The Use of AlphaScreen Technology in HTS: Current Status

Eglen, Richard M.; Reisine, Terry; Roby, Philippe; Rouleau, Nathalie; Illy, Chantal; Bossé, Roger; Bielefeld, Martina

doi:10.2174/1875397300801010002

Cited by 221 publications

(192 citation statements)

References 39 publications

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“…The bicycle and HECT were immobilized on streptavidin and anti-GST IgG coated donor and acceptor beads, respectively. In the Alpha assay, an optical signal is obtained when donor and acceptor are linked by ligands, and compounds that disrupt the binding result in a decreased signal (15). A counter screen using biotinylated GST to link the beads was used to eliminate compounds that interfere nonspecifically with the optical readout.…”

Section: Significancementioning

confidence: 99%

“…We found that these frequently targeted the E2 binding site and inhibited ligase activity in vitro. We then used Alpha screening technology (15) to identify molecules that disrupted the bicycle-Smurf2 interaction. We identified one compound, termed heclin (HECT ligase inhibitor), that inhibits a range of HECT ligases in vitro and in tissue culture cells, with IC 50 s in the low micromolar range.…”

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confidence: 99%

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Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

Proc. Natl. Acad. Sci. U.S.A.

129

133

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The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/ Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.inhibitor | ubiquitin | HECT ligase | phage display U biquitin ligases are involved in almost every important function of cells, including the removal of misfolded proteins, the regulation of signaling pathways, DNA repair, the cell cycle, and apoptosis (1-4). This broad range of functions is mediated by a large number of E3 ubiquitin ligases, which recognize a similarly large range of substrates. Ubiquitination begins with the ATP-dependent formation of a ubiquitin (Ub)-E2 enzyme thioester intermediate by the E1 enzyme. The E3 enzymes then transfer ubiquitin from this intermediate to a lysine residue on the substrate (3). There are two main classes of E3: the RING domain ligases and their relatives, of which there are several hundred in humans, and the HECT domain ligases, which number around 28 (5-7). Because of their diversity and numerous functions and the fact that some ligases are frequently overexpressed in cancer cells, ubiquitin ligases have attracted much attention as possible targets for therapeutic intervention (2,5,6,8).Despite this interest, it has proven challenging to make small molecule inhibitors of ubiquitin ligases. The action of these enzymes involves primarily protein-protein interactions among them, the Ub-E2 conjugate, and the substrate. For the RING enzymes, this is simultaneous, and the Ub moiety is transferred directly from E2 to substrate; for the HECT ligases, the interaction is sequential, with a thioester-linked Ub-E3 intermediate forming transiently. Such protein-protein interactions are considered difficult to block with small molecules. Added to this difficulty has been the complexity of the ubiquitin system, with ligases having many substrat...

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Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Mund

Lewis

Maslen

2014

Proc. Natl. Acad. Sci. U.S.A.

129

133

View full text Add to dashboard Cite

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“…1). When used in a homogenous AlphaLISA assay (Eglen et al 2008), this pair of antibodies detected a standard of Fc-EDA1 diluted in EDA-deficient serum with a sensitivity of about 0.1 ng/ml in as little as 2.5 µl of serum. Signals obtained in a panel of XLHED sera were consistently very low (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This light is quenched by a nearby Europium chelate that emits the final signal at 615 nm (Eglen et al 2008). This system is particularly suited for measures in sera, even hemolysed, because absorption spectra of hemoglobin and other serum proteins are low above 600 nm.…”

Section: Titles and Legends To Figuresmentioning

confidence: 99%

Ectodysplasin A in Biological Fluids and Diagnosis of Ectodermal Dysplasia

et al. 2016

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The TNF family ligand ectodysplasin A (EDA) is produced as two full-length splice variants, EDA1 and EDA2, that bind to EDA receptor (EDAR) and X-linked EDA receptor (XEDAR/EDA2R) respectively. Inactivating mutations in the genes EDA or EDAR genes cause hypohidrotic ectodermal dysplasia (HED) with malformation of teeth, hair and glands, while milder deficiencies affect teeth only. EDA acts early during development of ectodermal appendages as early as the embryonic placode stage, and also plays a role in adult appendage function. We detected three-to fourfold higher levels of circulating EDA in cord blood than in adult sera. A receptor binding-competent form of EDA1 was the main form of EDA, but a minor fraction of EDA2 was also found in fetal bovine serum. Sera of EDA-deficient patients contained either background EDA levels or low levels of EDA that could not bind to recombinant EDAR.Serum of a patient with a V262F missense mutation in EDA causing a milder form of X-linked HED (XLHED) contained low levels of EDA capable of binding to EDAR. In two mildly affected carriers, intermediate levels of EDA were detected, while a severely affected carrier had no active EDA in serum. Small amounts of EDA were also detectable in normal adult saliva.Finally, EDA could be measured in spots of wild-type adult or cord blood dried onto filter paper at levels significantly higher than in EDA-deficient blood. Measurement of EDA levels combined with a receptor-binding assay might be of relevance to aid diagnosis of total or partial EDA deficiencies.

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“…Alternative signal generation methods have been developed, such as electrochemiluminescence detection (Meso Scale Discovery), which provides a much greater dynamic range and sensitivity compared to HRP based signals (Zhao et al 2004). Fluorescent bead-based technologies, such as those developed by Luminex and PerkinElmer's AlphaScreen® are also alternatives to standard solid phase ELISAs (Kellar et al 2006;Eglen et al 2008). These are analogous to ELISA sandwich assays, but use suspended beads as the solid phase in the assay, rather than the plate surface.…”

Section: Introductionmentioning

confidence: 99%