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 mailto [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 mailto [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)
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

File(s) associated to this reference

Fulltext file(s):

FileCommentaryVersionSizeAccess
Limited access
Manoharan_Analbiochem2019_Accepted manuscript.pdfAuthor postprint2.68 MBRequest a copy

Bookmark and Share SFX Query

All documents in ORBilu are protected by a user license.