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• W2022204925 abstract "Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. The activation of the MAPK pathways p38, JNK, and ERK plays a crucial role in inflammatory diseases and cancer. COT 2The abbreviations used are: COTCancer Osaka thyroidNi-NTAnickel-nitrilotriacetic acidaaamino acidsDMSOdimethyl sulfoxide. kinase is the single kinase that mediates the activation of the Mek/Erk pathway downstream of the pro-inflammatory receptors of the IL-1/TLR/TNF/IL-17 receptor superfamilies, whereas NF-κB, p38, and JNK are turned on by the MAP3K Tak1. In cells, COT kinase forms a heterotrimeric complex together with NF-κB transcription factor p105 and A20-binding inhibitor of NF-κB (ABIN-2). COT kinase is activated by IκB kinase β (IKKβ), which triggers proteasome-dependent degradation of p105 to p50 and disassembly of the ternary complex (1.Beinke S. Robinson M.J. Hugunin M. Ley S.C. Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IκB kinase-induced proteolysis of NF-κB1 p105.Mol. Cell. Biol. 2004; 24: 9658-9667Crossref PubMed Scopus (171) Google Scholar). Furthermore, IκB kinase β phosphorylates COT kinase at position Ser400, which mediates binding of 14-3-3 protein (2.Ben-Addi A. Mambole-Dema A. Brender C. Martin S.R. Janzen J. Kjaer S. Smerdon S.J. Ley S.C. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E2394-E2403Crossref PubMed Scopus (29) Google Scholar, 3.Roget K. Ben-Addi A. Mambole-Dema A. Gantke T. Yang H.-T. Janzen J. Morrice N. Abbott D. Ley S.C. IκB kinase 2 regulates TPL-2 activation of extracellular signal-regulated kinases 1 and 2 by direct phosphorylation of TPL-2 serine 400.Mol. Cell. Biol. 2012; 32: 4684-4690Crossref PubMed Scopus (38) Google Scholar). In addition, the full activation of COT kinase requires phosphorylation of Thr290 in the activation loop by an unknown kinase. Dissociation of activated COT kinase from p105 and ABIN-2 allows substrate loop phosphorylation of the downstream substrate MEK. A number of other downstream targets of COT kinase have been described, and recent data show that as yet uncharacterized substrates required for posttranslational TNF processing are activated while COT kinase is residing in the ternary p105·ABIN-2 complex (4.Yang H.T. Papoutsopoulou S. Belich M. Brender C. Janzen J. Gantke T. Handley M. Ley S.C. Coordinate regulation of TPL-2 and NF-κB signaling in macrophages by NF-κB1 p105.Mol. Cell. Biol. 2012; 32: 3438-3451Crossref PubMed Scopus (52) Google Scholar). Cancer Osaka thyroid nickel-nitrilotriacetic acid amino acids dimethyl sulfoxide. COT kinase is widely expressed in the spleen, thymus, liver, brain, testis, intestine, kidney, skeletal muscles, lungs, and pancreas (5.Patriotis C. Makris A. Bear S.E. Tsichlis P.N. Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 2251-2255Crossref PubMed Scopus (147) Google Scholar). COT kinase has a function in hematopoietic lineages, with the best described role in pro-inflammatory cytokine production in macrophages. Its catalytic activity is required for both TNFα and IL-1β production (6.Dumitru C.D. Ceci J.D. Tsatsanis C. Kontoyiannis D. Stamatakis K. Lin J.H. Patriotis C. Jenkins N.A. Copeland N.G. Kollias G. Tsichlis P.N. TNF-α induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway.Cell. 2000; 103: 1071-1083Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar). Single cytokine neutralization of either TNFα or IL-1β by antibody is highly efficient in the clinic for multiple indications. The recently discovered role of COT kinase in IL-17 signaling (7.Xiao Y. Jin J. Chang M. Nakaya M. Hu H. Zou Q. Zhou X. Brittain G.C. Cheng X. Sun S.-C. TPL2 mediates autoimmune inflammation through activation of the TAK1 axis of IL-17 signaling.J. Exp. Med. 2014; 211: 1689-1702Crossref PubMed Scopus (59) Google Scholar, 8.Sriskantharajah S. Gückel E. Tsakiri N. Kierdorf K. Brender C. Ben-Addi A. Veldhoen M. Tsichlis P.N. Stockinger B. O'Garra A. Prinz M. Kollias G. Ley S.C. Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase.J. Immunol. 2014; 192: 3518-3529Crossref PubMed Scopus (32) Google Scholar) further adds weight to its therapeutic importance in IL-17-driven diseases such as multiple sclerosis and psoriasis and may explain its function in stromal cells that has been found in inflammatory disease models (9.Van Acker G.J. Perides G. Weiss E.R. Das S. Tsichlis P.N. Steer M.L. Tumor progression locus-2 is a critical regulator of pancreatic and lung inflammation during acute pancreatitis.J. Biol. Chem. 2007; 282: 22140-22149Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In cancer, COT kinase seems to have ambivalent roles by being able to act as a tumor suppressor (10.Gkirtzimanaki K. Gkouskou K.K. Oleksiewicz U. Nikolaidis G. Vyrla D. Liontos M. Pelekanou V. Kanellis D.C. Evangelou K. Stathopoulos E.N. Field J.K. Tsichlis P.N. Gorgoulis V. Liloglou T. Eliopoulos A.G. TPL2 kinase is a suppressor of lung carcinogenesis.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E1470-E1479Crossref PubMed Scopus (41) Google Scholar) or as a driver of tumorigenesis (11.Vougioukalaki M. Kanellis D.C. Gkouskou K. Eliopoulos A.G. Tpl2 kinase signal transduction in inflammation and cancer.Cancer Lett. 2011; 304: 80-89Crossref PubMed Scopus (73) Google Scholar). A COT kinase knock-out background has been shown to be beneficial in prostate cancer and breast cancer (12.Kim J.Y. Lim S.-C. Kim G. Yun H.J. Ahn S.-G. Choi H.S. Interleukin-33/ST2 axis promotes epithelial cell transformation and breast tumorigenesis via upregulation of COT activity.Oncogene. 2014Google Scholar, 13.Lee H.W. Cho H.J. Lee S.J. Song H.J. Cho H.J. Park M.C. Seol H.J. Lee J.-I. Kim S. Lee H.M. Choi H.Y. Nam D.-H. Joo K.M. Tpl2 induces castration resistant prostate cancer progression and metastasis.Int. J. Cancer. 2015; 136: 2065-2077Crossref PubMed Scopus (12) Google Scholar). In addition, COT kinase was described as an important resistance gene being able to drive MEK-ERK activation by circumventing rapidly accelerated kinase as upstream MAP3K (14.Johannessen C.M. Boehm J.S. Kim S.Y. Thomas S.R. Wardwell L. Johnson L.A. Emery C.M. Stransky N. Cogdill A.P. Barretina J. Caponigro G. Hieronymus H. Murray R.R. Salehi-Ashtiani K. Hill D.E. Vidal M. Zhao J.J. Yang X. Alkan O. Kim S. Harris J.L. Wilson C.J. Myer V.E. Finan P.M. Root D.E. Roberts T.M. Golub T. Flaherty K.T. Dummer R. Weber B.L. Sellers W.R. Schlegel R. Wargo J.A. Hahn W.C. Garraway L.A. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.Nature. 2010; 468: 968-972Crossref PubMed Scopus (1178) Google Scholar). Although the discovery of COT inhibitors has been reported in the literature (15.Hu Y. Cole D. Denny R.A. Anderson D.R. Ipek M. Ni Y. Wang X. Thaisrivongs S. Chamberlain T. Hall J.P. Liu J. Luong M. Lin L.L. Telliez J.B. Gopalsamy A. Discovery of indazoles as inhibitors of Tpl2 kinase.Bioorg. Med. Chem. Lett. 2011; 21: 4758-4761Crossref PubMed Scopus (19) Google Scholar, 16.Kaila N. Green N. Li H.Q. Hu Y. Janz K. Gavrin L.K. Thomason J. Tam S. Powell D. Cuozzo J. Hall J.P. Telliez J.B. Hsu S. Nickerson-Nutter C. Wang Q. Lin L.L. Identification of a novel class of selective Tpl2 kinase inhibitors: 4-alkylamino-[1,7]naphthyridine-3-carbonitriles.Bioorg. Med. Chem. 2007; 15: 6425-6442Crossref PubMed Scopus (27) Google Scholar, 17.Hu Y. Green N. Gavrin L.K. Janz K. Kaila N. Li H.Q. Thomason J.R. Cuozzo J.W. Hall J.P. Hsu S. Nickerson-Nutter C. Telliez J.B. Lin L.L. Tam S. Inhibition of Tpl2 kinase and TNF-α production with quinoline-3-carbonitriles for the treatment of rheumatoid arthritis.Bioorg. Med. Chem. Lett. 2006; 16: 6067-6072Crossref PubMed Scopus (53) Google Scholar, 18.Gavrin L.K. Green N. Hu Y. Janz K. Kaila N. Li H.Q. Tam S.Y. Thomason J.R. Gopalsamy A. Ciszewski G. Cuozzo J.W. Hall J.P. Hsu S. Telliez J.B. Lin L.L. Inhibition of Tpl2 kinase and TNF-α production with 1,7-naphthyridine-3-carbonitriles: synthesis and structure-activity relationships.Bioorg. Med. Chem. Lett. 2005; 15: 5288-5292Crossref PubMed Scopus (46) Google Scholar, 19.Wu J. Green N. Hotchandani R. Hu Y. Condon J. Huang A. Kaila N. Li H.-Q. Guler S. Li W. Tam S.Y. Wang Q. Pelker J. Marusic S. Hsu S. Perry Hall J. Telliez J.-B. Cui J. Lin L.-L. Selective inhibitors of tumor progression loci-2 (Tpl2) kinase with potent inhibition of TNF-α production in human whole blood.Bioorg. Med. Chem. Lett. 2009; 19: 3485-3488Crossref PubMed Scopus (38) Google Scholar, 20.Green N. Hu Y. Janz K. Li H.Q. Kaila N. Guler S. Thomason J. Joseph-McCarthy D. Tam S.Y. Hotchandani R. Wu J. Huang A. Wang Q. Leung L. Pelker J. Marusic S. Hsu S. Telliez J.B. Hall J.P. Cuozzo J.W. Lin L.L. Inhibitors of tumor progression loci-2 (Tpl2) kinase and tumor necrosis factor α (TNF-α) production: selectivity and in vivo antiinflammatory activity of novel 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles.J. Med. Chem. 2007; 50: 4728-4745Crossref PubMed Scopus (47) Google Scholar, 21.George D. Friedman M. Allen H. Argiriadi M. Barberis C. Bischoff A. Clabbers A. Cusack K. Dixon R. Fix-Stenzel S. Gordon T. Janssen B. Jia Y. Moskey M. Quinn C. Salmeron J.A. Wishart N. Woller K. Yu Z. Discovery of thieno[2,3-c]pyridines as potent COT inhibitors.Bioorg. Med. Chem. Lett. 2008; 18: 4952-4955Crossref PubMed Scopus (23) Google Scholar, 22.Cusack K. Allen H. Bischoff A. Clabbers A. Dixon R. Fix-Stenzel S. Friedman M. Gaumont Y. George D. Gordon T. Grongsaard P. Janssen B. Jia Y. Moskey M. Quinn C. Salmeron A. Thomas C. Wallace G. Wishart N. Yu Z. Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase and TNF-α production.Bioorg. Med. Chem. Lett. 2009; 19: 1722-1725Crossref PubMed Scopus (26) Google Scholar), difficulties in obtaining monodisperse protein and the lack of structural information have hampered the discovery of chemical compounds suitable for clinical studies. Careful selection of protein constructs and optimizations in the protein production procedures allowed us to obtain pure enzyme of exquisite biochemical activity. In this study, we additionally report on two new COT kinase inhibitors, 5-(5-(1H-indol-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3,4-oxadiazol-2-amine (2) and 5-(2-amino-5-(quinolin-3-yl)pyridin-3-yl)-1,3,4-oxadiazole-2(3H)-thione (3), that display nanomolar inhibitory activity of substrate phosphorylation in our biochemical assay. We also present the first atomic resolution x-ray crystal structures of the human COT kinase domain as complexes with the two inhibitors. These structures reveal that COT kinase adopts a unique and unexpected kinase domain fold. The P-loop (also known as glycine-rich loop) is preceded by a 15-amino acid insert, which turned out to be a central modulator of the size and chemical characteristics of the active site. In addition, the active site of COT kinase is structurally more flexible than previously anticipated. The results presented in this study provide a basis for enzyme production and the design of novel, potent, and selective COT kinase inhibitors. For protein production and crystallization, all chemicals, unless stated otherwise, were purchased from Sigma-Aldrich. The DNA sequence, containing codons optimized for insect cell expression, for full-length human COT kinase (UniProt P41279) was ordered from GeneArt and used as a template for creating truncated variants encompassing the kinase domain. Constructs coding for amino acids 66–395 and 30–404, having an N-terminal hexahistidine tag followed by a PreScission protease cleavage site, were designed and subcloned into a transfer plasmid containing a proximal polyhedrin promotor. Sf21 cells were co-transfected with pFlashbac virus DNA (Oxford Expression Technologies) for baculovirus generation according to the vendor's recommendation. Protein expression was carried out in 1-liter cell cultures of Sf21 cells with plaque-purified viruses. To improve the yields of overexpressed protein, compound 1 was added to the culture at a final concentration of 10 μm at 24 h after infection. Cells were harvested 72 h after infection, and the pellets were frozen and stored at −80 °C. The pellets of 50 g of cells containing overexpressed COT(aa66–395) or COT(aa30–404) were resuspended in 300 ml of lysis buffer (0.05 m Tris-HCl, pH 8, 0.003 m tris(2-carboxyethyl)phosphine (TCEP), 10% (v/v) glycerol, 0.4 m NaCl, 10 mm imidazole, 0.05% (v/v) Triton X-100, and Complete, EDTA-free (Roche Applied Science)). Compound 1 (dissolved in DMSO) was added to a final concentration of 20 μm followed by homogenization of the lysate. Cleared lysates were applied on a Ni-NTA column (Qiagen) equilibrated in buffer A (0.025 m Tris-HCl, pH 8, 0.003 m tris(2-carboxyethyl)phosphine, 10% (v/v) glycerol, 0.4 m NaCl, 0.01 m imidazole). The column was washed extensively with buffer A, followed by another wash step with modified buffer A containing 0.2 m NaCl. The protein was eluted by applying an imidazole gradient from 20 to 290 mm imidazole, and fractions containing the kinase were pooled. Exchange of compound 1 and cleavage of the His6 tag were carried out simultaneously overnight in the presence of 1 mm EDTA, PreScission protease and an excess of new ligand. The final purification step was carried out by size exclusion chromatography using a Superdex 75 HiLoad 16/60 column (GE Healthcare). Fractions containing the protein·ligand complex were pooled and concentrated to a final concentration of about 4 mg/ml. Both COT·ligand complexes were concentrated to 3.5 mg/ml using an Amicon Ultra centrifugal filter with 10,000 molecular weight cut-off (Millipore) before crystallization by vapor diffusion. Hanging drops consisting of an equal ratio of complex to reservoir solution (1 μl) were set up and equilibrated against 500 μl of reservoir solution containing either 0.1 m HEPES, pH 7.2, 24% (w/v) PEG monomethyl ether 500 (PEGMME500), and 0.05 m MgCl2 (COT·compound 2) or 8% (w/v) PEG4000, 12% (v/v) ethylene glycol and 7% (v/v) isopropyl alcohol (COT·compound 3) in a 24-well VDX plate (Hampton Research) at room temperature. Crystals grew within 2 days and were flash-frozen in liquid nitrogen, either directly after harvest from the mother liquor (COT·compound 2) or after a quick dip into a reservoir solution containing 10% (v/v) glycerol (COT·compound 3). Diffraction data were collected at 100 K at the Swiss Light Source beamline X10SA at 1.0 Å wavelength. Diffraction images were recorded on a MAR225 CCD detector (MAR Research) and processed and scaled with XDS and XSCALE (23.Kabsch W. Xds. Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (10.1107/S0907444909047337): 125-132Crossref PubMed Scopus (11225) Google Scholar), respectively (Table 1). The COT kinase·compound 2 structure was solved by molecular replacement. A BLAST search was carried out against the Protein Data Bank (PDB) using the human COT kinase amino acid sequence (UniProt P41279) including residues 66–395, and coordinates of the top 99 search results were downloaded. Sequence alignments of the downloaded hits with COT kinase residues 66–395 were created using FASTA (24.Pearson W.R. Lipman D.J. Improved tools for biological sequence comparison.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 2444-2448Crossref PubMed Scopus (9378) Google Scholar). To create the search models for molecular replacement, the coordinate files were prepared with Chainsaw, which is part of the CCP4 software suite (25.Winn M.D. Ballard C.C. Cowtan K.D. Dodson E.J. Emsley P. Evans P.R. Keegan R.M. Krissinel E.B. Leslie A.G.W. McCoy A. McNicholas S.J. Murshudov G.N. Pannu N.S. Potterton E.A. Powell H.R. Read R.J. Vagin A. Wilson K.S. Overview of the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 235-242Crossref PubMed Scopus (9205) Google Scholar), using the corresponding sequence alignments obtained from FASTA. The models obtained from Chainsaw were aligned in PyMOL (26.DeLano W.L. The PyMOL Molecular Graphics System. Schrödinger, LLC, New York2010Google Scholar), and large, protruding loops were removed. Molecular replacement searches with each input model were carried out with Phaser (27.McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14437) Google Scholar) and returned 85 solutions in space group C21 with three copies in the asymmetric unit. Three solutions with the highest log likelihood gain were used as a starting point for initial model building. The COT kinase·compound 2 structure was built using COOT (28.Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17075) Google Scholar) and refined with Phenix (29.Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. Richardson J.S. Terwilliger T.C. Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16434) Google Scholar) and AutoBuster (30.Bricogne G. Brandl M. Flensburg C. Keller P. Paciorek W. Roversi P Sharff A. Smart O.S. Vonrhein C. Womack T.O.B.E. Buster. Global Phasing Ltd., Cambridge, United Kingdom2011Google Scholar) to Rwork = 17.7% and Rfree = 20.7% at a high resolution limit of 2.3 Å. 96.4% of all the residues in the structure are in the favored outlier regions of the Ramachandran plot, and 0.3% are in the outlier regions of the Ramachandran plot. The COT kinase·compound 3 structure was solved by molecular replacement with Phaser using protein coordinates of the structure with compound 2 as input model. The solution from molecular replacement (space group P31 with three copies in the asymmetric unit) was rebuilt in COOT and refined with Phenix and AutoBuster to Rwork = 20.1% and Rfree = 24.0% at a high resolution limit of 2.9 Å. 94.8% of all the residues in the structure are in the favored regions of the Ramachandran plot, and 1.5% are in the outlier regions of the Ramachandran plot. PyMOL (26.DeLano W.L. The PyMOL Molecular Graphics System. Schrödinger, LLC, New York2010Google Scholar) was used to prepare all structural figures.TABLE 1Data collection and refinement statisticsCOT/compound 2COT/compound 3Data collectionSpace groupC2P3Unit cella, b, c (Å)147.978, 85.113, 85.964101.952, 101.952, 108.315α, β, γ (°)90.0 91.06 90.090.0 90.0 120.0Resolution (Å)73.8-2.388.3-2.9Wavelength (Å)1.00001.0000Unique reflections45,30728,213Completeness (%)99.0 (94.0)aValues in parentheses are for highest-resolution shells.100.0 (100.0)aValues in parentheses are for highest-resolution shells.〈I〉/σ(I)10.6 (3.3)aValues in parentheses are for highest-resolution shells.14.7 (3.7)aValues in parentheses are for highest-resolution shells.Rsym0.10 (0.39)aValues in parentheses are for highest-resolution shells.0.09 (0.45)aValues in parentheses are for highest-resolution shells.RefinementResolution (Å)34.1-2.388.3-2.9Reflections45,29328,212Rwork/Rfree0.177/0.2070.201/0.240No. of waters361167No. of protein atoms69396124No. of ligand atoms452268Wilson B (Å2)36.3776.04a Values in parentheses are for highest-resolution shells. Open table in a new tab All assays were performed in 384-well microtiter plates using automated liquid dispensing. The assay plates were prepared by the addition of 50 nl/well of compound solution in 90% (v/v) DMSO. The kinase reactions were started by the stepwise addition of 4.5 μl/well of peptide/ATP solution (50 mm HEPES, pH 7.5, 1 mm DTT, 0.02% (w/v) Tween 20, 0.02% (w/v) BSA, 10 mm β-glycerophosphate, 10 μm sodium orthovanadate, 14 mm MgCl2, 1 mm MnCl2, 306 μm ATP, 4 μm peptide (5-fluorescein-aminohexanoic acid-AGAGSGQLIDSNANSFVGTR-NH2, Biosyntan GmbH)) and 4.5 μl/well of enzyme solution (50 mm HEPES, pH 7.5, 1 mm DTT, 0.02% (w/v) Tween 20, 0.02% (w/v) BSA, 10 mm β-glycerophosphate, 10 μm sodium orthovanadate, 14 mm MgCl2, 1 mm MnCl2, and 17 nm COT kinase (encompassing amino acids 30–404)). Kinase reactions were incubated at 30 °C for 60 min and subsequently terminated by the addition of 16 μl/well of stop solution (100 mm HEPES, pH 7.5, 5% (v/v) DMSO, 0.1% (v/v) Caliper coating reagent, 10 mm EDTA, and 0.015% (w/v) Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated and quantified using the Caliper microfluidic mobility shift technology. A detailed protocol for the syntheses of (E)-3-(2-amino-5-(naphthalen-2-yl)pyridin-3-yl)acrylic acid (1), 5-(5-(1H-indol-3-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3,4-oxadiazol-2-amine (2) and 5-(2-amino-5-(quinolin-3-yl)pyridin-3-yl)-1,3,4-oxadiazole-2(3H)-thione (3) (see Fig. 1) can be found in the supplemental information. A series of DNA plasmids encoding hexahistidine-tagged human COT kinase domain with varying N and C termini was subjected to small scale expression tests in baculovirus-infected Sf21 insect cells. Two constructs, one encompassing amino acid residues 30–404 (aa30–404) and the other one encompassing amino acid residues 66–395 (aa66–395), were identified in a first round of expression experiments to yield soluble but heterogeneously phosphorylated protein. It has been reported previously that expression yields of kinases in baculovirus-infected insect cells can be improved by the addition of specific small molecular weight kinase inhibitors to the cell cultivation medium (31.Strauss A. Fendrich G. Horisberger M.A. Liebetanz J. Meyhack B. Schlaeppi J.M. Schmitz R. Improved expression of kinases in Baculovirus-infected insect cells upon addition of specific kinase inhibitors to the culture helpful for structural studies.Protein Expr. Purif. 2007; 56: 167-176Crossref PubMed Scopus (14) Google Scholar). For both constructs, the addition of the ATP-competitive inhibitor 1 during fermentation and initial purification was essential to increase the overall yield as well as to reduce heterogeneous phosphorylation. In the absence of compound 1, about 0.2 mg of 65% non-phosphorylated and soluble COT kinase (aa30–404) was isolated per liter of cell culture volume after a nickel affinity purification step. The addition of compound 1 greatly enhanced this yield to 4–6 mg of protein per liter of cell culture. Purified COT protein was found 90% non-phosphorylated after the addition of compound 1, suggesting that compound 1 blocks COT kinase autophosphorylation (Table 2). A second construct (aa66–395) was identified using a similar approach and yielded about 2 mg/liter of cell culture after the first purification step. It was possible to quantitatively exchange compound 1 by other ATP site binders by chromatography or to remove it completely to prepare apo-form enzyme (see “Experimental Procedures”).TABLE 2Optimization of human COT kinase (aa30–404) expression in Sf21 insect cellsCompound 1Form of additionTime of additionCultivation time [h]Ni-NTA yieldPhosphorylation in %0×1×2×μmmg0NANA640.2–0.365251030Diluted into medium, sterile filteredAt infection632.48416010DMSO stock diluted into cultureAt infection644.28911010DMSO stock diluted into culture24 h after infection64790100 Open table in a new tab The use of radioactive filter binding and homogeneous time-resolved fluorescence assays to measure COT kinase activity has been described before (32.Jia Y. Quinn C.M. Bump N.J. Clark K.M. Clabbers A. Hardman J. Gagnon A. Kamens J. Tomlinson M.J. Wishart N. Allen H. Purification and kinetic characterization of recombinant human mitogen-activated protein kinase kinase kinase COT and the complexes with its cellular partner NF-κ B1 p105.Arch. Biochem. Biophys. 2005; 441: 64-74Crossref PubMed Scopus (16) Google Scholar). For this study, we developed an assay that measures in vitro phosphorylation of a peptide substrate. Enzyme reactions were carried out in 384-well plates. We used the Caliper microfluidic mobility shift technology to quantitatively separate phosphorylated and non-phosphorylated substrate peptides (supplemental Fig. 1). With this experimental setup, the half-maximal inhibitory concentrations (IC50 values) of a large variety of compounds were measured at compound concentrations of up to 10 μm. Chemical derivation of a hit obtained from high throughput screening resulted in the discovery of the two COT inhibitors 2 and 3 discussed in this study (Fig. 1). Both compounds show low nanomolar inhibitory activity of COT kinase in the enzymatic Caliper assay with IC50 values of 13 nm for compound 2 and 17 nm for compound 3. Both protein variants mentioned above were subjected to extensive crystallization experiments, and COT kinase (aa66–396) yielded well diffracting crystals. We determined the x-ray co-crystal structures of the human COT kinase domain in complex with compound 2 at 2.3 Å resolution. The crystal structure of this complex has a refined model that includes residues 73–93, 103–139, and 143–392 (Table 1). Another co-crystal of the same human COT kinase domain was solved in complex with compound 3 at a resolution of 2.9 Å. The model of this co-crystal structure contains residues 73–92, 102–331, and 343–389 (Table 1). Both structures revealed a bilobed kinase domain fold (Fig. 2) with an N-terminal extension that is similar to the one observed in the crystal structure of nuclear factor κB-inducing kinase (PDB entry 4DN5) (33.Liu J. Sudom A. Min X. Cao Z. Gao X. Ayres M. Lee F. Cao P. Johnstone S. Plotnikova O. Walker N. Chen G. Wang Z. Structure of the nuclear factor κB-inducing kinase (NIK) kinase domain reveals a constitutively active conformation.J. Biol. Chem. 2012; 287: 27326-27334Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The N-terminal extension consists of an α-helix (αN2) and a short, parallel β-sheet (βN1 and βN2). Helix αN2 stacks against the central β-sheet and helix αC. βN1 and βN2 pack against and extend the central β-sheet of the N-lobe (Fig. 2A). The loop connecting the N-terminal extension with the N-lobe is disordered in both crystal structures as jud" @default.
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• W2022204925 title "The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold" @default.
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