Characterization of TistH, a multifunctional peptide from the scorpion Tityus stigmurus: Structure, cytotoxicity and antimicrobial activity

Accepted Manuscript Characterization of TistH, a multifunctional peptide from the scorpion Tityus stigmurus: Structure, cytotoxicity and antimicrobial activity Richele J.A. Machado, Andréia B. Estrela, Ana K.L. Nascimento, Menilla M.A. Melo, Manoela Torres-Rêgo, Edeltrurdes O. Lima, Hugo A.O. Rocha, Eneas Carvalho, Arnóbio A. Silva, Jr., Matheus F. Fernandes-Pedrosa PII:

S0041-0101(16)30156-8

DOI:

10.1016/j.toxicon.2016.06.002

Reference:

TOXCON 5387

To appear in:

Toxicon

Received Date: 12 April 2016 Revised Date:

31 May 2016

Accepted Date: 2 June 2016

Please cite this article as: Machado, R.J.A., Estrela, A.B., Nascimento, A.K.L., Melo, M.M.A., TorresRêgo, M., Lima, E.O., Rocha, H.A.O., Carvalho, E., Silva Jr., A.A., Fernandes-Pedrosa, M.F., Characterization of TistH, a multifunctional peptide from the scorpion Tityus stigmurus: Structure, cytotoxicity and antimicrobial activity, Toxicon (2016), doi: 10.1016/j.toxicon.2016.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterization of TistH, a multifunctional peptide from the scorpion Tityus stigmurus: structure,

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cytotoxicity and antimicrobial activity

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Richele J. A. Machadoa,b; Andréia B. Estrelaa; Ana K. L. Nascimentob; Menilla M. A. Meloa;

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Manoela Torres-Rêgoa; Edeltrurdes O. Limac; Hugo A. O. Rochab; Eneas Carvalhod; Arnóbio A.

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Silva Jra; Matheus F. Fernandes-Pedrosaa,b a

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Laboratório de Tecnologia e Biotecnologia Farmacêutica, Universidade Federal do

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Programa de Pós-Graduação em Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil

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Laboratório de Micologia, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, PB, Brazil

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Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil

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Running title: Multifunctionality of TistH from Tityus stigmurus.

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Rio Grande do Norte, Natal, RN, Brazil

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*Address correspondence to: Universidade Federal do Rio Grande do Norte, Av. Gal. Cordeiro de

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Farias, s/n, CEP 59012-570, Natal, RN, Brazil. Fax: +55 84 3342-9804. E-mail Address:

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[email protected] (M. F. Fernandes-Pedrosa).

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ACCEPTED MANUSCRIPT Abstract

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The presence of bioactive peptides in animal venoms has been targeted in scientific research for

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assessing biological activities, as well as mechanisms of action. A recent study by our group

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observed hypotensive action of TistH (Tityus stigmurus Hypotensin), a peptide deduced from the

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transcriptome of T. stigmurus venom gland. The present study aims to analyze TistH structure

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properties and to evaluate its toxicity on normal and tumor cells, its in vitro antimicrobial activity, as

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well as its inflammatory effect. Circular dichroism analyses of TistH showed a general

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predominance of α-helix conformation in TFE (20 to 70%) and structural stability to pH variations.

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TistH was not cytotoxic to normal cell lines (3T3, RAW and HEK), and also not to cancer cell lines

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(HeLa, B16, 786-0, SiHa and HepG2). The peptide did not present inflammatory activity up to 6

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hours after administered subcutaneously to Swiss mice. It was observed that concentrations of 4 -

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1024 µg/mL of TistH produced no inhibition against the bacteria Staphylococcus aureus, S.

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epidermidis e Pseudomonas aeruginosa. The results of antifungal assays showed a moderate

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activity of TistH against Candida albicans strain LM-108 and the filamentous fungus Trichophyton

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rubrum LM-640, with growth inhibition at a concentration of 1024 µg/mL. In contrast, the peptide

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presented a greater activity (MIC 128 µg/mL) against C. albicans LM-106, C. tropicalis ATCC

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13308 and Aspergillus flavus strains LM-247 and LM-26, fungi that cause oral and vaginal

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infections, candidiasis and respiratory allergies, respectively. The present data contribute to a

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better understanding of TistH and its possible use as a bioactive compound. This multifunctional

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peptide is capable of acting as anti-hypertensive, as well as to inhibit the growth of fungal strains,

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having low toxicity, which suggests its safety for using as a pharmacological agent.

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Keywords: Scorpion venom; Bioactive peptide; Antimicrobial; Circular dichroism.

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1. Introduction

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Tityus stigmurus (Thorell, 1876), also known as yellow scorpion, is a species of scorpion

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belonging to the Buthidae family. It is widely distributed in the Northeast of Brazil, being present in

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Alagoas, Pernambuco, Sergipe and Rio Grande do Norte, and causes most of the scorpionism

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accidents in the region (De Albuquerque et al., 2009; “Ministério da Saúde,” 2009).

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Scorpion venoms are used for defense and preying (Plessis et al., 2008); they contain

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polypeptides, nucleotides, lipids, mucoproteins, biogenic amines and unknown substances (Ortiz

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et al., 2015). Advanced methods of fractionation, chromatography and sequencing have made it

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possible to characterize scorpion venom toxins which are among the best-studied components

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and responsible for the pathogenesis of poisoning due to specific interaction with membrane ion

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channels (Plessis et al., 2008).

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The peptide content of scorpion venoms does not exceed 5% of the dry weight. This

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peptide fraction is usually divided into different groups based on the structure, action target,

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pharmacological relevance and toxicity to mammals, insects and crustaceans (Hmed et al., 2013).

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The peptides present in the venom can be further grouped into disulfide bridged peptides

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(DBPs) and non-disulfide bridged peptides (NDBPs) (Zeng et al., 2012). The majority of DBPs

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from scorpions are crosslinked by three to four disulfide bridges. They can specifically interact with

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different ion channels including Na+, K+, Ca2+ and CI− channels (Zeng et al., 2012). NDBPs are

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relatively small, containing between 13 and 56 amino acid residues, with varying sequence, being

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mostly cationic and having flexible structure. A variety of important activities have been described

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for these peptides, including antibacterial activity, antifungal, cytolytic, antiviral, antimalarial,

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anticancer, immunomodulatory and bradykinin potentiating (Ortiz et al., 2015). Most NDBPs from

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scorpions are found to be antimicrobial peptides (AMPs) which are α- helical and amphipathic

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(Zeng et al., 2012). Antimicrobial peptides (AMPs) are important components of the innate

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immune system, showing a broad spectrum of action against microbes (Melo et al., 2015).

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Scorpion venom also contains non-antimicrobial peptides (NAMPs) without disulfide

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bridges, shown to possess bradykinin-potentiating activity (Zeng et al., 2012). Recently a new

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hypotensin identified in T. stigmurus scorpion venom called TistH (T. stigmurus Hypotensin) had

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its three-dimensional structure resolved by homology modeling, in a study by our group (Machado

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et al., 2015). TistH presented vasorelaxant effect on mesenteric artery rings with endothelium, and

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bradykinin-potentiating activity in normotensive rats. TistH-induced hypotension was dependent of

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NO release, an independent of ACE inhibition. In addition, it showed no hemolytic activity

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(Machado et al., 2015).

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Several scorpion peptides demonstrate ample potential for therapeutic application in

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treating infectious diseases, cancers, erectile dysfunction, epilepsy and cardiovascular diseases

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(Guo et al., 2013; Hmed et al., 2013; Lima et al., 2010; Verano-Braga et al., 2010, 2008; Zeng et

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al., 2012). The present study aims to analyze TistH structure properties, evaluate its toxicity on

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normal and tumor cells, its in vitro antimicrobial activity, as well as its inflammatory effect in vivo.

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2. Materials and methods

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2.1 Peptide synthesis - The peptide named TistH was deduced from the cDNA clone TSTI0006C

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obtained from T. stigmurus transcriptome (Almeida et al., 2012). Synthetic mature TistH

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(ADMDFTGIAESIIKKIKETNAKPPA) was purchased from Thermo Scientific, USA. The synthetic

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peptide was purified by reverse-phase HPLC (>98% purity) and its molecular weight was

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confirmed by mass spectrometry performed by the supplier (Supplementary Fig. 1)

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2.2 Tityus stigmurus scorpion venom - T. stigmurus scorpion venom was obtained at our

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laboratory (Tecbiofar, UFRN) by electrostimulation of the telson. After lyophilization, the venom

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was weighed and dissolved in PBS. The scientific use of the material was approved by the

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Brazilian Genetic Heritage Management Council (CGEN) (Process 010844/2013-9).

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2.3 Cell Culture - 3T3 (ATCC CCL-164), HEK (ATCC CRL-1573), Raw (ATCC TIB-71), HeLa

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(ATCC CCL2), 786-0 (ATCC CRL-1932), HepG2 (ATCC HB-8065), B16 (ATCC CRL-6475), and

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SiHa (ATCC HTB-35) cells were obtained from American Type Culture Collection (ATCC,

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Rockville, MD, USA). Cultures were grown in DMEM (Dulbecco’s Modified Eagle’s Medium, except

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for 786-0 cells) or RPMI-1640 medium (786-0 cells), supplemented with 10% fetal calf serum,

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adding streptomycin (5000 mg/mL)/penicillin (5000 IU) in a sterile environment at 37°C with 5%

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CO2 and humidified atmosphere.

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2.4 Microorganisms - For antimicrobial assays, the following strains were used: Staphylococcus

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aureus ATCC- 6538, S. aureus M-177, S. aureus LM-197, S. epidermidis ATCC- 12228;

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Pseudomonas aeruginosa ATCC-9027, P. aeruginosa P-03, P. aeruginosa P-12, Candida

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albicans ATCC- 76645, C. albicans LM-108, C. albicans LM-106, C. tropicalis ATCC- 13803, C.

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ACCEPTED MANUSCRIPT tropicalis LM-14; C. krusei LM- 08, C. krusei LM-13, Trichophyton rubrum LM- 600, T. rubrum LM-

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640; Aspergillus flavus LM-26 e A. flavus LM-247.

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2.5 Experimental animals- Male and female Swiss mice (25–35 g), 6–8 weeks of age, from the

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vivarium of the Center for Health Sciences UFRN were used in the experiments. The animals were

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maintained under controlled temperature (22 ± 2°C), light/dark cycle of 12 hours and with free

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access to water and commercial feed. All procedures described in the present study are approved

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by the Committee for Ethics in Animal Experimentation of UFRN (protocol no.: 066/2015) and in

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accordance with the guidelines of the Brazilian Committee for Animal Experimentation (COBEA).

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2.6 Circular dichroism (CD) - The analyzes were conducted at a temperature of 25 °C in water,

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TFE 20%, 50% and 70% (v/v), or sodium phosphate buffer (200 mM, pH 7,4) using 0.1 cm

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pathlength quartz cuvette in a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) with Peltier-

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type temperature controller. Each spectrum corresponds to an average of five scans taken from

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185 to 260 nm, at a scan rate of 50 nm/min (Wallace et al., 2003; Whitmore and Wallace, 2008).

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Secondary structure of TistH was determined by deconvoluting CD data using SELCON3,

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CONTIN and CDSSTR at Dichroweb platform (Whitmore and Wallace, 2004).

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The structural stability of the peptide was evaluated in the pH range between 3 and 9. Citric acid

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/disodium phosphate buffer was used for pH from 3 to 7.4, and borate buffer for pH from 8 to 9.

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The thermal stability of TistH structure, at concentration of 0.3 mg/mL in 50% TFE, pH 7.4, was

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evaluated in a scan of wavelengths (182-260 nm), ranging the temperature from 5 to 95 °C,

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recording spectra each 5 °C.

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2.7 Cell viability assay- Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

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tetra-zolium bromide (MTT) assay. Cells were transferred into 96-well plates at a density of 5×103

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cells/well and allowed to attach overnight in 100 µL medium, at 37 °C and 5% CO2. After this

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period, TistH was added at different concentrations (10-4 - 10-10 M) for a period of 24h. After

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incubation, the peptide was removed, and 100 µL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-

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ACCEPTED MANUSCRIPT 2,5-diphenyltetrazoliumbromide) dissolved in DMEM were added and further incubated for 4 h at

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37 °C. Supernatants were removed and replaced by 100 µL of ethanol to solubilize formazan

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crystals (Mosmann, 1983), and absorbance was measured at 570 nm using a Multiskan Ascent

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Microplate Reader (Thermo Labsystems, Franklin, MA, USA).

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2.8 Nitric oxide determination in Raw cells - Nitric oxide production by Raw cells was

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determined by measuring nitrite in culture supernatants using the method by Green et al. (1982)

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(Green et al., 1982). Cells were first incubated for 24 h with increasing concentrations of TistH,

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with or without LPS. After incubation, 50 µL of culture supernatants were placed in 96-well plates

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(TPP - USA). Then, an equal volume of a mixture containing 1% sulfanilamide, 5% phosphoric

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acid, 0.1% naphthylethylenediamine and distilled water was added to each well. After 10 minutes

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incubation at room temperature, reading was carried out in a microplate reader (Bio-Tek

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PowerWave HT - USA) at 540 nm. The amount of nitrite was calculated using a standard curve of

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sodium nitrite (200 µM - 1.5625 µM).

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2.9 Inflammatory Activity in vivo - The effect of TistH was assessed in a model of air pouch

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inflammation in Swiss mice. All three groups received 5 mL of sterile air subcutaneously injected

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into the back of the animals. After three days, 2.5 mL of sterile air were injected into the cavity

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(Vigil et al., 2008; Yoon et al., 2005). Six days after the initial air injection, the animals were

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subjected to a subcutaneous injection of sterile saline solution (0.9 mg/mL), T. stigmurus venom

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(VTs) (0.4 mg.kg-1) or TistH (600 and 1200 µg/kg). After 6 hours, the animals were euthanized with

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an overdose of xylazine and ketamine (10 µg/kg – 100 µg/kg) and exudates were harvested from

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each air pouch by washing with 2 mL of sterile saline solution (0.9 mg/mL). The exudates collected

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from each mouse were centrifuged at 1500 rpm for 10 min at 4 °C. After staining with Turk’s

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solution, the total cell count was determined using a Neubauer chamber and the results were

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expressed as the total number of leukocytes (x106/ml).

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2.10 Determination of antimicrobial activity - The evaluation of the antimicrobial activity of TistH

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was performed by broth microdilution technique in 96-well plates (Hadacek and Greger, 2000;

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ACCEPTED MANUSCRIPT Queiroga et al., 2015). Growth media (100 µL) was placed in each well, being Nutrient Broth used

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for bacteria and RPMI 1640 for yeast and fungi. Then, 100 µL of serially diluted TistH (1024 µg/mL

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- 8 µg/ mL) and 10 µL (106 CFU/mL) of bacterial cells, yeast or fungi were added to the wells. The

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plates were incubated at 35 °C for 24-72 h (for bac teria and yeast strains), or at 28-30 °C for 7-10

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days (for filamentous fungi strains). After the appropriate incubation time, the turbidity was visually

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evaluated as a representation of growth. The antibiotic chloramphenicol (100 µg/ml) for bacteria,

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and antifungals nystatin (100 IU) for yeasts and fluconazole (100 µg/ml) for fungi were used as

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controls. A control with sterile culture medium was also included. Assays were performed in

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duplicate, and the results were expressed as the arithmetic mean of the MIC.

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2.11 Statistical analysis - All statistical analyses were performed using GraphPad Prism (v. 5.0,

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GraphPad Software, Inc.). Results were expressed as mean ± standard deviation. The comparison

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between groups was performed using unpaired "t" test or ANOVA (One-Way) followed by Tukey’s

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post-test. The difference between means was considered significant when p <0.05.

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3. Results

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3.1 TistH secondary structure evaluated by CD spectroscopy - The prediction of the

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secondary structure of the peptide TistH was performed by circular dichroism experiments,

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analyzing the behavior of the peptide in water, 200 mM sodium phosphate buffer pH 7.4, and in

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the presence of increasing concentrations of TFE (20%, 50% and 70%; v/v) (Fig. 1). In water and

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phosphate buffer, the peptide showed a typical spectrum of disordered structure with

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predominance of random regions and negative values of molar ellipticity (Fig. 1A). However, in the

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presence of TFE, the spectrum adopted a different pattern, with marked changes at 208 and 222

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nm, and a positive peak at approximately 190 nm (Fig. 1A), a typical characteristic of proteins with

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α-helix structure (Greenfield, 2007); this peak was even more pronounced in the presence of TFE

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50% (v/v). Accordingly, the deconvolution analyses show that the addition of increasing

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concentrations of TFE resulted in a higher percentage of α-helix structure (Fig. 1B).

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Fig. 1. TistH structure evaluation by Far-UV circular dichroism. (A) CD spectra in TFE 20%

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(dashed), 50% (dark gray) and 70% (light gray), 200 mM Phosphate buffer (gray) and water

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(dotted). The analyses were performed at a peptide concentration of 0.3 mg/mL. The spectra were

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recorded at wavelengths of 182-260 nm, with a speed of 50 nm.min-1, at intervals of 0.1 nm. Five

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readings from each sample were averaged to obtain the mean spectrum. The CD spectra are

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presented as molar ellipticity per residue [θ] in degrees cm2.dmol-1, plotted as a function of

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wavelength. (B) Percentage of secondary structures according to the deconvolution of CD spectra

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of TistH (0.3 mg/mL) under different conditions (TFE, phosphate buffer and water) using different

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algorithms (SELCON3, CONTIN and CDSSTR). Values presented are mean ± SD of the

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percentages obtained by the algorithms used.

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ACCEPTED MANUSCRIPT 3.2 Structural stability of TistH

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3.2.1 Structural stability to pH variations - In the pH stability analysis, we used different buffer

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solutions at pH values ranging between 3.0 and 9.0, generating scan spectra from 189 to 260 nm

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(Fig. 2A). One can observe the peptide stability to pH variations, since the variation in the

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structural percentage of the peptide was minimal (Fig. 2B). The characteristic valleys of alpha-

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helix structure at wavelengths 208 nm and 222 nm, as well as the characteristic beta-sheet valley

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at 217 nm, were selected for a more detailed analysis. Structural variation in the three regions

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showed a similar pattern of change, characterized by a modest tendency towards structure loss as

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pH increases, which was slightly more pronounced for beta-sheet (Fig. 2C).

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ACCEPTED MANUSCRIPT Fig. 2. Structural evaluation of TistH (0.3 mg/mL) in 50% TFE at pH values ranging between 3.0

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and 9.0. (A) CD spectra at pH 3.0 (dot), 4.0 (square), 5.0 (gray dash), 6.0 (dash and dot), 7.0

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(circle), 7.4 (black line), 8.0 (solid dark gray) and 9.0 (solid light gray) are shown. Citric acid

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/disodium phosphate buffer was used for pH from 3.0 to 7.4, and borate buffer for pH 8.0 and 9.0.

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(B) Deconvolution of TistH (0.3 mg/mL) CD data in TFE 50% at pH between 3.0 and 9.0 using

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SELCON3, CONTIN and CDSSTR algorithms. Values presented are the average ± SD of the

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percentages obtained with the algorithms. (C) Peptide molar ellipticity at 222 nm, 208 nm and 217

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nm as a function of pH. The wavelengths 208 nm and 222 nm were selected corresponding to the

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characteristic valleys of alpha-helix structure and 217 nm corresponding to the characteristic valley

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of beta-sheet, for a more detailed analysis.

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3.2.2 Structural stability to temperature variation - When evaluating peptide structural profile at

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a wavelength from 185 to 260 nm, the data of molar ellipticity increased proportionally to

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temperature, suggesting a partial loss of α-helix structure and increase of beta-sheet and random

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coil regions (Fig. 3 A and B). However, after returning to the original temperature, no significant

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structural changes were observed, indicating that the heat-induced structure loss was reversible

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(Table 1). The wavelengths 208 nm, 222 nm and 217 nm were chosen to further evaluate the

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thermal stability. As a result, it was detected an increase in the molar ellipticity proportional to the

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temperature increasing, confirming that the molecule partially lost its structure as temperature

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increases (Fig. 3C).

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ACCEPTED MANUSCRIPT Fig. 3. Structural CD profile of TistH as a function of temperature variation. (A) The molar ellipticity

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of TistH (0.3 mg/mL) in 50% TFE solution was measured as a function of temperature variation

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(2°C to 98°C) in wavelengths from 185 to 260 nm. CD spectra in temperature 5°C (dot), 25°C

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(square) 37°C (gray dash), 50°C (dash and dot) 75°C (solid dark gray) and 95°C (solid light gray)

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are shown. (B) Deconvolution of the CD spectra obtained as a function of temperature variation,

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in the wavelength range 185 to 260 nm. Percentage of secondary structures according to

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deconvolution of CD spectra using different algorithms (SELCON3, CONTIN and CDSSTR).

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Values presented are mean ± SD of the percentages obtained by the algorithms. (C) Peptide

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molar ellipticity at 222 nm, 208 nm and 217 nm as a function of temperature variation. The

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wavelengths 208 nm and 222 nm were selected corresponding to the characteristic valleys of

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alpha-helix structure and 217 nm corresponding to the characteristic valley of beta-sheet, for a

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more detailed analysis.

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Table 1. Structural stability of TistH to heating-cooling treatment (2 °C - 98 °C - 2 °C). Percentage

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of secondary structures (mean ± SD) according to deconvolution by SELCON3, CONTIN and

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CDSSTR algorithms, of CD spectra recorded at 25 °C before and after the treatment.

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Beta-sheet (%)

62.0 ± 3.5 61.2 ± 3.5

4.5 ± 0.9 3.5 ± 1.9

Random coil (%) 34.1 ± 6.4 36.4 ± 5.7

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Alpha-helical (%)

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Temperature conditions Preheating After-cooling

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ACCEPTED MANUSCRIPT 3.3 In vitro cytotoxicity- TistH was not cytotoxic to normal cells 3T3, RAW and HEK (Fig. 4), and

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also not to cancer cells HeLa, B16, 786-0, SiHa and HepG2 for 24 hours at all concentrations

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tested.

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ACCEPTED MANUSCRIPT Fig. 4. Effect of TistH on cell viability. Dose–response analysis of cell viability as measured by 3-

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(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in different cell lines. The

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viability of cells treated with TistH for 24 h was expressed as percentage viability relative to control

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untreated cells. *P<0.05; **P<0.01; ***P<0.001 compared to control.

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3.4 Nitric oxide release by macrophages Raw cells- The release of nitrite by RAW cells in the

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presence of increasing concentrations of TistH did not change significantly when compared to the

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negative control (Fig. 5).

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Fig.5. Determination of nitrite in RAW cell culture supernatants. Cells were incubated with

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increasing concentrations of TistH, in the presence or absence of LPS, for 24 hours. The quantity

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of nitrite was calculated using a standard curve of sodium nitrite (200 µM to 1.5625 µM).The

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experiment was performed in triplicate, and expressed as mean ± standard deviation.

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3.5 Inflammatory Activity in vivo – In the air-pouch inflammation model, TistH did not stimulate

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leukocyte migration at the evaluated concentrations (600 and 1.200 µg/kg), in contrast to the

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highly inflammatory effect of the crude venom from T. stigmurus scorpion, which stimulated

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leukocyte migration 91.5% higher than the negative control (saline group) (Fig. 6).

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Fig. 6. Inflammatory activity of TistH. The peptide was administered subcutaneously at two

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concentrations (600 µg/kg and 1.200 µg/kg) to Swiss mice in air pouch inflammation model. After

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six hours, the air pouch lavage was performed with PBS and the number of leukocytes was

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determined using a Neubauer chamber. Saline solution and T. stigmurus crude venom (VTs) were

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used as negative and positive controls, respectively. ***P<0.001, compared to VTs.

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3.6 Antimicrobial activity- It was observed that concentrations of 4 to 1024 µg/mL of TistH

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produced no inhibition against the bacteria Staphylococcus aureus, S. epidermidis and

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Pseudomonas aeruginosa (MIC > 1024 µg/mL) (Table 2). The results of antifungal assays showed

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a moderate activity of TistH against Candida albicans strain LM-108 and the filamentous fungus

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Trichophyton rubrum LM-640, with growth inhibition at the highest concentration tested (MIC =

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1024 µg/mL). In contrast, the peptide presented a greater activity (MIC 128 µg/mL) against C.

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albicans LM-106, C. tropicalis ATCC 13308 and Aspergillus flavus strains LM-247 and LM-26.

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Table 2- Antifungal activity of TistH. Assays were performed in duplicate and the result is

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expressed by the geometric mean of MIC values obtained in both tests. MIC (µg/mL)

C. albicans LM- 106

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C. tropicalis ATCC- 13803

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A. flavus LM-247 A. flavus LM-26

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4. Discussion

Scorpion venom has been used in traditional Chinese medicine for many centuries for

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treating the symptoms associated with convulsive spasm, cardiovascular and cerebrovascular

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diseases, tumors, inflammation and hepatopathy (Goudet et al., 2002; Wang et al., 2015). In

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recent decades, the toxins present in scorpion venom have attracted interest from researchers

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and the pharmaceutical industry. These toxins demonstrate therapeutic potential for the treatment

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of a variety of diseases (Goudet et al., 2002; Guo et al., 2013).

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The identification and characterization of these toxins from T. stigmurus scorpion venom

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have been the subject of studies by our group. Toxins with biotechnological potential were

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identified by a transcriptomic analysis from the venom gland of the scorpion T. stigmurus (Almeida

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et al., 2012). From this analysis, a new family of hypotensins was identified. After identification of

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the signal peptide cleavage point, the translated sequence in TSTI0006C cluster resulted in a

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peptide with 25 amino acid residues (ADMDFTGIAESIIKKIKETNAKPPA) and a molecular mass of

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2.7 kDa, which was named TistH (Machado et al., 2015).

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Predominant α-helix secondary structure of TistH was predicted by molecular modeling by

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our group (2) and confirmed in the present work experimentally by CD. The results of circular

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dichroism spectra analysis demonstrated that TistH presented predominant α-helix structure. Helix

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structure is common for most scorpion NDBPs (7). The structural stability of TistH was observed as a function pH and temperature variation.

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The peptide was stable to pH variation, whereas the increasing temperature provoked partial and

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reversible loss of structure, revealing decreased proportion of α-helix and, in parallel, increasing of

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beta-sheet and randomic regions. TistH sensitivity to temperature rise could be expected, because

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the peptide does not present cysteine residues in its primary structure and therefore does not form

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disulfide bonds. However, it must be highlighted that the structural changes were temporary, and

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the peptide returned to its original secondary structure composition after returning it to room

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temperature (25 °C). This stability is of utmost im portance for protein technology, targeting a

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biotechnological application of this molecule.

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The scorpion venom induces an intense inflammatory response and has high toxicity (Lima

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et al., 2014). Cytotoxicity and inflammation assays using TistH show that this peptide has low

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toxicity and is not pro-inflammatory, it had no effect on NO production by macrophages in vitro or

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leukocyte recruitment in vivo, which suggests it may be safe to approach its therapeutic potential.

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TistH did not affect the viability of normal or cancer cells within 24 hours. In contrast, an

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increasing number of experimental studies show that the venom of scorpions and some purified

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peptides have anticancer activities (Ding et al., 2014). Recently, an antimicrobial peptide identified

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in T. stigmurus scorpion venom (Stigmurin) was showed to be differentially toxic to cancer cells

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(SiHa), and to normal cells (Vero E6) (8). The peptides TsAP1 and TsAP2, isolated from scorpion

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T. serrulatus, are antimicrobial peptides that had the capacity to alter the viability of cancer cells.

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TsAP1 have low hemolytic activity (6.48%) at the highest concentration tested (160 × 10-6 M), but

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TsAP2 showed 100% hemolysis at concentrations 80 × 10-6, 120 × 10-6 and 160 × 10-6 M.

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ACCEPTED MANUSCRIPT NDBPs from scorpions display high diversity in primary and secondary structures. This

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suggests that NDBPs could possess diverse activities and functions. The majority of NDBPs from

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scorpions have been found to be antimicrobial peptides (Zeng et al., 2012). TistH activity against

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bacteria and fungi was evaluated in the present study. TistH was active upon different fungal

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strains, but showed no antibacterial activity. The strains C. albicans LM-106, C. tropicalis ATCC

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13308 and Aspergillus flavus LM-247 e LM-26 had their growth inhibited by TistH. Similar activities

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were observed in peptides such as Stigmurin, which also inhibited the growth of fungal strains C.

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albicans (ATCC 90028), C. krusei (ATCC 6258) and C. glabrata (ATCC 90030) (Melo et al., 2015).

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Comparable results were also obtained for the peptide BmKbpp from the venom of the scorpion

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Mesobuthus martensii, with activity against fungi Neurospora

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cinerea (ATCC 28387) and Fusarium culmorum (ATCC 32973) (Zeng et al., 2012). In addition,

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BmKbpp showed immune-regulatory and bradykinin-potentiating activities (Zeng et al., 2012).

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Similarly, TistH can be described as a multifunctional molecule with antifungal and bradykinin-

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potentiating activities.

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5. Conclusions

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2489), Botrytis

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TistH, a hypotensive agent identified in T. stigmurus scorpion venom, has predominant α-

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helix structure, as confirmed by circular dichroism; it exhibited structural stability to pH variation,

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and the ability to recover its original conformation after temperature denaturation. TistH also

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revealed antifungal activity, as well as a low cytotoxicity and absence of inflammatory effect. This

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study contributes to a better understanding of the structure and biological activity of TistH as a

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multifunctional molecule with therapeutic potential.

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Acknowledgments

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We would like to thank CAPES (Toxinology 063/2010) and FAPERN (PRONEM 2011) for

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the financial support. A.B.E acknowledges the BJT fellowship from CNPq. MFFP and HAOR are

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grateful to the support from CNPq.

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Conflict of interest statement There are no conflicts of interest.

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References

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Almeida, D.D., Scortecci, K.C., Kobashi, L.S., Agnez-Lima, L.F., Medeiros, S.R.B., Silva-Junior, A.A., Junqueira-de-Azevedo, I. de L.M., Fernandes-Pedrosa, M. de F., 2012. Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey. BMC Genomics 13, 1–11. doi:10.1186/1471-2164-13-362

381 382 383 384

De Albuquerque, C.M.R., Barbosa, M.O., Iannuzzi, L., 2009. Tityus stigmurus (Thorell, 1876) (Scorpiones; Buthidae): Response to chemical control and understanding of scorpionism among the population. Rev. Soc. Bras. Med. Trop. 42, 255–259. doi:10.1590/S003786822009000300004

385 386 387

Ding, J., Chua, P.-J., Bay, B.-H., Gopalakrishnakone, P., 2014. Scorpion venoms as a potential source of novel cancer therapeutic compounds. Exp. Biol. Med. 239, 387–393. doi:10.1177/1535370213513991

388 389 390

Goudet, C., Chi, C.W., Tytgat, J., 2002. An overview of toxins and genes from the venom of the Asian scorpion Buthus martensi Karsch. Toxicon 40, 1239–1258. doi:10.1016/S00410101(02)00142-3

391 392 393

Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of Nitrate , Nitrite , and [ 15N ] Nitrate in Biological Fluids. Anal. Biochem. 138, 131– 138.

394 395

Greenfield, N., 2007. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 1, 2876–2890. doi:10.1038/nprot.2006.202.Using

396 397 398 399

Guo, X., Ma, C., Du, Q., Wei, R., Wang, L., Zhou, M., Chen, T., Shaw, C., 2013. Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities. Biochimie 95, 1784–1794. doi:10.1016/j.biochi.2013.06.003

400 401 402

Hadacek, F., Greger, H., 2000. Testing of antifungal natural products: methodologies, comparability of results and assay choice. Phytochem. Anal. 11, 137–147. doi:10.1002/(SICI)1099-1565(200005/06)11:3<137::AID-PCA514>3.0.CO;2-I

403 404

Hmed, B., Serria, H.T., Mounir, Z.K., 2013. Scorpion peptides: potential use for new drug development. J. Toxicol. 2013, 1–15. doi:10.1155/2013/958797

405 406 407 408

Lima, M.C.J.D.S., Bitencourt, M.A.O., Furtado, A.A., Oliveira Rocha, H.A., Oliveira, R.M., SilvaJúnior, A.A. Da, Tabosa Do Egito, E.S., Tambourgi, D.V., Zucolotto, S.M., FernandesPedrosa, M.D.F., 2014. Ipomoea asarifolia neutralizes inflammation induced by Tityus serrulatus scorpion venom. J. Ethnopharmacol. 153, 890–895. doi:10.1016/j.jep.2014.03.060

AC C

EP

TE D

M AN U

SC

377 378 379 380

21

ACCEPTED MANUSCRIPT Lima, M.E., Borges, M.H., Verano-Braga, T., Torres, F.S., Montandon, G.G., Cardoso, F.L., Peixoto, K., Cardoso-Jr, H.C., Benelli, M.T., 2010. Some arachnidan peptides with potential medical application. J. Venom. Anim. Toxins Incl. Trop. Dis. 16, 8–33.

412 413 414 415

Machado, R.J. a., Junior, L.G.M., Monteiro, N.K.V., Silva, A. a., Portaro, F.C.V., Barbosa, E.G., Braga, V. a., Fernandes-Pedrosa, M.F., 2015. Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus. Toxicon 101. doi:10.1016/j.toxicon.2015.04.003

416 417 418 419 420

Melo, E.T., Estrela, A.B., Santos, E.C.G., Machado, P.R.L., Farias, K.J.S., Torres, T.M., Carvalho, E., Lima, J.P.M.S., Silva-Júnior, A.A., Barbosa, E.G., Fernandes-Pedrosa, M. de F., 2015. Structural characterization of a novel peptide with antimicrobial activity from the venom gland of the scorpion Tityus stigmurus: Stigmurin. Peptides 68, 3–10. doi:10.1016/j.peptides.2015.03.003

421 422 423

Ministério da Saúde [WWW Document], 2009. . Man. Control. Escorpiões. URL http://bvsms.saude.gov.br/bvs/publicacoes/manual_controle_escorpioes.pdf (accessed 11.18.15).

424 425 426

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. doi:http://dx.doi.org/10.1016/0022-1759(83)90303-4

427 428 429

Ortiz, E., Gurrola, G.B., Schwartz, E.F., Possani, L.D., 2015. Scorpion venom components as potential candidates for drug development. Toxicon 93, 125–135. doi:10.1016/j.toxicon.2014.11.233

430 431

Plessis, L.H., Elgar, D., du Plessis, J.L., 2008. Southern African scorpion toxins: An overview. Toxicon 51, 1–9. doi:http://dx.doi.org/10.1016/j.toxicon.2007.08.018

432 433 434

Queiroga, F., Guerra, S., Santos, R., Araújo, A. De, Sousa, J.P. De, Pereira, F.D.O., Mendonçajunior, F.J.B., Barbosa-filho, J.M., Lima, E.D.O., 2015. Evaluation of Antifungal Activity and Mode of Action of New Coumarin Derivative , against Aspergillus spp . 2015.

435 436 437 438 439

Verano-Braga, T., Figueiredo-Rezende, F., Melo, M.N., Lautner, R.Q., Gomes, E.R.M., MataMachado, L.T., Murari, A., Rocha-Resende, C., Elena de Lima, M., Guatimosim, S., Santos, R. a S., Pimenta, A.M.C., 2010. Structure-function studies of Tityus serrulatus Hypotensin-I (TsHpt-I): A new agonist of B(2) kinin receptor. Toxicon 56, 1162–1171. doi:10.1016/j.toxicon.2010.04.006

440 441 442 443

Verano-Braga, T., Rocha-Resende, C., Silva, D.M., Ianzer, D., Martin-Eauclaire, M.F., Bougis, P.E., de Lima, M.E., Santos, R. a S., Pimenta, a M.C., 2008. Tityus serrulatus Hypotensins: A new family of peptides from scorpion venom. Biochem. Biophys. Res. Commun. 371, 515– 520. doi:10.1016/j.bbrc.2008.04.104

444 445 446

Vigil, S.V.G., de Liz, R., Medeiros, Y.S., Fröde, T.S., 2008. Efficacy of tacrolimus in inhibiting inflammation caused by carrageenan in a murine model of air pouch. Transpl. Immunol. 19, 25–29. doi:10.1016/j.trim.2008.01.003

447 448

Wallace, B.A., Lees, J.G., Orry, A.J.W., Lobley, A., Janes, R.W., 2003. Analyses of circular dichroism spectra of membrane proteins. Protein Sci. 12, 875–884.

449 450 451

Wang, J., Zhang, H., Gao, S., Wang, J., 2015. Review on Bioactive Peptides and Pharmacological Activities of Buthus martensii Karsch. Biochem. Pharmacol. Open Access 04, 1–5. doi:10.4172/2167-0501.1000166

452 453 454

Whitmore, L., Wallace, B. a, 2004. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–73. doi:10.1093/nar/gkh371

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409 410 411

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ACCEPTED MANUSCRIPT Whitmore, L., Wallace, B.A., 2008. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases . Biopolymers 89, 392–400. doi:10.1002/bip.20853

458 459 460 461

Yoon, S., Kwon, Y., Kim, H., Roh, D., Kang, S., Kim, C., Han, H., Kim, K., Yang, I., Beitz, a, 2005. Intrathecal neostigmine reduces the zymosan-induced inflammatory response in a mouse air pouch model via adrenomedullary activity: Involvement of spinal muscarinic type 2 receptors. Neuropharmacology 49, 275–282. doi:10.1016/j.neuropharm.2004.12.024

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Zeng, X.-C., Wang, S., Nie, Y., Zhang, L., Luo, X., 2012. Characterization of BmKbpp, a multifunctional peptide from the Chinese scorpion Mesobuthus martensii Karsch: Gaining insight into a new mechanism for the functional diversification of scorpion venom peptides. Peptides 33, 44–51. doi:10.1016/j.peptides.2011.11.012

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Highlights

TistH revealed structure rich in α-helix as resolved by circular dichroism

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The peptide was not cytotoxic and did not show in vivo inflammatory activity

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The peptide presented antimicrobial activity capable of inhibiting fungal growth

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TistH is a peptide potentially safe to be used as a template for therapeutic agents

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