RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site.

Area of Science:

• Oncology
• Molecular Biology
• Protein Engineering

Background:

• RAS mutations are key drivers in many cancers, but developing effective inhibitors is challenging due to difficulties in identifying druggable pockets.
• Current therapeutic strategies targeting RAS proteins are limited, necessitating novel approaches for cancer treatment.

Purpose of the Study:

• To identify and characterize novel RAS-binding Affimer proteins as potential inhibitors of RAS signaling.
• To explore the potential of Affimer proteins in revealing previously uncharacterized druggable conformations of RAS.
• To assess the utility of Affimers as tools for pharmacophore identification in drug discovery for RAS-related cancers.

Main Methods:

• Affimer protein selection and characterization for RAS binding.
• Biochemical assays to assess inhibition of nucleotide exchange and downstream signaling.
• NanoBRET assays to study competitive binding with small molecules and identify pharmacophores.
• Structural analysis to understand Affimer-RAS interactions and identify druggable pockets.

Main Results:

• Two RAS-binding Affimer proteins, K3 and K6, were identified with distinct isoform and mutant profiles.
• Affimer K6 binds to the SI/SII pocket, while Affimer K3 inhibits the SII region, revealing a druggable SII/α3 pocket in wild-type RAS.
• Competitive NanoBRET assays demonstrated the potential of Affimers to identify pharmacophores for RAS-targeted drug discovery.

Conclusions:

• RAS-binding Affimer proteins represent a novel class of inhibitors for RAS-driven cancers.
• Affimers can uncover cryptic druggable pockets in challenging protein targets like RAS.
• This approach holds significant promise for developing therapeutics against historically undruggable proteins.