title: Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor.
abstract: BACKGROUND
  Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds.
  
  
  RESULTS
  A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods.
  
  
  CONCLUSIONS
  Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
authors.affiliation: Department of Molecular & Cellular Pharmacology, Abbott Laboratories, Worcester, MA, USA. maria.argiriadi@abbott.com
authors.forename: Maria A
authors.initials: MA
authors.lastname: Argiriadi
authors.forename: Eric R
authors.initials: ER
authors.lastname: Goedken
authors.forename: David
authors.initials: D
authors.lastname: Banach
authors.forename: David W
authors.initials: DW
authors.lastname: Borhani
authors.forename: Andrew
authors.initials: A
authors.lastname: Burchat
authors.forename: Richard W
authors.initials: RW
authors.lastname: Dixon
authors.forename: Doug
authors.initials: D
authors.lastname: Marcotte
authors.forename: Gary
authors.initials: G
authors.lastname: Overmeyer
authors.forename: Valerie
authors.initials: V
authors.lastname: Pivorunas
authors.forename: Ramkrishna
authors.initials: R
authors.lastname: Sadhukhan
authors.forename: Silvino
authors.initials: S
authors.lastname: Sousa
authors.forename: Nigel St John
authors.initials: NS
authors.lastname: Moore
authors.forename: Medha
authors.initials: M
authors.lastname: Tomlinson
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Voss
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.forename: Neil
authors.initials: N
authors.lastname: Wishart
authors.forename: Kevin
authors.initials: K
authors.lastname: Woller
authors.forename: Robert V
authors.initials: RV
authors.lastname: Talanian
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D015195:Drug Design; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D007191:Indazoles / Q000737:chemistry* / Q000494:pharmacology*; D053612:Janus Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017433:Protein Structure, Secondary; D059748:Proteolysis / Q000187:drug effects; D013329:Structure-Activity Relationship; D053634:TYK2 Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D007191:Indazoles; D047428:Protein Kinase Inhibitors; D053612:Janus Kinases; D053634:TYK2 Kinase