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Characterization of kinase inhibitors using a kinase binding assay on active/inactive TrkA/TrkB and a TrkB assay in neurons
Author(s) -
De Vries Luc,
Finana Frederic,
RaulyLestienne Isabelle,
Lestienne Fabrice,
Ailhaud MarieChristine,
Cussac Didier
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.567.1
TrkA/TrkB kinases play a crucial role in pain and human cancers and selective inhibitors are a favoured therapeutic approach for such disorders. While type I active‐kinase inhibitors inhibit the kinase activity in an ATP‐competitive manner, type II inactive‐kinase inhibitors bind to the ATP‐and an adjacent hydrophobic site, leading to increased selectivity. We validated tracer 236 in a Lanthascreen ® EU kinase binding assay with pKd for active/inactive forms of TrkA (8.1/8.0) and TrkB (8.3/7.4). Reference compounds have similar affinity (pKi) for both active and inactive TrkA: AZ23 (9.2/9.2), BMS9 (8.9/8.6), K252a (8.4/8.2), staurosporine (8.2/8.2) and PF02341066 (6.7/6.6). For TrkB, staurosporine (8.8/8.1), K252a (8.6/7.8) and PF02341066 (7.5/6.6) loose affinity between active and inactive forms, whereas AZ23 (9.7/9.4) and BMS9 (9.3/9.1) display similar affinities, suggesting enhanced selectivity. These compounds were also tested on isolated rat hippocampal neurons for inhibition of TrkB‐stimulated c‐Fos and Arc mRNA induction. The rank order of inhibitor potency: AZ23 > BMS9 > PF02341066 is in accordance with the results of our binding assay, whereas staurosporine and K252a are active at higher concentrations in this native model. Thus, our results represent an original combined approach to obtain more selective TrkA/TrkB inhibitors in diverse clinical therapeutics such as pain and cancer.