肽-寡核苷酸偶联物的化学研究综述-Chemistry of Peptide-Oligonucleotide Conjugates A Review

molecules

Review

ChemistryofPeptide-OligonucleotideConjugates:AReview

KristinaKlabenkova1,2,AlesyaFokina1,2,*andDmitryStetsenko

1,2o

checkfor

updates

Citation:Klabenkova,K.;Fokina,A.;Stetsenko,D.Chemistryof

Peptide-OligonucleotideConjugates:AReview.Molecules2021,26,5420.

/10.3390/

molecules26175420

AcademicEditors:HarriLönnbergandRogerStrömberg

Received:22July2021

Accepted:1September2021

Published:6September2021

Publisher’sNote:MDPIstaysneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffil-iations.

Copyright:©2021bytheauthors.LicenseeMDPI,Basel,Switzerland.ThisarticleisanopenaccessarticledistributedunderthetermsandconditionsoftheCreativeCommons

Attribution(CCBY)license(https://

/licenses/by/

4.0/).

1FacultyofPhysics,NovosibirskStateUniversity,630090Novosibirsk,Russia;k.klabenkova@g.nsu.ru(K.K.);d.stetsenko@nsu.ru(D.S.)

2InstituteofCytologyandGenetics,RussianAcademyofSciences,SiberianBranch,

630090Novosibirsk,Russia

*Correspondence:a.fokina@nsu.ru;Tel.:+7-383-363-4963

Abstract:Peptide-oligonucleotideconjugates(POCs)representoneoftheincreasinglysuccessfulalbeitcostlyapproachestoincreasingthecellularuptake,tissuedelivery,bioavailability,and,thus,overallefficiencyoftherapeuticnucleicacids,suchas,antisenseoligonucleotidesandsmallinter-feringRNAs.ThisreviewputsthesubjectofchemicalsynthesisofPOCsintothewidercontextoftherapeuticoligonucleotidesandtheproblemofnucleicaciddrugdelivery,cell-penetratingpep-tidestructuraltypes,themechanismsoftheirintracellulartransport,andthewaysofapplication,whichincludetheformationofnon-covalentcomplexeswitholigonucleotides(peptideadditives)orcovalentconjugation.ThemainstrategiesforthesynthesisofPOCsareviewedindetail,which

areconceptuallydividedinto(a)thestepwisesolid-phasesynthesisapproachand(b)post-syntheticconjugationeitherinsolutionoronthesolidphase,especiallybymeansofvariousclickchemistries.Therelativeadvantagesanddisadvantagesofbothstrategiesarediscussedandcompared.

Keywords:cell-penetratingpeptide;nucleicacidtherapeutic;antisenseoligonucleotide;smallinterferingRNA(siRNA);peptidenucleicacid(PNA);lockednucleicacid(LNA);phosphordiamidatemorpholinooligomer(PMO);cellularuptake;drugdelivery;clickchemistry

1.Introduction

Thepeptide-oligonucleotideconjugate(POC)isanameusuallyappliedtoasyntheticmoleculeconstitutingoneormoreresiduesofalinearor,lessoften,acyclicpeptidelinkedbyacovalentbondtoanoligonucleotideoritsanalog.Aschimericcompoundsthatincludean(oligo)peptidepartandanucleicacidpart,eachpeptide-oligonucleotideconjugate(POC)representsacombinationofitsparentbiomolecules,suchastheimmanentbase-pairingabilityofnucleicacidsandthemultifacetedbioactivityofthestructurallyandfunctionallydiversepeptides.AlthoughthecompoundsrelatedtoPOCsoccurinnatureasnucleopeptides[

1

–

3

],thisreview,asitisfocusedonthechemicalmethodsofconjugatingpeptidestooligonucleotides,willbenecessarilylimitedtosyntheticsubstancesonly.

Theinterestinpeptide-oligonucleotideconjugateswassparkedbytheadventofantisensetechnology[

4

],followedbythedevelopmentofthefirstgenerationoftherapeuticoligonucleotidesattheendofthe1980s[

5,

6]

.Afteraperiodofresearch,itwasgenerallyacceptedthatasuccessfulnucleicaciddrugoughttodemonstratebettercellularuptakethanwhatthemajorityoftheexploredto-dateoligonucleotidechemistriescanoffer[

7,

8]

.Thisunderstandingcoincidedwiththeserendipitousdiscoveryofwhatwaslatertobecalledcell-penetratingpeptidesinthemid-1990s[

9]

.

Clinicalapplicationoftherapeuticoligonucleotidesofficiallystartedin1998,whentheUSFoodandDrugAdministration(FDA)approvedthefirstnucleicaciddrugfomivirsen(Vitravene®)[

10

]forthetreatmentofcytomegalovirus-inducedblindingretinitisinAIDSpatients[

11]

.AftertheseminalworkonRNAinterference(RNAi)[

12

],ittookover20yearsforthefirstsmallinterferingRNA(siRNA)therapeuticpatisiran(Onpattro®)toappear[

13]

.Todate,theprogressinnon-clinicalandclinicalstudieswithsyntheticoligonucleotides

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haveacommonfeatureinthemechanismsoftheiraction,whichiscomplementarybasepairing[

68]

.

2.1.AntisenseOligonucleotides(ASOs)

Historically,antisenseoligonucleotides(ASOs)weretheearliestand,currently,thebest-studiedclassofnucleicacidtherapeutics.TheconceptofASOsoriginatedin1978,whenZamecnikandStephensondemonstratedthataspecific13-meroligodeoxynucleotideinhibitedRoussarcomavirusreplicationinchickenembryos[

4]

.ThemechanismofthetherapeuticeffectofASOsrestsontheabilityofsyntheticoligonucleotidesortheiranaloguestobindtoacomplementaryRNAthroughthecanonicalWatson–CrickduplextoalterthemetabolismofthecorrespondingRNAinoneofthefollowingways(Figure

1)

.

Figure1.Theaspectsoftheantisensemechanism.

AmoregeneralwayforASOstointerferewithRNAfunction,e.g.,theinitiationorelongationoftranslationofanmRNA,istophysicallyshieldaspecificfragmentofaregulatoryregionoftheRNA,e.g.,thetranslationinitiationsite,byformingaduplexwithASOs(stericblock)[

69

–

72]

.ThisapproachisparticularlyapplicablewhenoneneedstopreservethefunctionalRNA,e.g.,inthecaseofsplicingredirectionofapre-mRNAbyasplice-switchingoligonucleotide[

73

–

75]

.AnotherwayistoactivateenzymaticRNAdigestionbyrecruitingacellularRNase,mostcommonlyRNaseH[

76

],tohydrolyzetheRNAstrandoftheASO-RNAduplex[

77]

.

ThefirstASOstobeinvestigatedwerenativeoligodeoxynucleotides(Figure

2,

1a)thatprovedtoberapidlydigestedbynucleasesintheserumunlessprotectedbyatleastminimalchemicalmodification[

78,

79]

.Thus,unmodifiedoligonucleotidesprovedtobeunsuitableforinvivoapplications.Forthisreason,arangeofchemicalmodificationswereintroducedintoASOstorendertheprospectiveoligonucleotidetherapeuticssuf-ficientlyresistanttoenzymatichydrolysisoftheinternucleotidicphosphodiesterbond(Figure

2)[

80]

.Therefore,thefirst-generationASOsmaybesaidtoincorporatethemodi-fiedphosphatelinkages,suchasphosphorothioate(1b)[

81

],methylphosphonate(1c)[

82

],morerarelyphosphorodithioate(1d)[

83

]andboranophosphate(1e)[

84

],andrecentlyreportedmesylphosphoramidate(1g)[

85,

86

],aswellasmanyothers[

87,

88]

.AnothergroupofASOsconsistsofoligonucleotideswithmodificationsintheriboseringthatnotonlyofferavaryingdegreeofprotectionagainstnucleasesbut,evenmoreimportantly,increasethestabilityoftheASO-RNAduplex[

89

–

91

],notably2,-O-methyl(2b)[

92

–

94

],2,-O-(2-methoxy)ethyl(MOE)(2c)[

95,

96

],2,-deoxy-2,-α-fluoro(4)[

97

],and,especially,con-

Molecules2021,26,54204of36

strainedriboseanaloguessuchasbridged/lockednucleicacids(B/LNAs)(3)[

98

–

101

]andtricyclo-DNAs(5)[

102]

.AseparateclassofASOsencompassesoligonucleotideanalogs,inwhichthenaturalribose-phosphatebackboneisreplacedbyasuitablesurrogate;typ-icalexampleswouldbepeptidenucleicacids(PNAs)(6)[

103

]andphosphordiamidatemorpholinooligomers(PMOs)(7)[

104,

105]

.Thelatter,inparticular,gaverisetothethreesplice-switchingoligonucleotidedrugsforthetreatmentofDuchennemusculardystrophyapprovedbytheFDAin2016-2021:eteplirsen(Exondys51®)[

106

],golodirsen(Vyondys53®)[

107

],andcasimersen(Amondys45®)[

108]

.

Figure2.Oligonucleotidesandtheiranalogs:(1a)nativeDNA,(1b)phosphorothioate,(1c)methylphosphonate,(1d)phosphorodithioate,(1e)boranophosphate,(1f)mesylphosphorami-date,(2a)nativeRNA,(2b)2,-O-methylRNA,(2c)2,-O-(2-methoxy)ethylRNA,(3)bridged/lockednucleicacid(B/LNA),(4)2,-α-fluoroDNA,(5)tricyclo-DNA(tcDNA),(6)peptidenucleicacid(PNA),and(7)phosphordiamidatemorpholinooligomer(PMO).

2.2.SmallInterferingRNAs(siRNAs)

SmallinterferingRNAs(siRNAs)are(usually)double-strandedoligoribonucleotides(asinFigure

2,

2a)withalengthof20–25ntperstrand,whichwerefoundinplantsin1999[

109]

.Theyearbefore,FireandMellodiscoveredanaturalprocessofspecificgenesilencingtermed“RNAinterference”(RNAi)thatwasmediatedbyshortdouble-strandedRNAs(includingsiRNAs)viaamechanismthatisnotablydifferentfromtheantisensemechanism(theNobelPrizeinPhysiologyandMedicineof2006)[

12]

.Later,TuschlandcoworkersdemonstratedthatsyntheticsiRNAsareabletoinduceRNAiinmammals[

110]

. AtypicalsiRNAhasdinucleotideoverhangsatthe3,-endofeachstrand.OnestrandthatiscomplementarytoaspecificregionofthetargetmRNAisusuallycalledtheantisensestrand,whiletheotheroneiscalledthesenseorpassengerstrand[

111]

.Innature,thisstructureresultsfromtheactionoftheDicerenzyme,whichcleaveslongdouble-strandedRNAsorshorthairpinRNAsintosiRNAduplexes(Figure

3)[

112]

.Then,intheRNA-inducedsilencingcomplex(RISC)withtheparticipationoftheArgonautproteinAgo2,thesiRNAduplexisunwound,andthecomplementaryduplexoftheantisensestrandwith

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theconcomitantmRNAisformed,followedbydegradationofthelatter.ThisresultsinpotentexpressiondownregulationforthecorrespondinggeneviatranslationarrestatthemRNAlevel,similarlytothatoftheantisensemechanism(Figure

3)

.

Figure3.ThemechanismofRNAinterference(RNAi)mediatedbysmallinterferingRNAs(siRNAs).

Astheoriginandprogressionofmanydiseasesareassociatedwithupregulationofaparticulargene,theuseofsyntheticsiRNAsfortherapeuticgenesilencingisofgreatinterest[

113]

.However,siRNAdeliverytospecifictissues,withthenotableexceptionoftheliverviatherespectiveGalNacconjugates[

114

],remainsanobstacleonthewaytotheclin-ics.Nevertheless,therecentFDAapprovaloftwomoretherapeuticsiRNAs(apartfromthepioneeringpatisiran),givosiran(Givlaari®)[

115

]andlumasiran(Oxlumo®)[

116

],aswellasonemoreapprovedbytheEuropeanMedicinesAgency(EMA),inclisiran(Leqvio®)[

117

],isindicativeofthegreatpromiseofferedbythisparticularareaofdrugdevelopment.

2.3.CRISPR/Cas9

Theclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)werefirstdiscoveredinE.coliin1987[

118

],buttheirdetailedstudyonlybeganin1993byFranciscoMojica[

119]

.Later,Jansenetal.investigatedthatneartheCRISPRlocus,thereisalwaysasetofhomologousgenescalledCRISPR-associatedsystemsorCasgenesthatencodeendo-orexonucleases[

120]

.AlthoughCRISPR/Cassystemswerefoundinalargenumberofprokaryotes,almostnothingwasknownabouttheirfunctionuntil2005,whenMojicaetal.publishedapapershowingtherelationshipofCRISPRlociwithadaptiveimmunityinprokaryotes[

121]

.Severalfurtherstudieshaveshownthatbetweenrepeatsinloci,therearedifferentDNA“spacers”correspondingtopartsoftheviralgenomescorrespondingtopastparasitesofthesebacteria[

122]

.Thus,spacerscarryinheritedmemoriesofpastcellularinvasions.CRISPRRNA(crRNA)istranscribedfromthesespacersanddirectsCasproteinstotheforeignviruses,causingthecleavageoftheforeignDNA[

123]

.Inaddition,ithasbeenshownthatCasproteinsneedaspecialsequencelocalizednearthetargetDNA,calledaprotospaceradjacentmotif(PAM),forrecognitionandbindingtothetarget[

124]

. FromallthevarietyofCRISPR/Cassystems,scientistsweremostinterestedinthetypeIIsystemfromStreptococcuspyogenesfortherapeuticapplicationingeneticengineering,sinceonlyoneCas9proteinisrequiredforitsfulloperation[

125]

.InadditiontoCas9,thissystemrequiresthepresenceofcrRNAandtrans-activatingCRISPRRNA(tracrRNA)[

126

],whichtogetherformaduplexthatdirectsCas9endonucleasetothetarget.Later,DoudnaandCharpentierwithcolleaguesdesignedasystemthatincludedonlytwoelements,Cas9andchimericRNAcombinedfromtwomoleculescrRNAandtracrRNA,calledasingle-guideRNA(sgRNA)[

127]

.Withsuchasystem,itbecamepossibletodirectCas9toanyDNAsequenceforitscleavageonlybychangingthenucleotidesequenceofsgDNA.

TheworkwasdeemedsosignificantthatitwasawardedaNobelPrizeinChemistryin2020.ThepossibilityofusingtheCRISPR/Cas9systemineukaryoticcellshasbeendemon-strated[

128

–

130]

.Itwasalsoshownthatineukaryoticcells,afterCRISPR/Cas9-mediateddouble-strandedDNAbreaks,theDNAmoleculeisnotdegraded,butratherrepairedbytwomainpathways,namelynon-homologousend-joining(NHEJ)andhomology-directedrepair(HDR)[

131]

.HDRispreferredbecauseitallowsthedesirednucleotidesequencetobeobtainedbyusinganexogenoustemplateasarecombinationdonor.Currently,manyvariantsoftheCas9proteinhavebeendeveloped[

132

–

134]

.

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Today,inmostcases,aclinicalapplicationofCRISPRisbasedonexvivogeneeditingofcellswiththeirsubsequentre-introductionintothepatient[

132]

.Theexvivoeditingapproachishighlyeffectiveformanydiseases,includingcancerandsicklecelldisease.Inturn,invivoeditingislargelylimitedbythelackofavailabilityofthetargettissueororgan.Despitethis,recentlyaCRISPR-modifiedviruswasinjectedintothepatient’seyeinanattempttotreatLebercongenitalamaurosis[

133]

.However,beforewidespreadapplicationofCRISPRtechnologyinclinicalpractice,itisnecessarytocarryoutmanymoreexperimentstomakefinalconclusionsontheeffectivenessandsafetyofthismethodinvivo.

2.4.TheProblemofOligonucleotideDelivery

Incontradistinctiontosmall-moleculedrugs,oligonucleotidesaremacromolecules,andtheirphysicochemicalproperties,inparticular,theirpolarityandpolyanionicnatureoftheribosephosphatebackbone,essentiallypreventpassivediffusionthroughthephospho-lipidbilayerofabiologicalmembrane.Thus,overcomingaproblemofselectivedeliveryofanucleicaciddrugtotherightorgan/tissueaftersystemicorlocaladministration,followedbyefficienttransportintothespecificcellsand,onceinsidethecell,translocationtothecorrectcellularcompartmenttofinditsmoleculartarget,isakeystoneofoligonucleotide-basedtherapy.OnthewaytobindauniqueRNA,theoligonucleotideoughttocrossanumberofextracellularandintracellularbarriers,whichhavebeenextensivelyreviewedbyJulianoandcoauthors[

134

–

137

]andothers[

138]

.

Itisbelievedthatoligonucleotidesaretakenupintocellsviareceptor-mediatedendo-cytosis[

139]

.Therefore,thereisaneedforanoligonucleotidetherapeutictoescapefromendosomesintothecytosoltotriggerRNAi(forsiRNAs),orreachthenucleusforsplice-switchingandRNaseHactivation[

140,

141]

.Allthewayfromtheinitialadministrationtotheultimatesiteoftherapeuticactivity,theoligonucleotidemaybeattackedbyvariousexo-andendonucleases[

142

–

144]

.Thesearethemainobstaclesonthewaytothesuccessfulclinicalapplicationoftherapeuticoligonucleotides.

Thereby,itbecomesanimportanttasktodesignspecialdeliveryvectorsfortheeffectivetransportofnucleicaciddrugsintothecytosolandnucleus.Viral,e.g.,adenoviral,vectorshavebeendevelopedasspecificcarriersfornucleicacidsforgenetransferandgenetherapy[

145]

.However,despiteseveralapprovedto-dategenetherapies[

146,

147

],therearestillconsiderablelimitationsduetoimmunogenicityandsafetyconcerns.Mainly,theapplicationofaviralvectortodelivercargotohumancellsinducesanimmuneresponse.Thus,repeatedadministrationofthesameviralconstructsbecomesuseless[

148]

.

Thus,non-viralvectorshavereceivedwidespreadattentionasanalternativedeliverystrategythatcouldensuresafe,efficient,andaddressableoligonucleotidedelivery.Thenon-viralmethodstraditionallyincludetheuseofliposomes[

149

],polymers,dendrimers[

150

],inorganicnanoparticles,orconjugationtocertainsmallmolecules[

151]

.Amongtheabove,cell-penetratingpeptideshavebecomeoneofthemostpromisingcarrierstohelpoligonu-cleotidestotranslocatethroughcellularbarriersviaeithercovalent(peptideconjugate)ornon-covalent(peptideadditive)association.

3.Peptide-MediatedCellularDelivery:ABriefOverview

Theterm“cell-penetratingpeptide”(CPP)wasintroducedbyLangelandcoau-thors[

152

]andusuallyreferstoashort-tomedium-sizepeptidecontainingbetween5and40aminoacids.ACPPcanpassthroughcellmembranesthroughenergy-dependentorenergy-independentmechanisms,andmoreover,itcanfacilitatetheintracellulartrans-portofvariouscargomolecules,whicharepoorlyabletocrossthemembranesalone,suchasother(non-cell-penetrating)peptides,proteins,nanoparticles,ornucleicacids[

153]

.

ThefirstCPPwasdiscoveredover30yearsagoattheendofthe1980s.Tworesearchgroups,whenstudyingtheactivityofthetransactivationtranscriptionactivator(Tat)domainofHIV-1,independentlynoticedthatitcanbeefficientlyinternalizedbycellsinvitro

[154,

155]

.Afewyearslater,theProschiantzgroup,whenstudyingtheroleof

Molecules2021,26,54207of36

Drosophilahomeodomainproteinsinpost-mitoticneurons,discoveredthata60-amino-acidhomeodomainproteinsequenceoftheAntennapediagenewasabletocrossbiologicalmembranesbyanenergy-independentpathway.ThediscoveryledtothestudyoftheabilityofaseriesofsyntheticpeptidesderivedfromthethirdhelixoftheAntennapediahomeodomaintobeinternalizedbycells.Inparticular,itwasshownthata16-merpeptidenamedpenetratin(pAntp)successfullytranslocatedintocells,whileshorterpeptideswerenotinternalized[

156]

.

Later,LebleuandcoauthorsprobedthesequenceofTatproteintoascertainwhichsequencemayberesponsibleforitscellularuptake.Toachievethis,severalpeptidesfromresidues37–60oftheTatdomainweresynthesized.Asaresult,ashorterversionofTatpeptide13aminoacidsinlength,locatedfromaminoacids48to60,wasidentifiedasnecessaryforpenetrationintocells[

157]

.

In1998,thesuccessfulapplicationofpAntpforinvivodeliveryintoBowescellsof21-merPNAblockingtheexpressionofthegalaninreceptorwasdemonstrated[

158]

.Oneyearlater,theTatpeptidewasusedforinvivodeliveryofβ-galactosidase[

159]

.Thesestud-iesdemonstratedthepotentialofCPPsfortheinvivodeliveryofcargomacromolecules,whichisbeingextensivelystudieduptonowtotransportoligonucleotides,theiranalogs,andotherdifficult-to-deliverpotentialtherapeuticsacrosscellularmembranes[

160,

161]

.

4.Cell-PenetratingPeptides(CPPs):TypesandExamples

Atdifferenttimes,variouscriteriabasedonthesequence,function,orpenetrationmechanismhavebeenproposedforclassificationofCPPs.However,thereiscurrentlynosingletaxonomyofthesepeptides.TherearetwoCPPclassificationsintheliter-ature:onethatisbasedontheoriginofpeptidesandtheotheronebasedontheirphysicochemicalproperties.

Bytheirorigin,thepeptidesareclassifiedintoprotein-derivedones,suchasTatorpenetratin;synthetic,suchaspolyarginineR8;andchimeric,whicharecombinedfrompeptidefragmentswithdifferentproperties,suchastransportan.ThistypeofclassificationisnotquiteconvenientandismostlyhistoricalbecauseitdoesnotallowonetoevaluateCPPsfromthepointofviewoftheirinteractionwithcells.

Accordingtotheirphysicochemicalproperties,CPPsarebroadlydividedintothreemainclasses:cationic,amphipathic,andhydrophobicpeptides.

4.1.PolycationicCPPs

Polycationicpeptides,asthenamesuggests,consistpredominantlyofpositivelychargedaminoacidresidues,suchasArg,Lys,His,or,morerarely,Ornandothers.Thispolycationicnatureofpeptidesallowsthemtobeeffectivelyinternalizedbycells.OneofthefirstpolycationicpeptidescanberightfullyconsideredtheTatpeptide,whichcontainsthearginine-richRKKRRQRRRsequence.Anumberofstudieshavebeencarriedouttodeterminetheoptimalcompositionandamountofpositivelychargedaminoacidresidues.Thus,itwasfoundthat,first,peptidesrichinLys,His,orOrnresiduesarelessefficientlyabsorbedbycellsthanpeptidesrichinArg[

162]

.ThiscanberationalizednotonlybyahigherpKaofguanidinegroupsofarginine(pKaofca.13)butalsobytheirabilitytoformbidentatehydrogenbondswithnegativelychargedcarboxyl,sulfate,andphos-phategroupsofthecompoundspresentinthecellularmembrane,suchasphospholipids,acidicpolysaccharides,andproteins[

163]

.Second,theminimumrequiredamountofArgresiduesisnotlessthan6,buttoensureeffectivecellularuptake,theoptimalamountis8–10residues[

164]

.MostofthepolycationicCPPsareofnaturalorigin(Tat,penetratin),butsyntheticCPPshavealsobeendevelopedandincludeargininehomopolymers,pep-tidesofthePipseriesdevelopedbytheGaitgroup,andothers[

52

](moreexamplesinTable

1)

.

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4.2.AmphipathicCPPs

TheamphipathicclassisthemostextensiveamongallCPPs(about40%)[

165]

.Inadditiontopositivelychargedhydrophilicregions,amphipathicpeptidesalsocontainhydrophobicregionsrepresentedbyvaline,leucine,isoleucine,andalanineresidues[

166]

.DespitethefactthatmostamphipathicCPPsarechimericorsynthetic,therearealsorepre-sentativesderivedfromnaturalproteins.TheamphipathicCPPclassissubdividedintothreesubclasses:primary,secondary,andproline-richCPPs.Often,primaryamphipathicCPPsarechimericpeptidesobtainedbycovalentlybindingadomainconsistingofhy-drophobicaminoacids(necessaryforefficienttargetingofcellmembranes)withanuclearlocalizationsignal(NLS).AnNLSisashortcationicpeptidebasedonlysine,arginine,orproline-richmotivesdirectingpeptideconjugatestothecellnucleusthroughnuclearpores.RepresentativesofthissubclassareMPGpeptides[

167

]andPep-1[

168

],peptidesconsistingofahydrophilicpartNLSfromthelargeT-antigenofthesimianvacuolatingvirus40(SV40)andhydrophobicpartsglycoprotein41(gp41)ofthehumanimmunod-eficiencyvirus(HIV)oratryptophan-richcluster,respectively.NaturalrepresentativesofthissubclassaretheARF(1–22)peptidecorrespondingtotheN-terminaldomainofthetumorsuppressorproteinp14ARF[

169

],BPrPp(1–28)andMPrPp(1–30)derivedfromprionproteins[

170,

171

],andothers(formoreexamples,seeTable

1)

.SecondaryamphipathicCPPsusuallyhaveα-helicalconformationwithhydrophilicandhydrophobicresiduesgroupedonoppositesidesofthehelix.Examplesofsuchpeptidesarethemodelamphipathicpeptide(MAP)[

172

],transportan[

158

]oritsanalogueTP-10[

173

],CADYdesignedbycombinationaromatictryptophanandcationicarginineresidues[

174

],andothers.Itshouldbenotedthatamongthesecondaryamphipathicpeptides,therearealsoanionicrepresentatives,suchasanionicp28obtainedfromazurin[

175,

176]

.Thelasttypeofamphipathicpeptidesisproline-richCPPs.Duetoitssecondaryaminogroup,prolinecannotserveasadonorofahydrogenbondforeithertheα-helixortheβ-fold.Suchpeptidesusuallyformaleft-handedpolyprolineIIhelix(PPII).Anexampleofproline-richpeptidesisasyntheticderivativeofBac7(afragmentofantimicrobialproteinfromthebactenecinfamilycontaining59aminoacids,withfour14-merrepeats);thefunctionsofcellpermeabilityandantimicrobialactivityofBac7areconcentratedin24aminoacids(Bac1–24)[

177,

178]

.Otherexamplesaresyntheticproline-richpeptides(PPR)nand(PRR)n,wherenisintherangeof3to6[

179]

.

4.3.HydrophobicCPP

HydrophobicCPPsconsistofnon-polarorlow-chargedaminoacidresiduesandarethesmallestclassofCPPs.Themechanismsoftheircellularpenetrationarenotfullyunderstoodbutapparentlyoccur