Fluorescence polarization assays in high-throughput screening and drug discovery: a review

Hall, Matthew D.; Peryea, Tyler; Braisted, John C.; Jadhav, Ajit; Simeonov, Anton; Coussens, Nathan P.

doi:10.1088/2050-6120/4/2/022001

Cited by 176 publications

(151 citation statements)

References 109 publications

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“…The accurate determination of thermodynamic parameters of molecular interactions is performed by fast, but superficial high-throughput (HTP) methods. In the literature several HTP approaches are applied such as co-immunoprecipitation [2], yeast two hybrid and spot assays [3], pull-down assay [4], holdup assay [5] and direct fluorescence polarization/anisotropy [6]. In direct fluorescence polarization (FP) experiments, a fluorescent probe (usually a labeled peptide) is titrated with a globular partner.…”

Section: Introductionmentioning

confidence: 99%

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

Simon

Gógl

Ecsédi

et al. 2019

Preprint

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The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To assess their interactome, high-throughput, systematic analysis is indispensable, which allows one to get not only qualitative but quantitative insight into their protein-protein interactions (PPIs). We have chosen an unbiased assay, fluorescence polarization (FP) that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified (orphan). Our results demonstrate that in vitro FP assays are highly suitable for quantitative ligand binding studies of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family. Author summaryFunctional similarity among a protein family can be essential in order to understand proteomic data, to find biomarkers, or in inhibitor design. Proteins with similar functions can compensate the loss-offunction of the others, their expression can co-vary under pathological conditions, and simultaneous targeting can lead to better results in the clinics. To investigate this property one can use sequencebased approaches. However, this path can be difficult. In the case of the vertebrate specific, evolutionary young, S100 family, phylogenetic approaches lead to ambiguous results. To overcome this problem, we applied a high-throughput biochemical approach to experimentally measure the binding affinities of a large number of S100 interactions. We performed unbiased fluorescence polarization assay, involving the complete human S100ome (20 paralogs) and 13 known interaction partners. We used this measured 20x13 (260) protein-protein interaction array to reveal the functional relationships within the family. Our work provide a general framework for studies focusing on phenotype-based domain classification.

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Section: Introductionmentioning

confidence: 99%

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

Simon

Gógl

Ecsédi

et al. 2019

Preprint

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“…We have demonstrated that BI is an easily implementable local strategy, within the reach of current experimental techniques, that performs quasioptimally. The identification of quantum change points demonstrated in our experiment might be applicable in many practical situations, such as identifying domain walls in condensed matter systems [34] and detecting the changes of fluorescence polarization in some biological processes [35]. Our work can also be extended to high dimensional quantum states, mixed states, and multiple change points.…”

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confidence: 73%

Experimentally detecting a quantum change point via the Bayesian inference

Huang

Tang

et al. 2018

Phys. Rev. A

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Detecting a change point is a crucial task in statistics that has been recently extended to the quantum realm. A source state generator that emits a series of single photons in a default state suffers an alteration at some point and starts to emit photons in a mutated state. The problem consists in identifying the point where the change took place. In this work, we consider a learning agent that applies Bayesian inference on experimental data to solve this problem. This learning machine adjusts the measurement over each photon according to the past experimental results and finds the change position in an online fashion. Our results show that the local-detection success probability can be largely improved by using such a machine learning technique. This protocol provides a tool for improvement in many applications where a sequence of identical quantum states is required.

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“…In DSF, changes in protein stability due to small‐molecule binding are quantified by assessing thermal shifts in the presence of a dye, such as SYPRO® Orange, which becomes more fluorescent when bound to unfolded proteins. Applications of both FP and DSF in high‐throughput screening (HTS) and drug development are well known and have been thoroughly reviewed …”

Section: Figurementioning

confidence: 99%

Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening

2019

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High‐throughput small‐molecule screening in drug discovery processes commonly rely on fluorescence‐based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false‐negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment‐sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside‐based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment‐based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine‐based probe used for this high‐throughput screen has a KD value of 7.2 μm with the TcdB GT and shows a >30‐fold increase in fluorescence intensity upon binding. The ES‐based probe assay is benchmarked against two other screening approaches.

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Fluorescence polarization assays in high-throughput screening and drug discovery: a review

Cited by 176 publications

References 109 publications

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

Experimentally detecting a quantum change point via the Bayesian inference

Deploying Fluorescent Nucleoside Analogues for High‐Throughput Inhibitor Screening

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