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- Title
Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK α Kinase.
- Authors
Lopreiato, Mariangela; Di Cristofano, Samuele; Cocchiola, Rossana; Mariano, Alessia; Guerrizio, Libera; Scandurra, Roberto; Mosca, Luciana; Raimondo, Domenico; d'Abusco, Anna Scotto; Lymperopoulos, Anastasios
- Abstract
The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy- β -D-glucose (NAPA), was shown to inhibit the kinase activity of IKK α , 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 α 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 α and IKKtide, a 20 amino acid peptide substrate derived from IkB α 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 α and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK α kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min − 1 to 0.0076 mM·min − 1 . The computational analyses indicate the region between the KD, ULD and SDD domains of IKK α as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax 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.
- Subjects
NAPA (Calif.); GLUCOSAMINE; MOLECULAR docking; BINDING sites; MASS spectrometry; MOLECULAR dynamics; SERINE
- Publication
International Journal of Molecular Sciences, 2021, Vol 22, Issue 4, p1643
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms22041643