Utilization of Fluorescence Polarization and Time Resolved Fluorescence Resonance Energy Transfer Assay Formats for SAR Studies: Src Kinase as a Model System

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA®), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (μM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIA® assays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of Ki = 0.01nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.

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A Dual Readout Assay Based on Fluorescence Polarization and Time-Resolved Fluorescence Resonance Energy Transfer to Screen for RSK1 Inhibitors

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.b15-00808 ◽

2016 ◽

Vol 39 (4) ◽

pp. 547-555

Author(s):

Eun-mi Jeong ◽

Mi Young Lee ◽

Jeong Hyun Lee ◽

Byung Ho Lee ◽

Kwang-Seok Oh

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Fluorescence Polarization ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Fluorescence Resonance

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Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114526433 ◽

2014 ◽

Vol 19 (7) ◽

pp. 1060-1069 ◽

Cited By ~ 9

Author(s):

Nicolas Wyhs ◽

David Walker ◽

Hugh Giovinazzo ◽

Srinivasan Yegnasubramanian ◽

William G. Nelson

Keyword(s):

Energy Transfer ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Binding Domain ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Methylated Dna ◽

Domain Protein

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)–based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z′ factor = 0.58) emerged as a superior screening strategy compared with FP (Z′ factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET–based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.

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Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1–Nrf2 Protein–Protein Interaction

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220935816 ◽

2020 ◽

Vol 26 (1) ◽

pp. 100-112

Author(s):

Sumi Lee ◽

Dhulfiqar Ali Abed ◽

Lesa J. Beamer ◽

Longqin Hu

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Small Molecule ◽

Protein Interaction ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Protein Protein Interaction ◽

Nrf2 Protein

The transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), plays a major role in regulating the antioxidant defense system through the Kelch-like ECH-associated protein 1–Nrf2–antioxidant response element (Keap1–Nrf2–ARE) pathway. Small-molecule inhibitors targeting Keap1–Nrf2 protein–protein interaction (PPI) decrease the rate of Nrf2 degradation by the 26S proteasome and thus increase the intracellular level of Nrf2, which translocates into the nucleus, leading to upregulated expression of cytoprotective and antioxidant enzymes. Such inhibitors can be developed into potential preventive and therapeutic agents of diseases caused by oxidative damage. To more effectively identify promising Nrf2 activators through the inhibition of Keap1–Nrf2 PPI, a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed in this work by indirectly labeling the Keap1 Kelch domain protein with Tb-anti-His antibody as the donor and using, as the acceptor, fluorescein isothiocyanate (FITC)-labeled 9mer Nrf2 peptide amide, the same fluorescent probe that was used in an earlier fluorescence polarization (FP) assay. Assay conditions, including concentrations of the various components, buffer type, and incubation time, were optimized in the TR-FRET competition assay with known small-molecule inhibitors of Keap1–Nrf2 PPI. Under the optimized conditions, the Keap1–Nrf2 TR-FRET assay exhibited great sensitivity with a high dynamic range and considerable stability for as long as 5 h. The Z’ factor was determined to be 0.82, suggesting that the assay is suitable for high-throughput screening and lead optimization of inhibitors of Keap1–Nrf2 PPI. Furthermore, the TR-FRET assay is capable of differentiating potent inhibitors of Keap1–Nrf2 PPI down to the subnanomolar inhibition constant ( Ki) range.

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