4. Molecular targets for flavonoids

Many efforts have been done to describe flavonoids' main biological targets involved in cellular processes such as inflammation and cancer. Flavonoid mechanism of action in cellular responses includes the inhibition of proteins in cytoplasm medium such as IκB kinase (IKK) complex and mitogen-activated protein kinases (MAPKs), as well as in extracellular medium such as interleukins and other cytokines.

In the pathway that triggers the activation of nuclear factor κB (NF-κB) in tumor necrosis factor (TNF) stimulated cells (Figure 6), IKK complex is a target for many flavonoids such as apinegin [34], luteolin [34], acacetin [35], ochnaflavone [35], delphinidin [35], and morin [36].

Molecular Mechanism of Flavonoids Using Fluorescence Spectroscopy and Computational Tools DOI: http://dx.doi.org/10.5772/intechopen.84480

The inhibition of IKK complex results in failure in phosphorylation of Iκβ. As a consequence, NF-κB is not released to translocate to the nucleus. In the case of the flavonoids apinegin and luteolin, they can also inhibit NF-κβ activation by inhibition of MAPKs [34].

#### Figure 6.

NF-kB and AP-1 activation pathways induced by TNF.

Figure 7. NF-kB activation pathway induced by LPS.

In the pathway that triggers the activation of activator protein 1 (AP-1), c-Jun N-terminal kinase (JNK) is a target for flavonoids kaempferol and chrysin [34]. Such flavonoids bind to the protein and inhibit the activation of AP-1. Sumaflavone also suppresses the AP-1 activation, but the main target for this flavonoid has not been identified yet [35].

In LPS-stimulated cells (Figure 7), epigallocatechin-3-gallate (EGCG) [37] and morin [36] inhibited the activation of NF-κB activity by IKK complex inhibition. Besides that, EGCG also inhibited p38 MAP kinase activity [37]. Another molecular target in LPS-stimulated cells is JNK, which is inhibited for galangin [38].

The molecules genistein, kaempferol, quercetin, and daidzein inhibited the activation of STAT-1 and NF-kB in LPS-induced cells, while flavone, isorhamnetin, naringenin, and pelargonidin inhibited only the NF-kB activation. However the molecular targets for such flavonoids have not been identified yet [39, 40].

Cytokines can also be targets for flavonoids. As verified by Li and co-workers [41], baicalin is able to complex with a variety of chemokines such as SDF-1a, IL-8, MIP1-b, and MCP-2 and reduce the capacity of the cytokines to bind and activate their receptors.

Despite the works described above show which proteins are inhibited by flavonoids, the methodology used does not give information related to how strong is the affinity of the flavonoid for the protein, which amino acids of the protein participate in the interaction with flavonoids, which are the molecular driving forces involved in the interaction, the thermodynamic parameters of complex formation, how many binding sites there are in the protein for the flavonoids, and whether the protein presents cooperativity among their sites. In this way, the methodology of fluorescence spectroscopy and molecular docking described in Sections 2 and 3 come to help give this set of information that are of the utmost importance to drug discovery.

Among the target proteins mentioned above, NF-κβ (p50 and p65 subunits) has one tryptophan residue, while JNK have four tryptophan residues. Besides that, all the cytokines (SDF-1a, IL-8, and MCP-2) mentioned above have 1 tryptophan residue, except for MIP1-b which has 12. The presence of tryptophan makes the use of fluorescence spectroscopy possible, but the presence of more than one makes the interpretation of the spectral data more complicated. Different of the proteins mentioned above, AP-1 does not have tryptophan residue. On the other hand, there are tyrosine residues, which can also be followed in the experiments.

Besides the endogenous fluorescent probe, solubility is another important factor because fluorescence spectroscopy experiments are conducted with protein in solution. As the proteins mentioned above are found in cytoplasm or in extracellular medium, they are expected to be soluble in buffer.
