Probing the hras-1^Y i-motif with small molecules

Area of Science:

• Medicinal Chemistry
• Molecular Biology
• Genomics

Background:

• Non-B DNA structures, such as G-quadruplexes and i-motifs, are crucial regulators of gene expression.
• The HRAS oncogene promoter contains i-motif structures, which are less understood than G-quadruplexes.
• Targeting these atypical DNA structures offers a novel therapeutic strategy.

Purpose of the Study:

• To discover and characterize small organic compounds that selectively bind to the HRAS i-motif.
• To explore the structure-activity relationship (SAR) of these compounds.
• To understand the binding mechanism and its dependence on DNA conformation.

Main Methods:

• Small molecule microarray screening to identify potential binders.
• Surface plasmon resonance (SPR) and fluorescence titration to determine binding affinities.
• Chemical shift perturbation (CSP) to elucidate binding modes and conformational effects.

Main Results:

• Identified two lead compounds (1 and 2) with micromolar binding affinities to the HRAS i-motif.
• Developed analogues with submicromolar binding affinities.
• Demonstrated pH-dependent binding, confirming interaction with the folded i-motif.
• Observed distinct binding mechanisms: compound 1 altered i-motif structure, while compound 2 did not.

Conclusions:

• First report of small molecules directly interacting with the HRAS i-motif.
• Compounds exhibit selective and conformation-dependent binding.
• Potential for developing targeted therapies against HRAS-driven cancers by modulating i-motif structures.