A1 Refereed original research article in a scientific journal
High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors
Authors: Manoharan G.B., Kopra K., Eskonen V., Härmä H., Abankwa D.
Publisher: ACADEMIC PRESS INC ELSEVIER SCIENCE
Publication year: 2019
Journal: Analytical Biochemistry
Journal name in source: ANALYTICAL BIOCHEMISTRY
Journal acronym: ANAL BIOCHEM
Volume: 572
First page : 25
Last page: 32
Number of pages: 8
ISSN: 0003-2697
DOI: https://doi.org/10.1016/j.ab.2019.02.015(external)
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 sternness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel CaM-inhibitors as potential K-Ras sternness-signaling disruptors.A modulated time-resolved Forster resonance energy transfer (mTR-FRET)-assay was developed and bench-marked 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 Forster 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 sternness.
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 sternness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel CaM-inhibitors as potential K-Ras sternness-signaling disruptors.A modulated time-resolved Forster resonance energy transfer (mTR-FRET)-assay was developed and bench-marked 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 Forster 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 sternness.
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