanoBRET ligand binding assay is a valuable system for studying the ligand engagement of GALR1 in living cells, offering an alternative to neuropeptide radioligand binding assays.

Abstract Identifying the physiologically relevant targets of bioactive compounds is often the rate limiting step toward understanding their mechanism of action. To this end, we developed a streamlined chemical proteomic strategy that utilizes a novel photoreactive cleavable chloroalkane tag, which can be attached to bioactive compounds to isolate their respective cellular targets for identification by mass spectrometry. The tag does not significantly affect compounds potency and membrane permeability, allowing for binding interactions to be established within intact cells under appropriate physiological conditions. UV-induced covalent photo-crosslinking “freezes” the cellular interactions and prevents their dissociation upon cell lysis. Effective enrichment of the cellular targets is achieved through their selective covalent capture onto HaloTag coated particles and subsequent selective release by tag cleavage. Putative targets identified by mass spectrometry can be readily verified using resonance energy transfer. By exchanging the chloroalkane tag for a fluorophore, direct binding to the targets can be revealed through proximity of the fluorophore to a small NanoLuc luciferase genetically fused to the putative target. This approach was used to identify physiologically relevant targets for bioactive compounds including those with low affinity and/or low abundance as well as insoluble targets with multiple trans membrane domains. Citation Format: Tetsuo Uyeda, Robin Hurst, Sergiy Levin, Mike Rosenblatt, Kris Zimmermann, Rachel F. Ohana. Utilizing a photoreactive chloroalkane capture tag for target deconvolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5771.

Point-of-care tests are highly valuable in providing fast results for medical decisions for greater flexibility in patient care. Many diagnostic tests, such as ELISAs, that are commonly used within clinical laboratory settings require trained technicians, laborious workflows, and complex instrumentation hindering their translation into point-of-care applications. Herein, we demonstrate the use of a homogeneous, bioluminescent-based, split reporter platform that enables a simple, sensitive, and rapid method for analyte detection in clinical samples. We developed this point-of-care application using an optimized ternary, split-NanoLuc luciferase reporter system that consists of two small reporter peptides added as appendages to analyte-specific affinity reagents. A bright, stable bioluminescent signal is generated as the affinity reagents bind to the analyte, allowing for proximity-induced complementation between the two reporter peptides and the polypeptide protein, in addition to the furimazine substrate. Through lyophilization of the stabilized reporter system with the formulated substrate, we demonstrate a shelf-stable, all-in-one, add-and-read analyte-detection system for use in complex sample matrices at the point-of-care. We highlight the modularity of this platform using two distinct SARS-CoV-2 model systems: SARS-CoV-2 N-antigen detection for active infections and anti-SARS-CoV-2 antibodies for immunity status detection using chemically conjugated or genetically fused affinity reagents, respectively. This technology provides a simple and standardized method to develop rapid, robust, and sensitive analyte-detection assays with flexible assay formatting making this an ideal platform for research, clinical laboratory, as well as point-of-care applications utilizing a simple handheld luminometer.

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

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. One way to alleviate the burden of developing a specific tracer for each receptor is using promiscuous tracers, which is made possible by the intrinsic specificity of BRET. Here, we devised an integrated tracer discovery workflow that couples machine learning-guided in silico screening for scaffolds displaying promiscuous binding to GPCRs with a blend of synthetic strategies to rapidly generate multiple tracer candidates. Subsequently, these candidates were evaluated for binding in a NanoBRET ligand-engagement screen across a library of HiBiT-tagged GPCRs. Employing this workflow, we generated several promiscuous fluorescent tracers that can effectively engage multiple GPCRs, demonstrating the efficiency of this approach. We believe that this workflow has the potential to accelerate discovery of NanoBRET fluorescent tracers for GPCRs and other target classes.

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. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.

Enzyme-linked immunosorbent assays (ELISAs) are used extensively for the detection and quantification of biomolecules in clinical diagnostics as well as in basic research. Although broadly used, the inherent complexities of ELISAs preclude their utility for straightforward point-of-need testing, where speed and simplicity are essential. With this in mind, we developed a bioluminescence-based immunoassay format that provides a sensitive and simple method for detecting biomolecules in clinical samples. We utilized a ternary, split-NanoLuc luciferase complementation reporter consisting of two small peptides (11mer, 13mer) and a 17 kDa polypeptide combined with a luminogenic substrate to create a complete, shelf-stable add-and-read assay detection reagent. Directed evolution was used to optimize reporter constituent sequences to impart chemical and thermal stability, as well as solubility, while formulation optimization was applied to stabilize an all-in-one reagent that can be reconstituted in aqueous buffers or sample matrices. The result of these efforts is a robust, first-generation bioluminescence-based homogenous immunoassay reporter platform where all assay components can be configured into a stable lyophilized cake, supporting homogeneous, rapid, and sensitive one-step biomolecule quantification in complex human samples. This technology represents a promising alternative immunoassay format with significant potential to bring critical diagnostic molecular detection testing closer to the point-of-need.

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. More recently, the high specificity of resonance energy transfer has been leveraged toward the development of homogeneous cell-based proximity assays with capacity for real-time kinetic measurements. This suite of ligand binding protocols couples the specificity of bioluminescent resonance energy transfer (BRET) with the sensitivity afforded by the luminescent HiBiT peptide. The BRET format is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent Tracers. At the same time, high affinity complementation of HiBiT with the cell impermeable LgBiT limits the bright bioluminescence donor signal to the cell surface and eliminates luminescence background from unoccupied receptors present in intracellular compartments.

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. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the ?-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

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. Recently, we reported on a single-step method integrating recombinant protein purification with 2-cyanobenzothiazole (CBT) condensation for labeling a proteolytically exposed N-terminal cysteine. Here, using three growth factors, we show that unlike random lysine labeling, this site-specific approach yielded homogeneous populations of growth factors that were quantitatively labeled at their N-termini and retained their binding characteristics. We demonstrate the utility of this labeling method through the development of a novel assay that quantifies the capacity of antibodies to block receptor-ligand interactions (i.e. antibody blockade). The assay uses bioluminescence resonance energy transfer (BRET) to detect binding of CBT-labeled growth factors to their cognate receptors genetically fused to NanoLuc luciferase. The ability of antibodies to block these interactions is quantified through decrease in BRET. Using several antibodies, we show that the assay provides reliable quantification of antibody blockade in a cellular context. As demonstrated here, this simple method for generating uniformly-labeled proteins has potential to promote more accurate and robust ligand binding assays.

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. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.

Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pathways and molecular mechanisms. Although many assay methods exist for confirming whether an apoptotic response has occurred in vitro, most methods are destructive and involve laborious operator effort or specialized instrumentation. Here we describe a real-time, no-wash, microplate method which utilizes recombinant annexin V fusion proteins containing evolved binary subunits of NanoBiT™ luciferase. The fusion proteins, a time-released enzymatic substrate, a necrosis detection dye and exogenous calcium ions are delivered via an optimized and physiologically inert reagent directly to cells in culture at the time of treatment or dosing. Luminescent signals proportional to phosphatidylserine (PS) exposure and fluorescent signals generated as a result of loss of membrane integrity are then collected using a standard multimode plate reader at scheduled intervals over the exposure. The resulting luminescent and fluorescent data are then used to define the kinetics and magnitude of an apoptotic response. This study details our efforts to develop, characterize, and demonstrate the features of the assay by providing relevant examples from diverse cell models for programmed cell death.

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

Abstract Efficacious and durable anti-cancer responses are driven by both the selective death of malignant cells and the induction of immunostimulatory activities. Limited but promising clinical evidence suggests that provocation of an unknown balance of inflammatory- and non-inflammatory cell death may be key for orchestrating these positive outcomes. Therefore, identifying and characterizing new clinically useful small molecules and biologic inducers (or combinations thereof) which promote a spectrum of programmed cell death in in vitro screening environments remains critically important. Unfortunately, current screening methods are either insufficiently robust, cost- or resource-prohibitive, or provide no means for initial characterization of the kinetics of programmed cell death. To address this unmet need, we developed a real-time, live cell assay method that utilizes a fully homogeneous, bioluminescent annexin V reagent. The method does not require laborious washing and sample preparation steps associated with traditional annexin methods and is fully compatible with plate-based multimodal signal detection systems. The system contains two annexin proteins which have been engineered to contain separate and distinct complementing domains of a binary luciferase. Additionally, the system contains a novel time-released luciferase substrate and a cell impermeable, fluorogenic DNA dye for monitoring necrosis. Because the annexin-luciferase fusion pairs have only modest affinity for each other, luminescence remains low until phosphatidylserine exposure, a hallmark of the programmed cell death phenotype, brings annexin monomers into close proximity facilitating complementation of the luciferase sensor. The assay reagent can be applied at dosing for real-time measurement of the dose-dependency and magnitude of programmed cell death progression. This work describes our efforts to characterize and validate the performance of the bioluminescent annexin assay using relevant cell death induction models. First, the assay was shown to be functionally concordant with a flow cytometry annexin method in a dose-response model with bortezomib at exposure periods known to produce both early and late-apoptosis (with necrosis) phenotypes. Next, we assessed the performance of the assay using a limited training set of small molecule and biologic inducers of programmed cell death that utilize different mechanisms of action (i.e., apoptosis and necroptosis) and by using a number of diverse but representative cancer cell lines. We conclude that the bioluminescent annexin method provides a new kinetic approach to efficient and effective detection of programmed cell death mechanisms in real time in a convenient homogeneous format. Citation Format: Kevin Kupcho, Andrew Niles, John Shultz, Jamison Grailer, Wenhui Zhou, Robin Hurst, Jim Hartnett, Terry Riss, Dan Lazar, James Cali. A real-time annexin V method for monitoring programmed cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4296. doi:10.1158/1538-7445.AM2017-4296