*Article* **In Silico Evaluation of Quercetin Methylated Derivatives on the Interaction with Secretory Phospholipases A2 from** *Crotalus durissus terrificus* **and** *Bothrops jararacussu*

**Mariana Novo Belchor 1,2, Caroline Ramos da Cruz Costa 1,2, Airam Roggero <sup>2</sup> , Laila L. F. Moraes <sup>2</sup> , Ricardo Samelo <sup>2</sup> , Isabelly Annunciato <sup>2</sup> , Marcos Antonio de Oliveira 1,2 , Sergio F. Sousa <sup>3</sup> and Marcos Hikari Toyama 1,2,\***


**Abstract:** Quercetin derivatives have already shown their anti-inflammatory potential, inhibiting essential enzymes involved in this process. Among diverse pro-inflammatory toxins from snake venoms, phospholipase A2 is one of the most abundant in some species, such as *Crotalus durissus terrificus* and *Bothrops jararacussu* from the Viperidae family. These enzymes can induce the inflammatory process through hydrolysis at the sn-2 position of glycerophospholipids. Hence, elucidating the main residues involved in the biological effects of these macromolecules can help to identify potential compounds with inhibitory activity. In silico tools were used in this study to evaluate the potential of quercetin methylated derivatives in the inhibition of bothropstoxin I (BthTX-I) and II (BthTX-II) from *Bothrops jararacussu* and phospholipase A2 from *Crotalus durissus terrificus*. The use of a transitional analogous and two classical inhibitors of phospholipase A2 guided this work to find the role of residues involved in the phospholipid anchoring and the subsequent development of the inflammatory process. First, main cavities were studied, revealing the best regions to be inhibited by a compound. Focusing on these regions, molecular docking assays were made to show main interactions between each compound. Results reveal that analogue and inhibitors, Varespladib (Var) and p-bromophenacyl bromide (BPB), guided quercetins derivatives analysis, revealing that Leu2, Phe5, Tyr28, glycine in the calcium-binding loop, His48, Asp49 of BthTX-II and Cdtspla2 were the main residues to be inhibited. 3MQ exhibited great interaction with the active site, similar to Var results, while Q anchored better in the BthTX-II active site. However, strong interactions in the C-terminal region, highlighting His120, seem to be crucial to decreasing contacts with phospholipid and BthTX-II. Hence, quercetin derivatives anchor differently with each toxin and further in vitro and in vivo studies are essential to elucidate these data.

**Keywords:** natural compounds; inflammation; toxins; snake venoms; molecular docking

### **1. Introduction**

Flavonoids, compounds that are secondary metabolites from plants, have a nucleus which consists of A, B and C rings. A series of modification reactions, such as hydroxylation, glycosylation, prenylation, and methylation, can enhance multiple physiological functions corresponding to both their structural diversity and tissue specificities. The *O*-methylation of aglycone flavonoids, such as Quercetin (Q), results in the reduction of the molecular activity of a hydroxyl fraction and the consequent increase in lipophilicity, which modifies its intracellular compartmentalization. Furthermore, *O*-methylation provides a branch

**Citation:** Belchor, M.N.; Costa, C.R.d.C.; Roggero, A.; Moraes, L.L.F.; Samelo, R.; Annunciato, I.; de Oliveira, M.A.; Sousa, S.F.; Toyama, M.H. In Silico Evaluation of Quercetin Methylated Derivatives on the Interaction with Secretory Phospholipases A2 from *Crotalus durissus terrificus* and *Bothrops jararacussu*. *Pharmaceuticals* **2023**, *16*, 597. https://doi.org/ 10.3390/ph16040597

Academic Editor: Diana Roxana Pelinescu

Received: 6 March 2023 Revised: 7 April 2023 Accepted: 10 April 2023 Published: 15 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

point in the biosynthesis of several metabolic pathways, including production of modified flavonoids with increased antimicrobial properties [1]. Considered a post-modification product, these derivatives are formed through the methyl group fixation with oxygen at the flavonoid hydroxyl moiety. Due to the diverse hydroxyl groups in the flavonoid core, flavonoids' methylation positions are diverse and provide multiple health benefits, such as increased bioavailability compared to flavonoid precursors [2]. Therefore, these methylated flavonoids are potential candidates for use as anti-inflammatories by decreasing the enzymatic activity of enzymes such as cyclooxygenases (COX), pro-inflammatory interleukins, reactive oxygen species (ROS) and nitrogen (RNS) production [2–4]. Previous analyzes using ChEMBL and SwissTargetPrediction tools reveals that both Rhamnetin (Rhm; 7- *O*-Methylquercetin); 3-*O*-Methylquercetin (3MQ; 3-*O*-Methylquercetin) are compounds with an antioxidant capacity, besides to exhibits anti-inflammatory potential by strongly decreasing the cyclooxygenase (COX) and Lipoxygenase (LOX) activity. Rhamnazin (Rhz; 7,3'-Di-*O*-methylquercetin) exhibits two methylations, which seem to considerably increase the ability to sequester free radicals in cells. Methylated derivatives of Q are also found in some plant species, such as *Coriandrum sativum, Achyrocline satureioides*, and *Rhamnus petiolaris* with Rhm, 3MQ, and Rhz compounds, respectively, which have already shown different responses against BthTX-II activities [5].

Snake venoms consist of a complex mixture of biologically active molecules, and the phospholipase A2 (PLA2) group is one of the most studied toxins. There are two main groups of PLA2 (E.C. 3.1.1.4) in snake venoms: phospholipase A2-like (PLA2-like), such as the Lys49-PLA2 and the classic phospholipase A2, Asp49-PLA2. These proteins belong to the secreted PLA2 (sPLA2), a subgroup found in diverse secretions, body fluids and venom of bees, scorpions, and snakes [6]. Therefore, these macromolecules hydrolyze the sn-2 position of glycerophospholipids, leading to a release of fatty acids, such as arachidonic acid (AA) and lysophospholipids, triggering the inflammatory process [7,8]. Bothropstoxin II (BthTX-II) is an Asp49-PLA2 from *Bothrops jararacussu* (Bj), which belongs to the Viperidae family. This toxin is considered a PLA2-like due to some characteristics, such as the Ca2+ binding loop distortion, leading to changes in the C-terminal region. In this way, this protein shows a low phospholipase A2 activity besides to exhibit myotoxic, edematogenic, and hemolytic effects [9,10]. Bothropstoxin I (BthTX-I) is a Lys49-PLA2, which reveals high myotoxic activity with a lack of enzymatic activity. The myotoxic mechanism of these enzymes has already been demonstrated and includes mainly some amino acids from the C-terminal region, which interacts with membrane [8].

*Crotalus durissus terrificus* (Cdt) belongs to the same family and has an essential heterodimeric complex named Crotoxin in its venom, which is a potent β-neurotoxin with a phospholipase A2 activity. This toxin consists of two subunits: a basic pla2 with a weak neurotoxicity, named crotoxin B (CB) or Cdt PLA2, associated with a small acidic, nontoxic and nonenzymatic protein named crotapotin or crotoxin A (CA) [11]. CB presents four isoforms, CBa2, CBb, CBc, and CBd, performing 16 different CA-CB complexes. To date, there are three crystal structures from these isomers: one of them includes CA2 and CBb, the other with CBd (tetramer), and the structure used in this study consists of isoforms CBa2 and CBc (tetramer) [12].

Commercial inhibitors of PLA2, such as p-bromophenacyl bromide (BPB) and Varespladib (Var), have already shown their potential against PLA2 from diverse snake venoms. The classical inhibitor BPB has been used since 1970 to inhibit the catalytic PLA2 once it binds specifically with the His48 residue. However, this compound has already been shown to inhibit myotoxic activity of PrTX-I, a Lys49-PLA2 from *Bothrops pirajai*, through a covalent binding to His48, leading to a Ca2+-binding loop distortion and then, a C-terminus rearrangement [13]. Var (LY315920) is a synthetic molecule which was clinically tested to block the inflammatory cascade initiated by secreted PLA2. There are several studies that reveal the efficacy of this compound in the treatment of phospholipase A2-rich snake venoms [14]. In snake venoms, this compound has already been shown to inhibit the cytotoxic e myotoxic effect of MjTX-II from *Bothrops moojeni* through the physical blockage

of its allosteric activation [15]. In addition to this, a synergic effect of this compound with the antivenom was observed to decrease neuromuscular blockage induced by crotamine, highlighting the broad-spectrum effect of this drug [16].

Due to PLA2 role in the inflammatory process, the abundance of this toxin in the Cdt and Bj venoms, and the fact that these proteins show similar structures with sPLA2 from mammals [12], it is essential to find new inhibitors of this enzyme. Hence, in this study, we performed some steps to better understand how these compounds could interact with these three phospholipases A2. First, we aim to elucidate the main protein's regions to anchor with an inhibitor using CavityPlus. Afterwards, using the phosphonate transitionstate analogue from 1POB crystal structure, named L-1-0-octyl-2-heptylphos-phonyl-snglycero-3-phosphoethanolamine (Analogue) [17], we focus on the elucidation of the main residues that interact with each toxin. Moreover, two commercial inhibitors were used for comparison purposes to identify the main residues involved in the anchoring with all toxins. Finally, we intend to compare the compounds' interactions with phospholipase A2 from *Bothrops jararacussu* and *Crotalus durissus terrificus* with the analogue and the inhibitors. Thus, we analyze if compounds anchor equally with all toxins, and if not, we try to elucidate main differences and the reasons for that.

#### **2. Results**

#### *2.1. Properties of Compounds*

Firstly, compounds were evaluated concerning physical–chemical features to better understand their biological activity. Data obtained from SwissADME, Mollinspiration and ChEMBL support these results. Q reveals a molecular weight (MW) of 302.24 g/mol, Rhm and 3MQ show the same value, and Rhz, with two methylations, exhibits a higher MW. The topological polar surface area (TPSA) is higher in Q, the same in Rhm and 3MQ, and Rhz exhibits the smallest value (Table 1). A classical descriptor evaluated lipophilicity: the partition coefficient between *n*-octanol and water, which is essential to analyzing physicochemical properties for pharmacokinetics drug discovery [18].


**Table 1.** Physical–chemical characteristics from Q, Rhm, 3MQ and Rhz.

Each compound shows specific alerts related to chemical structures which reveals a high tendency to interact with a great number of molecules and macromolecules. Moreover, the physicochemical properties are exhibited in the second column, revealing specific differences between them, such as a higher amount of unsaturation in Q than the others, besides being more polar. In general, these compounds show that enzymes are their main target (Figure 1).
