*2.6. Study of Mammalian* α*-Amylase Inhibition*

Since magnificamide shared a high sequential and structural similarity with the competitive tight-binding inhibitor helianthamide, it was predicted to possess analogous kinetic features. For a quantitative assessment of tight-binding inhibitor potency, Morrison's method was applied [34,35]. This method uses the Morrison quadratic equation (Equation (1)) for fitting inhibitor-response data and determining Ki *app* as a nonlinear regression parameter.

$$\frac{\nu}{\nu\_o} = 1 - \frac{\left( [E] + [I] + K\_i^{app} \right) - \sqrt{\left( [E] + [I] + K\_i^{app} \right)^2 - 4[E][I]}}{2[E]\_o} \tag{1}$$

True Ki values are calculated from the Equation (2), suggesting a competitive mode of inhibition.

$$K\_i^{app} = K\_i \left( 1 + \frac{[S]}{K\_M} \right) \tag{2}$$

Recombinant magnificamide inhibition constants against porcine pancreatic α-amylase (PPA) and human salivary α-amylase (HSA) were determined. Kinetic assays revealed that recombinant magnificamide was indeed a potent nanomolar tight-binding inhibitor: Ki against PPA was 0.17 ± 0.06 nM; Ki against HSA was 7.7 ± 1.5 nM (Figure 6).

**Figure 6.** Amylase inhibition curves using r-magnificamide. Fixed concentrations of each enzyme (PPA on (**a**) and HSA on (**b**)) were mixed with increasing concentrations of r-magnificamide (displayed in nM). Each connecting line represents the best fits to the quadratic Morrison equation for tight binding inhibitors [35].

#### **3. Discussion**

Sea anemones are ancient sessile predators inhabiting the marine environment. They have specialized stinging cells which contain venom rich in peptides acting on different biological targets, mainly cytoplasmic membranes [36–38] and ion channels [39–44]. Venom with such a complex composition ensured the existence of sea anemones for millions of years [45]. Recently, it has been shown that sea anemones also present a source of pancreatic α-amylase inhibitors belonging to the β-defensin family [18,19]. In the venoms of sea anemones, the β-defensin fold is widely recruited to create toxins modulating ion channel activity, interestingly, often with little amino acid sequence identity, but with similar spatial structure. According to Mitchell and coauthors, cnidarian β-defensin-like toxins can be divided into four main groups: APETx-like, BDS-like, Nv1-like, and ShI-like [46]. Representatives of APETx-like and BDS-like groups interact with ASICs, hERG, voltage-gated sodium, and potassium ion channels [33,47–49]. Some of them, crassicorin I and II from *Urticina crassicornis*, reveal paralytic activity against crustaceans, as well as antimicrobial activity against Gram-positive and Gram-negative bacterial strains [33]. Nv1-like and ShI-like peptides are often a major content of sea anemones' venom and modulate voltage-gated sodium ion channels [50,51]. Helianthamide-like peptides represent a separate group [46] suggesting a different activity.

Taking into account the wide variety of sea anemone defensin-functions, we conducted a study of activity of magnificamide on various ion channels (Table 4) and found no activity. No antimicrobial activity against Gram-positive, Gram-negative bacteria, or fungi was observed either (Table 3). Thus, structural remoteness may occur due to narrow specialization of sea anemones' helianthamide-like peptides. The presence of numerous digestive enzyme (proteinases and amylases) inhibitors [19] in sea anemone venoms is per se an interesting defensive strategy, similar to plant protection from insects and herbivores.

From a practical point of view, pancreatic α-amylase inhibitors effectively control the influx of glucose into the bloodstream from the gastrointestinal tract [18,19]. Inhibitors of pancreatic α-amylase have a great pharmacological potential for the prevention and treatment of metabolic disorders and type 2 *diabetes mellitus*. In this work we have shown that magnificamide was an effective inhibitor of mammalian α-amylases. The homologue of magnificamide, helianthamide from *S. helianthus*, inhibited PPA with very close Ki (Table 5). Sea anemone inhibitors had great inhibitory activity against mammalian α-amylases (Table 5); the combination of such activity with a compact fold could be used to create new drugs.


**Table 5.** Mammalian α-amylase inhibitors from different sources.

Moreover, for the first time, sea anemone peptides' ability to inhibit HSA was clarified on the example of magnificamide, with Ki equal to 7.7 nM. Inhibition of salivary α-amylase allows for blocking the digestion of starch upon the first stages of entering the body. In addition, it may be useful for the treatment of diseases the of oral cavity, including caries. Caries is a multifactorial disease, a significant role in the development of which is played by oral *Streptococci*, capable of binding salivary α-amylase and using sugar that can be broken down by it for their own needs. The binding of *Streptococci* to salivary α-amylase also contributes to the formation of biofilms and the demineralization of teeth [52]. It has been shown that cherry and tea extracts exhibiting inhibitory activity for salivary α-amylase could inhibit the growth of oral *Streptococci* (in particular *Streptococcus mutans*) [53–55]. Given a stable structure and high activity of magnificamide, it may also find an application in the form of chewing gum, as was shown for cherry extract [53].

#### **4. Materials and Methods**
