Antifungal and antitumor models of bioactive protective peptides

18 Pages·2009·0.89 MB·English

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AnaisdaAcademiaBrasileiradeCiências(2009)81(3):503-520
(AnnalsoftheBrazilianAcademyofSciences)
ISSN0001-3765
www.scielo.br/aabc
Antifungal and antitumor models of bioactive protective peptides
ELAINE G. RODRIGUES1, ANDREY S. DOBROFF1, CARLOS P. TABORDA2 and LUIZ R. TRAVASSOS1
1UnidadedeOncologiaExperimental,DepartamentodeMicrobiologia,ImunologiaeParasitologia,UniversidadeFederaldeSãoPaulo
RuaBotucatu,862,8◦andar,04023-062SãoPaulo,SP,Brasil
2DepartamentodeMicrobiologia,InstitutodeCiênciasBiomédicas,UniversidadedeSãoPaulo
Av.Prof.LineuPrestes1374,CidadeUniversitária,05508-900SãoPaulo,SP,Brasil
ManuscriptreceivedonJune27,2008;acceptedforpublicationonMarch31,2009;
contributedbyLUIZR.TRAVASSOS*
ABSTRACT
Peptidesareremarkablyreactivemoleculesproducedbyagreatvarietyofspeciesandabletodisplayanumberof
functionsinuni-andmulticellularorganismsasmediators,agonistsandregulatingsubstances. Someofthemexert
cytotoxic effects on cells other than those that produced them, and may have a role in controlling subpopulations
and protecting certain species or cell types. Presently, we focus on antifungal and antitumor peptides and discuss
a few models in which specific sequences and structures exerted direct inhibitory effects or stimulated a protective
immuneresponse. Thekillerpeptide,deducedfromanantiidiotypicantibody,withseveralantimicrobialactivitiesand
otherIg-derivedpeptideswithcytotoxicactivitiesincludingantitumoreffects,aremodelsstudiedinvitroandinvivo.
Peptide10fromgp43ofP.brasiliensis(P10)andthevaccineperspectiveagainstparacoccidioidomycosisisanother
topicillustratingtheprotectiveeffect invivoagainstapathogenicfungus. Thecationicantimicrobialpeptideswith
antitumoractivitiesaremostlyreviewedhere. Localtreatmentofmurinemelanomabythepeptidegomesinisanother
modelstudiedattheExperimentalOncologyUnitofUNIFESP.
Keywords: bioactivepeptides,Paracoccidioidesbrasiliensis,tumorcells,killerpeptide,melanoma,apoptosis.
INTRODUCTION recognizedbycellsurfacereceptorsoractasligandsin-
teracting with intracellular compounds and subcellular
Bioactive peptides arise from proteins by the action of
structures. Peptidescanincludeepitopesrecognizedby
peptidases or are chemically synthesized based on cer-
antibodies and TCRs, and those called protective epi-
tain templates of natural sequences that have been se-
topeselicitaprotectiveimmuneresponse. Onfocusing
lected by a variety of screening methods. Peptides can
theactualfungalandtumormodels,peptidesthatdisplay
be designed aiming at enhanced functional activity by
direct cytotoxicity on target cells or elicit a protective
using amino acid substitutions and chemical modifica-
immuneresponseinanimalsexperimentallyinfectedor
tion. Owingtotheirgreatdiversityofbindingproperties,
challengedwithtumorcellshavebeeninvestigated.
peptides can play roles of biochemical reagents, phar-
macologicaldrugs, hormones, antibiotics, vaccinesand
mediatorsofneuralandimmunologicalsignaling. Pep- ANTIFUNGALPEPTIDES
tidesinteractwithmembranestructures,arespecifically Duringthepastdecades,anincreaseintheincidenceof
Incommemorationofthe75thanniversary fungal diseases has been recognized mainly caused by
ofEscolaPaulistadeMedicina/UniversidadeFederaldeSãoPaulo. Candida spp. and filamentous fungi such as Aspergil-
*MemberAcademiaBrasileiradeCiências
lus spp. (reviewed in Mavor et al. 2005 and Brakhage
Correspondenceto:Dr.LuizR.Travassos
E-mail:[email protected] 2005). Todate,therearenolicensedfungalvaccines,and
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504 ELAINE G. RODRIGUES et al.
the use of antimycotics is the only option for the treat- donotcoevolvewithpathogens(JigginsandKim2005).
mentoffungalinfections. Currentlyusedantimycotics, Incontrast,antimicrobialpeptides(AMPs)appeartoun-
however,frequentlyhavealimitedactivityspectrum,are dergoarapidadaptiveevolutioninvertebrates. Infrogs,
available only in intravenous formulations, favor resis- each species produces 10-20 AMPs that differ in size,
tance development, and cause serious side-effects (re- sequenceandspecificity,andthisrapiddiversificationis
viewed in François et al. 2005). Thus, the search for drivenbyevolutionaryselection(Dudaetal. 2002). Der-
newantifungaltherapiesisstronglystimulated,andthe maseptins,producedbyPhyllomedusasauvagii,aSouth
useofantifungalpeptidesisapromisingalternative. American frog, are lysine-rich linear peptides fungici-
Antifungal properties of peptides have been re- dal for A. flavus, A. fumigatus and F. oxysporum (Mor
viewed by De Lucca and Walsh (2000). There are 405 et al. 1994). Magainins are antifungal peptides pro-
peptides with antifungal properties described, compris- ducedbytheAfricanfrogXenopuslaevis(DeLuccaand
ing linear or cyclic, hydrophobic or amphipathic struc- Walsh 2000). They are not hemolytic and inhibit Can-
tures(http://aps.unmc.edu/AP/main.php). dida albicans (Zasloff 1987). Plant [DmAMP1 from
Theircytotoxicitymayinvolvebindingtoanddis- dahlia(Dahliamerckii),RsAFP2fromradish(Raphanus
ruptionofthemembrane(Shai1995),membranepene- sativus), HsAFP1 from coral bells (Heuchera sangui-
tration and interaction with the mitochondria (Helmer- nea),Psd1frompea(Pisumsativum),MsDef1fromal-
horst et al. 1999) or pore formation (Bechinger 1997). falfa (Medicago sativa) and MtDef2 from barrel medic
Antifungalpeptideshavebeenstudiedinbacteria,fungi, (Medicagotruncatula)],insects(Termicinfromtheter-
plants,insects,amphibiansandmammals. Relevantex- mite Pseudacanthotermes spiniger, Drosomycin from
amplesaregivennext. the fruitfly Drosophila melanogaster, Heliomicin from
β
Syringomycins, syringostatins and syringotoxins thetobaccobudwormHeliothisvirescens)andhuman[ -
β β
from Pseudomonas syringae are lipodepsipeptides defensin 1 (HBD1), -defensin 2 (HBD2), -defensin
highly lethal to Candida albicans, Aspergillus and Fu- 3 (HBD3)] defensins showed antifungal properties (re-
sarium species (De Lucca and Walsh 2000, De Lucca viewedinAertsetal. 2008). Althoughtherearenoclear
etal. 1999,Sorensenetal. 1996). Glycopeptidecepa- similaritiesinthemodeofactionofthesedefensins,the
cidines from Burkholderia cepacia are active against presenceofsphingolipidglucosylceramide(GlcCer)in
Candida sp., Aspergillus niger, Fusarium oxysporum fungalmembranesseemstoplayacentralroleintheac-
andCryptococcusneoformans (Leeetal. 1994,Limet tion of some defensins (Thevissen et al. 2004). Only
al. 1994). Antifungal peptidylnucleoside nikkomycins Psd1 was internalized in the fungal cell, affecting the
areproducedby Streptomycestendae, actbyinhibiting normalprogressionofthecellcycle(Loboetal. 2007),
chitin biosynthesis and were effective in murine infec- anditispossiblethattheotherdefensinsstayoutsidethe
tionsbyCoccidioidesimmitisandBlastomycesdermati- cellinducingfungalcelldeathafterinteractionwiththeir
tidis (Hector et al. 1990). Zeamatin, the 22 kilodal- target(e.g. sphingolipids)andmodulationofintracellu-
ton (kDa) peptide produced by Zea mays, permeabi- larsignalingcascades(Aertsetal. 2008). RsAFP2was
lizes the fungal membrane and kills C. albicans with alsoeffectiveinaninvivoprophylacticmodelofmurine
μ
aminimalinhibitoryconcentration(MIC)of0.5 g/ml candidiasis(Tavaresetal. 2008).
β
(Roberts and Selitrennikoff 1991). Cecropins from the -Defensinsincludeporcinecationic,cysteine-rich
silk moth Hyalopora cecropia are linear, lytic peptides protegrinswhich inhibitedC.albicans(Choetal. 1998).
effective against germinating conidia of F. oxysporum Gomesin, a cationic AMP isolated from the hemocytes
and A. fumigatus (De Lucca et al. 1998). Both the L- of the unchallenged Brazilian spider Acanthoscurria
andD-isomericformsofcecropinBwerefungicidal(De gomesiana (Silvaetal. 2000), isstructurallyrelatedto
Luccaetal.2000). Drosomycinisa44aminoacids(aa) protegrins and exerts microbicidal activity against fila-
induciblepeptideactiveagainstF.oxysporum(Lemaitre mentous fungi, yeast and parasites. Gomesin bound to
et al. 1997). There is no evidence of adaptive protein thesurfaceofCryptococcusneoformans,resultingincell
evolutioninthedrosomycingenes,suggestingthatthey deathbymembranepermeabilization. Fungalgrowth,in
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ANTI-FUNGAL AND ANTITUMOR PEPTIDES 505
the presence of the peptide, induced a decrease in cap- susceptible cells by various mechanisms, including the
suleexpression,renderingcellsmoresusceptibletobrain inductionofcation-selectiveionchannelsintheplasma
phagocytesand,inassociationwithfluconazole,incon- membrane, interference in the cell cycle (G1, G1/S, S
μ
centrations with low antimicrobial activity (0.1–1 M), arrest),chromosomalDNAsynthesisandanticodonnu-
inhibitedfungalgrowthandenhancedtheantimicrobial clease(SchmittandBreinig2006,SantosandMarquina
activityofbrainphagocytes(Barbosaetal. 2007). One 2004, Jablonowski and Schaffrath 2007, Klassen et al.
ofthemodelsdescribedinthepresentreviewisthatof 2004). Killer toxins can induce apoptosis mediated by
gomesin cytotoxicity in murine and human tumor cells yeastcaspaseYca1p,characterizedbyDNAfragmenta-
(Rodriguesetal. 2008). tion,andphosphatidylserineexternalmembraneexpres-
Amongtheantifungalpeptidesproducedbyfungi, sion. Thiscouldbeageneralcelldeathmechanismun-
the echinocandins interfere with the cell wall biosyn- dernaturalenvironmentalconditions(Paluszynskietal.
thesis (Denning 1997) and the pneumocandins, aculea- 2007,SchmittandReiter2008).
cins, WF11899, and mulundocandins have a modified The direct use of killer toxins in antifungal ther-
echinocandinBpeptidecore(DebonoandGordee1994, apy was discouraged owing to some of their proper-
KurtzandDouglas1997). Echinocandinsareproduced ties. They are generally heat-labile, protease-sensitive
byAspergillusnidulansandA.rugulosusandareeffec- andactwithinanarrowpHandtemperaturerange. They
μ
tiveagainstCandida(MIC=0.6 g/mlforechinocandin are antigenic and toxic, as shown for Pichia anomala
B and C. albicans) (reviewed in De Lucca and Walsh killertoxin(Pettoello-Mantovanietal. 1995). Toover-
2000). Clinical trials have started with molecules of comethesepitfallsofapotentialtherapeuticagent,im-
the echinocandin group, VER-002, FK463 and caspo- munological derivatives were generated on the basis of
fungin(MK-0991)modifiedforincreasedsolubilityand the idiotypic network that mimicked the toxic effect of
active against Candida spp. and Aspergillus spp. V- P. anomala killer toxin (Polonelli et al. 1991). Killer
echinocandinandFK463wereeffectiveinthetreatment antibodies with the internal image of the active site of
of esophageal candidiasis, the latter in AIDs patients akillertoxin, whichactedasantibiotics, werethenob-
(reviewed in De Lucca and Walsh 2000). Clinical tri- tained. They exerted significant therapeutic effects in
als with caspofungin (derived from pneumocandin), a experimental models of candidiasis, aspergillosis and
β
drugthatinhibits -1,3D-glucansynthase,haveshown pneumocystosis.
excellent results in the treatment of Candida infections Toxic effects were also obtained with single chain
and invasive aspergillosis refractory to other antifungal variablefragment(scFv)preparationsandtheywerefur-
agents(i.e.,conventionalorlipidformulationsofampho- therexaminedbysynthesizingoverlappingdecapeptides
tericin B and/or itraconazole). Aureobasidins are pro- which correspond to the light chain of antibodies (VL)
ducedbyAureobasidiumpullulans,interferewithsphin- and heavy chain of antibodies (VH) regions. These re-
golipid synthesis and are effective against murine can- gions include the complementary determining regions
didiasis(Nageicetal. 1997,Takesakoetal. 1993). (CDRs) that were tested in vitro against C. albicans.
Several peptides were active and one of them, corres-
KILLERTOXINSANDKILLERPEPTIDES pondingtotheframeworksequencewiththefinalthree
Killer yeasts secrete killer toxins that target suscepti- amino acids belonging to VL CDR1, was selected. It
blecellsinatwo-stepreceptor-mediatedmanner. They was very cytotoxic and the substitution of the N-ter-
bindtocellwallreceptorsandtranslocatetotheplasma minalglutamicacidbyalaninegeneratedapeptidewith
membrane. They can then interact with secondary the AKVTMTCSAS sequence that was several times
receptors or enter susceptible cells to exert a cytoci- moreactiveandwascalledkillerpeptide(KP).TheKP
β
dal effect (Magliani et al. 1997, Schmitt and Breinig interactedwith -glucanandthisbindingwasinhibited
2006). β-1,6 Glucan, α-1,3 mannoprotein and β-1,3 in a dose dependent manner by laminarin (Polonelli et
glucanarepossiblereceptors,thelatterforkillertoxins al. 2003). The peptide was as active as the killer an-
fromspeciesofPichiaandWilliopsis. Killertoxinskill tibody against a number of microbial pathogens in ad-
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506 ELAINE G. RODRIGUES et al.
dition to C. albicans, and was effective even in normal
and immunocompromised animals against vaginal and
systemic candidiasis (Polonelli et al. 2003), dissemi-
nated cryptococcosis (Cenci et al. 2004) and paracoc-
cidioidomycosis (Travassos et al. 2004a). The KP is
very stable forming dimers in non-reducing conditions
withoutlossofactivity(Maglianietal. 2004a,b).
TheremarkablecytotoxicityofKPwasalsoexam-
ined by electron microscopy. C. albicans cells treated
with KP showed important internal alterations, includ-
ing cell wall swelling with middle electron-dense re-
gion, collapse of the plasma membrane, condensation
andfragmentationofnuclearmaterial,andalterationof
mitochondriastructure(Fig. 1A).Inadividingcellwith
abigvacuoleandchromatincondensationandfragmen-
tation,cellularalterationswereseenbeyondtheseptum
separatingbothcells, withthedaughtercellalreadyaf-
fectedbytheKPshowinganalteredcellwall(Fig. 1B).
AMODELOFDIRECTANTIFUNGALEFFECT
OFAPEPTIDE
Fig. 1–Electronmicrographsshowingthecytotoxiceffectsofthe
Glucans,chitinandmannoproteins,inadditiontoplasma killer peptide (KP) on Candida albicans. (A) Normal untreated or
membranesterols,arenaturaltargetsofantifungaldrugs. treatedwiththeinactivescrambledpeptideC.albicansyeastcell(left)
Additionaltargetsareceramidemonohexosides,ubiqui- ascomparedwiththeKP-treatedyeastcell(right). Majoralterations
tously present on the fungal cell wall and displaying canbeseenastheswellingofthecellwall,plasmamembranecollapse,
severalrolesinfungalcells(Nimrichteretal. 2008). In chromatincondensationandnuclearfragmentation.(B)Anelongated
C.neoformans(Rodriguesetal. 2000),C.albicansand C.albicanscellwithabuddingcell,bothaffectedbyKPtreatment.
Pseudallescheria boydii, these glycolipids were identi- Thesamealterationsasin(A)areseenwithnuclearfragmentationand
fied as targets of human antibodies that inhibited fun- cytoplasmicblebsinvadingthedaughtercellsbeyondtheseptum.
galgrowth. Othertargetsaremelanin,adhesionfactors,
andcellwallenzymes. Thekillerdecapeptide(KP)de- apy with itraconazole, amphotericin or sulfamethoxa-
scribed above was synthesized and engineered demon- zole/trimethoprimareusedinclinicalpractice,relapses
stratingastrongcandidacidalactivityinvitroandcuring are a significant unsolved problem (Travassos et al.
ratvaginalinfectionscausedbyfluconazole-susceptible 2008b). VaccinationagainstPCMisnowaprospective
and-resistantC.albicansstrains(Polonellietal. 2003). goalafterP10,andfourotherpeptidesderivedfromthe
The fungicidal activity of KP in vitro against P. brasi- majordiagnosticantigengp43werefoundtobepromis-
liensis anditstherapeuticactivity invivo havebeenre- cuouslypresentedbyseveralhumanleukocyteantigens
ported(Travassosetal. 2004a). DR, MHC class II molecules (HLA-DR) (Iwai et al.
Paracoccidioidomycosis (PCM) is the prevalent 2003). Such a vaccine could function as an adjuvant
systemicmycosisinSouthAmericawithmostreported tochemotherapysignificantlyreducingthetimeoftreat-
cases in Brazil. It is a major cause of disability and ment(Travassosetal. 2008a,b).
deathamongyoungadultruralworkers. Sequelsarefre- Wide-spectrumantimicrobialpeptides,suchasKP,
quent. The evolution of the disease and the mortality might also be considered as an alternative adjuvant to
burden are influenced by the socio-economic status of chemotherapy, and the projected peptide immunother-
the patients. Although long periods of antifungal ther- apytoshortenthetimeoftreatmentandasanotherop-
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ANTI-FUNGAL AND ANTITUMOR PEPTIDES 507
tion in cases of anergy and drug resistance. Multiply- to the hypervariable domains called complementarity-
buddingyeastcellsof P.brasiliensis hadtheirviability determiningregions(CDRs). Thereare6CDRsinboth
hampered at 39 ng of KP/yeast in distilled water. The variable regions of light (VL) and heavy chains (VH)
D-isomeric form of KP was also active. Further, the with background variability on each side of the CDRs.
decapeptide was therapeutic in B10A mice infected in- The CDRs are named H1, H2, H3 and L1, L2, L3 in
travenously with 3 106 cells of P. brasiliensis Pb18 heavy and light chains, respectively. The framework
×
μ
isolate administered intraperitoneally at 3.3 g/g of sequences between CDRs can be similar or identical.
bodyweight, 1hafterinfectionand1and2dayslater. Although all CDRs are expected to contribute to anti-
Withthisprotocol,nocolonyformingunits(CFUs)were genbindingwithvariableaffinity,onlytheCDR3from
obtainedfromlung,spleenandliverafter8daysoffun- VH when tested as an isolated linear or cyclic peptide
galchallengeintheKPtreatedanimals. Intheseanimals was found to have the same specificity of the original
comparedtothoseinjectedwiththescrambledpeptide, antibody, sharingsomeofitsbiologicalproperties. VH
the liver granulomas were smaller and fewer with no CDR3(H3)peptideswithsuchpropertieshavethusbeen
visiblefungi. Thelungswerelessinfiltratedwithexten- calledmicro(mini)antibodies(Levietal. 1993, Bour-
siveareasofnormalalveoliandnovisiblefungi. Spleens geoisetal. 1998). Theycanevencompetewiththean-
alsowerelittleaffected,withnodetectablefungi. tibodyforbindingtoacertainantigen. TheotherCDRs
It was clear therefore that KP was an effective in- generally do not show a similar reactivity when tested
hibitor of P. brasiliensis in vitro and in vivo (Travassos asisolatedpeptides.
etal. 2004a). Recently we showed, in collaboration with Polo-
α
Itisstillnotclearwhether -1,3glucan,thepredom- nelli’sandPonton’sgroupsfromParmaandBilbao,re-
inant polysaccharide of yeast forms of P. brasiliensis, spectively, that, independently of the specificity of the
isatargetofKP.Thereis,however,evidencethatyeast nativeAb, CDRsotherthanH3maydisplay, withhigh
β
forms may have -glucans at the cell surface. Macro- frequency, antimicrobial, antiviral and antitumor activ-
phages from pentraxin 3 transgenic (PTX3 Tg) mice ities in a way reminiscent of molecules of early innate
showedimprovedopsonin-independentphagocytosisof immunity (Litman et al. 2005). The following mAbs
zymosanparticlesandyeastformsofP.brasiliensis. In were studied as sources of the CDRs: Ab (mAb C7),
the case of P. brasiliensis, an enhanced microbicidal raisedagainstaC.albicansantigen; mousemAbpc42,
activity accompanied by high production of nitric ox- sharing H1 and H2 with mAb C7; and human mAb
idewasobservedinmacrophagesfromtransgenicmice. HuA,sharingnoCDReitherwithmAbC7ormAbpc42,
β
Blockade of dectin-1 receptor for -1,3 glucan inhib- with specificity for difucosylated blood group A. All
itedthephagocytosisofzymosanparticlesbyPTX3Tg mAbs generated CDRs that, represented by synthetic
macrophages, pointing out the relevant role of dectin-1 peptides,showedinvitro,exvivoand/orinvivodifferen-
as the main receptor involved in zymosan and possibly tialantimicrobial(C.albicans),antiviral(HIV-1)and/or
alsoofP.brasiliensisuptake(Dinizetal. 2004). antitumoractivities(Polonellietal. 2008).
CDRsC7/pc42H2andHuAL1weredirectlycyto-
BIOACTIVEPEPTIDESEXPRESSEDAS toxicformelanomaandHL-60(humanleukemia)cells
IMMUNOGLOBULINISOLATEDCDRs causing caspase-dependent apoptosis. H2 peptide ac-
ThediscoverybyPolonellietal. (Polonellietal. 2003, tivity was receptor-mediated in melanoma cells. Both
Maglianietal.2004a,b)thatinternalsequencesofim- C7H2andHuAL1peptidesintheC-terminalamidated
munoglobulin variable regions may display antibiotic formwereactiveagainstlungcolonizationbymelanoma
propertiespromptedustoinvestigatetheactivityofmon- cells by intravenous injection (i.v.). Peptides were ad-
μ
oclonal antibody (mAb) CDRs tested as synthetic pep- ministered by intraperitonial injection (i.p.) (250 g)
tides. Immunoglobulins have polymorphic heavy and every other day for 11 days, starting on the 1st day af-
light chains and the idiotypic variability is related to ter tumor cell challenge. After 22 days and compared
thediversityoftheantigenbindingsiteandparticularly to the untreated control, the number of cancerous nod-
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508 ELAINE G. RODRIGUES et al.
ules in the lungs of peptide treated animals were very Caucasian HLA-DR molecules (Iwai et al. 2003).
few. Presumably, even better results could have been Additionalgp43peptideswerealsoidentifiedusingthe
obtained by optimization of the peptide administration TEPITOPE algorithm, which bound promiscuously to
protocol(Polonellietal. 2008). C7H3butnotC7/pc42 several HLA-DR molecules. As pointed out before
H2competedwithmAbC7forbindingtophosphatidyl- (Travassos et al. 2008a, b) this is an essential property
choline,theprobableligandofpolyreactiveC7(IgM)on of a vaccine peptide candidate considering the genetic
melanoma cells. This CDR (C7 H3) together with the diversityofthetargetimmunizablepopulation.
H3CDRsoftwoanti-melanomamAbs(A4andA4M), In 29 patients with PCM and submitted to
that competed with the antibodies for binding to mel- chemotherapy,79%ofthemrecognizedonepeptidese-
anomacells, werethreeexamplesofmicro(mini)anti- lected by the TEPITOPE algorithm. By pooling pep-
bodiesshowninourlaboratory(unpublishedresults). tides gp4345-59, gp43106-120, gp43181-195 or P10, and
gp43283-298,therecognitionfrequencyincreasedto86%
APEPTIDEVACCINEAGAINST (Iwaietal.2007). Overallfor25CaucasianHLA-DRs,
PARACOCCIDIOIDOMYCOSIS P10 and neighboring peptides were predicted to bind
ThemaindiagnosticantigenofP.brasiliensiswasiden- (TEPITOPE)to90%ormoreofthesemolecules. Very
tified in our laboratory in 1986 (Puccia et al. 1986; re- few healthy individuals had peripheral blood mononu-
viewedTravassosetal. 2004b). Glycoproteingp43re- clear cells (PBMC) proliferating with gp43 and even
acts with 100% sera of patients with paracoccidioido- fewerwithgp43derivedpeptides. Theymayhavebeen
mycosisfromavastregionofSouthAmerica, withthe exposed to P. brasiliensis on a trip to reserve areas of
possible exception of sera from certain Western areas. thefungusorcross-reactedwithrelatedfungalantigens,
β
It elicits an immune response that protects against the possiblyalsoexo- -1,3-D-glucanases. Sitehomologous
intratracheal challenge by virulent P. brasiliensis yeast butunidenticalsequences,incomparisonwithP10,were
β
cells. This molecule has been cloned and sequenced found in -1,3-glucanases from Aspergillus nidulans,
(Cisalpino et al. 1996). Apart from B cell epitopes, Histoplasmacapsulatum,Blastomycesdermatitidisand
which are beginning to be identified, the gp43 carries Lacazialoboi(agp43-likeprotein).
an immunodominant epitope that elicits a predominant The rationale for a peptide vaccine based on P10
IFN-γ-mediated Th-1 response. It is responsible for has been discussed recently (Travassos et al. 2008a).
γ
delayed type sensitive (DTH) reactions in infected ani- Basically: “StimulationofaneffectiveIFN- -producing
mals(RodriguesandTravassos1994). TheT-CD4+cell T-helper response can simultaneously trigger the pro-
epitope was mapped to a peptide called P10 with the duction of potentially protective antibodies and the ac-
QTLIAIHTLAIRYANsequence,theHTLAIRhexapep- tivation of CD8+ T cells in addition to activation of
tide core being essential for priming the immune re- phagocytic cells. In the presence of several immuno-
sponse (Taborda et al. 1998). P10 was as protective genicmoleculesofthefungalagent,stimulationofone
as the gp43 in intratracheal injection (i.t.) challenged armoftheimmunesystemmayalterastateofearlyorin-
mice,beingadministeredi.pwithcompleteFreund’sad- stalledimmunosuppression”. Sincetreatmentoffungal
juvant (CFA). The nucleotide sequence encoding P10 infectionsandparticularlyofPCMinvolveschemothera-
was conserved in a number of isolates (Travassos et peuticdrugs,apeptidevaccinecouldworkasanadjuvant
al. 2004b). to reduce the treatment period, which is usually long,
The T cell epitope in peptide P10 is presented by avoidrelapsesandreversethepotentiallylethalanergic
major histocompatibility complex (MHC) class II mo- cases. It also could help to treat those cases of fungal
leculesfromthreedifferentmousehaplotypes(Taborda drugresistance.
etal. 1998). PromiscuityofP10wasalsoobservedwith Totackletheaboveissueswhileusingexperimental
differentHLA-DRalleles,asthispeptideandaderivative PCMinBalb/cmice,P10immunizationwasassociated
(gp43180-194) without the C-terminal asparagine residue with chemotherapy in i.t. infected animals using two
and with N-terminal lysine bound to nine prevalent protocols. Inthefirstprotocol,infectedmiceweretreated
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ANTI-FUNGAL AND ANTITUMOR PEPTIDES 509
with P10 and/or a chemotherapeutic drug starting after resultssuggestthatP10immunizationcanbeprotective
48hofinfection. Inthesecondprotocol,P10and/ordrug inanergicpatients.
treatmentwasstartedafter30daysofinfection. Itaimed Deliveryofpeptidesforanefficientimmunization
atreproducingaconditionofestablishedinfectionasin hasalwaysbeenaconcernofourgroupbecauseprevious
patientswithPCM.Thetreatmentwasheldfor30days, experimentshavealwaysusedCFAasanadjuvant. The
duringwhichgroupsofmicereceivedi.p. dosesofitra- followingalternativesthereforehavebeeninvestigated.
conazole, fluconazole, ketoconazole, sulfamethoxazole Early studies have shown that immunization of
or trimethoprim-sulfamethoxazole at every 24 h. Am- Balb/cmicewithamammalianexpressionvector(VR-
photericin B was given at every 48 h. P10 was admin- gp43)carryingthefullgeneofgp43withCytomegalo-
istered weekly for 4 weeks, initially in CFA and three virus (CMV) promoter induced B and T cell-mediated
times in incomplete Freund’s adjuvant (Marques et al. immuneresponseswhichwereprotectiveagainstthei.t.
2006). challenge by virulent P. brasiliensis yeast forms (Pinto
Inallcases,therewasanadditiveprotectiveeffect etal. 2000). Thecellularimmuneresponseinmiceim-
withthecombinationofP10immunizationandchemo- munized with VR-gp43 was kept for at least 6 months
therapy. Animalstreatedwithsulfamethoxazoleshowed after immunization. A similar construction with P10
early protection followed by relapse. Significantly, the was made several years later. Immunization with the
association of sulfamethoxazole and P10 successfully P10minigeneinplasmidDNAaloneorassociatedwith
controlled the infection. In the second protocol, the a plasmid carrying mIL-12 insert was tested in Balb/c
fungal burden was examined after 60 and 120 days of mice i.t. infected with a virulent isolate (Pb18) of P.
infection. An additive protective effect of P10 immu- brasiliensis. Asignificantreductionoffungalburdenin
nizationanddrugtreatmentwasalsoobserved,with60 lung, spleen and liver was obtained with production of
γ
to 80% reduction in lung CFUs. Chemotherapy alone IL-12 and IFN- and reduction of IL-4 levels in lung
induced a predominant Th-2 response with increased homogenates(G.Rittneretal.,unpublishedresults).
production of IL-4 and IL-10 detected in lung homo- The construction of MAP (multiple antigen pep-
genates, whereas P10 vaccination stimulated a Th1 re- tide)wasalsotriedtodeliveratetravalentantigencon-
γ
sponse,richinIFN- andIL-12withoutsuppressingthe tainingP10sequence. MAP-10,orM10,hadfourequal
Th-2response(Marquesetal. 2006). Theseareencour- LIAIHTLAIRYAN(QT-lessP10)chainssynthesizedon
aging results in short term experiments. It is probable a branched lysine core. Lymph node cell proliferation
that an increased protective effect will be obtained in fromP10orM10-sensitizedmicewasidenticalwith in
long term trials in which the animals will have time to vitrostimulationwitheitherP10orM10. Immunization
completelyrecoverofthefungalinfection. with single dose of M10 without adjuvant was protec-
Theconditionofanergywasaddressedasfollows. tive with few lung, spleen and liver CFUs and few or
Balb/cmiceweretreatedwithdexamethasone-21phos- noyeastsinlunghistopathologicalsections(Tabordaet
phate added to drinking water. Negative DTH with P. al. 2006).
brasiliensis antigen was obtained after 30 days. Im- In Balb/c mice infected i.t. for 30 days, the pro-
munosuppressed mice (n=10), infected with virulent P. tective effect of P10 was tested alone or mixed with
brasiliensis,begantodie10daysafterinfection,andall adjuvants: alum, monophosphoryl lipid A or complete
animalsweredeadafter70days. Chemotherapyand/or Freund’sadjuvant(Travassosetal. 2008a,b). Unexpect-
P10 immunization of immunosuppressed animals was edly,P10administeredinphosphate-bufferedsalinewas
started15daysafteri.t. infectionandalltreatedanimals mosteffectivewithasignificantreductioninlungCFUs
survivedthereafter. ChemotherapyandP10immuniza- withnofungidetectedinspleensandlivers.
tionconferredadditiveprotection. Asignificantincrease The protective effect of P10 has also been tested
γ
inIL-12andIFN- anddecreaseofIL-4andIL-10were with anti-gp43 mAbs. Anti-gp70 mAbs have been de-
observedinmiceimmunizedwithP10aloneorassoci- scribedasprotectiveagainstexperimentalPCM(Mattos
atedwithantifungaldrugs(Marquesetal. 2008). These Grossoetal.2003). Inpatientsthatunderwentchemo-
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510 ELAINE G. RODRIGUES et al.
therapy,bothgp43andgp70aremarkersformonitoring tides (CAPs), are promising candidates for antitumor
successive treatment and cure through their decreased treatment.
antigenemia and specific antibody response (Marques CAPshavebeenfoundinallspeciesthathavebeen
da Silva et al. 2004, Silva et al. 2004). In the experi- tested so far, including bacteria, fungi, plants and ani-
mentalBalb/cmodelofPCMinfection,anti-gp43mAb mals,andtheyprobablyrepresentoneofthefirstevolved
3Eeffectivelyreducedthefungalburdenandpromoted forms of defense of eukaryotic cells against pathogens
phagocytosis in vitro (Buissa-Filho et al. 2008). The (Zasloff 2002). An updated list of CAPs can be found
recognized epitope in the gp43 was mapped to the inhttp://aps.unmc.edu/AP/main.php,with1,393entries.
sequence NHVRIPIGYWAV shared with Aspergillus MostCAPshaveabroadspectrumofantimicrobialac-
fumigatus,A.oryzaeandB.graminisinternalsequences tivities;only82ofthelistedCAPswereactive,however,
β
of -1,3-glucanases. This peptide could increase the againsttumorcells.
protective effect of P10 in a possible peptide vaccine Despite their diverse origins, antimicrobial pepti-
againstPCM. des have common biophysical parameters, including
Again, as stressed, we quote our own thought ex- smallsize,positivecharge,andamphipathicity,thatare
pressed before (Travassos et al. 2008a): “Short term likely important for peptide activity. These molecules
protocols (30 to 45 days) have the advantage of allow- are grouped according to structural characteristics, and
ing repeated experiments to define a certain response. are usually separated in three classes: (1) linear, often
However, longer periods of treatment and observation formingalpha-helicalstructures;(2)cysteinestabilized,
mayleadtoevenmoreeffectiveresults, aimingatster- beta-sheetstructures;and(3)peptideswithoneormore
ilizationinexperimentalmodelswithmassiveinfection predominantaminoacidresidues, butvariableinstruc-
loads”. ture(Yountetal. 2006). Asstatedbefore,notallCAPs
areabletokillcancercells,andtodate,ithasnotbeen
possible to predict an antitumor activity based on the
ANTITUMORPEPTIDES
peptidestructure.
Cancer remains as a major source of mortality and The short length and cationic/amphipathic proper-
morbidity around the world, despite numerous recent tiesofthesemoleculesenableCAPstointeractanddis-
advances in treatment alternatives. Chemotherapy and, rupt lipid membranes. Positively charged amino acid
morerecently,biochemotherapy,isstillthechoicetreat- residues, such as lysine and arginine, and hydropho-
ment for advanced and metastatic disease (Espinosa et bic residues are frequently found in large numbers in
al. 2003). It is, though, often associated with delete- CAPs (Hoskin and Ramamoorthy 2008). The high ex-
rious side effects caused by drug-induced damage to pressionofanionicmolecules,suchasphosphatidylser-
healthy cells and tissues (Buzaid and Atkins 2001). ineintheoutermembraneleafletofhumantumorcells
Quiescent or slowly proliferating cancer cells are re- (Utsugi et al. 1991, Dobrzynska et al. 2005), as well
fractorytothecytotoxiceffectofdrugsinterferingwith as O-glycosylated mucins (Yoon et al. 1996) on can-
DNA synthesis (Naumov et al. 2003) and, frequently, cercellmembranes,accountforthenetnegativecharge
cellular changes affected sensitivity to chemotherapeu- of these cells and their electrostatic interactions with
tic drugs by increased expression of drug-detoxifying cationic CAPs. In the case of magainin peptides, the
enzymes and/or drug transporters, altered interactions cytotoxicactivityfortumorcellswasabolishedbyelim-
between the drug and its target, increased ability to re- inating the electrical gradient across the plasma mem-
pairDNAdamageanddefectsintheapoptoticpathway brane. Apparently, the cellular potential is critical for
(GattiandZunino2005). Developmentofanewclassof peptidechannelformationintumorcellmembranesand
anticancerdrugsthatlacktoxicitytohealthycellsandare could determine the selective killing of tumor cells by
unaffectedbycommonmechanismsofresistancewould CAPs (Cruciani et al. 1991). The interaction between
beamajoradvanceincancerchemotherapy. Inthissense CAPs and normal cells is not favored because of the
bioactivepeptides,includingcationicantimicrobialpep- overall neutral charge conferred by the zwitterionic
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ANTI-FUNGAL AND ANTITUMOR PEPTIDES 511
major membrane components, such as sphingomyelin, tides can exhibit direct tumor cell cytotoxicity, act as
phosphatidylethanolamine and phosphatidylcholine immunomodulators or as antiangiogenic factors. For a
(Zachowski1993). reviewonthesepeptides,seeDaffreetal. (2008).
CAPsinteractionwithcancercellmembranesisnot
mediatedbyreceptors,sinceD-aminoacidpeptideana- AMODELOFANTITUMOREFFECTOFAPEPTIDE
logues displayed an activity similar to the all-L-amino Gomesin is a CAP isolated from hemocytes of the un-
acidpeptide(Rodriguesetal. 2008,Hetruetal. 2000). challenged Brazilian spider Acanthoscurria gomesia-
Another mechanism for cancer cell killing by na. Itisahairpin-liketwo-strandedantiparallelβ-sheet
CAPs is the induction of apoptosis by permeation of structureformedby18aminoacidresiduesandtwopost-
mitochondrial membrane after internalization, release translationalmodifications,theN-terminalpyroglutamic
of cytochrome c, leadind to caspase 9 and 3 activation acid (Z) and the C-terminal amidated arginine residue
(Pardo et al. 2001). Both cationic and hydrophobic (Silva et al. 2000, Mandard et al. 2002; Table I). A
amino acids play a role in the peptide permeation of rigid conformation is maintained by two internal disul-
mitochondrial membranes (Horton et al. 2008). Alter- fide bridges formed by four cysteine residues, Cys2-15
natively,apoptosismaybeinducedbyCAPsinteraction and Cys6-11, together with six hydrogen bonds in the
withcelldeathreceptors,suchasFasligand,leadingto central part of the molecule, as well as at each end of
caspase8activation. Interestingly,arginine,glycineand the β-sheet (Mandard et al. 2002). The peptide is am-
asparagine,integrinhomingdomain(RGD)-conjugated phypathic,withahydrophobicface(residuesLeu5,Tyr7,
tachyplesin induced both pathways, suggesting that Val12 andTyr14)andthreehydrophilicregionscontain-
some CAPs may have more than one effect on cancer ing positively charged and polar amino acids at the N-
cells(Chenetal. 2001). terminus(Arg3 andArg4),attheC-terminus(Arg16 and
Protein glycosylation may alter the secondary Arg18)andwithinthecanonical β-turn(Lys8,Gln9 and
structure of a membrane-associated protein or peptide, Arg10)(Fazioetal. 2006). Arepresentationofgomesin
and altered glycosylation of membrane proteins is fre- isdepictedonFigure2.
quentlyfoundinmalignantcells. Moreover,differential As stated before, gomesin has a broad and strong
branching and sialic acid content of N-linked glycans microbicidalactivity. ThepeptideisactiveagainstGram-
areassociatedwithanincreaseinthenetnegativecharge positiveandGram-negativebacteria,filamentousfungi,
in the membrane of many cancer cells. Interestingly, yeast(Silvaetal. 2000),Cryptococcusneoformans(Bar-
peptide-glycosylationwasassociatedwithincreasedpo- bosaetal. 2007)andparasites,suchasPlasmodiumfal-
tencyofdrosocininvitro(McManusetal. 1999). Itis ciparumandPlasmodiumberghei(Moreiraetal. 2007).
therefore likely that glycosylation of CAPs and/or can- The antitumor activity of gomesin was tested in
cer cell membrane proteins may influence the binding vitro and in vivo (Rodrigues et al. 2008). Gomesin ex-
affinityofsomeCAPsforthecancercell. erted direct cytotoxic effects on murine and human tu-
CAPs may be used in combination with conven- mor cells in vitro. The estimated IC50 for the murine
tionalchemotherapeuticantitumordrugsinordertore- melanoma cell line B16F10-Nex2 was 3.58 μM, and
duce effective doses, and thereby reduce harmful side- was below 10 μM for human tumor cell lines (Fig. 3).
effectsfrequentlyobservedintreatedpatients. Cecropin Humanendothelialcellswerealsosensitivetogomesin
A, in combination with 5-fluorouracil and cytarabine, in vitro, with an IC50 of 5.30 μM. The cytotoxic effect
showedasynergisticcytotoxiceffectonhumanleukemia wastime-anddose-dependent,andwasnotreversedaf-
cells(Huietal. 2002). terpeptideremoval. Theβ-hairpinstructureandtheam-
RepresentativenaturallyoccurringCAPswithanti- phipathicity of the peptide are important for antitumor
tumoractivitiesaredepictedonTableI. activity,sincesubstitutionofcysteineresiduesbyserine
Peptides with antitumor activities have also been ones(eliminatingoneorbothdisulfidebridges),ordis-
produced and/or identified from other sources, such as ruptionofthehydrophobicface(bysubstitutingresidues
phage-,bacterial-andcell-displaylibraries. Thesepep- Leu5 and/orVal12 byserineunits)reducedorabolished
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512 ELAINE G. RODRIGUES et al.
TABLEI
NaturallyoccurringCAPswithantitumoractivity.
Antitumor
Peptides AAsequence* Source Refs.
Activity
α
-helical
BMP27,BMP28 GRFKRFRKKFKKLFKKLSPVIPLLHL, Bovine Invitro Rissoetal.1998,
GGLRSLGRKILRAWKKYGPIIVPIIRI Cathelicidin- Rissoetal.2002
derived
CecropinA, KWKLFKKIEKVGQNIRDGIIKAG- Insectsand Invitro, Mooreetal.1994,
CecropinB PAVAVVGQATQIAKY mammals Xenogeneic Chanetal.1998,
KWKVFKKIEKMGRNIRNGIVKAG- modelinvivo Winderetal.1998,
PAIAVLGEAKAL Huietal.2002,
Yeetal.2004,
Suttmanetal.2008
LL-37/hCAP-18 LLGDFFRKSKEKIGKEFKRIVQRIK- Human Invitro Okumuraetal.2004,
DFLRNLVPRTES Lietal.2006
Magaininsand GIGKFLHSAKKFGKAFVGEIMNS Frogskin Invitro, Crucianietal.1991,
analogues (magainin2) Xenogeneic Soballeetal.1995,
modelinvivo Takeshimaetal.2003,
(localtherapy) Cruz-Chamoroetal.2006,
Lehmanetal.2006
Gaegurin5, FLGALFKVASKVLPSVKCAITKKC Frogskin Invitro Kimetal.2003,
Gaegurin6 FLPLLAGLAANFLPTIICFISYKC Wonetal.2006
Aurein1.2 GLFDIIKKIAESF Frogskin Invitro Rozeketal.2000
Citropin1.1 GLFDVIKKVASVIGGL Frogskin Invitro Doyleetal.2003
Melittin GIGAVLKVLTTGLPALISWIKRKRQQ Insectvenom Invitro,Invivo TostesonandTosteson1981,
(melittin- KillionandDunn1986,
avidin Sainietal.1999,
conjugate) Holleetal.2003
Epinicidin-1 GFIFHIIKGLFHAGKMIHGLV Fish Invitro Linetal.2009
Polybia-MP1 IDWKKLLDAAKQIL Waspvenom Invitro Wangetal.2008
β
-sheet
Defensins ACYCRIPACIAGERRYGTCIYQGRLWAFCC Human Invitro,Invivo Lichtensteinetal.1986,
HNP-1 CYCRIPACIAGERRYGTCIYQGRLWAFCC xenogeneic Mülleretal.2002,
HNP-2 DCYCRIPACIAGERRYGTCIYQGRLWAFCC model(HNP-1) McKeownetal.2006,
HNP-3 Xuetal.2008
Bovine FKCRRWQWRMKKLGAPSITCVRRAF milk Invitro,Invivo Yooetal.1997a,b,
Lactoferricin Xenogeneic Eliassenetal.2002,
model, Maderetal.2005,
antiangiogenic Eliassenetal.2006
TachyplesinI KWCFRVCYRGICYRRCR Crustacean Invitro, Lietal.2000,
hemocytes Invivo Chenetal.2001,
(RGD- Ouyangetal.2002,
tachyplesin) Chenetal.2005,
Shietal.2006
Gomesin ZCRRLCYKQRCVTYCRGR Insect Invitro,Invivo Rodriguesetal.2008
(localtherapy)
Linear,with
predominantAA
PR-39,Proline RRRPRPPYLPRPRPPPFFPPRLPPRIPP- Porcine Invitro Ohtakeetal.1999
arginine-rich GFPPRFPPRFP cathelicidin-
porcine derived
cathelicidin
*Aminoacid(AA)sequencesaregiveninone-lettercode. BoldindicateCysresiduesthatformdisulfidebonds.
AnAcadBrasCienc(2009)81(3)