Strategies for gastrin releasing peptide recepror targeted imaging in prostate cancer Carlucci, Giuseppe
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Carlucci, G. (2014). Strategies for gastrin releasing peptide recepror targeted imaging in prostate cancer.
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Chapter(2:(
( (
Multimerization(improves(targeting(of(peptide(
radio8pharmaceuticals(
(
(
!Giuseppe'Carlucci' 'Hildo'J.K.'Ananias' 'Zilin'Yu'' Christophe'Van'de'Wiele'' Rudi'A.J.O.'Dierckx'' Igle'J.'de'Jong' Philip'H.'Elsinga(
' (
' ' ' ' ' ' ' ' '
Current!Pharmaceutical!Design!2012;18(17):2501<16!
32
Abstract(
Multimerization' offers' unique' kinetic' and' thermodynamic' properties' to' molecules.' Multimeric' ligands,' characterized' by' multiple' similar' or' different' monomeric' molecules' tethered'together,'can'bind'several'receptors'simultaneously.'Multimerization'occurs'also' in' nature.' This' process' can' be' used' to' develop' molecules' with' high' diagnostic' and' therapeutic' value.' By' altering' parameters' as' linkers`' length' and' flexibility,' scaffold' and' backbones' insertion,' and' ligandsRreceptors' recognition,' it' is' possible' to' provide' high' selectivity' and' binding' affinity.' The' resultant' multimeric' ligand' has' a' more' favorable' binding'affinity'than'corresponding'monomeric'ligands.'
INTRODUCTION(
Multimerization' is' realized' by' linking' multiple' copies' of' a' specific' molecule' to' linear' or' dendritic' peptides' or' even' to' polymers,' liposomes' and' nanoparticles.' The' goal' of' this' multimerization'is'to'increase'the'binding'affinity'of'the'peptide'to'its'target.'The'process' of'multimerization'can''be'observed'in'nature'if'we'consider'phenomena'such'as'antibody' recognition,'the'DNARDNA'duplex'formation'by'interacting'nucleotides,'the'presentation' of'numerous'peptides'by'a'virus'or'the'binding'of'one'molecule'or'particle'with'numerous' sites' onto' a' target' (1R4).' Multivalency' as' key' in' some' biological' interactions' is' the' common' term' to' describe' multiple' interactions' (1);' (5R6).' The' binding' selectivity' of' a' multimer'is'strongly'connected'to'different'parameters'as'its'overall'size,'length,'flexibility' of'the'linker'between'ligand'and'target'and'the'density'of'the'receptor'(7).'
This' review' article' will' focus' on' the' influence' of' multivalency' on' biological' and' biochemical'interactions.'By'discussing'examples'of'multivalent'interaction'in'comparison' to' monovalent' interactions,' we' will' outline' thermodynamic' and' kinetic' aspects' of' multimerization' and' discuss' the' potential' role' of' multimerization' in' the' construction' of' cell'membrane'receptor'targeted'imaging'agents.'
' '
33
MULTIMERIZATION(IN(NATURE(
In' nature,' the' concept' of' multimerization' of' peptides' has' been' established' for' several' processes.'Multivalency'is'observed'not'only'in'binding'DNA'motifs'that'can'cluster'or'in' sugars'that'can'be'linked'to'form'oligosaccharides,'but'also'in'proteins'and'peptides'(8);' (10).' Multimerization' also' plays' a' key' role' in' ' immunological' phenomena' as' viral' infections'(11).'By'multimeric'interactions'some'viruses'can'recognize'and'attach'to'more' than' one' receptor' on' the' cell' surface.' Multivalent' binding' of' a' virus' serves' viralR membrane'fusion,'for'example'by'the'influenza'virus'invading'bronchial'epithelial'cells'by' interaction'between'multiple'trimers'of'hemagglutinin'(antigenic'glycoprotein)'and'sugar' residues' of' sialic' acid.' This' concept' also' concerns' other' viruses' such' as' the' Human' Immunodeficiency'Virus'type'1'(HIVR1)'and'the'Hepatitis'C'virus'(HCV).'Both'attach'to'the' host'target'cell'by'multimerization'of'core'proteins'during'the'initial'stage'of'the'infection' (11R12).'The'adhesion'occurs'by'changes'in'the'virus'attachment'proteins'(VAPs)'or'within' the' receptor.' By' an' additional' VAP' or' receptor' this' processes' probably' lead' to' an' increased' binding' affinity.' The' soRcalled' secondary' binding' is' probably' the' result' of' multivalent'binding'to'multiple'VAPs.'Multimerization'is'also'involved'in'the'adhesion'of' bacteria'to'the'cell'surface'and'in'mediating'the'intercellular'binding'process.'This'process' is' facilitated' by' proteinRcarbohydrate' interactions,' so' multivalent' binding' can' enhance' proteinRcarbohydrate'interactions'(13).'
Many' classes' of' antibodies' use' multivalency' to' recognize' antigens' and' ligands' on' the' surface' of' pathogens.' Antibodies' increase' their' binding' affinity' by' repeated' or' multiR epitopes.' This' results' in' an' increased' avidity' with' the' receptor.' This' multivalency' is' encountered' in' a' broad' spectrum' of' processes' such' as' the' formation' of' the' immuneR synapse' at' the' B' cellRT' cell' junction' and' the' activation' of' signal' transduction' pathways.' Multivalency'also'occurs'in'many'microscopic'entities'such'as'atoms,'molecules,'proteins,' cells'and'several'types'of'nanoparticles.'The'binding'chemistry'of'two'molecules'of'oxygen' or' nitrogen' is' considered' as' the' most' simple' example' of' multivalency' (14).' The' multivalent' interaction' can' be' clarified' by' the' soRcalled' “chelate' effect”.' This' demonstrates'that'the'affinity'of'chelating'ligands'for'a'metal'ion'is'higher'in'comparison'
34
to'that'of'a'nonchelating'monodentate'ligand.'In'other'words,'it'refers'to'the'advantage' of'the'binding'between'a'doubleRended'ligand'(bidentate'binding)'such'as'ethylendiamine' ligand'and'a'monodentate'separate'ligand,'such'as'methylamine.'In'chelate'binding'the' free'energy'will'be'higher.'This'means'that'the'formation'of'a'chelateRcomplex'between' ligand' and' correspondent' substrate' forms' a' favorable' enthalpic' and' thermodynamic' complex.' A' bidentate' ligand' may' have' twice' as' much' free' energy' compared' to' a' monodentate' analog.' Multivalent' binding' increases' the' avidity' of' the' binding' process' (15).'This'changes'the'grade'of'interactivity'and'uptake'in'the'ligandRreceptor'association.'' Increasing'the'number'of'cyclic'RGD'moieties'from'monomers'to'multimers,'Wängler'et' al.' have' demonstrated' avidity' differences' (16).' They' used' radiolabeled' cRGD' containing' the' sequence' RGD' (arginineRglycineRaspartic' acid)' for' imaging' of' integrin' αvβ3.' Their' in!
vitro'experiments'of'immobilized'αvβ3'integrin'in'U87MG'glioblastoma'cell'lines'showed'a' 100Rfold'increase'in'avidity'by'multimeric'RGD'compared'to'the'monomeric'cyclic'RGDfC' sequence.''
GENERAL(ASPECTS(
Theory(of(multimerization(
'
Multivalent'interactions'can'be'of'two'types:'intramolecular'and'intermolecular.'To'define' geometries'and'structures'with'a'relevant'role'in'multivalency,'combinations'of'intraR'and' intermolecular'binding'pathways'for'multivalent'entities'have'been'studied'and'classified' according'to'kinetics'and'thermodynamics'aspects'of'the'interactions'(Fig'1).((
Whitesides' et! al.' have' described' the' contribution' of' ' enthalpy' (∆H0)' and' entropy' of' binding'(∆S0)'in'multivalent'interactions'(1).'Mulder'et!al.'analyzed'the'architecture'of'a' multivalent'and'multimeric'structure'such'as'core'concept'in'evaluating'the'strength'and' mode' of' binding.' Multivalency' allows' assembling' stable' constructs' at' very' low' concentration'(5).'
'
35
'''''''''' '
'
Figure( 1:( Schematic' representation' of' multimeric' binding' pathways' for' multivalent' unities:' (A)' monovalent' complex;' (B)' intramolecular' binding' and' (C)' intermolecular' binding'pathways.'
'
Multivalent' ligands' consist' out' of' multiple' binding' domains,' so' called' ligands,' which' interact' by' linking' with' one' or' more' specific' receptors' (17).' The' receptor' is' the' natural' counterpart'of'the'ligand'and'shows'a'pocket'on'its'surface'allowing'tethering'of'ligands' via'a'backbone'scaffold.'When'presented'to'the'receptor'multivalent'ligands'are'coupled' in' a' single' molecule;' multiple' ligands' are' coupled' in' a' multimeric' molecule' by' specific' linkers.' The' ligands' are' recognized' by' specific' receptors.' In' some' cases,' the' involved' ligands'may'have'different'binding'properties.'''
Multivalency'can'be'considered'as'a'chemical'tool'to'design'specific'multimeric'ligands.' Designing'a'multivalent'construct'is'not'easy'because'of'the'necessity'of'several'physicoR chemical' elements.' (18).' It' is' difficult' to' synthesize' highRavidity' ligands' for' biological' targets' or' imaging' agents.' The' main' challenge' is' to' design' a' ligand' that' matches' the' geometry' and' selectivity' of' the' correspondent' receptor.' Other' important' factors' with' regards' a' ligandRreceptor' interaction' are' the' loss' of' entropy' by' the' linker' and/or' the' scaffold'and'the'sort'of'interaction'between'the'linker'and'the'receptor'(19).'
36
Wester' et! al' presented' different' mechanisms' to' evaluate' the' binding' affinity' in' a' multivalent'interaction'(20).'Mainly,'they'postulated'that'any'interaction'between'ligand' and' receptor' will' result' in' a' subsequent' relative' increase' in' ligand' concentration.' However,'the'binding'between'multivalent'ligands'and'receptors'results'in'an'increased' avidity'for'the'ligandRreceptor'binding.''
(
Thermodynamics((
'
Thermodynamics'are'necessary'to'establish'the'“modus!operandi”'of'multivalent'binding;' free'energy,'enthalpy'and'entropy'are'crucial.'Whitesides'et!al.'highlight'the'importance' of' free' multivalent' energy' as' result' of' enthalpic' and' entropic' components' (1).' They' studied'multivalent'interactions'between'an'increasing'number'of'ligands'and'a'variable' number' of' receptors.' While' entropy' plays' a' big' role' in' the' design' of' multivalent' molecules,'the'average'values'of'free'energy'for'a'multivalent'ligandRreceptor'interaction' and'the'analogous'monovalent'interaction'turned'out'to'be'different.'However,'several' other' studies' on' intramolecular' binding' involving' multivalent' constructs' indicate' that' entropy' is' not' the' only' important' parameter' (3);' (18);' (21).' The' total' entropy' of' a' multivalent'interaction'is'not'an'absolute'value,'but'the'sum'of'different'entropic'factors,' such' as' translational' and' rotational' entropy' values,' conformational' entropy' and,' very' importantly,' entropy' of' solvation.' All' these' parameters' play' a' role' in' multivalent' interaction.' Translational' and' rotational' entropies' are,' respectively,' the' “degree' of' freedom”' of' a' molecule' to' translate' or' to' rotate' in' space;' translational' and' rotational' entropies'are'strongly'related'to'the'mass'and'to'the'size'of'molecules'(1);'(21).'
The'standard'free'energy'for'multimeric'binding,'defined'as'∆G°'multimer,'can'be'defined' as:'
∆G°'multimer'='N∆G°'monovalent'interaction'+'∆G°'interaction.'''''''''''''''
The'standard'free'energy'of'interaction'is'the'result'of'the'tethering'effect;'N'indicates' the' valency' of' the' overall' complex.' ∆G°' interaction' indicates' the' balance' between' favorable'and'nonRfavorable'tethering'effects.'The'valency'can'be'defined'as'number'of' interactions' observed' during' a' simultaneous' binding' process' between' a' compound'
37
(ligand)' and' its' counterpart' (receptor)' (22).' Considering' all' the' events' involved' in' the' multivalent' process,' valency' can' be' subdivided' in' bivalency,' oligovalency,' multivalency.' Bivalency' is' characterized' by' two' interactions' (i=2),' oligovalency' involves' maximum' 10' different' interactions' (i≤10)' and' multivalency' is' characterized' from' a' number' of' interactions'higher'than'10'(i>10)'(23).'''
The' valency' is' a' function' of' the' binding' affinity,' so' influences' the' overall' avidity.' Whitesides'et!al.'refer'to'avidity'as'regulated'by'the'association'constant'of'a'multivalent' interaction' (1).' They' define' the' association' constant' for' a' multivalent' complex' and' the' association'constant'for'a'monovalent'ligand'as'Kmulti'and'Kmono.'The'ratio'between'these' two'association'constants'is'called'the'enhancement'factor'(β).'Multivalent'interactions' can' be' divided' in' synergistic,' additive' and' interfering' interactions' according' to' their' degree'of'cooperativity.'''
' '
' '
Figure( 2:' Model' for' cooperativity' based' on' energetic' interactions' between' subunits' (bound'or'unbound).'The'symbols'L,'K,'β'represent'ligand'concentration,'binding'affinity' of' ligand' and' degree' of' enhancement' factor.' β' measures' the' cooperativity.' Positive' cooperativity'occurs'as'a'synergistic'effect'and'with'β>1;'neutral'cooperativity'is'observed' with'β=1'and'negative'cooperativity'is'observed'with'β<1.'
Although' related,' multivalent' and' cooperative' binding' do' not' describe' the' same' modalities.'Cooperativity'is'a'special'form'of'allostery'and'occurs'when'the'binding'of'one' ligand'influences'receptor'properties'in'order'to'receive'a'second'ligand'for'subsequent' binding.' This' can' be' clarified' by' analysing' the' binding' process' between' haemoglobin,' which' is' a' tetramer,' and' four' molecules' of' oxygen;' in' this' process,' the' binding' of' one'
38
molecule'of'oxygen'at'one'site'increases'the'affinity'for'oxygen'at'the'other'sites'of'the' tetramer.'This'is'an'example'of'positive'cooperativity'or'allosteric'regulation'(24);'(Fig.3).'''''''''''''''''
''''''''' ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
'
Figure(3:(Symmetry'model'of'haemoglobin'to'explain'the'cooperativity'in'oxygen'binding' as'well'as'the'transitions'of'multimeric'proteins'and'peptides.'It'focuses'on'the'two'states' of'the'haemoglobin,'T'and'R.'T'is'“tense”'form'and'R'is'“relaxed”'form'of'receptor.'KR'and' KT' indicate' the' equilibrium' constants' for' T' and' R' states.' Kc' is' allosteric' equilibrium' constant'(T)/(R).'Allosteric'cooperativity'occurs'if'Kc<1'and'KR>KT.'The'binding'of'oxygen' at'one'site'will'increase'the'binding'affinity'in'other'active'sites'for'other'oxygen'binding.''' '
Kinetic'processes,'which'help'drive'multivalency,'are'totally'different'from'kinetic'values' observed'in'monomers'and'in'monovalent'interactions.''The'dissociation'rate'plays'a'key' role'in'the'kinetics'of'interactions'between'monomers'as'well'as'multimers.'It'is'known' that'the'dissociation'of'multivalently'linked'ligands'occurs'after'breaking'a'receptorRligand' bound'one.'This'means'that'the'dissociation'is'timeRrelated'to'the'type'of'interaction.'It' derives'that'the'event'of'dissociation'is'faster'in'monovalent'interaction'(23);'(25).''
'
Binding(mechanisms(and(ligands(choice(
'
39
Designing'a'ligand'is'still'a'challenge,'especially'if'one'realizes'that'controlling'molecular' recognition'has'been''a'utopia'for'more'than'fifty'years'(21);'(26).'Considering'the'types' of'ligands'that'can'be'used'to'date,'one'will'notice'that'a'large'library'of'molecules'can'be' used:'peptide'fragments,'carbohydrates'and'antibody'fragments'(27R30).'Antibodies'are' important' tools' in' radioRimmunoRimaging,' but' for' developing' multivalent' ligands' most' efforts' are' addressed' towards' small' peptides.' Because' of' their' chemical' and' affinity' properties' peptides' are' considered' to' be' optimal' targets' for' the' development' of' new' radiopharmaceuticals.'To'improve'specific'targeting'and'radioRimaging'using'both'PET'and' SPECT,'efforts'are'made'to'design'larger'peptide'molecules,'such'as'multimers'(31).(
This'specific'interest'in'peptides'as'radiopharmaceuticals'is'associated'to'their'favorable' pharmacokinetic' characteristics.' Peptides' show' a' high' penetration' into' tumor' vascular' endothelium'and'rapid'clearance'from'blood'and'nonRtarget'tissues.'Another'important' advantage'is'the'possibility'to'use'different'pH'values'and'temperatures'while'conjugating' them'during'the'radiolabeling'procedure.'Their'low'toxicity'and'low'immunogenicity'make' peptides' suitable' agents' for' tumor' targeting.' Many' peptides' have' already' been' introduced' as' radiopharmaceuticals' and' entered' clinical' studies' (32R34).' This' concerns' among' others' radioRiodinated' analogues' of' somatostatin' and' DOTATOC' (DOTARPhe(1)R Tyr(3)ROctreotide)'R'both'introduced'for'the'localization'of'neuroendocrine'tumors'R''and' the' soRcalled' “affibody' binding' proteins”,' which' have' been' successfully' introduced' for' PETRCT''imaging'of'breast'cancer'by'targeting'HERR2'receptors'(35R38).''
Designing' a' functional' protein' is' a' big' challenge' because' it' is' difficult' to' synthesize' a' multivalent'ligand'that'can'maintain'the'folded'structure'of'peptides'and'proteins'after' chemical' treatment.' Multivalent' ligands' can' be' synthesized' by' several' approaches' and' can'be'constructed'in'different'structural'conformations.'That'makes'multivalency'also'a' promising' tool' for' biomedical' applications,' for' example' in' vaccines,' molecular' imaging' and'drug'delivery'(21).'
To'build'up'an'efficient'multimeric'construct,'many'factors'need'to'be'considered.'Overall' size,' type' of' linker' or' backbone,' receptor' density,' receptor' geometry' and' distance' between' ligands' must' be' taken' into' account.' Designing' a' multivalent' ligand,' one' has' carefully'to'consider'the'several'ways'in'which'binding'can'occur.'Gillies'et'al.'defined'four'
40
mechanisms'to'obtain'a'ligandRreceptor'interaction:'the'first'is'the'chelate'effect.'This'can' typically'be'observed'in'carbohydrate'interactions'that'occur'between'a'multimeric'ligand' and'receptors'(Fig.'4a).'The'second'mechanism'is'known'as'a'statistical'effect'in'which'a' single'ligand'of'a'multimeric'scaffold'interacts'with'a'single'binding'site'(Fig.'4b).'The'third' can'occur'via'an'aggregative'effect'when'a'cluster'of'recruited'receptors'allow'multivalent' interactions'(Fig.'4c).'The'fourth'mechanism'is'known'as'subsite'binding'mode,'which'can' be'observed'when'a'simultaneous'binding'of'a'ligand'to'a'single'main'binding'side'occurs' (typical'of'enzymes)'(Fig.'4d);'(1);'(4);'(7).'
'
'''A' ''
'B'
'C'
' 'D'
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Figure(4:'Multimeric'binding'pathways'between'ligands'and'receptors:''a)'chelate'effect' occurs' between' multimeric' ligands' and' receptors;' b)' statistical' effect' occurs' when' only' one'ligand'of'a'multimeric'construct'interacts'with'a'single'receptor;'c)'receptor'clustering' occurs'as'a'flexible'phenomenon'where'receptors'move'through'the'membrane'to'bind' specific'ligands;'d)'subsite'simultaneous'binding'is'observed'for'ligand'carrying'subligands' (such'as'enzymes)'which'interact'with'the'receptor.'
'
Ligands'and'receptor'densities'are'two'critical'aspects'in'the'interaction.'This'process'is' known'as'the'“lockRandRkey”'postulate'which'is'at'the'basis'of'any'ligandRreceptor'binding' model'(85).'It'is'the'ligand'density'that'influences'the'majority'of'binding'processes.'The' receptors' spacing' rule' should' be' kept' in' mind' when' designing' a' multivalent' ligand' because'receptors'can'move'through'the'membrane;'the'receptor'plays'a'pivotal'role'in' this'choice'because'the'scaffold'should'match'the'symmetry'of'the'specific'receptor.' (
Linkers(and(scaffolds(
'
The' availability' of' scaffold' structures,' backbones,' linkers' and' spacers' preceded' a' new' wave' of' interest' in' designing' multivalent' peptides.' In' general,' a' synthetic' or' semiR synthetic'scaffold'is'the'backbone'of'the'multivalent'ligand.'Ligands,'identical'or'different,' are'attached'via'linkers'or'spacers.'The'linker'may'have'a'significant'effect'on'the'activity' of' the' multivalent' ligand.' So,' the' choice' of' the' most' appropriate' one' is' essential' when' designing'multimers'(25).''
Important'parameters'of'a'linker'are'its'length,'composition,'grade'of'rigidity,'chemical' and'biological'targets.'Linkers'can'have'many'different'shapes,'from'small'linear'chains'up' to' polymeric' and' dendritic' architectures.' A' good' linker' will' lead' to' a' high' avidity.' The' avidity'of'the'multivalent'binding'is'associated'to'the'linkers’'length;'a'too'short'one'will' not'allow'simultaneous'binding'between'multiple'ligands'and'their'receptors'and'too'long' linkers'will'lead'to'increased'conformational'entropy'(1).'As'a'consequence'it'is'possible' to' change' the' binding' affinity' between' ligand' and' receptor' by' using' different' lengths.' Even' in' the' presence' of' receptor' clusters' or' highly' expressed' receptor' densities,' short'
42
linkers' with' lowRmolecularRweight' multimers' (such' as' dimers,' tetramers,' and' octamers)' will' not' lead' to' good' multivalent' binding.' Relatively' longer' linkers' will' increase' the' stability'of'the'multivalent'binding'between'receptor'and'ligand'(7).'Linkers'can'also'be' chosen'based'on'their'flexibility.'A'rigid'linker'will'often'be'chosen'because'it'is'a'simple' approach'to'increase'affinity'(23).'However,'to'avoid'a'poor'fit'and'a'low'stability'of'the' linking,'a'careful'analysis'of'the'binding'sites'by'computational'studies'will'be'necessary.' Flexible'linkers'allow'multivalent'connection'because'of'their'specific'structure.'They'can' adopt' a' higher' number' of' possible' conformations' in' comparison' to' the' rigid' ones.'' Moreover,'the'possibility'of'steric'effects'will'be'reduced'(20);(21).'A'flexible'linker'has'a' much'stronger'entropy'than'a'rigid'one,'allowing'more'receptor'recruitment'and'stronger' binding'affinity.'With'regards'the'overall'construct'long,'flexible'linkers'provide'a'higher' capacity'of'making'stable'multivalent'reactions.''
Many'linkers'were'introduced'to'link'two'or'more'ligands,'for'example'glycine,'ethylene' glycol,' diacetylene,' alanine,' aminocaproic' acid' and' piperidine.' Short' or' long,' flexible' or' rigid,' linkers' will' lead' to' important' functional' changes' in' the' multivalent' construct' (17)' (Fig'5).'''''
''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''
' Figure(5:'Representation'of'rigid'and'flexible'linker.'A'rigid'linker'(A)'permits'are'preferred' in' case' of' tight' binding.' Flexible' linkers' (B)' have' high' capacity' to' form' strong' affinity' complex'by'association'with'receptors'by'adopting'a'higher'number'of'conformations.(((
'
Scaffolds' and' backbones' are' needed' for' coupling' multiple' ligands' as' multiple' peptides' and' proteins.' Several' scaffolds' of' different' sizes' and' molecular' structures' are' available.'
A) B)
43
They'are'divided'in'linear'polymers,'dendrimers,'dendritic'wedges,'liposome'and'micelles' (39);'(40);''(Fig.'6).'
' ' ' ' ' ' ' ' ' '
'
Figure(6:'Synthetic'scaffold'commonly'used'as'carriers'of'peptides'and'proteins.(a)'linear' polymer;'b)'dendrimer;'c)'dendritic'wedge;'d)'micelle;'e)'liposome''
'
The'choice'mainly'depends'on'practical'aspects.'Linear'and'branched'polymers'are'widely' used' as' scaffolds.' They' match' with' the' receptor' in' a' favorable' way,' avoiding' steric' repulsion'that'will'cause'a'mismatch.'The'importance'of'the'scaffold'was'demonstrated'in' two' comparative' studies' in' which' ligands' were' connected' only' by' linkers,' by' scaffolds' only'or'by'a'combination'of'them'(41R42).'It'concerned'several'molecules:'1,4Rphenylene' diamine,' trimesic' acid,' porphyrin,' pentacyclen,' poly(pRphenylene' ethynylene),' poly(acrylamide)' and' PAMAM' dendrimers.' It' was' demonstrated' that' proper' scaffolds' improved'targeted'applications'and'delivery.'Different'scaffolds'(dendrimers,'liposomes,' polymersomes,'and'microspheres),'were'reviewed'with'regards'their'possible'application' in'interacting'multivalently'with'target'cells'(43R44).'Polymeric'scaffolds,'for'example,'can'
A
) B
)
C )
D )
E
44
be'used'as'backbones'to'synthesize'multivalent'ligands'with'a'wide'spectrum'of'sizes.'This' is' the' reason' why' modern' polymer' chemistry' has' become' important' in' synthesizing' multivalent' ligands.' The' ligands' tailored' from' polymers' have' a' central' scaffold' with' multiple'copies'of'peptides,'proteins'or'epitope'tags.'Polymers'can'be'synthesized'via'1)' ringRopening' metathesis' polymerization' (ROMP)' of' ligandRfunctionalized' monomers' (as' described' by' Kiessling' et' al.),' 2)' by' a' single' step' reaction' or' 3)' by' using' a' preRformed' polymeric'scaffold'to'which'monovalent'ligands'can'be'attached'(3).'Polymers'synthesized' by'ringRopening'metathesis'polymerization'will'become'important'for'the'production'of' biologically' active' multivalent' ligands.' Besides' linear' and' random' coil' polymers,' other' common'molecules'will'be'used'as'backbones'are'liposomes'and'dendrimers.'Liposomes' are'nonRcovalent'scaffolds'used'to'synthesize'multivalent'ligands.'Matching'is'mediated' via'lipidRlipid'interactions'(45R47).''
Dendrimers' are' highly' branched' molecules' offering' optimal' characteristics' for' spatially' defined' multiple' ligands.' They' show' a' high' homogeneity' and' are' reliable' platforms' for' multimerization' reactions.' Their' wellRdefined' monodisperse' 3Rdimensional' structures' make'dendrimers'ideal'backbones'for'simultaneous'presentation'of'different'ligands'(7);' (20);'(34);'(47).'Their'structure'is'constituted'by'a'central'core'connected'to'an'internal' layer' made' of' repeated' branching' units' and' an' external' part' with' dispersed' surface' groups.'These'scaffolds'allow'a'stable'interaction'with'strong'avidity'and'their'structure'is' nonRtoxic' and' nonRimmunogenic.' Dendrimers' are' able' to' pass' biomembranes' and' are' resistant'to'biodegradation.'Boas'et!al.'described'the'specific'dendrimeric'properties'(40),' which' can' make' them' important' tools' in' biomedicine' (48),' drugRdelivery' (47);' (49)' and' cancer' therapy' (50R51).' Synthetic' scaffolds' have' been' employed' for' the' synthesis' of' multiple'antigen'peptides'and'for'the'development'of'RGD'tripeptides'(52).'The'biological' role' of' these' complex' structures' has' been' studied' extensively' (14);' (48);' (49);' (51).' Important'and'clinically'relevant'are'the'polyamidoamine'(PAMAM)'dendrimers.'They'act' as' multimodal' molecular' imaging' agents' with' excellent' properties' in' combination' with' MRI,'PET,'CT,'scintigraphy'and'provide'optical'imaging'(54R56)'(Fig.'7).'
'
45
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Figure( 7:' Representation' of' dendrimers' used' as' multimodal' synthetic' probes.( These' scaffolds' are' engineered' to' carry' ligands,' radioisotope' tags' and' a' second' imaging' molecule'to'provide'combinatorial'molecular'imaging'agents.'
SYNTHESIS(OF(MULTIMERS(
Multimers' can' be' obtained' via' either' covalent' or' nonRcovalent' methods.' The' choice' between'these'two'methods'depends'on'the'characteristics'required'for'their'construct.'If' the'aim'is'to'design'a'wellRdefined'and'stable'structure,'the'best'choice'for'the'synthesis' is'the'covalent'way.'When'the'aim'is'to'design'a'structure'that'can'easily'be'adapted'to' the'conformation'and'valency'of'its'counterpart,'a'nonRcovalent'method'will'be'preferred' (3);'(7);'(20).'Combinatorial'multivalent'ligand'synthesis'is'an'emerging'method,'but'still' only'a'few'synthetic'strategies'have'been'devised'(57).'Several'chemical'reactions'can'be' used'to'link'the'peptides'and'proteins.'In'general,'a'primary'amine'of'a'lysine'or'a'cysteine' with' its' sulfhydryl' group' is' used.' Chemoselective' conjugation' based' on' bioRorthogonal' synthetic' strategies' can' facilitate' the' design' of' a' well' folded' bioactive' multimeric' complex.'Because'the'chemoselective'conjugation'is'regulated'by'bioRorthogonality,'this' strategy'will'lead'to'a'high'receptor'binding'affinity'(58).'The'DielsRAlder'cycloRaddiction,' imine'formation,'thiolRene'addiction,'Staudinger'ligation'and'Cu(I)Rcatalyzed'azide/alkyne' cycloaddiction'are'all'specific'tools'in'peptide'and'protein'design'chemistry.'Currently,'the' most' widely' used' approach' to' design' multimers' from' unprotected' molecules' is' native' chemical' ligation' (59R61)' (Fig' 8).' This' reaction' is' fast' and' selective' in' buffered' aqueous'
46
media.'Two'peptides'react'via'a'CRterm'thioRester'and'an'NRterminal'cysteine'residue'in' side'chain'position'(62R65).''
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Figure( 8:( Native' chemical' ligation' occurs' between' a' peptide' containing' a' CR terminal'thioester'and' a' second' peptide' a' cysteine'residue' at' the' NRterminal.' A' thiol' is' required'as'catalyst.'In'the'first'reversible'step,'a'thioesterRlinked'intermediate'is'obtained' via' transthioesterification;' in' the' second' irreversible' step,' this' thioesterRlinked' intermediate'reacts'to'yield'a'native'peptide'bond'at'the'ligation'site.'
'
The' reaction' is' catalyzed' by' addition' of' a' thiol.' ' CRterminal' thioRester' peptides' can' be' obtained'via'tertRbutoxycarbonyl'(Boc)Rmediated'peptide'synthesis'by'mercaptopropionic' acid'synthesized'resin.''
47
Expressed'chemical'ligation'improves'the'native'chemical'one.'This'phenomenon'is'also' known'as'inteinRmediated'protein'ligation.'By'this'approach'a'CαRthioester'reacts'with'NR term' cysteine' to' form' a' native' bond.' This' method' is' especially' applied' ' in' site' specific' modifications' of' proteins,' generation' of' cyclic' proteins' and' peptides,' cytotoxic' proteins' and'intermolecular'protein'interactions'(62);'(66).''
Another'widely'used'functional'group'in'bioRorthogonal'chemistry'is'azide.'By'promoting' the'reaction'between'azide'and'a'phosphine,'the'formation'of'azaRylide'will'be'mediated.' This' reaction' is' known' as' Staudinger' ligation' (67R68)' (Fig' 9a).' Azides' can' also' mediate' cycloaddiction' reactions' such' as' the' Huisgen' 1,3Rdipolar' cycloaddition.' These' reactions' occur'between'azides'and'alkynes'(Fig'9b).'''
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48
Figure( 9:' Staudinger' ligation' (A)' and' copper' catalyzed' click' Huisgen' 1,3Rdipolar' cycloaddiction' (B):( A)' The'Staudinger' reaction'or'reduction'occurs' between' an'azide' and' a'phosphine'activated'molecule'to'form'a'iminophosphorane'intermediate.'In'the'second' step,'the'hydrolysis'of'the'azaRylide'intermediate'produces'an'amide'bond.'B)'In'copper' catalyzed' click' Huisgen' 1,3Rdipolar' cycloaddiction' an' alkyle' containing' peptide' and' an' azide'containing'peptide'react'to'form'a'copperRazideRacetylide'complex.'The'reaction'is' assisted'by'the'copper'and'proceeds'efficiently'at'the'physiological'conditions'at'neutral' pH.'
A' recent' publication' describes' an' interesting' chemical' strategy' to' develop' homoR' and' heteroRmultimers' of' lung' cancer' specific' peptoids' (69).' The' authors' analyze' oligoRNR substituted' glycines' (peptoids)' which' are' protease' resistant,' more' cell' permeable' and' conformational'adaptable'to'several'structures.'A'few'peptoid'dimers'have'already'been' reported' as' good' candidates' for' future' targeting' probes.' Currently' binding' studies' are' carried' out' to' evaluate' the' effects' of' these' multimers' on' HCC4017' lung' cancer' cells' in' comparison'to'the'corresponding'monomeric'ligands'(69).'
Using' microwaveRassisted' click' chemistry' several' multivalent' c(RGDfk)' peptides' with' a' fourRfold'affinity'increase'for'tetravalent'peptides,'have'already'been'synthesized'(7).' Another' example' of' successful' synthesis' of' multivalent' peptide' and' protein' dendrimers' was' reported' by' van' Baal' et' al.' They' added' oligopeptides' and' recombinant' proteins' to' dendrimers.'In'that'way''cysRdendrimers'were'synthesized'by'the'reaction'of'the'amine' groups' of' poly(propyleneimine)' dendrimers' with' succinimideRactivated' and' tritylR protected'cysteine'motifs.'With'this'approach'it'was'possible'to'conjugate'dendrimers'to' the' CRterminus' of' a' protein.' The' overall' protein' function' was' not' affected' by' this' procedure'(70)'(Fig.'10).'
'
49
( Figure( 10:' Synthesis' of' cysteineRfunctionalized' poly(propyleneimine)' dendrimers:' amine' end' ' groups' of' poly(propyleneimine)' dendrimers' can' react' with' ' succinimideRactivated'' and''tritylRprotected''cysteines'to'form'cysRdendrimers.''
STUDIES(WITH(MULTIVALENT(TARGETS(AND(RADIOLABELED(PROBES(
Multimerization'can'be'used'in'the'development'of'targeting'molecules'or''radiolabelled' probes.'In'that'way'cellRspecific'binding'affinity'and'radioRimaging'can'be'improved'(71);' (72).'Many'versatile'chelators,'such'as'NOTA'(1,4,7RtriazacyclononaneR1,4,7Rtriacetic'acid)' or' DOTA' (1,4,7,10RtetraazacyclododecaneR1,4,7,10Rtetraacetic' acid)' have' been' synthesized' to' form' stable' multimeric' complexes;' polyethylene' glycol' has' been' introduced'to'improve'in!vivo'pharmacokinetics'(73R76).'''''
Widely' studied' multivalent' ligands' are' carbohydrate' molecules.' Using' a' rigid' synthetic' scaffold' Merritt' et! al.' ' found' multivalent' receptorRbinding' antagonists' against' cholera' toxin'and'heatRlabile'enterotoxin'from'Escherichia!coli''(77).'These'constructs,'based'on'a' series' of' galactose' branches' connected' via' flexible' linkers,' showed' much' higher' affinity' than'the'monovalent'counterpart.'The'pentavalent'ligand'was'designed'with'a'symmetric' core' surrounded' by' different' linkers' and' monovalent' units' for' matching' the' toxin’s' receptor'binding'site.'
The'synthesis'and'application'of'multivalent'peptides'and'proteins'has'been'impeded'by' all' the' difficulties' in' maintaining' a' final' folded' structure' during' chemical' synthesis.'
50
However,' there' are' already' many' examples' of' multivalent' peptides' based' on' synthetic' scaffolds.'A'wellRknown'example'of'multivalent'application'is'the'use'of'multiple'antigen' peptides'as'immunogens'for'systemic'induction'in!vivo.'These'peptides'are'multivalently' assembled' via' lysineRbased' dendritic' wedges' by' a' solid' phase' peptide' synthesis.' Today' they'are'available'as'immunogens'for'routine'antibody'production'and'cytotoxic'immune' responses.'Other'approaches'for'multivalency'include'the'use'of'nonRcovalent,'dynamic' scaffolds'as'well'as'the'selfRassembly'of'peptides'into'liposomes'(78).''
Multimerization' is' becoming' more' and' more' important' in' angiogenesis,' an' important' process' for' tumor' development' and' tumor' growth' (1);' (7);' (13).' There' is' a' growing' interest'in'techniques,'which'visualize'angiogenesis'in'a'nonRinvasive'way.'Several'nuclear' imaging' probes' for' nonRinvasive' visualization' of' tumor' angiogenesis' have' already' been' developed.' Very' promising' are' those' tracers' in' which' multimerization' plays' a' key' role.' RGD'is'an'extensively'described'peptide'used'for'the'nuclear'imaging'of'angiogenesis'(20);' (41).' E(c(RGDfK))2Rbased' dimers' were' the' first' cyclic' RGD' multimers' developed' and' labeled'with'64Cu'or'18F'for'positron'emission'tomography'(PET)'imaging.'E(c(RGDyK))2'was' labeled'with'18F'via'a'prosthetic'4R(18F)fluorobenzoyl'moiety'(Fig.'11).'(18F)FBRE(c(RGDyK))2' showed'a'significant'higher'tumor'uptake'and'prolonged'tumor'retention'compared'to'its' monomeric' analog' (18F)FBRc(RGDyK).' ' Although' the' monomeric' analog' was' excreted' preliminary' through' the' biliary' route,' the' dimeric' showed' a' better' renal' excretion' than' the'monomeric'one'(7);'(79).''
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51
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Figure( 11:' Multimeric' RGD' peptides' (monomer,' dimer,' tetramer)' labeled' with'18F' via' a' prosthetic'4R(18F)fluorobenzoyl'moiety.(
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52
Thumshirn' et' al.' designed' the' pentapeptide' cyclo(RRGDfER)' for' multimerization' of' RGD' peptides' (80).' They' used' aminohexanoic' acid' (Ahx)' or' heptaethylene' glycol' (HEG)' as' spacers.'Subsequently,'these'RGD'monomeric'units'were'linked'via'lysine'to'form'dimeric' and'tetrameric'RGD'peptides.'The'final'18FRlabeling'step'was'performed'by'oxime'ligation' using' 4R(18F)' fluorobenzaldehyde.' In! vitro' studies' by' this' radiotracer' showed' increased' affinity'of'the'multimers'for'αvβ3Rintegrin.'αvβ3Raffinities'of'the'cyclo(RRGDfER)Rmonomer,' dimer,'and,'tetramer'containing'HEG'spacer'units'were'increased'by'a'factor'of'10'with' each'duplication'of'binding'units'(80).'Poethko'et!al.,'showed'in'mice'with'αvβ3Rpositive' M21Rmelanoma'that'the'uptake'of'the'18FRlabeled'RGD'peptides'in'tumor'tissue'increased' in' the' order' monomer' <' dimer' <' tetramer.' Due' to' lower' uptake' of' the' tetramer' in' the' other'organs'(compared'to'the'dimer),'the'tumorRtoRorgan'ratios'were'highest'for'the'18FR labeled' RGDRtetramer' (81).' The' main' problem' with' these' multimers' is' that' they' bind' several'αvβ3'integrins'simultaneously.'The'distance'between'the'RGD'units'is'very'short.' Obviously,' this' cannot' be' considered' as' a' potential' benefit' of' multivalent' probes.' The' distance'between'the'cyclo(RGDfE)'moieties'may'be'long'enough'to'bind'adjacent'αvβ3' integrins' simultaneously' and' multivalently' by' using' Ahx' or' HEG' as' spacers.' Using' PEG4' linker'Liu'et!al.'solved'this'problem'by'extending'the'distance'between'two'RGD'motifs' (82).'It'resulted'in'a'higher'affinity'and'a'significantly'higher'tumor'uptake,'compared'with' the'dimer'without'linkers.''
Dijkgraaf' et' al.' investigated' how' multimerization' can' change' the' binding' affinity' if' multivalent'RGD'peptides'are'used.'In'their'work'DOTARconjugated'monomeric,'dimeric' and'tetrameric'RGD'peptide'were'synthesized'(Fig.'12)'and'radiolabeled'with'111In.'In!vitro' and'in!vivo!results'show'an'increase'of'binding'affinity'by'multimerization'and'a'significant' higher' tumor' uptake' of' tetrameric' RGD' peptides' compared' to' monomeric' and' even' dimeric'counterparts'(41);'(73);'(83).''
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53
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Figure(12:(DOTARconjugated'dimeric'and'tetrameric'RGD.' '
Various' other' RGD' dimers,' tetramers,' and' even' octamers' labeled' with' different' radionuclides'have'been'developed'and'studied'in!vitro'and'in!vivo.'Many'research'groups' have'demonstrated'that'increasing'the'peptide'multiplicity'can'significantly'enhance'the' integrin'αvβ3Rbinding'affinity'of'RGD'peptides'and'improve'the'tumorRtargeting'capability'
54
of' the' radiotracer.' Obviously,' the' incorporation' of' the' appropriate' spacer' between' the' RGD'motifs'can'enhance'affinity'and'binding'capacities.'
HOMO8(VERSUS(HETERO8MULTIMERS(
The' majority' of' investigator' groups' consider' multivalency' as' the' most' important' interaction'between'a'ligand'and'its'corresponding'receptor'(1);'(5);'(21).'It'is'relevant'to' distinguish'homoRmultivalency'and'heteroRmultivalency.'Multivalency'is'in'the'first'place' classified'on'the'basis'of'the'number'of'interactions'between'the'multivalent'ligand'and' its' specific' targeting' receptor,' and' then' on' the' basis' of' types' of' ligands' involved' in' the' same'binding'process.''
Homodimers,'or,'more'in'general,'homomultimers'are'constructs'presenting'two'or'more' identical'ligands'that'are'crossRlinked'with'high'avidity'values'(21);'(84).'The'best'example' of'a'homomultimer'concerns'RGD'peptide'multimers'radiolabeled'with'18F,'68Ga,'64Cu'and' evaluated' in' both' in! vitro' and' in! vivo' studies' (82);' (85R86).' There' is' a' controversy' with' regards' the' best' tumor' imaging' agent:' homomultimers' or' heteromultimers.' Because' cancer'is'characterized'by'simultaneous'expression'of'different'peptide'receptors,'many' authors' mention' heteromultimers' the' best' imaging' agents.' Others' consider' homomultimers'more'efficient'because'of'their'imaging'properties.'They'target'only'one' species'of'receptor'and'do'not'allow'the'targeting'of'other'sites'expressing'other'kinds'of' receptors.' Many' studies' focus' on' the' improvement' of' peptide' binding' affinity' via' heteromultimerization.' Currently' a' few' heterodimers' such' as' bombesinRRGD' or' DeltRIIR MSH(7)' are' under' investigation' (87).' ' A' bombesinRRGD' peptide' heterodimer' shows' promising'results'with'regards'patients'with'androgenRindependent'prostate'cancer'(82).' Imaging' results' gained' by' two' different' peptides' linked' in' the' same' multimer' may' improve'the'diagnostic'process.''Yan'et'al.'showed'that'a'BNRRGD'peptide'heterodimer,' linked' via' a' glutamate' linker' and' labeled' with' 18FRSFB' (N<succinimydilR4R(18F)' fluorobenzoate),'has'high'binding'affinities'for'both'GRPR'and'αvβ3'receptors,'but'displays' a' slower' washout' than' monomeric' peptide' counterparts' (82).' Recently,' Liu' et! al.,' synthesized' a' novel' BNRRGD' heterodimer' by' using' an' orthogonal' Alloc' protected' glutamate,'which'is'linked'to'aminocaproic'acid'(Aca)'of'AcaRBN(7R14).'After'removal'of'αR
55
allyl'ester'and'coupling'to'RGD'peptide'by'using'lysine'aminoacid,'a'miniRPEG'spacer'was' attached'to'the'glutamate'linker;'introduction'of'this'spacer'lead'to'an'improved'yield'of' the' radio' labeling' (Fig.' 13).' Subsequent' tumor' and' tissue' uptake' studies' revealed' the' same' binding' affinities' of' BNRRGD' heterodimer,' but' a' faster' renal' clearance' due' to' the' attachment'of'miniRPEG'spacer'(85).'''
'
' Figure( 13:' BNRRGD' peptide' heterodimer,' linked' via' a' miniRPEG' spacer' attached' to' the' glutamate'linker'and'labeled'with'18FRSFB'(N<succinimydilR4R(18F)'fluorobenzoate)'
'
Yan'et!al.'developed'a'new'BNRRGD'heterodimer'by'introducing'the'AEADP'linker'[3,3`R(2R aminoethylazanediyl)' dipropanoic' acid]' (88).' This' linker' contains' one' amino' and' two' functional' carboxylic' acid' ' groups.' This' AEADPRBNRRGD' heterodimer' showed' good' receptor' binding' affinities' for' integrin' αvβ3' and' GRPR' receptors.' These' data' were' collected' by' in' vitro' cellRbinding' assays' on' U87MG' human' glioblastoma' cells' and' PCR3' human'prostate'carcinoma'cells.'In!vivo'blocking'studies'demonstrated'that'the'receptor' binding' of'18FRFBRAEADPRBNRRGD' for' integrin' αvβ3' and' GRPR' was' not' affected' by' the' AEADP'linker.'Substituting'the'glutamate'linker'with'AEADP'linker'did'not'show'any'other' effect'on'the'biological'activities'of'the'BNRRGD'heterodimer.'Both'GluRAcaRBN(7R14)'and' AEADPRBNRRGD' tracers' had' a' high' tumor' uptake,' but' also' a' nonRspecific' tissue' binding'
56
uptake.' The' main' advantage' of' AEADPRBNRRGD' tracer' was' its' efficient' synthesis.' Other' studies'on'BNRRGD'heterodimers'were'performed'by'attachment'of'bifunctional'chelators' as'DOTA'and'NOTA,'and'radiolabeling'by'64Cu'and'68Ga'(82);'(85).'64CuRNOTARBNRRGD'was' documented' as' an' imaging' agent' with' good' binding' affinity,' high' binding' kinetics' and' showed'a'good'quality'of'imaging'and'low'backgrounds,'even'after'late'time'points'(Fig.' 14).''Radio'labeling'of'NOTARBNRRGD'by'68Ga'had'also'promising'results;'it'showed'a'high' tumor'uptake'and'a'lower'efflux'ratio'(82);'(85).''
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Figure(14:'DOTA'and'NOTA'BNRRGD'heterodimers.'These'ligands'have'been'evaluated'as' imaging'agents'with'good'binding'affinity,'high'binding'kinetics'and'showed'both'a'good' quality'of'imaging'and'low'backgrounds'even'after'late'time'points.'
'
In'2008,'Handl'et!al.'described'the'binding'properties'of'DeltRIIRMSH(7)'heterodimers'(87).'' This'heterodimer'is'composed'of'ligands'connected'by'semiRrigid'or'flexible'linkers'and'is' complementary' to' the' human' melanocortin' receptor' Type' 4' (hMC4R)' and' the' cholecystokininR2' receptor' (CCKR2R).' The' receptor' binding' properties' of' ' DeltRIIRMSH(7)' was' evaluated' by' two' different' cell' lines:' human' embryonic' kidney' 293' (Hek293)' expressing'only'one'of'the'complementary'receptors'(Hek293/hMC4R'and'Hek293/CCKR 2R)' and' Hek293/hMC4R/CCKR2R.' Cells' expressing' both' complementary' receptors' had' a' twentyRfold'higher'affinity'compared'to'those'expressing'only'one'receptor.''
Neurotensin'(8–13)'peptide'homomultimer'is'well'documented'with'regards'its'synthesis' and'structure'(Fig.'15).''A'series'of'NT(8–13)'derivatives'with'a'multivalent'core'structure' have' been' synthesized' and' evaluated' via' linking' of' NT(8–13)' units' using' the' CR' or' NR termini' of' the' NT(8–13)' residues' (89).' The' attachment' of' the' NT(8–13)' moieties' to' the' core'unit'was'the'most'crucial'part'of'the'synthesis.'More'in'detail'the'dimeric'analogues' showed' an' approximately' two' times' higher' IC50' value' compared' to' the' tetrameric' compound.' Subsequent' radio' labeling' by' fluorineR18' showed' promising' results' of' the' sulfydrylRfunctionalized' peptides' using' (18F)FBAM.' However,' the' results' were' not' comparable'with'(18F)SFB'or'(18F)FBA'as'labeling'agents'because'both'reactions'strongly' depended'on'the'peptide'concentration'(89).'
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Figure( 15:' Neurotensin' (8–13)' peptide' homomultimers.' Monomer,' homodimer' and' homotetramer'have'been'synthesized'and'radiolabelled.'
59 '
Recently' DOTARconjugated' monomeric,' dimeric,' and' tetrameric' (Tyr3)octreotideRbased' analogues' (Fig.' 16)' have' been' evaluated' (90)' A' relatively' long' retention' of' radiolabeled(Tyr3)octreotide' dimeric' conjugate' was' measured' in' sc' AR42J' inoculated' tumors' in' BALB/c' nude' mice.' Current' studies' address' the' receptor' binding' affinity' by' investigating' the' effects' of' different' kinds' of' spacer.' In' this' way' the' high' lipophilic' character'of'the'multimeric'conjugate'may'be'reduced.''
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Figure( 16:' DOTARconjugated' monomeric,' dimeric,' and' tetrameric' (Tyr3)octreotideRbased' analogues.'
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Specific'uptake'and'affinity,'washout'and'clearance,'imaging'and'therapeutic'value'are'all' important;' they' have' been' extensively' explained' in' the' previous' paragraphs.' When' the' goal' is' to' design' a' new' (radio)Rligand,' all' these' characteristics' need' to' be' evaluated' a!
priori'(Fig.'17).''
( Figure( 17:' Summary' of' important' characteristics' of' monomers,' multimers' and'
polymers/dendrimers.((Although'the'monomers'offer'best'imaging'properties,'dendrimers' and'highly'branched'multimers'have'the'best'therapeutic'applications.'They'are'able'to' deliver'a'payload'to'tumors.'''
NANOTECHNOLOGIES(AND(NEW(PERSPECTIVES(
The'thermodynamic'and'kinetic'characteristics'of'multivalent'interactions'have'influenced' the'field'of'supramolecular'nanofabrication'dramatically.'It'has'been'demonstrated'that' selfRassembled' hybrid' monolayers' as' well' as' nanoparticles' and' dendrimers' prefer' multivalent'binding'and'intermolecular'binding'(25).'
61
Multivalency'is'one'of'the'most'extensively'studied'themes'of'nanotechnology.'Recently,' supramolecular'multivalent'interactions'have'been'studied'systematically'by'Stoddart'et' al.'(91).'By'increasing'the'number'of'repeats'of'the'original'sequence,'multivalency'may' play' an' important' role' in' modifying' the' binding' activity' of' a' given' peptide.' Such' multimerization' can' be' accomplished' using' the' simple' tandem' repeat' of' the' original' sequence.'''
Oren' and' Notman' applied' a' multipleRrepeatRbased' strategy' on' both' phage' display' selected'platinum'and'quartz'binder'(7'and'12'amino'acid'sequences'respectively)'and'cell' surface'selected'gold'binders'of'14'aminoacids'each'(92).'They'showed'that'the'increase' of'the'number'of'repeated'peptides'was'not'always'associated'with'an'enhancement'of' binding' activity' and,' interestingly,' that' peptides' were' changed' when' used' in' multipleR repeat'forms.'So,'one'may'assume'that'the'molecular'structure'is'relevant'and'that'there' is' a' correlation' between' conformational' arrangement' and' binding' ability' (93).' Linkers' must'be'incorporated'between'two'or'more'following'sequences'to'conform'the'overall' multipleRrepeated'peptides'for'binding'and'other'biological'functions.'
Potentially' the' soRcalled' dock' and' lock' (DNL)' method' may' provide' a' large' number' of' bioactive' molecules' by' multivalency' (94).' This' approach' may' not' only' overcome' the' limitations'of'current'technologies'with'regards'the'production'of'antibodyRbased'agents,' but' also' be' useful' for' multimerization' in' general.' The' limitations' of' the' DNLRmethod' include'high'manufacturing'cost,'low'expression'yields,'instability'in'serum,'formation'of' aggregates'or'dissociated'subunits,'undefined'batch'composition'due'to'the'presence'of' multiple' product' forms,' contaminating' sideRproducts,' reduced' functional' activities' or' binding'affinity/avidity'attributed'to'steric'factors'or'altered'conformations.'
A'very'new'approach'is'nanoRengineering'by'gold'probes.'Gold'nanoparticles'are'similar'in' size'to'proteins,'biocompatible,'and'can'be'modified'using'functional'groups.'Rotello'et!al.' made' libraries' of' gold' nanoparticles' by' ligand' exchange' reactions' of' several' aminoacidR' and' peptideRfunctionalized' alkene' thiols' on' 1RpentanethiolRcoated' gold' nanoparticles' (95).'Changes'in'the'charge,'hydrophobic'index,'and/or'chirality'of'the'pending'moieties' caused'different'binding'affinity'capacity'toward'protein'surfaces.''
62
Recently,' a' potent' inhibition' of' HIV' fusion' by' gold' nanoparticles' coated' with' a' therapeutically'inactive'small'molecule'related'to'TAKR779'has'been'described'(96).'TAKR 779' is' a' specific' inhibitor' of' HIVR1' entry.' This' study' demonstrates' how' small' organic' molecules'can'be'converted'into'highly'active'drugs'by'conjugating'them'to'multivalent' gold' nanoparticles' as' scaffold.' So,' multivalent' attachment' of' small' molecules' to' nanoparticles'can'be'a'valid'tool'to'increase'specific'binding'affinity.'This'tool'may'also' facilitate' the' development' of' ' nanomaterials' for' potential' application' in' drug' discovery' and'drug'delivery'as'well'as'in'the'diagnosis'and'treatment'of'human'diseases'in'general'' Undoubtedly,'nanomaterials'will'also'be'developed'for'designing'novel'anticancer'drugs' (95);'(97);'(98).''Several'are'already'commercially'available'and'used'to'attach'peptides'as' diagnostic'imaging'agents'for'targeting'inflammation,'and'other'angiogenic'processes.'
CONCLUSIONS(
In'the'future,'multivalency'may'become'a'prominent'tool'for'designing'and'developing'a' wide' library' of' new' compounds' to' use' as' targeting' agents' for' radioimaging' and' radionuclide' therapy.' This' review' described' and' evaluated' parameters' which' play' a' critical'role'in'designing'a'functional'construct.'Knowing'that'in'the'next'future'the'design' principles' and' the' basics' of' multivalency' need' extensive' investigations,' the' results' reported'by'many'research'groups'show'that'both'homoR'and'heteroRmultimers'provide' higher' binding' affinities' than' the' respective' monomeric' counterparts.' The' designing' strategies'all'requires'series'of'computational'studies'to'distinguish'within'the'multitude' of'known'backbones,'linkers,'receptors'and'especially'ligands.''
' ' ' ' ' ' '
63
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