title: Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biochemistry, Abbott Laboratories, Worcester, MA USA. maria.argiriadi@abbott.com
authors.forename: Maria A
authors.initials: MA
authors.lastname: Argiriadi
authors.forename: Silvino
authors.initials: S
authors.lastname: Sousa
authors.forename: David
authors.initials: D
authors.lastname: Banach
authors.forename: Douglas
authors.initials: D
authors.lastname: Marcotte
authors.forename: Tao
authors.initials: T
authors.lastname: Xiang
authors.forename: Medha J
authors.initials: MJ
authors.lastname: Tomlinson
authors.forename: Megan
authors.initials: M
authors.lastname: Demers
authors.forename: Christopher
authors.initials: C
authors.lastname: Harris
authors.forename: Silvia
authors.initials: S
authors.lastname: Kwak
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hardman
authors.forename: Margaret
authors.initials: M
authors.lastname: Pietras
authors.forename: Lisa
authors.initials: L
authors.lastname: Quinn
authors.forename: Jennifer
authors.initials: J
authors.lastname: DiMauro
authors.forename: Baofu
authors.initials: B
authors.lastname: Ni
authors.forename: John
authors.initials: J
authors.lastname: Mankovich
authors.forename: David W
authors.initials: DW
authors.lastname: Borhani
authors.forename: Robert V
authors.initials: RV
authors.lastname: Talanian
authors.forename: Ramkrishna
authors.initials: R
authors.lastname: Sadhukhan
mesh_terms: D019943:Amino Acid Substitution; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D015195:Drug Design; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D000099190:MAP-Kinase-Activated Kinase 2