Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study
1 Department of Biochemistry, Abbott Laboratories, Worcester, MA USA
2 Department of Molecular Pharmacology, Abbott Laboratories, Worcester, MA USA
3 Department of Molecular Cell Biology, Abbott Laboratories, Worcester, MA USA
4 Present address : Department of Physical Biochemistry, Biogen Idec, Cambridge, MA USA
5 Present address : Department of Process Sciences, Abbott Laboratories, Worcester, MA USA
6 Present address : 119 North Swain Street, Raleigh, NC 27601, USA
7 Present address : Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA USA
8 Present address : Department of Biologics, Abbott Laboratories, Worcester, MA USA
9 Present address : D. E. Shaw Research, New York, NY USA
BMC Structural Biology 2009, 9:16 doi:10.1186/1472-6807-9-16Published: 18 March 2009
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.