High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• The KRAS gene is frequently mutated in human cancers, making it a key target for cancer drug development.
• The interaction between K-Ras C-terminus and calmodulin (CaM) is a newly identified target for inhibiting K-Ras-driven cancer cell stemness.

Purpose of the Study:

• To develop high-throughput screening assays for identifying novel calmodulin inhibitors.
• To discover compounds that disrupt K-Ras-driven cancer cell stemness signaling.

Main Methods:

• Development of a modulated time-resolved Förster resonance energy transfer (mTR-FRET) assay and a fluorescence anisotropy (FA) assay.
• Utilized CaM-binding peptides labeled with Eu(III)-chelate or fluorescein as reporter probes.
• Established a cell lysate-based FRET assay to detect covalent CaM inhibitors.

Main Results:

• Both mTR-FRET and FA assays successfully detected peptidic and small molecule calmodulin inhibitors with nanomolar to micromolar affinities.
• A peptide derived from the K-Ras C-terminus was identified as a CaM binder.
• The cell lysate-based FRET assay identified ophiobolin A as a slow, residue-specific covalent inhibitor of CaM.

Conclusions:

• A panel of fluorescence-based assays has been successfully developed for identifying calmodulin inhibitors.
• These assays can distinguish between conventional and covalent inhibitors.
• The developed assays hold potential for discovering novel therapeutics targeting K-Ras-driven cancer stemness.