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Search of effective protein phosphatases inhibitors using nanochemical approaches and evaluation of their biological activity in silico Karpov P.A., Samo falo va D.A.*, Blume Ya.B.
Institute of Food Biotechnology and Genomics, Natl. Academy of Sci. of Ukraine, Osipovskogo str., 2a, Kyiv, 04123, Ukraine, e-mail: samofalova.dariya@gmail.com

Protein phosphorylat ion and dephosphorylat ion is an important regulatory mechanisms invo lved in the control of many cellular funct ions and, in particular, transcript ion, t ranslat ion, cell divisio n, lipid metabo lism, gluconeogenesis, muscle contraction, etc. [1] By reversing the phosphorylat ion of key regulatory proteins mediated by protein kinases, phosphatases serve as an important complement to kinases and att enuate activated signal transduction pathways. [1, 2]. Considering such fundamental role, it seems appropriate to have a set of co mpounds that select ively inhibits individual protein phosphatases activit y. [3]. Our strategic goal was structure-based design and discovery of novel select ive protein phosphatase inhibitors. In total, 63 experimentally resolved structures were found in Protein Data Bank, and we creat ed a local database, t hat store informat ion on Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae and Enterobacteria phage lambda protein phosphatase catalyt ic subunits. We performed spatial structure ho mology modeling of animal (12), fungi (10), plant (22), bacterial (1), protozoan (6) and viral (4) phosphatase catalyt ic subunit s of based on selected templates. In particular, using structures of known anima l prot ein phosphatases 1 and 2A, we performed ho mology modeling o f respect ive plant protein phosphatases. Built structure models was optimized (MD) and verified (Mo lPro bit y, PROCHECK, etc.). We have per formed search for known and potential (based on structure) protein phosphatase inhibitors and build up compound libraries for High-Throughput Screening (HTS) and docking. In PDB, it was found 10 serine-threonine and 11 tyrosine-specific PP in complexes wit h inhibitors. Analysis of protein-ligand co mplexes reveal structure of binding sites, role o f water mo lecules in a ligand binding (CCDC Relibase) and relevant pharmacophores (LigandScout). As there are significant difference in animal and plant phosphatomes, undoubted ly t here are considerable target variat ions. To answer this question, based on available data about human protein-ligand co mplexes (control), we performed an chemogeno mic analysis of specificit y o f known phosphatase inhibitors in Arabidopsis thaliana and Physcomitrella patens. Based on jo int clustering of exper imental and t heo ret ical binding sites profiles, we ident ify t he group o f plant


phosphatases forming co mmon clades wit h experimentally proven (PDB) pro tein-ligand complexes. So, we consider selected plant proteins as the most probable targets of stud ied inhibitors of mammalian protein phosphatases. To ident ify alternat ive ligand targets and binding sites, we carried out the blind docking (Hex 6.3) test for each o f studied catalyt ic subunit. Due to applicat ion o f GPU (CUDA), t he durat ion o f the blind docking has been reduced by more tha n 10 times. The potential binding sites were checked by a flexible docking in CCDC GOLD Suite v.5.1. These methods were applied to search for new PP1 and PP2A inhibitors. Select ive PP1 and PP2A inhibitor - okadaic acid (CID 446512) was used as a reference structure. Based on PubChem and ZINK struct ure search, were selected 26 perspective co mpounds. The affinit y o f the ligand was confirmed by molecular dock ing ( evaluat ion fu nct ions) and mo lecu lar dynamics ШG calculation). As a result, we selected five compounds (CID10437923, CID44288019, CID3053530, CID10930902, CID44451975), which had not previously been reported as potential PP1 and PP2A inhibitors. Ackno wledgements: This study was supported by NASU-STCU grant #5215: "Search of effect ive protein phosphatases inhibitors using nanochemical approaches and evaluation of their bio logical act ivit y in silico." 2011-2012.

1. F.A. Barr, P.R. Elliott, U. Gruneberg (2011) Protein phosphatases and the regulat ion o f mito sis, J Cell Sci, 124 (Pt 14): 2323-2334. 2. G.B. Moorhead, V. De Wever, G. Templeton, D. Kerk (2009) Evolution o f protein phosphatases in plants and animals, Biochem J, 417 (2): 401-409. 3. L. Cui, X.-P. He, L.-X. Gao, J. Li, G.-R. Chen (2013) Click Synt hesis o f Triazo lyl Phenylalaninyl and Tyrosinyl Derivat ives as New Protein Tyrosine Phosphatase Inhibitors, J of Heterocy Chem, 50 (3): 684­688.