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Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK? Kinase.

ABSTRACT: The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-?-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK?, 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? 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? and IKKtide, a 20 amino acid peptide substrate derived from IkB? 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? and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK? kinase activity with an IC₅₀ 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-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKK? 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.

SUBMITTER: Lopreiato M

PROVIDER: S-EPMC7915277 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<i>α</i> Kinase.

Lopreiato Mariangela M Di Cristofano Samuele S Cocchiola Rossana R Mariano Alessia A Guerrizio Libera L Scandurra Roberto R Mosca Luciana L Raimondo Domenico D Scotto d'Abusco Anna A

International journal of molecular sciences 20210206 4

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, 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 IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and ...[more]

PMID: 33562013

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Dataset Information

Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK? Kinase.

Publications

Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<i>α</i> Kinase.

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