Roger Bossé

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.

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.

Current Screens Based on the AlphaScreen Technology for Deciphering Cell Signalling Pathways.

Global deciphering of signal transduction pathways represents a new challenge of the post-genomic era. However, for the majority of these signaling pathways the role(s), the function(s) and the interaction(s) of the signaling intermediates remain to be characterized in an integrated fashion. The global molecular study of cell signaling pathways and networks consequently requires sensitive, robust technologies which may allow in addition multi-parallel and highthroughput applications. The Alphascreen™ technology, relying on a bead-based homogenous approach, constitutes a valuable tool to detect and quantify a wide range of signaling events such as enzymatic activities or biomolecular interactions. In this article, we exhaustively review the literature and report the broad spectrum of Alphascreen™-based applications in the study of signal transduction pathways.

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. (Journal of Biomolecular Screening 2003:191-197)

Homogeneous and Nonradioactive High-Throughput Screening Platform for the Characterization of Kinase Inhibitors in Cell Lysates

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.

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.

Development of Nonseparation Binding and Functional Assays for G Protein-Coupled Receptors for High Throughput Screening: Pharmacological Characterization of the Immobilized CCR5 Receptor on FlashPlate(r)

G protein-coupled receptors (GPCRs) represent a very important class of drug targets. The development of microformatted nonseparation assays constitute a key step in the process of assay development for high throughput drug screening (HTS). We have developed a microformatted nonseparation assay for membrane preparations containing the CCR5 GPCR using FlashPlate® microplates (Packard Instrument Company, Meriden, CT). The pharmacodynamic (radioligand-binding) and functional (agonist-stimulated [35S]GTPγS binding) properties of this receptor observed in FlashPlate-based assays were compared with standard filtration assays. Saturation binding experiments performed using either assay platform revealed identical Kd for [125I]-MIP-1 β (0.7 nM). Comparable signal-to-noise ratios (SNR), similar affinities (Ki), and identical order of potency (RANTES ≅ MIP-1β > MIP-1α) were observed following competition binding assays in both platforms. In functional assays, the order of potency for different agonists were similar in both platforms with RANTES ≅ MIP-1β ≥ MIP-1α, which correspond to the relative affinities determined for the three ligands in competition binding experiments. Because similar pharmacologic properties were obtained in both FlashPlate microplates and standard filtration platforms, we conclude that FlashPlate microplates could provide a valuable nonseparation platform for primary and secondary HTS for this and possibly other GPCRs.

Phosphorylation of Serine Palmitoyltransferase Long Chain-1 (SPTLC1) on Tyrosine 164 Inhibits Its Activity and Promotes Cell Survival

Background: Increasing evidence points toward the role of tyrosine phosphorylation in the regulation of ER homeostasis. Results: BCR-ABL-mediated tyrosine phosphorylation of SPTLC1 at Tyr164 attenuates SPT activity, thus preventing ceramide-dependent cell death. Conclusion: Tyrosine phosphorylation is critical for regulating SPT activity in the ER. Significance: This is a novel mechanism of oncogenic protein kinase-dependent control of sphingolipid metabolism. In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr164 by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr164 to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.

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.

AlphaScreen®-Based Characterization of the Bifunctional Kinase/RNase IRE1α 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, IRE1α, 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 IRE1α. 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.