Engineering subtilisin proteases that specifically degrade active RAS.

Area of Science:

• Biochemistry
• Molecular Biology
• Protease Engineering

Background:

• RAS proteins are critical signaling molecules.
• Mutated RAS drives approximately one-third of human cancers.
• Targeting active RAS presents a therapeutic opportunity.

Purpose of the Study:

• To engineer subtilisin proteases capable of degrading active RAS.
• To achieve high specificity for the RAS target sequence.
• To explore cofactor-dependent protease mechanisms.

Main Methods:

• Subtilisin active site modification for cofactor dependence (imidazole or nitrite).
• Engineering of protease sub-sites for substrate-cofactor binding linkage.
• In vitro assays, bacterial reporter systems, and human cell culture to assess protease activity.

Main Results:

• Engineered proteases demonstrate specific cleavage of active RAS in vitro.
• Protease activity leads to depletion of RAS levels in a bacterial reporter system.
• Degradation of RAS observed in human cell culture.

Conclusions:

• Novel cofactor-dependent proteases effectively and specifically degrade active RAS.
• The 2-step mechanism involving cofactor binding enables selective proteolysis.
• The fundamental design principles are adaptable for targeting other proteins.