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

Argiriadi, Maria A.; Sousa, Silvino; Banach, David; Marcotte, Douglas; Tao Xiang; Tomlinson, Medha J.; Demers, Megan; Harris, Christopher; Kwak, Silvia; Hardman, Jennifer; Pietras, Margaret; Quinn, Lisa; DiMauro, Jennifer; Ni, Baofu; Mankovich, John; Borhani, David W.; Talanian, Robert V.; Sadhukhan, Ramkrishna
January 2009
BMC Structural Biology;2009, Vol. 9, Special section p1
Academic Journal
Background: Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed. Results: Our construct design strategy had four tactics: N- and C-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort. Conclusion: Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.


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