peptides 2016 1 - eth z · 1. occurrence and functions of peptides in nature and every day life...
TRANSCRIPT
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
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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.