toxins

Article The Aromatic Head Group of Toxin Polyamines Influences Toxicity to Cancer Cells

David Wilson 1, Glen M. Boyle 2 ID , Lachlan McIntyre 3, Matthew J. Nolan 1 ID , Peter G. Parsons 2 ID , Jennifer J. Smith 4, Leon Tribolet 1, Alex Loukas 1, Michael J. Liddell 3, Lachlan D. Rash 4,5 ID and Norelle L. Daly 1,*

1 Centre for Biodiscovery and Molecular Development of Therapeutics, AITHM, James Cook University, Cairns, QLD 4878, Australia; [email protected] (D.W.); [email protected] (M.J.N.); [email protected] (L.T.); [email protected] (A.L.) 2 QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia; [email protected] (G.M.B.); [email protected] (P.G.P.) 3 Centre for Tropical Environmental and Sustainable Sciences, James Cook University, Cairns, QLD 4878, Australia; [email protected] (L.M.); [email protected] (M.J.L.) 4 Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; [email protected] (J.J.S.); [email protected] (L.D.R.) 5 School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia * Correspondence: [email protected]; Tel.: +61-7-4232-1815

Academic Editor: Hang Fai (Henry) Kwok Received: 5 September 2017; Accepted: 23 October 2017; Published: 27 October 2017

Abstract: Spider constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA366, containing a hydroxyphenyl-based structure is present in the venom of several species of , and has selective toxicity against MCF-7 breast cancer cells. By contrast, a polyamine from an Australian funnel-web spider venom, which contains an identical polyamine tail to PA366 but an indole-based head-group, is only cytotoxic at high concentrations. Our results suggest that the ring structure plays a role in the cytotoxicity and that modification to the polyamine head group might lead to more potent and selective compounds with potential as novel cancer treatments.

Keywords: spider venom; NMR spectroscopy; polyamine; cancer; cytotoxicity

1. Introduction The discovery of new compounds from nature is still one of the most efficient methods for finding lead molecules for the development of pharmaceuticals [1,2]. These new compounds range from small molecules to large biologics, and several approaches have facilitated their discovery. Screening using NMR spectroscopy and recent advances in metabolomics have been valuable for characterising novel small molecules, whereas advances in genomics and proteomics, as well as high-throughput screening approaches are providing novel methods for the discovery of peptides and proteins [3,4]. In nature, venomous creatures are a rich source of new bioactive compounds. In particular, spider venoms have enormous compound diversity, much of which has yet to be explored. More than four million compounds are estimated to be present in the venom of the 46,000 different spider species [5,6]. Spider venom components are present for prey capture and defence purposes; however, some venom compounds have been shown to have therapeutic potential, such as in the treatment of pain and inflammation [7]. While the majority of compounds present in spider venoms are disulfide-rich

Toxins 2017, 9, 346; doi:10.3390/toxins9110346 www.mdpi.com/journal/toxins Toxins 2017, 9, 346 2 of 13 peptides [5], small molecules such as the sulfated nucleosides found in brown recluse [8] and polyamines, found in many spider species (reviewed in [9]), are also present. Polyamines are cationic compounds widespread throughout nature and are found in both prokaryotic and eukaryotic organisms. They can modulate a range of cellular processes that include cell proliferation, signalling and ion channel function [10]. The biological activities of polyamines are mediated through interaction with anionic molecules including DNA, RNA and proteins. However, such structural diversity exists amongst the polyamines that some have been shown to be involved in tumorigenesis [11], and some have shown potential in treating diseases such as cancer [12]. Polyamines with potential for treating diseases include examples from marine sponges and fungi that inhibit carbonic anhydrase IX, a cancer drug target [13]. One study that showed the conjugation of certain polyamines to chloramphenicol enhanced anti-cancer and anti-bacterial activity [14]. The polyamine toxins present in spider venoms are part of a sub-class of polyamines characterised by an aromatic head group with a polyamine chain that can block ionotropic glutamate (iGlu) receptors [12,15]. These receptors have been considered as promising drug targets for neurological and psychiatric disorders [16]. Using a combination of NMR and bioassay screening, we have identified and characterised a novel polyamine (PA366) present in a range of tarantula (Theraphosidae) venoms. PA366 contains a hydroxyphenyl-based aromatic head group and has more potent cytotoxicity against MCF-7 breast cancer cells than SK-MEL-28 melanoma or neonatal foreskin fibroblast (NFF) primary cells. Potential protein binding interactions were probed using protein array analysis to investigate the mechanism of action, and a comparison with an indole-based polyamine from Australian funnel-web spider venom provided insight into the structure/function relationships.

2. Results

2.1. Cytotoxicity Assays and Characterisation of Bioactive Compound Crude venoms from female specimens of 31 species of tarantula (Theraphosidae) were screened for activity against the MCF-7 breast cancer cell line. Crude venom from funnel web spiders (male Sydney Funnel-web spider (Atrax robustus), female Hadronyche valida, Hadronyche cerberea and Hadronyche infensa- including species variants), and the wolf spider Hogna carolinensis, was also tested to allow comparison with venom from two other families (Hexathelidae and Lycosidae). Substantial cytotoxicity activity (defined as greater than 50% decrease in absorbance relative to the negative control) was observed for 17 of the species tested including examples from all three families tested (see Table S1 for the complete list of species screened). The cytotoxicity observed with crude female Phlogius sp. spider venom is shown in Figure1A. Venom from eight species that displayed cytotoxicity were chosen for further study based on availability of sample, and are listed in Table1. Crude venom from these eight species were fractionated using reversed-phase high performance liquid chromatography (RP-HPLC), and one-minute fractions were collected. The fractions were subsequently tested against two cancer cell lines, MCF-7 and SK-MEL-28 cells (melanoma cell line) in a clonogenic-type assay. These cell lines were chosen to cover different origins of cancer as MCF-7 are epithelial based and the SK-MEL-28 cells are from neural crest. NFF cells were also tested to represent normal cells with a doubling time similar to cancer cell lines. Variation was observed in activity between the fractionated venoms, and is consistent with venom component variations between spiders as shown previously for related species [5,17]. Despite this variation, all eight species showed early eluting fractions in the RP-HPLC chromatograms that demonstrated activity against the MCF-7 breast cancer cell line. Toxins 2017, 9, 346 3 of 13 Toxins 2017, 9, 346 3 of 13

Figure 1. Cytotoxicity of Phlogius sp. crude venom and a purified component, PA366. (A) Dose Figure 1. Cytotoxicity of Phlogius sp. crude venom and a purified component, PA366.(A) Dose response forresponse cytotoxic for cell cytotoxic killing bycellPhlogius killing sp.by Phlogius crude venom sp. crude compared venom tocompared vehicle. Crudeto vehicle. venom Crude (squares) venom or vehicle(squares) (circles) or vehicle was incubated (circles) withwas incubated MCF-7 breast with cancer MCF-7 cells breast at the cancer indicated cells at volumes the indicated for 5 days, volumes before assayfor for5 days, cell survival before assay using for the sulforhodaminecell survival using B(SRB) the sulforhodamine assay. Cell survival B (SRB) percentages assay. Cell normalised survival to percentages normalised to untreated cells are indicated. (B) Dose response for cytotoxic cell death of untreated cells are indicated. (B) Dose response for cytotoxic cell death of PA366 compared to vehicle in PA366 compared to vehicle in MCF-7, SK-MEL-28 or NFF cells. Cells were treated with the indicated MCF-7, SK-MEL-28 or NFF cells. Cells were treated with the indicated concentrations of purified PA366 concentrations of purified PA366 for 5 days, before assay for cell survival using SRB. Cell survival for 5 days, before assay for cell survival using SRB. Cell survival percentages normalised to untreated percentages normalised to untreated cells are indicated. Representative data from a single experiment cells are indicated. Representative data from a single experiment with triplicate readings are shown. with triplicate readings are shown.

a TableTable 1. Spider1. Spider venoms venoms with with cytotoxic cytotoxic activity activityand and thethe presencepresence or absence of of two two polyamines polyamines a. .

SpeciesSpecies PA366 PA366PA389 PA389 AcanthoscuriaAcanthoscuria geniculata geniculata × × CeratogyrusCeratogyrus darlingi darlingi × × ChilobrachyChilobrachy penang penang × × HisterocrastesHisterocrastes gigas gigas NhanduNhandu chromatus chromatus × PhlogiusPhlogius sp.sp. × irminia × PsalmopoeusAtrax robustus irminia × × a Atrax robustus × Crude venoms were screened for the presence of PA366 or PA389 using NMR spectroscopy. a Crude venoms were screened for the presence of PA366 or PA389 using NMR spectroscopy.

DueDue to to the the availability availability of of suitable suitable quantities quantities ofof thethe PhlogiusPhlogius sp. s spiderpider venom, venom, this this species species was was chosenchosen for for larger larger scale scale purification purification and characterisationand characterisation of the of active the active component(s). component(s). The active The fractionsactive fromfractions the Phlogius from thesp. Phlogius spider venomsp. spider were venom analysed wereusing analysed mass usin spectrometryg mass spectrometry and analytical and analytical RP-HPLC, andRP-HPLC, shown to and contain shown a singleto contain major a single component. major component. This purified This component, purified component, termed PA termed366, was PA tested366, forwas activity tested in for the activity clonogenic-type in the clonogenic-type assays. PA assays.366 showed PA366 activityshowed againstactivity against MCF-7 MCF-7 cells, and cells, limited and activitylimited against activity the against SK-MEL-28 the SK-MEL-28 and primary and NFFprimary cells NFF when cells compared when compared to vehicle to controlsvehicle controls as shown as in Figureshown1B. in Figure 1B. PAPA366 366is highlyis highly hydrophilic hydrophilic and and is oneis one of theof the first first peaks peaks of crudeof crudePhlogius Phlogiussp. sp. spider spider venom venom to eluteto fromelute a C from18 RP-HPLC a C18 RP-HPLC column column (Figure (Figure2A). Analysis 2A). Analysis of the of homonuclear the homonuclear and heteronuclearand heteronuclear NMR NMR data data of PA366 indicated the presence of a ring structure connected to a spermine polyamine tail, as of PA366 indicated the presence of a ring structure connected to a spermine polyamine tail, as shown in Figureshown2 inB. ThisFigure structure 2B. This was structure elucidated was elucidat based oned correlations based on correlations observed inobserved the HSQC, in the HMBC HSQC, and

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Toxins 2017, 9, 346 4 of 13 HSQC-TOCSY spectra, and coupling constants and chemical shifts measured from one-dimensional HMBC and HSQC-TOCSY spectra, and coupling constants and chemical shifts measured from one- 1H spectra. The multiplicities of the 1H NMR signals are reported as: d, doublet; t, triplet; p, pentet; dimensional 1H spectra. The multiplicities of the 1H NMR signals are reported as: d, doublet; t, triplet; m, multiplet; br, broad. Coupling constants (J values) are reported in hertz (Hz). HRMS: calculated p, pentet; m, multiplet; br,+ broad. Coupling constants 1(J values) are reported in hertz (Hz). HRMS: for C19H34N4O3 [M + H] 367.2631, found 367.2653. H NMR (600 MHz, H2O/10% D2O): δ 8.06 calculated for C19H34N4O3 [M + H]+ 367.2631, found 367.2653. 1H NMR (600 MHz, H2O/10% D2O): δ (1H, br t, J = 6.21 Hz, H*), 7.16 (2H, d, J = 8.71 Hz, H ), 6.85 (2H, d, J = 8.71 Hz, Ha), 4.41 (1H, t, 8.06 (1H, br t, J = 6.21 Hz, H*), 7.16 (2H, d, J = 8.71 Hz, Hb), 6.85 (2H, d, J = 8.71 Hz, Ha), 4.41 (1H, t, J = J = 5.96 Hz,H ), 3.31–3.25 (2H, m, H ), 3.20–3.08 (6H, m, H ,H ,H ), 3.02–2.93 (4H, m, H ,H ), 2.73 5.96 Hz, Hd),d 3.31–3.25 (2H, m, He), e3.20–3.08 (6H, m, Hn, Hhn, Hl),h 3.02–2.93l (4H, m, Hc, Hk), c2.73 k(2H, J 13 (2H,br brt, J t,= 8.72= 8.72 Hz, HzHg),,H 2.08g), (2H, 2.08 br (2H, p, H brm), p, 1.78–1.71 Hm), 1.78–1.71 (6H, br m, (6H, Hi br, H m,j, Hf H). i13,HC NMRj,Hf). (150C MHz, NMR H (1502O/10% MHz, δ H2DO/10%2O): δ 179.0, D2O): 156.7,179.0, 133.3 156.7, (2C), 130.7, 133.3 118.3 (2C), (2C), 130.7, 73.7, 118.3 48.8, (2C), 48.6, 73.7, 46.7, 48.8,46.2, 40.4, 48.6, 38.4, 46.7, 37.4, 46.2, 27.1, 40.4, 25.5, 38.4, 37.4,24.3 27.1, (2C). 25.5, The 24.3 proton (2C). assignment The protons assignmentsare displayed are on displayed the one-dimensional on the one-dimensional spectrum in spectrumFigure 3. in FigureMS/MS3. MS/MS fragmentation fragmentation resulted resultedin two primary in two fragment primary fragmentions of m/ ionsz 129.26 of mand/z m129.26/z 293.37, and consistentm/z 293.37, consistentwith fragmentation with fragmentation at the sites at labelled the sites in labelled Figure 2B. in FigureOther fragment2B. Other ions fragment were also ions evident were also including evident includingm/z 58.15,m /mz/z58.15, 112.23m and/z 112.23 m/z 222.28, and m and/z 222.28,are consistent and are with consistent the fragmentation with the fragmentation patterns observed patterns in observedsimilar inmolecules similar molecules[18]. The theoretical [18]. The theoreticalcalculated mass calculated for the mass derived for structure the derived of PA structure366 is 366.2631 of PA366 is 366.2631Da, and a Da, m/z and 367.2653 a m/z (exact367.2653 mass (exact 366.2573 mass Da) 366.2573 was observed Da) was by observed MALDI-MS by MALDI-MS in reflector inpositive reflector positiveion mode. ion mode.

Figure 2. Characterisation of spider venom polyamines. (A) RP-HPLC chromatogram of crude female Figure 2. Characterisation of spider venom polyamines. (A) RP-HPLC chromatogram of crude female Phlogius sp. venom with the peak corresponding to the polyamine PA highlighted in red (Thermo Phlogius sp. venom with the peak corresponding to the polyamine PA366366 highlighted in red (Thermo Scientific Hypersil GOLD aQ 250 × 10 mm, 5 µm column; 1 mL/min flow rate; Solvent A H O/0.05% Scientific Hypersil GOLD aQ 250 × 10mm, 5µm column; 1 mL/min flow rate; Solvent A H2O/0.05%2 TFA, Solvent B 90% ACN/H O/0.045% TFA; 5–80% solvent B in 75 min, 80–90% solvent B in 5 min, TFA, Solvent B 90% ACN/H22O/0.045% TFA; 5–80% solvent B in 75 min, 80–90% solvent B in 5 min, 90%90% solvent solvent B forB for 5 min,5 min, and and 90–5% 90–5% solvent solvent BB inin 22 min;min; absorbance at at 214 214 nm); nm); (B (B) )SCIEX SCIEX TOF/TOF™ TOF/TOF ™

58005800 MALDI MALDI MS/MS MS/MS spectrum spectrum of ofPA PA366366 using CHCA matrix, matrix, and and the the de determinedtermined chemical chemical structure structure withwith relevant relevant fragment fragment ions ions highlighted; highlighted; ((CC)) RP-HPLCRP-HPLC chromatogram of of crude crude female female A.A. robustus robustus

venomvenom with with the the peak peak corresponding corresponding to to thethe polyaminepolyamine PAPA389389 highlightedhighlighted in in red red (Thermo (Thermo Scientific Scientific HypersilHypersil GOLD GOLD aQ aQ 250 250 ×× 10mm,10 mm, 5µm 5 µ column;m column; 1 mL/min 1 mL/min flow rate; flow Solvent rate; SolventA H2O/0.05% A H2 TFA,O/0.05% Solvent TFA, SolventB 90% B ACN/H 90% ACN/H2O/0.045%2O/0.045% TFA; 5–80% TFA; solvent 5–80% B solventin 75 min, B in80–90% 75 min, solvent 80–90% B in solvent 5 min, 90% B in solvent 5 min, 90%B solventfor 5 min, B for and 5 min, 90–5% and solvent 90–5% B solvent in 2 min; Bin absorbance 2 min; absorbance at 214 nm); at ( 214D) SCIEX nm); ( DTOF/TOF™) SCIEX TOF/TOF 5800 MALDI-™ 5800

MALDI-MS/MSMS/MS spectrum spectrum of PA389of using PA389 CHCAusing matrix, CHCA and matrix, the determined and the determined chemical structure chemical with structure relevant with relevantfragment fragment ions highlighted. ions highlighted.

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1 1 Figure 3.3. ChemicalChemical structurestructure and and HH NMR NMR spectrum spectrum of of PA PA366366. The. The assignments assignments were were derived derived based based on two-dimensionalon two-dimensional NMR NMR spectra spectra and and confirmed confirmed using using mass mass spectrometry spectrometry fragmentation fragmentation analysis. analysis.

2.2. NMR Screening of Crude SpiderSpider VenomsVenoms

Following thethe characterisationcharacterisation ofof purifiedpurified PAPA366366, crude venom from thethe sevenseven otherother spidersspiders thatthat showedshowed earlyearly elutingeluting fractionsfractions withwith cytotoxiccytotoxic activityactivity werewere analysedanalysed usingusing one-dimensionalone-dimensional NMRNMR spectroscopyspectroscopy toto screenscreen forfor thethe presencepresence ofof PAPA366366.. The one-dimensionalone-dimensional NMR spectraspectra ofof selectedselected examples are shownshown inin FigureFigure4 4.. AlthoughAlthough spiderspider venomsvenoms havehave numerousnumerous disulfide-richdisulfide-rich peptides,peptides, thethe smallsmall molecules molecules have have sharper sharper peaks peaks in the in 1Dthe spectra, 1D spectra, making making them easilythem discernibleeasily discernible from peptide from signals.peptide Thissignals. analysis This analysis confirmed confirmed the presence the presence of PA366 ofin PA the366 crude in the venom crude fromvenom the from spider the species spider Acanthoscuriaspecies Acanthoscuria geniculata, geniculata, Chilobrachys Chilobrachy penang, ands penang, Psalmopoeus and Psalmopoeus irminia, in addition irminia, toin the additionPhlogius tosp. the PhlogiusAnalysis sp. of the NMR spectra of the crude venom of darlingi and A. robustus indicated they doAnalysis not contain of the PA NMR366, despitespectra demonstratingof the crude venom cytotoxic of Ceratogyrus activity in darlingi the assays. and The A. robustus lack of PA indicated366 was confirmedthey do not by contain mass spectrometry PA366, despite analysis, demonstrating and instead cytotoxic a mass activity of 389 Dain wasthe assays. present. The The lack structure of PA of366 thiswas activeconfirmed component by mass in A. spectrometry robustus venom analysis, was determined and instead following a mass further of 389 purification Da was bypresent. RP-HPLC, The andstructure subsequent of this analysisactive component using NMR in spectroscopy A. robustus andvenom MS/MS. was Thedetermined analysis following determined further that A.purification robustus venom by RP-HPLC, contains a and polyamine subsequent previously analysis characterised using NMR from spectroscopy the venom of aand trap-door MS/MS. spider The (analysisHebestatis determined theveniti) andthat aA. tarantula robustus (venomHarpactirella containssp.), a andpolyamine termed previously Het389 [19 ].characterised Subsequent from analysis the ofvenom the C. of darlingi a trap-doorvenom spider also ( confirmedHebestatis theveniti the presence) and a of tarantula Het389. Given(Harpactirella the presence sp.), and of Hettermed389 across Het389 multiple[19]. Subsequent genera, weanalysis suggest of the the C. new darlingi identifying venom term also PAconfirmed389 is more the appropriate. presence of Het The389 structure. Given the of PApresence389 is shown of Het in389 Figure across2D andmultiple the 1D genera, NMR spectrawe suggest in Figure the4 .new The theoreticalidentifying calculated term PA exact389 is massmore forappropriate. PA389 is 389.2791 The structure Da, and of a PAm/389z 390.2443 is shown (exact in Figure mass 2D 389.2364 and the Da) 1D was NMR observed spectra by in MALDI-MS Figure 4. The in reflectortheoretical positive calculated ion mode.exact mass In comparison for PA389 is to389.2791 the hydroxyphenyl Da, and a m/z based390.2443 head (exact group mass in PA389.2364366, PA Da)389 haswas anobserved indole headby MALDI-MS group and identicalin reflector spermine positive tail. ion The mode. cytotoxicity In comparison of PA389 tois the shown hydroxyphenyl in Figure5 andbased shows head this group polyamine in PA only366, PA displays389 has cytotoxicityan indole head at millimolar group and concentrations. identical spermine tail. The cytotoxicity of PA389 is shown in Figure 5 and shows this polyamine only displays cytotoxicity at millimolar concentrations.

Toxins 2017, 9, 346 6 of 13 Toxins 2017, 9, 346 6 of 13

FigureFigure 4.4. One-dimensionalOne-dimensional NMRNMR spectraspectra ofof selectedselected crudecrude spiderspider venomsvenoms andand purifiedpurified polyamines.polyamines. 11HH NMRNMR spectraspectra ofof crudecrude venomvenom fromfrom PhlogiusPhlogius sp.,sp., A.A. robustusrobustus andand C.C. darlingidarlingi recordedrecorded atat 600600 MHz,MHz,

showingshowing thethe presencepresence ofof PAPA366366 inin thethe PhlogiusPhlogius sp.sp. venom,venom, andand PAPA389389 inin thethe A.A. robustusrobustus andand C.C. darlingidarlingi 11 venom.venom. TheThe H NMR spectra ofof purifiedpurified PAPA366366 and PA 389 recordedrecorded at at 600 600 MHz MHz are are also also shown. shown.

Toxins 2017, 9, 346 7 of 13 Toxins 2017, 9, 346 7 of 13

Toxins 2017, 9, 346 7 of 13

Figure 5. Cytotoxicity of PA389 against cancer cell lines. Dose response for cytotoxic cell killing of PA389 Figure 5. Cytotoxicity of PA389 against cancer cell lines. Dose response for cytotoxic cell killing of PA389 in MCF-7 (circles) or SK-MEL-28 (squares) cells. Cells were treated with the indicated concentrations in MCF-7 (circles) or SK-MEL-28 (squares) cells. Cells were treated with the indicated concentrations of purified PA389 for five days, before assay for cell survival was assessed using SRB. Cell survival of purified PA389 for five days, before assay for cell survival was assessed using SRB. Cell survival percentagesFigure 5. Cytotoxicity normalised ofto PAuntreated389 against cells cancer are indicatecell lines.d. Dose Representative response for data cytotoxic from cella single killing experiment of PA389 percentages normalised to untreated cells are indicated. Representative data from a single experiment within triplicateMCF-7 (circles) readings or SK-MEL-28 are shown. (s quares) cells. Cells were treated with the indicated concentrations with triplicateof purified readings PA389 for are five shown. days, before assay for cell survival was assessed using SRB. Cell survival percentages normalised to untreated cells are indicated. Representative data from a single experiment The venom from H. gigas and N. chromatus did not appear to contain either PA366 or PA389, despite with triplicate readings are shown. displayingThe venom cytotoxic from activity.H. gigas H.and gigasN. appears chromatus to containdid not primarily appear peptide to contain based either molecules, PA366 whereasor PA389 , despitethe spectra displaying from cytotoxicN. chromatus activity. is dominatedH. gigas appears by small to molecules contain primarily but with peptide different based shifts molecules, to the The venom from H. gigas and N. chromatus did not appear to contain either PA366 or PA389, despite whereaspolyaminesdisplaying the spectra characterisedcytotoxic from activity.N. in chromatus H.this gigas study. appearsis dominatedComparison to contain by primarily of small the molecules peptide1D spectra based but of molecules, withA. geniculata different whereas and shifts toPamphobeteus thethe polyamines spectra antinousfrom characterised N., chromatusa tarantula inis thisdominatedspecies study. that by Comparison did small not molecules display of the cytotoxicbut 1D with spectra activity,different of A. highlightsshifts geniculata to the the and Pamphobeteusdifferencespolyamines in antinous thecharacterised venom, a tarantula NMR in “profiles”this species study. in thatComparisonthe pr didesence not ofand display the absence 1D cytotoxic spectra of small of activity, molecules,A. geniculata highlights as shownand the differencesin FigurePamphobeteus in6. theThe venomantinousNMR spectra NMR, a tarantula “profiles”of A. geniculataspecies in thethat ispresence dominateddid not anddisplay by absence PA cytotoxic366, whereas of small activity, P. molecules, antinous highlights shows as the shown no evidence of small molecules. Instead, the venom of P. antinous appears to contain primarily peptides in Figuredifferences6. The in NMR the venom spectra NMR of “profiles”A. geniculata in theis pr dominatedesence and absence by PA 366of small, whereas molecules,P. antinous as shownshows nobased evidencein Figure on the of 6. large smallThe NMRnumber molecules. spectra of peaks of Instead,A. andgeniculata significan the is venom dominatedt peak of dispersionP. by antinous PA366, whereasinappears the 6–9.5 P. antinous to ppm contain range. shows primarily These no peptidespeptidesevidence based are of likely on small the to molecules. large be disulfide-rich number Instead, of peaksbasedthe venom andon previous of significant P. antinous analyses peak appears of dispersion spider to contain venoms in primarily the [20]. 6–9.5 peptides ppm range. based on the large number of peaks and significant peak dispersion in the 6–9.5 ppm range. These These peptides are likely to be disulfide-rich based on previous analyses of spider venoms [20]. peptides are likely to be disulfide-rich based on previous analyses of spider venoms [20].

Figure 6. One-dimensional NMR spectra of selected crude spider venoms from P. antinous and A. Figure 6.1 One-dimensional NMR spectra of selected crude spider venoms from P. antinous and A. Figuregeniculata 6. One-dimensional. H NMR analysis NMR of the spectra crude ofvenom selected from crude P. antinous spider shows venoms a fromcompositionP. antinous that andis geniculata. 1H NMR analysis of the crude venom from P. antinous shows a composition that is A.primarily geniculata peptides,. 1H NMR based analysis on the of number the crude of peaks venom and from dispersionP. antinous in theshows amide a region; composition this venom that is doesprimarily not show peptides, any evidence based on of thecytotoxicity number of agains peakst MCF-7,and dispersion SK-MEL-28 in the or amide NFF cells.region; In this contrast, venom the primarily peptides, based on the number of peaks and dispersion in the amide region; this venom spectrumdoes not of show cytotoxic any evidence crude venom of cytotoxicity from A. againsgeniculatat MCF-7, is dominated SK-MEL-28 by or the NFF peaks cells. corresponding In contrast, the to does notspectrum show of any cytotoxic evidence crude of venom cytotoxicity from A. againstgeniculata MCF-7, is dominated SK-MEL-28 by the peaks or NFF corresponding cells. In contrast, to PA366. the spectrumPA366. of cytotoxic crude venom from A. geniculata is dominated by the peaks corresponding to PA366.

Toxins 2017, 9, 346 8 of 13

2.3. Protein Array Analysis

A sample of PA366 was biotinylated for protein array analysis to identify any protein binding interactions. Biotinylated PA366 was shown by MS analysis to have a molecular weight of 706 Da, confirming biotin binding to the primary amine at the end of the polyamine chain. Analysis of the biotinylated PA366 binding to a protein array demonstrated interaction with numerous proteins, presumably via non-specific interactions. Signals with Z-scores above three were considered to be significant. The polyamine bound to MAPK1 and MAP3K2; both giving Z-scores of 3.7. These proteins have previously been shown to have roles in tumour development and breast cancer [21]. The protein with the highest score however, based on the signal intensity, was a zinc finger protein (ZNF501 Z-score 13.93).

3. Discussion In the current study, we have used NMR spectroscopy and MS to characterise the novel polyamine PA366, which has selective cytotoxicity for MCF-7 breast cancer cells. Comparison with a related polyamine, PA389, provided insight into the important structural features. The two polyamines consist of a spermine tail functionalised with an aromatic head group, and are identical except PA366 has a 2-hydroxy-3-(4-hydroxyphenyl)propanal aromatic head group, while PA389 contains a 2-hydroxy-3-(1H-indol-3-yl)propanal based head group. PA389 only displays cytotoxicity at high concentrations in contrast to PA366, indicating that the aromatic head group is important for cytotoxicity. Using ProtoArray technology, analysis of the protein binding properties of PA366 indicates non-specific binding to a range of proteins. However, the protein with the highest Z-score in the protein array analysis, which is likely to have the most significant interaction, is zinc finger protein 501, which belongs to the Krüppel-like c2h2 type zinc finger protein family. Zinc finger proteins are one of the most common families of DNA-binding transcription factors [22] and a recent study suggested they can be involved in tumour development [21]. Interestingly, the mammalian polyamines putrescine, spermidine and spermine alter binding of DNA to zinc finger transcription factors [23], which is consistent with a role for zinc finger protein 501 in the bioactivity of PA366. However, additional interactions might also be involved given that MAPK1 and MAP3K2, mitogen-activated protein kinases (MAPK), were also significant hits in the ProtoArray analysis. Several MAPK genes are associated with breast cancer [24], and activation of MAPK can lead to cell proliferation, invasion and metastasis, making these kinases potential targets for cancer treatment [25,26]. To provide greater insight into the possible targets of PA366 it is important to determine the membrane permeability, which will influence the targets accessible to the molecule. The cytotoxicity we observed for PA366 is consistent with the role of mammalian polyamines in cell death. Numerous links have been identified between mammalian polyamines and apoptotic pathways. For example, the production of hydrogen peroxide produced during polyamine catabolism is thought to be involved in cell death [27]. However, alteration in polyamine levels can have an impact on numerous cellular functions such as DNA-protein interactions, protein-protein interactions and mitochondrial integrity which can lead to cell death. Morphological analysis of the cells following incubation with PA366 indicates the presence of apoptosis but further study is required to confirm this hypothesis. Although PA366 displays cytotoxicity against a cancer cell lines, this is unlikely to be related to its function in the spider venom. The primary target of venom polyamine toxins is thought to be the ionotropic glutamate receptors [28]. PA389 has previously been shown to cause rapid but reversible paralysis to lepidopteran insect larvae, suggesting a role in prey capture [19]. Given the structural and functional similarities between PA389 and PA366, it is likely that the latter also displays some form of bioactivity against insect prey. The known distribution of PA366 and PA389 determined from previous work and this study is shown in the phylogenetic tree in Figure7. PA 366 is present in a range of geographically diverse spider Toxins 2017, 9, 346 9 of 13

Toxinsspecies 2017 from, 9, 346 the family Theraphosidae, indicating that differences such as diet and environmental9 of 13 conditions are unlikely to influence the production of this polyamine. Similarly, PA389 is present thein the venom venom of ofspiders spiders from from the the Theraphosidae Theraphosidae family, family, but but also also in in the the venom venom of of species species from from the the Hexathelidae and Ctenizidae families. In this study, we have confirmed the presence of PA389 in the Hexathelidae and Ctenizidae families. In this study, we have confirmed the presence of PA389 in venomthe venom of the of Sydney the Sydney Funnel-web Funnel-web spider, spider, Atrax robustusAtrax robustus, as the undefined, as the undefined 3-(3-indoyl)lactic 3-(3-indoyl)lactic acid and spermine complex suggested by Sutherland [29] and proposed as identical to PA389 by Skinner et al. [19]. acid and spermine complex suggested by Sutherland [29] and proposed as identical to PA389 by It is likely that PA366 is also present in other spider families but further analysis is required to confirm Skinner et al. [19]. It is likely that PA366 is also present in other spider families but further analysis is thisrequired hypothesis. to confirm this hypothesis.

Figure 7. Phylogenic tree of some spider venoms showing the distribution of PA366 and PA389. A Figure 7. Phylogenic tree of some spider venoms showing the distribution of PA366 and PA389. phylogeneticA phylogenetic tree treeof crude of crudespider spidervenoms venoms that demonstrated that demonstrated cytotoxicity cytotoxicity on MCF-7 or on SK-MEL-28 MCF-7 or cells in this study, and the presence of the polyamines PA366 and PA389. The two spider species, SK-MEL-28 cells in this study, and the presence of the polyamines PA366 and PA389. The two Hebestatisspider species, thevenetiHebestatis and Harpactirella theveneti sp.,and fromHarpactirella the original sp.,identificati fromon the and original characterisation identification study and of

PAcharacterisation389 are also included study of PA[19]389 (Photographare also included credits: [19 ]Acanthoscurria (Photograph credits:geniculataAcanthoscurria, Ceratogyrus geniculata darlingi, , HarpactirellaCeratogyrus sp., darlingi , Harpactirella gigas, sp.,and NhanduHysterocrates chromatus gigas—Bastian, and NhanduRast; Chilobrachys chromatus —Bastianpenang— MuhammadRast; Chilobrachys Ashraf; Psalmopoeus penang—Muhammad irminia—Edward Ashraf; Evans;Psalmopoeus Hebestatis theveneti irminia—Edward—Marshal Hedin; Evans; PhlogiusHebestatis sp. theveneti and Atrax—Marshal robustus Hedin;—DavidPhlogius Wilson).sp. and Atrax robustus—David Wilson).

OneOne outcome outcome of of this this study study is is NMR spectrosco spectroscopypy proved proved an an effective effective way way of screening and detecting the presence of the polyamines PA366 and PA389 without the need for fractionation of the detecting the presence of the polyamines PA366 and PA389 without the need for fractionation of the venom.venom. AlthoughAlthough the the spider spider venoms venoms contain contain a large a numberlarge number of disulfide-rich of disulfide-rich peptides, thepeptides, polyamines the polyamineshave low molecular have low weights molecular and weights provide muchand provide sharper much spectral sharper peaks, spectral and can peaks, be detected and can when be detectedonly low when microgram only low amounts microgram of material amounts are of presentmaterialin are contrast present toin thecontrast larger topeptides. the larger Thispeptides. type This type of NMR spectroscopy screening has also been shown to be useful for the discovery of new small molecules from other sources. For example, unfractionated extracts from insects examined using 2D NMR spectroscopy led to the discovery of tricyclic pyrones from a ladybird beetle including the identification of ring systems that had not previously been found in nature [1].

Toxins 2017, 9, 346 10 of 13 of NMR spectroscopy screening has also been shown to be useful for the discovery of new small molecules from other sources. For example, unfractionated extracts from insects examined using 2D NMR spectroscopy led to the discovery of tricyclic pyrones from a ladybird beetle including the identification of ring systems that had not previously been found in nature [1]. Despite the conservation of PA366 in several venoms, our study also serves to highlight the compound diversity and broad range of biological activities found in spider venom. N. chromatus has early eluting peaks from RP-HPLC that display cytotoxicity but the venom does not appear to contain PA366 or PA389. The 1D NMR spectrum indicates the presence of small molecules, and it is likely there are several, related polyamines that might account for our observed activity. Although there is evidence of small molecules in the crude venom of H. gigas, the venom is dominated by peptides/proteins in contrast to the venoms such as Phlogius sp., A. robustus and C darlingi. Furthermore, P. antinous appears to show only peptide related peaks in the amide region, and does not display cytotoxicity. This compound and bioactivity diversity is one of the features that has led to the extensive study of spider venom for the discovery of novel compounds for therapeutic or agricultural applications [20]. In summary, we have shown that a spider venom polyamine can have relatively selective cytotoxicity activity against specific cancer cell lines and that the aromatic head group appears to be involved in conferring cytotoxicity. Insight into the distribution of spider venom polyamines across species and genera has also been gained from screening using NMR spectroscopy.

4. Materials and Methods

4.1. Venom Collection and Purification A majority of the crude venoms were purchased from the commercial supplier Alphabiotoxine Laboratory, Montroeul-au-bois, Hainaut, Belgium. Crude venom from Pterinochilus meridionalis, Vitalius roseus and Hogna carolinensis was purchased from SpiderPharm, Yarnell, AZ, USA. Crude venom was also collected from a colony of housed specimens of female Australian (Phlogius sp. and Coremiocnemis tropix), Macrothele gigas, and fasciata and Poecilotheria metallica by electrostimulation of the venom glands. Male Sydney funnel-web spider (Atrax robustus) venom (~5 mg) was supplied by the Australian Park (Gosford, NSW, Australia). Crude venom was fractionated by reversed-phase HPLC (RP-HPLC) using an Agilent 1260 Infinity HPLC system (Agilent, Santa Clara, CA, USA) and a Thermo Scientific Hypersil GOLD aQ (250 × 10 mm, 5 µm) column (Thermo Fisher Scientific, Waltham, MA, USA). Fractionation of the venom components was achieved using a linear gradient of two mobile phases: H2O/0.05% trifluoroacetic acid (TFA; Auspep, Tullamarine, VIC, Australia) [solvent A], and 90% acetonitrile (ACN; Sigma-Aldrich, St. Louis, MO, USA)/H2O/0.045% TFA [solvent B]. Separation used a gradient of 5–80% solvent B in 75 min, 80–90% solvent B in 5 min, 90% solvent B for 5 min, and 90–5% solvent B in 2 min at a flow rate of 6 mL/min. Venom component elution was monitored at 214 nm and 1 min fractions were collected.

4.2. Cytotoxicity Assays All cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated foetal calf serum (Life Technologies, Carlsbad, CA, USA) in RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 3 mM HEPES at 5% CO2, 99% humidity at 37 ◦C. Routine mycoplasma tests were performed using Hoechst stain or PCR and were always negative. The cytotoxicity of compounds was determined using clonogenic survival assays of MCF-7 (breast adenocarcinoma), SK-MEL-28 (melanoma) and human neonatal foreskin fibroblasts (NFF) cells [30,31]. The cell lines were purchased from ATCC. Cells were plated into 96-well microtitre plates at 5 × 103 cells/well, and allowed to adhere overnight. Compounds were added to culture medium at the indicated final concentrations, and plates incubated under the above conditions for five days. Cell survival was then assayed using sulforhodamine B (SRB; Sigma, St. Louis, MO, USA). Briefly, the culture medium was removed from the 96-well microtitre plates and the plates washed Toxins 2017, 9, 346 11 of 13 twice with phosphate buffered saline (PBS), before the cells were fixed with methylated spirits for 15 min. The plates were then rinsed with tap water and the fixed cells stained with 50 µL/well of SRB solution (0.4% sulforhodamine B (w/v) in 1% (v/v) acetic acid) over a period of 1 h. The SRB solution was removed from the wells and the plates rapidly washed two times with 1% (v/v) acetic acid. Protein bound dye was then solubilised with the addition of 100 µL of 10 mM unbuffered Tris, and incubated for 15 min at 25 ◦C. Plates were then read at 564 nm on a VERSA max tuneable microplate reader (Molecular Devices, Sunnyvale, CA, USA). Negative (vehicle only) and positive (cisplatin) controls were run with every cell survival experiment to ensure the quality of the data. Vehicle only control results in <5% change in cell survival whereas the positive control inhibited cell survival at nanomolar concentrations as expected.

4.3. Mass Spectrometry and NMR Analysis 1H NMR and 13C NMR spectra were recorded at 600.13 MHz and 150.91 MHz at 298 K on a Bruker Avance III 600 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with a cryoprobe. Samples were dissolved in 90% H2O/10% D2O(v/v) (100 µM). D2O (99.9%) was obtained from Cambridge Isotope Laboratories, Woburn, MA, USA for 1H NMR measurements. Spectra were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS; Cambridge Isotope Laboratories, Woburn, MA, USA). Two-dimensional spectra included TOCSY, NOESY, DQF-COSY, HSQC, HMBC and HSQC-TOCSY. TOCSY and NOESY mixing times of 80 ms and 500 ms respectively were used. Spectra were analyzed using TOPSPIN (Bruker, Billerica, MA, USA). Mass spectrometry (MS) was performed using a SCIEX TOF/TOF™ 5800 MALDI, and high resolution mass spectrometry (HRMS) using direct infusion on a SCIEX QSTAR Elite equipped with a nanospray (SCIEX, Framingham, MA, USA). MALDI-MS samples were spotted on 384-well stainless steel target plates using 0.5 µL of sample and 0.5 µL of either α-cyano-4-hydroxycinnamic acid (CHCA; Sigma-Aldrich, St. Louis, MO, USA) matrix at 7.5 mg/mL in 50% ACN/0.1% TFA, or 2,5-dihydroxybenzoic acid (DHB; Sigma-Aldrich, St. Louis, MO, USA) matrix at 10 mg/mL in 50% ethanol/0.1% TFA. Calibration was performed before spectra collection for each sample using Calibration Mix solution 2 (SCIEX, Framingham, MA, USA). Spectra were acquired in reflector positive ion mode from m/z 300 to 1000 Da, and averaged over 2000 laser shots. QSTAR Elite MS samples were infused at 2 µL/min. MS spectra were acquired in positive ion mode from m/z 200 to 1000 Da and spectra were externally calibrated using SCIEX renin standard. MS/MS spectra were acquired for the product ions 367.26 Da and 390.2 Da from m/z 10 to 500 Da. All spectra were acquired with an accumulation time of 1 s, using an ion-spray voltage of 3700 V, a declustering potential of 80 V, and a focussing potential of 280 V.

4.4. Protein Array Analysis

Biotinylation of PA366 for protein interaction analysis was achieved utilising a 20-fold excess of EZ-Link® Sulfo-NHS-LC-biotin (ThermoFisher Scientific, Waltham, MA, USA), and following the manufacturer’s instructions. Successful labelling of PA366 was confirmed by molecular weight analysis using a SCIEX TOF/TOF™ 5800 mass spectrometer (SCIEX, Framingham, MA, USA), followed by RP-HPLC purification of labelled PA366 to remove excess non-reacted and hydrolyzed biotin reagent from the solution. The purified sample was dried and resuspended in 100 µL of 1.0% PBS (pH 7.2) to achieve a required final amount of 10 µg. Interactions between biotinylated PA366 and more than 9000 human proteins were investigated employing a ProtoArray® Human Protein Microarray v5.0 Protein-Protein Interaction (PPI) kit (Invitrogen™, Carlsbad, CA, USA), and significant interactions detected using a GenePix® 4000Bmicroarray scanner (Molecular Devices, Sunnyvale, CA, USA), according to the manufacturer’s instructions. Toxins 2017, 9, 346 12 of 13

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6651/9/11/346/ S1. Table S1: Crude spider venoms tested for cytotoxicity against MCF-7 cells. Acknowledgments: This work was funded in part by a grant from the National Breast Cancer Foundation (D.W., G.M.B., P.G.P., L.D.R., N.L.D.). Fellowship support was provided to AL from NHMRC (1020114) and N.L.D. from the Australian Research Council (FF110100226). The authors are grateful to Volker Herzig (I.M.B., Q.L.D.) for providing crude venom from Macrothele gigas, Coremiocnemis tropix, Poecilotheria fasciata, and Poecilotheria metallica, the Australian Reptile Park (Gosford, NSW) for the provision of crude male Sydney funnel-web spider venom, and to Bastian Rast, Edward Evans, Marshal Hedin, and Muhammad Ashraf for the provision of photographs. Author Contributions: D.W., L.D.R., and N.L.D. designed the study. D.W., G.M.B., L.M., M.J.N., P.G.P., J.J.S., L.T., A.L., M.J.L., L.D.R., and N.L.D. carried out experiments. D.W. and N.L.D. wrote the paper. All authors analysed the results and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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