Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Structure-based drug design (SBDD) is crucial for guiding drug discovery programs.
• Robust methods for protein construct design, expression, purification, crystallization, and diffraction are essential for iterative inhibitor design.
• Previous structure-based methods for human MAPKAP kinase 2 (MK2) lacked reproducibility, necessitating improved approaches.

Purpose of the Study:

• To establish robust methods supporting SBDD for the oral anti-cytokine drug target, human MK2.
• To obtain high-quality diffraction data for a diverse set of lead compounds targeting MK2.
• To overcome limitations in reproducibility of existing MK2 structural methods.

Main Methods:

• Implemented a construct design strategy including N-/C-terminal variations, surface mutations, and activation loop modifications.
• Utilized high-throughput cloning and expression coupled with automated liquid handling for rapid crystallization screening.
• Developed a novel, customized robotic crystallization screen for MK2/inhibitor complexes.

Main Results:

• Generated 44 MK2 constructs and tested approximately 500 crystals for diffraction.
• Determined approximately 30 high-resolution MK2 structures, providing critical data for drug design.
• Achieved successful crystallization of MK2/inhibitor complexes in seven distinct crystal forms under various conditions.

Conclusions:

• Prioritizing constructs based on performance criteria and early, high-throughput exploration are key to SBDD success.
• Customized crystallization screens are valuable tools for structure determination.
• The developed methods significantly advanced the MK2 drug design effort through high-impact structural data.