Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase
The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-<inline-formula><math display="inline"><semantics><mi>β</mi></semantics></math></inline-formula>-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK<inline-f...
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| Language: | English |
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MDPI AG
2021-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/4/1643 |
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| author | Mariangela Lopreiato Samuele Di Cristofano Rossana Cocchiola Alessia Mariano Libera Guerrizio Roberto Scandurra Luciana Mosca Domenico Raimondo Anna Scotto d’Abusco |
| author_facet | Mariangela Lopreiato Samuele Di Cristofano Rossana Cocchiola Alessia Mariano Libera Guerrizio Roberto Scandurra Luciana Mosca Domenico Raimondo Anna Scotto d’Abusco |
| author_sort | Mariangela Lopreiato |
| collection | DOAJ |
| description | The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-<inline-formula><math display="inline"><semantics><mi>β</mi></semantics></math></inline-formula>-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula>, 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<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> 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<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> and IKKtide, a 20 amino acid peptide substrate derived from I<span style="font-variant: small-caps;">k</span>B<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> 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<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> kinase activity with an IC<sub>50</sub> of 0.5 mM, to decrease the K<sub>m</sub> value from 0.337 mM to 0.402 mM and the V<sub>max</sub> from 0.0257 mM·min<inline-formula><math display="inline"><semantics><msup><mrow></mrow><mrow><mo>−</mo><mn>1</mn></mrow></msup></semantics></math></inline-formula> to 0.0076 mM·min<inline-formula><math display="inline"><semantics><msup><mrow></mrow><mrow><mo>−</mo><mn>1</mn></mrow></msup></semantics></math></inline-formula>. The computational analyses indicate the region between the KD, ULD and SDD domains of IKK<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between K<sub>m</sub> and K<sub>i</sub> whereas there is a statistically significant difference between the two V<sub>max</sub> 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. |
| first_indexed | 2024-03-09T05:22:58Z |
| format | Article |
| id | doaj.art-3b6c0061717b4061b6b080d2cecf8050 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T05:22:58Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3b6c0061717b4061b6b080d2cecf80502023-12-03T12:38:34ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01224164310.3390/ijms22041643Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> KinaseMariangela Lopreiato0Samuele Di Cristofano1Rossana Cocchiola2Alessia Mariano3Libera Guerrizio4Roberto Scandurra5Luciana Mosca6Domenico Raimondo7Anna Scotto d’Abusco8Department of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyDepartment of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, ItalyDepartment of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyDepartment of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyDepartment of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyDepartment of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyDepartment of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyDepartment of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, ItalyDepartment of Biochemical Sciences, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Rome, ItalyThe glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-<inline-formula><math display="inline"><semantics><mi>β</mi></semantics></math></inline-formula>-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula>, 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<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> 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<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> and IKKtide, a 20 amino acid peptide substrate derived from I<span style="font-variant: small-caps;">k</span>B<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> 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<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> kinase activity with an IC<sub>50</sub> of 0.5 mM, to decrease the K<sub>m</sub> value from 0.337 mM to 0.402 mM and the V<sub>max</sub> from 0.0257 mM·min<inline-formula><math display="inline"><semantics><msup><mrow></mrow><mrow><mo>−</mo><mn>1</mn></mrow></msup></semantics></math></inline-formula> to 0.0076 mM·min<inline-formula><math display="inline"><semantics><msup><mrow></mrow><mrow><mo>−</mo><mn>1</mn></mrow></msup></semantics></math></inline-formula>. The computational analyses indicate the region between the KD, ULD and SDD domains of IKK<inline-formula><math display="inline"><semantics><mi>α</mi></semantics></math></inline-formula> as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between K<sub>m</sub> and K<sub>i</sub> whereas there is a statistically significant difference between the two V<sub>max</sub> 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.https://www.mdpi.com/1422-0067/22/4/1643IKKNAPAosteoarthritischondrocytesUPLC-MSmolecular docking |
| spellingShingle | Mariangela Lopreiato Samuele Di Cristofano Rossana Cocchiola Alessia Mariano Libera Guerrizio Roberto Scandurra Luciana Mosca Domenico Raimondo Anna Scotto d’Abusco Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase International Journal of Molecular Sciences IKK NAPA osteoarthritis chondrocytes UPLC-MS molecular docking |
| title | Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase |
| title_full | Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase |
| title_fullStr | Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase |
| title_full_unstemmed | Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase |
| title_short | Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKK<em>α</em> Kinase |
| title_sort | biochemical and computational studies of the interaction between a glucosamine derivative napa and the ikk em α em kinase |
| topic | IKK NAPA osteoarthritis chondrocytes UPLC-MS molecular docking |
| url | https://www.mdpi.com/1422-0067/22/4/1643 |
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