G-quadruplex deconvolution with physiological mimicry enhances primary screening: Optimizing the FRET Melt² assay

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Non-B-DNA G-quadruplex (G4) structures are promising molecular targets for cancer therapeutics by modulating oncogenic transcription.
• Current G4-stabilizing compounds often lack cellular activity or in vivo efficacy due to challenges in predicting G4-mediated mechanisms.
• There is a critical need for improved predictive screening methods to identify compounds with both in vitro G4 activity and in vivo efficacy.

Purpose of the Study:

• To investigate the impact of physiological conditions on G-quadruplex (G4) formation and compound screening using the MYC promoter G4.
• To identify factors that influence the reliability of G4 screening assays and compound hit rates.
• To develop an enhanced screening assay, FRET Melt², for more accurate identification of G4-targeting drug candidates.

Main Methods:

• Examined the effects of varying annealing conditions (cooling rate, heat/cool cycles), co-solvents (glucose, glycerol, etc.), and nucleoplasm on MYC promoter G4 formation.
• Assessed G4 formation and compound screening under different buffer conditions, including the presence of potassium and glycerol.
• Modified the FRET Melt assay to create the FRET Melt² assay, incorporating insights from physiological G4 formation principles.

Main Results:

• Hit rates for G4-stabilizing compounds significantly decreased under conditions mimicking physiological environments (e.g., presence of potassium, glycerol, and multiple rapid annealing cycles).
• Observed discrepancies in compound activity (active, inactive, non-G4-mediated) correlated with specific screening conditions, highlighting assay limitations.
• Identified specific co-solvents and annealing protocols that negatively impact G4 screening accuracy.

Conclusions:

• Physiological conditions significantly alter G-quadruplex (G4) formation and compound screening efficiency, impacting the identification of potential therapeutics.
• The study underscores the limitations of current screening methods and the necessity of optimizing assays to reflect in vivo conditions.
• The developed FRET Melt² assay and understanding of G4 formation principles will enhance the discovery of effective G4-targeting drugs for cancer treatment.