* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Supplementary Information (doc 662K)
Biochemical cascade wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Butyric acid wikipedia , lookup
MTOR inhibitors wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Western blot wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Biosynthesis wikipedia , lookup
Genomic library wikipedia , lookup
Signal transduction wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Lipid signaling wikipedia , lookup
Paracrine signalling wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Metalloprotein wikipedia , lookup
Expression vector wikipedia , lookup
Mitogen-activated protein kinase wikipedia , lookup
Point mutation wikipedia , lookup
Protein purification wikipedia , lookup
Biochemistry wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Peptide synthesis wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Proteolysis wikipedia , lookup
Discovery and development of neuraminidase inhibitors wikipedia , lookup
Supporting Information Centrosomal Kinase Nek2 Cooperates with Oncogenic Pathways to Promote Metastasis Tirtha K. Das4, Dibyendu Dana1, Suneeta S. Paroly1, Senthil K. Perumal2, Satyakam Singh3, Hugo Jhun1, Jay Pendse4, Ross L. Cagan4, Tanaji T. Talele3, Sanjai Kumar1,* 1 Department of Chemistry and Biochemistry, Queens College and the Graduate Center of the City University of New York, Queens, New York 11367-1597, USA 2 Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA 3 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY 11439, USA 4 Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029, USA *Corresponding author: Tel: 718-997-4120, Fax: 718-997-5531 Email: [email protected] 1. Abbreviations NMP - N-methyl-2-pyrrolidone; TIS – Triisopropylsilane; BOP - Benzotriazol-1yloxy)tris(dimethylamino)phosphonium Hydroxybenzotriazole; hexafluorophosphate; HoBT - 1- DTT – Dithiothreitol; DMSO - Dimethyl sulfoxide; BSA - Bovine serum albumin; 2. (a) The sequence alignments of serine/threonine kinase hNek2, and receptor tyrosine kinase EGFR and HER2 kinases is shown in Figure S.1 below. (b) Computational Validation of In Silico Docking Methodology Using Existing Nek2 Inhibitors: To ensure that our in silico docking methodology would yield a reasonable outcome, a two-pronged approach was undertaken. First, we validated the ability of our docking algorithm to reproduce the co-crystallized pose of the pyrroleindolinone Nek2 inhibitor. Indeed a good agreement between the docked and the crystal structure conformation was observed, as evident from the root mean square (rms) deviation values of 0.20 Å when heavy atoms were superimposed, and 1.14 Å, when heavy atoms along with hydrogen atoms were superimposed. This confirmed the reliability of the Glide docking procedure in reproducing the experimentally observed binding mode for Nek2 inhibitor and instilled confidence that the parameters set for Glide docking were reasonable to provide a meaningful insight into the predicted binding mode of EGFR and HER2 inhibitor within the active site of Nek2. To further validate our computational approach, we undertook a second approach. We did this to establish if our Glide docking methodology would be able to discriminate between the reported potent and weakly active Nek2 inhibitors. We therefore carried out in silico docking experiments on a “validation set” of 10 reported Nek2 inhibitors (For structure and their reported IC-50 values see Table S1). These included eight potent Nek2 inhibitors exhibiting IC-50 of 20 nM to 570 nM and two moderately potent Nek2 inhibitors with IC-50 of 1300 nM and 4900 nM. A good correlation in the reported experimental IC-50 values and their corresponding Glide Scores (reported in kCal/mol) validated the correctness of our docking algorithm. Notably, this investigation ranked four of the most potent Nek2 inhibitors reported to date as top hits with the Glidescores ranging from -6.9 to -8.1 kcal/mol (Table S1). This process instilled confidence in our virtual screening methodologies, and allowed us to move forward with the virtual screening of existing EGFR/HER2 inhibitors as potential Nek2 inhibitors. Table S1. Validation of Our Computational Docking Strategy Using 10 known Nek2 inhibitors Structures and Chemical Names of Known Sets of Glide-Score IC-50 Existing Nek2 Kinase Inhibitors (kcal/mol) (nM) S.N. O Cl N 1 HN -8.07 20 -6.93 25 -7.99 60 -7.53 130 N O H 5-(2-Chloro-acetyl)-3-(5-methyl-3H-imidazol4-ylmethylene)-1,3-dihydro-indol-2-one N N O S NH2 O O 2 F N F F 5-[6-(1-Methyl-piperidin-4-yloxy)-benzoimidazol-1-yl]-3[1-(2-trifluoromethyl-phenyl)-ethoxy]-thiophene-2-carboxylic acid amide O Cl N 3 HN N O H 5-(2-Chloro-acetyl)-3-(3H-imidazol-4-ylmethylene)-1,3-dihydroindol-2-one O Cl N 4 HN N O H 5-(2-Chloro-acetyl)-3-(2-ethyl-5-methyl-3H-imidazol -4-ylmethylene)-1,3-dihydro-indol-2-one O 5 N NH2 N N OH O -7.50 200 -7.70 310 -7.05 380 -6.95 570 O O 1-[3-Amino-6-(3,4,5-trimethoxy-phenyl)-pyrazin-2-yl] -2,3-dimethyl-piperidine-4-carboxylic acid S N 6 N H N O N NH N N O N-(3-{5-[2-(3-Morpholin-4-yl-phenylamino)-pyrimidin-4-yl] -imidazo[2,1-b]thiazol-6-yl}-phenyl)-2-phenyl-acetamide N N NH2 O O 7 O F F F N 4-[5-(3-Dimethylamino-propoxy)-benzoimidazol-1-yl] -2-[1-(2-trifluoromethyl-phenyl)-ethoxy]-benzamide N N O 8 NH2 O O F F F N 4-[5-(1-Methyl-piperidin-4-yloxy)-benzoimidazol-1-yl] -2-[1-(2-trifluoromethyl-phenyl)-ethoxy]-benzamide O O O O HO N 9 O -5.85 1300 -5.15 4900 N H2 N 4-[3-Amino-6-(3,4,5-trimethoxy-phenyl) -pyrazin-2-yl]-2-methoxy-benzoic acid O Cl N 10 HN N O 5-(2-Chloro-acetyl)-3-(2-ethyl-5-methyl-3H-imidazol4-ylmethylene)-1-methyl-1,3-dihydro-indol-2-one (c) Selection Criteria for Choosing Seven Potential Nek2 Inhibitory Candidates from Focused EGFR/HER2 Inhibitory Library: The presence of key interactions in docked structures previously reported for inhibitor-bound Nek2 structure was the primary criterion for assigning compounds a high score (1-3). Some of these key interactions included hydrogen bonding interaction with the backbone of Cys89 and Asp159, electrostatic interaction with Lys37, ionic interaction with Asp93, hydrophobic interaction with Met86, Phe148, and Tyr88, and packing of aromatic ring against α-T helix residues. This analysis resulted in selection of 14 compounds from the library for further evaluation. A visual inspection of these 14 virtual hits with respect to presence of substituents at the 4-, 6- and 7-positions (exception lapatinib) of the quinazoline or quinoline ring (hinge loop binding scaffold), Michael acceptor alone and/or capped with basic tertiary amine group, basic amine group substituent instead of Michael acceptor at 6-position of the quinoline or quinazoline ring resulted in selection of 7 representative compounds (See Table S2 below) for in-vitro screening. Table S2. Structures of the Potential Nek2-Inhibitory Lead Compounds Identified from the In Silico Screening of Existing EGFR/HER2 Inhibitors Glide-Score S.N. Structures and Chemical Name (kcal/mol) O N HN N O 1 N HN Cl -7.93 O NERATINIB N 4-Dimethylamino-but-2-enoic acid {4-[3-chloro-4-(pyridin-2-ylmethoxy)phenylamino]-3-cyano-7-ethoxy-quinolin-6-yl}-amide O N HN N 2 O N HN Cl -7.86 PELITINIB F 4-Dimethylamino-but-2-enoic acid [4-(3-chloro-4-fluorophenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide N N H N 3 O S O HN Cl O O LAPATINIB F [3-Chloro-4-(3-fluoro-benzyloxy)-phenyl]-(6-{5-[(2-methanesulfonyl-ethylamino)-methyl]-furan-2-yl}-quinazolin-4-yl)-amine -7.65 O O N H HN N 4 N HN O Cl -7.23 AFATINIB F 4-Dimethylamino-but-2-enoic acid [4-(3-chloro-4-fluorophenylamino)-7-[{(3S){tetrahydro-furan-3-yloxy}-quinazolin-6-yl]amide O N O N N HN 5 O CANERTINIB HN Cl -7.38 F N-[4-(3-Chloro-4-fluoro-phenylamino)-7(3-morpholin-4-yl-propoxy)-quinazolin-6-yl]-acrylamide O O 6 N N N O HN GEFITINIB Cl -7.30 F (3-Chloro-4-fluoro-phenyl)-[7-methoxy-6(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine 7 O O O O ERLOTINIB N N HN -7.52 [6,7-Bis-(3-methoxy-propoxy)-quinazolin4-yl]-(3-ethynyl-phenyl)-amine 3. Expression of Active Wild Type Human Nek2 Kinase Human Nek2 plasmid was a generous gift from Prof. Andrew M. Fry, University of Leicester. The supplied plasmid contained Nek2 cDNA in a pGEM-3ZF (-) vector with ampicillin resistant cassette as the selection marker. Following plasmid amplification in DH5α cells, we prepared and isolated the plasmid. Human Nek2 plasmid was a generous gift from Prof. Andrew M. Fry, University of Leicester. The supplied plasmid contained Nek2 cDNA in a pGEM-3ZF (-) vector with ampicillin resistant cassette as the selection marker. Following plasmid amplification in DH5α cells, we prepared and isolated the plasmid. The gene encoding the wild-type Nek-2 kinase was PCR amplified from a pGEM plasmid containing the Nek2 gene CAGGACGTCATATGCCTTCCCGGGCTGAGGACTATG-3′ using 5′- and 5′- GACATGCCTCGAGGCGCATGCCCAGGATCTGTCTGC-3′ as the forward and reverse primers respectively. The underlined sequences denote the NdeI and XhoI restriction sites, respectively, which were used to clone the PCR amplified product into the pET32a expression vector. The C-terminally (His)6-tagged Nek2 enzyme was achieved by incorporating the Nek2 gene into pET32a that supplies a hexahistidine tag at the C-terminus of the protein connected to the open reading frame of Nek2 by a two residue amino acid linker. The recombinant plasmids were transformed into E. coli XL-1 Blue cells and clones were selected on nutrient agar plates containing kanamycin (30 μg/ml). The identity of the clones was verified by dideoxy sequencing. The gene encoding the wild-type lambda protein phosphatase (LPP2) was PCR amplified from the lambda genomic GATATACATATGCGCTATTACGAAAAAATTGA-3′ DNA using and 5′5′- CCAGACTCGAGTCATGCGCCTTCTCCCTGTACCTG-3′ as the forward and reverse primers respectively. The underlined sequences denote the NdeI and XhoI restriction sites, respectively, which were used to clone the PCR amplified product into the pET22b expression vector. The recombinant plasmids were transformed into E. coli XL-1 Blue cells and clones were selected on nutrient agar plates containing ampicillin (50 μg/ml). The identity of the clones was verified by dideoxy sequencing. The recombinant Nek2 protein was expressed under IPTG induction by a slight modification of an earlier published protocol (4). The vectors containing the Nek2 and LPP2 genes were transformed into BL21 (RIPL) codon plus E. coli cells and single colony selected on a plate containing kanamycin (30 μg/ml) and ampicillin (50 μg/ml) was used to inoculate a 50 ml Luria Broth (LB) overnight culture. A 10 ml of the overnight culture was diluted into two 1L culture in two liter flasks containing kanamycin (30 μg/ml), chloramphenicol (30 μg/ml) and ampicillin (50 μg/ml). The cells were grown at 37 °C until the absorbance reached ~1.0 at 600 nm. The cells were then cooled down to 18 °C and 0.2 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) and continued to grow overnight. The cells were harvested by ultracentrifugationa at 5000 rpm at 4 °C. The harvested cells were then resuspended in 50 ml of lysis buffer containing 20 mM Tris-HCl (pH: 7.5), 300 mM NaCl, 5 mM imidazole in the presence of an EDTA-free protease inhibitor cocktail tablet and lysed by sonication. The cell debris was removed by ultracentrifugation at 15000 rpm at 4 °C for 30 min. The cell-free lysate thus obtained was loaded on to a Ni-NTA column, equilibrated with lysis buffer. The protein was eluted with 100 ml of a linear gradient of imidazole (5-150 mM). The protein fractions were separated on a 12% SDS-PAGE gel, and the fractions containing the protein were pooled together and dialyzed overnight against 2L of buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mM EDTA, 2 mM dithiothreitol (DTT), 5% glycerol. The protein was then applied to a SP HP column at rate of 2 ml/min. The protein was then eluted with a linear gradient of 0.15-0.8 M NaCl in buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM DTT, and 5% glycerol. The fractions containing the protein analyzed by 12% SDS-PAGE were pooled together and concentrated to 27 µM and frozen aliquots were stored at -80 °C. Protein concentration was calculated using the extinction coefficient of 34840 M-1 cm-1. Enzymatic activity of purified hNek2 was evaluated by monitoring the incorporation of [γ32P]ATP into dephosphorylated β-casein (Sigma-Aldrich Inc.), a known substrate of hNek2. As anticipated, we observed efficient phosphorylation of βcasein by hNek2 protein (Figure S.3). 4. Synthesis of a Nek2 Peptide Substrate for In vitro Kinase Assay All chemical reagents and resins for the synthesis were purchased from Advanced ChemTech Inc., Louisville Phospholemma-derived KY, Nek2 USA. peptide substrate, Ac-IRRLSTRRR-CONH2, was synthesized on solid support using standard solid phase peptide synthesis (SPPS) protocol (5). Rink amide resin (Creosalus Inc.) with an amino group loading of 0.62 mmol/g was used. Briefly, the following typical procedure was adopted for coupling of the first amino acid, arginine to the resin matrix. The resin (200 mg, 0.124 mmol) was swelled in NMP (3 ml) solvent for five minutes. To this was added a cocktail of Fmoc-Arg(Pbf)-OH (241 mg, 0.372 mmol), BOP (164.5 mg, 0.372 mmol), HoBt (50.2 mg, 0.372 mmol), and 4-methylmorpholine (125 mg, 1.24 mmol) in NMP (5 ml). The resulting mixture was shaken vigorously at room temperature for 2 hours. The excess reagents were removed by vacuum filtration and the resin was thoroughly washed with NMP, isopropanol, and dichloromethane successively (3 times each). A negative Kaiser test indicated that the amino acid coupling to resin matrix was successful. The Fmoc-group was then removed using 30% piperidine in NMP (30 minutes shaking) and resin was washed again as described before. Coupling of subsequent amino acids was performed in a similar manner. Acetylation of N-terminal isoleucine was performed by addition of acetic anhydride (253.1 mg, 2.48 mmol) and 4methylmorpholine (251 mg, 2.48 mmol) to the resin in NMP (30 min shaking). The resin was thoroughly washed and the final Nek2 peptide substrate was cleaved using a cleavage cocktail (95% TFA, 2.5% TIS, and 2.5% water, 6 ml, 3hrs). The volatile components were evaporated to dryness under nitrogen and the peptide was precipitated out using cold ethyl ether (15 ml). The crude peptide was dissolved in water and the purification was achieved on an RP-HPLC (Waters Inc) column, using a linear gradient of water/acetonitrile containing 0.1% TFA (67% yield). The final characterization of Nek2 peptide substrate was achieved using electro-spray ionization mass spectrometry (FLEET, Thermo Scientific Inc.). Expected mass calculated for quadruply-charged parent ion C51H99N25O12: 314.6; Observed: 314.8). dd_120424184756 #30 RT: 0.09 AV: 1 NL: 7.56E4 T: ITMS + p ESI Full m s [200.00-1300.00] 314.83 100 95 90 85 80 75 70 65 Relative Abundance 60 55 50 45 40 419.25 35 30 343.08 628.17 25 20 15 10 5 0 200 456.92 252.25 300.58 300 367.08 400 495.00 500 684.58 668.00 550.00 599.58 600 703.75 760.67 700 798.50 855.75 894.00 800 900 973.58 1032.83 1000 1131.67 1100 1204.75 1255.08 1200 1300 m /z 5. In vitro Inhibitor Screening and IC-50 Determination All in-vitro Nek2 kinase assays were performed in triplicate at 25 °C at pH 7.5 in kinase assay buffer (50 mM Tris HCl buffer containing 2 mM DTT, 10 mM MgCl2, 0.3mg/ml BSA, 0.5 mM Na2EDTA) using wt human Nek2. All inhibitor stock solutions were made in DMSO at a net concentration of 10 mM and stored at -20 °C when not in use. Net DMSO concentrations in all assays were maintained at 5%. Initial screening of small molecule drug library was performed in a 96-well format at an inhibitory concentration of 10 μM. Total assay volume in each well was restricted to 50 μl. A phospholemma-derived peptide, Ac-Ile-Arg-Arg-Leu-Ser-Thr-Arg-Arg-Arg-CONH2, synthesized as described earlier, was used as the hNek2 substrate. The following typical assay procedure was adopted: To a 46 μl kinase assay buffer solution containing 25 μM cold ATP supplemented with 2 μCi of [γ-32P] ATP (Perkin Elmer Inc., USA) was added 2.5 μl of Inhibitor solution (net inhibitor concentration 5 μM) and 2.5 μl enzyme (net hNek2 concentration 20 nM). The plate was then incubated for seven minutes. The enzymatic reaction was initiated by the addition of 2 μl of Nek2 peptide substrate (net concentration 50 μM) to the assay mixture. After the reaction was allowed to run for seven minutes, it was quenched by the addition of 100 μl of 6% phosphoric acid. After five minutes, 45 μl of the quenched reaction mixture was transferred onto a Whatman 350 Unifilter plate (P81 cellulose phosphate paper) and incubated for thirty minutes. Each well of the plate was then sequentially washed with 0.1% phosphoric acid (3 × 100 μl) and water (3 × 100 μl) using a vacuum manifold (Millipore Corp.). After the plate was dried on vacuum for thirty minutes, 50 μl of scintillation fluid (EcoScintA) was added. The top and bottom of the plate was sealed using TopSeal A (Perkin Elmer Inc.). The sealed plate was then loaded into TopCount NXT (Perkin Elmer Inc.) for scintillation counting. The percentage inhibition was assessed upon direct comparison of counts from wells that did not have any inhibitor present in it (control well). For determination of IC50 values, similar assay protocols were followed except that relative activity was determined in a dose-dependent manner. The experimental data thus obtained were plotted using SigmaPlot software and fitted using Enzyme Kinetics module to obtain IC50 values. References 1. Henise JC, Taunton J. Irreversible Nek2 kinase inhibitors with cellular activity. Journal of medicinal chemistry. 2011;54(12):4133-46. Epub 2011/06/02. 2. Solanki S, Innocenti P, Mas-Droux C, Boxall K, Barillari C, van Montfort RL, et al. Benzimidazole inhibitors induce a DFG-out conformation of never in mitosis gene A- related kinase 2 (Nek2) without binding to the back pocket and reveal a nonlinear structure-activity relationship. Journal of medicinal chemistry. 2011;54(6):1626-39. Epub 2011/03/04. 3. Whelligan DK, Solanki S, Taylor D, Thomson DW, Cheung KM, Boxall K, et al. Aminopyrazine inhibitors binding to an unusual inactive conformation of the mitotic kinase Nek2: SAR and structural characterization. Journal of medicinal chemistry. 2010;53(21):7682-98. Epub 2010/10/13. 4. Rellos P, Ivins FJ, Baxter JE, Pike A, Nott TJ, Parkinson DM, et al. Structure and regulation of the human Nek2 centrosomal kinase. The Journal of biological chemistry. 2007;282(9):6833-42. 5. Kumar S, Liang F, Lawrence DS, Zhang ZY. Small molecule approach to studying protein tyrosine phosphatase. Methods. 2005;35(1):9-21. Epub 2004/12/14.