Verbascoside Inhibits Promastigote Growth and ... - ACS Publications

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Verbascoside Inhibits Promastigote Growth and Arginase Activity of Leishmania amazonensis Claudia C. Maquiaveli,*,† Joaõ F. Lucon-Júnior,‡ Simone Brogi,§ Giuseppe Campiani,§ Sandra Gemma,§ Paulo C. Vieira,† and Edson R. Silva*,‡ †

Department of Chemistry, Universidade Federal de São Carlos, Rod. Washington Luís, Km 235, 13565-905 São Carlos, SP, Brazil Department of Veterinary Medicine, Universidade de São Paulo, Avenida Duque de Caxias Norte, 225, 13635-900 Pirassununga, SP, Brazil § European Research Centre for Drug Discovery and Development (NatSynDrugs) and Department of Biotechnology, Chemistry, and Pharmacy, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy ‡

S Supporting Information *

ABSTRACT: Verbascoside (1) is a phenylethanoid glycoside that has antileishmanial activity against Leishmania infantum and Leishmania donovani. In this study, we verified the activity of 1 on Leishmania amazonensis and arginase inhibition. Compound 1 showed an EC50 of 19 μM against L. amazonensis promastigotes and is a competitive arginase inhibitor (Ki = 0.7 μM). Docking studies were performed to assess the interaction of 1 with arginase at the molecular level. Arginase is an enzyme of the polyamine biosynthesis pathway that is important to parasite infectivity, and the results of our study suggest that 1 could be useful to develop new approaches for treating leishmaniasis.

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utaneous leishmaniasis is a neglected tropical disease that has affected 1 million people in the last five years.1 The first effective drug to treat Leishmania infection was developed 100 years ago. Traditional medicine in Latin America first discovered several plants that have effects against Leishmania. One of them is Stachytarpheta cayennensis (Rich.) Vahl (Verbenaceae), which is used in the Brazilian and Peruvian Amazon basin.2−4 Verbascoside (1; Figure 1) is a phenyl-

Arginase is a binuclear manganese hydrolase that converts Larginine into L-ornithine and urea.15 Arginase plays a pivotal role in Leishmania infection because the amino acid L-ornithine is a key precursor of polyamines that are involved in the production of the antioxidant compound trypanothione.16−18 In this study, we have characterized, for the first time, the kinetics of the arginase inhibition by 1 and verified its effect against L. amazonensis promastigotes. Arginase enzyme is considered a promising drug target for the development of antileishmanial compounds because it is important to parasite infection.16,17 Arginase from L. amazonensis is inhibited by natural compounds containing the catechol group.5,6,19 The Ki for arginase inhibition was determined using three different concentrations for both inhibitor and L-arginine. The constant of arginase inhibition was determined by the analysis of the Dixon20 plot, resulting in a Ki value of 0.7 ± 0.1 μM. The kinetics of enzyme inhibition reveals that 1 is a competitive arginase inhibitor (Figure 2). The biological effects of compound 1 were evaluated in L. amazonensis promastigotes and showed an EC50 = 19 μM. This result is similar to the EC50 value described for L. donovani amastigotes, which was found to be 14 μM (8.7 μg/mL).10 The interference of 1 on L. amazonensis growth at 100 μM was

Figure 1. Chemical structure of compound 1. The structure represents the compound verbascoside (PubChem CID: 5281800; molecular formula: C29H36O15; molecular weight: 624.587 14 g/mol).

ethanoid glycoside that has two catechol groups, a pharmacophoric moiety described to be involved in the inhibition of the activity of Leishmania amazonensis arginase.5−7 Compound 1 can be found in S. cayennensis,8 Buddleja davidii,9 and Phlomis brunneogaleata10 as well as in olive oil mill wastewater.11 Antiprotozoal activity of 1 was reported against Leishmania infantum,12 Leishmania donovani, and Trypanosoma brucei rhodesiense.10 Plants with high amounts of 1 were used in folk medicine as an antimicrobial and anti-inflammatory.13,14 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 14, 2015

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DOI: 10.1021/acs.jnatprod.5b00875 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 2. Kinetics of the arginase inhibition by 1. The mechanism of action was determined by analysis of the Dixon (A) and Cornish−Bowden plot (B), and the Ki constant was measured using Dixon plots (A). The concentrations of L-arginine used were 100 mM (▲), 50 mM (■), and 25 mM (●). The inhibitor concentrations varied from 0.25 to 1 μM. Each point drawn represents the mean of three independent experiments (n = 3) performed in duplicate.

them through its catechol system, as indicated by the ligand interaction diagram reported in Figure 5B. Moreover the hydrogens of the hydroxyl groups can establish two H-bonds with D245 and H154, while the phenethyl region interacts with H154 and H139 by a double stacking (π−π stacking with H139 and cation−π stacking with the protonated H154). The central glucopyranose is able to target different arginase side chains (H139, S150, N152, and D194) by H-bond interactions. Furthermore, the caffeic acid moiety of 1 is exposed to the surface of the enzyme and establishes two H-bonds with the residue D195, keeping the molecule anchored to the site, allowing a strong interaction with the protein. This pattern of interaction accounts for a docking score (Glide XP) of −9.73 kcal/mol, whereas the crystallized ligand, correctly accommodated into the binding site by using the presented docking protocol, showed a docking score of −6.77 kcal/mol. Next, the docked pose was submitted to the estimation of free binding energy (ΔGbind) to better evaluate the affinity of 1 for its target.25−28 Following this procedure we have found a ΔGbind for 1 of −100.56 kcal/mol, while for the crystallized ligand Nωhydroxy-nor-arginine (nor-NOHA), the ΔGbind was found to be −73.85 kcal/mol, indicating the stronger affinity of 1 for arginase with respect to nor-NOHA (Ki ≈ 50 μM).22 The analysis performed on the homology model obtained by Prime (Prime version 3.1, Schrödinger, LLC, New York, NY, 2012) of arginas from L. amazonensis gave us superimposable results (data not shown), as expected due to the similarity of both arginases. In order to gain some insight into the potential selectivity of 1 with respect to the human arginase, we have performed a docking calculation employing this latter protein. Human and parasite proteins were found to be quite similar to a sequence identity of 39.10% and a sequence similarity of about 69% (calculated by the application align in the UniprotKB Web site using Clustal Omega http://www.uniprot.org/align/). Due to this sequence similarity, the superposition of these enzymes highlights a similar arrangement in the binding site with few differences. In particular the residues A192 and D195 in Leishmania are replaced by D181 and P184 human sequence, respectively. As mentioned above the two binding sites are

observed 72 h after treatment and differed significantly in relation to the control (p < 0.01) (Figure 3).

Figure 3. Cell viability of L. amazonensis treated with 1 after 24, 48, and 72 h. The data represent two independent experiments in triplicate. The bars represent control (white), amphotericin B at 6 μM (black), and 1 at 100 μM (gray). The results are expressed as mean ± SEM. **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01 vs amphotericin B.

In order to investigate the possible binding mode of inhibitor 1 to arginase, we have performed molecular docking studies focused on the substrate binding site, using a crystal structure of arginase belonging to Leishmania mexicana (PDB ID 4IU1).22 We used the crystal structure of L. mexicana instead of a homology model of arginase from L. amazonensis since, as highlighted in Figure 4, their sequences have a high degree of conservation. In fact by using the Clustal Omega program23 to perform the alignment between the two sequences, we have found that they showed a 99.4% sequence identity, and only two residues, not involved in the binding site, were different. The ligand and the protein, prepared as reported in the Experimental Section, were used in a molecular docking protocol by using Glide software.24 The grid for the docking calculation was centered on the crystallized ligand present in the 4IU1 crystal structure.22 The molecular docking output regarding the competitive inhibitor onto the active site is reported in Figure 5A. Verbascoside (1) strongly interacts with the active site of arginase by a series of polar and hydrophobic contacts. In particular, the phenethyl moiety reaches the region containing metal ions and is able to complex at least one of B



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Figure 4. L. amazonensis and L. mexicana arginase sequence alignment as performed by means of the Clustal Omega program accessible via the UniprotKB Web server (http://www.uniprot.org/align). *Consensus sequence.

Figure 5. (A) Docked solution of 1 (green sticks) into Leishmania arginase binding site (PDB ID 4IU1; light yellow cartoon) as found by Glide software (Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012). Metals are represented as gray spheres. Key residues of the binding site are represented by lines. H-bonds are represented as black dotted lines, while the metal coordination bonds are represented by lines. The picture was generated by means of PyMOL (The PyMOL Molecular Graphics System, v1.6-alpha; Schrodinger, LLC, New York, 2013). (B) Ligand interaction diagram (Maestro version 9.3, Schrödinger, LLC, New York, NY, 2012).

strongly similar, so molecular docking calculation provided a similar accommodation of 1 into human protein (see Figure S1 in the Supporting Information for further details). Interestingly, despite the similar pattern of interactions of 1 in both human and Leishmania arginase, the computational scores calculated for 1 in the human enzyme allows us to predict a potential lower affinity with respect to the Leishmania enzymes (Glide XP −8.23 kcal/mol, ΔGbind −86.08 kcal/mol). In conclusion, we have characterized verbascoside as a new inhibitor of the arginase enzyme of L. amazonensis and showed that it is also active against the promastigote forms of L. amazonensis. In addition, a docking analysis showed a potential interaction of 1 with L. amazonensis arginase at the molecular level and its interaction with human arginase I with lower energy of binding in the catalytic site. Compound 1 is active against Leishmania infantum12 and Leishmania donovani.10

Traditional medicine in the Brazilian and Peruvian amazon basin uses S. cayennensis, which contains 1, 8 against tegumentary leishmaniasis. To use pure compound 1 against human leishmaniasis, new evidence must be obtained in animal models of the infection.

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EXPERIMENTAL SECTION

Materials. Compound 1 (cat. 89289) was purchased from Phytolab GmbH & Co (Vestenbergsgreuth, Germany). The compound is a primary reference substance with assigned absolute purity (96% considering chromatographic purity, water, residual solvents, and inorganic impurities). Amphotericin B (Unianf) was ́ purchased from União Quimica (São Paulo, Brazil). Inhibition Kinetics. Purified recombinant arginase was prepared and used to assay arginase inhibition as previously described.5,6 Briefly, the inhibitor was dissolved at 1 mM in methanol, and the assay was performed using 50 mM L-arginine in 50 mM CHES buffer (pH 9.5) C



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for 15 min at 37 °C. The 50% inhibitory concentration (IC50) was obtained using serial dilution of the inhibitors from 100 to 0.01 μM. Three independent assays were performed, in duplicate. The constant Ki for the formation of the enzyme−inhibitor complex and mechanism of arginase inhibition were determined by altering the inhibitor and substrate concentrations simultaneously,6 and the data were used to obtain Dixon20 and Cornish−Bowden plots21 by linear regression and calculate the Ki value. Promastigote Culture. The MHOM/BR/1973/M2269 strain of L. amazonensis was used throughout this study. Promastigotes were grown in M199 medium (Life Technologies) supplemented with 10% fetal bovine serum, 50 μg/mL of streptomycin, and 100 U of penicillin at 25 °C. Cells were grown until they reached the stationary phase, and 998 μL of the culture at 5.0 × 105 cells/mL was incubated with 2 μL of the inhibitors (50 to 0.0005 mM) dissolved in DMSO to obtain final concentrations of 100 to 0.001 μM. The positive control of the promastigote growth was performed with DMSO at 0.2%. Amphotericin B was used as a control of the cell growth inhibition in a concentration varying from 60 to 0.0006 μM dissolved in DMSO. The tests were performed in a conical microtube with a volume of 1500 μL. After 24, 48, and 72 h of incubation the surviving cells were counted using a Neubauer chamber using 0.4% of the trypan blue exclusion solution for determination of cell viability, and the EC50 values were calculated with a sigmoidal (log EC50) model using GraphPad Software 5.0 for Windows (San Diego, CA, USA). Two independent assays were performed in triplicate. Molecular Docking Studies. Molecule Preparation. The threedimensional structure of 1 (CHEMBL231853) was taken from the ChEMBL Web site (https://www.ebi.ac.uk/chembl/) and imported into Maestro (version 9.3, Schrödinger, LLC, New York, NY, 2012). Molecular energy minimizations were performed by means of MacroModel using the optimized potentials for liquid simulations-all atom (OPLS-AA) force field 2005.29 The solvent effects are simulated using the analytical generalized-Born/surface-area (GB/SA) model,30 and no cutoff for nonbonded interactions was selected. The Polak− Ribiere conjugate gradient (PRCG) method with 1000 maximum iterations and 0.001 gradient convergence threshold was employed. The compounds (1 and nor-NOHA, extracted from PDB 4IUB1) were treated by LigPrep application (LigPrep version 2.5, Schrödinger, LLC, New York, NY, 2012), implemented in Maestro suite 2012, generating the most probable ionization state of any possible enantiomers and tautomers at a cellular pH value (7 ± 0.5). Protein Preparation. The three-dimensional structures of the L. mexicana arginase (PDB ID: 4IU1) and the human counterpart (PDB ID: 3KV2) were taken from PDB and imported into the Schrödinger Maestro molecular modeling environment. The structures were submitted to the Protein Preparation Wizard implemented in Maestro suite 2012 (Protein Preparation Wizard workflow 2012). This protocol, through a series of computational steps, allowed us to obtain a reasonable starting structure of the proteins for molecular docking calculations. In particular, we performed three steps to (1) add hydrogens, (2) optimize the orientation of hydroxyl groups, Asn, and Gln and the protonation state of His, and (3) perform a constrained refinement with the impref utility, setting the max RMSD of 0.30. The impref utility consists of cycles of energy minimization based on the impact molecular mechanics engine and on the OPLS_2005 force field. Molecular Docking. Docking studies were carried out by Glide (Grid-Based Ligand Docking with Energetics) (Glide, version 5.8, Schrödinger, Release 2012) using the ligands and the protein prepared as mentioned above, applying the Glide extra precision (XP) method. Energy grids were prepared using a default value of the protein atom scaling factor (1.0 Å) within a cubic box centered on the crystallized ligand nor-NOHA for both enzymes. After grid generation, the ligands were docked into the enzymes with default parameters (no constraints were added). The number of poses entered in the postdocking minimization was set to 100. The Glide XP score was evaluated. The XP method correctly accommodated the crystallized inhibitor into the binding site (data not shown).

Estimated Free Binding Energy. The Prime/MM-GBSA method implemented in Prime software (Prime version 3.1, Schrödinger, Release 2012) consists in computing the change between the free and the complex state of both the ligand and the protein after energy minimization. The technique was used on the docking complexes of the compounds presented in this study. The software was employed to calculate the free binding energy (ΔGbind) as previously reported.31−33

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00875. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +55(19)3565-4305. E-mail: [email protected]. *Tel: +55(19)3565-4371. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grant #14/18642-1, São Paulo Research Foundation (FAPESP). C.C.M. acknowledges a fellowship from Conselho Nacional de Desenvolvimento ́ Cientifico e Tecnológico (CNPq - 168248/2014-0), and J.F.L.J. acknowledges a fellowship from Coordenaçaõ de ́ Aperfeiçoamento de Pessoal de Nivel Superior (CAPES). The authors thank Dr. L. M. Floeter-Winter for providing L. amazonensis and Raw 264.7 cells, and Dr. F. Vieira Meirelles and Dr. A. A. Mendes Maia for sharing their laboratories.

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REFERENCES

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Verbascoside Inhibits Promastigote Growth and ... - ACS Publications

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