Small-molecule anti-HIV-1 agents based on HIV-1 capsid proteins

Article abstract of DOI:10.3390/biom11020208

The capsid of human immunodeficiency virus type 1 (HIV-1) is a shell that encloses viral RNA and is highly conserved among many strains of the virus. It forms a conical structure by assembling oligomers of capsid (CA) proteins. CA dysfunction is expected to be an important target of suppression of HIV-1 replication, and it is important to understand a new mechanism that could lead to the CA dysfunction. A drug targeting CA however, has not been developed to date. Hydrophobic interactions between two CA molecules via Trp184/Met185 in CA were recently reported to be important for stabilization of the multimeric structure of CA. In the present study, a small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the interaction site, was synthesized and its significant anti-HIV-1 activity was confirmed. Structure activity relationship (SAR) studies of its derivatives were performed and provided results that are expected to be useful in the future design and development of novel anti-HIV agents targeting CA.

Full text of DOI:10.3390/biom11020208

biomolecules
Communication
Small-Molecule Anti-HIV-1 Agents Based on HIV-1 Capsid Proteins
Takuya Kobayakawa 1, Masaru Yokoyama 2 , Kohei Tsuji 1 , Masayuki Fujino 3, Masaki Kurakami 1,
Sayaka Boku 1, Miyuki Nakayama 1, Moemi Kaneko 1, Nami Ohashi 1,†, Osamu Kotani 2, Tsutomu Murakami 3,*,
Hironori Sato 2,* and Hirokazu Tamamura 1,
*
1
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku,
Tokyo 101-0062, Japan; tkobmr@tmd.ac.jp (T.K.); ktsuji.mr@tmd.ac.jp (K.T.);
m-kurakami@hhc.eisai.co.jp (M.K.); ma190086@tmd.ac.jp (S.B.); ma190076@tmd.ac.jp (M.N.);
kanekomo@nissanchem.co.jp (M.K.); ohashi@ac.shoyaku.ac.jp (N.O.)
Pathogen Genomics Center, National Institute of Infectious Diseases, Musashimurayama,
Tokyo 208-0011, Japan; yokoyama@nih.go.jp (M.Y.); konioo@nih.go.jp (O.K.)
AIDS Research Center, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan;
fmasa@niid.go.jp
2
3
*
Correspondence: tmura@niid.go.jp (T.M.); hirosato@niid.go.jp (H.S.); tamamura.mr@tmd.ac.jp (H.T.);
Tel.: +81-3-4582-2816 (T.M.); +81-42-561-0771 (H.S.); +81-3-5280-8036 (H.T.)
Current address: Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
†
Abstract: The capsid of human immunodeficiency virus type 1 (HIV-1) is a shell that encloses
viral RNA and is highly conserved among many strains of the virus. It forms a conical structure
by assembling oligomers of capsid (CA) proteins. CA dysfunction is expected to be an important
target of suppression of HIV-1 replication, and it is important to understand a new mechanism
that could lead to the CA dysfunction. A drug targeting CA however, has not been developed to
date. Hydrophobic interactions between two CA molecules via Trp184/Met185 in CA were recently
reported to be important for stabilization of the multimeric structure of CA. In the present study, a
small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the
interaction site, was synthesized and its significant anti-HIV-1 activity was confirmed. Structure
activity relationship (SAR) studies of its derivatives were performed and provided results that are
expected to be useful in the future design and development of novel anti-HIV agents targeting CA.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kobayakawa, T.;
Yokoyama, M.; Tsuji, K.; Fujino, M.;
Kurakami, M.; Boku, S.; Nakayama, M.;
Kaneko, M.; Ohashi, N.; Kotani, O.;
et al. Small-Molecule Anti-HIV-1
Agents Based on HIV-1 Capsid
Proteins. Biomolecules 2021, 11, 208.
https://doi.org/10.3390/biom11020208
Keywords: anti-HIV; capsid; dipeptide mimic; in silico screening
Received: 25 November 2020
Accepted: 12 January 2021
Published: 3 February 2021
1. Introduction
As a retrovirus, human immunodeficiency virus type 1 (HIV-1) can infect CD4-positive
T-cells or macrophage, eventually causing acquired immunodeficiency syndrome (AIDS).
To date, many anti-HIV-1 drugs [
1–3], such as inhibitors of reverse transcriptase [4], pro-
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
tease [ ] and integrase [ ], have been developed for the therapeutic treatment of HIV-1-
5
6,7
infected individuals and AIDS patients. The utilization of the above drugs in combination
with antiretroviral therapy (cART) has brought remarkable success to the chemotherapy
of HIV infectious diseases [1–3]. There are serious defects however, which have not been
escaped. These contain the appearance of mutant viral strains with multi-drug resistance,
emergence of severe side effects and costs of the dosed drugs. In an effort to solve these
problems and enhance the repertoire of anti-HIV-1 drugs, we have sought drugs with
Copyright:
© 2021 by the authors.
different mechanisms of action such as coreceptor CXCR4 antagonists [
ics [15 19], fusion inhibitors [20 23], integrase inhibitors [24 26] and inhibitors of viral
uncoating and viral assembly [27–30].
HIV-1 capsid (CA) proteins [31 32], which are generated from the Gag precursor pro-
8–14], CD4 mim-
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
–
–
–
,
tein Pr55Gag and are composed of N- and C-terminal domains (NTD/CTD), are highly
conserved among many HIV strains. These proteins are structurally assembled by oligomer-
ization of hexamers and pentamers [33], to form a CA core with a conical structure [34,35],
Biomolecules 2021, 11, 208. https://doi.org/10.3390/biom11020208
https://www.mdpi.com/journal/biomolecules

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which encapsulates the HIV-1 RNA genome, the integrase and reverse transcriptase. Matrix
(MA) proteins also result from Pr55Gag, located inside viral membranes, and contribute
to the assembly of the virion shell [36
to be great targets for inhibition against viral replication, and some MA- and CA-derived
peptides with anti-HIV activity have been reported to date by our group [27 30] and oth-
ers [38 41]. Since viral uncoating based on the MA/CA degradation and viral assembly as a
,37]. Both the MA and CA proteins are considered
–
–
consequence of MA/CA protein oligomerization are performed inside host cells, inhibitors
must have cell membrane permeability to be able to suppress viral uncoating and assem-
bly. Consequently, an octa-arginyl group [42] was incorporated into the above peptide
inhibitors to add cell membrane permeability [27
–30]. However, small compounds that
are found to have inhibitory activity against viral uncoating and assembly might have cell
membrane permeability. To date, several small compounds have been discovered [43
but, except for GS-6207 [57], none has progressed to clinical trials.
–59]
2. Materials and Methods
2.1. General Information
All reactions utilizing air- or moisture-sensitive reagents were performed in dried
glassware under an atmosphere of nitrogen, using commercially supplied solvents and
reagents unless otherwise noted. CH2Cl2 (DCM) was distilled from CaH2 and stored
over molecular sieves 4A. Thin-layer chromatography (TLC) was performed on Merck
60F254 precoated silica gel plates (Merck, Darmstadt, Germany) and was visualized by
fluorescence quenching under UV light and by staining with phosphomolybdic acid, p-
anisaldehyde, or ninhydrin. A solvent system consisting of 0.1% TFA in H2O solution
(v/v, solvent A) and 0.1% TFA in MeCN (v/v, solvent B) were used for HPLC elution. For
analytical HPLC, a Cosmosil 5C18-ARII column (4.6
×
250 mm, Nacalai Tesque, Inc., Kyoto,
Japan) was employed with a linear gradient of B at a flow rate of 1 cm3min−1 on a JASCO
PU-2089 plus (JASCO Corporation, Ltd., Tokyo, Japan), and eluting products were detected
by UV at 220 nm. Preparative HPLC was performed using a Cosmosil 5C18-AR II column
(20
×
250 mm, Nacalai Tesque, Inc.) on a JASCO PU-2086 plus (JASCO Corporation, Ltd.)
in a suitable gradient mode of B at a flow rate of 10 cm3min−1. Optical rotations were
measured on a JASCO P-2200 polarimeter (JASCO Corporation, Ltd.) operating at the
◦
sodium D line with a 100 mm path length cell at 25 C, and were reported as follows: [
α
]
D
(concentration (g/100 mL), solvent). Infrared (IR) spectra were measured on a JASCO
FT/IR 4100 (JASCO Corporation, Ltd.) and recorded as wavelength (cm−1). 1H- and 13C-
NMR spectra were recorded using a Bruker AVANCE III 400 spectrometer or AVANCE 500
spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are reported in
δ (ppm) relative
to Me4Si (in CDCl3, or MeOH-d4) as an internal standard. High-resolution mass spectra
were recorded on a Bruker Daltonics micrOTOF focus (ESI) mass spectrometer (Bruker) in
the positive detection mode. For flash chromatography, silica gel 60 N (Kanto Chemical
Co., Inc., Tokyo, Japan) was employed.
The details of synthesis and characterization data of the compounds are available in
the Supplementary Materials.
2.2. In Silico Screening of Antiviral Candidates
To perform the in silico screening, we first obtained the structure of the dimer of CA
proteins (PDB ID: 3J34) from the Protein Data Bank (https://www.rcsb.org/). The structure
of the dimer of CA proteins was thermodynamically optimized by the energy minimization
using MOE and the Amber10: EHT force field [60,61]. The one monomer of the dimer of
CA proteins was fixed as a receptor, whereas the residues of the other monomer were
removed except for Typ184 and Met185 residues, of which the side chains play a key role
of the dimer formation. Using the complex composing of the CA monomer as a receptor
and Trp184Met185 dipeptide as a ligand, we searched for the compounds having a higher
affinity than the dimer formation. To do this, the main chain backbone replacement of
the dipeptide on the CA protein was performed by the Scaffold Replacement application

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in MOE using the linker database of MOE and the Amber10: EHT force field, whereas
the side chains of the dipeptide were fixed. From the result, we selected the compounds
having higher scores of the binding affinity for receptor (London dG), ligand efficacy, and
topological polar surface area (TPSA).
2.3. Evaluation of Anti-HIV-1 Activity and Cytotoxicity
For virus preparation, 293T/17 cells (Invitrogen), which are maintained in Dulbecco’s
modified Eagle medium (DMEM) containing 10% FBS, in a T-75 flask were transfected with
10
µg of the pNL4-3 construct by the calcium phosphate method. The supernatant was col-
lected 48 h after transfection, passed through a 0.45
µ
m filter, and stored at −80 ◦C as a stock
virus. Inhibitory activities of test compounds against X4-HIV-1 (NL4-3 strain)-induced
cytopathogenicity in MT-4 cells [62], which are maintained in RPMI-1640 containing 10%
FBS, were assessed by the MTT assay. Various concentrations of test compound solutions
were added to HIV-1-infected MT-4 cells at multiplicity of infection (MOI) of 0.001, and
placed in wells of a 96-well microplate. The test compounds and the reference compounds
such as AZT and AMD3100 were diluted by two-fold and five-fold, respectively. After
5 days’ incubation at 37 ◦C in a CO2 incubator, the number of viable cells was determined
by the MTT assay. Cytotoxicities of the test compounds were determined based on reduc-
tion of the viability of MT-4 cells by the MTT assay. The p24 antigen content in the culture
supernatant was measured using HIV-1 p24 antigen enzyme-linked immunosorbent assay
(ELISA) kit according to manufacturer’s instructions (Zeptometrix, Buffalo, NY, USA). A
reverse transcriptase inhibitor, AZT, a CXCR4 antagonist, AMD3100 (Sigma Aldrich, St.
Louis, MO, USA) were employed as positive control compounds with anti-HIV activity.
3. Results
3.1. In Silico Screening to Find Drug Leads Targeting CA Proteins
3.1.1. Structural Analysis of CA Proteins
Structural analysis of CA proteins [33–35,63] revealed a hydrophobic interaction
between two CA molecules in Helix 9 of CTD involving Trp184 of one molecule and Met185
of the other molecule. This interaction is important for stabilization of the multimeric
structure forming the CA core (Figure 1). In addition, viral mutants with Trp184Ala and
Met185Ala mutations have no infectivity because the dimeric interaction between two
CA molecules of the mutants is weakened, thereby causing abnormal morphology of
the viral particles [64]. Furthermore, a CA-derived fragment peptide covering Helix 9,
which includes Trp184 and Met185, was previously found to have significant anti-HIV
activity [29], and the Trp184-Met185 dipeptide is known to be highly conserved among
natural HIV/simian immunodeficiency virus (SIV) strains [65]. In this study, based on the
above information, the site of the hydrophobic interaction between two CA molecules via
Trp184 of one molecule and Met185 of the other molecule might be considered a valid drug
target for CA dysfunction. A novel small molecule, which might bind to the above site,
was designed by in silico screening. This drug candidate and several of its derivatives were
synthesized, and their anti-HIV activity and cytotoxicity were evaluated.
3.1.2. Results of the In Silico Screening
Initially, a series of dipeptide mimics of Trp184 and Met185 were designed using the
Molecular Operating Environment (MOE) (Chemical Computing Group Inc., Montreal,
QC, Canada). Briefly, using the structure of the CA protein dimer (PDB ID: 3J34), the
structure of one monomer molecule was fixed as a receptor side, and the main chain of
Trp184 and Met185 of the other monomer molecule was removed. The side chains of these
two residues were fixed in place, and the backbone structures crosslinking two side-chain
functional groups were screened against the linker database provided by MOE to bind to
the receptor side (Figure 2). Antiviral candidates of dipeptide mimics were selected using
the Scaffold Replacement application in MOE and based on the following scores showing
binding affinity for receptors, ligand efficacy (London dG), topological polar surface area,

Biomolecules 2021, 11, 208
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molecular weight, log of the octanol/water partition coefficient (SlogP) and an estimate of
the synthetic feasibility [66]. Many London dG values mean binding affinities of available
compounds for target proteins, and smaller values show higher binding affinity. The
London dG value of the dimer of CA proteins is approximately
London dG values of less than 6 have higher binding affinity for a CA molecule when
compared to the interaction between two CA molecules. This screening served to identify
some candidates with useful binding affinity, including MKN-1 ( ), whose London dG
value, ligand efficiency, topological polar surface area (TPSA) and SlogP value are 9.134
kcal/mol, 0.2559, 69.73 Å2 and 4.022, respectively. The structure of MKN-1 (
) is completely
different from that of known small compounds, which were previously developed [43 59].
−
6, and compounds with
−
1
−
1
–
Figure 1. Interaction between two hexamers of capsid (CA) proteins (
mol-ecules via Trp184 of one molecule and Met185 of the other molecule: enlarged view of the box shown in A (
3J34). In ( ) blue: N-terminal domain (NTD), orange: C-terminal domain (CTD), red: Trp (W) 184 and Met (M) 185 in Helix
9; In (B) orange: CTD, red: carbon atom, blue: nitrogen atom, yellow: sulfur atom.
A
) and the hydrophobic interaction between two CA
B
) (PDB ID:
A
Figure 2. The interface of a CA monomer molecule as a receptor side (brown) and the Trp184 and
Met185 residues (green) of the other monomer molecule in the CA dimer ( ), and docking simulation
of MKN-1 ( , green) on a CA monomer ( ), and the design of dipeptide mimic of Trp184 and Met185
(MKN-1 ( )) by in silico screening ( ). Red: oxygen atom; blue nitrogen atom; yellow: sulfur atom;
grey: hydrogen atom.
A
1
B
1
C

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3.2. Synthesis of Novel CA-Targeting Anti-HIV Agents
3.2.1. Synthesis of MKN-1 (1)
A possible synthesis of MKN-1 (
1) was outlined. For its construction, the structure of
1, with two chiral centers was divided into three segments (Scheme 1).
Scheme 1. Retrosynthetic analysis of MKN-1 (1).
Initially, Segment I was prepared as a mixture of racemates. Treatment of 1,3-butanediol (
withp-toluenesulfonyl(tosyl)chloride(TsCl)inthepresenceofacatalyst, 4-dimethylaminopyridine
(DMAP) gave the tosylated alcohol ( ) [67], and subsequent treatment with sodium
methanethiolate led to a sulfide ( ) that corresponds to Segment I, in 96% yield over
two steps (Scheme 2a). The synthesis of Segment II is shown in Scheme 2b. Treatment of
the indole ( ) with iodine in the presence of potassium hydroxide yielded the 3-iodinated
indole ( ), and subsequent N-Boc-protection gave a Boc-protected indole ( ) in 60% yield
with n-butyllithium and isopropoxyboronic acid pinacol es-
) corresponding to Segment II, in 50% yield (Scheme 2b) [68].
2)
3
4
5
6
7
over two steps. Treatment of
ter produced a pinacol ester (
7
8
Segment III was stereoselectively synthesized using a Strecker reaction [69]. Treatment of
o-bromobenzaldehyde ( ) with (S)-(-)-1-(4-methoxyphenyl)-ethyl amine (10) and sodium
cyanide gave (S,S)- -aminonitrile (11) in a highly diastereoselective reaction, in 56% yield.
The acid hydrolysis of 11 led to an enantiopure (S)- -arylglycine (12), and the subsequent
methyl esterification with thionyl chloride and N-Boc-protection of the -amino group in
13) produced compound 14 that corresponds to Segment III, in 69% yield after three steps
(Scheme 2c).
Using these three segments, MKN-1 (
shown in Scheme 2d. Compounds (Segment II) and 14 (Segment III) were condensed by a
9
α
α
α
(
1
) and its diastereomer were synthesized as
8
Suzuki-Miyaura cross coupling reaction using tetrakis(triphenylphosphine)palladium (0) to
obtain compound 15 in 98% yield. Saponification of compound 15 with lithium hydroxide
yielded an acid (16), and the subsequent condensation with compound
ethyl-3-(3-dimethylaminopropyl) carbo-diimide hydrochloride (EDCI HCl) in the presence
of a catalyst (DMAP) gave an ester (17). The deprotection of the two N-Boc groups of 17 by
HCl/dioxane gave MKN-1 ( ) and its diastereoisomer, which were separated and purified
4 (Segment I) by 1-
·
1
by preparative HPLC to yield a first eluent MKN-1A (1A) and a second eluent, MKN-1B
(1B), in yields of 3% and 2%, respectively, over three steps (Scheme 2d).
3.2.2. Stereoselective Synthesis of MKN-1 (1)
Next, the stereoselective synthesis of MKN-1 (
Segment I, (S)-(+)-1,3-butanediol (18) was used as a starting material, and a chiral alcohol
20) was obtained in 63% in two steps in a manner (Scheme 3) similar to that shown in
Scheme 2a. Saponification of the ester (15), the subsequent condensation with an alcohol
20) and the N-Boc deprotection followed by HPLC purification obtained diastereoselec-
tively the target compound MKN-1 ( ) in 12% yield over three steps in a pathway similar
1) was performed. In the synthesis of
(
(
1
to that shown in Scheme 2d (Scheme 3). In HPLC analysis, MKN-1 (1) corresponds to
MKN-1A (1A) (Supplementary Materials).

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Scheme 2. Synthesis of Segment I (compound
4
) (
a
), Segment II (compound
8
) (
b
), Segment III (compound 14) (
c
), and
#
MKN-1 (1) and its diastereomer (
d
). HPLC purification using 0.1% (v/v) trifluoroacetic acid (TFA) containing eluents after
the reaction. Absolute configurations of MKN-1A and MKN-1B were determined in Section 3.2.2.

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Scheme 3. The stereoselective synthesis of MKN-1 (1 .
). # HPLC purification using 0.1% (v/v) TFA containing eluents after the reaction
3.2.3. Synthesis of MKN-1 Derivatives
Synthesis of MKN-1 Derivatives with Aryl Ring Substitution 22, 24, 27, and 28
MKN-1 ( ) has two pharmacophoric functional groups: the indolyl and sulfidyl
1
groups. Initially, MKN-1 derivatives, in which the indolyl moiety has been replaced,
were designed. In general, naphthyl groups and benzofuranyl and benzothiophenyl
groups are used as useful analogues of an indolyl group. Thus, MKN-1 derivatives,
with naphthyl, benzofuranyl and benzothiophenyl groups in the place of the indolyl
group, were synthesized. In the synthesis of MKN-1 derivatives with a 1-naphthyl or
2-naphthyl group, the Suzuki-Miyaura cross coupling of a phenylglycine derivative (14
)
with 1-naphthylboronic acid or 2-naphthylboronic acid, led to compound 21 or 23, both
in 42% yield (Scheme 4a,b). Subsequent saponification, condensation with compound 20
deprotection of the N-Boc group and HPLC purification gave compound 22 in 12% yield
or 24 in 10% yield, over a three-step route similar to Schemes 2 and 3 (Scheme 4a,b).
In the synthesis of MKN-1 derivatives with a benzofuranyl or benzothiophenyl group,
the Suzuki-Miyaura cross coupling of a phenylglycine derivative (14) with benzofuran-
3-boronic acid or benzo[b]thiophene-3-boronic acid followed by the treatment described
above gave compound 27 in 5% yield or 28 in 2% yield, in four steps (Scheme 4c).
,
Synthesis of MKN-1 Derivatives with Sulfide Substitution 30, 34–36, and 38
Next, MKN-1 derivatives, in which the sulfidyl moiety was replaced, were designed.
MKN-1 derivatives, with methoxy, tert-butyl sulfidyl, iso-propyl sulfidyl, benzenesulfidyl
and methanesulfonyl groups in the place of the methanesulfidyl group, were synthesized.
In the synthesis of an MKN-1 derivative with a methoxy group, the alcohol bearing a
methoxy group (29) was prepared by methylation of compound 18 in 17% yield, and
saponification of compound 15. Subsequent condensation with alcohol 29, deprotection
of the N-Boc group and HPLC purification gave compound 30 in 6% yield over a three-
step pathway similar to that shown in Schemes 2 and 3 (Scheme 5). In the synthesis of
MKN-1 derivatives with a sulfidyl group, the alcohols bearing tert-butyl sulfidyl, iso-propyl
sulfidyl and benzenesulfidyl groups (31–33) were prepared in 77%, 70% and 88% yields,
respectively, by treatment of compound 19
2-propanethiolate or sodium thiophenolate. Then, condensation of the hydrolysate of
compound 15 with alcohols 31 33, followed by deprotection of the N-Boc group and HPLC
purification gave compounds 34 36 in 12%, 19% and 9% yields, respectively, in three steps
[70] with sodium tert-butylthiolate, sodium
–
–
similar to those described above in Scheme 5. In the synthesis of an MKN-1 derivative with
a methanesulfonyl group, the condensation of the hydrolysate of compound 15 with the
alcohol (20) led to the ester (37) in 35% yield in two steps, and subsequent oxidation of the
sulfidyl group of 37 with m-chloroperoxybenzoic acid (mCPBA), deprotection of the N-Boc
group and HPLC purification gave compound 38 in 8% yield over two steps (Scheme 5).

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Scheme 4. Synthesis of MKN-1 derivatives 22, 24, 27, and 28. (a) Synthesis of 22 which has 1-naphthyl group; (b) synthesis
of 24 which has 2-naphthyl group; (
c
) synthesis of 27 and 28, which have benzofuranyl and benzothiophenyl groups,
#
respectively, in the place of the indolyl group of MKN-1 (
1). HPLC purification using 0.1% (v/v) TFA containing eluents
$
after the reaction. For 26, H2O2 aq. was not added.
Scheme 5. Synthesis of MKN-1 derivatives, with methoxy (30), tert-butyl sulfidyl (34), iso-propyl sulfidyl (35), benzenesul-
#
fidyl (36) and methanesulfonyl groups (38) in the place of the methanesulfidyl group of MKN-1 (
1
). HPLC purification
using 0.1% (v/v) TFA containing eluents after the reaction.

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3.3. Evaluation of Anti-HIV Activity and Cytotoxicity of the Synthesized Compounds
The anti-HIV activity of the synthesized compounds was assessed based on protec-
tion against HIV-1 (NL4-3 strain)-induced cytopathogenicity in MT-4 cells by an MTT
assay [27–30]. The cytotoxicity of these compounds was determined based on reduction of
the viability of MT-4 cells determined by an MTT assay. These results are shown in Table 1.
Table 1. Anti- human immunodeficiency virus type 1 (HIV) activity and cytotoxicity of the synthe-
sized compounds.
MTT Assay
p24 ELISA
EC50 (µM) c
Compound
EC50 (µM) a
CC50 (µM) b
MKN-1A (1A)
MKN-1B (1B)
8.0 (2) e
24 (2)
38 (2)
37 (2)
14 (1)
22 (1)
MKN-1 (1)
4.5 ± 2.3 (10)
26 ± 8.8 (10)
33 ± 1.5 (3)
30 ± 2.0 (3)
34 (2)
45 (2)
> 50 (3)
17 (2)
18 (2)
17 (2)
> 50 (2)
79 ± 42 (7)
> 50 (10)
8.5 (1)
31 ± 8.5% at 25 µM d (3)
40 ± 4.6% at 25 µM d (3)
11% at 25 µM d (2)
31% at 25 µM d (2)
26 ± 2.9 (3)
22
24
27
28
30
34
35
36
38
not tested
not tested
not tested
not tested
not tested
not tested
not tested
not tested
not tested
0.047 (2)
0.029 (2)
12 (2)
10 (2)
11 (2)
34% at 25 µM d (2)
0.024 ± 0.015 (10)
0.027 ± 0.0077 (10)
AZT
AMD3100
a
EC50 values are the concentrations for 50% protection from HIV-1 (NL4-3 strain)-induced cytopathogenicity in
b
c
MT-4 cells. CC50 values are the concentrations for 50% reduction of the viability of MT-4 cells. EC50 values are
the concentrations for 50% inhibition against the viral p24 antigen production in NL4-3 strain-infected MT-4 cells.
d
The data are shown as a mean value of at least two independent experiments. % inhibition at 25 or 50
µ
M. e The
number of experiments for each compound is shown inside parentheses, and the data are shown as a mean value
or mean value ± standard deviation in case of more than three experiments.
MKN-1A (1A), which is exactly MKN-1 (1), showed significant anti-HIV activity,
and its diastereoisomer MKN-1B (1B) showed moderate anti-HIV activity, suggesting
that chiral recognition by a target molecule, possibly a CA protein, might be important.
These compounds were also evaluated by a different method, using an enzyme-linked
immune sorbent assay (ELISA) based on their inhibitory effect against the viral p24 antigen
expression in NL4-3 strain-infected MT-4 cells. The results agree with the MTT assay
in that MKN-1A (1A) showed higher anti-HIV activity than MKN-1B (1B). MKN-1 (
which was stereoselectively synthesized and corresponds to MKN-1A (1A), showed high
anti-HIV activity. The cytotoxicities of MKN-1 ( ) (MKN-1A (1A)) and MKN-1B (1B) at
essentially the same level were moderate and weak. MKN-1 derivatives with 1-naphthyl,
2-naphthyl, benzofuranyl and benzothiophenyl groups (22 24 27, and 28) showed weak
anti-HIV activity, indicating that the indolyl group is critical and cannot be modified.
These MKN-1 derivatives (22 24 27, and 28) showed relatively weak cytotoxicity similar
to that of MKN-1 ( ), MKN-1A (1A) and MKN-1B (1B). An MKN-1 derivative with a
1),
1
,
,
,
,
1
methoxy group in place of the methanesulfidyl group (30) showed weak anti-HIV activity,
suggesting that a sulfur atom is important for significant anti-HIV activity, although an
oxygen atom in this position retains minor activity. Compound 30 however failed to
exhibit any cytotoxicity below 50
than a sulfur atom in terms of low cytotoxicity. MKN-1 derivatives with tert-butyl sulfidyl,
iso-propyl sulfidyl and benzenesulfidyl groups (34 36) showed higher anti-HIV activity
than the 1-naphthyl, 2-naphthyl, benzofuranyl and benzothiophenyl group-substituted
derivatives (22 24 27, and 28) but lower activity than the parent compound MKN-1 (
(MKN-1A (1A)). These derivatives (34 36) exhibited relatively high cytotoxicity, compared
µM, indicating that an oxygen atom is more suitable
–
,
,
1)
–
with other derivatives. This suggests that sulfidyl groups are critical for significant anti-HIV
activity but sulfidyl groups which are bulky are not suitable in terms of cytotoxicity. A
sulfone-substituted derivative (38) showed weak anti-HIV activity and failed to exhibit

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any cytotoxicity below 50
µM, indicating that sulfidyl but not sulfonyl groups are critical
for significant anti-HIV activity.
4. Discussion
Taken together, two pharmacophore functional groups (indolyl and sulfidyl groups)
of MKN-1 (1), which was originally designed as a dipeptide mimic of Trp184 and Met185,
are both important for high anti-HIV activity and should not be modified. The indolyl
moiety cannot be changed into a 1-naphthyl, 2-naphthyl, benzofuranyl or benzothiophenyl
group. The methanesulfidyl moiety can be converted into other sulfidyl groups, such as
tert-butyl sulfidyl, iso-propyl sulfidyl and benzenesulfidyl with a slight decrease of anti-HIV
activity, and into a methoxy group with a significant decrease of anti-HIV activity, but
when changed into a methanesulfonyl group leads to total loss of activity. The Trp184 and
Met185 residues are extremely conserved among the proteins of various HIV-1 subtypes
circulating in nature, possessing the Shannon entropy scores of 0.0012 and 0.0014 for W184
and M185, respectively, which are even much lower values as compared with those of
highly conserved active sites of HIV-1 integrate [71]. The data indicate exceedingly strong
selective constraints against changes in the Trp184-Met185 dipeptides and suggest potential
therapeutic benefits in targeting them to reduce the risk of emergence of drug resistance
variants. As described in Section 3.1.1, viral mutants with Trp184Ala and Met185Ala
mutations has no infectivity, causing abnormal morphology of the viral particles [64].
Therefore, the dipeptide mimic might have advantages when it is used with the lead
compounds targeting capsid proteins, such as GS-6207, having different site of actions [57].
Such combinational use of compounds could increase antiviral effects via synergistic effects
for disturbing capsid assembly/disassembly during HIV-1 replication in the cells and
would reduce risk of emergence of drug resistance variants more significantly than the
single-compound use would. However, to obtain more potent lead compounds based on
the present results, pharmacophore models on the target site, i.e., the CA-CA interface,
can be constructed via molecular dynamics study. The approach may be beneficial to
perform alternative screening, de novo design and optimization of lead compounds for
obtaining compounds with high antiviral activity. In addition, de novo design of the
candidates can be performed by using structural information of a few amino acid residues
flanking the W184M185 residues of the CA protein dimer. This approach may improve the
binding affinity.
5. Conclusions
In conclusion, this study presents a new class of small molecules, which was designed
by in silico screening as a dipeptide mimic of Trp184 and Met185 at the hydrophobic
interaction site between two CA molecules that have been reported to be important for
stabilization of the multimeric structure of CA. The designed compound MKN-1 (1) has
significant anti-HIV-1 activity, and its diastereoisomer MKN-1B (1B) has lower activity.
Structure activity relationship (SAR) studies of MKN-1 derivatives reveal the importance
of two pharmacophore groups: indolyl and sulfidyl. In this study, whether MKN-1 (
actually binds to the CA protein was not confirmed, but the chiral recognition of its target
molecule was confirmed according to the difference of potencies between MKN-1 ( ) and
1)
1
its diastereoisomer. The present results should be useful in the future design of a novel
class of anti-HIV agents.
Supplementary Materials: The details of synthesis and characterization data of the compounds (1H,
13C-NMR, [
α]D, IR, HRMS) including analytical HPLC charts to check the purity of the compounds
are available online at https://www.mdpi.com/2218-273X/11/2/208/s1.
Author Contributions: Conceptualization, T.K. and M.Y.; methodology, T.K. and M.Y.; software, M.Y.
and O.K.; validation, K.T. and N.O.; formal analysis, M.K. (Masaki Kurakami); investigation, M.F.,
M.K. (Masaki Kurakami), S.B., M.N. and M.K. (Moemi Kaneko); writing—original draft preparation,

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T.K.; writing—review and editing, H.T.; supervision, T.M., H.S. and H.T. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported in part by the grant for Research Program on HIV/AIDS, Japan
Agency for Medical Research and Development (AMED) under Grant Number 18fk0410004h0503
(T.M., H.S. and H.T.); JSPS KAKENHI Grant Numbers 20H03362 and 19K22488 (H.T.); 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.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available as Supplementary
Materials. These include the details of synthesis and characterization data of the compounds (1H,
13C-NMR, [
α
]D, IR, HRMS) including analytical HPLC charts to check the purity of the compounds.
Conflicts of Interest: The authors declare no conflict of interest.
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