Switch II Pocket Inhibitor Allosterically Freezes KRAS^G12D Nucleotide-binding Site and Arrests the GTPase Cycle

Area of Science:

• Molecular Biology
• Oncology
• Biochemistry

Background:

• KRAS G12D mutations are common drivers in many cancers.
• KRAS G12D inhibitors like MRTX1133 show promise but require mechanistic understanding.
• Understanding MRTX1133's action is crucial for cancer therapy and resistance prediction.

Purpose of the Study:

• To elucidate the molecular mechanisms of KRAS G12D inhibition by MRTX1133.
• To characterize the biochemical and biophysical effects of MRTX1133 binding.
• To identify potential resistance mechanisms against MRTX1133.

Main Methods:

• Hydrogen/deuterium exchange mass spectrometry to assess protein conformational changes.
• Biochemical assays to study GTPase cycle activity (nucleotide exchange and hydrolysis).
• Analysis of KRAS-effector (BRAF) interactions.

Main Results:

• MRTX1133 binding to KRAS G12D induces conformational rigidification, stabilizing nucleotide-binding regions.
• The inhibitor completely arrests the KRAS G12D GTPase cycle, blocking both nucleotide exchange and GTP hydrolysis.
• MRTX1133 attenuates KRAS G12D interaction with BRAF, but binding to Switch I allows partial reversal of inhibition.
• MRTX1133 overcomes mutant-specific KRAS G12D interactions with BRAF.

Conclusions:

• MRTX1133 allosterically 'freezes' KRAS G12D, arresting its GTPase cycle and providing a mechanistic basis for its efficacy.
• The study offers insights into the action of Switch II pocket (SII-P) inhibitors.
• This work provides a framework for understanding MRTX1133 efficacy and potential acquired resistance in KRAS G12D-driven cancers.