In vitro activity: Src Inhibitor 1 ( Src-I1) is a novel, potent and selective dual site Src tyrosine kinase inhibitor which binds competitively to both the ATP- and peptide-binding sites with IC50 values of 44 nM for Src and 88nM for Lck. Src-I1, is found to be a potent inhibitor of Src (IC50=0.18 μM), but also inhibited other Src family members, such as Lck, Csk and Yes with similar potency to Src, and RIP2 (IC50=0.026 μM) with even greater potency. In addition, it inhibited CHK2 with similar potency to Src, and Aurora B with slightly lower potency.

Kinase Assay: All assays (25.5 μl volume) were carried out robotically at room temperature (21 °C) and were linear with respect to time and enzyme concentration under the conditions used. Assays were performed for 30 min using Multidrop Micro reagent dispensers (Thermo Electron Corporation, Waltham, MA, U.S.A.) in a 96-well format. The concentration of magnesium acetate in the assays was 10 mM and [γ-33P]ATP (800 c.p.m./pmol) was used at 5, 20 or 50 μM as indicated, in order to be at or below the Km for ATP for each enzyme. Protein kinases assayed at 5 μM ATP were: MKK1, ERK1, p38γ MAPK, p38δ MAPK, ERK8, PKBα, PKCζ, PRK2, GSK3β, CK2, MARK3, IKKβ, DYRK3, PIM2, EF2K, PLK1, Aurora C, HIPK2 and PAK4. Protein kinases assayed at 20 μM ATP were: JNK1, JNK2, p38β MAPK, PDK1, SGK1, S6K1, PKA, ROCK2, PKCα, MSK1, MAPKAP-K2, MAPKAP-K3, PRAK, CaMKKα, CaMKKβ, CHK1, CHK2, CDK2, Aurora B, CK1, PIM1, PIM3, NEK7, MST2, HIPK3, PAK5, PAK6, CSK, Yes and FGF-R1. Protein kinases assayed at 50 μM ATP were: Eph-A2 (Ephrin-A2 receptor), ERK2, JNK3, p38α MAPK, RSK1, RSK2, PKBβ, PKD1, MNK1, MNK2, AMPK, CaMK1, smMLCK, PHK, BRSK2, MELK, DYRK1a, DYRK2, NEK2a, NEK6, SRPK1, Src, Lck, IKKε and TBK1. Protein kinases assayed at 0.1 mM ATP were RIP2, GAK, c-Raf and B-Raf.