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Abstract The cyclic GMP-AMP synthase-stimulator of the interferon gene (cGAS-STING) signaling pathway is considered an essential pattern recognition and effector pathway in the natural immune system and is mainly responsible for recognizing DNA molecules present in the cytoplasm and activating downstream signaling pathways to generate type I interferons (IFN-I) and other inflammatory factors. STING, a crucial junction protein in the innate immune system, exerts an essential role in host resistance to external pathogen invasion. The DNA introduced by pathogens or tumors is recognized by the cytoplasmic nucleic acid receptor cGAS, and a second messenger, cGAMP, is generated using intracellular guanosine triphosphate (GTP) and adenosine triphosphate (ATP). Furthermore, cellular and extracellular cGAMP concentrations are also controlled by ENPP1, an enzyme that breaks down cGAMP to AMP and GMP. Therefore, the role of the cGAS-STING signaling pathway has generated great interest in inflammatory and cancer research. To advance our understanding of innate immune system and in particular the STING pathway, we have developed a homogeneous, bioluminescent cGAMP detection assay that is very sensitive and highly selective against other nucleotides, cyclic nucleotides, and dicyclic nucleotides. The assay can be also used to monitor the activity of cGAS and ENPP1 to enable the development of inhibitors of both enzymes which might be used for therapeutic applications.

Abstract 3′,5′-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5′-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

Abstract 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.

KRAS is one of the most heavily mutated oncogenes in cancer and targeting mutant KRAS with drugs has proven difficult. However, recent FDA approval of the KRAS G12C selective inhibitor sotorasib (AMG-510), has breathed new life into the drive to develop mutant KRAS inhibitors. In an effort to study RAS inhibitors in cells and identify new compounds that inhibit Ras signaling, western blotting and ELISA assays are commonly used. These traditional immunoassays are tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we applied Lumit immunoassay technology to analyze RAS signaling pathway activation and inhibition through the detection of phosphorylated ERK. The assay we developed was used to rank order potencies of allele specific inhibitors within cell lines harboring various activating KRAS mutations. An inhibition profile was obtained indicating various potencies and selectivity of the inhibitors, including MRTX-1133, which was shown to be highly potent against KRAS G12D signaling. MRTX-1133 had approximately 40 and 400 times less inhibitory potency against G12C and G12V mutant KRAS, respectively, while no inhibition of WT KRAS was observed. The potency of PROTAC compound LC-2 targeting selective degradation of KRAS G12C was also tested using the Lumit pERK immunoassay, and a maximal decrease in RAS signaling was achieved. Lumit immunoassays provide a rapid, homogeneous platform for detecting signaling pathway activation and inhibition. Our results demonstrate that this bioluminescent technology can streamline the analysis of signaling pathways of interest, such as RAS-dependent pathways, and be used to identify much needed inhibitors. The results further imply that similar assay designs could be applied to other signaling pathway nodes.

Abstract Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.

The lipid second messengers phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) and sphingosine 1-phosphate (S1P) are well recognized to play important roles in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. Disruption of lipid signaling pathways often leads to human cancers, making lipid kinases attractive drug targets. In order to develop novel drugs against these enzymes, an assay that monitors their activity and amenable to high-throughput scale for screening large number of compounds is essential. The newly developed ADP-Glo™ assay is such an assay that measures kinase activity of lipid kinases by detecting the formation of ADP using a highly robust and sensitive bioluminescence approach. We evaluated this technology for studying lipid kinases, class I PI3 kinases, and sphingosine kinases and we show that the assay exhibits good tolerance to different lipids substrates. It generates kinetic parameters for substrates and inhibitors similar to those reported in the literature using other published assay formats. The sensitivity and robustness of this assay allow the detection of 5 of substrate conversion with Z′ values >0.7 making it attractive for high-throughput screening (HTS) applications. It is noteworthy that ADP-Glo assay addresses the need for a single integrated platform to comprehensively measure all classes of lipid and protein kinases. The selected inhibitors of lipid kinases can be screened against the panel of desired protein kinases, making ADP-Glo assay a simple, inexpensive platform for HTS and profiling of lipid kinases. [ABSTRACT FROM AUTHOR]

ADP-Glo™ is a novel bioluminescent, homogeneous assay for monitoring ADP producing biochemical reactions and thus it is an ideal assay for detecting enzyme activity using a wide variety of substrates. It is a universal assay that can be used with protein kinases, lipid kinases, sugar kinases, and many more kinases as well as ATPases. Because of its high sensitivity, it is suitable for monitoring enzyme activities at very early substrate conversions requiring very low amount of enzymes. Furthermore, as the assay is applicable to a broad range of ATP and substrate concentrations, it is optimal for enzymes that require high ATP and substrate concentrations. This is critical since inhibitor potency has to be demonstrated at the cellular level where ATP is present at millimolar concentrations. ADP-Glo is performed in 2 steps upon completion of kinase reaction: a combined termination of kinase reaction and depletion of remaining ATP in the first step, and conversion of generated ADP to ATP and the newly produced ATP to light output using luciferase/luciferin reaction in the second step. The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the kinase activity. Due to its high signal to background and luminescent readout, this assay is less susceptible to generation of false hits and thus it is applicable to not only primary and secondary screening but also kinase profiling. [ABSTRACT FROM AUTHOR]

Kinases continue to be one of the most important targets in today’s drug discovery efforts. Following the identification of lead compounds through screening efforts, it is important to profile these leads against other kinases within that family, as well as from other families, to ascertain potential off-target effects. Because many kinase assays require the use of different substrates, optimization time and costs during profiling can be prohibitive. Here we demonstrate the versatility of a luminescent ADP accumulation assay, where one set of reagents can be used for a wide variety of kinases with differing Km appfor ATP and substrates. Assay sensitivity allows for the use of low enzyme concentrations and small percent ATP conversion levels while still maintaining high signal:background ratios. We have used a simple, inexpensive automated pipetting system to automate the entire process from enzyme optimization through generation of compound IC50values. Agreement with literature values proves this combination of chemistry and instrumentation provides a simple, yet robust solution for automated kinase profiling. [ABSTRACT FROM AUTHOR]

Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive assay that accurately measures their activities. The authors present a novel, homogeneous, and nonradioactive assay to measure the enzyme activity of low concentrations of several protein phosphatases (phosphoserine/phosphothreonine phosphatases and phosphotyrosine phosphatases). The assay is based on the use of fluorogenic peptide substrates (rhodamine 110, bis-phosphopeptide amide) that do not fluoresce in their conjugated form, which is resistant to cleavage by aminopeptidases. However, upon dephosphorylation by the phosphatase of interest, the peptides become cleavable by the protease and release the highly fluorescent-free rhodamine 110. The assay is rapid, can be completed in less than 2 h, and can be carried out in multiwell plate formats such as 96-, 384-, and 1536-well plates. The assay has an excellent dynamic range, high signal-to-noise ratio, and a Z′ of more than 0.8, and it is easily adapted to a robotic system for drug discovery programs targeting protein phosphatases. [ABSTRACT FROM AUTHOR]

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 ± 5 fmol [mean ± standard error of the mean] of cAMP per well) and reproducibility (Z′ of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferaseluciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Gαsor Gαiforms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50 effective concentration (EC50) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC50values for agonists and 50 inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Gαsand Gαi). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications. [ABSTRACT FROM AUTHOR]

The success of immunotherapy treatment in oncology ushered a new modality for treating a wide variety of cancers. However, lack of effect in some patients made it imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. It has been recently recognized that adenosine levels increase significantly in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressive and attenuating several effector T cell functions. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and produces AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. Towards the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. We have developed very sensitive and homogenous assays that enabled us to monitor the activity of both enzymes and demonstrate selectivity of known inhibitors as well as monoclonal antibodies. This should speed up screening for novel inhibitors that might lead to more effective cancer therapy.