**8. Application of Microviridins**

Proteases play an important role in the regulation of biological processes in all living organisms by controlling the maintenance, recovery, development and modification of tissues, which may be beneficial or harmful. They may modulate protein–protein interactions that create bioactive molecules involved in DNA replication and transcription [78]. In plants, these enzymes lead to the maturation and degradation of a series of unique proteins relevant to the environmental condition and the stage of growth [79]. Thus, molecules with inhibitory activity against these enzymes have an immense biotechnological potential with a multitude of applications (Figure 10).

**Figure 10.** Potential applications of microviridins.

Tyrosinase is a multifunctional and metalloenzyme widely distributed among plants, microorganisms and animals, where it plays a key role in the development of melanin [80]. The excessive synthesis of this photoprotective pigment may lead to a condition known as hyperpigmentation, which may lead to an esthetic problem where one part of the skin is more pigmented than the other [81]. In addition, this disorder has been linked to many diseases, such as skin cancer and Parkinson's disease [82]. Molecules with the capacity to interfere with the catalytic activity of tyrosinase have been extensively studied as a skinwhitening agent. They can also be used as food additives, reducing the browning process

of mushrooms and fruits caused by tyrosinase [83]. Some of these commercially available compounds exhibit low stability and safety [84]. Among the microviridins reported in the literature, microviridin A was demonstrated to be tyrosinase inhibitor. However, its action mechanism is not clear, and information regarding its toxicity to human cells has never been accessed (Table 3) [21]


**Table 3.** Inhibitory activities of microviridins.

\* IC50 not defined. ND, not determined.

The mechanism of coagulation is regulated by proteases. Serine protease inhibitors therefore function as essential regulators in this pathway, such as proteins in the serpin superfamily. Malfunctioning of one of these elements can lead to coagulation disorders. Excessive blood clotting can lead to a disorder known as thrombosis, where the blood flow is blocked by thrombus [85]. Thrombin is one the major target of anticoagulant drugs, since it acts in the conversion of soluble fibrinogen into insoluble filamentous of fibrins, which, together with platelets, are responsible for a hemostatic plug formation, impeding the bleeding. The thrombin inhibition by microviridin B is superior to the positive control Leupeptin, possessing an EC50 (half maximal effective concentration) value equal to 4.58 μM. This value was inferior than that encountered for Micropeptin K139, a serine protease also detected in cyanobacteria. In contrast, microviridins D-F do not affect thrombin activity, most likely due to the absence of an indole motif, which is encountered in microviridin B, suggesting its role as a recognition motif for thrombin [86].

Human neutrophil elastase is a proteolytic enzyme that belongs to the serum protein family of chymotrypsin-like. This highly active enzyme has revealed a wide substrate specificity and is one of the few proteases capable of degrading the extracellular matrix protein elastin, resulting in the enzyme's name [87]. The elastase overactivity is involved in tissue destruction and inflammation characteristic of various diseases, such as chronic obstructive hereditary emphysema, pulmonary disease, cystic fibrosis, adult respiratory distress syndrome and ischemic-reperfusion injury [88]. Pharmaceuticals already use elastase inhibitors for the treatment of diseases related to this enzyme, such as the drug Sivelastat, which has been cogitated in the treatment of COVID-19 [89].

A study by Masahiro Murakami et al. (1997) [25] evaluated the elastase inhibitory effect of some microviridins, synthesized by the cyanobacterium *Nostoc insulare* (NIES-26). The results of the analysis showed two new peptides, microviridin G and microviridin H. In the experiment, both IC50 were compared with the values of other microviridins already described in the literature. Microviridin A showed no inhibitory effect on elastase; microviridins D and F had the weakest values for inhibiting this enzyme. Microviridins G and H had the best results, followed by microviridins B and C, respectively. After the work

of Murakami et al. [25], three new microviridins were described as elastase inhibitors: I, LH1667 and 1777 (Table 3) [26,30,32].

Proteases are also essential for the growth of insects, such as in the larval and adult stages, where they are present in the intestine and play an important role in digestion [90]. For example, silkworms, near the final stage of their metamorphosis, produce cocoonase, a serine protease capable of hydrolyzing silk protein, which enables the adult moth to emerge [91]. During the embryony phase, proteases digest egg-specific proteins, such as vitellin, for the amino acid release, which are utilized as a nitrogen source [92]. Serine protease can also confer protection against predation. The South American Saturniid caterpillars belonging to the genus *Lonomia* harbor in their hemolymph a toxic protease that some mammals who come in contact with can have, as a consequence, bleeding disorders [93]. Nowadays, various studies have focused on the search for a protease inhibitor whose target are proteases produced by insects involved in disease transmission, such as the *Aedes aegypti*, one of the largest vectors of arboviruses, being responsible for the propagation of the dengue virus, yellow fever, zika virus and chikungunya fever [94].

Microviridins have already shown to be good inhibitors of enzymes present in microcrustaceans intestines, such as those of the genus *Daphnia* and *Thamnocephalus* [32,50]. For instance, in the presence of microviridins B, C, I, J, L and SD1652, the enzyme trypsin has its activity negatively affected [23,26,27,29,31] (Table 3). They act by inhibiting enzymes that are closely related to the diet of these crustaceans. In insects, many of these serine proteases are located in the intestines as well [95], sharing similar functions. Two of these serine proteases are trypsin and chymotrypsin, well-known targets of some microviridins (Table 3).

Plants have served as a great heterology expression system for bioactive peptides. Hilder et al. (1987) [96] were the first to use these organisms to express serine protease inhibitors with the potential to kill predatory larvae insects. There is a considerable number of works employing plants as hosts of ribosomally synthesized and post-translationally modified peptides [97–99]. Plant-based microviridins have promises for future applications, since they can replace the use of pesticides to help control insect pests with low costs and low environmental impacts. Furthermore, they can be easily purified with a high yield, retaining full activity.

One of the bottlenecks for microviridin production and the evaluation in cyanobacteria is a low yield of this peptide. Several extraction approaches in different genus of this phylum demonstrated that these organisms are not well-suited for industrial applications when considering both the time and volume of the cultivations. A heterologous expression in *E. coli* demonstrated to be an efficient method for microviridin biosynthesis resulted in a yield of 60 ◦C 70 mg of microviridin L per 100 g of dried cells after five hours of cultivation. In comparison, about 0.87 mg.g−<sup>1</sup> of microviridin A was obtained from *Microcystis viridis* (NIES-102) by a cultivation period of 10 ◦C for 14 days. In filamentous cyanobacteria, this yield was even lower, with a production of 9.1 mg.g−<sup>1</sup> of microviridin E after the same period of incubation of *Planktothrix* in 400 L [21,25,31].

However, the problems related to microorganism cultivation, heterologous expression and laborious purification can still be tackled, and techniques have been developed to overcome these barriers in order to explore the diversity of the microviridins. As a consequence, the development of microviridins obtained from environmental DNA can be accomplished by the synthesis of the solid-phase peptide of the core peptide coupling with MdnC and -B enzymes fused to the leader peptide in the N-terminal. This chemoenzymatic approach allows an in vitro production of a fully processed microviridin, demonstrating the efficiency during production of different variants of this peptide [34]. Another approach for microviridin production in vitro is to provide the LP in trans for both MdnB and -C, also achieving a tricyclic microviridin J [36].

Another important feature for the biotechnological application of microviridin is its binding affinity to serine proteases. Microviridin J demonstrated a *K*<sup>D</sup> value of 0.68 μM from its interaction with trypsin. This mode of inhibition is similar to a cyclic depsipeptide A90720A produced by a nonribosomal peptide synthetase [34,37]. However, the NRPS pathways are not well-susceptible to genetic engineering compared to RiPPs. Thus, in addition to the possibility of genetic modifications, the microviridin biosynthetic cluster is smaller than NRPS, facilitating a heterologous expression [37].

The crystallography structure of trypsin bound to N-acetylated tricyclic microviridin J (PDB codes: 4KTU and 4KTS; pH 6.5 and 8.5, respectively) has been determined to better understand the relationship between the microviridin structure and its enzymatic target (Figure 11). It was therefore possible to observe that the N-terminal of microviridin J was flexible and bound to the hydrophobic surface of trypsin [100]. As far as the catalytic domain is concerned, both crystallized structures showed the interface as a substrate-like trypsin-binding motif. The threonine residue at position 4 of microviridin J interacts with Leu99 at the S2 pocket through its methyl group. At the S1 pocket, Asp189 coordinates the side chain of arginine at position 5, which is located between the residues making ester and amide bonds. A van der Waals contact by the aliphatic region of Lys6 of microviridin is made with the disulfide bond between Cys43 and Cys58 of the S1' subsite. Finally, the C-terminal of microviridin J, Ser9-Trp14, stabilizes a helical structure of trypsin by the intramolecular covalent linkages of this inhibitor. The interaction between these two structures showed a *K*<sup>D</sup> value of 0.68 μM, which is similar to the NRPS cyanobacterial inhibitor A90720A [100].

**Figure 11.** Interaction between microviridin J and trypsin at pH 8.5 (Protein Data Bank (PDB) code: 4KTS). (**A**) 3D representation of the interaction. (**B**) 2D view of the major interaction between microviridin J and trypsin. Hydrogen bonds are in green, while the hydrophobic interactions are in red.

As far as the Ser-His-Asp triad of trypsin is concerned, these three residues are located in the direction of the Arg5 carbonyl of microviridin. However, the peptide bond between Arg5 and Lys6 was not affected, suggesting that the rings and the compact structure of microviridin J neutralize the cleavage of this tricyclic structure [100].

According to the crystallized structures, it could be concluded that position 5 of the microviridins is essential to its inhibitory activity due to the interaction of the trypsin triad. This hypothesis supports the mutagenesis approach to the modification of residue 5 of microviridin L, which modified both the specificity and the inhibitory activity against different proteases. The wild-type microviridin L has its most potent inhibition against subtilisin (IC50 = 5.8 μMol.L−1); however, the replacement of Phe5 for other amino acids changed this activity. The F5L mutant improved the elastase inhibition; in comparison, F5R increased its inhibition toward trypsin. The F5Y variant shifted its activity against chymotrypsin, and F5M had an IC50 = 0.09 μMol.L−<sup>1</sup> toward subtilisin. In contrast, the exchange of amino acids at positions 7, 9 and 11 did not boost the inhibitory activity at the same scale, nor did any inhibition cease [20,100].

The G2A variant coupled with the shift in position 5 of microviridin J not only enhanced the post-translational modification performances but, also, increased the inhibitory function. The positive charged residues of Arg and Lys at position 5 had superior activity towards subtilisin and trypsin, while the latter was the only variant with a low micromolar activity against plasmin [100]. Similar findings were also observed with microviridin B variants L5R and L5K [38]. In addition, the hydrophobic residues of Leu and Val also demonstrated the inhibition of subtilisin and inhibition of elastase inhibitory activity, with minor variations compared to the single mutants at position 5 [100]. As a result, the amino acids at position 5 of the microviridins have shown great potential to be the focus for therapeutic development, with the goal of enhancing and defining the inhibitory action of microviridins by different synthetic chemical techniques.

#### **9. Final Considerations**

Microviridins are one of the largest oligopeptides present in cyanobacteria. While they were firstly identified in this community of microorganisms, the genomic approach has revealed gene clusters for these metabolites in bacteria belonging to another phyla. Their production is affected by abiotic and biotic factors such as temperature, pH, nutritional content and quorum sensing. This latter is poorly explored in cyanobacteria and can act as a powerful tool in the control of these microorganisms in the environment. Due to their protease inhibitory property, microviridins can be utilized for various purposes, such as an anticoagulant and as whitening agent, as well as in the control of disease vectors.

One of the greatest bottlenecks to the commercial application of microviridins is the low yield and the absence of information about their use in humans and animals. The former issue can be mitigated with the utilization of a heterologous expression system, which has well-described in the literature for this oligopeptide, mainly in the model organism *E. coli.* Others approaches would be the in vitro chemoenzymatic synthesis or the variations in the culture conditions, which could serve as an elicitor, leading to an upregulation of the metabolite. The strong inducible insertion of the promoter may make this process less laborious. In relation to health risks, further studies are required to better develop the inhibitory mechanism of microviridins, as well as their toxicity to humans. However, since they are a ribosomally synthesized and post-translationally modified peptide, they possess a certain plasticity for engineering that can reduce their risks and increase their specificity. The bulk of the cyanobacterial gene cluster remains undiscovered. The ability of these photosynthetic microorganisms to generate biomolecules is greater than was assumed before the genome age. Future studies will disclose new microviridins, as well as knowledge on their biological significance.

**Author Contributions:** Conceptualization, S.C.d.A., P.R.M., L.P.X. and A.V.S. Investigation, S.C.d.A., P.R.M. and G.M.S. Writing—original draft preparation, S.C.d.A., P.R.M., J.d.S.P.N. and G.M.S. Writing—review and editing, S.C.d.A., P.R.M., A.V.S. and L.P.X. Supervision, E.C.G., A.V.S. and L.P.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001 and Fundação Amazônia Paraense de Amparo a Estudos e Pesquisas (FAPESPA)—03/2019.

**Acknowledgments:** The authors would like to thank Pró-Reitoria de Pesquisa e Pós-Graduação da Universidade Federal do Pará (PROPESP/UFPA).

**Conflicts of Interest:** The authors declare no conflict of interest.
