**1. Introduction**

*O*-glycoside hydrolases are involved in the degradation of various poly- and oligosaccharides that serve as a source of carbon and energy for organism's growth, as well as performing various functions in organisms. Modification or blocking of these functions by powerful selective inhibitors underlies the treatment of a number of infectious diseases, malignant tumors and genetic disorders [1]. Inhibitors of enzymes are molecules that reduce or completely block the catalytic activity of an enzyme, causing either complete death of a cell or modification in the metabolic pathways. The marine sponges are important sources of enzyme inhibitors [2,3].

α-D-galactosidases ( α-D-galactoside galactohydrolases, EC 3.2.1.22) catalyze the hydrolysis of non-reducing terminal α-D-galactose (Gal) from α-D-galactosides, galactooligosaccharides and polysaccharides. α-D-galactosidases are widespread among terrestrial plants, animals, human organs and tissues, as well as microorganisms [4]. The enzymes occur frequently in marine bacteria, especially in γ-Proteobacteria and Bacteroidetes [5–8]. The marine enzyme α-D-galactosidase ( α-PsGal) was

isolated from the cold-adaptable marine bacterium *Pseudoalteromonas* sp. KMM 701 inhabiting in the cold water of the Sea of Okhotsk [9]. The enzyme attracted our attention due to its ability to reduce the serological activity of B red blood cells [9]. The enzyme also interrupted the adhesion of *Corynebacterium diphtheria* to buccal epithelium cells at the neutral pH [10], and regulated the growth of biofilms of some pathogenic bacteria [11]. According to the carbohydrate active enzymes' classification (CAZy) [12] that is based on the amino acid sequence, α-PsGal belongs to the glycoside hydrolases (GHs) family 36.

<sup>α</sup>-*<sup>N</sup>*-Acetylgalactosaminidases (EC 3.2.1.49) catalyze the hydrolysis of the terminal α-linked *N*-acetylgalactosamine residues from the non-reducing ends of various complex carbohydrates and glycoconjugates. In the marine environment, <sup>α</sup>-*N*-acetylgalactosaminidases have been isolated from the liver and digestive organs of marine invertebrates, fishes, and marine bacteria of the genus *Arenibacter* [13,14]. The <sup>α</sup>-*N*-acetylgalactosaminidase from the marine bacterium *Arenibacter latericius* KMM 426<sup>T</sup> (α-NaGa) was successfully applied for the complete conversion of A- into *O*-erythrocytes [15]. According to the CAZy classification, α-NaGa belongs to the GH109 family and is NAD+-dependent *O*-glycoside hydrolase as <sup>α</sup>-*N*-acetylgalactosaminidase from a clinical pathogen *Elizabethkingia meningoseptica* [14]. Thus, α-PsGal and α-NaGa were found to be potential tools for blood transfusion as well as for structural studies in glycobiology and infection diseases. Therefore, screening and studying the natural inhibitors of these enzymes should be helpful for understanding the molecular machinery of their function and designing a method for their removal from the reaction for medical purposes.

Some of the secondary metabolites from marine sponges, which are biologically active compounds, were found to be applicable for pharmacology as the inhibitors of different classes for enzymes [3,16]. To date, a number of alkaloids of unique structures have been isolated from the marine sponge *Monanchora* sp. [17]. Their antitumor activity and mechanism of action have been shown [18–22]. The effect of pentacyclic guanidine alkaloids monanchomycalin B, monanchocidin A and normonanchocidin A isolated from the marine sponge *Monanchora pulchra* on the activity of exo-β-1,3-D-glucanases from the marine filamentous fungus *Chaetomium indicum* and endo-β-1,3-D-glucanase LIV from the marine bivalve mollusk *Spisula sachalinensis* was investigated [23]. In the present study, we focus our attention on the effect of the marine sponge secondary metabolites with a good therapeutic potential on the activity of two well-characterized α-glycosidases to elucidate the mechanism of their inhibitor action.

The present article aimed to compare of the effects of monanchomycalin B, monanchocidin A and normonanchocidin A on the activities of recombinant α-galactosidase from the marine bacterium *Pseudoalteromonas* sp. KMM 701 of the GH36 family and <sup>α</sup>-*N*-acetylgalactosaminidase from the marine bacterium *Arenibacter latericius* KMM 426<sup>T</sup> of the GH109 family.

#### **2. Results and Discussion**

#### *2.1. Identification of the Compounds*

The samples of the marine sponge *M. pulchra* were collected in the Sea of Okhotsk (Kuril Islands region). The ethanol (EtOH) extract of the sponge *M. pulchra* sample N 047-243 was concentrated. The ethanol-soluble materials were further subjected to flash column chromatography on YMC\*GEL ODS-A and high-performance liquid chromatography (HPLC) to obtain the pure monanchomycalin B (1). Monanhocidin A (**2**) and normonanhocidin A (**3**) were isolated from the EtOH extract of the sponge *M. pulchra* sample N 047-28 by the same method. The structure of the compounds **1**, **2** and **3** were assigned through the comparison of their spectral data with those reported in the references [20–22], respectively. Structural formulas of the pentacyclic guanidine alkaloids are shown on Figure 1.

The compounds isolated from the sponge *M. pulchra* have the same "vessel" part and differ in the structure of the "anchor" part of the molecule. The "anchor" part is presented by spermidine residue in monachomycalin B (**1**), by the tetra-substituted morpholinone derivative in monanchocidin A (**2**), and by the monosubstituted diaminopropane in normonanchocidin A (**3**).

**Figure 1.** Structural formulas of pentacyclic guanidine alkaloids. "Vessel" part is on the left, and the "anchor" part is on the right of the molecule formula.

#### *2.2. Effect of Monanchomycalin B, Monanchocidin A, and Normonanchocidin A on Activity of Two Glycosidases*

The results of the pretreatment of two marine bacterial glycosidases with pentacyclic guanidine alkaloids within 30 min (Table 1) showed that all three compounds inhibited the activity of recombinant GH36 α-PsGal and had no effect on the recombinant GH109 α-NaGa.

**Table 1.** Residual activity v/v0 (%) of the glycosidases after incubation with monanchomycalin B, monanchocidin A, or normonanchocidin A 1.


1 Concentration of compounds in each sample was 0.2 mM, the enzyme was preincubated with an inhibitor for 30 min, 20 ◦C, pH 7.0.

It was previously shown that all three compounds significantly activated *Sps*LamIV endo-β(1→3)-D-glucanase of the mollusk *Spisula sachalinensis* and completely inhibited *Chin*Lam exo-β(1→3)-D-glucanase of the marine fungus *Chaetomium indicum* [23].

We have shown with the example of monanchomycalin B that pentacyclic guanidine alkaloids irreversibly inactivate the α-PsGal. The activity of the enzyme did not recover after dialysis against the buffer solution for 72 h (Table 2). The decrease of free enzyme activity by 2.6 times was observed, probably, due to the enzyme α-PsGal thermolability [24] and instability at the low concentrations (data not shown).

The study of the inhibitory effect of monanchomycalin B, monanchocidin A, and normonanhocidin A at different concentrations and incubation times showed that the IC50 values of compounds decreased with increasing of the incubation time of α-PsGal with inhibitors (data not shown). The results of kinetic studies on the α-PsGal inactivation by pentacyclic guanidine alkaloids are shown on Figure 2. The curves of the dependences of the residual activity v/v0 on the time in semilogarithmic coordinates are shown in Figure 2a,c,e.


**Table 2.** The activity of α-PsGal after treating with monanchomycalin B 1.

<sup>1</sup> The activity of α-PsGal after dialysis (72 h, 4 ◦C, 0.02 M sodium phosphate buffer (pH 7.0)) is presented considering the dilution of the enzyme sample. The results are average of three parallel measurements.

**Figure 2.** The results of kinetic studies of the α-PsGal inactivation by pentacyclic guanidine alkaloids: (**a**) the kinetic change of the residual activity of the enzyme (v/v0) in semilogarithmic coordinates at 1.3 μM (1), 2.66 μM (2), (3) 5.69 μM, (4) 11.8 μM, and (5) 23.9 μM of monanchomycalin B; (**b**) the inactivation rate constants (*k*obs) dependence on the concentrations of monanchomycalin B; (**c**) the kinetic change of the residual activity of the enzyme in semilogarithmic coordinates at 2.7 μM (1), 5.4 μM (2), (3) 10.7 μM, (4) 21.4 μM, and (5) 42.9 μM of monanchocidin A; (**d**) the inactivation rates (*k*obs) dependence on the concentrations of monanchocidin A; (**e**) the kinetic change of the residual activity of the enzyme in semilogarithmic coordinates at 1.49 μM (1), 2.98 μM (2), 5.97 μM (3), and 11.9 μM (4) of normonanchocidin A; (**f**) the inactivation rates (*k*obs) dependence on the concentrations of normonanchocidin A. All of the experiments were performed in duplicates.

The α-PsGal inactivation developed relatively slowly, within a few minutes under these experimental conditions. In this case, the inhibitory activity of the compounds can be more accurately described by the inactivation rate constant (*k*inact, min–1) and equilibrium inhibition constant *K*i [25]. The values of *k*obs increased together with the compound concentrations. Sigmoid curves of *k*obs dependences on concentration of the inhibitors (Figure 2b,d,f) mean that the process of the enzyme (E) inactivation by slowly-binding irreversible inhibitors (I) has a cooperative character, and occurs in two stages: (i) the formation of a reversible enzyme-inhibitor complex [E In] and (ii) irreversible inactivation of the enzyme in the E-In complex. The kinetic Equation (1) describes the irreversible slow inhibition of α-PsGal under the action of the pentacyclic guanidine alkaloids:

$$\begin{array}{ccc} \mathbb{K}\_{\text{i}} & k\_{\text{inact}} \\ \mathrm{E} + \mathrm{nI} \Longrightarrow \{\mathrm{E} \ I^{\mathrm{n}}\} \rightarrow \mathrm{E} \cdot \mathrm{I}^{\mathrm{n}}, \end{array} \tag{1}$$

where n is coefficient of cooperativity, which is interpreted as the number of identical binding sites; *K*i is an equilibrium constant of inhibition (μM). The experimental dependences of *k*obs on the concentration of the compounds (I) (Figure 2b,d,g) are approximated by the Hill's Equation (2).

$$k\_{\rm obs} = k\_{\rm incact} \,\mathrm{I}^{\mathrm{n}} / (\mathrm{K}\_{\mathrm{i}}^{\mathrm{n}} + \mathrm{I}^{\mathrm{n}}),\tag{2}$$

The results of the experimental data fitting with theoretical curves are shown in Table 3.

**Table 3.** The α-PsGal inhibition constants for monanchomycalin B, monanchocidin A, and normonanchocidin A.


According to the values of *K*i and *k*inact, the alkaloids can be arranged in descending of binding-affinity as normonanchocidin A > monanchomycalin B > monanchocidin A, but in increasing inactivation rate in the following order: monanchomycalin B > monanchocidin A> normonanchocidin A.

Thus, based on the results of the kinetic studies, we have suggested that the pentacyclic guanidine alkaloids are slow-binding inhibitors for α-PsGal similarly to the *Chin*Lam glucanase [23]. It is accepted that slow-binding inhibition is observed whenever an enzyme-inhibitor complex forms or undergoes further conversion, at a slower rate relative to the overall reaction rate [26]. Inhibitors of peptidases [27], monoamine oxidases, and acetylcholinesterases [28,29] are examples of the slow-binding. Previously, the property of monanchocidin A as a slow-acting biologically active compound was shown for cancer cells [30]. For α-PsGal, the chlorine and bromine echinochrome derivatives from a sea urchin have been previously shown to be slow-binding inactivators as well [31]. Moreover, we have found that the inhibition rate increases with the binding of at least four molecules of the compounds.

D-galactose being a competitive inhibitor for α-galactosidases of GH36 family [32,33] decreased the activity of α-PsGal on 50% at 0.7 mM. The active-site-directed nature of the inactivation was proven by demonstration of the enzyme's protection against inactivation by D-galactose (Figure 3).

From the Figure 3, it is evident that this monosaccharide significantly protects α-PsGal from the inactivation.

Regardless of the inhibitor concentration, *k*obs Gal decreased on average by 50% in the presence of the reaction product D-galactose (Table 4). The monosaccharide partially protects the enzyme from inactivation. This suggests that the inhibitor interacts with the enzyme molecule in the region of the active center.

**Figure 3.** Protection of α-PsGal activity by D-galactose (0.7 mM) against monanchomycalin B inactivation: curves 1 and 2 show the effect of the enzyme activation rate on incubation time with the inhibitor (11.4 μM and 14.2 μM, respectively) in the presence of D-galactose in semi-log coordinates; curves 3 and 4 represent the rates of enzyme inactivation at the same inhibitor concentration and incubation time without D-galactose. All of the experiments were performed in duplicates.

**Table 4.** Protection of α-PsGal activity by D-galactose against monanchomycalin B inactivation.


*k*obs Gal—in the presence of D-galactose.

Taking into account that the active center of the enzyme and the "vessel" part of the molecules of the compounds are identical in all the experiments, their inhibitory properties towards α-PsGal are determined by the structure of the "anchor" part. In this case, the diaminopropane residue has the greatest affinity, but more slowly penetrates to the active center of the GH36 α-PsGal from the marine bacterium. The monosubstituted diaminopropane has been shown to be also the best inhibitor for the *Chin*Lam exo-(1→3)β-D-glucanase from a marine fungus as well [23]. However, these compounds did not show inhibitor properties towards the GH109 α-NaGa from the marine bacterium *A. latericius* as well as the GH16 endo-(1→3)β-D-glucanase from the marine bivalve mollusk *S. sachalinensis* [23].

#### *2.3. Theoretical Model of the Guanidine Alkaloids Complexes with α-Galactosidase*

ǂ

The enzyme α-PsGal is a typical *O*-glycoside hydrolase of the GH36 family. It was previously shown that its molecule consists of two identical subunits [9,10]. One subunit is a three-domain protein. The active center is located in the central (β/α)8 domain. Asp 451 and Asp 516 are catalytic residues [24].

Figure 4 shows 2D-diagrams of the α-PsGal complexes with the guanidine compounds. The "vessel" part identical for the all compounds (Figure 4a), and the "anchor" parts of monanchomycalin B (Figure 4b), monanchocidin A (Figure 4c), and normonanchocidin A (Figure 4d) complexes with the active center of α-PsGal were built by molecular docking of the program Molecular Operating Environment version 2018.01 (MOE) [34].

**Figure 4.** *Cont*.

**Figure 4.** 2D-diagrams of the α-PsGal complexes with the guanidine alkaloids: (**a**) 2D-diagram of α-PsGal—"vessel" part complex; (**b**) 2D-diagram of the α-PsGal-spermidine residue of monachomycalin B; (**c**) 2D-diagram of the α-PsGal-tetra-substituted morpholinone derivative of monanchocidine A; (**d**) 2D-diagram of α-PsGal—the monosubstituted diaminopropane of normonanchocidine A.

The "anchor" parts of the compounds are the spermidine residue in monachomycalin B, tetra-substituted morpholinone derivative in monanchocidine A, and monosubstituted diaminopropane in normonanchocidine A.

Two different binding sites for the "vessel" and "anchor" parts of alkaloids in the molecule of α-PsGal were found. The carboxyl groups of the catalytic residues Asp451 and Asp516 in the active site of α-PsGal take part directly in the interaction with amino groups of "anchor" parts of the compounds.

The molecules of the test compounds consist of two polar nitrogen-containing residues connected by hydrophobic polymethylene chains. In this case, the "anchor" part of the molecule is very mobile. Based on the simulation results, the "vessel" part of the molecule binds near the crater of the active center and does not influence the activity of the enzyme, but directs and promotes an increase in the affinity of the "anchor" part; thus, the binding of the latter occurs more slowly and leads to the loss of enzyme activity. D-galactose located in the active center prevents the spermidine residue of monachomycalin B from entering to the catalytic site what can slow down the inactivation of the enzyme (Figure S1). In accordance with the results of a 3D-superposition of the α-PsGal active site with D-galactose and anchor parts of the compounds, the monosubstituted diaminopropane of normonanchocidine A penetrates most deeply into the pocket of the active center of α-PsGal (Figure S1c).

#### **3. Materials and Methods**
