Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKKα Kinase

Lopreiato, Mariangela; Di Cristofano, Samuele; Cocchiola, Rossana; Mariano, Alessia; Guerrizio, Libera; Scandurra, Roberto; Mosca, Luciana; Raimondo, Domenico; d’Abusco, Anna Scotto

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy- [Formula: see text]-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK [Formula: see text] , one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKK [Formula: see text] by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKK [Formula: see text] and IKKtide, a 20 amino acid peptide substrate derived from IkB [Formula: see text] kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKK [Formula: see text] and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK [Formula: see text] kinase activity with an IC(50) of 0.5 mM, to decrease the K(m) value from 0.337 mM to 0.402 mM and the V(max) from 0.0257 mM·min [Formula: see text] to 0.0076 mM·min [Formula: see text]. The computational analyses indicate the region between the KD, ULD and SDD domains of IKK [Formula: see text] as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between K(m) and K(i) whereas there is a statistically significant difference between the two V(max) values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.