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

Springer Science and Business Media LLC - 2009
Maria A Argiriadi1,2, Silvino Sousa1,3, David Banach2,1, Douglas Marcotte1,4, Tao Xiang5,1, Medha J Tomlinson3,1, Megan Demers6,1, Christopher Harris2,1, Silvia Kwak2,1, Jennifer Hardman2,1, Margaret Pietras3,1, Lisa Quinn1,7, Jennifer DiMauro1,5, Baofu Ni3,1, John Mankovich1,8, David W Borhani9,1, Robert V Talanian2,1, Ramkrishna Sadhukhan3,1
1Department of Biochemistry, Abbott Laboratories, Worcester, USA
2Department of Molecular Pharmacology, Abbott Laboratories, Worcester, USA
3Department of Molecular Cell Biology, Abbott Laboratories, Worcester, USA
4Department of Physical Biochemistry, Biogen Idec, Cambridge, USA
5Department of Process Sciences, Abbott Laboratories, Worcester, USA
6Raleigh, USA
7Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, USA
8Department of Biologics, Abbott Laboratories, Worcester, USA
9D. E. Shaw Research, New York, USA

Tóm tắt

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. 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. 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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