The IKB kinase (IKK) complex, involved in several cellular pathways, comprises two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO. Globally, the organization of the two kinases is similar, even if significant differences can be observed. IKKβ, associated with NEMO, activates the canonical pathway of NF-κB, while IKKα mainly activates the non-canonical pathway. NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-b-D-glucose, is a glucosamine derivative synthetized and tested in vitro in our lab on human primary chondrocytes isolated from cartilage of patients with Osteoarthritis (OA) the most common inflammatory joints disease. The NF-κB kinases have a prominent role in the activation of inflammatory processes in OA such as the stimulation of several molecules, matrix metalloproteinases (MMPs) and transcriptional factors.
Considering that the IKKα kinase activity resulted inhibited by NAPA, as shown in our previous work, the aim of this study is to explore the interaction between NAPA and IKKα through in vitro kinase assay. The IKKα enzymatic activity was studied performing an in vitro assay using a recombinant IKKα kinase and a synthetic peptide, IKKtide, containing the two serine residues. Interestingly, in relation of these reaction conditions, IKKα was able to phosphorylate the IKKtide preferable on one serine only, as pIKKtide was largely exceeding the ppIKKtide amount revealed by UPLC/MS. To evaluate the inhibitory effect of NAPA on the IKKtide phosphorylation, different concentrations of NAPA were incubated with IKKα, ATP and IKKtide, as substrate. The NAPA IC50 was found to be 0.5 ± 0.086 mM. that could seem high, anyway, it has to be considered that a strong inhibition of IKKα could result in a detrimental effect for cells and could display side effects in humans. To verify if NAPA inhibited IKKα interacting with the active site of the kinase, the assay was performed using different amount of ATP, but all these concentrations were unable to revert the inhibition of NAPA, suggesting that NAPA did not interact with the ATP binding site of IKKα. To confirm this hypothesis, a kinetic assay was performed using a fixed amount of enzyme and increasing concentrations of substrate, IKKtide. The same experiment was conducted in presence of different concentrations of NAPA. The difference between Km and Ki obtained resulted not statistically significant, in contrast, the difference between the Vmax values obtained in absence or in the presence of NAPA strongly suggested that the inhibition could be non-competitive.
It can be concluded that this inhibition of IKKα kinase activity makes NAPA a very appealing molecule considering that phosphorylation has a central role in biological regulation of intracellular pathways. Our findings demonstrated that NAPA does not bind to the ATP binding site and made this molecule extremely interesting and particularly suitable for long-term treatments, such as those required for OA
