Subsequently, we analyzed the number of H-bonds and their stability during the MD simulations from 150 ns to 300 ns. from 0.0257 mMminto 0.0076 mMminas 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. and IKKand IKKproteins share high amino JC-1 acid sequence identity, almost 50%, and their 3D structure presents a kinase catalytic domain (KD) at N-terminal region, a ubiquitin-like domain (ULD) in the middle, followed by an also contains a Nuclear Localization Sequence (NLS) . A detailed structural analysis of the IKKKD revealed that this domain, when dephosphorylated, shows conformations that are not compatible with its substrates, thus the enzymatic activity is inactive [4,5]. The ULD is required for the activation of kinase activity and, together with SDD, it is involved in the exact positioning of the kinase substrate, which is recruited by NEMO. The interdependence of the three domains is reflected by their intramolecular interactions [5,6]. On the other hand, the IKKdimerization depending on SDD is required for binding with NEMO GIII-SPLA2 but not for kinase activity after the phosphorylation of the activation loop . To date, the signal transmission leading to IKK activation is still a matter of debate. The presence of kinases that phosphorylate the activation loop or IKK trans-autophosphorylation are both hypothesized [7,8]. In the inactive IKKformation is available. Analyses performed by gel filtration revealed that the complex has a molecular weight of about 700C900 kDa, and equilibrium sedimentation experiments suggest that NEMO has a tetrameric structure. Thus, the hypothesis is that the IKK complex stoichiometry could be IKKand IKKinto proximity . The IKKcrystal structure analysis suggests that this kinase can form homodimers and tetramers even if to a lesser extent, and that the oligomerization can revert in solution [5,6]. Moreover, other complexes might exist, considering that it has been demonstrated that NEMO can interact with IKKand IKKhomodimers [9,10]. Recently, the crystal structure of IKKhas been solved, showing both the protein domains and the supramolecular organization . Like IKKforms a dimer and each monomer contains KD, ULD and SDD domains. KD shows both inactive and active conformations, ULD serves for interaction with substrates and SDD is involved in the interaction between the monomers, finally, the dimer interfaces of IKKand IKKare highly similar . Globally, the organization of the two kinases is similar, but significant differences can be observed, mainly regarding the orientation of KD with respect to SDD and ULD. In IKKreduces the kinase activity. Most of these JC-1 residues are conserved in IKKexcept for Trp434 and Phe111 . The single particle electron cryo-microscopy (cryo-EM) method showed the presence of three predominant conformational states of IKKdomains. Moreover, X-ray crystal (PDB: 5EBZ) and cryo-EM structures (PDB codes of IKKdimer solved by cryo-EM: 5TQW, 5TQX, and 5TQY) together revealed the presence of distinct conformers of IKKsimilar to those observed in IKK. Finally, JC-1 Polley et al. observed the formation of further higher order oligomers consisting of three dimers forming an hexamer. The observed interactions were KD-KD, KD-SDD and SDD-SDD . This organization was found only in vitro in X-ray and cryo-EM experiments, whereas the analysis of cellular extracts by Superose size-exclusion chromatography showed the presence of dimers and of an uncharacterized large complex likely containing IKKand NEMO . The two kinases in vivo show different functions. When IKKis associated with NEMO, it activates the canonical pathway of NF-kB [12,13] through the phosphorylation of the isoform alpha of NF-kB inhibitor protein (IkBphosphorylates IkB proteins, which can be recognized by the ubiquitin ligase machinery, leading to their polyubiquitination and subsequent degradation, allowing NF-kB transcriptional factors to migrate into nuclei and bind specific promoters or enhancer regions of the target genes. IKKactivates the non-canonical pathway , even if IKKsupports the activation of the NF-kB canonical pathway through the phosphorylation of p65 subunit and H3 histone in the nucleus . Moreover, IKKneeds to be associated with the kinase NIK to activate the non-canonical NF-kB pathway. The point of contact corresponds to the sites used by IKKdimers to form the hexamer, His578 and Tyr580, and Asn408 and Tyr409. The mutation of these sites induces the inhibition of non-canonical pathway . In our lab, we synthesized and studied the effects of a glucosamine-derivative, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-activates the non-canonical pathway, in support of IKKaction and is also responsible for NF-kB-independent processes, such as the stimulation of several molecules, JC-1 matrix metalloproteinases (MMPs), transcriptional factors and others, involved in the progression of.