Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange

Area of Science:

• Oncology
• Molecular Biology
• Drug Discovery

Background:

• Ras mutations drive numerous human cancers, yet targeted therapies remain limited.
• Inhibiting Ras nucleotide exchange is a potential therapeutic strategy requiring further mechanistic understanding.

Purpose of the Study:

• To describe DARPin K27, an antibody mimetic designed to inhibit Ras nucleotide exchange.
• To evaluate the efficacy and specificity of K27 as a Ras inhibitor.

Main Methods:

• Characterization of K27 binding affinity and selectivity for Ras GDP.
• Intracellular expression of K27 in HCT116 cells.
• Assessment of downstream signaling, including phosphorylated ERK levels.
• Evaluation of cell proliferation in soft agar assays.

Main Results:

• K27 demonstrated high affinity (4 nM Kd) for the inactive Ras GDP form, with selectivity confirmed by structural studies.
• Intracellular K27 expression reduced active Ras levels and inhibited downstream signaling pathways, notably phosphorylated ERK.
• K27 treatment slowed the growth of HCT116 cells in soft agar.
• The inhibitor showed consistent efficacy across various Ras isoforms, including wild-type and oncogenic mutants.

Conclusions:

• DARPin K27 is a potent, non-covalent inhibitor of Ras nucleotide exchange.
• K27 effectively reduces active Ras signaling and inhibits cancer cell growth.
• This antibody mimetic represents a promising new avenue for developing Ras-targeted cancer therapies.