Thiol-ene enabled preparation of S -lipidated anti-HBV peptides

Article abstract of DOI:10.1039/d0ob01997f

Despite significant efforts made towards treatments for Hepatitis B virus (HBV), a long-term curative treatment has thus far eluded scientists. Recently, the Sodium Taurocholate Co-Transporting Polypeptide (NTCP) receptor has been identified as the entry pathway of HBV into hepatocytes. Myrcludex B, an N-terminally myristoylated 47-mer peptide mimic of the preS1 domain of the Hepatitis B virion, was identified as a potent protein-protein interaction (PPI) inhibitor blocking HBV fusion (IC50 = 140 pM). Herein we report an optimised chemical synthesis of Myrcludex B and a series of novel analogues. Employing a small modification to the Cysteine Lipidation of a Peptide or Amino acid (CLipPA) thiol-ene reaction, a library of S-lipidated Myrcludex B and truncated (21-mer) analogues were prepared, providing novel chemical space to probe for the discovery of novel anti-HBV peptides. The S-lipidated analogues showed an equivalent or a slight decrease (～2-fold) in binding effectiveness to NTCP expressing hepatocytes compared to Myrcludex B. Three S-lipidated analogues were highly potent HBV inhibitors (IC50 0.97-3.32 nM). These results demonstrate that incorporation of heteroatoms into the lipid 'anchor' is tolerated by this antiviral scaffold and to the best of our knowledge constitutes the first report of potent S-lipidated antiviral peptides. Interestingly, despite only moderate reductions in binding effectiveness, truncated analogues possessed dramatically reduced inhibitory activity thus providing new insights into the structure activity relationship of these hitherto unreported antiviral S-lipopeptides.

Full text of DOI:10.1039/d0ob01997f

Organic &
Biomolecular Chemistry
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Thiol–ene enabled preparation of S-lipidated anti-
HBV peptides†
Cite this: DOI: 10.1039/d0ob01997f
Oscar A. Shepperson,a,b,c Alan J. Cameron,
*
a,b,c Carol J. Wang,b
a,b,c
Paul W. R. Harris, a,b,c John A. Taylorb,c and Margaret A. Brimble
*
Despite significant efforts made towards treatments for Hepatitis B virus (HBV), a long-term curative treat-
ment has thus far eluded scientists. Recently, the Sodium Taurocholate Co-Transporting Polypeptide
(NTCP) receptor has been identified as the entry pathway of HBV into hepatocytes. Myrcludex B, an
N-terminally myristoylated 47-mer peptide mimic of the preS1 domain of the Hepatitis B virion, was
identified as a potent protein–protein interaction (PPI) inhibitor blocking HBV fusion (IC50 = 140 pM).
Herein we report an optimised chemical synthesis of Myrcludex B and a series of novel analogues.
Employing a small modification to the Cysteine Lipidation of a Peptide or Amino acid (CLipPA) thiol–ene
reaction, a library of S-lipidated Myrcludex B and truncated (21-mer) analogues were prepared, providing
novel chemical space to probe for the discovery of novel anti-HBV peptides. The S-lipidated analogues
showed an equivalent or a slight decrease (∼2-fold) in binding effectiveness to NTCP expressing hepato-
cytes compared to Myrcludex B. Three S-lipidated analogues were highly potent HBV inhibitors (IC50
0.97–3.32 nM). These results demonstrate that incorporation of heteroatoms into the lipid ‘anchor’ is tol-
erated by this antiviral scaffold and to the best of our knowledge constitutes the first report of potent
S-lipidated antiviral peptides. Interestingly, despite only moderate reductions in binding effectiveness,
truncated analogues possessed dramatically reduced inhibitory activity thus providing new insights into
the structure activity relationship of these hitherto unreported antiviral S-lipopeptides.
Received 30th September 2020,
Accepted 1st November 2020
DOI: 10.1039/d0ob01997f
rsc.li/obc
in patients. Thus, there is a need to develop new or novel com-
binatorial treatments that can eliminate persistent viral infec-
Introduction
Hepatitis B virus (HBV) is responsible for chronic infection in tion in chronic HBV sufferers.3,4
millions of people worldwide and is a major factor in the
development of liver cirrhosis and hepatocellular carcinoma.1
HBV infects hepatocytes via interaction of the preS1 region
of the HBV large surface protein with the virus entry receptor,
Currently, HBV infection is treated with either nucleoside the Sodium (Na) Taurocholate Co-transporting Polypeptide
analogues, non-/PEGylated interferon-α or a combination of (NTCP).5 Identification of NTCP as an HBV receptor elucidates
the two.2,3 Although these current HBV treatments exist, their the fusion process as an attractive target for drug development.
cost often limits availability in the countries of highest need. While searching for new methods of treatment for chronic
Furthermore, while treatment is effective in suppression of HBV, Gripon et al. developed a 47-mer N-lipidated peptide ana-
viral replication to low or undetectable levels, it is rarely cura- logue of the N-terminal sequence of the preS1 domain with
tive, requiring lifelong therapy to maintain a virus-free status the ability to inhibit HBV infection.6,7 Subsequent studies
involving variation of the peptide sequence and the nature of
the lipid established Bulervitide, coined ‘Mycludex B’, as the
lead analogue for clinical trials, with IC50 potency of 140 pM
aSchool of Chemical Sciences, The University of Auckland, 23 Symonds St,
towards HBV.8,9 Myrcludex B is a “first-in-class” drug that acts
Auckland 1010, New Zealand
as a competitive protein–protein interaction (PPI) inhibitor of
HBV and the NTCP receptor.10 Urban et al. determined that
lipidation was essential for the inhibitory activity of Myrcludex
B, and proposed it acts as a membrane ‘anchor’ increasing
local concentration through membrane affinity.5 Further
investigations by Schulze et al. determined that a myristoylated
and truncated sequence of Myrcludex B (21-mer) retained
activity, albeit with reduced potency (IC50 = 3 nM).11 A similar
bSchool of Biological Sciences, The University of Auckland, 3A Symonds St,
Auckland 1010, New Zealand
cMaurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland,
3A Symonds St, Auckland 1010, New Zealand. E-mail: m.brimble@auckland.ac.nz,
alan.cameron@auckland.ac.nz
†Electronic supplementary information (ESI) available: Experimental procedures
and characterisation of intermediate and final compounds (1H, 13C spectra,
RP-HPLC traces, and high and low-res MS) along with biological data. See DOI:
10.1039/d0ob01997f
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Scheme 1 3-MPA modified CLipPA lipidation of a model peptide (blue) via a radical intermediate leading to the desired S-lipidated product; the
undesired bis-adduct is not observed under our optimized conditions.15
approach using lipopeptides as fusion inhibitors has recently S-lipidated Myrcludex B and truncated Myrcludex B analogues
been investigated for coronaviruses and HIV.12,13
bearing distinctive lipid moieties prepared from a modified
We recently reported a novel lipidation technology, based on Myrcludex B scaffold using the CLipPA thiol–ene reaction, by
thiol–ene chemistry, termed “CLipPA” – Cysteine Lipidation on a incorporation of the thiol handle, 3-mercaptopropionic acid
Peptide or Amino Acid, that enables a lipid to be installed selec- (3-MPA). The analogues were additionally prepared as fluores-
tively onto a peptide upon UV irradiation (Scheme 1).14–17 This cently labelled variants for the determination of binding
strategy has previously been applied to the preparation of efficiency and ultimately for correlation with HBV inhibitory
S-lipidated peptides with a range of therapeutic properties, potency. To the best of our knowledge this is the first report of
including antibacterial lipopeptides, self-adjuvating vaccines and potent S-lipidated antiviral peptides and the first study
calcitonin gene-related peptide (CGRP) receptor antagonists.18–23 attempting to correlate NTCP binding with inhibitory activity.
The CLipPA reaction is carried out by irradiation (365 nm)
of an NMP solution containing a peptide bearing a thiol
handle, a vinyl ester containing the lipid of a desired chain
length and a photoinitiator, 2,2-dimethoxy-2-pheynlacetophe-
none (DMPA). The sulfur radical species thus generated
Results/discussion
Library design
quickly reacts with the unsaturated vinyl group of the ester to The S-lipidated analogue series was designed to incorporate
give a stabilised radical intermediate. Hydride transfer by tert- two unique peptide sequences, one mimicking the truncated
nonylthiol (tNonSH) and triisopropylsilane (TIPS) then affords sequence reported by Schulze et al. (residues 2–21 of
the desired S-lipidated product (Scheme 1). In the absence of a Myrcludex B) and one equivalent to Myrcludex B (47 residues)
hydride reagent, an undesired bis-adduct forms together with (Fig. 1).9,11 By replacing Gly1 at the N-terminus with 3-mercap-
the desired product. The apparent pH of the reaction is topropionic acid (3-MPA), these sequences were thus functio-
lowered using trifluoroacetic acid (TFA) to protonate electron nalised with the necessary thiol handle required for
rich side-chain residues, thus preventing propagation of implementation of the desired chemo-selective S-lipidation
radical species by single-electron transfer. The reaction con- strategy. Towards this end, 3-MPA was chosen rather than
ditions are mild, generally high-yielding, atom economical, cysteine to avoid the addition of a positively charged amino
and chemo-selective. Thus, fully deprotected peptides are group (at physiological pH) to the hydrophobic region at the
amenable to CLipPA.
N-terminus of the peptides. A series of four lipids (Fig. 1) were
We envisaged the CLipPA lipidation strategy could be used selected to encompass a variety of different functionalities,
for the facile preparation of a library of S-lipidated Myrcludex such as aromatic groups, branched lipids and long chain alkyl
B analogues from a common precursor, in which the lipid lipids similar to the lipid in the parent sequence. This would
portion was varied to encompass structural diversity, including enable a direct comparison of the effects imparted by the
aromatic moieties. We herein report a library of potent heteroatoms present in the CLipPA bridging unit.
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Fig. 1 Library of envisioned analogues for truncated/full length/labelled and non-labelled peptides. Lipidation highlighted in blue. Amino acids rep-
resented as single letter code; X = 3-mercaptopropanoyl.
A series of 5(6)-carboxyfluorescein (5(6)-CF) labelled ana- resin (residues 27 to 47), ESI-MS revealed masses of +66 m/z
logues were additionally prepared for binding assays with and +132 m/z (relative to the desired mass), attributed to piper-
NTCP expressing hepatocytes. As the literature in the past has idide adduct(s) formed on aspartic acid residue(s) during itera-
not been definitive as to the site of fluorescent labelling, we tive Fmoc removals with piperidine/DMF (1 : 4 v/v) (2 × 5 min, rt).
determined the position of our tag from previous SAR studies. Michels et al. reported that the addition of formic acid (5% v/v)
Given that the C-terminus is reported to be less essential for to the Fmoc deprotection solution was effective in suppres-
activity, the side chain of Lys45 near the C-terminus of the full- sing the formation of these difficult to separate adducts.24,25
length peptides was labelled. As the truncated sequence pos- While implementing this modification to our synthesis proved
sessed no Lys in close proximity to the C-terminus, a β-Ala successful in eliminating the formation of piperidide adducts,
spacer followed by a Lys residue were inserted at the the synthesis ultimately remained unsuccessful, with a mini
C-terminus to enable fluorescent labelling (3 and 3a–d, Fig. 1). cleavage of the peptide at residue 23 revealing multiple del-
etions and complete absence of the desired peptide mass
(Fig. 2, lower). Upon switching from a polystyrene based resin
(0.94 mmol g−1) to an aminomethyl ChemMatrix® resin
(0.62 mmol g−1), little improvement was achieved (Fig. 2,
The truncated sequence (1), the parent compound
(Myrcludex B, 2) and their 5(6)-CF labelled derivatives (3 and 4)
were prepared using the native myristic acid as the lipid com-
ponent to serve as controls during biological evaluations. In
addition, a non-lipidated fluorescently labelled 47-mer peptide
(5) (see ESI: S5.20†) was prepared as a further control for the
binding assays.
middle), however, the use of the lower loading (0.27 mmol g−1
)
TentaGel® S NH2 resin resulted in a highly successful syn-
thesis of Myrcludex B (2) (Fig. 2, upper). Implementing this
modified synthesis, crude 47-mer lipopeptide (2) was yielded
in ∼85% purity (Fig. 2, upper) after resin cleavage and global
Peptide synthesis
deprotection using
a
cocktail of TFA/EDT/H2O/TIPS
Controls and CLipPA precursors. Our synthesis began with
the reference peptide Myrcludex B (2) employing 9-fluorenyl-
methoxycarbonyl solid phase peptide synthesis (Fmoc-SPPS),
where couplings were performed with 2-(7-aza-1H-benzotria-
(94 : 2 : 2 : 2 v/v/v/v) for 3 h at rt. Having optimised an effective
synthetic procedure, the amended protocol was adapted for
use with an automated synthesiser, in which the base was
swapped from DIPEA to N-methylmorpholine (NMM)
(Scheme 2) for improved miscibility in DMF, with little to no
effect on the success of the synthesis. Following this pro-
cedure, the truncated analogue (1) and CLipPA precursor
zole-1-yl)-1,1,3,-tertramethyluronium
hexafluorophosphate
(HATU) and N,N-diisopropylethylamine (DIPEA) (1 h, rt). Fmoc
removal was performed with piperidine/dimethylformamide
(DMF) (1 : 4 v/v) (2 × 5 min, rt). During the attempted syn-
thesis, upon mini-cleavage of the partially elongated peptidyl peptide (2s) in which the N-terminal glycine was replaced with
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RP-HPLC and ESI-MS upon resin mini-cleavage, in accordance
with the literature).27 Upon resin cleavage with TFA/EDT/H2O/
TIPS (94 : 2 : 2 : 2 v/v/v/v) (3 h, rt) the crude native myristoylated
peptides (3 and 4) and thiol bearing CLipPA precursors (3s
and 4s) were afforded in >70% purity. These fluorescently
labelled peptides were purified via semi-preparative RP-HPLC
to afford the desired peptides in >95% purity with yields
ranging from 12%–30%.
Peptide S-lipidation
The desired lipid moieties were functionalised as vinyl esters
(Scheme 4), following the procedures reported by Magrone
et al.28 Purified peptides (2s/3s and 4s) were subjected to the
CLipPA thiol–ene reaction with each of the vinyl esters (12a–
12d). Each peptide, the photoinitiator (DMPA) and the CLipPA
additives were dissolved in a solution of NMP with the desired
vinyl ester and the reaction mixture irradiated under UV
(365 nm) for one hour to effect CLipPA S-lipidation.16 After
irradiation, the reaction mixture containing the crude CLipPA
product was triturated with diethyl ether prior to purification,
thereby removing non-peptidyl by-products (Fig. 3).
CLipPA conjugation of vinyl esters (12a–d) with peptide pre-
cursors 2s and 4s afforded S-lipidated 47-mer Myrcludex B ana-
logues 2a–d and 4a–d, respectively, while conjugation with
peptide precursors 3s afforded truncated analogues (3a–d)
(Schemes 2 and 3) with conversions ranging 60%–90% and iso-
lated yields ranging 6%–37%. Conversion rates of the
S-lipidation were moderately dependent on the precursor
peptide, vinyl ester and reaction scale. The 47-mer thiol func-
tionalised peptides 2s and 4s and the straight chain vinyl ester
Fig. 2 RP-HPLC traces of initial attempts to synthesise Myrcludex B (2).
(lower) Polystyrene based resin, (middle) aminomethyl ChemMatrix®
resin and (upper) TentaGel® S NH2 resin. Desired final product only
observed for TentaGel® S NH2. Other syntheses were aborted mid
sequence due to the presence of many unidentified by-products.
3-mercaptopropionic acid were successfully prepared with (vinyl myristate, 12a) provided the greatest conversion rates.
crude purities >75%. The crude peptides were then purified to The S-lipidated peptide analogues were then purified via semi-
>95% by RP-HPLC to afford the pure peptides (1) and (2s) in preparative RP-HPLC to >95% purity.
21% and 30% yield respectively, for either direct use in biologi-
Lipopeptide binding to HepG2-NTCP cells
cal assays or CLipPA S-lipidation as appropriate. While not
necessary for successful CLipPA thiol–ene lipidation, the thiol The binding of the lipopeptides to hepatocytes expressing
bearing precursor peptides were first purified to facilitate reac- NTCP was assessed rather than the isolated receptor, as the
tion monitoring and comparison of the conversion rates for lipid is thought to play an important role as an ‘anchor’ in the
the four structurally diverse lipids.
cell membrane.5,29 All ten fluorescently labelled peptides (3,
5(6)-Carboxyfluorescein labelled peptides. By incorporating 3a–d and 4, 4a–d) were titrated (800 nM–5 nM) in triplicate
a Lys residue with the fully orthogonal (1-(4,4′-dimethyl-2,6- experiments against HepG2 NTCP cells and their relative
dioxocyclohexylidene)-3-ethyl) Dde as a side chain protecting receptor binding affinity measured by flow cytometry, employ-
group in the aforementioned positions of the sequence, fluor- ing Myrcludex B (4) as a positive control. Lipopeptide 4 bound
escently labelled analogues of each biological control peptides exclusively to NTCP expressing HepG2 cells and not to the par-
(3 and 4) and CLipPA precursor peptides (3s and 4s) were pre- ental HepG2 cell line that lacks expression of the HBV recep-
pared (Scheme 3) alongside the non-lipidated biological tor. The non-lipidated 47-mer peptide (5) was incubated at 400
control peptide (5, shown in ESI, S5.20†).26 After complete nM with both HepG2 and HepG2 NTCP cell lines, showing no
sequence elongation, the Dde protecting group of each pepti- binding with either cell line, confirming N-terminal lipidation
dyl-resin was then selectively removed by treatment with a as a prerequisite for peptide–receptor binding. Observation by
solution of hydrazine in DMF (2% v/v) (3 × 3 min, rt). Having fluorescent microscopy confirmed these findings (Fig. 4).
liberated the lysine ε-amino group, 5(6)-CF was coupled under
Upon titration of the S-lipidated analogues, a concentration
base free conditions using 6-chloro-1-hydroxylbenzotriazole dependant relationship was observed. Increasing peptide concen-
(6-Cl-HOBt) and N,N′-diisopropylcarbodiimide (DIC) (3 h, rt), trations produced greater mean fluorescence, with the saturation
followed by three treatments with the standard Fmoc removal at ∼200 nM for the majority of lipopeptides. No further increase
solution to effect aminolysis of the 5(6)-CF groups polymerised in mean fluorescence was observed upon increasing peptide con-
onto the peptidyl resin via the 5(6)-CF phenol (as observed by centrations to 800 nM. These experiments were used to derive
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Scheme 2 Preparation of Myrcludex B analogues.
EC50 values to compare the binding effectiveness of each peptide ‘anchor’ during NTCP binding. Interestingly, the truncated
for NTCP expressing HepG2 cells. The S-lipidated analogues analogue bearing native myristoylation (3) appeared to bind
demonstrated comparable binding (Table 1) to the native myris- most strongly of the lipopeptides. This was surprising given
toylated sequences (Tables 1 and 2 and Fig. 5 and 6). The 47-mer previous literature reported the viral inhibitory effect of the
lipopeptides (Table 1, Fig. 5) demonstrated EC50 values ranging truncated analogue to be ∼20-fold less than that of the full-
45.69 nM to 79.85 nM and in general bound more effectively length 47-mer.11 While a possible explanation could be that
than their equivalently S-lipidated truncated analogues [EC50 incorporation of the relatively large fluorescent tag favoured
values ranging 40.55 nM to 213.1 nM] (Table 2 and Fig. 6).
interaction with the NTCP receptor, this observation did not
For both the full length 47 residue and truncated analogue hold true for our S-lipidated analogues (vide supra), which
series, the S-lipidated 8-phenyloctanoate analogue showed the bound less effectively than their corresponding 47-mers.
best binding [3c, EC50 = 101.0 nM and 4c, EC50 = 59.72 nM], Overall, the binding experiments were encouraging and the
suggesting aromatic moieties can be well-tolerated by the lipid S-lipidated compound series were progressed for evaluation of
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Scheme 3 Preparation of 5(6)-carboxyfluorecein labelled Myrcludex B analogues.
inhibitory activity in order to examine the correlation between secreted into the cell culture medium 9 days post-infection, by
binding and inhibitory potency.
enzyme-linked immunosorbent assay (ELISA), and data nor-
malised to a control infection in the absence of an inhibitor.
The truncated lipopeptide (1) and its 5(6)-CF labelled counter-
Antiviral activity of lipopeptides
Eight lipopeptides were investigated for their HBV inhibitory part (3) were first evaluated for inhibitory activity (Table 3,
effect. Inhibition was determined by measurement of HBeAg Fig. 7), to investigate if the fluorescent tag may have contribu-
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Scheme 4 Synthesis of vinyl esters for CLipPA chemistry. Reagents and
conditions: (Hg(OAc)2 (0.025 equiv.), conc. H2SO4 (cat., 15 μL), 74 °C;
reactions were conducted on gram scale with vinyl acetate (10 equiv.) as
solvent. Reaction time and % yield stated below. 12b structure indicative
only (mixture of vinyl neodecanoate isomers).
Fig. 4 Fluorescent microscopy of 5(6)-CF labelled peptides; (lower)
HepG2 cells treated with 400 nM of Myrcludex B (4); (middle) HepG2
NTCP cells treated with 400 nM non-lipidated peptide (5); (upper)
HepG2 NTCP cells treated with 400 nM Myrcludex B (4). Nuclei in blue
and 5(6)-CF labelled peptides in green. Bar = 50 μm.
Table 1 EC50 values for binding of Myrcludex B (4) and S-lipidated ana-
logues, 4a–d to HepG2 NTCP cells
Peptide
EC50 (nM)
4
4a
66.99
4b
79.85
4c
59.72
4d
64.86
45.69
Table 2 EC50 values for binding of truncated Myrcludex B (3) and
S-lipidated analogues, 3a–d to HepG2 NTCP cells
Peptide
EC50 (nM)
3
3a
112.4
3b
213.1
3c
101.0
3d
107.1
40.55
Fig. 3 RP-HPLC traces for CLipPA addition of vinyl myristate (12a) to
precursor peptide (2s), yielding S-lipidated peptide 2a.
ted to the unexpectedly strong binding of 3. Their inhibitory
effect was also compared against the native myristoylated
47-mer (2, Myrcludex B), which demonstrated an IC50 of 0.056
nM, in accordance with previous work from Schulze et al.11
Both the truncated lipopeptides 1 and 3 exhibited reduced
inhibitory activity (∼800-fold and ∼500-fold respectively) com-
pared to the corresponding 47-mer (2). Their inhibitory effect
Fig. 5 EC50 plots for 47-mer of 5(6)-CF labelled lipopeptides (4 and
4a–d) binding HepG2 NTCP cells. Error bars correspond to 1 SE.
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reported less crucial for inhibition.11 Given the poorer inhibi-
tory potency of truncated lipopeptides
1 and 3, their
S-lipidated analogues (1a–d) were not further investigated.
The S-lipidated 47-mer analogues (2a–d) were however,
assayed for their inhibitory activity (Table 4, Fig. 8), exhibiting
IC50 values in the nM range. Analogues 2a, 2b and 2d exhibited
IC50 values of 1.23 nM, 3.33 nM, and 0.97 nM respectively,
within ∼17–60-fold the potency of Myrcludex B (2) (IC50 0.056
nM). The CLipPA S-lipidation approach is therefore tolerated
moderately well for these antiviral lipopeptides, and the novel
thioether analogues retain remarkable potency despite the
presence of the additional heteroatoms in their lipid ‘anchor’.
The 47-mer analogue (2c) bearing the S-linked 8-phenylocta-
noic lipid (12c), exhibited significantly reduced potency [IC50
50.32], in the same range as the truncated analogues 1 and 3,
despite being the most effective binder of NTCP expressing
HepG2 cells. Importantly, the results from this aromatic
S-lipidated analogue further corroborate our findings from
truncated lipopeptides 1 and 3, that the anti-HBV potency is
poorly correlated to binding of NTCP expressing hepatocytes.
Lipids have previously been identified as membrane
‘anchors’ that support peptides in maintaining a high local
concentration at the membrane and thereby facilitate inter-
action with the relevant receptor.29 A similar role has been pro-
posed for the lipid of Myrcludex B (2).5 Despite numerous ana-
logues (4a–d) binding to NTCP expressing hepatocytes with
similar effectiveness to 5(6)-CF labelled Myrcludex B (4), this
doesn’t correlate with their inhibitory activity (2, 2a–d). This
phenomenon was exemplified by the S-linked 8-phenyloctano-
ate analogue (4c), which demonstrated the most effective
Fig. 6 EC50 plots for truncated 5(6)-CF labelled lipopeptides (3, 3a–d)
binding HepG2 NTCP cells. Error bars correspond to 1 SE.
Table 3 IC50 values for native (myristoylated) lipopeptides 1–4 on HBV
replication
Peptide
IC50 (nM)
1
2
3
4
47.11
0.056
30.37
0.645
Table 4 IC50 values for 47-mer S-lipidated analogues 2a–d on HBV
replication
Peptide
IC50 (nM)
2a
1.230
2b
3.320
2c
50.32
2d
0.970
Fig. 7 HBV replication inhibitory potency of native (myristoylated), lipo-
peptides. Error bars correspond to 1 SE.
therefore did not correlate with the strong binding of 3 and
the 5(6)-CF label appeared to have little effect upon inhibitory
potency. The drastic reduction in potency upon truncation is
particularly interesting given it produced only a small (approx.
two-fold) reduction in their EC50 for binding to NTCP expres-
sing HepG2 cells. Somewhat surprisingly, for the 47-mer
peptide, incorporation of the C-terminal 5(6)-CF label (4)
reduced inhibitory potency ∼10-fold, despite this region being
Fig. 8 HBV replication inhibitory potency of S-lipidated 47-mer ana-
logues (2a–d). Error bars correspond to 1 SE.
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binding of all 47-mer lipopeptides whereas the equivalent lipo- cated in the experimental procedures, according to elution
peptide (2c) exhibited by far the lowest inhibitory potency, times and peak profiles obtained during analytical analysis.
reduced by 15–50-fold compared to the other S-lipidated Analytical thin-layer chromatography (TLC) was performed on
analogues (2a,b,d) and 900-fold compared to Myrcludex B (2). Kieselgel F254 200 μm silica plates. The compounds were then
This discrepancy between binding and inhibitory potency visualised by ultraviolet fluorescence or by staining with pot-
suggests the lipid may play an additional role in blocking the assium permanganate or vanillin followed by heating of the
NTCP–HBV interaction, beyond simply acting as a membrane plate. Column chromatography was performed using Grace
anchor.
Davison
Discovery
Sciences
LC60A
40-63
Micron
Chromatographic Silica Media with the indicated eluent. Low-
resolution mass spectrometry was performed on a Waters
Quattro micro API Mass Spectrometer in ESI positive mode.
Conclusions
Ultraviolet irradiation was performed using
a handheld
We report the first SPPS-based synthesis of Myrcludex B that Spectroline UV lamp at a peak wavelength of 365 nm. Nuclear
includes a detailed and optimised synthetic procedure. Using magnetic resonance (NMR) experiments were recorded as
the 3-MPA modified CLipPA thiol–ene approach, a series of shown on a Bruker AVANCE 400 spectrometer operating at
1
S-lipidated peptides bearing structurally diverse lipids were 400 MHz for H nuclei and 100 MHz for 13C nuclei. Chemical
prepared chemoselectively from a common precursor, in mod- shifts were reported in parts per million (ppm) on the δ scale
erate to excellent conversions. These PPI inhibitors appear to from tetramethylsilane (TMS), at 0.00 ppm. Shifts were refer-
constitute the first report of potent S-lipidated antiviral agents, enced to residual solvent peaks of CDCl3 at δ = 7.26 ppm for
with three analogues demonstrating IC50 values ranging 1H NMR, and CDCl3 δ = 77.16 ppm for 13C NMR or DMSO-d6
1
0.97–3.32 nM. With the potency retained by these analogues, at δ = 2.50 ppm for H NMR and δ = 39.52 ppm for 13C NMR.
the incorporation of heteroatoms within the lipid ‘anchor’ war- The 1H NMR shift values are reported as chemical shift δ, mul-
rants further investigation into the pharmacological properties tiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
of these S-lipidated peptides. In particular, the presence of multiplet, dd = doublet of doublets, td = triplet of doublets,
both an ester and thioether within the lipid portion of these qd = quartet of doublets), coupling constant (J in Hz), relative
compounds favours solubility and may also provide a poten- integral and assignments.
tially beneficial metabolic route, allowing the lipid to dis-
sociate from the peptide through ester hydrolysis. be 95% or greater by analytical HPLC using
The purity of all final tested compounds was confirmed to
μm
a
5
Interestingly, the effectiveness with which these lipopeptides Phenomenex Luna reverse phase C18 column (4.6 mm ×
bind NTCP expressing hepatocytes was found to correlate very 250 mm) (ESI Fig. S5.1–S5.20†).
poorly with their inhibitory potency and, to the best of our
Vinyl ester synthesis
knowledge, also represents the first investigation of this inter-
relationship. This apparent discrepancy highlights how the
Vinyl myristate (12a). Myristic acid (2.00 g, 8.76 mmol) was
exact role of the proposed lipid ‘anchor’ remains unclear and added to an excess of vinyl acetate (8.07 ml, 87.6 mmol),
warrants further investigation.
mercury acetate (60 mg, 0.219 mmol) and fuming sulfuric acid
(H2SO4, 15.0 μL). The reaction proceeded under reflux (74 °C)
under a nitrogen atmosphere for 4 hours until completion, as
judged by TLC monitoring of the reaction mixture. Upon com-
pletion of the reaction the reaction mixture was cooled, the sul-
furic acid quenched portion-wise with NaOAc·3H2O (3 ×
Experimental section
General procedures
Analytical reverse phase high-performance liquid chromato- 25.0 mg) and filtered through Celite. Excess vinyl acetate was
graphy (RP-HPLC) was performed on either a Waters Alliance evaporated and the crude reaction mixture purified by flash
analytical HPLC or Dionex Ultimater 3000 equipped with a column chromatography on silica gel with an eluent of ethyl
Phenomenex Luna C18 column (100 A, 5 μm, 4.6 mm × acetate : petroleum ether (1 : 99), to yield vinyl myristate (12a) a
250 mm) operated at rt, with chromatograms recorded at 210/ colourless oil (1.52 g, 68%). δH (400 MHz, CDCl3) 7.25–7.32
214 nm and 254 nm. Semi-preparative RP-HPLC was per- (1H, m), 4.85 (1H, dd, J = 4.4, 2.0), 4.55 (1H, dd, J = 6.4, 2.0),
formed on a Waters 1525 Binary HPLC pump equipped with a 2.38 (2H, t, J = 10), 1.65 (2H, t, J = 5.6), 1.26 (20H, s), 0.88 (3H,
Waters 2489 UV/visible detector using either a Waters XTerra t, J = 8.4). δC (100 MHz, CDCl3) 171.0, 141.4, 97.5, 34.1, 32.1,
Prep MS C18 OBD column (125 A, 10 μm, 300 × 19 mm) or 29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.2, 24.8, 22.8, 14.3.
Waters XTerra Prep MS C18 OBD column (125 A, 10 μm, 250 × Spectroscopic data was consistent with that reported in
10 mm). For both analytical and semi-preparative RP-HPLC, literature.30
solvents used were as follows: solvent A = 0.1% TFA in water
Vinyl neodecanoate (12b). Neodecanoic acid (98%, mixture
(MQ H2O) and solvent B = 0.1% TFA in MeCN. For analytical of isomers) (2.17 ml, 11.6 mmol) was added to an excess of
RP-HPLC the gradient employed was 5–95% of solvent B over vinyl acetate (10.7 ml, 116 mmol), mercury acetate (92 mg,
30 minutes at flow rate 1 ml min−1, unless specified otherwise. 0.290 mmol) and fuming sulfuric acid (H2SO4, 15.0 μL). The
For semi-preparative RP-HPLC gradients were adjusted as indi- reaction proceeded under reflux (74 °C) under a nitrogen atmo-
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sphere for 4 hours until completion, as judged by TLC moni- m), 0.88 (3H, s). δC (100 MHz, CDCl3) 163.8, 149.4, 141.5,
toring of the reaction mixture. Upon completion of the reac- 130.1, 128.6, 126.4, 97.9, 36.1, 31.9, 31.1, 29.4, 29.3, 29.2, 22.7,
tion the reaction mixture was cooled, the sulfuric acid 14.1. ESI-HRMS: m/z [M + Na]+ calculated for C17H24NaO2:
quenched portion-wise with NaOAc·3H2O (3 × 25.0 mg) and fil- 283.1662, observed 283.1669.
tered through Celite. Excess vinyl acetate was evaporated and
the crude reaction mixture purified by flash column chromato-
Peptide synthesis
graphy on silica gel with an eluent of ethyl acetate : petroleum
General procedures. Linear peptides were synthesised by
ether (1 : 99), to yield vinyl neodecanoate (12b) a colourless oil 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide syn-
(1.44 g, 63%). νmax/cm−1: 2962, 2935, 2875, 1745, 1645, 1464, thesis (SPPS) at rt on a 0.1 mmol scale either manually using
1212, 1138. δH (400 MHz, DMSO-d6) 7.25–7.17 (1H, m), a fritted glass reaction vessel or using a PS3 automated
4.92–4.87 (1H, m), 4.67–4.65 (1H, m). Other signals unable to peptide synthesiser. TentaGel®
S NH2 Resin (0.370 g,
be clearly assigned due to overlaps from a mixture of neodeca- 0.100 mmol, 0.270 mmol g−1 loading) was swollen in CH2Cl2
noic acid isomers that originate from the commercial starting (3 ml) for 5 min followed by DMF (3 ml) for 5 min. Fmoc-
material (see Fig. S4.5†). δC (100 MHz, DMSO-d6) 174.3, 141.4, Rink amide linker (216 mg, 0.4 mmol, 4 equiv.) was used as a
98.2. Other signals unable to be clearly assigned due to over- linker to the solid TentaGel support and was coupled to the
laps from a mixture of neodecanoic acid isomers that originate resin using the same method as the subsequent amino acids.
from the commercial starting material. ESI-HRMS: m/z [M + Using the PS3 Peptide Synthesiser, all amino acids were
Na]+ calculated for C12H22NaO2: 221.1514, observed: 221.1514.
Vinyl 8-phenyloctanoate (12c). 8-Phenyloctanoic acid acids were coupled using
coupled in single, hour long cycles at rt. Protected amino-
mixture of Fmoc-AAx-OH
a
(1.58 mg, 7.72 mmol) was added to an excess of vinyl acetate (0.4 mmol, 4 equiv.), HATU (144 mg, 0.38 mmol, 3.8 equiv.)
(7.22 ml, 77.2 mmol), mercury acetate (55 mg, 0.193 mmol) and NMM (1 mmol, 10 equiv.) in DMF (3 ml). The Fmoc pro-
and fuming sulfuric acid (H2SO4, 15.0 μL). The reaction pro- tecting group was removed with a solution of 20% piperidine
ceeded under reflux (74 °C) under a nitrogen atmosphere for in DMF (v/v) containing 5% formic acid (v/v) (3 ml, 2 ×
8 hours until completion, as judged by TLC monitoring of the 5 min). Between couplings and following Fmoc removal, the
reaction mixture. Upon completion of the reaction the reaction resin was washed with DMF (6 × 5 ml).
mixture was cooled, the sulfuric acid quenched portion-wise
Method for coupling of 3-(tritylthio)propionic acid (3-MPA)
with NaOAc·3H2O (3 × 25.0 mg) and filtered through Celite. to the N-terminus. A mixture of 3-(tritylthio)propionic acid
Excess vinyl acetate was evaporated and the crude reaction (0.139 mg, 0.4 mmol, 4 equiv.) HATU (144 mg, 0.38 mmol, 3.8
mixture purified by flash column chromatography on silica gel equiv.) and DIPEA (175 μL, 1 mmol, 10 equiv.) in DMF (3 ml)
with an eluent of ethyl acetate : petroleum ether (1 : 99), to was coupled for 2 hours at rt to the deprotected N-terminus of
yield vinyl 8-phenyloctanoate (12c) a colourless oil (0.300 g, the peptidyl resin.
16%). νmax/cm−1: 3027, 2929, 2856, 1753, 1646, 1294, 1266,
Method for coupling of myristic acid (MA). A mixture of myr-
1138. δH (400 MHz, CDCl3) 7.25–7.31 (5H, m), 7.15–7.18 (1H, istic acid (90 mg, 0.4 mmol, 4 equiv.) HATU (144 mg,
m), 4.87 (1H, dd, J = 12.8, 1.6), 4.55 (1H, dd, J = 5.2, 1.6), 2.60 0.38 mmol, 3.8 equiv.) and DIPEA (175 μL, 1 mmol, 10 equiv.)
(2H, t, J = 8.0), 2.37 (2H, t, J = 7.6), 1.55–1.67 (4H, m), 1.34 (6H, in DMF (3 ml) was coupled for 1 hour at rt to the deprotected
s). δC (100 MHz, CDCl3) 142.8, 170.9, 141.2, 128.4, 128.3, N-terminus of the peptidyl resin.
125.6, 97.5, 35.9, 33.9, 31.4, 29.1, 28.9, 24.6. ESI-HRMS: m/z [M
+
Method of removal of Dde protecting group from amino
Na]+ calculated for C16H22NaO2: 269.1507, observed acid side chain. The resin-bound peptide was treated with 2%
269.1512.
hydrazine monohydrate in DMF v/v (3 × 3 min) at rt and vigor-
Vinyl 4-octylbenzoate (12d). 4-Octylbenzoic acid (1.55 mg, ously agitated.
6.60 mmol) was added to an excess of vinyl acetate (6.09 ml,
Method for coupling of 5(6)-carboxyfluorescein (5(6)-CF).
66.0 mmol), mercury acetate (53 mg, 0.165 mmol) and fuming After removal of the sidechain Dde group, the resin-bound
sulfuric acid (H2SO4, 15.0 μL). The reaction proceeded under peptide was treated with a mixture of 5(6)-CF (151 mg,
reflux (74 °C) under a nitrogen atmosphere for 8 hours until 0.4 mmol, 3 equiv.) 6-Cl HOBT (50.8 mg, 0.400 mmol, 3 equiv.)
completion, as judged by TLC monitoring of the reaction and DIC (46.0 μL, 0.4 mmol, 3 equiv.) in DMF and vigorously
mixture. Upon completion of the reaction the reaction mixture agitated for 4 hours at rt. The resin was then washed with 20%
was cooled, the sulfuric acid quenched portion-wise with piperidine in DMF (v/v) containing 5% formic acid (v/v) (3 ml,
NaOAc·3H2O (3 × 25.0 mg) and filtered through Celite. Excess 3 × 3 min).
vinyl acetate was evaporated and the crude reaction mixture
Method for cleavage of peptide from resin. After washing
purified by flash column chromatography on silica gel with an the resin with CH2Cl2 (3 × 5 ml) a cleavage mixture of TFA/TIS/
eluent of ethyl acetate : petroleum ether (1 : 99), to yield vinyl EDT/H2O (10 ml, v/v/v/v; 94/2/2/2) was added to the on-resin
4-octylbenzoate(12d) a colourless oil (0.710 g, 42%). νmax/cm−1
:
peptide and agitated for 2 hours at rt. The cleavage mixture
2925, 2856, 1730, 1645, 1611, 1294, 1260, 1178, 1138, 1087. δH was drained and chilled diethyl ether (45 ml) was used to pre-
(400 MHz, CDCl3) 8.01 (2H, d, J = 8), 7.51 (1H, dd, J = 8, 6.0), cipitate the peptide. The peptide was isolated by centrifugation
7.28 (2H, d, J = 8.4), 5.05 (1H, dd, J = 12.0, 1.6), 4.68 (1H, dd, J and the pellet washed with diethyl ether (45 ml), dissolved in
= 4.4, 1.6), 2.67 (t, J = 8), 1.65 (2H, t, J = 7.2), 1.26–1.31 (10H, MeCN : MQ H2O (1 : 1) and lyophilised.
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Paper
CLipPA lipidation of peptides with vinyl esters. The desired HBV production
thiol-containing peptide (1 equiv.) was added to a solution of
HepAD38 cells were grown to 80% confluence whereupon the
media was replaced with doxycycline-free DMEM containing 5%
heat inactivated FCS (day 0). Cells continued to produce virus
for 15 days with media being changed every 3–4 days. Every 3–4
days from day 15 the supernatant media was harvested. Every
second harvest, virus was concentrated and stored at −80 °C. To
concentrate the supernatant, it was filtered through a sterile
0.45 μm filter and a solution of 40% polyethylene glycol (PEG)
added to result in a final solution with 6% PEG v/v. After inver-
sion this solution was stored overnight at 4 °C. Remaining at
4 °C the PEG solution was centrifuged at a fixed angle for
1 hour (10 000 rcf). Post centrifugation the supernatant was
removed, and the pellet resuspended in 1/50 of the volume in a
solution of PBS/FCS (90%/10% v/v). The viral suspension in
PBS/FCS was rotated and inverted overnight at 4 °C. To remove
insoluble precipitate the viral suspension was centrifuged twice
at 4 °C for 10 minutes (3000 rcf). The final viral suspension was
aliquoted, and the supernatant stored at −80 °C after quantifi-
cation by quantitative Polymerase Chain Reaction (qPCR).
NMP (volume adjusted to ensure peptide concentration of
2.50 μM) together with DMPA (0.5 equiv.), tert-nonyl mecaptan
(80 equiv.), TIPS (80 equiv.) and TFA (5% v/v of final volume).
To this solution the respective vinyl ester (35 equiv.) was added
and the reaction mixture agitated at rt under UV light
(365 nm) for 60 min. Upon completion of the reaction, as
determined by RP-HPLC, the peptidyl products were precipi-
tated out by addition of diethyl ether (45 ml), isolated by cen-
trifugation, and washed with diethyl ether (2 × 45 ml). The
final crude CLipPA peptide was dissolved in a 1 : 1 solution of
MeCN : MQ H2O and lyophilized. The lyophilized crude
peptide was dissolved into a solution of MeCN : MQ H2O and
purified using semi preparative RP-HPLC using linear gradi-
ents determined from observation of the analytical RP-HPLC
traces (Table 5). For each peptide the linear gradient for purifi-
cation can be found in the ESI.†
Cell culture
HepG2 (HB8065) cells were obtained from ATCC. HepG2-NTCP
cells were kindly provided by Stephan Urban, University
Hospital Heidelberg. Both cell lines were maintained in
DMEM supplemented media with 10% v/v FCS and 0.1% v/v P/
S with additional supplementation of 4 μg ml−1 G418 for
HepG2-NTCP. Cell were cultivated at 37 °C and 5.0% CO2 and
subcultured every 3–5 days at a ratio from 1 : 3 to 1 : 6.
HepAD38 cells were kindly provided by Stephen Locarnini,
Victorian Infectious Diseases Reference Laboratory and grown
in DMEM containing 5% heat inactivated FCS and 0.3 μg ml−1
of doxycycline.31
Lipopeptide binding assays
24-Well microtitre plates were seeded with HepG2 NTCP or
HepG2 cells at a density of 2 × 105 per well in a growth media
of DMEM containing 10% heat inactivated FCS and 0.1% P/S
(v/v) and incubated overnight at 37 °C in a 5% CO2 atmosphere
until cells were adhered and confluent. The cells were washed
twice with PBS. Peptide solutions were prepared in identical
growth media. A dilution series (800 nM–5 nM) was made
across the plate from standard solutions prepared from the
target peptide in DMSO stock, with the final DMSO concen-
tration being less than 0.1% v/v. For each peptide one
additional well was treated with a control peptide at fixed con-
centration as an internal standard, and one well treated with
media devoid of peptide. The plates were then incubated for
30 minutes once again at 37 °C in a 5% CO2 atmosphere. Cells
were then twice washed with PBS and trypsinised. Cells were
centrifuged at 500 rcf in growth media initially and twice in
PBS to be finally suspended in a solution of 1% FCS in PBS
(FACS buffer). The final cell suspension solutions were then
each analysed using a BD Accuri Flow Cytometer and the raw
data gated and analysed with FlowJo® to acquire mean fluo-
rescent values. All peptides were analysed in triplicate. The
data was normalised relative to a standard of Myrcludex B at a
concentration (400 nM) exceeding the saturation point of
binding. Using Prism 8.0.2 the mean fluorescent values of
each titration were used to create EC50 curves and EC50 values
for each peptide. All fluorescence assays were performed in
duplicate and repeated at least twice independently.
Table 5 Synthetic peptide ESI-MS, % yield and purity
Peptide
Calc. m/z
Observed m/za
% yieldb
% purity
1
2
2394.10
5496.70
5317.48
5571.70
5515.64
5563.64
5577.65
2950.43
2773.20
3027.44
2971.38
3019.38
3033.40
5854.75
5676.50
5929.75
5873.69
5921.69
5935.70
5644.55
2394.20
5496.57
5317.40
5572.70
5516.27
5564.40
5580.73
2950.80
2771.40
3026.40
2970.20
3017.80
3032.40
5855.38
5676.20
5931.90
5474.87
5923.73
5936.52
5644.00
21
30
23
37
19
22
18
35
12
6
17
10
10
14
22
12
12
15
9
95.7
95.0
99.1
97.5
99.0
99.0
95.8
96.1
96.5
97.7
97.5
97.7
98.5
98.3
97.5
99.2
99.4
96.5
98.2
>99.5
2s
2a
2b
2c
2d
3
3s
3a
3b
3c
3d
4
4s
4a
4b
4c
4d
5
HBV inhibition assay
Peptides (500 nM–0.0064 nM final concentration) were diluted
in a 96 well plate in DMEM containing 2% heat inactivated
FCS, 0.1% P/S, 2% DMSO, 1% NEAA and 4% PEG v/v and
1000 genome equivalent units of HBV. HepG2 NTCP cells (5 ×
104 per well) were added and infection initiated by spinocula-
29
a Deconvoluted mass observed. b All yields reported as isolated yield
after purification by RP-HPLC; yield for peptides 1–5 and purified
CLipPA intermediate peptides 2s–4s based initial resin loading; yields
of 2a–d, 3a–d, 4a–d reported for CLipPA reaction.
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tion as described.32 Cells were incubated for up to 12 days
with media replaced at days 1,3,6 and 9 with the peptide
dilution series maintained during the 12-day incubation
period. Peptide inhibition of HBV replication was determined
by HBeAg chemiluminescence immunoassay of secreted
HBeAg, 50 μL of culture media collected at day 9 were applied
to 96-well plates of a chemiluminescence immunoassay (CLIA)
according to the manufacturer’s instructions (Autobio
Diagnostics Co., Zhengzhou, China). Results were normalised
to peptide-free controls. All competition assays were performed
in duplicate and repeated at least twice independently.
References
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Abbreviations
ˇ
3-MPA
5(6)-CF
3-Mercaptopropionic acid
5(6)-Carboxyfluorescein
6-Cl-HOBt 6-Chloro-1-hydroxylbenzotriazole
approx.
CLIA
CLipPA
Dde
Approximately
Chemiluminescence immunoassay
Cysteine lipidation on a peptide or amino acid
1-(4,4′-Dimethyl-2,6-dioxocyclohexylidene)-3-ethyl
N,N′-diisopropylcarbodiimide
N,N-Diisopropylethylamine
Dulbecco’s Modified Eagle’s Medium
1,2-Ethanedithiol
DIC
DIPEA
DMEM
EDT
Et2O
Diethylether
Et(OAc)2
FA
Ethylacetate
Formic acid
HATU
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,-tertramethyl-
uronium hexafluorophosphate
9-Fluorenylmethoxycarbonyl
Fluorescence activate cell sorting
HBV E antigen
Fmoc
FACS
HBeAg
Hg(OAc)2 Mercury acetate
MeCN
MQ
Acetonitrile
Milli-q water
NEAA
NMM
NTCP
Non-essential amino acids
N-Methylmorpholine
Sodium
(Na)
taurocholate
co-transporting
performance liquid
polypeptide
RP-HPLC Reverse-phase
chromatography
high
17 World
Intellectual
Property
Organization,
WO2014207708A2, 2014.
SE
Standard error
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A. Chakraborty, S. Swift, A. Cameron, S. A. Ferguson,
M. K. Knottenbelt, I. Kavianinia and G. Cook, Chem. Sci.,
2020, 11, 5759–5765.
SPPS
tNonSH
Solid-phase peptide synthesis
tert-Nonylthiol.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank the Maurice Wilkins Centre for Molecular
Biodiscovery for financial support.
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