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Key Event Title
Inhibition of Fyna
|Level of Biological Organization|
Key Event Components
|protein tyrosine kinase activity||tyrosine-protein kinase fyna (zebrafish)||decreased|
Key Event Overview
AOPs Including This Key Event
Key Event Description
Src family kinases (SFKs) include nine members (i.e., Src, Fyn, Yes, Blk, Yrk, Fgr, Hck, Lck and Lyn) and regulate multiple signal transduction pathways involved in growth, proliferation, differentiation, migration, metabolism and apoptosis, interacting with a diverse array of molecules, including growth factor receptors, cell–cell adhesion receptors, integrins and steroid hormone receptors (Schenone et al., 2007). Protein kinases enable transfer of γ phosphate of ATP to specific amino acids of protein substrates (tyrosine, serine, threonine, or even histidine residues) (Saito, 2001). There are two major groups of tyrosine kinases: receptor and nonreceptor tyrosine kinases. Nonreceptor tyrosine kinases (cytoplasmatic proteins) are important components of signaling pathways through different receptors such as receptor tyrosine kinases, G-protein coupled receptors, or T-cell receptors (TCR) on the cell surface (Hanrs & Hunter, 1995).
Fyna (Src family tyrosine kinase A) is a nonreceptor tyrosine kinase. It is involved in several processes, including adherens junction maintenance and gastrulation. Fyna localizes to the cytosol and the nucleus. It is expressed in the central nervous system, olfactory placode, peripheral olfactory organ, and retina. Human ortholog(s) of this gene are implicated in Alzheimer's disease and schizophrenia. Zebrafish Fyna gene is orthologous to human FYN (FYN proto-oncogene, Src family tyrosine kinase) (ZFIN Gene: Fyna, n.d.) and shares 89% sequence identity (full length sequence and kinase domain) with the human FYN gene (Challa & Chatti, 2013).
The Src family kinases are of a modular nature, consisting of a unique N-terminal sequence, three protein modules including the SH3, SH2, and kinase domains, and a C-terminal tail. The modules play an important role in enzyme reactions (Lamers et al., 2003). Fyna kinase activity, like that of other Src family kinases, is regulated by intramolecular interactions that depend on equilibrium between tyrosine phosphorylation and dephosphorylation. In the basal state, catalytic activity is constrained by engagement of the SH2 domain by a phosphorylated C-terminal tyrosine 531 (Krämer-Albers & White, 2011), after activation enzymes transmit signals from several surface receptors to target proteins by phosphorylating tyrosine residues (Sen and Johnson, 2011). Crystal structures of the SH2 and SH3 domains of the Fyna kinase reveal its binding specificity for peptide inhibitors (Morton et al., 1996; Mulhern et al., 1997). Binding of inhibitors to fyna domains inhibits its activity. Research in inhibition of Fyna kinase is mostly due its role in Alzheimer's disease (AD) and anti-inflamatory therapy (Löwenberg et al., 2005).
There are multiple known Fyna kinase inhibitors (see Evidence for perturbation by stressor).
How It Is Measured or Detected
Changes in activity of Fyna kinase can be evaluated with enzyme-linked immunosorbent assay (ELISA). This type of ELISA method is based on kinase phosphorylation of an immobilized substrate, which is detected using anti-tyrosine phosphate antibody. The extent of the substrate phosphorylation can be measured by absorbance, fluorescence, or fluorescence polarization (Jelić et al., 2007).
Adp-GloTM Kinase Assay can be used to measure Fyna kinase activity. The assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinases, making it ideal for both primary screening as well as kinase selectivity profiling (Zegzouti et al., 2009).
Domain of Applicability
Theoretically, this MIE is applicable to any organisms with the Fyna kinase (zebrafish and other vertebrate models). While, in zebrafish Fyna inhibition was achieved with kinase dead (KD) point mutant of Fyna (St. Clair et al., 2018), chemical inhibition most pertaining to this AOP was mostly studied in human (Green et al., 2009; Jelić et al., 2007; Kinoshita et al., 2006; Lamers et al., 2003; Toullec et al., 1991) and mouse (Morisot et al., 2019; Nygaard et al., 2014) cell lines.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Evidence for perturbation of Fyna is available for rosmarinic acid (Jelić et al., 2007), stauroporine (Kinoshita et al., 2006; Lamers et al., 2003) and AZD0530 (Green et al., 2009, Morisot et al., 2019).
Rosmarinic acid, a secondary metabolite of herbal plants, was discovered as a new Fyna kinase inhibitor using immunochemical and in silico methods. Kinetic data implicate that rosmarinic acid inhibits Fyna kinase in a linear-mixed type of inhibition. In this type of reversible inhibition, a compound can interact both with the free enzyme and with the enzyme-substrate complex at a site other than the active site (Jelić et al., 2007).
Dual SFK inhibitors (e.g., dasatinib, bosutinib and saracatinib) already approved for therapy or are in clinical trials (Musumeci et al., 2012). Saracatinib (AZD0530) is a small molecule inhibitor of Src family kinases, inhibiting Src, Fyna, Yes and Lyn, with 2 to 10 nM potency. AZD0530 specific inhibition of Fyna and other SFKs has led to its development as therapy for solid tumors, because Src family kinases regulate tumor cell adhesion, migration and invasion, and also regulate proliferation (Hennequin et al., 2006). AZD0530 has numerous desirable properties. For example, AZD0530 inhibits Fyna activity in the low nM range (Green et al., 2009) has a half‐life of 16 hours in the mouse and is highly brain penetrable (Kaufman et al., 2015).
Staurosporine is a microbial alkaloid isolated from Streptomyces sp. (Omura, S.,1977) which has been shown to be a potent broad-range inhibitor competing with ATP, but not peptide substrate, for binding to protein kinases including Fyn kinase. Staurosporine binds to the ATP-binding site of Fyn (Kinoshita et al., 2006) and has nanomolar potency against most protein kinases including Fyn. This level of selectivity has already enabled staurosporine to be used as a lead inhibitor for the design of specific potent protein kinase inhibitors (Toullec et al., 1991).
Challa, A. K., & Chatti, K. (2013). Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology. 10(3). https://doi.org/10.1089/zeb.2012.0781
Green, T. P., Fennell, M., Whittaker, R., Curwen, J., Jacobs, V., Allen, J., Logie, A., Hargreaves, J., Hickinson, D. M., Wilkinson, R. W., Elvin, P., Boyer, B., Carragher, N., Plé, P. A., Bermingham, A., Holdgate, G. A., Ward, W. H. J., Hennequin, L. F., Davies, B. R., & Costello, G. F. (2009). Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530. Molecular Oncology, 3(3), 248–261. https://doi.org/10.1016/j.molonc.2009.01.002
Hanrs, S. K., & Hunter, T. (1995). The eukaryotic protein kinase superfamily: idnase. (catalytic) domam structure and classification. https://doi.org/10.1096/fasebj.9.8.7768349
Jelić, D., Mildner, B., Koštrun, S., Nujić, K., Verbanac, D., Čulić, O., Antolović, R., & Brandt, W. (2007). Homology modeling of human Fyn kinase structure: Discovery of rosmarinic acid as a new Fyn kinase inhibitor and in Silico study of its possible binding modes. Journal of Medicinal Chemistry, 50(6), 1090–1100. https://doi.org/10.1021/jm0607202
Kinoshita, T., Matsubara, M., Ishiguro, H., Okita, K., & Tada, T. (2006). Structure of human Fyn kinase domain complexed with staurosporine. Biochemical and Biophysical Research Communications, 346(3), 840–844. https://doi.org/10.1016/j.bbrc.2006.05.212
Krämer-Albers, E.-M., & White, R. (2011). From axon-glial signalling to myelination: the integrating role of oligodendroglial Fyn kinase. Cell. Mol. Life Sci. https://doi.org/10.1007/s00018-010-0616-z
Lamers, M. B. A. C., Antson, A. A., Hubbard, R. E., Scott, R. K., & Williams, D. H. (2003). ￼Structure of the Protein Tyrosine Kinase Domain of C-terminal Src Kinase (CSK) in Complexwith Staurosporine. J. Mol. Bi(285), 713–725. papers2://publication/uuid/CBF6FE3B-FE88-4A68-9E4A-EC387CF85D43
Löwenberg, M., Tuynman, J., Bilderbeek, J., Gaber, T., Buttgereit, F., Van Deventer, S., Peppelenbosch, M., & Hommes, D. (2005). Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn. Blood, 106(5), 1703–1710. https://doi.org/10.1182/blood-2004-12-4790
Morisot, N., Berger, A. L., Phamluong, K., Cross, A., & Ron, D. (2019). The Fyn kinase inhibitor, AZD0530, suppresses mouse alcohol self-administration and seeking. Addiction Biology, 24(6), 1227–1234. https://doi.org/10.1111/adb.12699
Nygaard, H. B., Van Dyck, C. H., & Strittmatter, S. M. (2014). Fyn kinase inhibition as a novel therapy for Alzheimer’s disease. Alzheimer’s Research and Therapy, 6(1), 1–8. https://doi.org/10.1186/alzrt238
Saito, H. (2001). Histidine phosphorylation and two-component signaling in eukaryotic cells. Chemical Reviews, 101(8), 2497–2509. https://doi.org/10.1021/cr000243+
Sen, B., Johnson, F.M., 2011. Regulation of Src Family Kinases in Human Cancers. J. Signal Transduct. 2011, 1–14. https://doi.org/10.1155/2011/865819
St. Clair, R. M., Emerson, S. E., D’Elia, K. P., Weir, M. E., Schmoker, A. M., Ebert, A. M., & Ballif, B. A. (2018). Fyn-dependent phosphorylation of PlexinA1 and PlexinA2 at conserved tyrosines is essential for zebrafish eye development. FEBS Journal, 285(1), 72–86. https://doi.org/10.1111/febs.14313
Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D., & Kirilovsky, J. (1991). The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. Journal of Biological Chemistry, 266(24), 15771–15781. https://doi.org/10.1016/s0021-9258(18)98476-0
Zegzouti, H. et al. (2009) ‘ADP-Glo: A bioluminescent and homogeneous adp monitoring assay for Kinases’, Assay and Drug Development Technologies, 7(6), pp. 560–572. doi: 10.1089/adt.2009.0222.
ZFIN Gene: fyna. (n.d.). Retrieved March 14, 2021, from https://zfin.org/ZDB-GENE-030903-5#phenotype