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Anal Biochem. 2019 May 1;572:25-32. doi: 10.1016/j.ab.2019.02.015. Epub 2019 Feb 27.

High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors.

Author information

1
Cancer Cell Biology and Drug Discovery Group, Life Sciences Research Unit, University of Luxembourg, 4362, Esch-sur-Alzette, Luxembourg; Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520, Turku, Finland.
2
Materials Chemistry and Chemical Analysis, University of Turku, 20500, Turku, Finland.
3
Cancer Cell Biology and Drug Discovery Group, Life Sciences Research Unit, University of Luxembourg, 4362, Esch-sur-Alzette, Luxembourg; Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, 20520, Turku, Finland. Electronic address: daniel.abankwa@uni.lu.

Abstract

The KRAS gene is highly mutated in human cancers and the focus of current Ras drug development efforts. Recently the interface between the C-terminus of K-Ras and calmodulin (CaM) was proposed as a target site to block K-Ras driven cancer cell stemness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel CaM-inhibitors as potential K-Ras stemness-signaling disruptors. A modulated time-resolved Förster resonance energy transfer (mTR-FRET)-assay was developed and benchmarked against an identically designed fluorescence anisotropy (FA)-assay. In both assays, two CaM-binding peptides were labeled with Eu(III)-chelate or fluorescein and used as single-label reporter probes that were displaced from CaM upon competitor binding. Thus, peptidic and small molecule competitors with nanomolar to micromolar affinities to CaM could be detected, including a peptide that was derived from the C-terminus of K-Ras. In order to detect CaM-residue specific covalent inhibitors, a cell lysate-based Förster resonance energy transfer (FRET)-assay was furthermore established. This assay enabled us to measure the slow, residue-specific, covalent inhibition by ophiobolin A in the presence of other endogenous proteins. In conclusion, we have developed a panel of fluorescence-assays that allows identification of conventional and covalent CaM-inhibitors as potential disruptors of K-Ras driven cancer cell stemness.

KEYWORDS:

Calmodulin; Cancer stemness; FRET; Fluorescence anisotropy; KRAS; Z′-factor

PMID:
30825429
DOI:
10.1016/j.ab.2019.02.015

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