A novel small-molecule inhibitor of IL-6 signalling

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 23, 2010, Pages 7029-7032

  Zinzalla, Giovanna ^a   Haque, Mohammad R ^a   Piku Basu, B ^b   Anderson, John ^b   Kaye, Samantha L ^a   Haider, Shozeb ^a   Hasan, Fyeza ^b   Antonow, Dyeison ^a   Essex, Samantha ^a   Rahman, Khondaker M ^a   Palmer, Jonathan ^a   Morgenstern, Daniel ^b   Wilderspin, Andrew F ^a   Neidle, Stephen ^a   Thurston, David E ^a  

^a IMPERIAL CANCER RESEARCH FUND   (United Kingdom)

^b UNIVERSITY COLLEGE LONDON   (United Kingdom)

Author keywords

IL 6 signalling; Inhibitor; Small molecule; STAT3

Indexed keywords

ANTINEOPLASTIC AGENT; INTERLEUKIN 6; INTERLEUKIN 6 SIGNALLING INHIBITOR; POLYCYCLIC AROMATIC HYDROCARBON DERIVATIVE; PYRROLIDINE DERIVATIVE; STAT3 PROTEIN; UNCLASSIFIED DRUG;

ARTICLE; CANCER CELL CULTURE; CYTOSTASIS; DRUG CYTOTOXICITY; DRUG SELECTIVITY; DRUG STRUCTURE; DRUG SYNTHESIS; HELA CELL; PROTEIN PHOSPHORYLATION; REACTION ANALYSIS; SIGNAL TRANSDUCTION; STRUCTURE ANALYSIS;

BREAST NEOPLASMS; CELL LINE, TUMOR; CYTOSTATIC AGENTS; FEMALE; HELA CELLS; HUMANS; INTERLEUKIN-6; PHOSPHORYLATION; PYRROLIDINES; SIGNAL TRANSDUCTION; SMALL MOLECULE LIBRARIES; STAT3 TRANSCRIPTION FACTOR; STRUCTURE-ACTIVITY RELATIONSHIP; SULFONES;

EID: 78149281633     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.09.117     Document Type: Article

References (19)

1. D.R. Hodge, E.M. Hurt, and W.L. Farrar Eur. J. Cancer 41 2005 2502
2. J. Scheller, and N. Ohnesorge Scand. J. Immun. 63 2006 321
3. T. Ara, and Y.A. DeClerck Eur. J. Cancer 46 2010 1223
4. F. Blanchard, L. Duplomb, M. Baud'huin, and B. Brounais Cytokine Growth Factor Rev. 20 2009 19
5. S.H. David, S.A. Laura, and K. Razelle Cancer 110 2007 1911
6. T. Hirano, K. Ishihara, and M. Hibi Oncogene 19 2000 2548
7. H. Yu, D. Pardoll, and R.S. Jove Nat. Rev. Cancer 9 2009 798
8. Y. Adachi, N. Yoshio-Hoshino, and N. Nishimoto Curr. Pharm. Des. 14 2008 1217
9. K. Mitsuyama, S. Matsumoto, J. Masuda, H. Yamasaki, K. Kuwaki, H. Takedatsu, and M. Sata Anticancer Res. 27 2007 3749
10. S. Rose-John, G.H. Waetzig, J. Scheller, J. Gratzinger, and D. Seegert Expert Opin. Ther. Targets 11 2007 613
11. 3 (2 equiv) followed by heating for 5-20 min at 100 °C under microwave irradiation. The products (6a-v) were isolated in 29-95% yields and >98% purity by column chromatography on silica gel.
12. Full characterisation data for 6a-v are provided in Supplementary data.
13. General procedure for the MTS assay: 20,000 MDA-MB-231 or A4 cells were plated into a 96-well plate, and ∼80% confluency achieved by incubating overnight. Inhibitors were added and the cells incubated for a further 24 h. MTS reagent (CellTiter 96® Aqueous One Solution Cell Proliferation Assay MTS Solution; Promega) was added in accordance with the manufacturer's protocol. After 3 h, absorbance readings were taken at 490 nm and assumed to be directly proportional to the number of living cells. Absorbance values for cells treated with either vehicle or media alone were used as controls. The MTS data (calculated as percentage of control values) were calculated from triplicate measurements in at least two separate experiments to allow SDs to be calculated.
14. General procedure for Trypan Blue assay: MDA-MB-231 cells were plated into 24 well plates and allowed to incubate overnight to achieve 80% confluency. Inhibitors were then added, and the cells incubated for a further 24 h. After addition of Trypan Blue, unstained (viable) and stained (non-viable) cells were counted and calculated as a percentage of total cells using a haemocytometer.
15. General procedure for the Luciferase Reporter assay: HeLa cells containing the relevant plasmid were plated-out and incubated overnight to achieve 80% confluency. The medium was then changed to a serum-free type, and Oncostatin M (100 ng/ml) was added at the same time as an inhibitor. The cells were allowed to incubate for 8 h, and then cell lysates were prepared and luciferase activities measured using dual luciferase kits (Promega) in accordance with the manufacturer's protocol.
16. 6cells were plated into 2% FCS media in 6-well plates and allowed to incubate overnight. The medium was then changed to a serum-free type, and IL-6 (20 ng/ml) was added at the same time as an inhibitor. Whole cell extracts were then prepared using RIPA buffer (Thermo Scientific) with protease and phosphatase inhibitors. Extracts were dissolved in SDS buffer and run on a 10% PAGE gel for 1 h at 100 V (4 °C). Blots were probed with the following antibodies: STAT3, P-STAT3, STAT1, P-STAT1 and GAPDH (Cell Signalling Inc.).
17. G. Regis, S. Pensa, D. Boselli, F. Novelli, and V. Poli Semin. Cell Dev. Biol. 19 2008 351
18. M.J. Boulanger, D.C. Chow, E.E. Brevnova, and K.C. Garcia Science 300 2003 2104
19. Molecular modeling methodology: The ligand was generated via the PRODRG server (http://davapc1.bioch.dundee.ac.uk/prodrg/ ). The non-polar hydrogen atoms were merged, rotatable bonds assigned and partial atomic charges calculated as implemented in the AutoDockTools package (Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew R. K.; Olsen, A. J. J. Comp. Chem. 1998, 19, 1639). After removing IL-6, Grid maps were then generated on the receptor interfaces where the IL-6 α-receptor and gp130 should reside. The grid was large enough to encompass the entire IL-6 binding interface on the receptors. The grid points were thus chosen to be 118 × 118 × 118 with a grid spacing of 0.375. Automated docking was carried out using Autodock 4.0. A Lamarckian genetic algorithm (LGA) was applied to investigate plausible interactions between the ligand and receptor. The Solis and Wets algorithm (Solis, F. J.; Wets, J. B. Math. Oper. Res. 1981, 6, 19) was employed to carry out the local search. Values for all other docking parameters were kept as standard. Multiple docking runs can enhance the performance of docking programs (Wang, R.; Lu, Y.; Wang, S. J. Med. Chem. 2003, 46, 2287), and so 100 independent docking runs were carried out. The best docked conformation at each interface was then extracted based on interaction energies.