Characterization of the Humoral Immune Response to Porcine Epidemic Diarrhea Virus Infection under Experimental and Field Conditions Using an AlphaLISA Platform.

Coronavirus infections are a continuous threat raised time and again. With the recent emergence of novel virulent strains, these viruses can have a large impact on human and animal health. Porcine epidemic diarrhea (PED) is considered to be a reemerging pig disease caused by the enteropathogenic alphacoronavirus PED virus (PEDV). In the absence of effective vaccines, infection prevention and control through diagnostic testing and quarantine are critical. Early detection and differential diagnosis of PEDV infections increase the chance of successful control of the disease. Therefore, there is a continuous need for development of reduced assay-step protocols, no-wash, high-throughput immunoassays. This study described the characterization of the humoral immune response against PEDV under experimental and field conditions using a rapid, sensitive, luminescent proximity homogenous assay (AlphaLISA). PEDV IgG and IgA antibodies were developed toward the beginning of the second week of infection. PEDV IgG antibodies were detected for at least 16 weeks post-exposure. Remarkably, the serum IgA levels remained high and relatively stable throughout the study, lasting longer than the serum IgG response. Overall, AlphaLISA allows the detection and characterization of pathogen-specific antibodies with new speed, sensitivity, and simplicity of use. Particularly, the bridge assay constitutes a rapid diagnostic that substantially improves upon the "time to result" metric of currently available immunoassays.

Catalytic specificity of human protein tyrosine kinases revealed by Peptide substrate profiling.

Out of the 90 human protein tyrosine kinases, 81 were assayed with short peptides derived from well-characterized [CDK1(Tyr15), IRS1(Tyr983), and JAK1(Tyr1023)] or generic [polyGlu:Tyr(4:1) and poly-Glu:Ala:Tyr(1:1:1)] substrates. As expected, the CDK1 peptide is a substrate for all Src family kinases. On the other hand, some of the activities are novel and lead to a better understanding of the function of certain kinases. Specifically, the CDK1 peptide is a substrate for many of the Eph family members. Interestingly, profiling of nearly all the human protein tyrosine kinases revealed a distinct pattern of selectivity towards the CDK1 and IRS1 peptides.

AlphaScreen-based characterization of the bifunctional kinase/RNase IRE1alpha: a novel and atypical drug target.

Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the authors investigated and characterized the biological properties of a novel target, IRE1alpha, a bifunctional kinase/RNase stress sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology AlphaScreen to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1alpha. They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using AlphaScreen were biologically relevant. Preliminary characterization of assay robustness indicates that both AlphaScreen assays should be useful in HTS for the identification of IRE1 activity modulators.

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Expression and purification of PI3 kinase alpha and development of an ATP depletion and an alphascreen PI3 kinase activity assay.

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal His-tag and the p85alpha regulatory domain in Sf9 insect cells. The complex consisting of p110alpha and p85alpha was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110alpha/p85alpha. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110alpha/p85alpha and determined the IC(50) for wortmannin, a known PI3 kinase inhibitor. The IC(50) for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4, 5-biphosphate turned out to be the best substrate at a concentration of 50 microM. p110alpha/p85alpha underwent autophosphorylation most prominently at the p85alpha subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors.

The use of AlphaScreen technology in HTS: current status.

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions.Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems.Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.

Biochemical clustering of monomeric GTPases of the Ras superfamily.

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen and FlashPlate, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases.

AlphaScreen kinase HTS platforms.

Kinases represents one of the most important family of targets in high throughput drug screening. Tyrosine kinases and serine/threonine kinases are known to play key roles in signal transduction as well as in cell growth and differentiation. Intense screening campaigns are underway in all major pharmaceuticals and large biotech companies to find kinase inhibitors for the treatment of inflammatory diseases, immunological disorders and cancer. The present contribution describes models that were developed to produce kinase assays amenable to HTS using AlphaScreen. Because of the flexibility allowed by AlphaScreen, kinase assays can be developed using direct or indirect approaches. Tyrosine kinase assays are usually performed with a direct format involving generic anti-phosphotyrosine antibodies while serine/threonine kinase assays are performed with an indirect format where specific antibodies are captured using protein A conjugated Acceptor beads. Streptavidin-coated Donor beads are used to capture either generic (ex. poly GT) or specific biotinylated substrates. Herein, are presented different methods to perform screening for inhibitors acting on the soluble beta-insulin receptor tyrosine kinase (IRKD), and on p38, a member of the MAP kinase family.

Development of a versatile platform for nuclear receptor screening using AlphaScreen.

The interaction between nuclear receptors (NRs) and their coactivators, a key step in transcription regulation, requires a short consensus sequence called the LXXLL motif found in the coactivators' structure. Using the AlphaScreen technology, the authors have taken advantage of this receptor-coactivator interaction to develop a highly sensitive assay to identify and characterize compounds modulating NR activity. Estrogen and retinoic acid receptors were chosen as models to demonstrate the versatility of the AlphaScreen technology: (1) the assay can be designed using different antibodies to capture either full-length receptors or receptor domains that have been tagged, (2) the assay can differentiate between ligands that act as agonists or antagonists because only agonists will allow recruitment of the coactivator sequence-derived peptide, and (3) the assay gives the opportunity to screen for antagonists targeting the ligand-binding site or the dimerization interface between the receptor and the coactivator. Titration of the receptor and biotinylated peptide indicates that AlphaScreen is highly sensitive, requiring nanomolar concentration of reagents. Competition isotherms performed with known receptor antagonists demonstrate that the assay is a useful tool to rank the antagonists according to their order of potency. Overall, the results presented here indicate that the versatility, sensitivity, robustness, and ease of execution of the AlphaScreen NR assay will allow for efficient screening of NR modulators.