**1. Introduction**

Type 2 diabetes mellitus is a widespread disease (~8% of adults), often resulting from a metabolic disorder caused by over-feeding, an unhealthy diet, and physical inactivity [1,2]. It covers all age groups of the population, and recently it has spread epidemiologically among children and adolescents [3,4]. The blood glucose levels of diabetic patients reaches abnormally high values, which leads to serious damage to many body systems, especially nerves and blood vessels, causing heart and kidney diseases, blindness, and even the amputation of limbs [5]. The preferred way to maintain good health for people with type 2 diabetes or prediabetes is a control of the input of glucose from the digestive tract into the blood stream [6–8]. For this purpose, the medicines based on inhibitors of pancreatic α-amylase are used. GlucobayTM, the active ingredient of which, acarbose, inhibits porcine pancreatic α-amylase (PPA) and human saliva α-amylases (HSA) with Ki's of 0.797 and 1.265 μM, respectively, is one of the most common of that type of drug [9]. In some countries, medicinal plants are traditionally used to

treat diabetes; the study of their composition revealed the presence of low molecular weight substances inhibiting mammalian α-amylases [10,11]. Since the effectiveness of currently existing drugs is limited and they have some side effects, the search for new highly effective inhibitors of pancreatic α-amylase is an attractive goal in the field of drug discovery.

The medicines based on proteins and peptides are poorly represented in the pharmacological market, but attract the interest of specialists due to their high selectivity and effectiveness, combined with relative safety and good tolerability. A large number of proteinaceous α-amylase inhibitors have been isolated from plants, but they are highly specific and interacted with plant α-amylases to control the breakdown of stored starch or with insect α-amylases for defense [12]. Several very effective proteinaceous inhibitors of mammalian, but not plant or microbial α-amylases were found in bacteria belonging to the genus *Streptomyces* [13–16]. However, it was shown that α-amylase inhibitors isolated from bacteria, for example, tendamistat (Ki 9−200 pM), have a high immunogenicity due to their β-sandwich fold and cannot be used in clinical practice [17].

Among animals, amylase inhibitors were found only in sea anemones, ancient sessile predators inhabiting marine environment. Helianthamide (PPA, Ki = 100 pM; human pancreatic α-amylase (HPA), Ki = 10 pM), the first representative of a new group of α-amylase inhibitors belonging to the β-defensins family, was isolated from *Stichodactyla helianthus* in 2016 [18]. This inhibitor is very active, and in contrast to tendamistat, has a more compact structure, which significantly decreases the likelihood of an immune response. Recently, as a result of the proteomic analysis of the sea anemone *H. magnifica* mucus, we have revealed that α-amylase inhibitors are major components, numbering dozens isoforms [19]. Major α-amylase inhibitor, magnificamide, was identified and sequenced (44 aa, 4770 Da) [19]. It shares 84% of sequence identity to helianthamide (44 aa, 4716 Da). The biological relevance of the presence of inhibitors of α-amylases in the mucus of Cnidaria, such as the sea anemone *H. magnifica*, remains largely unexplained. It is hypothesized that inhibition of α-amylase activity intervenes with the metabolism of starch, which forms a major source of nutrition for many organisms. Organisms exposed to α-amylase inhibitors, therefore, suffer from a reduced availability of carbohydrates that serve as an energy resource.

The results presented here are a continuation of an in depth study of magnificamide, more precisely, of a recombinant analog of the peptide with a detailed investigation of its biological activity.
