Bivalent HIV-1 fusion inhibitors based on peptidomimetics

Article abstract of DOI:10.1016/j.bmc.2020.115812

Membrane fusion is a valid target for inhibition of HIV-1 replication. A 34-mer fragment peptide (C34), which is contained in the HIV-1 envelope protein gp41, has significant anti-HIV activity. Previously, a dimeric derivative of C34 linked by a disulfide bridge at its C-terminus was found to have more potent anti-HIV activity than the C34 peptide monomer. To date, several peptidomimetic small inhibitors have been reported, but most have lower potency than peptide derivatives related to C34. In the present study we applied this dimerization concept to these peptidomimetic small inhibitors and designed several bivalent peptidomimetic HIV-1 fusion inhibitors. The importance of the length of linkers crosslinking two peptidomimetic compounds was demonstrated and several potent bivalent inhibitors containing tethered peptidomimetics were produced.

Full text of DOI:10.1016/j.bmc.2020.115812

Journal Pre-proofs
Bivalent HIV-1 fusion inhibitors based on peptidomimetics
Takuya Kobayakawa, Kento Ebihara, Kohei Tsuji, Takuma Kawada,
Masayuki Fujino, Yuzuna Honda, Nami Ohashi, Tsutomu Murakami,
Hirokazu Tamamura
PII:
S0968-0896(20)30642-8
https://doi.org/10.1016/j.bmc.2020.115812
BMC 115812
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 September 2020
29 September 2020
3 October 2020
Please cite this article as: T. Kobayakawa, K. Ebihara, K. Tsuji, T. Kawada, M. Fujino, Y. Honda, N. Ohashi, T.
Murakami, H. Tamamura, Bivalent HIV-1 fusion inhibitors based on peptidomimetics, Bioorganic & Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115812
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Bivalent HIV-1 fusion inhibitors based on peptidomimetics
a,#
a,#
a
a
b
Takuya Kobayakawa , Kento Ebihara , Kohei Tsuji , Takuma Kawada , Masayuki Fujino , Yuzuna
a
a,$
b,*
a,*
Honda , Nami Ohashi , Tsutomu Murakami , Hirokazu Tamamura
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10
Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
bAIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo
1
62-8640, Japan
#
$
equally contributed present address: Showa Pharmaceutical University, Machida, Tokyo 194-8543,
Japan
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
*To whom correspondence should be addressed; tmura@nih.go.jp, phone; +81-3-4582-2816, fax; +81-3-
5
285-5037, or tamamura.mr@tmd.ac.jp, phone; +81-3-5280-8036, fax; +81-3-5280-8039

Abstract
Membrane fusion is a valid target for inhibition of HIV-1 replication. A 34-mer fragment peptide (C34),
which is contained in the HIV-1 envelope protein gp41, has significant anti-HIV activity. Previously, a
dimeric derivative of C34 linked by a disulfide bridge at its C-terminus was found to have more potent
anti-HIV activity than the C34 peptide monomer. To date, several peptidomimetic small inhibitors have
been reported, but most have lower potency than peptide derivatives related to C34. In the present study
we applied this dimerization concept to these peptidomimetic small inhibitors and designed several
bivalent peptidomimetic HIV-1 fusion inhibitors. The importance of the length of linkers crosslinking two
peptidomimetic compounds was demonstrated and several potent bivalent inhibitors containing tethered
peptidomimetics were produced.
Keywords
anti-HIV-1 activity
bivalent fusion inhibitor
peptidomimetic
gp41

1
. Introduction
Many anti-human immunodeficiency virus (HIV)-1 drugs are currently available to treat HIV-1-infected
1
2
3
4
individuals and AIDS patients, and inhibitors of reverse transcriptase, protease and integrase in
combination antiretroviral therapy (cART) have brought great success to the chemotherapy of HIV
infectious diseases. Serious drawbacks have been noted however, including the emergence of mutant viral
strains with multi-drug resistance, the appearance of significant side effects, and the expense of the
prescribed medicines. In an attempt to enlarge the repertoire of anti-retroviral drugs and address the above
problems, we have developed drug candidates with different mechanisms of action, including co-receptor
5
6
7
8
CXCR4 antagonists, CD4 mimics, fusion inhibitors, integrase inhibitors and inhibitors involving viral
9
uncoating and viral assembly. Theviral entry steps are attractive targets for drug discovery because these
are points at which an HIV-1 invasion could be entirely blocked.
Entry of HIV-1 into target cells is manipulated by two glycoproteins in the HIV-1 envelope, which
consists of a surface subunit gp120 and a non-covalently associated transmembrane subunit gp41.10
Sequential binding of gp120 to its cell receptor CD4 and to a co-receptor such as CCR5 or CXCR4, can
1
1
trigger a conformational rearrangement of gp41. The gp41 protein forms trimer structures which mainly
1
0
consist of the N- and C-terminal heptad repeat (NHR and CHR) helical regions. Fusion is caused by
assembly of these NHR and CHR trimers into six-helical bundles according to the conformational
rearrangement of gp41. In this process, the membranes of the virus and the host cell approach each other,
and the final result is fusion between viral and cellular membranes.1,11 To date, several peptides mimicking
NHR or CHR have been synthesized and their inhibition of HIV-1 fusion has been studied. CHR peptides
interact competitively with NHR, and this can prevent the dynamic formation of six-helical bundles.12
1
3
T20, a 36-mer fragment peptide derived from CHR, has been used clinically since 2003. The brand
name of this peptide, manufactured by Roche/Trimeris, is enfuvirtide and for AIDS therapy, it is
1
4
administered by injection. The site at which T20 interacts with NHR is the C-terminal side of the CHR
1
5
region, which is designated as a lipid binding domain (LBD). The N-terminal side of the CHR region,
which is composed of hydrophobic amino acids and has been identified as a pocket binding domain
3

1
6
(
PBD), is a more important NHR interaction site. A 34-mer peptide, C34, which contains amino acid
1
7
sequences of PBD, shows higher inhibitory activity than T20. The hydrophobic interactions between
the side chains of the amino acids in C34 and NHR are essential for the formation of six-helical bundles.18
C34 is a useful tool in terms of its high anti-HIV activity, but it is highly hydrophobic and insoluble, and
therefore unlikely to be useful as a drug. C34 derivatives with conjugated sterols in the C-terminus for
1
9
7
membrane attachment have been reported, and as noted in our previous study, the solubility of the C34
peptide was increased by addition of a triplet repeat of arginine and glutamic acid (RERERE) to the C-
terminus of wild type C34, the NL4-3 strain. This new C34 derivative was designated C34REG (Fig. 1).7
C34REG peptides conjugated with a polyethylene glycol (PEG)-linker to construct a dimer (1) or trimer
of C34REG, and a dimeric derivative of C34REG linked by a disulfide bridge in its C-terminus (2), were
7
found to have remarkably more potent anti-HIV activity than the C34REG monomer. The dimeric
structure could therefore be a critical unit for the interaction with viral NHR. These C34REG dimeric
derivatives might interact with two sites of the NHR trimer, possibly enhancing the stability of the
complex structure interacting with the NHR trimer, and more strongly suppressing 6-helical bundle
formation by the NHR trimer and the CHR trimer compared to the monomer peptides.
4

On the other hand, small molecules (3–5) based on the amino acid sequence of C34 have been
synthesized by Whitby et al. in an attempt to find drugs whose administration does not require injection
C34 (HIV-1 NL4-3 strain)
628
661
H N-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-REREREG-COOH
2
O
C34REG
OH
SR
H N
2
SR
H N GCGG
O
H
N
O
CH3
C34REG
N
3 O
N
2
H
O
O
CHR
NHR
SR
H N GCGG
O
H
O
N
N
dimeric
inhibitors
HIV
gp41
C34REG
2
H
O
O
SR
R =
C34
NH2
1
SAcm
H N GCGG
O
H
N
C34REG
2
OH
O
S
SAcm
H N GCGG
S
O
OH
C34REG
2
N
H
O
2
Figure 1. Design of dimeric inhibitors based on the gp41-C34 peptide, a C-terminal pegylated dimer
1) and a C-terminal disulfide bridged dimer (2): the SH groups of the Cys residues at the N-terminal
second position are protected by acetamido (-CH CONH ) and acetamidomethyl (Acm) groups,
(
2
2
respectively.
2
0
(
Fig. 2). Compounds such as 3–5 however, failed to show high potency compared to C34 or T20 peptide
derivatives. Thus, we applied our above dimerization concept to these compounds and designed bivalent
HIV-1 fusion inhibitors based on peptidomimetics. Recently, bivalent small gp41 inhibitors with
enhanced potency were reported, but the length of the linker tethering the two inhibitors was not
2
1
considered, and relatively short linkers were used. Thus, in this study the appropriate length of linkers
to crosslink two peptidomimetic compounds was investigated in the search for potent bivalent inhibitors.
5

2
2
. Results and discussion
.1. Design of compounds
Initially, bivalent derivatives of 3 were designed and synthesized. The site of crosslinking between
compound 3 and a linker had been determined to be the terminal amino group in earlier structure-activity
2
0
relationship studies according to the report by Whitby et al. Compound 3 partially mimics the -helical
structure of C34, and its terminal amino group can be treated as if it is an N-terminus of peptides. As a
linker, a PEG-linker was adopted because such a choice suppresses an increase in hydrophobicity and
non-specific binding, and permits simple preparation of linkers of various lengths because PEG-linkers
can easily be elongated by incorporation of additional ethylene glycol moieties. Since both the compounds
that are to be linked are required to interact with PBDs, the length of the linker between these two
compounds is critical for high inhibitory activity. No reports however, have focused on the distance
between two PBDs and consequently, based on the six-helical bundle structure consisting of three N36
and three C34 peptides, bivalent inhibitors crosslinking two molecules of compound 3 with PEG-linkers
of various lengths (6a–6g) were designed and synthesized (Fig. 3). In this design, succinic acid was
H
H
N
N
O
OH
O
O
O
N
H
OH
OH
H N
2
O
O
N
O
N
N
H
H
H
H2N
O
O
H2N
O
O
N
N
H
H
NH
O
O
N
H
OH
N
OMe
N
H
H
3
4
5
Figure 2. Structures of small molecules based on the amino acid sequence of C34: H-Trp-[Trp]-Leu-
OH (3), H-Asp-[Trp]-Trp-OH (4) and H-Tyr(Me)-[Trp]-Trp-OH (5).
Figure 3. (left) The six-helical bundle structure consisting of three N36 and three C34 peptides (PDB:
2
Z2T) and the distance between two PBDs. (right) The designed structures of bivalent inhibitors
crosslinking two molecules of compound 3 with PEG-linkers of various lengths (6a–6g).
6

introduced between the terminal amino group of compound 3 and PEG-linkers, and the suitable length of
PEG-linkers was investigated according to data from the C34REG dimeric derivatives. Bivalent
compounds of 4 or 5 containing the PEG-linker with the suitable length were then designed and
synthesized.
2
2
.2. Chemistry
.2.1. Synthesis of PEG-linkers (12a–12g)
The bis-azide derivatives (9b, 9c) were synthesized from commercially available diethylene glycol (7b)
and tetraethylene glycol (7c) respectively, by tosylation and azidation (Scheme 1). The bis-azide
2
2
derivatives 9d–9g were synthesized by condensation of bis-tosylates (8b, 8c) and azides (10b, 10c). Bis-
hemisuccinates (12b–12g) were synthesized from the bis-azides (9b–9g), respectively, by a Staudinger
reaction followed by hemi-succination. The hemisuccinate (12a) was synthesized from 1.2-
ethylenediamine (11a).
7

a
b
O
O
O
HO
n
OH
TsO
O
n
OTs
N3
n
N3
7
7
b (n = 1)
c (n = 3)
8b (n = 1)
8c (n = 3)
9
9
b (n = 1)
c (n = 3)
c
O
n
+
O
TsO
OTs
HO
n
N3
N3
n
N3
8
b (n = 1)
10b (n = 1)
10c (n = 3)
9
9
9
9
d (n = 5): 8b + 10b x 2
e (n = 7): 8c + 10b x 2
f (n = 9): 8b + 10c x 2
8c (n = 3)
g (n = 11): 8c + 10c x 2
O
O
e
d
O
n
O
HO
O
OH
N3
N3
H N
n
NH2
N
H
n
N
H
2
O
O
1
1a (n = 0)
12a (n = 0)
9
b (n = 1)
c (n = 3)
d (n = 5)
e (n = 7)
f (n = 9)
g (n = 11)
11b (n = 1)
11c (n = 3)
11d (n = 5)
11e (n = 7)
11f (n = 9)
1
1
1
1
1
1
2b (n = 1)
2c (n = 3)
2d (n = 5)
2e (n = 7)
2f (n = 9)
9
9
9
9
9
11g (n = 11)
2g (n = 11)
Scheme 1. Synthesis of PEG-linkers; (a) TsCl, Et N, DCM, 0 °C to rt, 1 d; (b) NaN , EtOH, reflux,
3
3
overnight; (c) NaH, THF, 0 °C to rt, overnight; (d) PPh , H O, THF, reflux; (e) succinic anhydride,
3
2
Et N, THF, rt, overnight.
3
2
.2.2. Synthesis of the bivalent compounds (6a–6g)
Condensation of the tert-butyl ester (13) of compound 3 and various PEG-linkers (12a–12g), followed by
deprotection of the tert-butyl groups yielded the bivalent compounds (6a–6g), respectively (Scheme 2).
8

O
O
O
t
O Bu
HO
OH
O
N
N
O
O
O
O
N
H
a, b
H
H
H
H
H2N
O
O
N
O
N
O
N
N
N
n
N
N
H
H
H
H
H
O
O
O
O
O
NH
HN
6a (n = 0)
6b (n = 1)
NH
N
N
N
H
H
H
6
6
6
6
6
c (n = 3)
d (n = 5)
e (n = 7)
f (n = 9)
13
g (n = 11)
Scheme 2. Synthesis of the bivalent compounds (6a–6g); (a) N,N′-diisopropylcarbodiimide (DIPCDI),
Oxyma Pure, 12a or 12c–12f, DMF, rt, 2 d for 6a or 6c–6f; or 1-[bis(dimethylamino)methylene]-1H-
1
,2,3-triazolo[4,5-b]pyridinium
3-oxide
hexafluorophosphate
(HATU),
1-hydroxy-7-
azabenzotriazole (HOAt), 12b or 12g, DMF, rt, 2 h for 6b or 6g; (b) anisole, 4 M HCl/dioxane, 0 °C
to rt, 1 h.
2
.2.3. Synthesis of the monovalent compound (16)
Condensation of the tert-butyl ester (13) of 3 with the hemisuccinate (15), which was derived from PEG-
linker (12f), and subsequent deprotection of the tert-butyl group yielded the monovalent compound (16)
(Scheme 3).
O
O
O
a
b
O
tBuO
tBuO
O
O
OH
H N
2
NH2
9
N
NH2
N
NH
9
9
H
H
O
O
O
12f
14
15
O
OH
O
O
O
N
c, d
H
H
HO
O
N
O
N
N
N
9
H
H
H
O
O
O
16
NH
N
H
Scheme 3. Synthesis of the monovalent compound (16); (a) mono-tert-butyl succinate, DIPCDI,
DCM, rt, overnight; (b) succinic anhydride, Et N, THF, 0 °C to rt, 2 d; (c) DIPCDI, Oxyma Pure, 13,
3
DMF, rt, 2 d; (d) anisole, TFA, 0 °C to rt, 0.5 h.
2
.2.4. Synthesis of the bivalent compounds (19, 20)
9

Condensation of the di-tert-butyl ester (17) of compound 4 with the PEG-linker (12f), and subsequent
deprotection of the tert-butyl groups yielded the bivalent compound (19) (Scheme 4). Condensation of
the tert-butyl ester (18) of compound 5 with the PEG-linker (12f), and subsequent deprotection of the
tert-butyl groups yielded the bivalent compound (20) (Scheme 5).
H
N
O
t
O Bu
O
N
H
a, b
H N
2
O
N
H
O
O
t
O Bu
N
H
H
17
H
N
N
O
O
HO
OH
N
O
O
O
O
N
H
H
H
H
O
N
O
N
O
N
N
H
N
N
H
O
9
H
H
O
O
O
O
O
OH
19
OH
N
N
H
H
Scheme 4. Synthesis of the bivalent compound (19); (a) DIPCDI, Oxyma Pure, 12f, DMF, rt, 2 d; (b)
anisole, TFA, DCM, 0 °C to rt, 1 h.
1
0

H
N
O
t
O Bu
O
N
a, b
H
H N
2
O
N
H
O
OMe
N
H
18
H
H
N
N
O
O
HO
OH
N
O
O
O
O
N
H
H
H
H
O
N
O
N
O
N
N
N
N
9
H
H
H
H
O
O
O
O
20
N
MeO
OMe
N
H
H
Scheme 5. Synthesis of the bivalent compound (20); (a) DIPCDI, Oxyma Pure, 12f, DMF, rt, 2 d; (b)
anisole, TFA, DCM, 0 °C to rt, 1 h.
2
.3. Evaluation of the anti-HIV-1 activity and the cytotoxicity of the bivalent compounds (6a–6g)
The inhibitory activities of test compounds against infection of HIV-1 (NL4-3 strain) in TZM-bl cells
2
3
were assessed by a luciferase assay, and the results are shown in Table 1. The bivalent compounds (6a–
g) showed remarkably higher anti-HIV activity than the original monomer compound (3). It is
noteworthy that 6f showed the highest anti-HIV activity among the bivalent compounds (6a–6g).
6
20
According to the report by Whitby et al., the potency of compound 3 (IC (cell-cell fusion assay) = 5
5
0
M) is relatively high, but the anti-HIV assay condition is different because the present assay is a cell-
based infection assay, and the absolute value might not be significant. The cytotoxicity of the bivalent
compounds (6a–6g) in TZM-bl cells was not observed at concentrations below 50 M. The monomer
compound with a PEG-linker (16), which is the same length of PEG-linker as 6f, did not show high
potency, but almost the same level of potency as the original monomer compound 3. This suggests that
the key to the increased the anti-HIV activity of 6f is not primarily a PEG unit, but two molecules of
compound 3, which are separated by a suitable distance. All of the bivalent ligands (6a–6g) possess a
great advantage in that they have two PBD interaction sites, and the binding affinity of 6a–6g for the NHR
1
1

trimer is much higher than that of the monomers (3 and 16) and the dissociation is slower. In particular,
the length of the PEG-linker in 6f is critical for the binding because two molecules of compound 3 might
fit in two PBDs. The interaction of 6f with two PDBs was therefore investigated by docking simulations.
Table 1. The anti HIV-1 activity and the cytotoxicity of the bivalent compounds (6a–6g).
a
b
compound
(monomer)
n (PEG repeat) linker length (Å)
-
IC (M)
CC (M)
5
0
50
-
3
1% inhibition (at 50 M)
20.5 ± 1.7
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
6
a
0
1
15.0
15.8
23.8
30.6
36.8
43.9
51.1
6
b
23.8 ± 1.3
6
c
3
22.8 ± 4.9
6
d
5
17.3 ± 1.3
6
e
7
17.0 ± 1.4
6
f
9
2.1 ± 0.4
6
g
11
15.0 ± 0.7
1
6 (monomer
with PEG)
9
43.9
7% inhibition (at 50 M)
> 50
> 5
T20
-
-
-
-
0.145 ± 0.043
0.118 ± 0.045
AZT
> 100
a
b
IC values are based on luciferase signals in TZM-bl cells infected with HIV-1 (NL4-3 strain). CC
5
0
50
values are based on the reduction of the viability of TZM-bl cells.
2
.4. Docking simulation of compound 6f
Molecular modeling calculations were performed using compound 6f docked into the N36 trimer (PDB
ID: 2Z2T) with MOE (version 2014.0901). The docked structures show two molecules of compound 3,
which are contained in 6f, and seem to fit tightly into two PBDs of the N36 trimer with the clasped PEG-
A
B
C
Figure 4. The structures of the bivalent inhibitor 6f docked in the N36 trimer (PDB ID: 2Z2T). (A)
the whole structure, red: N36, green: 6f, (B) the C-terminal region of the N36 trimer (upper-right
region of A), gray: the surface of N36, (C) view of B from the C-terminus.
1
2

linker (Fig. 4). This suggests that the length of the PEG-linker of 6f is optimal and critical for the binding
to two PBDs of the N36 trimer.
2
.5. Evaluation of anti HIV-1 activity and cytotoxicity of the bivalent compounds (19, 20)
The anti HIV-1 activity of the bivalent compounds (19, 20) with a PEG-linker of the same length as that
of 6f was evaluated to investigate whether the length of the PEG-linker is suitable. The reproducibility of
the data from the bivalent compound (6f), which is derived from two molecules of 3, was established
(Table 2). The bivalent compound (20), derived from two molecules of compound 5, showed significantly
higher anti-HIV activity than the monomer (5). In this assay anti-HIV activity of compound 5 was not
2
0
observed at concentrations below 50 M (cell-based infection assay), although Whitby et al. reported
that the potency of compound 5 is relatively high, with IC (cell-cell fusion assay) = 8 M, but the anti-
5
0
HIV assay employs different conditions and the absolute value might have no significant meaning. The
effectivity of bivalent binding and inhibition in the bivalent compound (20) was confirmed. On the other
hand, the bivalent compound (19), which is derived from two molecules of compound 4, showed
significantly lower anti-HIV activity than the monomer (4). The anti-HIV activity of compound 4 is much
higher (IC (cell-based infection assay) = 1.6 M) than that of 19, and this is compatible with the previous
5
0
2
0
data; the anti-HIV activity of compound 4 is relatively high (IC (cell-cell fusion assay) = 6 M). The
5
0
effectivity of bivalent binding and inhibition in the bivalent compound (19) was not observed. According
2
0
to Whitby et al., compound 3 functions as a CHR -helix mimic and binds to NHR, and compound 4
functions as a reversed CHR -helix mimic and binds to NHR in the opposite direction. In the bivalent
molecule of 3, two compounds of 3 are crosslinked in the terminal amino groups with the succinyl PEG-
linker. Since compounds 3 and 4 interact with NHR in different directions, the suitable crosslink point or
the optimal linker length of the bivalent molecule of 3 is not the same as in the bivalent molecule of 4.
Thus, to search for potent bivalent molecules of 4, investigations of the suitable crosslink point in
2
0
compound 4 and the optimal linker length are necessary. According to Whitby et al., compound 5
1
3

functions as a shifted CHR -helix mimic and binds to NHR in a one-residue slipped manner. Therefore,
the optimal linker length of the bivalent molecule of 3 is not valid for the bivalent molecule of 5. Since
the bivalent compound (20) does not have the optimal length linker, the anti-HIV activity of compound
2
0 is lower than that of the bivalent compound (6f). The optimal linker length of the bivalent molecule
derived from 5 might be two-amino acids length shorter because when compared with compound 3,
compound 5 binds to NHR in a one-residue slipped manner.
Table 2. The anti HIV-1 activity and the cytotoxicity of the bivalent compounds (19, 20).
compound
(monomer)
IC (M)
CC (M)
5
0
50
3
4
5
2% inhibition (at 50 M)
1.7 ± 0.6
> 50
6
f
> 50
> 50
> 50
> 50
> 50
> 5
(monomer)
1.6 ± 0.6
1
9
36.3 ± 4.2
(monomer)
0% inhibition (at 50 M)
10.2 ± 0.6
2
0
T20
0.145 ± 0.043
0.118 ± 0.045
AZT
> 100
3
. Conclusion
In this study, several bivalent HIV-1 fusion inhibitors tethered to peptidomimetic compounds were
developed based on our dimerization concept, which had previously produced a C34 dimeric derivative
with potent anti-HIV activity. Dimerization of peptidomimetic compounds causes a remarkable increase
in anti-HIV-1 activity. As drug candidates, these bivalent inhibitors have great advantages which are not
found in simple peptides. The length of PEG-linkers crosslinking two peptidomimetic compounds was
found to be critical for the binding to two PBD binding sites in the NHR and for the anti-HIV-1 activity,
and the suitable crosslink point and the optimal linker length of the bivalent molecules could be adjusted
according to the structures of small peptidomimetic compounds. These results are useful for the design of
HIV-1 fusion inhibitors in future.
1
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4
. Experimental
Chemistry
All of the compounds were synthesized as shown in Schemes 1–5. Detailed procedures and data are
provided in the Supplementary data.
Virus preparation
For virus preparation, 293T/17 cells in a T-75 flask were transfected with 10 g of the pNL4-3 construct
2
3
by the calcium phosphate method. The supernatant was collected 48 h after transfection, passed through
a 0.45 m filter, and stored at -80 °C as a stock virus.
Anti-HIV-1 assay
4
For the viral fusion inhibitory assay, TZM-bl cells (1 x 10 cells/100 L) were cultivated with the NL4-3
virus (10 ng of p24) and serially diluted peptides. After cultivation for 48 h, cells were lysed and the
luciferase activity was determined with the Steady-Glo luciferase assay system (Promega, WI, USA).23
Cytotoxicity assay
The cytotoxic effects of peptides were determined by the CellTiter 96 Non-Radioactive Cell Proliferation
assay system (Promega) under the same conditions as the anti-HIV assay but in the absence of viral
infection.
Declaration of interest
Conflicts of interest: none.
1
5

Acknowledgements
This work was supported in part by JSPS KAKENHI Grant Numbers 20H03362 and 19K22488
H.T.), 20J13977 (K.E.), 19K16312 (T.K.), JP18H07158 (T.M. and H.T.); Japan Agency for Medical
(
Research and Development (AMED) JP20am0101098 (Platform Project for Supporting Drug Discovery
and Life Science Research, BINDS) (H.T.). This research is based on the Cooperative Research Project
of Research Center for Biomedical Engineering. K.E. was supported by JSPS Research Fellowships for
Young Scientists.
Appendix A. Supplementary data
Supplementary data (synthetic procedures and characterization data of compounds) associated with this
article can be found, in the online version, at doi: .bmc. .
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Declaration of interest
Conflicts of interest: none.
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