Vicinal disulfide constrained cyclic peptidomimetics: A turn mimetic scaffold targeting the norepinephrine transporter

Article abstract of DOI:10.1002/anie.201304660

Loopy peptides: Peptide turn mimetics of a clinically relevant norepinephrine reuptake inhibitor were developed employing a high-throughput synthesis approach to generate peptide thioesters, with subsequent cyclization through native chemical ligation. The vicinal disulfide constrained cyclic peptidomimetics (see scheme) show high structural and functional similarity to the parent peptide, though with superior metabolic stability. Copyright

Full text of DOI:10.1002/anie.201304660

Angewandte
Chemie
DOI: 10.1002/anie.201304660
Cyclic Peptides Hot Paper
Vicinal Disulfide Constrained Cyclic Peptidomimetics: a Turn Mimetic
Scaffold Targeting the Norepinephrine Transporter**
Andreas Brust,* Ching-I. A. Wang, Norelle L. Daly, Joe Kennerly, Mahsa Sadeghi,
Macdonald J. Christie, Richard J. Lewis, Mehdi Mobli,* and Paul F. Alewood*
[
1]
The design of peptidomimetics of peptide–protein or
protein–protein interactions needs to take into consideration
the specific amino acid interactions between the ligand and its
The c-conotoxin mrIa (1, sequence NGVCCGYKL-
CHOC; Scheme 1A) noncompetitively inhibits the norephi-
nephrine transporter (NET), and a closely related analogue
Xen2174 (1a, sequence UGVCCGYKLCHOC where
[
2]
protein receptor. Peptide turns play an essential role in
many recognition events, including receptor recognition in
the most abundant receptor class, G protein coupled recep-
tors (GPCRs), with many bioactive peptides adopting g or b
turn conformations. Strategies for making peptide turn
mimetics range from local peptide amino acid modification to
the development of small-molecule turn mimetics.
ever, these approaches often generate overly flexible scaf-
folds that reduce receptor selectivity and potency , or
require complex synthetic strategies. In the absence of
precise three-dimensional structures for ligands in complex
with membrane proteins , the introduction of conforma-
tional constraints (cyclization and turn mimetics) is a promis-
ing approach for the development of bioactive peptidomi-
[
2]
[3]
[
3]
[
1–2,4]
How-
[5]
[6]
[
7]
[8]
[9]
metics. Such strategies have produced clinically relevant
and potent peptidomimetics of somatostatin, angiotensin II
receptor antagonists, integrin inhibitors, and oxytocin and
[
3]
vasopressin analogues.
To date, there is no design strategy that can generate
readily accessible diverse sets of turn mimetics with proper-
ties appropriate for further drug development. Here, we
describe
a broadly applicable combinatorial chemistry
approach that addresses some of these shortcomings, and
use it to design a norepinephrine reuptake inhibitory peptide
mimetic of exceptional metabolic stability.
[
10]
Scheme 1. A) NMR structure of c-conopeptide 1 highlighting the
pharmacophore of this norepinephrine reuptake inhibitor. B) Pharma-
cophore (GYKL) of 1 represented by an inverse turn. C) Design of N-
to-C cyclic vicinal disulfide constrained peptide turn mimetics with X
indicating the modification sites.
[*] Dr. A. Brust, Dr. C.-I. A. Wang, J. Kennerly, Prof. R. J. Lewis,
Prof. P. F. Alewood
Institute of Molecular Bioscience, The University of Queensland
St Lucia QLD 4072 (Australia)
E-mail: a.brust@imb.uq.edu.au
U = pyroglutamyl) is currently under development for the
[10a,11]
p.alewood@imb.uq.edu.au
treatment of severe pain.
These c-conotoxins contain the
Dr. M. Mobli
sequence GYKL, which is essential for high-affinity NET
[10a]
Centre for Advanced Imaging, The University of Queensland
St Lucia 4072 QLD (Australia)
E-mail: m.mobli@uq.edu.au
inhibition.
Structural studies reveal that the rigid frame-
work formed by two disulfide bonds between the cysteine
residues (1–4, 2–3) induces a b hairpin with two antiparallel
strands (Scheme 1A) connected by an inverse g turn com-
M. Sadeghi, Prof. M. J. Christie
Discipline of Pharmacology, University of Sydney (Australia)
[
10b]
prising GYK,
thereby further constraining the pharmaco-
Prof. N. L. Daly
phore (Scheme 1B).
Norelle Daly Centre for Biodiscovery and Molecular Development of
Therapeutics, School of Pharmacy and Molecular Sciences
James Cook University, Cairns (Australia)
Our initial design strategy involved incorporating the
YKL pharmacophore into a cyclic peptide. To minimize the
size of the ring and restrict flexibility, we incorporated
a vicinal disulfide bond as a further constraint (Scheme 1C)
and then varied the amino acids (indicated as X in
Scheme 1C) adjacent to the YKL pharmacophore to modu-
[
**] We acknowledge financial support by NHMRC (Program Grant No.
351446).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201304660.
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late structure and gain additional structure–activity informa-
tion. Vicinal disulfide bonds are found in the active sites of
thiol resin 3 was then used to produce the cyclic-peptide
libraries (Scheme 1C) of type 8 (Scheme 2) in a split-and-mix
fashion using standard peptide coupling conditions. After
chain assembly was complete, the thioester-loaded lanterns 4
were transferred together into a single HF cleavage vessel,
which allowed removal of the side-chain protecting groups,
while the HF stable SCAL maintained the deprotected
thioester on the resin (4!5). The free thioester peptides 7
were obtained after sorting the lanterns into individual vials
[12]
[12,13]
enzymes and near the binding sites of receptors,
as in toxin peptides. In proteins, this unique 8-membered
as well
[
14]
ring prefers a type VIII turn with a distorted trans-amide
[
12]
conformation, while in small model peptides, cis or trans
[
15]
amide bond conformations are observed.
vicinal disulfide bonds may function as regulatory switches
through changes in their oxidation states.
In receptors,
[
12,16]
We used intramolecular native chemical ligation (NCL) to
access these designed turn mimetics. Conveniently, the NCL
approach allows access to a diverse range of cyclopeptides
without the need for complex side chain protection strategies.
To produce cyclic peptide libraries where X is variable
and adding NH I/DMS/TFA, which both reduces the SCAL
4
sulfoxide (5!6) and renders the substituted benzhydryl-
[
17]
amine linkage TFA labile, while oxidizing the vicinal disulfide
[
18–19]
bond to yield 7.
The crude peptide thioesters 7 were
readily N-C cyclized in NH HCO buffer (0.1m) to yield 8, the
4
3
(
Scheme 1C), we employed tert-butoxycarbonyl (Boc)
formation of which is autocatalyzed by the thiol released from
the linker moiety during cyclization (see the Supporting
Information).
chemistry to synthesize the peptide thioesters necessary for
the NCL cyclization. Although Boc chemistry is well estab-
lished and robust, throughput is limited by the final HF
cleavage step. To overcome this bottleneck, we employed
the combination of a safety-catch amide linker and a resin
support that is compartmentalized to allow easy manual
handling and sorting (Scheme 2).
Cyclic peptides 8a–8j were tested using a functional
human norepinephrine transporter assay. Table 1 shows that
our design strategy successfully generated active mimetics of
peptide 1, with the vicinally constrained cyclic heptapeptide
8a (CCGYKLG) being equipotent to 1. Other analogues
showed that we could manipulate potency by varying either
the pharmacophoric residues or residues at positions X. To
confirm that the vicinal disulfide bond is a structurally
[
17]
Starting with Mimotopes aminomethylated lanterns,
[
18]
a safety-catch linker (SCAL) was attached (2), followed
by a Gly spacer and a mercaptopropionic acid linker. The
Scheme 2. High-throughput synthesis of peptide thioester 7 and intramolecular native chemical ligation, yielding cyclic peptide turn mimetics (8):
A combination of a safety-catch linker/thiol linker (2!3) together with compartmentalized resin (lanterns) allows for split-and-mix Boc synthesis
(
3!4) and parallel hydrogen fluoride deprotection (!5). After sorting and safety-catch linker activation (5!6), peptide thioester 7 is obtained.
Native chemical ligation yields cyclic peptides of type 8. HBTU=O-(benzotriazol-l-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate,
DIEA=diisopropylethylamine, Trt=trityl, TFA= trifluoroacetic acid, TIPS=triisopropylsilane, DMS=dimethyl sulfide, Pg=protecting group,
PS-AM-SCAL=safety-catch amide linker on aminomethylated polystyrene.
2
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Chemie
Table 1: Norepinephrine reuptake inhibition (IC50 ꢁSEM) by vicinal
disulfide constrained cyclic peptidomimetics 8a–8j, and c-conopeptide
1
(1–4, 2–3 disulfide bond). Reduced and alkylated products 9 and 10
show decreased activity owing to loss of the vicinal disulfide constraint.
Entry
Sequence
NGVCCGYKLCHOC
IC50 [mm]
[
a]
1
2.45ꢁ0.07
2.55ꢁ0.18
3.9ꢁ0.2
2.3ꢁ0.3
31.6ꢁ4
18ꢁ1.2
117ꢁ2.8
72ꢁ6
[
a]
1
8
8
8
8
8
8
8
8
8
8
9
1
a
a
b
c
d
e
f
g
h
i
UGVCCGYKLCHOC
(CCGYKLG)
[
b]
(CCGYK[CHA]G)
(CCGYKL-)
(CCGYK[CHA]-)
(CCEYKLG)
[
b]
(CCFYKLG)
(CCH(Dnp)YKLG)
(CCLYKLG)
[
c]
24ꢁ11
63ꢁ20
28ꢁ11
87ꢁ14
20ꢁ3
(CCWYKLG)
(CCFYKL-)
([C-Saa][C-Saa]GYKLG)
j
[
d]
[
e]
0
([C-SMe][C-SMe]GYKLG)
49ꢁ8
[
a] (U=pyroglutamic acid, O=hydroxyproline), [b] CHA=cyclohexyl
alanine. [c] Dnp=2,5-dinitrophenyl, [d] Saa=S-acetamidated cysteine
=S-CH CONH ), [e] SMe=S-methylated cysteine.
(
2
2
essential element for the cyclopeptide bioactivity, we reduced
peptidomimetic 8a using tris(2-carboxyethyl)phosphine
(
TCEP), and S-alkylated the cysteine residues with iodoacet-
amide or iodomethane. The cyclic and S-alkylated products (9
and 10, Table 1) showed an approximately 10-fold loss in
activity, confirming the importance of the vicinal disulfide
bond.
Scheme 3. A) NMR structure of vicinal disulfide constrained cyclic
peptidomimetic 8a. B) Superposition of the pharmacophore region of
c-conopeptide (1, gray) with mimetic 8a (red).
The structure of mimetic 8a in water (with 5% D O) was
2
1
determined by 1D H NMR and 2D NMR spectroscopy
TOCSY, NOESY, H- C HSQC) at a H frequency of
1
13
1
(
9
00 MHz. Structure calculations were performed using the
albumin adsorption (see the Supporting Information), and
remained predominantly intact for at least 50 h (Scheme 4).
This stability can be attributed to the N-to-C cyclization but
the stability of the disulfide bond (redox potential of 8a
ꢀ313 mV, see the Supporting Information), which forms
[
20]
torsion angle dynamics approach of CYANA v3.0
automated NOE assignments. The NOE data was supple-
mented with backbone angle restraints based on H and
chemical shift data using TALOS + .
with
[
23]
1
13
C
[
21]
Sufficient NOE
interactions enabled determination of a high-resolution
structure of 8a (Scheme 3A). The vicinal disulfide bond in
8
a results in a distorted trans-amide bond (418 from
planarity), and this observation is consistent with previous
[22]
reports (see also the Supporting Information). This geom-
etry allows the formation of a hydrogen bond between Cys1-
(
CO) and Tyr4-(NH), an interaction that stabilizes the CCGY
region in a b turn conformation (Scheme 3A). Superposition
of the YKL side chains of the parent compound 1 and mimetic
8
a revealed a high degree of overlap, suggesting that the
mimetic retained the pharmacophore of 1 (root-mean-square
deviation (RMSD) over Ca atoms: 0.06 ꢀ and Cb atoms:
0
.22 ꢀ, see Scheme 3B). In contrast, NMR analysis of the
Scheme 4. Stability of cyclic peptidomimetics 8a (c c) and 8b
&
reduced and methylated 10 produced results consistent with
a random coil, with no long-range interactions, thus confirm-
ing that the vicinal disulfide bond provides a key structural
constraint (see the Supporting Information).
(c~c) in rat plasma compared with clinical candidate 1a
(c*c).
readily even under highly acidic conditions, may also con-
We further evaluated the suitability of analogues 8a and
b as clinical leads. Unlike many peptides, we found that both
tribute. The redox potential of 8a compared well with values
[
15]
8
reported for similar transoid vicinal disulfides,
and it is
peptidomimetic 8a, which is stable in trypsin (see the
Supporting Information), and peptidomimetic 8b had excep-
tional stability in plasma that was not derived from serum
surprisingly more stable than many naturally occurring
[
24]
disulfides.
We also established that mimetic 8a inhibits
NET in isolated rat brain slices (IC = 4.2 mm; see the
5
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.
Angewandte
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Supporting Information), thereby indicating stability not only
in plasma but also in brain tissue.
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In conclusion, we herein describe a synthesis of peptide
turn mimetics that combines a novel high-throughput combi-
natorial strategy for peptide thioesters, and their subsequent
conversion into backbone cyclic peptides containing a vicinal
disulfide constraint. Our results demonstrate that it is possible
to graft an active peptide turn onto a vicinal disulfide
constrained cyclic scaffold to improve both chemical and
biological stability while maintaining full potency. Although
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we
believe that this approach could be broadly applied to the
design of turn mimetics for other peptide turns that are less
well studied. This is due to the fact that it is the topological
orientation of the pharmacophore side chains that largely
[25]
determines bioactivity, rather than the specific turn type.
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257 – 1267.
Received: May 30, 2013
Revised: August 2, 2013
Published online: && &&, &&&&
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[
[
[
Keywords: biological activity · cyclic peptides ·
.
combinatorial chemistry · drug discovery · peptidomimetics
[
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4
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Angew. Chem. Int. Ed. 2013, 52, 1 – 5
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Angewandte
Chemie
Communications
Cyclic Peptides
Loopy peptides: Peptide turn mimetics of
a clinically relevant norepinephrine reup-
take inhibitor were developed employing
a high-throughput synthesis approach to
generate peptide thioesters, with subse-
quent cyclization through native chemical
ligation. The vicinal disulfide constrained
cyclic peptidomimetics (see scheme)
show high structural and functional sim-
ilarity to the parent peptide, though with
superior metabolic stability.
A. Brust,* C.-I. A. Wang, N. L. Daly,
J. Kennerly, M. Sadeghi, M. J. Christie,
R. J. Lewis, M. Mobli,*
P. F. Alewood*
&&&& — &&&&
Vicinal Disulfide Constrained Cyclic
Peptidomimetics: a Turn Mimetic
Scaffold Targeting the Norepinephrine
Transporter
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!