Identification and characterization of binders to a cryptic and functional pocket in KRAS

Area of Science:

• Oncology
• Molecular Biology
• Drug Discovery

Background:

• RAS proteins are crucial for cell proliferation, and their activating mutations are common in cancer.
• Existing RAS-targeted therapies face resistance, necessitating the identification of new druggable sites.
• The small size and globular nature of RAS proteins have historically made them challenging drug targets.

Purpose of the Study:

• To identify novel targetable pockets in RAS proteins beyond existing drug-binding sites.
• To develop new therapeutic strategies to overcome acquired resistance to RAS-targeted drugs.
• To explore new approaches for targeting both active (GTP-bound) and inactive (GDP-bound) RAS states.

Main Methods:

• Utilized macrocyclic peptides mRNA and nanobody yeast display screening platforms.
• Conducted in vitro and cellular experiments using novel KM12 and KM12-AM nanobodies.
• Performed mutagenesis experiments for orthogonal validation of the discovered binding pocket.

Main Results:

• Discovered a novel, targetable ligand-induced pocket in RAS proteins.
• Identified KM12 and KM12-AM nanobodies that inhibit RAS by displacing cRAF via the cRAF CRD domain.
• Demonstrated that the discovered pocket allows simultaneous targeting of both GTP-bound and GDP-bound RAS states.
• Confirmed that the identified approach does not affect the SwII pocket, allowing for combinatorial therapies.

Conclusions:

• A new druggable pocket in RAS proteins has been identified, offering a promising avenue for cancer therapy.
• The discovered nanobodies provide a novel mechanism for RAS inhibition, potentially overcoming existing resistance.
• This approach enables simultaneous targeting of different RAS states and combinatorial strategies with existing drugs.