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See detailHigh-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors
Manoharan, Ganesh Babu UL; Kopra, Kari; Eskonen, Ville et al

in Analytical Biochemistry (2019), 572

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 ... [more ▼]

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. [less ▲]

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See detailFRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases
Manoharan, Ganesh Babu UL; Enkvist, Erki; Kasari, Marje et al

in Analytical Biochemistry (2015)

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See detailMixing two differently labeled substrates in one immobilized metal assay for phosphochemicals assay to improve data quality.
Jezequel-Sur, Sylvie; Murray, Justin; Lucumi Moreno, Edinson UL et al

in Analytical biochemistry (2007), 360(2), 312-4

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