Synthesis and biological evaluation of: S -lipidated lipopeptides of a connexin 43 channel inhibitory peptide

Article abstract of DOI:10.1039/d0md00172d

The synthesis and biological activity of 42 novel S-lipidated analogues of a connexin 43 channel inhibitory Peptide5 is described. Unmodified Peptide5 moderates hemichannels and gap junctions that are both implicated in the progression of neurological disease. Peptide5 was site-specifically modified with a cysteine residue, which then underwent thiol-ene mediated S-lipidation to afford S-lipidated Peptide5 analogues containing straight-chain, branched, or aromatic lipids. The modified peptides were assessed for their effect on hemichannel opening and the most promising candidates were evaluated in serum stability studies.

Full text of DOI:10.1039/d0md00172d

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RESEARCH ARTICLE
Synthesis and biological evaluation of S-lipidated
lipopeptides of a connexin 43 channel inhibitory
peptide†
Cite this: DOI: 10.1039/d0md00172d
abd
Sung-Hyun Yang,‡ab Connor A. Clemett,‡c Margaret A. Brimble,
c
abd
*
*
Simon J. O'Carroll and Paul W. R. Harris
The synthesis and biological activity of 42 novel S-lipidated analogues of a connexin 43 channel inhibitory
Peptide5 is described. Unmodified Peptide5 moderates hemichannels and gap junctions that are both
implicated in the progression of neurological disease. Peptide5 was site-specifically modified with a
cysteine residue, which then underwent thiol–ene mediated S-lipidation to afford S-lipidated Peptide5
analogues containing straight-chain, branched, or aromatic lipids. The modified peptides were assessed for
their effect on hemichannel opening and the most promising candidates were evaluated in serum stability
studies.
Received 25th May 2020,
Accepted 18th June 2020
DOI: 10.1039/d0md00172d
rsc.li/medchem
registered lipopeptide drugs including the anti-diabetic
Introduction
agents semaglutide (Ozempic®),12 liraglutide (Victoza®)13 and
Biologically active peptides attract much attention as
potential drug candidates due to their intrinsic nature of
selective high binding affinity for their receptors with high
potency and low toxicity.1 However, the use of peptides as
therapeutics is limited by poor chemical and physical stability
such as proteolytic degradation.2 A plethora of chemical
methodologies are available for the modification of native
peptides to generate peptide mimetics with improved
pharmacokinetic and pharmacodynamic properties. Common
methods include substitution with unnatural (D)-amino acid3/
insulin detemir (Levemir®).14
Connexin (Cx)43 is a protein widely expressed within the
central nervous system where it acts to provide syncytial
formation and a high degree of intercellular communication,
particularly in endothelial and glial cells.15 Membrane-bound
connexin family members assemble into both homo- and
heteromeric hexamers referred to as connexons; like a pore,
these are dynamic in their ability to regulate the opening of a
non-proteinogenic
amino
acid,4
beta-amino
acid,5
cyclisation,6 N-methylation of the amide bond,7 PEGylation,8
glycosylation,9 phosphorylation10 and lipidation.11 Lipidation
of native peptides is an effective strategy to improve
pharmacokinetic and pharmacodynamic properties of native
peptides by increasing proteolytic stability, receptor selectivity
and potency. This strategy has been verified by the FDA-
a School of Chemical Sciences, The University of Auckland, 23 Symonds St,
Auckland, New Zealand. E-mail: paul.harris@auckland.ac.nz
b School of Biological Sciences, The University of Auckland, 3a Symonds St, Private
Bag 92019, Auckland, New Zealand
c Department of Anatomy Medical Imaging, School of Medical Sciences, Faculty of
Medical and Health Sciences, and Centre for Brain Research, University of
Auckland, Private Bag 92019, Auckland, New Zealand.
E-mail: s.ocarroll@auckland.ac.nz
d Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland,
3a Symonds St, Auckland, New Zealand
† Electronic supplementary information (ESI) available: Experimental details for
the synthesis of all peptides, analytical LC and MS, bioluminescence assay. See
DOI: 10.1039/d0md00172d
Fig.
1 Multimerisation of membrane anchored Cx43 protein. Six
monomers form a hemichannel connexon which then dock with a
neighbouring connexon to form a gap junction.
‡ These authors contributed equally to this work.
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central channel (Fig. 1). Two connexons of neighbouring cells
appose one another, forming a gap junction to link the
respective intracellular spaces.16 Perturbation of this system
can occur through tissue injury, metabolic failure, or
inflammatory activation. This leads to disruption of existing
gap junctions and the opening of unopposed connexons,
referred to as hemichannels, thereby creating a continuation
of cytoplasm with the extracellular space.17 Pathological
opening of hemichannels can lead to cell swelling,18
excitotoxic cell death,19 and vascular haemorrhage.20
Opening of hemichannels leads to the release of molecules
into the extracellular space. An example of this is adenosine
triphosphate (ATP), which when released from Cx43
hemichannels activates purinergic signalling21 and the
inflammasome pathway, ultimately leading to the secretion
of proinflammatory cytokines.22 Cx43 hemichannels are
therefore a potential drug target for treatment of neurological
conditions.23
Peptide5 (1) induces hemichannel closure with reduced gap
junction disruption,28 which makes it a better candidate for
the specific targeting of Cx43 hemichannels. However,
further work is required to maximise both the efficacy and
serum stability of Peptide5-based candidates to demonstrate
their potential as agents to treat human disease.
Our previous alanine scanning studies have identified key
amino acids required for the observed biological activity of
Peptide5 (1) and provided insight into potential sites for
further modification.26 In the current work, we report the
preparation of a library of S-lipidated analogues of Peptide5
(1), informed by our alanine scanning work, and enabled by
our ‘CLipPA’ (cysteine lipidation of peptides or amino acids)
lipidation technology. Six amino acids in the native Peptide5
(1) sequence were substituted sequentially with the thiol-
containing amino acid cysteine or a thiol acid which then
underwent S-lipidation with a range of lipidated vinyl esters
creating a unique 42-member library of novel lipopeptides. It
was found that employing high molecular weight lipids,
particularly in the SRPTEKT region within Peptide5 impaired
the ability of Peptide5 to induce hemichannel closure in an
endothelial ischaemic injury model. Pleasingly, some
substitutions that conserved the approximate mass and
polarity of the native side chains demonstrated improved
functional efficacy.
Small peptide mimetics to each of the Cx43 extracellular
loops (EL1 and EL2) have been a source of interest in
targeting the pharmacological closure of hemichannels in
cases of tissue injury as they act to mimic the EL–EL
interaction of an opposing connexon prompting channel
closure.24
A
promising
candidate,
Peptide5
(1),
(VDCFLSRPTEKT), derived from EL2 in Cx43 has been
demonstrated to provide therapeutic benefit in models of
inflammatory nervous tissue and vascular injury (Fig. 2).25
Peptide5 was found to restrict hemichannel leakage of ATP
into the extracellular space following ischaemic injury
in vitro26 providing a mechanism for its protective effects.
Existing gap junction blockers such as carbenoxolone (CBX)
have been shown to be protective in a number of disease
models27 and in limiting ATP leakage in vitro,26 but also
interfere with beneficial connexin gap-junctions. Importantly,
Results and discussion
Synthesis of peptide scaffolds (2)–(8)
We have developed a facile process to directly install lipids
onto
a
thiol-containing peptide, coined CLipPA.29,30,35
CLipPA involves a conventional thiol–ene coupling initiated
by a radical reaction between a sulphur atom and sp2–sp2
unsaturated carbon bond bearing a lipid chain (Scheme 1).
CLipPA displays high levels of chemoselectivity with
a
broad range of functional group tolerance and results in
exclusively anti-Markovnikov regioselectivity. The scope of
the direct S-lipidation of peptides using the CLipPA
technique has been established by the synthesis of
lipidated peptide hormones,30 rapid preparation of
antimicrobial peptides,29b cyclic lipopeptides,29d synthesis of
TLR-2 agonists for cancer vaccines29c,e and novel stapled
peptides.29f
We hypothesised that adding a lipid to Peptide5 (1)31 may
help to anchor the resultant lipopeptide to hydrophobic
Scheme
1 S-Lipidation of fully unprotected peptides by CLipPA
Fig. 2 Origin of Peptide5 derived from the extracellular loop 1 (EL1) of
the trans membrane domains of Cx43. CL1 = cytoplasmic loop.
reaction employing vinyl ester containing lipids under UV light. All
amino acid side chains are unprotected and remain unmodified.
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cellular membranes thereby improving its gap junction
function. Lipidation may also serve to increase the half-life of
the unmodified Peptide5 (1), which has a short half-life,
thereby limiting its clinical usefulness. Based on our previous
alanine scanning study of Peptide5 (1), we synthesised seven
scaffold peptides bearing a thiol-containing amino acid or a
thiol alkanoic acid (Table 1). Six analogues, 2–7 possessed a
single mutation in place of the native amino acid residues
coupling reagent/base for the condensation of the incoming
Fmoc-amino acid. Racemisation of Na-protected cysteine is a
well-known side reaction during chain assembly, hence
racemisation suppression conditions were employed for the
coupling of Fmoc-CysIJTrt)-OH and Fmoc-CysIJAcm)-OH by
using HATU/HOAt, and 2,4,6-trimethylpyridine in DCM/DMF
(1 : 1, v/v).33 A representative synthesis of peptide scaffold 2 is
depicted (Scheme 2).
Peptides (2–8) were then released from the resin with
concomitant removal of side chain protecting groups by
treatment of the peptidyl-resin with the cleavage cocktail,
TFA : H2O : EDT: TIPS (91.5 : 5 : 2.5 : 1, v/v/v/v). All peptides
were obtained with high purity which did not require any
further purification prior to lipidation. With peptide scaffolds
2–8 easily accessible, the S-lipidation of the free thiol was
next examined.
(1Val, Phe, Ser, Thr, 10Glu or 12Thr) employing cysteine as
a convenient thiol handle. For peptide 8 the N-terminus of
the peptide sequence was readily capped with
3-mercaptopropionic acid. Peptide5 (1) contains a cysteine
residue at the 3 position that needs to be preserved. To avoid
S-lipidation at this site, a temporary acetamidomethyl (Acm)
protecting group was introduced, which was removed
following S-lipidation on all thiol-containing peptide
scaffolds.
4
6
9
Peptide scaffolds 2–8 were synthesized by Fmoc solid
phase peptide synthesis (SPPS) on aminomethyl polystyrene
resin32 using an automated peptide synthesizer with the
reagents depicted in Scheme 2. Commercially available Fmoc-
ThrIJtBu)-HMPP (hydroxylmethylphenoxypropionic acid) was
used to immobilise the first amino acid-linker for the
synthesis of peptides 2–6 and 8, while 2-chlorotrityl resin
used to anchor the racemisation-prone cysteine residue using
non-activating conditions for peptide 7. Elongation of the
peptide chain on both loaded resins was then achieved
employing 20% (v/v) piperidine/DMF for Nα-Fmoc protecting
group removal and HATU/N,N′-diisopropylethylamine as the
S-Lipidation of peptide scaffolds using CLipPA chemistry
Readily available diverse vinyl esters were chosen as the lipid
component for the CLipPA reaction. Vinyl esters were
selected containing lipids that included straight chain alkyl
groups (propyl, nonyl, pentadecyl, heptadecyl), a branched
alkyl group (tert-butyl) and an aryl group (4-tert-butylphenyl).
These were selected as it has been reported that conjugation of
bioactive peptides with lipids affects the activity and stability
of the resultant lipopeptide that is dependent on the type
(length, bulkiness) and composition (polarity) of the lipid.34
Our optimised reaction conditions employed the vinyl ester
1
Table 1 Structures of the 42-member library of S-lipopeptide analogues of Peptide5 (1) with assorted lipid tails at the mutation sites of Val, 4Phe, 6Ser,
9Thr, 10Glu, 12Thr and N-terminus
1Val-2Asp-3Cys-4Phe-5Leu-6Ser-7Arg-8Pro-9Thr-10Glu-11Lys-12Thr
Peptide5 1
2 (62)
3 (65)
4 (52)
5 (55)
9 (62)
10 (65)
11 (52)
12 (55)
16 (64)
17 (63)
18 (58)
19 (51)
23 (55)
24 (62)
25 (60)
26 (55)
30 (53)
31 (45)
32 (55)
33 (62)
37 (55)
38 (64)
39 (49)
40 (53)
44 (58)
45 (48)
46 (55)
47 (61)
6 (62)
13 (58)
20 (52)
27 (51)
34 (51)
41 (55)
48 (51)
7 (65)
8 (52)
14 (60)
15 (52)
21 (57)
22 (55)
28 (51)
29 (52)
35 (57)
36 (51)
42 (58)
43 (51)
49 (53)
50 (55)
a
b
Parentheses indicate isolated yields of crude peptide following the resin cleavage. Parentheses indicate isolated yields following lipidation,
Acm removal and RP-HPLC purification.
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Biological evaluation of S-lipopeptide analogues of Peptide5
(1)
Limiting injury associated with Cx43 hemichannel opening
is a key mechanism for the anti-inflammatory behaviour of
Peptide5 (1) in both in vivo, and simple in vitro models of
cell or tissue slice culture.23 To test the effect of the peptide
modifications on Peptide5 activity, we employed a model of
ischemic insult on
a cell culture model of the brain
microvasculature to replicate tissue injury and damage
responses, including that of Cx43 hemichannel opening and
ATP release.26,28 This model is also applicable in the case of
a systemically delivered therapy as it is the inflammatorily
activated vasculature that likely sees the highest levels of
administered drug. The leakage of intracellular ATP into
media is a readily quantifiable means of describing the
extent of injury-induced hemichannel opening.15b To detect
this,
immortalized
human
cerebral
microvascular
endothelial cells (Applied Biological Materials, Canada; cat #
T0259) were incubated in Hypoxic-Acidic Ion-shifted Ringers
(HAIR) solution, designed to replicate the disrupted pH and
ion-balance in damaged vasculature. As ATP leaks via
hemichannels into this solution, it is easily sampled and
quantified fluorometrically by the firefly luciferase/D-
luciferin reaction (see ESI,† method 9). Consistent with
previous observations, Peptide5 (1) treatment provided a
significant reduction of ATP release over untreated controls
(53.8 3.1%) as did carbenoxolone (CBX; 23.3 3.4%), an
inhibitor of both Cx43 hemichannels and gap junctions
(Fig. 3A).
Scheme
2 The representative synthesis of peptide scaffold 2 and
S-lipopeptide 9. Conditions and reagents. (a) Fmoc-ThrIJtBu)-HMPP (2
equiv.), DIC (2 equiv.), DCM : DMF (9 : 1, v/v), r.t., overnight.; (b) iterative
Fmoc-SPPS using
a Tribute® automated peptide synthesizer i) 20%
Of the 42 S-lipopeptide analogues (9–50) of Peptide5 (1)
tested, the presence of a butyl lipid chain at each amino acid
position on Peptide5 (1) did not interfere with its bioactivity in
preventing ATP leakage compared to unmodified Peptide5 (1)
(Fig. 3B). Peptide 13 (32.0 5.1%, p = 0.0139), and 15 (28.8
4.2%, p = 0.0029) demonstrated improved bioactivity by further
limiting ATP release compared to Peptide5 (1) (53.8 3.1%). By
way of contrast, it was observed that the substitution of high
molecular weight, longer/straight chain aliphatic lipids (nonyl,
pentadecyl, and heptadecyl) did not improve the bioactivity of
Peptide5 (1) and lipidation at positions 9 (analogues 26, 33), 10
(analogues 20, 27, 34), and 12 (analogues 28, 35) reduced the
efficacy of hemichannel closure below that of the native
Peptide5 (1) sequence as demonstrated by an increase in ATP
release (Fig. 3C–E, Table 2). Given that the shorter butyl chain
is also non-polar although lesser so, but did not demonstrate
such functionality this may be suggestive of steric hindrance by
longer lipid chains occurring at the peptide–Cx43 interface.
Indeed, ATP release following treatment with modified
Peptide5 mimetics correlates positively to the similarity of the
molecular mass of the newly introduced side chains at amino
acid positions Phe-4, Glu-10, and Thr-12 (Fig. 3H). Indeed, our
previous alanine substitution studies identified that amino
piperidine/DMF (v/v), 7 min × 2, r.t.; ii) Fmoc-AA-OH (5 equiv.), HATU (4.75
equiv.), DIPEA (10 equiv.), DMF, 50 min; for either Fmoc-CysIJTrt)-OH or
Fmoc-CysIJAcm)-OH coupling; (5 equiv.), HATU (4.75 equiv.), HOAt (4.75
equiv.), 2,4,6-trimethylcollidine (8 equiv.), DCM : DMF (1 : 1, v/v), 50 min ×
2;. iii) 20% Ac2O/DMF (v/v), 1 min, r.t.; (c) TFA : H2O:EDT:TIPS (91.5:5:
t
2.5 : 1, v/v/v/v), r.t. 2 h; (d) vinyl butyrate (70 equiv.), DMPA (3 equiv.),
-
nonylmercaptan (80 equiv.), TIPS (80 equiv.), 5% TFA (v/v), NMP, 365 nm
UV irradiation, r.t. 1 h; (e) i) AgOAc (50 equiv.), MeCN : H2O (+0.1% TFA, 1:1
v/v), overnight; ii) DTT (300 equiv.), 6.0 M Gn·HCl, r.t., 1 h.
(70 equiv.), triisopropylsilane (80 equiv.), 5% (v/v) TFA in
N-methylpyrrolidinone
(NMP),
2,2-dimethoxy-2-
phenylacetophenone (DMPA) as
a
photo-induced radical
initiator, under UV light irradiation at 365 nm and an
exogenous thiol as a radical quencher. Recently, we replaced
the malodorous tert-butylmercaptan thiol quencher by the
more agreeable tert-nonylmercaptan with no effect on the
reaction and this modified procedure was also adopted in the
present work.35 Pleasingly, S-lipidation of all peptide
scaffolds 2–8 proceeded smoothly and lipidated peptides
could be obtained directly following precipitation from
diethyl ether requiring no further purification.35 The Acm
protecting group on 3Cys was then removed by treatment
with AgOAc (ref. 36) and the library of forty-two
S-lipopeptides 9–50 were obtained in moderate to high yield
after a single RP-HPLC purification step (Table 1).
6
acids within the SRPTEKT sequence (positioned at Ser–12Thr)
within Peptide5 (1) were key to elicit closure of Cx43
hemichannels.26
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Fig. 3 Modification of peptide5 side chains modulates functional behaviour in a mass- and position-dependent manner. (A) ATP release assays in
hCMVEC cultures exposed to model ischaemic injury (HAIR) alone or in combination with pan-Cx43 inhibitor CBX or Peptide5 (1) (both 10 μM).
ATP release assay in hCMVEC cultures treated with HAIR + Peptide5 mimetics 9–50 (10 μM) modified with (B) propyl, (C) nonyl, (D) pentadecyl, (E)
heptadecyl, (F) tert-butyl, (G) 4-tert-butylphenyl side-chains. A line at 53.75% on each figure indicates the level of Peptide5 (1) control. All values
represent mean SEM% relative to untreated injury (HAIR). n = 3 (S-lipopeptides), n = 16 (HAIR, NR, HAIR + CBX, HAIR + 1. (H) Linear regression of
lipid MW at AA sites 4, 10, and 12 within Peptide5 vs. the degree of ATP release when applied to injured cerebral endothelium). One-way ANOVA
with multiple comparisons vs. P5IJ1), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0001. * indicates a change above P5, # indicates a change below
Peptide5.
Table 2 Addition of high MW straight aliphatic lipid chains to the Peptide5 (1) at AA-positions 9, 10, and 12 increases ATP release in ischaemic
conditions
S-Lipopeptide analogues
Side chain modification
Mutation site
ATP (% HAIR)
p-Value vs. P5 (1)
p-Value vs. HAIR
Peptide 5 (1)
n/a
—
53.8 3.1
102.0 21.0
102.2 8.1
96.0 7.4
114.3 22.1
83.7 13.2
131.4 13.0
129.2 21.8
—
<0.0001
0.9998
0.9997
0.9994
0.8012
0.4192
0.0091
0.0294
26
33
20
27
34
28
35
Pentadecyl
Heptadecyl
Nonyl
Pentadecyl
Heptadecyl
Pentadecyl
Heptadecyl
Thr-9
Thr-9
Glu-10
Glu-10
Glu-10
Thr-12
Thr-12
<0.0001
<0.0001
<0.0001
<0.0001
0.0066
<0.0001
<0.0001
Thus, it appears that the presence of the native amino
acid side chains is required to elicit hemichannel closure in
some instances. This is consistent with the above findings
wherein it was found that altering Peptide5 (1) at Thr-9, Glu-
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10, or Thr-12 with lipids other than propyl, disrupts its mode Conclusions
of action. The addition of long aliphatic chains to Peptide5
In conclusion,
a
library of S-lipopeptide analogues of
(1) at these sites constitutes a significant change to the
overall hydrophobicity of the molecule and, likely too, its
ability to bind with an opposing Cx43 EL domain. At each
of the tested positions, substitution with the aromatic 4-tert-
butylphenyl, a linear butyl, or the branched tert-butyl lipid
chain did not significantly impair Peptide5 (1) hemichannel
blockade (Fig. 3B, F and G). However, the addition of the
tert-butyl lipid chain significantly improved peptide-induced
hemichannel closure when introduced at Phe-4 (analogue
Peptide5 (1) were synthesized using efficient “in-house”
CLipPA S-lipidation chemistry based on Fmoc SPPS. The
effect of varying the nature of the lipid chain substituents
on Cx43 hemichannel opening and ATP release was
examined using luminescence assay in vitro and proteolytic
stability was assessed in
a rat serum stability assay.
Generally, analogues possessing shorter lipid chains
displayed greatly enhanced functional efficacy activity, while
half-lives were not dramatically improved relative to the
non-lipidated Peptide5 (1). The tools developed in this
study will be used to further design lipidated analogues of
Peptide5 (1) to improve proteolytic stability and maintain
gap junction function.
38, 23.4
2.7%, p = 0.0006) (Fig. 3F). This substitution
maintains the polarity and approximate mass of the native
amino acid at this position. These data demonstrate that
chemical modification of the Peptide5 (1) structure alters its
functional behaviour in a modification- and amino acid
sequence specific-manner. The work reported herein
suggests that alteration of sites within the core Peptide5 (1)
structure to gain functional improvement can be achieved
but due care must be taken to avoid significantly disrupting
key binding interactions between it and the target Cx43
domains.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank the Auckland Medical Research Foundation (AMRF)
for generous financial support of this research (Grant Ref.
1117016). Mr Clemett was funded by a doctoral scholarship
generously provided by The CatWalk Trust.
Serum stability assay
Proteolytic degradation of peptides by proteases leads to low
tissue accumulation,37 and this is one of the inherent
limiting factors posed by peptide-based drug candidates. To
overcome this, many strategies have been employed to
improve the proteolytic stability while maintaining activity.
Peptide lipidation is of interest as the resulting lipopeptide
shows a tendency to exhibit an extended half-life time by
exploiting the long half-life of the most abundant serum
protein, human serum albumin (HSA, ∼19 days), by binding
via the lipid to fatty acid binding sites that are present on
serum albumin.38 In order to understand the effect of
S-lipidation on the proteolysis rate, the proteolytic stability of
the native Peptide5 (1) and representative lipopeptide
analogues was carried out in vitro by measuring the half-life
of the subjected peptides at 37 °C (pH 7.4) in rat serum.39
The half-life was estimated by analysis of reverse-phase high
performance liquid chromatography (RP-HPLC) data by
taking of aliquots at different time points. Synthetic
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