Discussion
Claire Tebbutt, Hayley Angove, Katie Chapman, Debbie Sanders, Gary Newton, Trevor Perrior, Jim Reid, , Mark Stewart.
Domainex Ltd, 162 Cambridge Science Park, Milton Road, Cambridge, Cambridgeshire, CB4 0GH
Tank-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKε) are closely related kinases with a homology of over 99%. Toll-like receptors (TLR), one of the key receptors on the surface of sentinel cells are involved in the innate immune response. They detect infection by the recognition of conserved bacterial or viral constituents (1). This in turn activates a number of key kinases including TBK1 and IKKε (2), which then phosphorylate transcription factors of the IRF family. This brings about the release of pro-inflammatory cytokines such as IFN-β, IFNα and IP10 (3,4).
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the US. There is heightened expression of IP10 along with its receptor CXCR3 in the submucosa and epithelium from patients with COPD compared to healthy individuals. This results in an increase in recruitment of T cells, which is linked to disease severity (5). Therefore inhibition of the TLR signalling pathway through chemical modulation of TBK1 and IKKε activity provides an opportunity to demonstrate a novel treatment for inflammatory diseases including COPD (6).
Initially a HTS-cell free kinase activity assay was established for TBK1 and IKKε. These assays were performed at Km and 30 x Km ATP allowing identification of ATP competition. In silico screening identified a series of novel low nM novel TBK1/IKKε compounds that were further developed through chemical modification. As TBK1 and IKKε are very homologous, these two activities generally track very closely, although some compounds showed up to 10-fold greater potency towards TBK1 than IKKε. The compounds were further profiled in a THP1 cell line, measuring inhibition of IP10 secretion after lipopolysaccharide (LPS) stimulation. The inhibition of IP10 correlates to the cell-free kinase data. This work paves the way for the discovery of a new drug to be used against COPD.
References:
1) Akira, S., Kawai, T., and Kumar, H. (2009) Biochemical and Biophysical Research Communications 388, 621-625.
2) Cho, J.Y., Jeong, D., Oh, J., Yang, Y., Yi, Y., and Yu, T. (2012) Mediators of Inflammation 2012.
3) Fitzgerald, K. A., McWhirter, S.M., Faia, K.L., Rowe, D.C., Latz, E., Golenbock, D.T., Coyle, A.J., Liao, S.M., and Maniatis, T. (2003) Nature Immunology, 491-496.
4) Miyahira, K.A., Shahangian, A., Hwang, S., Sun, R., and Cheng, G. (2009) The Journal of Immunology, 182, 2248-2257.
5) Saetta, M., Mariani, M., Panina-Bordignon, P., Turato, G., Buonsanti, C., Baraldo, S., Bellettato, C.M., Papi, A., Corbetta, L. , Zuin, R., Sinigaglia, F., and Fabbri, L.M. (2002) American Journal of respiratory and critical care medicine, 165, 1404-9
6) Wnag, Q., Nagarkar, D.R., Bowman, R.E., Schneider, D., Gosangi, B., Lei, J., Zhao, Y., McHenry, L.C., Burgens, V.R., Miller, J.D., Sajjan, U., and Hershenson, B.M. (2009) Journal of Immunology, 183, 6989-6997.
