Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT–phosphoprotein interaction

Abstract

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot-spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (Cterminal portion of BRCA1, a key tumor suppressor protein with various functions), and phosphorylated protein (Abraxas, BACH1, CtIP) implicated in DNA damage response and repair pathways, should prove useful in studies of BRCA1's role in cancer and to potentially sensitize tumors to chemotherapeutic agents. We developed and miniaturized to 1536-well format and 3 μL final volume a pair of fluorescence polarization (FP) assays utilizing fluorescein-and rhodaminelabeled pBACH1 fragment. In order to minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members were each assayed against both the fluorescein-and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations, following the qHTS paradigm (Inglese et al, PNAS, 103, 1147(2006). Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS we were able to confidently select a number of actives for further studies.

BRCA1-directed, enhanced and aberrant homologous recombination

“…Recently, a high-throughput assay based on fluorescence polarization to identify small molecules that bind to the BRCA1 BRCT domain was developed. 63,64 An initial screen of the NCI diversity database led to a single hit with an IC 50 of 10 μM. Later, a dual fluorescence screen of 75,000 compounds identified 16 inhibitors with the lowest IC 50 values in the single digit micromolar range.…”

A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction

“…In order to maintain robust fluorescence intensity signal with this fluorophore, the red probe concentration was increased slightly to 15 nM. In protein titration experiments, the FP signal change observed with the red-labeled peptide was higher, in the range of 180-190 mP, as previously experienced with this fluorophore [54] (Fig. 8A).…”

Protein–protein interactions as therapeutic targets in neuropsychopharmacology

“…Advances in our understanding of KOPr system plasticity will provide important insights regarding the role of opioid peptides systems in normal brain function and various psychiatric disorders. (Simeonov et al, 2008;Wells and McClendon, 2007). Much of the groundwork to suggest that targeting 'hot-spots' could result in either loss or gain of cellular function is found in the cancer field (Simeonov et al, 2008;Wells and McClendon, 2007).…”

“…(Simeonov et al, 2008;Wells and McClendon, 2007). Much of the groundwork to suggest that targeting 'hot-spots' could result in either loss or gain of cellular function is found in the cancer field (Simeonov et al, 2008;Wells and McClendon, 2007). For example, the interaction between the C-terminal domain of the breast cancer gene 1 (early onset; BRCA1) protein and BRCA1-associated carboxyl terminal helicase (BACH1) protein is essential for DNA damage-induced checkpoint control.…”

“…For example, the interaction between the C-terminal domain of the breast cancer gene 1 (early onset; BRCA1) protein and BRCA1-associated carboxyl terminal helicase (BACH1) protein is essential for DNA damage-induced checkpoint control. A competitive, high-throughput assay has allowed the identification of small molecule BRCA1-BACH1 inhibitors, which are currently being validated in cell-based assays and ultimately in preclinical studies to improve the efficacy of breast and ovarian cancer therapeutics (Simeonov et al, 2008).…”