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. 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 ß-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.

Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent.

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.

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. 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.

A real-time, bioluminescent annexin V assay for the assessment of apoptosis.

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.

Application of BRET to monitor ligand binding to GPCRs.

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.

Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor.

Monitoring the expression of cell-surface receptors, their interaction with extracellular ligands, and their fate upon ligand binding is important for understanding receptor function and developing new therapies. We describe a cell-based method that utilizes bioluminescent protein complementation technology to interrogate binding of a cellular receptor with its extracellular protein ligand, specifically LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Purified, full-length tagged PCSK9 is added to assay wells containing cells that stably express LDLR with an extracellular complementary tag. When the tagged PCSK9 binds the receptor, a bright luminescence signal is generated. The interaction is detected at the cell membrane with add-and-read simplicity, no wash steps, and flexibility, allowing data to be collected in endpoint format, kinetically, or with bioluminescent imaging. The assay is flexible, is rapid, and reports accurate biology. It is amenable to 96-well and 384-well formats, and the robustness allows for screening of new drug candidates (Z' = 0.83). The assay reports correct potencies for antibody titrations across a 50%-150% potency range and detects potency changes due to heat stress, suggesting that it may be useful during drug development. This assay technology can be broadly applied when studying other receptors with their extracellular ligands, whether protein or small-molecule binding partners.

Coelenterazine analogues emit red-shifted bioluminescence with NanoLuc.

We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.

Three Efficient Methods for Preparation of Coelenterazine Analogues.

The growing popularity of bioluminescent assays has highlighted the need for coelenterazine analogues possessing properties tuned for specific applications. However, the structural diversity of known coelenterazine analogues has been limited by current syntheses. Known routes for the preparation of coelenterazine analogues employ harsh reaction conditions that limit access to many substituents and functional groups. Novel synthetic routes reported here establish simple and robust methods for synthesis and investigation of structurally diverse marine luciferase substrates. Specifically, these new routes allow synthesis of coelenterazine analogues containing various heterocyclic motifs and substituted aromatic groups with diverse electronic substituents at the R(2) position. Interesting analogues described herein were characterized by their physicochemical properties, bioluminescent half-life, light output, polarity and cytotoxicity. Some of the analogues represent leads that can be utilized in the development of improved bioluminescent systems.

A luminescent assay for real-time measurements of receptor endocytosis in living cells.

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.

Identification of known drugs targeting the endoplasmic reticulum stress response.

The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response. In this study, we have developed a stable ER stress reporter cell line that stably expresses a ß-lactamase reporter gene under the control of the ER stress response element (ESRE) present in the glucose-regulated protein, 78 kDa (GRP78) gene promoter. This assay has been optimized and miniaturized into a 1536-well plate format. In order to identify clinically used drugs that induce ER stress response, we screened approximately 2800 drugs from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC library) using a quantitative high-throughput screening (qHTS) platform. From this study, we have identified several known ER stress inducers, such as 17-AAG (via HSP90 inhibition), as well as several novel ER stress inducers such as AMI-193 and spiperone. The confirmed drugs were further studied for their effects on the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the X-box-binding protein (XBP1) splicing, and GRP78 gene expression. These results suggest that the ER stress inducers identified from the NPC library using the qHTS approach could shed new lights on the potential therapeutic targets of these drugs.

Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins.

Intracellular pathways transduce signals through changes in post-translational modifications (PTMs) of effector proteins. Among the approaches used to monitor PTM changes are immunoassays and overexpression of recombinant reporter genes. Genome editing by CRISPR/Cas9 provides a new means to monitor PTM changes by inserting reporters onto target endogenous genes while preserving native biology. Ideally, the reporter should be small in order not to interfere with the processes mediated by the target while sensitive enough to detect tightly expressed proteins. HiBiT is a 1.3 kDa reporter peptide capable of generating bioluminescence through complementation with LgBiT, an 18 kDa subunit derived from NanoLuc. Using HiBiT CRISPR/Cas9-modified cell lines in combination with fluorescent antibodies, we developed a HiBiT-BRET immunoassay (a.k.a. Immuno-BRET). This is a homogeneous immunoassay capable of monitoring post-translational modifications on diverse protein targets. Its usefulness was demonstrated for the detection of phosphorylation of multiple signaling pathway targets (EGFR, STAT3, MAPK8 and c-MET), as well as chromatin containing histone H3 acetylation on lysine 9 and 27. These results demonstrate the ability to efficiently monitor endogenous biological processes modulated by post-translational modifications using a small bioluminescent peptide tag and fluorescent antibodies, providing sensitive quantitation of the response dynamics to multiple stimuli.

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. Regardless of the application, bioluminescence analyses have leaned heavily on the use of luminometers to measure the light output of a system. Current luminometers report the light output of a sample in relative units, limiting the ability to compare data between instruments and preventing the absolute power of a bioluminescent system from being quantified. Luminescent solution calibrants comprising luciferases and their cognate luciferins that have been characterized for absolute light output would enable calibration of any given luminometer for absolute photon counting. To this end, we have built a custom light detection apparatus and used it alongside wavelength-matched LED light sources emitting at 450 and 561 nm to characterize the absolute power of a series of NanoLuc and firefly luciferase solutions, respectively. This approach revealed that these two common luciferases produce 3.72 × 10-18 and 7.25 × 10-20 watts/molecule, respectively. Components of these luminescent solution calibrants are commercially available and produce stable bioluminescent signals over 2-5 min, enabling any luminometer to be calibrated for power measurements of bioluminescence emitted by these two luciferases in units of watts or photons per second.

Simultaneous inhibition of DNA-PK and PolÏ´ improves integration efficiency and precision of genome editing.

Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (PolÏ´). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.

Development of a rapid, simple, and sensitive point-of-care technology platform utilizing ternary NanoLuc.

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.

Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides.

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 (ß9 and ß10) 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.

Design, synthesis, and structure-activity relationship studies of thiophene-3-carboxamide derivatives as dual inhibitors of the c-Jun N-terminal kinase.

We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. Intriguingly, the compounds have a dual inhibitory activity by functioning as both ATP and JIP mimetics, possibly by binding to both the ATP binding site and to the docking site of the kinase. Several of such novel compounds display potent JNK inhibitory profiles both in vitro and in cell.

Identification of a new JNK inhibitor targeting the JNK-JIP interaction site.

JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics.

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. 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.

CDK Family PROTAC Profiling Reveals Distinct Kinetic Responses and Cell Cycle-Dependent Degradation of CDK2.

Targeted protein degradation using heterobifunctional proteolysis-targeting chimera (PROTAC) compounds, which recruit E3 ligase machinery to a target protein, is increasingly becoming an attractive pharmacologic strategy. PROTAC compounds are often developed from existing inhibitors, and assessing selectivity is critical for understanding on-target and off-target degradation. We present here an in-depth kinetic degradation study of the pan-kinase PROTAC, TL12-186, applied to 16 members of the cyclin-dependent kinase (CDK) family. Each CDK family member was endogenously tagged with the 11-amino-acid HiBiT peptide, allowing for live cell luminescent monitoring of degradation. Using this approach, we found striking differences and patterns in kinetic degradation rates, potencies, and Dmax values across the CDK family members. Analysis of the responses revealed that most of the CDKs showed rapid and near complete degradation, yet all cell cycle-associated CDKs (1, 2, 4, and 6) showed multimodal and partial degradation. Further mechanistic investigation of the key cell cycle protein CDK2 was performed and revealed CDK2 PROTAC-dependent degradation in unsynchronized or G1-arrested cells but minimal loss in S or G2/M arrest. The ability of CDK2 to form the PROTAC-mediated ternary complex with CRBN in only G1-arrested cells matched these trends, despite binding of CDK2 to TL12-186 in all phases. These data indicate that target subpopulation degradation can occur, dictated by the formation of the ternary complex. These studies additionally underscore the importance of profiling degradation compounds in cellular systems where complete pathways are intact and target proteins can be characterized in their relevant complexes.

Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors.

A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site.