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Structure modeling, molecular screening and docking of mammalian AMPK and its plant homolog KIN10 for new ATP-competitive inhibitors Raevsky A.V.1, Kap ov P.A.2, B lume Ya.B.3
In stitu te of Food Biotechnology and G enomics NAS o f Ukra ine, Osipovskogo str., 2a , Kiev-123, 04123 , U kraine ra yevsky85 @gma il.com 1, ka rpov.p.a@gmail.com 2, yab lume@univ.kiev.u a
3

Recently, mammalian micro tubule cytoskeleton was identified as a sensitive target of AMPactivated protein kinase (AMPK).[PM ID:23316058] It was shown that phosphorylation of the microtubule (MT) plus end protein CLIP-170 by AMPK is required for MT dyn amics and the regulation o f directional cell migration.[1] Now, mammalian AMPK are the one of th e mo st in terestin g targets associated with MT growth and responsible fo r cell activation under metabolic stresses such as nutrien t starvation , heat shock, ischemia/h ypo xia, etc. [PMID:12203120] It serves as an energy sensor and is considered as p ro mising dru g target for treatmen t of typ e II d iabetes and obesity. [PM ID: 19273282] In present, our attention to AMPK is caused b y the role of th eir plant homologue - SnRK kinase in cytoskeleton regu lation . Sucrose nonfermenting 1-Related p rotein Kinase (SnRK) is homologous of SNF1 and AMP-activated protein kinases (AMPK), wh ich widely exists in plan t and in volves in a variety o f signaling pathways. Particularly, Sn RK1 plays important roles in the transcription regulation, signaling and plan t develop ment. [PM ID: 17766403 ] A self-regulation kinase do main in the N-terminal is the common structural characteristic o f SnRK protein kin ase family.[2 ] The region is a highly variable, and can interact with other protein. Compared with other protein kin ase, there is a conserved amino acid-th reonine in the activation region. Subunit of a p robab le heterotrimeric complex consisting of an alpha catalytic (K IN10 or KIN11 ) subunit, and a beta (KINB) and a gamma (KING or SNF4) n on-catalytic regu lato ry subunits. Is probable th at catalytic subunit of the prob able trimeric SnRK-related complex, may play a role in a signal transduction cascade regulating gene expression and carbohydrate metabo lism in higher plan ts [PMID : 1 0220464; 11387208]. Based on these d ata we decided to bu ild alph a subunit (KIN10) and identify probable ATP-co mpetitive inh ib itors of this plant kinase. First o f all a template structure of AMPK fro m Rattus norvegicus [PDB ID 2Y94] was derived from Pro tein Data Bank. Basing on the sequence (Un iProt Q38997) of Kin 10 from Arabodopsis thaliana a co mplete 3D-structure model was reconstructed. Next part o f preparation assumed several


steps of energy minimization and structure relaxation . In itially our model was optimized du ring 20 ns with Generalized Bo rn Implicit Solven t calculation method of mo lecu lar dyn amics in Gromacs 4.5 p ackage to increase a nu mber o f confo rmation al states. The MD trajectory was an alysed and clustered in 15 separate groups basin g on the RMSD values with an average structure for each cluster. After validation of th e structures with Molprobity server one o f th em was selected. It was relaxed during 80 ns in exp licit solven t en vironmen t with amber99sb forcefield . RMSF for separate residues was calcu lated with g_ rmsf tool. The con fo rmation of active site can be in open o r closed state so it had to be op timized for screening. To cope with this task a molecule of staurosporine from mamalian complex [2Y94 ] was docked in the active site o f relaxed KIN10 model. For this pu rposes we app lied Go ldSco re/ASP pro tocol of CCDC Gold 5.0 software. To estimate stability o f the ligand pose a short molecular dyn amics simulation of 5 n s was carried ou t for bo th mamalian and p lant kinase complexes with a ligand. A topo lo gy file fo r staurosporine was generated with Antechamber too l of Amb erTools 12 package. The comparison o f both dynamics was based on ligand and protein RMSD and number of hyd rogen bonds. Several frames of the most stab le complexes were extracted from the trajectory and visualized in PyMol 1.5. The last step assumed a screenin g search o f a lib rary (~ 3000 compounds) against p lant homo logue to find pu tative inhibitors o f KIN10. Dock 6 was used as a screening tool to find 96 compounds with conjectu ral inhibito ry activity and CCDC Gold was applied fo r more exh austive docking to reduce this number to the end point number of 7 compounds. All these co mpounds are fallin g in the volume of a staurosporine, h ave good enough scores for the next stage of the study as prospective AMPK inhibito rs. 1 ) Q.A. Acton (2012 ) Advances in Cytoskeleton Research and App lica tion. 2011 Ed ition. Scho larlyEd itions. ­ 100 p. 2 ) D. Xue-Fei; C. Na; W. Li; Z. Xiao-Cui; Q. Bo; Li Tian-Lai; Z. Guo -Lian g (2012) The Sn RK Protein Kinase Family and the Function o f SnRK1 Pro tein Kinase, In t. J. Agric. Bio l., 14: 5 75579.