| Reference : High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors |
| Scientific journals : Article | |||
| Life sciences : Biochemistry, biophysics & molecular biology | |||
| http://hdl.handle.net/10993/38999 | |||
| High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors | |
| English | |
Manoharan, Ganesh Babu  [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >] | |
| Kopra, Kari [University of Turku] | |
| Eskonen, Ville [University of Turku] | |
| Harma, Harri [University of Turku] | |
| Abankwa, Daniel [University of Luxembourg > Faculty of Science, Technology and Communication (FSTC) > Life Science Research Unit >] | |
| Feb-2019 | |
| Analytical Biochemistry | |
| Elsevier | |
| 572 | |
| 25-32 | |
| Yes (verified by ORBilu) | |
| International | |
| 0003-2697 | |
| 1096-0309 | |
| Atlanta | |
| FL | |
| [en] 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. | |
| http://hdl.handle.net/10993/38999 | |
| 10.1016/j.ab.2019.02.015 |
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