An assay for human telomeric G-quadruplex DNA binding drugs

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• G-quadruplexes in human telomeres are crucial targets for cancer therapy.
• Inhibiting telomerase activity, often linked to G-quadruplex stabilization, offers a therapeutic strategy.
• High-throughput screening is essential for identifying potent G-quadruplex binding compounds.

Purpose of the Study:

• To develop and validate a novel, high-throughput assay for screening compounds with high affinity for human telomeric G-quadruplexes (HTG).
• To utilize a fluorescence displacement assay employing a novel thiazole orange-neomycin conjugate probe.

Main Methods:

• Development of a fluorescence displacement assay using a thiazole orange fluorescent reporter conjugated to neomycin.
• The probe combines thiazole orange's base stacking with neomycin's groove binding for high affinity.
• Assay validation for determining relative binding affinities of HTG ligands in a high-throughput format.

Main Results:

• The developed assay demonstrates high affinity for detecting HTG binders.
• The assay is robust and applicable in the presence of biologically relevant ions (K+ or Na+).
• Facilitates high-throughput screening for potential telomerase inhibitors.

Conclusions:

• The novel thiazole orange-neomycin probe assay is effective for high-throughput screening of human telomeric G-quadruplex binders.
• This assay represents a valuable tool for identifying novel anti-cancer drug candidates targeting telomerase.