3.5.5. Molecular Docking Analysis

In order to ge<sup>t</sup> a prediction of the interaction of the Kelch domain of Keap1 with **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E,** docking simulation was performed (Figure 7). The free energy of dissociation of the assemblies calculated after a simulation of 50 ns showed that peptide **E-11-F** has no binding affinity with the target protein. On the contrary, peptides **A-17-E**, **Q-14-R** and **N-15-M** formed stable assemblies

with the Kelch domain of Keap1. More in details, the first Serine (Ser 3) of **A-17-E** formed hydrogen bonds with a serine (Ser 363) and an arginine (Arg 415), the first **A-17-E** asparagine (Asn 9) formed a hydrogen bond with a tyrosine (Tyr 572) and the inner glutamic acid (Glu 14) formed hydrogen bonds with another arginine (Arg 336) (Figure 7A'). The **Q-14-R** peptide formed hydrogen bonds between its backbone and asparagine (Asn 382), arginine (Arg 380), glutamine (Gln 530), threonine (Thr 576) and histidine (His 575) of the Keap1 pocket; there were also hydrogen bonds between the asparagine (Asn 7) of the peptide and asparagine (Asn 387), arginine (Arg 380) and histidine (His 432) of Kelch domain, the aspartic acid (Asp 10) formed a hydrogen bond with a tyrosine (Tyr 525) and the inner glutamine of the peptide (Gln 11) formed hydrogen bonds with an arginine (Arg 415) and a serine (Ser 508) (Figure 7B'). The backbone of the **N-15-M** formed hydrogen bonds with an asparagine (Asn 382) a glycine (Gly 509), a tyrosine (Tyr 572) and three arginines (Arg 336, Arg 380, Arg 415); the first threonine of the peptide (Thr 2) formed a hydrogen bond with a Serine (Ser 602), the lysine (Lys 6) of the peptide form hydrogen bonds with a glycine (Gly 433) and an isoleucine (Ile 435), the glutamine of the peptide (Gln 11) formed a hydrogen bond with a threonine (Thr 576) (Figure 7C'). It is crucial to notice that there is a certain recurrence among the residues of the Kelch domain involved in the interaction with the peptides. Moreover, these residues are also implicated in the interaction between Kelch domain and Nrf2 [37] (Table 4).

**Figure 7.** Molecular docking analysis of the interaction between peptides and Keap1 Kelch domain. (**A–C**) Binding geometry of **A-17-E**, **Q-14-R** and **N-15-M** in the pocket of Keap1. (**A'–C'**) Magnification of the interaction of Keap1 Kelch domain with **A-17-E** (**A'**), **Q-14-R** (**B'**) and **N-15-M** (**C'**). Amino acids involved in the hydrogen bond formation were connected with orange dashed lines and highlighted in Table 4.


**Table 4.** Residues involved in the binding of Keap1 with Nrf2 or the analyzed peptides.
