A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors
Tekijät: Manoharan G.B., Kopra K., Eskonen V., Härmä H., Abankwa D.
Kustantaja: ACADEMIC PRESS INC ELSEVIER SCIENCE
Julkaisuvuosi: 2019
Journal: Analytical Biochemistry
Tietokannassa oleva lehden nimi: ANALYTICAL BIOCHEMISTRY
Lehden akronyymi: ANAL BIOCHEM
Vuosikerta: 572
Aloitussivu: 25
Lopetussivu: 32
Sivujen määrä: 8
ISSN: 0003-2697
DOI: https://doi.org/10.1016/j.ab.2019.02.015
Tiivistelmä
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.
Turun yliopiston Tutkimustietojärjestelmän portaali