A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

Zhang, Ji-Hu; Chung, Thomas D.Y.; Oldenburg, Kevin R.

doi:10.1177/108705719900400206

Cited by 6,176 publications

(6,213 citation statements)

References 8 publications

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“…An assay with ideal reproducibility displays a Z′ factor of 1, whereas a Z′ factor greater than 0.5 is considered acceptable for a good high-throughput assay. 22 The Z′ factor calculated for our fluorescence polarization assay is 0.89, confirming that these assay conditions are suitable for HTS.…”

supporting

confidence: 63%

Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

Lei

et al. 2015

ACS Med. Chem. Lett.

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Prolyl hydroxylase domain 2 (PHD2) enzyme, a Fe II and 2-oxoglutarate (2-OG) dependent oxygenase, mediates key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). PHD2 has been shown to have the therapeutic potentials for conditions including anemia and ischemic disease. Currently, many activity-based assays have been developed for identifying PHD2 inhibitors. Here we report an affinity-based fluorescence polarization method using FITC-labeled HIF1α (556−574) peptide as a probe for quantitative and site-specific screening of small molecule PHD2 inhibitors. KEYWORDS: PHD2, FITC-labeled probe, affinity-based, fluorescence polarization assay H ypoxia is linked to human diseases such as anemia and ischemia. 1,2 In humans, the response to hypoxia is mediated by up-regulation of the hypoxia inducible transcription factor (HIF). 3−5 HIF is a heterodimeric transcription factor composed of an oxygen-dependent α-subunit and constitutively expressed β-subunit. Under normoxic conditions, HIFα, the regulatory subunit of HIF dimer, is constitutively produced with a half-life of approximately 5 min. It is hydroxylated by prolyl hydroxylases1−3 (PHD1−3), recognized by von Hippel Lindau protein (pVHL), and then rapidly ubiquitylated and subsequently degraded by the 26S proteasome. 6 PHDs are members of the dioxygenase family that require O 2 , Fe II , and 2-oxoglutarate (2-OG) for their catalytic activity, which are responsible for the C4 trans hydroxylation of HIFα at Pro402 and Pro564 that initiates the path to protein degradation. 7 It is currently believed that PHD2 plays a dominant role in controlling the cellular HIFα levels. 8 Inhibitor of PHD2 has been pursued as a promising therapy for conditions including anemia and ischemic disease.To discover small molecules that can regulate PHD2 activity, many activity-based assays have been developed. The development of activity-based assay was based on the catalytic activity of PHD2, which utilizes 2-OG and oxygen as cosubstrates to catalyze the prolyl hydroxylations. 9−11 This property has led to the development of several generic activity-based assays, which detected the activity by measuring the ratio of HIFα peptide and its hydroxylated product, such as fluorescence-based assay using o-phenylenediamine, 12 MALDI-TOF MS, 13 AlphaScreen assay, 14 homogeneous time-resolved fluorescence assay, 15 and fluorescence polarization assay based on HIF-von Hippel− Lindau protein-Elongin B−Elongin C (VBC) interaction. 16 However, activity-based assays are not always well-suited to the initial stages of medicinal chemistry, for example, for fragmentbased screening, and are only possible when substrates are available. Recently, affinity-based assays that utilize nondenaturing electrospray ionization mass spectrometry (ESI-MS), 17 affinity selection mass spectroscopy assay (AS-MS), 15 or nuclear magnetic resonance (NMR) 18 technology have been developed for studying the binding of metal ions and small molecules with PHD2 protein. Amo...

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supporting

confidence: 63%

Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

Lei

et al. 2015

ACS Med. Chem. Lett.

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“…The Z'-factor is a widely accepted statistical parameter for evaluation and validation of HTS assays [36,37]. The Z'-factor describes an assay's ability to distinguish between the mean values for a positive and negative control, taking into account the data variability for both sample sets.…”

Section: Resultsmentioning

confidence: 99%

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

Grube

Roy

2017

RNA Biology

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Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

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“…In order to assess the reproducibility of the recorded data we have calculated Z´-factor for all the reporter cell lines in the Gal4/LBD reporter format (Table , ). Z´-factor exceeding 0.5 is commonly accepted criterion for the satisfactory assay quality for the HTS [30]. Four out of eight Gal4/LBD reporter assays exhibit Z´-factor in the range of 0.76 and 0.80 which is a sign of an excellent cell-based assay very well adapted to HTS.…”

Section: Resultsmentioning

confidence: 99%

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

Sedlák¹,

Paguio²,

Bartůněk

2011

CCHTS

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Steroid hormone receptors represent a major target in drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. Here we describe two panels of potent and selective luciferase reporter cell lines based on cells with low endogenous steroid receptor activity (U2OS). The panels contain reporter cell lines for estrogen receptors α and β, androgen, glucocorticoid, mineralocorticoid, and progesterone receptors. In the first panel, the activation of either synthetic, steroid response elements containing promoter or viral promoter is mediated by full-length steroid receptors. The second panel is based on the expression of the chimeric receptor, which was created by the replacement of the N-terminal part of the molecule by Gal4 DBD and that binds to multiple UAS sites in the reporter promoter. Both panels were extensively characterized by profiling 28 ligands in dose response manner in agonist and antagonist mode. We have analyzed and compared the responses to tested ligands from both panels and concluded that in general both systems generated similar qualitative response in terms of potency, efficacy, partial agonism/antagonism, mixed agonistic/antagonistic profiles and the rank of potencies was well conserved between both panels. However, we have also identified some artifacts introduced by the Gal4/LBD reporter assays in contrast to their full-length receptor reporter counterparts. Keeping in mind the advantages and drawbacks of each reporter format, these cell lines represent powerful and selective tools for profiling large compound libraries (HTS) and for detailed study of mechanisms by which compounds exert their biological effects.

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A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays

Cited by 6,176 publications

References 8 publications

Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

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