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

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.Structure-based drug design (SBDD) can be an effective contributor to the identification and optimization of drug candidates by providing a structural rationale for the design of improved compounds. Protein crystallization, structure determination, and the rapid determination of multiple protein/ligand complexes can be expensive and time-consuming. Major variables include protein construct design, mutations, and post-translational modifications, the nature of protein impurities (chemical or conformational), the choice of ligands or even proteins for co-crystallization, and the crystallization conditions themselves. These variables represent an enormous matrix of experimental possibilities that is difficult or impossible to explore systematically. Despite these challenges, the availability of structural information at the preliminary stages of a drug discovery program is critical to maximize impact. Therefore, efficient methods developments, techniques and strategies to de