1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis 3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices 4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides (Cetrorelix, Fuzeon) b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry

Syllabus

30 Prof. H. Wennemers

Prof. H. Wennemers 31

Ac-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2

Fuzeon/Enfuvirtide

O

ONH

OH

OHN

OH ONH

OH OHN

ONH

OHN

NHN

ONH

OHOH

N

ONH

OHN

OHO

ONH

OHO

OHN

OHO

NH

OH2N

OHN

ONH2

ONH

OH2N

O

HN

O

H2N

ONH

HOO

HN

NH2

O

NHO

NH2

O

O NH

OHO

ONH

OH2N

O HN

OHO

ONH

O HN

OHO

ONH

O HN

OHO

ONH

NH2

O HN

HNO

NH

O HN

HOONH

O HN

HNO

NH

OH2N

O HN

HNO

NH

H2N

t1/2 = 3.8 h, dose: 2 x 90 mg daily

Prof. H. Wennemers 32

Lead Structures

...and hormones

Toxins: nerve or muscle

A.  V.  Schally:  Biochem.  Biophys.  Res.  Commun.  1971,  43,  393  and  822.  Science  1976,  191,  195.    Int.  J.  Pep9de  Protein  Res.  1988,  32,  425  33  

HNNH

HN

NH

HN

NH

HN

N

HNN

NH

OO

O

O

O

O

O

O

ONH

OH

OH

NH

NH2HN

OHN

NH2

O

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Hormone isolated from the pituitary gland that controls the secretion of the luteinizing hormone (LH-RH)

Cetrorelix Lead structure:

Edmann Abbau

H2N NH

HN

O

O

O

CH3

OHPh

N C SHN N

H

HN

O

O

O

CH3

OHHN

SPh

H3NHN

O

OOHN

SNH

Ph

CH3

OPh NH

NH

OHS CH3

O

N NHPh

O

S

CH3

thiohydantoin

H+

+H2O (H+)

34 Prof. H. Wennemers

A.  V.  Schally:  Biochem.  Biophys.  Res.  Commun.  1971,  43,  393  and  822.  Science  1976,  191,  195.    Int.  J.  Pep9de  Protein  Res.  1988,  32,  425  35  

HNNH

HN

NH

HN

NH

HN

N

HNN

NH

OO

O

O

O

O

O

O

ONH

OH

OH

NH

NH2HN

OHN

NH2

O

HN

NH

HN

NH

HN

NH

HN

N

O

O

O

O

O

O

O

OH

OH

NH

NH2HN

OHN

NH2

ON

Cl

NH

O

O

NH

NH2O

Me

(pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Hormone isolated from the pituitary gland that controls the secretion of the luteinizing hormone (LH-RH)

Ac-DNal-DPhe(4Cl)-DPal-Ser-Tyr-DCit-Leu-Arg-Pro-DAla-NH2

Cetrorelix Lead structure:

Triazoles as Amide Bond Isosteres

36

518 NATURE CHEMISTRY | VOL 3 | JULY 2011 | www.nature.com/naturechemistry

NN

N

NH

O

OH

Fmoc

N3

O

NH O

HN

Ph

NH

NH

N

HN

HN

O

O

Ph

NN

O

NN

N

O

Ph

CuI, DIPEA2,6-lutidine

(>90%)

Alternative macrolactamization approach

SPPS

(66%)

NNN

H2N

O

HN

ONH N N

N

O NH

HO

OPh

Ph

Cyclic peptide nanotube

PyBOPDIPEA(65%)

Tandem dimerization-macrocyclization

N3O

HN

O

NH O

HN

NH

4

O

NN

N

O

HN

NH

O

NHON

H

O4

O

HN

NH

O

NHON

H

O4

N NN

NH

O

NO

NHHN

O

ON

MeO

O

4

CuI, DIPEA2,6-lutidine

MeCN (0.0002 M),TBTA

(50%)

trans-trans-trans-trans

Apicidin

NH

O

NO

NHHN

O

ON

MeO

O

4

trans-trans-trans-transcis-trans-trans-trans

cis-trans-trans-trans

N3O

HN

O

O

O4

FmocHN

[Cp*Ru(cod)Cl](20 mol%)

toluene, 45 °C

N

O

HN

O

O

O4

NN

NHFmocHO

O HN

O

O4

NN N

HNO

NH2

HN

(1) NMP, piperidine(2) Fmoc-Trp-OH

HBTU, DIPEA

(3) NMP, piperidine(4) TFA, CH2Cl2

HATU, DIPEADMF (0.0005 M)

(>95%)

P

ONH

OHN

ONH

Ph PhO

N3 HN

NNN

NH

NH

PPh

PhO

O

O

O

HO

S

OHHO

S

OH

NH

N

HN

NH

O

NNOH

O

O

OH

S

P

O

OtBu

Ph Ph P

ONH

OHN

ONH

Ph PhO

N3

S

OtBu

OtBu

O

TFA

(48%)

P

N NN

O PhPh

NNH

NN

O

O

O

NN

RON3

ON

OHN

ON

OBnCuBr, DBU

Toluene(0.0001 M)

reflux(56%)

R = Bn H2, Pd/CMeOH, CH2Cl2

(91%)

a b

R = H

c

SPPS

d

∆

tBuO

Figure 6 | Azide–alkyne cycloadditions in the synthesis of peptide macrocycles. a, A click-mediated macrocyclization of Tyr-Pro-Val-Pro. b, Synthesis of a cyclic peptide nanotube through either a high-yielding tandem dimerization-macrocyclization approach by two tandem click reactions of an azido-dipeptide alkyne (top) or through a less efficient conventional macrolactamization approach (bottom). c, The synthesis of triazole-modified analogues of the cyclic tetrapeptide, apicidin. Top: A ruthenium catalysed formation of a 1,5-disubstituted 1,2,3-triazole on solid-phase is followed by a macrolactamization to yield an analogue resembling the biologically active conformation of apicidin. Bottom: A Cu(i)-catalysed intramolecular azide–alkyne cycloaddition to yield an analogue of apicidin resembling its predominant conformation in solution. d, Synthesis of a cyclic tetrapeptide analogue containing a 1,5-disubstituted 1,2,3-triazole through an intramolecular cyclative cleavage of a solid-support-bound azidopeptidylphosphorane. HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; NMP, N-methyl-2-pyrrolidone; TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine.

REVIEW ARTICLE NATURE CHEMISTRY DOI: 10.1038/NCHEM.1062

© 2011 Macmillan Publishers Limited. All rights reserved

Bock, Speijer, Hiemstra, van Maarseven Org. Biomol. Chem. 2007, 5, 971. See also: Raines et al. J. Am. Chem. Soc. 2007, 129, 12670.

Tyrosinase inhibitor cyclo-[Pro-Val-Pro-Tyr] (isolated from L. helveticus)

Inhibitory activity of the tyrosinase: all amide bonds: IC50 1.5 mM triazole analogue: IC50 0.5 mM

Prof. H. Wennemers

Cyclic Peptides

Oxytocin

NH

OO

HN

CH3CH3

NHO

H2NS S

NH2

O

NH

NH

NO

OO

NH2

O

NH

OHN

H2NO

O

OH

HN

SS

NHO

NH2Ph

O O NHO

NHO

NH

HN

O

NH

OHO

HO

Ph

HN

H2N

OH

Octreotide (Somatostatin Analogon)

Cyclosporin

37

HN

O N

O

NO

HN

O NO

HN

OHNN

O

O

NO

N

N

O

O

HO

!"#$%%$&'(")*+,*( %+)**-"-./ 01"% $223"*%4 5#) *6$723*4 ,#,1* $-,"'89: $+,";",& #5 ,1* +")+<3"-%4 ,1* -*<)#,*-%"- $-,$.'#-"%7 #5 +&+3#2%&+1#,)"(*4 $-( ,1* 1$*7#3&,"+ $+,";",& #5:"#3$2*2,"(*4 =1"3* ,1* <,*)#,#-"+ $+,";",& #5 >$3$,$ ?@ A)%,+$7* ,# 3".1, 5)#7 $-*+(#,$3 -$,";* 7*("+"-* )*2#),%/ B33 #5,1*%* $+,";","*% %**7 ,# 1$;* ,1* +#77#- 5*$,<)* #5 2#%%"!3&!*"-. $%%#+"$,*( =",1 7*7!)$-* ("%)<2,"#-4 !<, $)* 7#%,3">*3& #-3& %"(* *55*+,% #5 ,1* -$,<)$3 5<-+,"#- "- 23$-,%4=1"+1 "% $37#%, +*),$"-3& $% (*5*-+* 7#3*+<3*%/ 9-(**(4,1"% "% +#-%"%,*-, =",1 )*2#),% #5 ,1*") $-,"7"+)#!"$3 $+,";",&C0$7 *, $3/4 @DDDE/

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LMM@E/ 01* %"6 +#-%*);*( J&% )*%"(<*% $)* $- *%%*-,"$35*$,<)* #5 ,1"% 7#,"54 $-( !*+$<%* #5 ,1*") )#3* $% -#(*% 5#)("%<3A(* +)#%%'3"->%4 $)* "72#),$-, ,#2#3#."+$3 7$)>*)% 5#)(*A-"-. %"6 3##2% "- ,1* 2*2,"(* !$+>!#-* CF"./ @E/ N<2*)'"72#%*( #- ,1*%* 3##2% $)* ,1* *62#%*( $7"-# $+"( %"(*+1$"-% )*%2#-%"!3* 5#) ,1* (";*)%* $+,";","*% #5 ,1* +&+3#',"(*%/

!" #$% &'&()& &'*+),% -,.+ /0012 3.+)4

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!"#" $%&'( ) *+,'-+. /0 123345 46307464/@R@M

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Cyclotides

Prof. H. Wennemers

J.  S.  Desgrosellier  and  D.  A.  Cheresh,  Nature  2010,  10,  9-­‐22;  M.  Barczyk,  S.  Carracedo  &  D.Gullberg  ,  Cell  Tissue  Res  2010,    339,  269–280  

Collagen receptors (GFOGER)

β3

β5

β6

β8

RG

Dre

cept

ors

α9

α4

β2

β7

Leukocyte - specificreceptors

β4

Laminin receptors

α1α2 α10

α11

α3α6

α7

αV

αllb

α5

α8

β1

αE

αL

αM αX

αD

Fig. 2 Representation of theintegrin family. In vertebrates,the integrin family contains 24heterodimers. Isolated speciesthat have undergone genomeduplication (e.g., Danio rerio)have more integrin familymembers. In higher vertebrates,the integrin family has 24 pro-totypical members

α 1 2 I domain 3 4 5 6 7 Thigh Calf-1 Calf-2

β -propeller domain repeats

β I domain 1 2 3 4

PSI I-EGFdomain domains

Hybrid domain

A

α I

H PSIThigh

C1

C2

β I

α β

β TM

B

β− propeller

EI-4

Fig. 3 Representation of a prototypical αI-domain-containing integrinheterodimer. Nine out of the 18 integrin α chains contains an αIdomain, as shown, but all integrins contain a βI domain in the β

subunit. A Representation of the domains in αI domain-containingintegrin (stars divalent cation-binding sites). B Representation ofarrangement of domains in αI-domain-containing integrin

272 Cell Tissue Res (2010) 339:269–280

Binding  domain  

Cyclic Peptides – Cyclic RGD

• Arg-Gly-Asp (RGD) is a common ligand for integrins

• Integrins are transmembrane receptors that modulate various signaling pathways (e.g. Cell migration, differentiation, immune and non-immune defenses, wound healing, angiogenesis)

angiogenesis = process by which new blood vessels are formed from pre-existing vessels

Integrins are interesting targets for Cancer Therapy

Prof. H. Wennemers 38

Review  arPcle:  H.  Kessler  et  al.,  An9-­‐Cancer  Agents  in  Medicinal  Chemistry,  2010,  10,  753-­‐768  

Peptide IC50(µM) αvβ3 IC50(µM) αIIbβ3 SelectivityαIIbβ3/αvβ3

GRGDSK 1.2 ± 0.27 5.4 ± 2.0 4.5

c(RGDfV) 0.0049 ± 0.0001 1.7 ± 0.38 347

Linear versus Cyclic RGD

c(RGDfV) Cilengitide

H2N

H

O

NH O

HN

H2N NH

HN

H

O

NH O

OHO

HN

O

OH

NH

OH

O

NH2

H2N

NH

HN

O NHNH

O

OHN

OH

O

O

NO

NH

Prof. H. Wennemers 39

Cyclic Peptides – Synthesis Recent review: Yudin, Nature Chemistry 2011, 509

40

H2N

NHO

HNBoc

OO Fm

NHO

HN

OO Fm

Boc-DLeu-(Trp-DLeu)3

1. Boc deprotection (TFA)

2. Fm deprotection (PIP)

3. Cyclization (BOP)

4. Cleavage (HF)

HN

HN

HN

HNNHNH

NH

NHO

OO

O

O

O O

O

NH2

O

NH

HN

NH

Example

Ghadiri, et al., Nature 1993, 366, 324. Nature 1994, 369, 301

Prof. H. Wennemers

Cyclo1des  –  Discovery  

•  Use  of  Oldenlandia  affinis  as  an  herbal  infusion  to  facilitate  childbirth  by  Congolese  women  

–  Reported  in  the  1960’s  

•  “Kalata  B1”  isolated  from  O.  affinis  by  Gran  in  1970‘s  

–  Kalata-­‐Kalata  indigenous  name  of  the  plant  –  Isolated  from  chromatographic  fracPon  B1    

•  Presence  of  cycloPdes  in  other  plants  has  been  discovered  

–  about  1g  can  be  isolated  from  1kg  of  plant  Pssue  

•  Structure  elucidaPon  in  the  1990‘s  –  CombinaPon  of  Enzyme  digesPon,  mass  spectrometry  and  

NMR    

 •  Term  “CycloPdes”  coined  by  Craik  in  1999  

–  Cyclic  pepPdes  

Oldenlandia  affinis    

Kalata  (2002)  by  Julian  Voss-­‐Andreae.  The  pictured  sculpture  is  based  on  the  atomic  coordinates  of  kalata  B1  

D.  K.  Craik  et  al.,  J  Org.  Chem.,  2011,  76,  4805-­‐4817.  U.  Göransson  et  al,  J  Nat.  Prod.,  2014,  77,  724-­‐736.    

development of plants and plant cells as protein expressionsystems.A protocol for the isolation of a highly purified polypeptide

fraction from plant biomass was developed.20 In particular, thisprotocol was used for the isolation of the first suite of cyclotidesfrom one species, and the results confirmed them as a family ofpeptides.5 Hence, we reconnected to Sandberg’s discoveries inAfrica some decades earlier. The peptide family has grownquickly since then, and the collective term cyclotidesaftercyclo-peptideswas suggested by one of the leaders in the field,Professor David Craik, in 1999.1 Since then, our group hasfocused our efforts to understand the chemistry and biology ofcyclotides and to explore their possible use for biotechnological,pharmaceutical, and agricultural applications.

■ LOCKING IN THE STRUCTUREAlready at an early stage it was clear to us that cyclotides fallinto two main subfamilies.5 In a landmark paper, Craik and co-workers then defined them as the Mobius and braceletsubfamilies.1 These subfamilies are distinguished by thepresence or absence, respectively, of a cis-Pro peptide bond.With an increasing number of cyclotides identified, the twosubfamilies now seem to merge, and several “hybrids”, i.e.,peptides containing features from both subfamilies, have nowbeen reported. Bracelet and Mobius cyclotides differ in size andamino acid content, with bracelets being the larger and morestructurally diverse subfamily of the two; to date according toCybase (the database of cyclic proteins),21,22 approximatelytwo-thirds of the known cyclotides belong to the braceletsubfamily. (Cybase can be accessed at http://www.cybase.org.au.)Figure 2 displays prototypic cyclotides. Apart from the six

conserved cysteines, some residues are found in all or nearly allcyclotides: the Glu residue in loop 1 and the (Asn/Asp)-Glypair in loop 6. The former has a key structural role, furtherrestricting flexibility and condensing the overall structure

through a network of hydrogen bonds with loop 3;23,24 thelatter residues are involved in the post-translational ringclosure.25 The remaining residues are interchangeable, andalthough there are relatively few amino acids in a cyclotidesequence, variations are immense.The current definition of a cyclotide is strictly based on the

structural motif, but this definition has become blurred lately bythe discovery of a few atypical variants. Until recently, only two

Figure 1. Ribbon structure of a cyclotide. The structure shows thecrystal structure of varv F (PDB-file 3E4H). Note the unique featuresof the CCK motif: a cyclic backbone with sequence loops (1−6) andthree stabilizing disulfide bonds. These disulfides are arranged in acystine knot, constituted by two disulfides that form a ring structuretogether with the backbone connecting the four cysteines (I−IV; II−V) and a third disulfide that threads through the ring (III−VI). Theunique cyclotide structure forces hydrophobic residues to be exposedon the surface of the protein, thereby increasing its amphipathicproperties.

Figure 2. (A) Surface representation of prototypic cyclotides fromeach subfamily: the bracelet cycloviolacin O2 (cyO2) and the Mobiuskalata B1. The backbone aligned models display the distribution ofhydrophobic/hydrophilic regions on the protein surface and theiramphipathic structure. Hydrophobic residues (Ala, Leu, Ile, Pro, Trp,Phe, Val) are in green, cationic (Arg, Lys) in blue, and anionic (Glu) inred. (B) Representative sequences of cyclotides from the twosubfamilies and a circular trypsin inhibitor. Cys residues arehighlighted in gray. (C) The sequences of the bracelet and Mobiussubfamilies summarized as sequence logos, generated by Weblogo.131

The overall height of the stack indicates the sequence conservation atthis position, while the height of symbols within the stack indicates therelative frequency of each amino or nucleic acid at this position.

Journal of Natural Products Review

dx.doi.org/10.1021/np401055j | J. Nat. Prod. 2014, 77, 724−736725

development of plants and plant cells as protein expressionsystems.A protocol for the isolation of a highly purified polypeptide

fraction from plant biomass was developed.20 In particular, thisprotocol was used for the isolation of the first suite of cyclotidesfrom one species, and the results confirmed them as a family ofpeptides.5 Hence, we reconnected to Sandberg’s discoveries inAfrica some decades earlier. The peptide family has grownquickly since then, and the collective term cyclotidesaftercyclo-peptideswas suggested by one of the leaders in the field,Professor David Craik, in 1999.1 Since then, our group hasfocused our efforts to understand the chemistry and biology ofcyclotides and to explore their possible use for biotechnological,pharmaceutical, and agricultural applications.

■ LOCKING IN THE STRUCTUREAlready at an early stage it was clear to us that cyclotides fallinto two main subfamilies.5 In a landmark paper, Craik and co-workers then defined them as the Mobius and braceletsubfamilies.1 These subfamilies are distinguished by thepresence or absence, respectively, of a cis-Pro peptide bond.With an increasing number of cyclotides identified, the twosubfamilies now seem to merge, and several “hybrids”, i.e.,peptides containing features from both subfamilies, have nowbeen reported. Bracelet and Mobius cyclotides differ in size andamino acid content, with bracelets being the larger and morestructurally diverse subfamily of the two; to date according toCybase (the database of cyclic proteins),21,22 approximatelytwo-thirds of the known cyclotides belong to the braceletsubfamily. (Cybase can be accessed at http://www.cybase.org.au.)Figure 2 displays prototypic cyclotides. Apart from the six

conserved cysteines, some residues are found in all or nearly allcyclotides: the Glu residue in loop 1 and the (Asn/Asp)-Glypair in loop 6. The former has a key structural role, furtherrestricting flexibility and condensing the overall structure

through a network of hydrogen bonds with loop 3;23,24 thelatter residues are involved in the post-translational ringclosure.25 The remaining residues are interchangeable, andalthough there are relatively few amino acids in a cyclotidesequence, variations are immense.The current definition of a cyclotide is strictly based on the

structural motif, but this definition has become blurred lately bythe discovery of a few atypical variants. Until recently, only two

Figure 1. Ribbon structure of a cyclotide. The structure shows thecrystal structure of varv F (PDB-file 3E4H). Note the unique featuresof the CCK motif: a cyclic backbone with sequence loops (1−6) andthree stabilizing disulfide bonds. These disulfides are arranged in acystine knot, constituted by two disulfides that form a ring structuretogether with the backbone connecting the four cysteines (I−IV; II−V) and a third disulfide that threads through the ring (III−VI). Theunique cyclotide structure forces hydrophobic residues to be exposedon the surface of the protein, thereby increasing its amphipathicproperties.

Figure 2. (A) Surface representation of prototypic cyclotides fromeach subfamily: the bracelet cycloviolacin O2 (cyO2) and the Mobiuskalata B1. The backbone aligned models display the distribution ofhydrophobic/hydrophilic regions on the protein surface and theiramphipathic structure. Hydrophobic residues (Ala, Leu, Ile, Pro, Trp,Phe, Val) are in green, cationic (Arg, Lys) in blue, and anionic (Glu) inred. (B) Representative sequences of cyclotides from the twosubfamilies and a circular trypsin inhibitor. Cys residues arehighlighted in gray. (C) The sequences of the bracelet and Mobiussubfamilies summarized as sequence logos, generated by Weblogo.131

The overall height of the stack indicates the sequence conservation atthis position, while the height of symbols within the stack indicates therelative frequency of each amino or nucleic acid at this position.

Journal of Natural Products Review

dx.doi.org/10.1021/np401055j | J. Nat. Prod. 2014, 77, 724−736725

D.  K.  Craik  et  al.,  J  Org.  Chem.,  2011,  76,  4805-­‐4817.  U.  Göransson  et  al,  J  Nat.  Prod.,  2014,  77,  724-­‐736.    

Cyclo1des  –  Structural  Proper1es  

Molecular  Grading  •  Myelin  oligodendrocyte  glycopepPde  (MOG)    

–  AcPvity  against  MulPple  Sclerosis  (MS)  –  Poor  instability  and  bioavailability    

•  Graded  parts  of  MOG35-­‐55  epitope  to  Kalata  B1  as  loop  5  –  Graded  cycloPde  retained  structure  and  stability  of  parent  scaffold  –  Displayed  potency  to  prevent  disease  development  in  MS  mice  

 

Cyclo1des  –  Molecular  GraCing  

Molecular Grafting onto a Stable Framework Yields Novel CyclicPeptides for the Treatment of Multiple SclerosisConan K. Wang,†,∥ Christian W. Gruber,†,‡,∥ Masa Cemazar,† Christopher Siatskas,§ Prascilla Tagore,†

Natalie Payne,§ Guizhi Sun,§ Shunhe Wang,§ Claude C. Bernard,§ and David J. Craik*,†

†Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia‡Center for Physiology and Pharmacology, Medical University of Vienna, Schwarzspanierstrasse 17, Vienna 1090, Austria§Multiple Sclerosis Research Group, Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia

*S Supporting Information

ABSTRACT: Multiple sclerosis (MS) is an inflammatory disease ofthe central nervous system (CNS) and is characterized by thedestruction of myelin and axons leading to progressive disability.Peptide epitopes from CNS proteins, such as myelin oligodendrocyteglycoprotein (MOG), possess promising immunoregulatory potentialfor treating MS; however, their instability and poor bioavailability is amajor impediment for their use clinically. To overcome this problem,we used molecular grafting to incorporate peptide sequences fromthe MOG35−55 epitope onto a cyclotide, which is a macrocyclicpeptide scaffold that has been shown to be intrinsically stable. Usingthis approach, we designed novel cyclic peptides that retained the structure and stability of the parent scaffold. One of the graftedpeptides, MOG3, displayed potent ability to prevent disease development in a mouse model of MS. These results demonstratethe potential of bioengineered cyclic peptides for the treatment of MS.

Multiple sclerosis (MS) is an inflammatory disorder of thecentral nervous system (CNS) characterized by focal

demyelinating lesions,1 where both the cellular and humoralarms of the immune system seem to play a pivotal role in thepathogenesis of disease.2 The distinguishing pathologicalfeatures of MS are localized, episodic, and progressive CNSdemyelination, as well as axonal damage.3,4 There is nowconsiderable experimental evidence suggesting that CNSmyelin proteins might be relevant target autoantigens. Amongthese, myelin oligodendrocyte glycoprotein (MOG) stands out,not only because it is located on the outmost lamella of themyelin sheath, but also because it is expressed exclusively inCNS myelin.1,5

With the FDA approval of interferon beta-1b around 20 yearsago, the landscape of MS therapeutics changed dramatically,giving further impetus to develop safer and more effectivetreatment strategies. Although there are currently several drugsapproved for the treatment of MS and several others at late-stage clinical trial, the available therapeutics generally engagenon-specific mechanisms of immune suppression, leavingpatients susceptible to opportunistic pathogens.6 As an exampleof the inherent dangers in these approaches, a clinical trial ofNatalizumab (Tysabri) led to the deaths of several participantsfrom progressive multifocal leukoencephalopathy, a viralinfection of the brain.7 In view of the side effects of currenttherapeutics, antigen-specific strategies offer a promisingalternative as they can potentially block the deleterious effectsof specific immune components, while maintaining the abilityof the immune system to clear nonself antigens.8 A novel and

more specific approach to the treatment of MS would thereforebe the design of antigen-specific therapies directed towardMOG.Peptides have long been implicated as valuable compounds

for the development of antigen-specific therapies because theyoffer many advantages over other modalities, including highactivity and specificity. However, the clinical use of antigenicpeptide sequences is limited because of their intrinsic in vivoinstability. An emerging approach to overcome this challenge isto insert peptides into a scaffold of high stability, i.e., moleculargrafting. In terms of peptide drug design, cyclotides9 representa particularly attractive scaffold for molecular grafting becauseof their exceptional stability, which is attributed to their uniquestructural framework, comprising a cyclic backbone and acystine knot motif (Figure 1a). There are now several successfulexamples showing that the cyclotide framework can be used todesign drug leads for chronic diseases.10−15

In this study we generated several chimeric moleculesconsisting of a partial sequences of MOG grafted onto theprototypic cyclotide kalata B1, a peptide scaffold with highstability, and tested their potential to prevent diseasedevelopment in an experimentally induced mouse model ofMS. We identified a novel grafted molecule with potent in vivo

Received: July 20, 2013Accepted: October 22, 2013Published: October 22, 2013

Articles

pubs.acs.org/acschemicalbiology

© 2013 American Chemical Society 156 dx.doi.org/10.1021/cb400548s | ACS Chem. Biol. 2014, 9, 156−163

Terms of Use

D.  K.  Craik,  et  al.,  ACS  Chem  Bio,  2014,  9,  156-­‐163.    D.  K.  Craik  et  al.,  J  Org.  Chem.,  2011,  76,  4805-­‐4817.  U.  Göransson  et  al,  J  Nat.  Prod.,  2014,  77,  724-­‐736.    

EAE  =  experimental  autoimmune                          encephalomyeliPs  

44

Molecules 2012, 17 12542

peptide resin or as a reagent during nucleophilic cleavage. For the incorporation of the thioester after chain assembly, the peptide has to be synthesized on an ‘ultra-acid-labile’ resin (e.g., a TGT resin) from which the peptide can be cleaved with all side-chain protecting groups intact. To that fully protected peptide the thiol is coupled forming the desired thioester [88]. Although this methodology is compatible with the common Fmoc-strategy of peptide synthesis, it is subjected to imponderabilities due to the unpredictable solubility of fully protected peptides, especially peptides of that size. Moreover, undesired carboxyterminal racemization may occur during synthesis [88,89]. This problem can be overcome very elegantly by choosing a glycine as aminoterminal and a cysteine as carboxyterminal residue as the site of macrocyclization, because glycine is the only non-chiral amino acid and glycine-cysteine combinations exist in a number of cystine-knot peptides (Figure 1) [88]. Installation of a thioester as on-resin cleavable linker seems more elegant, as no special modification is needed [86]. Unfortunately, piperidine that is a common reagent used in Fmoc-SPPS for N-terminal deprotection is not compatible with that linker as its nucleophilic attack at the thioester results in the cleavage of the peptide chain from the resin [86]. A combination of non-nucleophilic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1-hydroxybenzotriazole (HOBt) helps to overcome that problem on the cost of an enhanced aspartimide formation [90]. Interestingly, it has not been checked so far, whether 2-methylpiperidine could solve these problems as its utility for the synthesis of peptides with piperidine-labile tyrosine sulfate esters was demonstrated [91]. The third possibility, though not yet elaborated, might be the usage of safety-catch linkers (e.g., hydrazinobenzoyl) which can be cleaved by a respective nucleophile after suitable activation [92,93].

Figure 5. Proposed cyclization via native chemical ligation.

After successful incorporation of a carboxyterminal thioester, NCL frequently provides cyclic peptides in excellent conversions or yields, respectively (Table 2) [86,88]. Although to date the mechanism is not fully understood and not all intermediates are precisely characterized, it is commonly accepted that intramolecular thioesterifications of the internal thiol groups and the carboxyterminus take place (Figure 5) [46]. This “thia-zip” rearrangement gradually increases ring size and eventually brings both termini in close proximity. As a consequence, an irreversible S, N-acyl transfer is induced, finally leading to the cyclic product [46,84]. This model is supported by various studies, in which the aminoterminus was acetylated, a linker introduced or the ring-chain tautomeric equilibrium

Prof. H. Wennemers

Cyclotides – Synthesis

D. K. Craik et al., J Org. Chem., 2011, 76, 4805-4817.

Conotoxins  (CTX‘s)  

•  Disulfide-­‐rich  venom-­‐derived  pepPdes  of  cone  snails  (genus:  Conus)    •  Structurally  related  to  CycloPdes  

–  Linear  backbone  –  Median  size:  26  AA  (6-­‐50)  –  1-­‐5  disulfide  bond(s)  

•  More  diverse  than  CycloPdes  

Conus  marmoreus  

P.  Alewood  et  al.,  Chem.  Rev.  2004,  114,    5815-­‐  5847.    R.J.  Lewis  et  al.,  Nat.  Rev.  Drug  Discovery,  2003,  2,  790-­‐802.    

•  Biological  acPvity  in  cone  snails:  –  Defence  or  prey  capture  –  Paralysis  of  the  envenomed  animal    

•  Target:  CNS  

•  Biological  acPvity  in  humans:  –  Chronic  pain  relievers  

•  Marketed  drug:  Prilat®  (INN:  ZiconoPde)  –  FDA  approved  2004  –  Intrathecal  –  directly  into  cerebrospinal  fluid  by  a  catheter  –  Non-­‐addicPve  alternaPve  to  opiates  

–  Neurological:  Epilepsy,  Alzheimer‘s,  Parkinson‘s  –  Cardiac  Arrhythmia  

 

P.  Alewood  et  al.,  Chem.  Rev.  2004,  114,    5815-­‐  5847.  M.  Brönstrup  et  al.,  Nat.  Prod.  Rep.,  2014,  31,  35-­‐60.  Picture  taken  from:  S.  Becker  and  H.  Terlau  ,  Appl.  Microbiol.  and  Biotech.,  2008.    

Conotoxins  (CTX‘s)  –  Biological  Ac1vity    

effector platform can enable the harvesting of multiple clinicaldrug candidates. It also suggests that the exploration of addi-tional effector principles is a highly rewarding eld for futurenatural product research.

5 Synthetic supply of unmodifiednatural productsIn fortunate cases, the pharmaceutical prole (in terms of effi-cacy vs. side effects) of an isolated natural product is so favorablethat it can be used as a drug without further structural modi-cation. Roughly 20% of natural product-based drugs fall into thiscategory.1 Even without a need for lead optimization, thedaunting challenge to assure a large-scale supply of (GMP-quality) drug substance remains to be solved by naturalproduct biologists and chemists. For natural products withcomplex structures, the fermentation of microbial metabolites,or the horticulture of plant metabolites, followed by purecompound isolation, has been the method of choice. In thefollowing paragraphs, we present successfully realized alterna-tives to this ‘mainstream’ – although not at all trivial – approach.

The rst two marketed marine natural product drugs zicono-tide (Prialt™, Elan Pharmaceuticals) and trabectedin (Yondelis™,PharmaMar)191 did not have a sustainable biological productionsource; consequently, chemists have developed a viable access, bytotal- and semisynthesis, to the products (chapters 5.1 and 5.2,respectively). In order to become independent from the uctua-tions of horticulture for the supply of the antimalarial productartemisinin, a combination of molecular biology, creating a novelproducer strain for a central drug intermediate, with organicsynthesis has been realized (chapter 5.3).

5.1 Ziconotide

Ziconotide is the synthetic form of a 25-amino acid peptide thatwas originally isolated in 1979 as u-conotoxin MVIIA 70 (Fig. 6)in the venom of the cone snail, Conus magnus.192 Toxic peptides(conotoxins) produced by the venomous sh-hunting conesnails (Conus spp.) can be divided into several classes thatattack various critical functions of the neuromuscular system ofthe prey. All conotoxins share several common features: they arerelatively small (13 to 29 amino acids), strongly basic peptides,which are highly cross-linked by disulde bonds.193

As the animal sources provided only minute quantitiesof peptides, total synthesis approaches for the conotoxinswere required to ensure a sustainable supply for proling. Arst solid-phase peptide synthesis of 70 was accomplished in1987 on p-methylbenzhydrylamine resin using the Merrieldprotocol. Protection of side-chain amino acid functions wasrealized by standard methods for the Boc/Bzl strategy, in the

case of the cysteine residues as p-methoxy benzyl (Mob) orN-acetyl aminomethyl (Acm) derivatives. Aer building theu-conotoxin MVIIA sequence on the resin, the completelydeprotected linear peptide with the 6 Cys residues in reducedsulydryl form was obtained following cleavage with hydrogenuoride-based cocktails. Formation of the disulde bond cross-linking in the correct connectivity was achieved by simple airoxidation at neutral pH in aqueous solution to give correctlyfolded 70. The yields of the folding step could be improved bythe use of additives, such as cysteine and DTT.

In further studies, N-type voltage-sensitive calcium channelblocking was identied as the mode of action of 70.194 Devel-opment of the compound as an antinociceptive agent wasinitiated through its high effectiveness in a rat model ofneuropathic pain.195 In 2005, ziconotide was launched in theUSA for the treatment of severe chronic pain. Due to a thera-peutic scheme with doses as low as 2.4–21.6 mg day!1,196 themultistep total synthesis on solid phase remains an economi-cally viable supply of the drug substance.

5.2 Trabectedin

Trabectedin 71 (ecteinascidin-743, ET-743), launched in Europein 2007, was the rst anti-cancer drug from a marine source.Since its discovery and development and the underlyingsynthesis efforts have recently been outlined in an excellentreview article197 we will only provide a brief summary. Itsdiscovery dates back to 1969 when antitumour activities of anextract of the marine tunicate Ecteinascidia turbinata were rstdescribed.198 However, the minute quantities of isolated ectei-nascidins hampered the nal identication and structure eluci-dation of representatives of this compound class until the1990s.199 The ecteinascidins showed exceedingly potent activityin several tumour models in mice.200 ET-743 71 was selected forfurther development due to its relatively high abundance –though the yields from the tunicate were still very low ("10 ppm)in absolute terms.199 Total synthesis enabled the delivery of morematerial for in vitro and in vivo studies: the rst synthesis pub-lished in 1996 by the Corey group provided 71 in >30 steps (0.75%total yield).201 Improvements were achieved by a more efficientand effective preparation of a key intermediate,202 but the adap-tation to a large scale synthesis still remained a major challenge.

Although the establishment of aquacultures of E. turbinatafor the production of 71 proved to be very challenging andrequired considerable efforts, this approach has provided thebatches required for clinical trials.203,204 It was neverthelessconsidered necessary to establish a sustainable, long-term drugsupply capable of serving the needs for commercialization ofthe drug. A breakthrough was nally achieved by the develop-ment of a semisynthetic process starting from the readilyavailable fermentation product cyanosafracin B 72, which canbe obtained from Pseudomonas uorescens in multi-kilogramquantities.205 As shown in Scheme 4, 72 represents a well-suited scaffold for the construction of the more complex 71and its analogs, since its structure is comprised of two of thethree fused tetrahydroisoquinoline rings present in the ectei-nascidins and displays a favourable arrangement of functionalFig. 6 The amino acid sequence of u-conotoxin MVIIA 70.

50 | Nat. Prod. Rep., 2014, 31, 35–60 This journal is © The Royal Society of Chemistry 2014

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ZiconoPde  

•  Linear  chain  assembly    •  OxidaPve  Folding  

–  Intrinsic  informaPon  or  Directed  Folding  (PG  Strategies)    

Conotoxins  (CTX‘s)  –  Chemical  Synthesis  

P.  Alewood  et  al.,  Chem.  Rev.  2004,  114,    5815-­‐  5847.