*2.3. Molecular Modeling*

The spatial structure models of magnificamide were generated using the homology modeling approach with MOE 2016.08 software (Montreal, QC, Canada) [21]. The atom coordinates of the helianthamide from *S. helianthus*, extracted from the complex with porcine pancreatic α-amylase (PDB ID 4XON) were used as a template. Then the solvated models were optimized using the 400 ns MD simulations in Amber10: EHT force field and the most energetically favorable state of magnificamide was selected (Figure 5b). The molecule has a characteristic fold stabilized by 3 disulfide bridges, including a β-sheet, formed by four strands, an α-helix, and several loops, as well as sufficiently mobile C and N-terminal regions. The content of secondary structure elements agrees well with the data of CD spectroscopy of the native peptide. Despite the relatively high RMSD value for 44 Cα atoms of magnificamide model relative to the prototype—2.46 Å, the model quality assessment showed no conformational constraints (ψ and ϕ-angles) of amino acid residues, which indicates the good quality of the generated model. It turned out that the largest deviation (from 2 Å to 5.6 Å) involved the flexible parts of the structure in the sequence regions 1–4, 10–12, 18–20, and 32–33. It should be noted that these areas either included variable amino acid residues or were localized in close proximity to those (Figure 5a). The mapping of magnificamide variable residues revealed an interesting feature. In fact, the variability affected only one part of the molecule, while another one remained conservative.

**Figure 5.** *Cont*.

**Figure 5.** (**a**) Alignment of sea anemone α-amylase inhibitors: magnificamide *H. magnifica* [18] and helianthamide from *S. helianthus* [17] amino acid sequences and their spatial structures. (**b**) The ribbon diagrams of magnificamide and helianthamide spatial structures are colored according to the structure elements; the side chains of the variable residues magnificamide are shown as sticks and labeled. Molecular dipole and hydrophobic moments are indicated by blue and green arrows, respectively. (**c**) Magnificamide and helianthamide molecular surfaces are colored according to surface charge distribution.

Using the MOE 2016.08 program, the physicochemical characteristics of the inhibitor were evaluated and the surface properties of magnificamide were analyzed to compare them with helianthamide (Table 2). It was shown that, despite its greater compactness, this molecule was characterized by a larger hydrophobic surface area, as well as a redistribution of the localization of charged regions (Figure 5c). This is manifested in a change in both the magnitude and direction of the dipole, and in the hydrophobic moments of the molecules (Figure 5b; Table 2).


**Table 2.** Physico-chemical characteristics of the α-amylase inhibitors.

#### *2.4. Study of Antimicrobial Activity*

Since the main function of defensins in most organisms-producers is the protection against microorganisms [22–28], we performed a screening of potential antimicrobial activity displayed by r-magnificamide. It did not reveal activity against fungi, Gram-positive or Gram-negative bacteria (Table 3).


**Table 3.** Antimicrobial activity of r-magnificamide.
