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.

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure. We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family. With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein.ARIP4 is a nuclear protein and comprises 1466 amino acids. It interacts with AR in vitro and in cultured yeast and mammalian cells. ARIP4 can be labeled with 8-azido- [␥-32 P]ATP and exhibits DNA-dependent ATPase activity. Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner. With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation. In transient cotransfection assays, ARIP4 modulates AR function in a promoterdependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters. ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays. INTRODUCTIONThe androgen receptor (AR) belongs to the superfamily of nuclear receptors that are ligand-activated transcription factors capable of regulating transcription of genes containing appropriate response elements, usually within or around the proximal promoter regions (Beato et al., 1995;Quigley et al., 1995;Perlmann and Evans, 1997). After hormone binding, the receptors associate with their cognate DNA motifs and modulate transcription initiation. Nuclear receptors may interact directly with the basal transcription factors associated with RNA polymerase II, such as TFIIB (Ing et al., 1992;Blanco et al., 1995;Hadzic et al., 1995), TFIIF (McEwan and Gustafsson, 1997), and TFIIH (Lee et al., 2000), or elicit their actions indirectly via auxiliary regulatory proteins, called coactivators and corepressors (Torchia et al., 1998;Freedman, 1999;McKenna et al., 1999;Glass and Rosenfeld, 2000).DNA is folded in the nucleus into a tight chromatin structure that often renders important regulatory sequences inaccessible for sequence-specific transcription factors, including steroid receptors (Kingston et al., 1996;Näär et al., 2001). As a consequence, different chromatin-modifying complexes are required to counteract this repressive effect (Bjö rklund et al., 1999;Kingston and Narlikar, 1999;Lemon and Freedman, 1999;Vignali et al., 2000). The protein complexes can be classified into two main categories: 1) ATP-dependent chromatin-remodeling complexes, which use the energy of ATP hydrolysis to alter the association of histones with DNA; and 2) complexes that alter chromatin by covalent modification of its components. These modifications include histone acetylation, methylation, phosphorylation, and ADP-ribosylation. Yeast SWI/SNF was the first ATP-dependent complex shown to facilitate the function of gene regulatory proteins in a...

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