Cell penetrating peptides : methods and protocols

627 Pages·2021·14.972 MB·English

by  Ülo Langel (editor)

Methods in
Molecular Biology 2383
Ülo Langel Editor
Cell Penetrating
Peptides
Methods and Protocols
Third Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
Forfurther volumes:
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indexedinPubMed.
Cell Penetrating Peptides
Methods and Protocols
Third Edition
Edited by
€
Ulo Langel
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
Editor
€
UloLangel
DepartmentofBiochemistry
andBiophysics
StockholmUniversity
Stockholm,Sweden
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-1751-9 ISBN978-1-0716-1752-6 (eBook)
https://doi.org/10.1007/978-1-0716-1752-6
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Preface
In this brief introduction to the third edition of the series, I reprint the preface to the
previous edition on cell-penetrating peptides (CPPs) in Methods of Molecular Biology
[1] (see below). It is a short scientific history of CPPs, with key definitions, and is still a
goodstartingpointfor furtherexplorationofthedepthofthefield.Nonetheless,sincethe
secondedition,ourknowledgeofCPPshasexpandedandourmethodologiestostudythem
haveimprovedconsiderably,asrepresentedinthisrecentmonograph[2].Thehighimpact
of CPP utilization as scientific tools and in drug development has been impressively estab-
lished,withover5300scientificpublicationsavailableaccordingtoarecentPubMedsearch
“cellpenetratingpeptide”(March2021).Thisthirdeditionofthebookbringstogetherthe
mostcurrentachievementsinthefieldasacollectionofreviewsandprotocols,samplingthe
mostexcitingtopics.TheseincludeCPPmechanisms,applicationsinvitroandinvivo,CPP
targeting, and peptide or proteinand oligonucleotide delivery. Itis my hope that bringing
together these forty superb contributions from leaders in the field will facilitate the
continuedgrowthofourknowledgeoftheseuniquepeptides.
This book was initiated and written by the contributors during the COVID-19 pan-
demic (2020–2021). I am grateful to all the contributors for their excellent work in this
unusualtime.IamalsogratefultoProfessorJohnRobinsonforcorrectingmyEnglish.
€
Stockholm,Sweden UloLangel
March2021
References
€
1. Langel U (2015) Cell-penetrating peptides. Preface. Methods Mol Biol 1324:v–viii.
https://doi.org/10.1007/978-1-4939-2806-4
€
2. Langel U (2019) CPP, cell-penetrating peptides. Springer Nature Singapore Pte Ltd,
Singapore.ISBN:978-981-13-8747-0
v
Preface to the Second Edition [1]
Inthelate1980sandearly1990s,itbegantobecomeevidentthattheolddogmaasserting
the impermeability of the cell plasma membrane to proteins and peptides was not valid in
multipleimportantcases[3].First,in1988,twoindependentresearchgroupsdemonstrated
theshuttlingpropertiesforanHIVtattrans-activator protein[4,5].Second,in 1991,the
group of Alain Prochiantz reported [6] on cellular internalization of the 60 aa home-
odomain of Antennapedia (a Drosophila homeoprotein), followed in 1994 by the report
on a short 16 aa peptide, pAntp(43–58), later named penetratin, which was necessary and
sufficientfor thistranslocation[7].
These reports are usually considered the starting events for the research field of cell-
penetrating peptides, or CPPs.1 My personal preference tends to also see 1994 as another
defining year with the publication of the first scientific report on 16 aa pAntp/penetratin.
On the other hand, the sequence of pAntp was patented and studied several years before
1994, so the exact date for the CPP field is, hence, more difficult to define by any single
event.
TodeclareastartingmomentforCPPresearchisevenmorecomplicatedifwerecallthat
severalshortpeptideswereearlierreportedtoinduceintracellulareventsinreceptordepen-
dent or independent manner when exposed to cell cultures. It had been speculated even
before the “era of CPPs” that toxins or cell surface receptor ligands should be good drug
candidatesontheirownorasdeliveryvectorsforconnecteddrugs(reviewedforblood-brain
barrier delivery by TfR in [8]). We can refer to examples such as the peptide toxins
mastoparan (from wasp venom), melittin (from bee venom), as well as neuropeptides
bradykinin and substance P, which were demonstrated to activate mast cells receptor
independently by interaction with plasma membranes or initiating G-protein activation
(reviewedin[9]).Recentreports[10]demonstratetheinvolvementofglycosaminoglycans
in cellular uptake of the arginine-rich pituitary adenylate-cyclase-activating polypeptide
(PACAP),anendogenouspeptideneuro-hormone,anditsplasmamembranetranslocation
inaspecificreceptor-independentmanneraswellasitsefficientmediationoftheuptakeof
variouscargoesbeingevenmoreefficientthanTATpeptide.Hence,infuture,theseknown
peptides and hormones might be defined as novel members of CPP family, although it
soundsunconventionalyet.
A comprehensive work was carried out in the 1980s (and perhaps even before) to
characterize these peculiar effects of the short peptides. The many antimicrobial peptides
are other cases where short peptides dose-dependently influence cells by processes such as
forming membrane pores or through interacting directly with intracellular targets such as
DNA, RNA, and components of protein synthesis [11], suggesting their possible use as
drug delivery vectors at nontoxic doses. A recent report on cell-penetrating synthetic
nontoxic antimicrobial peptides gives promise to further development of the field [12].
Moreover, several of these peptides were later connected to different bioactive cargos, and
intracellulardelivery/bioactivityofthesecargoshasbeendemonstratedsuchasinthecases
ofmastoparan[13]orsubstanceP(discussedin[14]).Itseemsingeneralthatseveralcell-
1Alsoknownasprotein/peptidetransductiondomains(PTD),Trojanpeptides,etc.
vii
viii PrefacetotheSecondEdition
penetrating peptides carry more than a single function exemplified by antimicrobial pep-
tides,neuropeptides,orshortproteinmimics.
Obviously,thisrepertoireofinternalizedcargosshouldbeenlargedtoincludesmallor
largefluorescentlabelsconjugatedtoCPPs,currentlyusedoftenastoolsforvisualizationof
internalizationandintracellular/tissuelocalization.Therefore,althoughusuallynotdefined
asCPPs,alltheseshortpeptidescouldbeseenascellpenetratingandthetermCPPmight
needtoberedefinedinorder tocover morepropertiesandfunctions,evenifuncertainties
remain in characterizing the specific translocating mechanisms of CPPs. A definition that
wouldincorporatetheseexpandedpropertieswouldbeasfollows[1].
Cell-penetratingpeptides(CPPs)arerelativelyshortpeptides,4–40aa,withtheabilityto
gainaccesstothecellinteriorbymeansofdifferentmechanisms,mainlyincludingendocytosis,
and/orwiththecapacitytopromotetheintracellulareffectsbythesepeptidesthemselves,orbythe
deliveredcovalentlyor noncovalentlyconjugatedbioactivecargoes.
TheinclusionintheabovedefinitionofCPPs[3]ofthesentence“withthecapacityto
promotetheintracellulareffectsbythesepeptidesthemselves”iscertainlynecessarydueto
multiple demonstrations that short peptides contain motifs that might influence multiple
intracellular interactions. CPP mechanisms are very likely to be multiple as well, including
the spectra from direct cell membrane penetration to endocytotic internalization of the
peptidesorCPP/cargocomplexesandconjugates.Inthelattercase,onemightarguethat
there is little difference as compared to cellular signaling cascades initiated by classical
ligand-receptor interactions. However, I believe that this difference between CPPs and
classicalligandsisobviousduetothedemonstrationsthat,inmanycases,endosomalescape
ofthepeptidesorconjugatedcargoistheprerequisiteforinitiationofintracellulareffects.It
hasbeendemonstratedthatsuchendosomalescapeoccursincasesofCPP-initiateddelivery,
and hence the promotion of endosomal escape seems to be the essential characteristics
ofCPPs.
This leads to an intriguing, and even philosophical question: how does one define/
distinguishthecargoandCPP?SomeshortCPPsareconcealedinthesequenceofaprotein
like in case of pAntp/penetratin or Tat peptides, being also called peptide/protein trans-
ductiondomains(PTD).SuchPTDsare,tomypersonalpreference,animportantsubclass
of CPPs, and they have been an excellent tool in CPP research. However, the existence of
PTDsmakesitmorecomplicatedtodefineaCPP,sincewemustdecidewhether theshort
peptideorthewholeproteinshouldbecalledtheCPP/PTD.Toavoidhavingthedefinition
becomediffuse,wehaveintroducedtherequirementthatCPPsbearelativelyshort,4–40aa
peptide, well understanding that such a definition is somewhat arbitrary, at least for now.
Thelowerlengthofthepeptides,4aa,issetbyanovelcell-permeableantioxidantpeptide,
SS31 [15] and the others; the higher length, 40 aa, is here defined as an arbitrary border
between the peptides and proteins, although everybody knows that such a border is very
diffuse.
Anotherquestionofdefinitionconcernsthecargo.ThestatementthatCPPs“promote
the intracellular effects by these peptides themselves, or ... conjugated bioactive cargoes”
willhopefullycover theproblemswithCPPdefinitionsaswellasenableustoincludeeven
smallfluorescentlabelsasintracellularlyactivecargos.
Ofcourse,thereremainsmuchtodiscoverconcerningCPP-aidedcargodeliveryinvivo.
Critically,theCPPfieldissufferingfromalackofproper understandingofhowtomanage
with in vivo administration of CPP-based drug candidates. Although there are multiple
reports on in vivo administration of different peptides with CPP properties or as delivery
vectorsofbioactivecargos,severalhurdlesexistonthewaytotranslatetheresearchresults
PrefacetotheSecondEdition ix
into clinicaltrials, such astoxicity and degradation problems,optimization of theadminis-
tration, tissue targeting, and others. Careful in vivo studies are underway that seeks to
address these issues. Hopefully, this revised edition of the book will enable us to get a
senseofwherewestandandwillinspirefurtherapplicationsofCPPsindrugdevelopment.
The ideas are discussed for turning CPP-based strategies into drugs. Tumor-selective
targeting with flexible CPP technologies has been fueling the CPP research for years, and
nowthefirstfruitofthesestudieshasbecomeavailable.Additionalorgan-selectivedelivery
strategies are also described, demonstrating that the combination of CPPs with novel
nanoparticles and polymer systems is an efficient method for drug delivery. This point is
underscored by the contributions of authors based at pharma companies who have con-
tributedtheirideasaboutCPPapplicationsindrugdevelopmenttothishandbook.
In summary, the short history of research on cell-penetrating peptides has clearly
demonstrated that CPPs havehelpedus to expand beyond severallong-held dogmas.This
presentsus withsuperb opportunities tostudy innew waysmanyintracellular mechanisms
andpromotethefuturedevelopmentofnoveldrugs.
€
Stockholm,Sweden UloLangel
March2015
References
€
1. Langel U (2015) Cell-penetrating peptides. Preface. Methods Mol Biol 1324:v–viii.
https://doi.org/10.1007/978-1-4939-2806-4
€
2. Langel U (2019) CPP, cell-penetrating peptides. Springer Nature Singapore Pte Ltd,
Singapore.ISBN:978-981-13-8747-0
€
3. LangelU(2011)Cell-penetratingpeptides.Methodsandprotocols.Preface.Methods
inmolecularbiology,vol683.HumanaPress,NewYork,ppv–vi.ISBN:978-1-60761-
918-5
4. FrankelAD,PaboCO(1988)Cellularuptakeofthetatproteinfromhumanimmuno-
deficiencyvirus.Cell55(6):1189–1193
5. Green M, Loewenstein PM (1988) Autonomous functional domains of chemically
synthesized human immunodeficiency virus tat trans-activator protein. Cell 55
(6):1179–1188
6. Joliot A, Pernelle C, Deagostini-Bazin H, Prochiantz A (1991) Antennapedia homeo-
box peptide regulates neural morphogenesis. Proc Natl Acad Sci U S A 88
(5):1864–1868
7. Derossi D, Joliot AH, Chassaing G, Prochiantz A (1994) The third helix of the
Antennapedia homeodomain translocates through biological membranes. J Biol
Chem269(14):10444–10450
8. PardridgeWM(2015)Blood-brainbarrierdrugdeliveryofIgGfusionproteinswitha
transferrinreceptor monoclonalantibody.ExpertOpinDrugDeliv12(2):207–222
x PrefacetotheSecondEdition
9. Mousli M, Bueb JL, Bronner C, Rouot B, Landry Y (1990) G protein activation: a
receptor-independent mode of action for cationic amphiphilic neuropeptides and
venompeptides[seecomments].TrendsPharmacolSci11(9):358–362
10. TchoumiNereeA,NguyenPT,ChatenetD,FournierA,BourgaultS(2014)Secondary
conformational conversionisinvolved in glycosaminoglycans-mediatedcellular uptake
ofthecationiccell-penetratingpeptidePACAP.FEBSLett588(24):4590–4596
11. GuilhelmelliF,VilelaN,AlbuquerqueP,DerengowskiLdS,KyawCM(2013)Antibi-
otic development challenges: the various mechanisms of action of antimicrobial pep-
tidesandofbacterialresistance.FrontMicrobiol4:353
12. Sharma A, Pohane AA, Bansal S, Bajaj A, Jain V, Srivastava A (2015) Cell penetrating
synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of
mycobacterium by targeting its DNA. Chemistry 21(9):3540–3545. https://doi.
org/10.1002/chem.201404650
13. JonesS,HowlJ(2012)Enantiomer-specificbioactivitiesofpeptidomimeticanalogues
of mastoparan and mitoparan: characterization of inverso mastoparan as a highly
efficientcellpenetratingpeptide.BioconjChem23:47–56
14. Oehlke J, Lorenz D, Wiesner B, Bienert M (2005) Studies on the cellular uptake of
substanceP,andlysine-rich,KLA-derivedmodelpeptides.JMolRecogn18:50–59
15. ChoS,SzetoHH,KimE,KimH,TolhurstAT,PintoJT(2007)Anovelcell-permeable
antioxidantpeptide,SS31,attenuatesischemicbraininjurybydown-regulatingCD36.
JBiolChem282(7):4634–4642