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.

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.

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 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.

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.

A Simple and robust automated kinase profiling platform using luminescent ADP accumulation technology.

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.

Evaluating the utility of a bioluminescent ADP-detecting assay for lipid kinases.

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.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy.

Aim: To investigate the effects of mutations in domain III of the hepatitis C virus (HCV) internal ribosome entry sequences (IRES) on the response of chronic HCV genotype 4a patients to interferon therapy.

Methods: HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain III was cloned and 15 clones for each patient were sequenced.

A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP.

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.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro.

Aim: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro.

Methods: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture.

Antisense oligonucleotide inhibition of hepatitis C virus genotype 4 replication in HepG2 cells.

Background: Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is desperately needed.

A homogeneous, nonradioactive high-throughput fluorogenic protein phosphatase assay.

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).

A homogeneous, nonradioactive high-throughput fluorogenic protein kinase assay.

Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, it is important to develop assay systems that are not only sensitive but also homogeneous, fast, simple, nonradioactive, and cost-effective.