Homogeneous and bioluminescent biochemical and cellular assay for monitoring cGAMP and enzymes that generate and degrade cGAMP.

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).

A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

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 G-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.

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.

A Homogeneous Bioluminescent System to Monitor Cyclic Guanosine Monophosphate.

Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is mediated through highly regulated phosphodiesterase activities. cGMP metabolism has been targeted for the treatment of several diseases, including erectile dysfunction, hypertension, and heart failure.

Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets.

Traditional immunoassays to detect secreted or intracellular proteins can be tedious, require multiple washing steps, and are not easily adaptable to a high-throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. This bioluminescent immunoassay does not require washes or liquid transfers and takes less than 2 h to complete in a homogeneous "Add and Read" format.

High throughput bioluminescent assay to characterize and monitor the activity of SARS-CoV-2 methyltransferases.

The fast rate of viral mutations of SARS CoV-2 result in decrease in the efficacy of the vaccines that have been developed before the emergence of these mutations. Thus, it is believed that using additional measures to combat the virus is not only advisable but also beneficial. Two antiviral drugs were authorized for emergency use by the FDA, namely Pfizer's two-drug regimen sold under the brand name Paxlovid, and Merck's drug Lagevrio.

Profiling oncogenic KRAS mutant drugs with a cell-based Lumit p-ERK immunoassay.

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.

Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors.

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.

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels.

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.

A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2.

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE.

Assessing Pretreatment Effectiveness for Particulate, Organic and Biological Fouling in a Full-Scale SWRO Desalination Plant.

In this study, the removal of particulate, organic and biological fouling potential was investigated in the two-stage dual media filtration (DMF) pretreatment of a full-scale seawater reverse osmosis (SWRO) desalination plant. Moreover, the removal of fouling potential in two-stage DMF (DMF pretreatment) was compared with the removal in two-stage DMF installed after dissolved air floatation (DAF) (DAF-DMF pretreatment). For this purpose, the silt density index (SDI), modified fouling index (MFI), bacterial growth potential (BGP), organic fractions and microbial adenosine triphosphate (ATP) were monitored in the pretreatment processes of two full-scale SWRO plants.

Monitoring Biofouling Potential Using ATP-Based Bacterial Growth Potential in SWRO Pre-Treatment of a Full-Scale Plant.

Several potential growth methods have been developed to monitor biological/organic fouling potential in seawater reverse osmosis (SWRO), but to date the correlation between these methods and biofouling of SWRO has not been demonstrated. In this research, the relation between a new adenosine triphosphate (ATP)-based bacterial growth potential (BGP) test of SWRO feed water and SWRO membrane performance is investigated. For this purpose, the pre-treatment of a full-scale SWRO plant including dissolved air flotation (DAF) and two stage dual media filtration (DMF) was monitored for 5 months using BGP, orthophosphate, organic fractions by liquid chromatography coupled with organic carbon detection (LC-OCD), silt density index (SDI), and modified fouling index (MFI).

A homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation.

Monitoring cellular signaling events can help better understand cell behavior in health and disease. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple steps, and are not easily adaptable to high throughput screening (HTS). Here, we describe a new immunoassay approach based on bioluminescent enzyme complementation.

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39.

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.

Bioluminescent High-Throughput Succinate Detection Method for Monitoring the Activity of JMJC Histone Demethylases and Fe(II)/2-Oxoglutarate-Dependent Dioxygenases.

The modification of a diverse array of substrates by Fe(II)/2-oxoglutarate-dependent dioxygenases is central to the modulation of distinct biological processes such as epigenetics, hypoxic signaling, and DNA/RNA repair. Of these, JumonjiC domain-containing histone lysine demethylases (JMJCs) and prolyl hydroxylases are potential drug targets due to their relevance to human diseases. Thus, assays to interrogate this enzyme superfamily are needed to identify selective and potent inhibitors as leads for drug development and that could also be useful research tools.

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes.

A High-Throughput Bioluminescent Assay to Monitor the Deamidation of Asparagine and Isomerization of Aspartate Residues in Therapeutic Proteins and Antibodies.

Since the introduction of Herceptin and Rituximab in 1986, therapeutic antibodies have gained tremendous momentum in the treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions that may result in compromised bioactivity and safety profile.

A Flexible Workflow for Automated Bioluminescent Kinase Selectivity Profiling.

Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activities. However, cost and logistical limitations prevent profiling activities from being performed in-house. We describe the development of an automated and flexible kinase profiling workflow that combines ready-to-use kinase enzymes and substrates in convenient eight-tube strips, a bench-top liquid handling device, ADP-Glo Kinase Assay (Promega, Madison, WI) technology to quantify enzyme activity, and a multimode detection instrument.

A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins.

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process.

Methyltransferase-Glo: a universal, bioluminescent and homogenous assay for monitoring all classes of methyltransferases.

Aim: To develop a homogenous, nonradioactive, antibody-free and universal assay for diverse families of methyltransferases and monitor the activity of these enzymes in a high-throughput format.

Materials & Methods: The assay conditions are optimized for monitoring the enzymatic activity of a broad range of methyltransferases regardless of the chemical structure or nature of the enzyme substrate in a low- and high-throughput-formatted protocols. The assay detects S-adenosyl-L-homocysteine, the universal reaction products of all methyltransferases.

Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling.

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described.

Using Bioluminescent Kinase Profiling Strips to Identify Kinase Inhibitor Selectivity and Promiscuity.

The advancement of a kinase inhibitor throughout drug discovery and development is predicated upon its selectivity towards the target of interest. Thus, profiling the compound against a broad panel of kinases is important for providing a better understanding of its activity and for obviating any off-target activities that can result in undesirable consequences. To assess the selectivity and potency of an inhibitor against multiple kinases, it is desirable to use a universal assay that can monitor the activity of all classes of kinases regardless of the nature of their substrates.

A Homogenous Bioluminescent System for Measuring GTPase, GTPase Activating Protein, and Guanine Nucleotide Exchange Factor Activities.

GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators has been challenging due to paucity of convenient assays.

ADP-Glo: A Bioluminescent and homogeneous ADP monitoring assay for kinases.

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.