A "two-Birds-One-Stone" Approach toward the Design of Bifunctional Human Immunodeficiency Virus Type 1 Entry Inhibitors Targeting the CCR5 Coreceptor and gp41 N-Terminal Heptad Repeat Region

Article abstract of DOI:10.1021/acs.jmedchem.1c00781

Previous studies have reported the stepwise nature of human immunodeficiency virus type 1 (HIV-1) entry and the pivotal role of coreceptor CCR5 and the gp41 N-terminal heptad repeat (NHR) region in this event. With this in mind, we herein report a dual-targeted drug compound featuring bifunctional entry inhibitors, consisting of a piperidine-4-carboxamide-based CCR5 antagonist, TAK-220, and a gp41 NHR-targeting fusion-inhibitory peptide, C34. The resultant chimeras were constructed by linking both pharmacophores with a polyethylene glycol spacer. One chimera, CP12TAK, exhibited exceptionally potent antiviral activity, about 40- and 306-fold over that of its parent inhibitors, C34 and TAK-220, respectively. In addition to R5-tropic viruses, CP12TAK also strongly inhibited infection of X4-tropic HIV-1 strains. These data are promising for the further development of CP12TAK as a new anti-HIV-1 drug. Results show that this strategy could be extended to the design of therapies against infection of other enveloped viruses.

Full text of DOI:10.1021/acs.jmedchem.1c00781

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Article
A “Two-Birds-One-Stone” Approach toward the Design of
Bifunctional Human Immunodeficiency Virus Type 1 Entry Inhibitors
Targeting the CCR5 Coreceptor and gp41 N‑Terminal Heptad
Repeat Region
Chao Wang,*,∥ Xinling Wang,∥ Huan Wang, Jing Pu, Qing Li, Jiahui Li, Yang Liu,* Lu Lu,*
and Shibo Jiang*
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ABSTRACT: Previous studies have reported the stepwise nature of human immunodeficiency virus type 1 (HIV-1) entry and the
pivotal role of coreceptor CCR5 and the gp41 N-terminal heptad repeat (NHR) region in this event. With this in mind, we herein
report a dual-targeted drug compound featuring bifunctional entry inhibitors, consisting of a piperidine-4-carboxamide-based CCR5
antagonist, TAK-220, and a gp41 NHR-targeting fusion-inhibitory peptide, C34. The resultant chimeras were constructed by linking
both pharmacophores with a polyethylene glycol spacer. One chimera, CP12TAK, exhibited exceptionally potent antiviral activity,
about 40- and 306-fold over that of its parent inhibitors, C34 and TAK-220, respectively. In addition to R5-tropic viruses, CP12TAK
also strongly inhibited infection of X4-tropic HIV-1 strains. These data are promising for the further development of CP12TAK as a
new anti-HIV-1 drug. Results show that this strategy could be extended to the design of therapies against infection of other
enveloped viruses.
and cumulative toxicities, collectively motivating the search for
novel therapeutic methodologies.5,6 An alternative to these
drug cocktail, or fixed-dose, combinations is a simplified
multitarget-directed ligand (MTDL) design strategy. In this
approach, a single molecule can interact with multiple proteins,
thereby offering higher therapeutic efficiency and delayed drug
resistance, while circumventing the pitfalls of combination
therapy. As such, MTDLs have gained increasing attention
from drug discovery researchers.1,7,8 Here, we exploit MTDLs
for the treatment of HIV-1 infection. With significant progress
in elucidating HIV-1 entry into host cells at the molecular
INTRODUCTION
■
Drug discovery in the treatment of many multifactorial
diseases, such as infections, cancers, and degenerative CNS
disorders, is gradually moving from the “one molecule−one
target−one disease” paradigm to the development of
therapeutics able to simultaneously manipulate multiple
biological targets to generate satisfactory efficacy.1,2 Focusing
on human immunodeficiency virus type 1 (HIV-1) infection,
combinations of two or more antiretroviral medications that
act on different aspects of the HIV-1 life cycle, termed
combinatorial antiretroviral therapy (cART), fundamentally
changed the nature of HIV-1/AIDS treatment.3,4 The powerful
cART approach, which relies on a mixture of monotherapies,
has had considerable success in improving drug efficacy and
attenuating the development of drug resistance compared with
single-target antiviral drug utilities. Still, such drug combina-
tion formulations are hindered by possible drug−drug
interactions, pharmacokinetic/pharmacodynamic complexity,
Received: April 29, 2021
https://doi.org/10.1021/acs.jmedchem.1c00781
© XXXX American Chemical Society
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A

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Article
Figure 1. Schematic representation of HIV-1 gp41 and related virus entry inhibitors. (A) HIV-1 gp41 contains different functional domains,
including fusion peptide, NHR, CHR, transmembrane domain, and cytoplasm domain. The amino acid sequences of peptide fusion inhibitors T20
and C34, along with their respective target N36, are also shown. The pocket-binding domain (PBD) in C34 peptide is highlighted in red. (B)
Structure of small-molecule CCR5 antagonist maraviroc. (C) TAK-220 and its carboxylic acid derivative TAK. (D) Dual-target design strategy. In
this study, HIV-1 fusion inhibitor C34 and CCR5 antagonist TAK-220 pharmacophores have been incorporated into the chimera. Suitable PEG
spacers of different lengths were used in which n represents the number of PEG units in the linker.
level, events in this highly orchestrated process of HIV-1
invasion were selected as targets for our multitarget inhibitor
design.
trimer and have been shown to potently curtail HIV-1 activity.
One of these C-peptides, enfuvirtide (Fuzeon, T20),20 received
FDA approval in 2003 as the first HIV-1 fusion inhibitor-based
anti-HIV drug (Figure 1A,B). However, the low potency and
low genetic barrier for drug resistance of T20 have caused a
growing number of patients to fail in their response to T20
therapy, thus limiting its clinical use.21
The accepted mechanism of HIV-1 infection involves initial
binding of viral envelope glycoprotein (Env) surface subunit
gp120 to the cellular receptor CD4, which, in turn, triggers a
series of conformational changes of gp120 that allow it to
associate with a chemokine coreceptor, CCR5 or CXCR4, on
the target cell.9,10 Coordinated engagement of CD4 and the
chemokine receptor activates the fusion machinery in Env
transmembrane subunit gp41. Then, a gp41-central six-helix
bundle (6-HB) is subsequently formed through condensation
of its N-terminal heptad repeat (NHR) and C-terminal heptad
repeat (CHR) regions, providing the critical driving force that
facilitates the fusion between the viral envelope and host cell
membrane.11,12
Inhibition of HIV-1 entry can be achieved by disruption of
each of these stages in the early viral life cycle.9 For example,
intensive development of CCR5 antagonists able to disrupt
gp120−CD4 coreceptor interaction led to the first marketed
CCR5 inhibitor, maraviroc, for treatment-experienced pa-
tients.13,14 However, maraviroc is active only against R5 HIV-1
strains.15 Consequently, maraviroc therapy failed against
preexisting minor CXCR4-tropic X4 HIV-1 strains in the
early stage of HIV-1 infection and the incidence of CXCR4-
using variants associated with the disease progression.16
Another approach inhibited the binding of the gp41 CHR to
NHR, thus blocking the formation of the fusogenic 6-HB core
structure.9,11,12 During gp41-mediated HIV-1 infection, a fairly
long-lived intermediate conformation exists in which NHR
helices form a trimeric coiled coil with three hydrophobic
grooves that make perfectly susceptible drug targets.12 More
specifically, peptide-based inhibitors are derived from the gp41
CHR region.17−19 Designated as C-peptides, these inhibitors
act by competitively binding to the exposed grooves on NHR-
Inspired by the multistep nature of the HIV-1 entry process
and multiple viable molecular targets involved in this pathway,
we herein describe the rational design of MTDLs able to
inhibit two crucial stages of HIV-1 cell fusion and entry: the
binding of HIV-1 gp120 to its coreceptor CCR5 and gp41 6-
HB core formation. The design of such a dual-targeted drug
compound features bifunctional entry inhibitors, a CCR5
small-molecule antagonist (TAK-220) and a C-peptide fusion
inhibitor (C34) tethered via a poly(ethylene glycol) (PEG)
linker. As anticipated, one chimera, CP12TAK, exhibited
exceptionally potent antiviral activity, about 40- and 306-fold
over that of its parent inhibitors, C34 and TAK-220,
respectively. This dual-targeted compound has anti-HIV-1
activity in picomolar concentration against a panel of R5-tropic
viral isolates. Unlike its parent CCR5 antagonist TAK-220, it
has activity against X4-tropic and dual-tropic viruses.
CP12TAK also retained high potency against T20-resistant
viruses, conferring cross-resistance to the parental C34 peptide.
Our study provides tantalizing insights into developing
MTDLs that govern the HIV-1 entry process. Considering
the common cascade of events in viral entry,22,23 our
multitarget design strategy in the context of HIV-1 invasion
has the potential for application to therapeutic intervention
against other enveloped viruses.
DESIGN AND CHEMISTRY
Some key issues in the design of CCR5/gp41 dual-targeted
inhibitors include selection of suitable CCR5 antagonists and
■
B
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a
Scheme 1. Synthesis of TAK
a
Reagents and conditions: (a) N,N′-carbonyldiimidazole, THF, r.t.; (b) NaH, 1-bromo-3-chloropropane, DMF, 0 °C to r.t.; (c) (EtO)3P; (d) 1-
Boc-4-piperidone, NaH, THF; (e) H2, Pd/C, MeOH; (f) HCl/EtOAc, MeOH; (g) KI, K2CO3, DMF/CH3CN, reflux; (h) 1 M NaOH, dioxane
Figure 2. Schematic representation of the strategy used for the preparation of bifunctional molecules.
peptidic fusion inhibitors, identification of proper linkage
points of small-molecule CCR5 inhibitors to peptides, and
optimization of the length of PEG spacers. In the early 1990s,
exploration of peptide fusion inhibitors started with several
synthetic peptides overlapping gp41 NHR and CHR regions,
including DP-107, SJ-2176, and DP-178 (it was renamed T20
later).17−19 Two C-peptides, T20 and C34,11,12 effectively
blocked HIV-1 fusion in a low nanomolar range and have been
extensively exploited. Interestingly, although T20 was
developed as the only membrane fusion inhibitor for clinical
use 18 years ago, its mechanism of action has been a matter of
debate until recently.24,25 In contrast, the specific structural
properties of C34 in complex with an NHR-derived target
mimic peptide, N36, have been finely delineated by crystallo-
graphic analysis.12 C34 has a well-defined mechanism of action
and higher HIV-1 inhibitory potency compared with T20.
Consequently, C34 is widely used as a design template for the
engineering of novel fusion inhibitors.26 Following this logic,
we chose to use C34 as the fusion inhibitor portion of our
dual-target scaffold. For the coreceptor part of this hybrid
compound, we selected TAK-220, a piperidine-4-carboxamide
CCR5 antagonist, first disclosed by Takeda, based on its
conformational flexibility and highly potent anti-HIV-1
activity.27 In selecting a point of attachment for the linker on
TAK-220, replacement of the amide group in the right-side
phenyl ring with a carboxylic acid moiety was chosen, owing to
anticipated low interference with the antiviral potency of TAK-
220 after covalent conjugation of the free-acid form of TAK
and PEG spacers (Figure 1C).28 Previous studies suggested the
need for antiparallel orientation of C-peptides relative to the
viral NHR region and the importance of C-terminal
derivatization for their activity pattern.29−31 Based on the
above considerations, TAK was covalently linked to the C-
terminus of C34 peptides using flexible PEG linkers with
various lengths (hereinafter termed PEGn in which n
represents the number of PEG units in the linker) (Figure
1D). A PEG spacer was selected for the multitargeted ligand
design because its hydrophilicity would be beneficial to
aqueous solubility of the chimeric inhibitors and, hence,
avoid any nonspecific hydrophobic contact with either of the
target proteins.32
The synthetic pathway to TAK is illustrated in Scheme 1.
The aniline was coupled with commercially available 1-
acetylpiperidine-4-carboxylic acid to afford compound 1. In
C
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a
Table 1. Inhibition of Dual-Target Entry Inhibitors on Laboratory-Adapted HIV-1 R5 Virus Infection
b
compound
CP0TAK
CP4TAK
CP8TAK
CP12TAK
CP24TAK
C34
TAK-220
C34/TAK-220
CP12
CP12/TAK-220
P26TAK
sequence
EC50 (nM)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(−----TAK)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(PEG4-TAK)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(PEG8-TAK)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(PEG12-TAK)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(PEG24-TAK)
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL
0.25 0.05
0.63 0.23
0.49 0.43
0.03 0.01
0.89 0.35
1.19 0.41
9.18 5.78
0.58 0.16
3.29 1.73
2.08 1.69
>50
c
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(PEG12-Ac)
c
NNYTSLIHSLIEESQNQQEKNEQELLK(PEG12-TAK)
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
T20
1.58 0.27
a
The assay was performed in triplicate, and the data are expressed as the mean standard deviation. CEMx174 5.25M7 cells expressing both
b
CCR5 and CXCR4 on the surface were infected with the HIV-1 BaL strain (subtype B, R5). For these peptides, the N-termini were acetylated,
and the C-termini were amidated. Molar ratios of C34/TAK-220 or CP12/TAK-220 in combination were 1:1.
c
the presence of NaH, N-alkylation with 1-bromo-3-chloropro-
pane accessed chloride 2. Olefin 3 was prepared through the
Arbuzov reaction and Horner−Wadsworth−Emmons reaction,
using literature procedures27 and converted into 4 after
hydrogenation followed by deprotection of the Boc group.
Chlorides were reacted with 4-substituted piperidines in the
presence of potassium iodide and potassium carbonate to give
compound 5. By saponification, the methyl ester intermediate
5 was converted into TAK.
We employed an Fmoc solid-phase peptide synthesis
protocol to construct a modified version of C34 peptides in
which a lysine residue with a 1-(4,4-dimethyl-2,6-
dioxocyclohexylidene)ethyl (Dde) side-chain-protecting
group was incorporated into the C-terminus of C34 to enable
ligation with the carboxyl group of the PEG linkers after
selective removal of the Dde group on resin by 2%
hydrazinehydrate/ N,N-dimethylformamide (DMF)-mediated
deprotection. TAK was then conjugated with PEG spacers, and
the corresponding bifunctional molecules were obtained after
removal of all peptide protection groups and cleavage of the
peptides from resin using TFA (Figure 2).
laboratory-adapted HIV-1 BaL strain, CP12TAK had an
EC50 value of 0.03 nM against HIV-1 IIIB on CEMx174
5.25M7 cells that express both CCR5 and CXCR4 receptors,
which is approximately 84-fold more potent than that of the
1:1 mixture of C34 with TAK-220 (Table 2). We then assessed
Table 2. Inhibition of CP12TAK on Laboratory-Adapted X4
a
HIV-1 IIIB Infection
b
EC50 (nM)
HIV-1 strain
(subtype,
tropism)
TAK-
220 C34/TAK-220
c
CP12TAK
C34
IIIB (B, X4)
0.03 0.01
1.62 0.32
>50
2.54 0.85
a
The assay was performed in triplicate, and the data are expressed as
b
the mean standard deviation. CEMx174 5.25M7 cells expressing
both CCR5 and CXCR4 on the surface were infected with the HIV-1
IIIB strain (subtype B, X4). The molar ratio of C34/TAK-220 in
c
combination was 1:1.
the potential cytotoxicity of the CP12TAK peptide on target
cells used for viral inhibition assays and found that the
concentration for 50% cytotoxicity (CC50) was beyond 5000
nM (Figure S1), suggesting that CP12TAK is an effective HIV-
1 entry inhibitor with low or no in vitro toxic effect.
RESULTS
■
Dual-Targeted Peptides Effectively Inhibited Infec-
tion of Laboratory-Adapted HIV-1 Strains. First, inhib-
itory activity of dual-targeted peptides on infection by R5 HIV-
1 strain BaL (subtype B) on CEMx174 5.25M7 cells was
measured. As shown in Table 1, C34, TAK-220, and their 1:1
molar combination exhibited anti-HIV-1 activity with half-
maximal effective concentration (EC50) values at 1.2, 9.2, and
0.6 nM, respectively. Chimeric inhibitors without the PEG
spacer and with PEG4-, PEG8-, and PEG24-based linkers
possessed potency similar to that of the 1:1 mixture of the
individual C34 and TAK-220. Strikingly, CP12TAK with 12
PEG units showed high efficacy in inhibiting HIV-1 BaL
infection with an EC50 value of 0.03 nM, which is 52-fold more
potent than that of T20 (EC50 = 1.58 nM). Deletion of the N-
terminal PBD motif in the C34 portion of CP12TAK, or the
CCR5 small-molecule antagonist part of the chimera, resulted
in a remarkable decrease of antiviral activity. We further
evaluated the antiviral activity of CP12TAK against X4 HIV-1
strain IIIB (subtype B) infection, in comparison with C34,
TAK-220, and a mixture of C34 with TAK-220 (1:1 ratio,
unlinked). Surprisingly, consistent with the results of
Potent Antiviral Activity of CP12TAK against Clinical
HIV-1 Isolates and T20-Resistant Strains. In addition to
the laboratory-adapted HIV-1 BaL and IIIB strains, we also
tested the inhibitory activity of CP12TAK against a panel of
HIV-1 clinical isolates of subtypes A, B, C, D, F, O, and A/E.
As shown in Table 3, CP12TAK exhibited highly potent
activity against divergent R5 HIV-1 clinical isolates, including
91US_4, 00TZ_A125, and BCF02, with EC50 values of 0.02 to
2.1 nM, which are about 3.5- to 465-fold more potent than that
of the 1:1 mixture of C34 and TAK-220. After further testing
on X4 HIV-1 clinical isolates, including 92UG024, BZ167/
GS010, and MN/H9, CP12TAK was found to be 4.2- to 6.9-
fold more potent than that of C34. CP12TAK also had potent
inhibitory activity against two R5/X4 dual-tropic isolates. The
EC50 values were 1.9 and 1.6 nM against 93/BR/020 and
97TH_NP1525, respectively. Furthermore, the inhibitory
activity of CP12TAK on the replication of T20-resistant
strains was determined. T20 could potently inhibit infection by
a T20-sensitive strain with an EC50 value of 4.3 nM, but it was
much less effective against the T20-resistant strains, including
HIV-1NL4‑3(D36G)V38E/N42S (EC50 = 261 nM), HIV-
D
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a
Table 3. Inhibitory Effect of CP12TAK against Infection by Primary HIV-1 Isolates
b
EC50 (nM)
HIV-1 isolate
subtype
tropism
C34
TAK-220
C34/TAK-220
CP12TAK
91US_4
00TZ_A125
BCF02
92UG024
BZ167/GS010
MN/H9
B
R5
R5
R5
X4
X4
X4
X4/R5
X4/R5
74.5 33.3
24.0 11.9
6.9 2.0
30.2 17.5
22.1 4.3
2.1 1.7
20.1 6.7
62.8 11.8
31.2 3.3
>100
>100
>100
7.3 2.7
27.7 20.4
9.3 3.6
42.9 22.6
25.3 6.9
7.1 6.9
39.5 10.6
19.4 2.8
2.1 0.9
0.3 0.1
0.02 0.01
4.4 1.6
3.2 2.3
0.5 0.1
1.9 0.9
1.6 0.8
C
O
D
B
A
F
93/BR/020
97TH_NP1525
7.6 1.2
18.4 1.2
>100
>100
CRF01_AE
a
b
All assays were performed in triplicate, and the data are expressed as the mean standard deviation. CEMx174 5.25M7 cells expressing both
CCR5 and CXCR4 on the surface were used in this study.
a
Table 4. Inhibitory Activity of CP12TAK against Infection by T20-Resistant HIV-1 Strains
b
EC50 (nM)
HIV-1 strain
T20
4.31 0.89
261.22 63.40 (60.6)
672.86 24.50 (156.1)
655.63 227.48 (152.1)
C34
CP12TAK
c
D36G
3.65 0.56
0.66 0.33
d
V38E/N42S
13.74 6.28 (3.7)
20.35 5.13 (5.6)
12.97 6.64 (3.6)
0.39 0.07 (0.6)
0.52 0.19 (0.8)
0.55 0.35 (0.8)
d
N42T/N43K
d
V38A/N42T
a
The assay was performed in triplicate, and the data are expressed as the mean standard deviation. Values in parentheses indicate relative changes
b
(n-fold) in the EC50 compared with the EC50 in the presence of the D36G substitution. CEMx174 5.25M7 cells expressing both CCR5 and
CXCR4 on the surface were used in this study. T20-sensitive strain. T20-resistant strain.
c
d
1NL4‑3(D36G)N42T/N43K (EC50 = 673 nM), and HIV-
1NL4‑3(D36G)V38A/N42T (EC50 = 656 nM). Consistent with
previous studies,33 these T20-resistant HIV-1 variants also
conferred cross-resistance to the C34 peptide. Impressively,
CP12TAK retained high potency against these resistant
viruses. The double mutations resulted in considerable high
fold-changes of resistance to T20 and C34, but they had little
effect on the potency of CP12TAK (Table 4).
CP12TAK Inhibited HIV-1 Infection by Interfering
with Its Entry Step. A time-of-addition experiment was
performed to determine the target stage of HIV-1 replication
that is interfered by CP12TAK. The chimeric inhibitor was
incubated with target cells during different intervals post-
infection, and its inhibitory activity against HIV-1 BaL
replication was measured. We found that addition of
CP12TAK, together with virus, to the target cells had a
profound inhibitory effect on HIV-1 replication (91%
inhibition rate). However, a significantly decreased inhibitory
effect was observed when CP12TAK was added to infected
cells at 3 h or later (<37% inhibition rate), suggesting that
CP12TAK is only effective at the entry stage of the viral
lifecycle (Figure 3).
CP12TAK Could Bind to the NHR Region of gp41.
Subsequently, multiple biophysical and functional approaches
were performed to understand the mechanism of action of
these chimeras. We first determined whether CP12TAK could
interact with the gp41 NHR target surrogate N36 peptide to
form a complex, using native N-PAGE analysis, as described
previously.25,34 As shown in Figure 4A, N36 alone exhibited no
band in the gel because it carries net positive charges and may
move up and off the gel, which is consistent with previous
observations.25,34 Each of the peptides, including C34, CP12,
and CP12TAK, displayed a band at different positions in the
gel, depending on the net negative charge and molecular size of
each one, while all mixtures of C34/N36, CP12/N36, and
CP12TAK/N36 showed new bands at the upper position in
Figure 3. Time-of-addition experiment to clarify the stage at which
dual-targeted inhibitors blocked HIV-1 infection. CP12TAK (20 nM)
as a representative was added to CEMx174 5.25M7 cells at different
time points postinfection of HIV-1 BaL strain. ***p < 0.001 and
****p < 0.0001 (two-way analysis of variance (ANOVA)). Data are
presented as the mean standard deviations (n = 3).
the gel, indicating that CP12 and CP12TAK, like peptide C34,
could form complexes. As analyzed by sedimentation velocity
analysis (SVA), the sedimentation coefficient of the
CP12TAK/N36 complex was 2.08 s, corresponding to 26.4
kDa, in agreement with the theoretical molecular mass of a
CP12TAK/N36 6-HB (29.1 kDa) (Figure 4B). These results
therefore suggested that CP12TAK could interact with N36 to
form a 6-HB core structure. Next, circular dichroism (CD)
experiments were performed to investigate the secondary
structure and thermostability of the resulting CP12TAK/N36
hexamer (C34/N36 as a control). Peptides CP12TAK, C34,
and N36 alone exhibited random coil structures. As expected,
similar to C34, the chimeric inhibitor CP12TAK formed
typical coiled coil after incubation with N36. CP12TAK and
C34 interacted with N36 resulting in 58.7% and 75.5% α-
helical content in the complex, respectively. Although
CP12TAK induced slightly less α-helix content than C34,
E
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Figure 4. Determination of 6-HB formation between CP12TAK and N36. (A) Visualization of the binding of CP12TAK to the NHR peptide N36
by N-PAGE. (B) SVA of CP12TAK/N36 mixture. Sedimentation coefficient (s) and molecular mass (kDa) of each peak are indicated. The α-
helicity (C) and thermostability (D) of CP12TAK/N36 6-HB. The α-helicity and Tm values are shown in parentheses. The final concentration of
each peptide in PBS at 10 μM.
the CD thermal denaturation experiment showed a Tm value
of 66 and 64 °C for CP12TAK/N36 and C34/N36 complexes,
respectively, indicating that CP12TAK-based and C34-based
6-HBs have similar thermostability (Figure 4C,D).
cells expressing both CCR5 and CXCR4 (Figure 5). In
contrast, C34, as the control, showed similar EC50 values on
CP12TAK Had Antagonistic Activity on RANTES-
Induced Ca2+ Mobilization in CCR5-Expressing Cells.
Having demonstrated the interaction of CP12TAK with the
gp41 NHR-trimer at the prehairpin fusion-intermediate state,
we next probed the binding of this dual-targeted inhibitor to
cellular coreceptor CCR5. First, HIV-1 IIIB infection
inhibition assay using MT-2 target cells expressing no CCR5
was carried out. We found that the potency enhancement of
CP12TAK against X4 HIV-1 IIIB strain is target-cell
coreceptor-dependent. When inhibiting HIV-1 IIIB infection
on MT-2 cells expressing only CXCR4, CP12TAK behaved
essentially the same as the 1:1 noncovalent mixture of C34 and
TAK-220 (Table 5), which was different from its behavior in
inhibiting HIV-1 IIIB infection in CEMx174 5.25M7 target
Figure 5. Antiviral potency of CP12TAK against X4-tropic HIV-1
IIIB infection in CEMx174 5.25M7 cells and MT-2 cells. Inhibitory
activity of CP12TAK against X4-tropic HIV-1 IIIB infection in
CEMx174 5.25M7 cells that express both CCR5 and CXCR4 and
MT-2 cells which express only CXCR4 was compared. ***p < 0.001
and ****p < 0.0001 (two-way ANOVA). Experiments were
performed in triplicate, and the data are expressed as mean
standard deviations.
Table 5. Anti-HIV-1IIIB Activity of CP12TAK in MT-2
a
Cells
b
EC50 (nM)
both CEMx174 5.25M7 and MT-2 target cells. To further
investigate the coreceptor-targeting function of CP12TAK, we
adapted a calcium (Ca2+) mobilization assay to evaluate the
antagonistic activity of the chimeric inhibitor on RANTES-
induced Ca2+ mobilization via CCR5 in HEK293 cells. As
shown in Figure 6, maraviroc, as a positive control, exhibited
inhibitory activity against CCR5 and dose-dependently
blocked the specific RANTES-induced intracellular Ca2+ flux
with a half-maximal inhibitory concentration (IC50) value of
0.82 nM. TAK-220 also showed activity toward CCR5 with an
IC50 value of 1.49 nM. As expected, CP12TAK containing the
coreceptor-inhibitor component maintained the potent antag-
HIV-1 strain
TAK- C34/TAK-
c
(subtype, tropism)
CP12TAK
C34
220
220
IIIB (B, X4)
0.62 0.33
(20.6)
1.34 0.37
(0.8)
>50 0.63 0.08
(0.2)
a
The assay was performed in triplicate, and the data are expressed as
b
the mean standard deviation. HIV-1 IIIB strain infecting MT-2
cells expressing only CXCR4, but not CCR5, on the surface. Values in
parentheses indicate relative changes (n-fold) that were calculated by
dividing the EC50 of a compound when it was tested in MT-2 cells by
that of the same compound tested in CEMx174 5.25M7 cells that has
been shown in Table 2. The molar ratio of C34/TAK-220 in
combination was 1:1.
c
F
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CP12TAK, and the corresponding effects on inhibition of
HIV-1 infection were evaluated. The N-terminal PBD
truncation of CP12TAK into P26TAK (Table 1) leads to a
dramatic loss of antiviral potency. Cocrystallization of C34
with its target N36 sequence has revealed that the PBD motif
within the C34 peptide penetrates into the gp41 NHR primary
pocket and plays a critical role in maintaining its inhibitory
activity.12 Thus, loss of biological activity of CP12TAK in the
absence of PBD suggests that the C34 part of this chimeric
inhibitor is also indispensable. Consistently, truncation of
CP12TAK into CP12 resulted in remarkable reduction of
antiviral activity by the loss of the CCR5 inhibitor moiety
(Table 1). These structure−activity relationship (SAR) studies
imply that increased antiviral potency results from both
components attached as one molecule. Moreover, our
biophysical studies with N-PAGE, SVA, and CD spectroscopy
indicated that CP12TAK, like C34 peptide, could bind to the
exposed grooves on the gp41 NHR-trimer to form
heterologous 6-HB, thereby preventing HIV-1−cell membrane
fusion. Using MT-2 cells expressing only CXCR4 as target
cells, we observed the loss of synergistic potency of CP12TAK
against HIV-1 IIIB infection, indicating that the chimera could
bind to the CCR5 coreceptor. This finding was further
confirmed by calcium flux assay. These observations from
biophysical studies and functional experiments, combined with
prior SAR analysis in CP12TAK, have demonstrated that each
moiety in this bifunctional molecule maintains the capacity to
interact with its specific target and, consequently, produce
pharmacological responses that synergistically halt the cascade
of HIV-1 fusion and entry. One hypothesis to explain the
cooperative antiviral effects of two pharmacophores in one
molecule is that blockage of the coreceptor via the CCR5
antagonist leads to delayed kinetics of the viral fusion process
and increased its sensitivity to the C34 peptide.39,40 Recently,
numerous reports have shown that fusion inhibitor localization
to membrane microdomains where fusion occurs via lipid
conjugation can effectively enhance the inhibitory potency of
these fusion-inhibitory peptides.21,30,41 Therefore, another
possible explanation is that membrane localization of
CP12TAK by binding of the TAK-220 pharmacophore to
the CCR5 coreceptor, much like lipids, such as cholesterol and
fatty acids, raised the local concentration of the C34 peptide at
the target site and thus increased the association rate for C34
binding to its viral NHR region target within the short-lived
prehairpin intermediate of gp41. The third speculative
reasoning is that the favorable entropic contribution could be
responsible for the intramolecular cooperation of the two
inhibitors.42 In this scenario, when a fusion inhibitor and a
CCR5 antagonist are tethered in a single molecule using a
PEG12 linker, the smaller loss of entropy upon target binding
compared to the sum of entropy cost resulted from binding of
two separate components to their individual targets may
account for their synergistically inhibited HIV-1 infection. A
much looser PEG24 linker between C34 and TAK-220
resulted in a dramatic decrease in antiviral potency of
CP24TAK, possibly because its large loss of entropy for target
binding is similar to that of the unlinked combination of the
individual inhibitors.
Figure 6. Concentration-dependent inhibition of RANTES-induced
intracellular Ca2+ mobilization by HIV-1 inhibitors in HEK293 cells
stably expressing CCR5. Fluo-4-loaded cells were treated with serial
dilutions of HIV-1 inhibitors, including maraviroc, TAK-220,
CP12TAK, and CP12, and then stimulated with RANTES at 4 nM.
Experiments were performed in triplicate, and the data are expressed
as means the standard deviations (error bar). Percent inhibition of
the compounds and IC50 values were calculated.
onism of CCR5 with an IC50 value of 55.5 nM. Deletion of the
CCR5 coreceptor-inhibitor portion of CP12TAK resulted in
the loss of its inhibitory activity, that is, CP12 peptide
exhibited no CCR5 antagonistic activity at the concentration
up to 10 μM. These experiments provide evidence suggesting
that CP12TAK could bind to the cellular CCR5 through its
CCR5 antagonist pharmacophore.
DISCUSSION
■
HIV-1 entry proceeds through a cascade of three interdepend-
ent steps, including the attachment of the HIV-1 envelope
glycoprotein gp120 to CD4 receptor on T-cells and macro-
phages, gp120 and coreceptor binding, and gp41-mediated
membrane fusion. Each of these stages has afforded attractive
targets for the development of viral entry inhibitors. Previous
drug−drug combination studies have shown that using viral
entry inhibitor combinations acting at two different steps, for
example, the combination of a gp41-targeting fusion inhibitor
with a gp120-targeting attachment inhibitor, or coreceptor
antagonist, has resulted in strong synergistic inhibition of HIV-
1 entry.35−38 These findings encouraged us to engineer a novel
entry inhibitor through incorporation of a small-molecule
CCR5 antagonist, for example, TAK-220, and a peptide HIV-1
fusion inhibitor, such as C34, into one molecule. Strikingly, the
covalent linkage of these two pharmacophores resulted in
increased anti-HIV-1 potency of the chimeric inhibitors, and
the change of the linker length affected their overall antiviral
activity. Of note, CP12TAK peptide with a 12-unit PEG spacer
was demonstrated as the most active inhibitor against infection
of R5 HIV-1 BaL strain with an EC50 value of 0.03 nM, which
is 39- and 306-fold higher than that of C34 alone and Tak-220
alone, respectively. We also demonstrated that covalent
attachment led to a dramatic increase of the chimeric
inhibitor’s potency over that of the 1:1 (mol/mol) noncovalent
mixture of C34/TAK-220 or CP12/TAK-220 that mimicked
the molar ratio between inhibitors in the chimeric bifunctional
compound. These data strongly suggest that HIV-1 fusion
inhibitor C34 and CCR5 antagonist, when acting together, but
on different targets within the same entry cascade, provide a
synergistic antiviral effect.
One possible concern over long-term administration of the
CCR5 antagonist maraviroc is the coreceptor switch between
X4-tropic HIV-1 strains and R5X4 dual-tropic viruses to gain
entry and infect the host cells.15,43 As expected, the CCR5
antagonist TAK-220 showed no inhibition at the concentration
To investigate whether the gp41 NHR-targeted fusion
inhibitor moiety and small-molecule CCR5 antagonist portion
within the bifunctional entry inhibitor indeed act on their
targets accordingly, a series of mutations were made to
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as high as 100 nM against infection by either X4-tropic or dual-
tropic viruses in CEMx174 5.25M7 cells that express both
CCR5 and CXCR4 receptors. The C34 peptide is active
because of its ability to bind the gp41 NHR region. When the
CCR5 blocker TAK-220 was incorporated into the peptide, a
54-fold increase in potency against X4-tropic IIIB strain
infection was noted in comparison to C34, but this synergism
diminished in the mixture state. Further testing on X4 and R5
clinical isolates confirmed that CP12TAK was dramatically
more potent than C34. One may question why this
bifunctional entry inhibitor composed of one CCR5 antagonist
linked to a fusion inhibitor is more effective in inhibiting X4-
tropic and X4R5 dual-tropic strains than either of the
components alone. Consistently, previous studies have
reported that a chimeric HIV-1 entry inhibitor composed of
a CCR5 natural ligand RANTES variant covalently linked to a
gp41 peptide fusion inhibitor, designated 5P12-linker-C37,
fully retains the anti-X4 activity of the fusion inhibitor, and this
anti-X4 potency can be further enhanced when the target cells
coexpress CCR5 on the surface.44 Another bifunctional HIV-1
entry inhibitor named BFFI in which two peptide fusion
inhibitors fused to the C-terminal ends of the heavy chain of a
mAb targeting CCR5 also inhibited X4 HIVNL4‑3 with
dramatically increased potency in comparison to fusion
inhibitor alone on target cell lines expressing both CCR5
and CXCR4 coreceptors.45 Based on these observations, it can
be speculated here that the dramatically increased antiviral
efficacy of CP12TAK against X4-tropic HIV-1 on
CD4+CCR5+CXCR4+ cells could be explained by its CCR5
interaction. There have been reports showing that chemokine
receptors CCR5 and CXCR4 form heterodimers on the cell
surface.46,47 Therefore, CP12TAK increases the potency of the
fusion inhibitor moiety probably because of anchoring of the
coreceptor portion of this hybrid molecule to CCR5. The
localization to CCR5 on the host cell membrane brings the
fusion inhibitor to its target in gp41 protein from X4-tropic
HIV-1 who use nearby CXCR4 coreceptor for their entry. In
accordance with this speculation, a noncovalent mixture of
C34 and TAK-220 only exhibited anti-X4 activities at a similar
level to their individual components alone on
CD4+CCR5+CXCR4+ cells. Furthermore, the chimeric inhib-
itor CP12TAK inhibited X4-tropic HIV-1 with antiviral activity
similar to that observed for C34 alone in cell lines expressing
only CXCR4 but not CCR5. CP12TAK was able to inhibit
infection of CD4+CCR5−CXCR4+ cells with X4-tropic HIV-1
IIIB, possibly because the small-molecule CCR5 inhibitor
portion has a minimal steric hindrance to gp41 NHR target
binding of the peptide fusion inhibitor, and consequently no
adverse effects on the anti-HIV-1 activity of C34 were
observed. Indeed, further investigation of the mechanisms of
action of the chimeric inhibitor on X4- and R5/X4-tropic
viruses is warranted.
problem of T20 resistance.48 Su et al. have designed an
analogous peptide of HP23, HP23-E6-IDL, by adding an IDL
(Ile-Asp-Leu) anchor to the C-terminus of the HP23 peptide.
The newly designed peptide exhibited high potency against
HP23-resistant strains.49 These findings are consistent with
that seen for CP12TAK, suggesting construction of molecules
that can hit multiple target sites could significantly improve a
peptide’s resistance profile. In addition to efficiently inhibiting
the existing inhibitor-resistant HIV-1 variants, an ideal next-
generation HIV-1 entry inhibitor should possess a high genetic
barrier to resistance. Compared with T20, C34 is less
susceptible to the evolution of resistant viruses.48 In viral
selection experiments, viruses resistant to TAK-220 could not
yet be isolated in vitro.50 Moreover, extensive studies have
shown that incorporation of an extra motif, for example, M-T
hook structure, to C34, or sifuvirtide, could significantly
increase the genetic barrier of inhibitors to the development of
drug resistance.48 Thus, we expect that CP12TAK may lead to
delayed drug-resistant mutations in HIV-1 variants.
Overall, CP12TAK, as an outstanding MTDL lead
compound, has exquisite potency against R5-tropic, X4-tropic,
and dual-tropic HIV-1 strains, and it is substantially greater
than that of its individual components alone or in combination.
Moreover, the incorporation of two inhibitors into one
molecule could overcome some drawbacks of the parent
single-target drugs, for example, the treatment failures of the
CCR5 antagonist associated with patients infected by non-R5
viruses and rapid emergence of viral strains resistant to T20.
The current emerging severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) merits mention in the context of
our de novo dual-targeted compound. In particular, the 6-HB
inhibition strategy has been successfully applied to SARS-CoV-
2, which utilizes a class I fusion protein, similar to that of HIV-
1, for the discovery and development of effective fusion
inhibitors.51 In addition to inhibition of the cascade of
conformational changes of fusion proteins, interference of the
priming of SARS-CoV-2 spike protein by blocking trans-
membrane protease serine 2 also opens a new avenue for
preventing the entry process between viral and cellular
membranes.52 Therefore, based on the results obtained from
the present study, combined with these significant advances in
the development of SARS-CoV-2 entry inhibitors, the
approach of making MTDLs by linking two active ligands
together could be proposed as a promising strategy for the
discovery of SARS-CoV-2 entry inhibitors with reliable efficacy
and safety.
Recently, a series of multitargeted compounds has been
designed via linking together a peptide fusion inhibitor and a
CCR5-specific monoclonal antibody or variants of natural
ligands which interfere with gp120-CCR5 binding.44,45
Compared with these biologics-based dual-binding site entry
inhibitors, CP12TAK has several strengths. First, CP12TAK
can be easily synthesized, thus having lower production cost
than chimeric molecules containing large proteins. Second,
CP12TAK is very amenable to site-specific modification to
discover and develop new lead compounds with a novel
scaffold and higher potency. Third, CP12TAK can inhibit X4-
tropic viruses on cell lines expressing only the CXCR4
coreceptor, whereas the monoclonal antibody-based entry
inhibitors can only exert an antiviral effect on cells
coexpressing CCR5. Fourth, small-molecule CCR5 antago-
nists, unlike natural ligands for CCR5, would not induce cell
signaling or internalization,43 thus potentially lessening the
Drug resistance is the major disadvantage for current clinical
application of T20. It easily induces T20-resistant mutations in
the GIV motif (residues 36 to 45 GIVQQQNNLL) in the
gp41 NHR region.26 C34 is much more potent in inhibiting
T20-resistant strains, even though some of these strains still
exhibit resistance to this PBD-containing peptide. To explain,
it is possible that C34 also contains the GIV-motif-binding
domain. Strikingly, CP12TAK has dramatically increased
inhibition against infection of T20-resistant HIV-1 mutants.
Chong et al. found that addition of an M-T hook to the N-
terminus of C34 could allow it to effectively overcome the
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mixture was diluted with water (50 mL) and extracted with EtOAc (3
× 50 mL). The organic layer was washed with brine (3 × 50 mL) and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/MeOH 1:0 to 9:1) followed
by recrystallization from EtOAc/Et2O to afford 2 (0.65 g, 63%) as a
white solid. 1H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 8.0 Hz, 1H),
7.19 (d, J = 2.2 Hz, 1H), 6.99 (dd, J = 8.0 Hz, J = 2.2 Hz, 1H), 4.52
(d, J = 13.2 Hz, 1H), 3.77 (t, J = 6.6 Hz, 3H), 3.52 (t, J = 6.6 Hz, 2H),
2.85 (t, J = 13.1 Hz, 1H), 2.43 (s, 3H), 2.77 (m, 2H), 2.05 (s, 3H),
2.00 (m, 2H), 1.75 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 174.10,
168.68, 140.77, 135.62, 135.29, 132.00, 128.29, 126.16, 47.52, 45.38,
42.17, 40.54, 39.19, 30.68, 28.70, 28.19, 21.26, 19.70. MS (m/z):
calcd for C18H24Cl2N2O2, 370.12. LC−MS (m/z (rel intens)): 371.12
(M + H, 100).
risks associated with the use of CCR5 natural ligand variants as
coreceptor pharmacophores in dual-functional drug design.
CONCLUSIONS
■
In conclusion, we provide an example of MTDL design
strategy for creating dual-targeting HIV-1 entry inhibitors via
combining a gp41 peptide fusion inhibitor and a small-
molecule CCR5 antagonist into one compound. We
demonstrated that this dual-functional compound preserved
the specific targeting properties of the two distinct
pharmacophores and, at the same time, exhibited highly
potent antiviral activity against laboratory-adapted HIV-1
strains and primary HIV-1 isolates with diverse subtypes and
T20-resistant mutants. Also, the exceptional potency of the
dual-targeting ligands benefits from synergy between the
coreceptor antagonist and gp41 inhibitor. Overall, our
designed CP12TAK provides a therapeutic lead molecule for
developing new anti-HIV-1 drugs. Moreover, this work
highlights the development of MTDLs for the treatment of
complex infectious diseases caused by enveloped viruses, such
as HIV, MERS-CoV, and SARS-CoV-2.
Synthesis of Compound 3. Using literature procedures,27
compound 3 was prepared (70.4% yield). 1H NMR (400 MHz,
CDCl3) δ 7.99 (d, J = 7.7 Hz, 2H), 7.26 (d, J = 7.7 Hz, 2H), 6.37 (s,
1H), 3.91 (s, 3H), 3.52 (t, J = 5.8 Hz, 2H), 3.41 (t, J = 5.8 Hz, 2H),
2.46 (t, J = 5.8 Hz, 2H), 2.35 (t, J = 5.8 Hz, 2H), 1.48 (s, 9H). 13C
NMR (100 MHz, CDCl3) δ 166.89, 154.67, 142.18, 140.62, 129.48,
128.77, 127.90, 123.83, 79.62, 51.99, 45.25, 44.39, 36.26, 29.29, 28.39.
MS (m/z): calcd for C19H25NO4, 331.18. LC−MS (m/z (rel intens)):
354.16 (M + Na, 27).
Synthesis of Compound 4. Using literature procedures,27
compound 4 was prepared (94.9% yield). 1H NMR (400 MHz,
CDCl3) δ 9.49 (b, 1H), 9.04 (b, 1H), 7.97 (d, J = 7.9 Hz, 2H), 7.21
(d, J = 7.9 Hz, 2H), 3.90 (s, 3H), 3.35 (m, J = 12.8 Hz, 2H), 2.82 (m,
J = 12.4 Hz, 2H), 2.65 (t, J = 6.7 Hz, 2H), 1.82 (d, J = 12.4 Hz, 3H),
1.60 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 166.89, 144.30,
129.84, 128.99, 128.50, 52.04, 43.90, 42.28, 35.93, 28.47. MS (m/z):
calcd for C14H19NO2, 233.14. LC−MS (m/z (rel intens)): 234.13 (M
+ H, 100).
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise stated, all materials used were
commercially obtained and used as supplied. Thin layer chromatog-
raphy was performed on silica gel GF254 plates. Silica gel (200−300
mesh) from Qingdao Haiyang Chemical Company was used for
1
column chromatography. H (400 MHz) and 13C (100 MHz) NMR
spectra were measured on a JNM-ECA-400 spectrometer using TMS
as the internal standard. Chemical shifts for the proton-magnetic-
resonance (1H NMR) spectra were quoted in parts per million (ppm)
and referenced to the signals of residual chloroform (7.29 ppm) or
methanol (3.30 ppm). All 13C NMR spectra were reported in ppm
relative to deuterochloroform (77.00 ppm) or methanol (49.00 ppm).
For small molecules, mass spectra (MS) were measured on an API-
150 mass spectrometer with an electrospray ionization source from
ABI Inc. For compounds tested in biological assay, the purity of each
compound was confirmed to be ≥95% by analytical reversed-phase
high-performance liquid chromatography (RP-HPLC) (Shimadzu
analytical HPLC system). Such information is provided in the
Supporting Information (Tables S2 and S3). The molecular weight of
the peptides was characterized by matrix-assisted laser desorption
ionization−time-of-flight mass spectrometry (Autoflex III, Bruker
Daltonics Inc., Billerica, MA).
Synthesis of Compound 5. To a mixture of compound 2 (1.38
g, 3.72 mmol), compound 4 (1.0 g, 3.72 mmol), and K2CO3 (2.05 g,
14.8 mmol) in DMF/acetonitrile (80 mL, 1:1, v/v) was added KI
(0.62 mg, 4.5 mmol), and the mixture was stirred at reflux for 12 h.
After cooling to room temperature, the mixture was filtered, and the
filtrate was concentrated in a vacuo. The residue was diluted with
EtOAc, washed with water and brine, dried with MgSO4, filtered, and
concentrated in vacuo. Purification silica gel chromatography
(EtOAc/MeOH 1: 0 to 5: 1) afforded 9 as a white foam (0.63 g,
30.3% yield). 1H NMR (400 MHz, CDCl3) δ 11.14 (b, 1H), 9.12 (b,
1H), 7.97 (d, J = 7.7 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.21 (m, 3H),
7.03 (dd, J = 8.0 Hz, J = 2.2 Hz, 1H), 4.52 (d, J = 13.3 Hz, 1H), 3.90
(s, 3H), 3.79 (m, 2H), 3.64 (b, 3H), 3.07 (m, 2H), 2.89 (m, 1H),
2.65 (m, 4H), 2.41 (b, 5H), 2.08 (s, 3H), 1.99 (m, 2H), 1.86 (m,
2H), 1.74 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 174.95, 169.79,
166.83, 160.95, 160.58, 144.15, 139.72, 137.29, 135.58, 132.28,
129.78, 128.92, 128.45, 127.88, 126.24, 117.09, 114.21, 54.82, 53.25,
52.97, 51.97, 46.44, 45.47, 41.81, 40.88, 38.92, 35.81, 29.00, 28.61,
27.95, 22.68, 20.63, 19.63. MS (m/z): calcd for C32H42ClN3O4,
267.29. LC−MS (m/z (rel intens)): 568.28 (M + H, 100).
Synthesis of Compound TAK. To a stirred solution of
compound 5 (1.5 g, 2.65 mmol) in dioxane (15 mL) was added 1
M NaOH (15 mL). The mixture was stirred at room temperature for
1 h, quenched with 1 M HCl (5 mL), extracted with DCM (15 mL ×
3), dried, filtered, and concentrated. Purification by silica gel
chromatography (1:1 EtOAc/methanol) afforded TAK as a white
solid (0.94 g, 64.1% yield). 1H NMR (400 MHz, CD3OD) δ 7.96 (d, J
= 8.2 Hz, 2H), 7.45 (m, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.22 (dd, J =
2.1 Hz, 1H), 4.43 (d, J = 13.4 Hz, 1H), 3.87 (d, J = 13.4 Hz, 1H),
3.77 (m, 2H), 3.56 (d, J = 12.2 Hz, 2H), 3.09 (t, J = 7.7 Hz, 2H), 2.93
(t, J = 12.2 Hz, 3H), 2.70 (d, J = 6.6 Hz, 2H), 2.51 (m, 1H), 2.41 (s,
3H), 2.39 (m, 1H), 2.04 (s, 3H), 1.96 (m, 5H), 1.71 (m, 3H), 1.59
(m, 3H). 13C NMR (100 MHz, CD3OD) δ 177.66, 171.90, 170.20,
146.63, 142.11, 138.85, 136.94, 134.03, 131.45, 130.81, 130.60,
130.07, 128.35, 55.92, 54.51, 48.18, 47.14, 43.30, 42.32, 41.20, 37.00,
31.03, 30.36, 29.80, 24.05, 21.61, 20.26. MS (m/z): calcd for
C31H40ClN3O4, 553.27. LC−MS (m/z (rel intens)): 554.26 (M + H,
100).
Synthesis of Compound 1. To a solution of 1-acetylpiperidine-
4-carboxylic acid (1 g, 5.84 mmol) in THF (15 mL) was added N,N′-
carbonyldiimidazole (1.42 g, 8.76 mmol) at room temperature. After
stirring at room temperature for 4 h, 3-chloro-4-methylaniline was
added, and the mixture was stirred for 24 h. The mixture was
concentrated in vacuo, and the residue was diluted with dichloro-
methane (DCM). The organic layer was washed with aqueous
NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by column chromatography (EtOAc/MeOH
1
50: 1 to 30: 1) to give 1.41 g (82%) of 1 as a white solid. H NMR
(400 MHz, CDCl3) δ 8.11 (b, 1H), 7.62 (d, J = 2.2 Hz, 1H), 7.30
(dd, J = 8.2 Hz, J = 2.2 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 4.56 (d, J =
13.5 Hz, 1H), 3.87 (d, J = 13.5 Hz, 1H), 3.11 (t, J = 12.4 Hz, 1H),
2.69 (t, J = 12.4 Hz, 1H), 2.52 (m, 1H), 2.31 (s, 3H), 2.11 (s, 3H),
1.95 (m, 2H), 1.79 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 172.47,
169.17, 135.72, 134.33, 131.84, 130.93, 120.54, 118.25, 45.74, 43.57,
41.00, 28.70, 28.45, 21.34, 19.39. MS (m/z): calcd for C15H19ClN2O2,
294.11. LC−MS (m/z (rel intens)): 295.11 (M + H, 100).
Synthesis of Compound 2. To an ice-cooled stirred solution of
1 (1.0 g, 2.8 mmol) in DMF (15 mL) was added NaH (60% in oil,
0.70 g, 29.2 mmol), and the mixture was stirred at 0 °C for 30 min.
To the mixture was added 1-bromo-3-chloropropane (0.89 g, 5.7
mmol), and the mixture was stirred at room temperature for 1 h. The
I
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Peptide Synthesis. Peptides were synthesized using standard
Fmoc solid-phase synthesis techniques with a CS biopolypeptide
synthesizer. Rink Amide resin, with a resin loading of 0.53 mmol/g,
was selected as the solid support. DMF, DCM, N-methyl-2-
pyrrolidone, methanol, piperidine, and other reagents used in the
reaction process were anhydrous reagents or dried prior to use. The
template peptides containing a deprotected lysine residue at their C-
terminus required a special deprotection step (four 3-min washes of
2% hydrazinehydrate in DMF). This enabled the conjugation of a
small-molecule moiety, which was performed by the addition of three
equivalents of small molecules, three equivalents of HBTU, and six
equivalents of DIEA in DMF to the resin followed by stirring for 2 h.
The peptides were cleaved from the resin and deprotected with
reagent K, which contained 85% trifluoroacetic acid, 5% thioanisole,
5% m-cresol, and 5% water. The carboxyl termini were amidated upon
cleavage from the resin, and the amino termini were capped with
acetic acid anhydride. All crude peptides were purified by RP-HPLC
(Shimadzu preparative HPLC system), and the purity of each peptide
was confirmed to be ≥95% by analytical RP-HPLC (Shimadzu
analytical HPLC system). The molecular weight of the peptides was
characterized by matrix-assisted laser desorption ionization−time-of-
flight mass spectrometry (Autoflex III, Bruker Daltonics Inc., Billerica,
MA).
ellipticity change by applying a thermal gradient of 2 °C/min from 20
to 90 °C.
Calcium Mobilization Assay. Intracellular calcium fluxes were
measured with the FLIPR Tetra system. HEK293 cells stably
expressing Gα15 and CCR5 were seeded onto 384-well plates and
incubated at 37 °C in 5% CO2 overnight. Cells were loaded with 4
μM Fluo-4 Direct (Invitrogen, Cat# F10471) in Assay Buffer [Hanks’
balanced salt solution (HBSS):HEPES = 49:1, v/v] and incubated for
50 min at 37 °C with 5% CO2 followed by an incubation for 10 min at
room temperature. After washing, 10 μL HBSS containing various
concentrations of testing compounds was added. After incubation at
room temperature for 10 min, 10 μL HBSS containing RANTES-
(CCL5) (final concentration 4 nM) was added to the cell plate, and
the intracellular calcium change was recorded at an excitation
wavelength of 490 nm and an emission wavelength of 515 nm. The
IC50 values of the compounds were determined with GraphPad Prism
software by constructing their dose−response curves.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00781.
■
sı
Inhibition of HIV-1 Infection. Inhibitory activities of peptides
against HIV-1 infection were determined as previously described.49,53
Briefly, 100 TCID50 HIV-1 strains were infected with 104 CEMx174
5.25M7 or MT-2 cells at 37 °C overnight with tested compounds that
were diluted with fetal bovine serum (FBS)-free RPMI 1640 medium.
The medium was replaced with fresh RPMI 1640 medium containing
10% FBS the next morning. After an additional 6 days for CEMx174
5.25M7 cells and 3 days for MT-2 cells, the culture supernatants from
each well were mixed with equal volumes of 5% Triton X-100. P24
antigen was detected using ELISA assay. The IC50 values were
calculated using CalcuSyn software.54
HPLC method used for the purification of peptide
compounds; HPLC method used for the analysis of
peptide compounds; potential cytotoxicity of CP12TAK
on CEMx174 5.25M7 and MT-2 cells; MALDI-TOF-
1
MS and Analytical HPLC of designed peptides; H and
13C NMR spectra of small-molecule compounds; and
ESI-TOF-MS spectra of small-molecule compounds
(PDF)
Molecular formula strings (CSV)
Cytotoxicity Assay. Serial dilutions of CP12TAK were mixed
with CEMx174 5.25M7 cells. Then the culture medium was replaced
with fresh RPMI 1640 medium with 10% FBS 10 h later. The
cytotoxicity of CP12TAK was tested after an additional 48 h using the
Cell Counting Kit-8 (Dojindo, Japan).
Native-PAGE. Peptides at a final concentration of 50 μM,
including the N- and C-terminal free peptides, as well as their
equimolar mixtures, were dissolved in PBS (1×, pH 7.4) and
incubated at 37 °C for 30 min. After mixing the above peptides with
Tris-glycine native sample buffer (BioRad, Hercules, CA) in a 1:1
ratio, the samples were loaded (20 μL in each well) onto 10% Tris-
glycine gels. Gel electrophoresis was run under a constant voltage of
120 V at room temperature for 4 h. The obtained gel was then stained
with Coomassie Blue R250. The images were taken using a
ChampGel 6000 Imaging System (Sage Creation Ltd., Beijing,
China).
SVA. SVA was performed using a Proteomelab XL-A/XL-I
analytical ultracentrifuge (Beckman Coulter, Fullerton, CA) equipped
with a three-channel cell and an An-60 Ti rotor. All samples were
prepared at a final concentration of 150 μM in PBS (1×, pH 7.4). The
C-peptides/N36 mixtures were incubated at 37 °C for 30 min and
were initially scanned at 3000 rpm for 10 min. Data were obtained at
a wavelength of 280 nm after centrifugation at 60,000 rpm and 20 °C
for 7 h. Weight-averaged molecular weights were calculated and fitted
by processing with the SEDFIT program.
CD Spectroscopy. The C-peptides and N36 were dissolved in
PBS (1×, pH 7.4) and ddH2O, respectively, at a concentration of 10
μM. The equimolar mixtures were incubated at 37 °C for 30 min at a
final concentration of 10 μM. The CD spectra were recorded on a
MOS-450 system (BioLogic, Claix, France) with the following
conditions: temperature, 4 °C; wavelength, 190−260 nm; resolution,
0.1 nm; path length, 0.1 cm; response time, 4.0 s; and scanning speed,
50 nm/min. The obtained results were processed to obtain the mean
residue ellipticity. A mean residue ellipticity [θ] of −33,000° cm2/
dmol at a wavelength of 222 nm was considered to be 100% α-
helicity. Thermal denaturation was monitored at 222 nm by the
AUTHOR INFORMATION
Corresponding Authors
■
Chao Wang − State Key Laboratory of Toxicology and
Medical Countermeasures, Beijing Institute of Pharmacology
and Toxicology, Beijing 100850, China; orcid.org/0000-
0002-7673-0638; Phone: 86-10-6693-0640;
Email: chaow301@sina.com; Fax: 86-10-6821-1656
Yang Liu − Key Laboratory of Structure-based Drug Design &
Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenyang
110016, China; orcid.org/0000-0002-9284-1944;
Phone: 86-24-4352-0227; Email: y.liu@syphu.edu.cn
Lu Lu − Key Laboratory of Medical Molecular Virology
(MOE/NHC/CAMS), School of Basic Medical Sciences and
Shanghai Public Health Clinical Center, Shanghai Institute of
Infectious Diseases and Biosecurity, Fudan University,
Shanghai 200032, China; Phone: 86-21-5423-7671;
Email: lul@fudan.edu.cn; Fax: 86-21-5423-7465
Shibo Jiang − Key Laboratory of Medical Molecular Virology
(MOE/NHC/CAMS), School of Basic Medical Sciences and
Shanghai Public Health Clinical Center, Shanghai Institute of
Infectious Diseases and Biosecurity, Fudan University,
Shanghai 200032, China; orcid.org/0000-0001-8283-
7135; Phone: 86-21-5423-7673; Email: shibojiang@
fudan.edu.cn; Fax: 86-21-5423-7465
Authors
Xinling Wang − Key Laboratory of Medical Molecular
Virology (MOE/NHC/CAMS), School of Basic Medical
Sciences and Shanghai Public Health Clinical Center,
Shanghai Institute of Infectious Diseases and Biosecurity,
Fudan University, Shanghai 200032, China
J
https://doi.org/10.1021/acs.jmedchem.1c00781
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
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Huan Wang − State Key Laboratory of Toxicology and
Medical Countermeasures, Beijing Institute of Pharmacology
and Toxicology, Beijing 100850, China
Jing Pu − Key Laboratory of Medical Molecular Virology
(MOE/NHC/CAMS), School of Basic Medical Sciences and
Shanghai Public Health Clinical Center, Shanghai Institute of
Infectious Diseases and Biosecurity, Fudan University,
Shanghai 200032, China
Qing Li − State Key Laboratory of Toxicology and Medical
Countermeasures, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China
Jiahui Li − Key Laboratory of Structure-based Drug Design &
Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenyang
110016, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00781
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Author Contributions
∥C.W. and X.W. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported, in part, by grants from the
National Natural Science Foundation of China (21877127 to
C.W.; 81630090 to S.J.; and 81822045 to L.L.) and Shanghai
Clinical Research Center for Infectious Disease (HIV/AIDS)
(20MC1920100).
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