Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase
Acta Pharmacol Sin
Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase1
Li DU 0
Ya-xue ZHAO 1
Liu-meng YANG 2
Yong-tang ZHENG 2
Yun TANG 1
Xu SHEN 0 1
Hua-liang JIANG 0 1
0 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 201203 , China
1 School of Pharmacy, East China University of Science and Technology , Shanghai 200237 , China
2 Laboratory of Molecular Immunopharmacology, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences , Kunming 650223 , China
Aim: To characterize the functional and pharmacological features of a symmetrical 1-pyrrolidineacetamide, N,N?-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide, as a new anti-HIV compound which could competitively inhibit HIV-1 integrase (IN) binding to viral DNA. Methods: A surface plasma resonance (SPR)-based competitive assay was employed to determine the compound's inhibitory activity, and the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide cell assay was used to qualify the antiviral activity. The potential binding sites were predicted by molecular modeling and determined by site-directed mutagenesis and a SPR binding assay. Results: 1-pyrrolidineacetamide, N,N?-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide could competitively inhibit IN binding to viral DNA with a 50% inhibitory concentration (IC50) value of 7.29?0.68 ?mol/L as investigated by SPR-based investigation. Another antiretroviral activity assay showed that this compound exhibited inhibition against HIV-1(IIIB) replication with a 50% effective concentration (EC50) value of 40.54 ?mol/L in C8166 cells, and cytotoxicity with a cytotoxic concentration value of 173.84 ?mol/L in mock-infected C8166 cells. Molecular docking predicted 3 potential residues as 1-pyrrolidineacetamide, N,N?-(methylene-di-4,1-phenylene)bis-1pyrrolidineacetamide binding sites. The importance of 3 key amino acid residues (Lys103, Lys173, and Thr174) involved in the binding was further identified by site-directed mutagenesis and a SPR binding assay. Conclusion: This present work identified a new anti-HIV compound through a new IN-binding site which is expected to supply new potential drug-binding site information for HIV-1 integrase inhibitor discovery and development.
An essential step in the HIV-1 life cycle is the
integration of the reverse-transcribed viral genome into
host chromosomal DNA by the virally-encoded integrase
]. The fact that its inactivation either by
mutagenesis or inhibition might block the productive
infection by HIV-1[
] implies that HIV-1 IN is an attractive
target for antiviral drug discovery. IN catalyzes 2 steps of
reaction of the integration process. In the first step, termed
3?-processing, IN cleaves the 2 terminal nucleotides from
each 3? end of the viral DNA. The second step is called
strand transfer, in which IN transfers both extremities of
the viral DNA into the target DNA with the help of some
cellular enzymes by a 1-step transesterification reaction,
resulting in full-site integration[
HIV-1 IN is composed of 3 distinct structural and
functional domains: the N-terminal domain (residues 1?50)
that contains a conserved HHCC zinc-binding motif, the
core domain (residues 51?212) that contains the catalytic
site with 3 spatially conserved and invariable amino
acids (D64, D116, and E152), and the C-terminal domain
(residues 213?270), which is suggested to be responsible
to multimer formation and non-specific binding to DNA[
To date, crystallographic or Nuclear Magnetic Resonance
(NMR) structural data have been available for each of the
IN individual domains, and 2-domain crystal structures
(either the core and C-terminal domains or N-terminal and
the core domains) have been determined[
]. However, in
the 2-domain integrase structures, the positioning of both
the N- and C-terminal domains in relation to the catalytic
core domain (CCD) may not correspond to that assumed
when viral DNA is bound. Efforts to obtain a structure
of the full-length IN have been impeded by poor protein
The structural and biochemical understanding of IN has
led to the discovery and development of diverse classes of
active compounds against IN. The most promising drug
candidate is of ?-diketo type, which is the only class of IN
inhibitors with a clear inhibition mechanism[
the group, S-1360 and L-870, 810 entered phase II clinical
trials in 2003 and 2004[
] and failed later. However,
although various kinds of IN inhibitors have been reported,
and some have been used in clinical trials, only 1 IN
inhibitor, raltegravir, was approved by the Food and Drug
Administration (FDA) in 2007[
]. Therefore, the
discovery of a novel IN potent inhibitor is still an alluring
It is well known that the structural information detailing
the association between IN and potential inhibitors is
of highly-therapeutic importance. Identification of the
key amino acid residues involved in the binding site of
candidate drugs would help to predict drug-resistant viral
strains and provide specific information for inhibitor
In this work, we report a small molecular compound
(compound 1; Figure 1) 1-pyrrolidineacetamide,
that could competitively inhibit IN binding to viral DNA
and show moderate antiretroviral activity. Site-directed
mutagenesis with molecular docking analyses revealed
that compound 1 binds to IN with key residues at the CCD
dimer interface. Our current study is expected to provide
some useful information for the discovery of IN-based
Materials and methods
Chemistry Compound 1 was purchased from SPECS
Bank (Delft, Netherlands).
Plasmid construction The wild-type HIV-1 IN DNA
coding for HIV-1 integrase (GenBank No AF 040373)
was synthesized with an Applied Biosystems DNA
synthesizer (Shanghai Sangon Biological Engineering and
Technology and Service, Shanghai, China) and cloned into
glutathione S-transferase (GST) expression vector
pGEX4T-1 to construct the plasmid pGEX-4T-1-IN. The F185K
substitution was introduced to construct the mutant plasmid
pGEX-4T-1-IN (F185K) to increase the solubility[
plasmid pGEX-4T-1-IN (F185K) was used as the template
DNA to construct the deletion mutant pGEX-4T-1-IN52?210,
which encodes the residues of the HIV-1 I core domain
(amino acids 52?210, IN52?210). Site-directed mutagenesis
was performed based on the plasmid pGEX-4T-1-IN
using the QuikChange site-directed mutagenesis system
(Stratagene, La Jolla, CA, USA). Codons for Lys103,
Lys173, and Thr174 were mutated to alanine by using the
following duplex oligonucleotides: K103A: 5?-CA GCA
TAC TTT CTC TTA GCA TTA GCA GGA AGA TGG-3?,
K173A: 5?-GAT CAG GCT GAA CAT CTT GCG ACA
GCA GTA CAA ATG GC-3?, and T174A: 5?-CAG GCT
GAA CAT CTT AAG GCA GCA GTA CAA ATG GCA
G-3?. The mutated codon is underlined. All clones were
verified by sequencing.
Protein preparation The proteins IN, IN52?210, and
IN mutants K103A, K173A, and T174A were expressed
and purified according to the GST Gene Fusion System
Handbook (Amersham Bioscience, Pittsburgh, PA, USA).
In brief, Escherichia coli BL21(DE3) cells transformed
with wild-type or mutated HIV-1 IN expression plasmids
were grown at 37 ?C in Lysogeny Broth (LB) medium
containing 100 ?g/mL ampicillin until the optical density at
600 nm reached 0.6?0.8. Proteins were expressed for 5?8
h at 25 ?C after induction with 0.5 mmol/L
isopropyl-?-Dthiogalactopyranoside (IPTG). The cells were harvested
by centrifugation, resuspended in 1?precooled
phosphatebuffered saline (PBS; 140 mmol/L NaCl, 2.7 mmol/L
KCl, 10 mmol/L Na2HPO4, and 1.8 mmol/L KH2PO4, pH
7.4), and lysed by sonication in an ice bath. The lysate
was centrifuged for 30 min at 21 000?g at 4 ?C. The
supernatant was loaded on a glutathione?Sepharose 4B
column (Amersham?Pharmacia, Pittsburgh, PA, USA)
equilibrated with PBS at 4 ?C. The column was washed
with 120?200 mL of 1? PBS and then eluted with 10 mL of
20 mmol/L reduced glutathione. The elution fraction was
applied on a Superdex 75 column on an AKTA instrument
(Amersham?Pharmacia, Pittsburgh, PA, USA) for further
purification. IN and IN mutants K103A, K173A, and
T174A were purified in GST fusion form. For IN52?210,
the GST-fusion protein was digested on the glutathione?
Sepharose 4B column with 50 U thrombin for 16 h at 4 ?C
to remove the GST fusion tag. The purity of all proteins
was confirmed by SDS?PAGE.
Competitive inhibition assay The compound?s
competitive inhibition assay against IN/viral DNA binding
was performed by using the surface plasma resonance
(SPR) biosensor technology-based Biacore 3000 system
(Biacore AB, Uppsala, Sweden) as previously reported[
During the assay, a 21 bp 5?-biotinylated oligonucleotide
with a non-biotinylated complementary oligonucleotide
was immobilized on the streptavidin matrix-coated
sensor chip (SA chip), and 200 nmol/L IN incubated with
0?0.2 mmol/L compounds for 1 h at 4 ?C flowed over
the chip surface. A 21 bp DNA with random sequence
was immobilized to the reference flow cell as the control.
The compound inhibition against IN binding to DNA
was demonstrated by monitoring the response unit (RU)
decrease with the addition of the compounds at different
concentrations. All the sensorgrams were processed by
using automatic correction for non-specific bulk refractive
Binding assay The binding affinity of the compound
to HIV-1 IN, IN52?210, IN(K103A), IN(K173A), and
IN(T174A) in vitro was determined by using SPR
technology. The measurement was performed using the
dual flow cell Biacore 3000 instrument. Immobilization
of the wild-type and mutant IN proteins to the hydrophilic
carboxymethylated dextran matrix of the sensor chip CM5
(Biacore, Sweden) was carried out by the standard primary
amine coupling method. The protein to be covalently
bound to the matrix was diluted in 10 mmol/L sodium
acetate buffer (pH 4.5) to a final concentration of 0.2
mg/mL, and the resonance signal reached approximately
8500 RU. Equilibration of the baseline was completed by
a continuous flow of HBS?EP buffer (10 mmol/L HEPES,
150 mmol/L NaCl, 3 mmol/L EDTA and 0.01% P20,
pH 7.4) through the chip for 4?5 h. For the GST fusion
protein, IN, IN(K103A), IN(K173A), and IN(T174A)
binding assays, the reference flow cell surface was
immobilized at a parallel level (4500 RU) using GST as
a control. All the sensorgrams were processed by using
automatic correction for non-specific bulk refractive index
effects. The specific binding profiles of the compounds to
the immobilized protein were obtained after subtracting the
response signal from the control flow cell. All the Biacore
data were collected at 25 ?C with HBS?EP as the running
buffer at a constant flow of 30 ?L/min. The equilibrium
dissociation constants (KD) evaluating the protein?ligand
binding affinity were determined using the 1:1 binding
model (Langmuir), and the curve fitting efficiency was
checked by residual plots and ?2.
Antiretroviral activity assay The C8166 cells
were grown and maintained in RPMI-1640 medium
supplemented with 10% heat-inactivated fetal calf serum,
2 mmol/L L-glutamine, 0.1% sodium bicarbonate, and
20 ?g gentamicin per mL. HIV-1(IIIB) was obtained
from Medical Research Council, AIDS Reagent Project
(London, UK). The inhibitory effect of the compound
on HIV-1 replication was monitored by the inhibition of
virus-induced cytopathicity in the C8166 cells for 5 d after
infection as described. Cytotoxicity of the compounds
against the C8166 cells was determined by measuring the
viability after 5 d of incubation[
Molecular modeling The computational molecular
modeling studies were carried out using a Dell Precision
670 workstation (Austin, TX, USA) running Redhat Linux
WS 3.0 (Redhat, Raleigh, NC, USA). The 3-D structure of
compound 1 was constructed and energetically minimized
with Gasteiger?H?ckel charges[
] and the Tripos force
] in molecular modeling software package SYBYL
]. Both the nitrogen atoms in the pyrrolidines
were protonated, which is consistent with the condition of
the binding assay (pH 7.4). The 3-D crystal structure of
HIV-1 IN CCD was obtained from the Protein Data Bank[
(PDB) with entry code 1QS4[
]. Only chains A B of 1QS4
were used in this study. After all of the hydrogen atoms
were added, the Glu131 residues in the surface of the CCD
dimer were mutated back to Trp. The hydrogen atoms and
the mutated Glu131 residues were then minimized using
the Kollman all-atom force field[
] with Kollman all-atom
] in SYBYL.
GOLD version 3.0.1 (Cambridge Crystallographic
Data Centre, Cambridge, UK) was used to investigate the
reasonable binding site[
]. In the docking, the ligand was
flexible, and the protein remained rigid. All the protonation
of the histine residues were maintained as in crystal
structure without special treatment. The whole CCD dimer
was treated as the binding pocket by defining 2 active
center atoms with a 30 ? radius first, respectively. Then
only the potential binding site of the CCD was included
in the following docking. During the GOLD docking, the
default parameters of genetic algorithms were applied to
search the reasonable binding conformation of compound
1. To ensure the atoms? type, both ligand and protein were
turned on in the option ?set atom type?. To find more
accurate geometries, the option ?allow early termination?
was turned off. The GOLDScore function was used to
evaluate the docking results[
Compound 1 could inhibit HIV-1 integrase binding
to viral DNA as a catalytic core domain binder
Compound 1 was identified from other compounds by random
screening against HIV-1 IN. The SPR technology-based
competitive inhibition assay revealed that compound 1
could compete the binding of IN to the immobilized viral
DNA in a dose-dependent manner. As shown in Figure 2A,
the RU values of IN binding to the viral DNA significantly
decreased with the increase of compound concentrations.
The IC50 value for compound 1 was therefore evaluated
as 7.29?0.68 ?mol/L (Figure 2B) by fitting the inhibition
data to a dose-dependent curve using a logistic derivative
equation (Origin 6.1; Northampton, MA, USA).
In addition, the SPR binding assay also showed
that compound 1 could directly bind to HIV-1 IN at the
CCD (Table 1). Therefore, compound 1 inhibited HIV-1
integrase binding to viral DNA by acting as a CCD binder.
Antiretroviral activity The antiviral activity of
compound 1 against the HIV-induced cytopathic effect
(CPE) in the C8166 cell culture was determined by
]. The results showed that compound
1 could inhibit HIV-1(IIIB) replication by an EC50 value
of 17.05 ?g/mL in the C8166 cells, and cytotoxicity was
observed with a 50% cytotoxic concentration value of
73.11 ?g/mL in mock-infected C8166 cells.
Binding site identified with molecular docking
Molecular docking was used to identify the potential
binding site of the compound 1 at the HIV-1 IN CCD
with PDB code 1QS4. According to the docking results
of GOLD (Figure 3A), compound 1 bound to the CCD
dimer interface instead of the DDE (D64, D116 and E152)
motif. The residues of both chains of the homodimer
were involved in the compound 1/IN CCD interaction. As
shown in Figure 3B, an N-H bond of compound 1 forms
N-H-O hydrogen bond with the O atom of Thr-174 in
chain A. This hydrogen bond might greatly determine
the orientation of compound 1 in the binding site. The
positive-charged side chain of Lys173 in chain A interacts
with the right benzene ring of compound 1 by a cation??
interaction. Moreover, a well-defined salt bridge ring
exists around the 2 NH3+ groups of Lys103 in both chains.
The docking results revealed that residues Thr174, Lys173,
and Lys103 in chain A, and Lys-103 in chain B, might play
important roles in the interaction between compound 1 and
Binding site validated by site-directed mutagenesis
To validate the binding site of compound 1 to IN CCD,
a site-directed mutagenesis technique-based assay was
performed. Three of the important residues which are
involved in the interaction between compound 1, and
HIV1 IN CCD were selected for mutation: Lys103 (involved in
salt bridge, although it indirectly interacts with compound
1), Lys173 (mainly involved in a cation?? interaction and
hydrogen bond), and Thr174 (only involved in hydrogen
bond). During the test, Lys103, Lys173, and Thr174
were substituted by alanine, respectively. As indicated in
Table 1 and Figure 4, the SPR binding assay suggested
that substitution of alanine for Lys103 could significantly
reduce the binding of compound 1 to IN, while the alanine
mutation for Lys173 and Thr174 (K173A and T174A)
almost abolished the binding of compound 1 to IN.
Therefore, our mutagenesis experiments confirmed that
residues Lys103, Lys173, and Thr174 were involved in the
IN interaction with compound 1, supporting the docking
To date, many IN inhibitors and related binding sites
have been reported[
]. However, few have been used
in clinical trials so far and many of the IN inhibitors
belong to the ?-diketo-like acid (DKA) family. Previous
research revealed that 2 different binding sites (the donor
and the acceptor sites) for DKA may coexist in the active
site of IN[
], while in the case of the azidothymidine
(AZT) analog, K156, K159, and K160 were identified as
key residues involved in nucleotide binding[
an acetylated-inhibitor binding site K173 was recently
]. It was discovered that the acetylated inhibitor
specifically bound at an architecturally-critical region that
was located at the IN CCD dimer interface with K173 as a
The current work indicated that compound 1 could
inhibit IN binding to viral DNA as a potential IN
competitive inhibitor. Molecular docking, site-directed
mutagenesis and the SPR technology-based assay revealed
that compound 1 could bind to the interface of the HIV-1
IN CCD dimer. The ablation of interaction to compound 1
for IN CCD when the residues Lys173 and Thr174
were mutated to alanine illustrates their importance for
compound 1/IN CCD interaction. The significant reduction
binding affinity between them when the residue Lys103
was mutated to alanine indicates that the salt bridge ring is
important for their binding. When considering that CCD
contains the catalytic triad and interacts with substrate viral
DNA, it could be presumed that compound 1 might prevent
DNA binding once bound to IN CCD functioning as an IN
Since it has been identified that 3 key residues, Lys103,
Lys173, and Thr174, are involved in the binding of
compound 1 to CCD and compound 1 does not contain
methyl ester group, we excluded the possibility that it
could interact with Lys173 as an acetylated inhibitor as
]. Thus it can be tentatively concluded
that compound 1 might exhibit a different inhibition
mechanism from the above-mentioned cases for
acetylatedinhibitor and DKA.
Recently, Li et al reported a peptide IN inhibitor, NL-6,
which corresponds to IN residues 97?108[
Alaninescanned analog results showed a decrease in inhibitory
potency when Lys103 was substituted by Ala (NL-6-K7A),
which indicated that Lys103 might be an important site
for IN inhibitor binding[
]. Interestingly, our mutagenesis
assay also confirmed that the substitution of Lys103 with
Ala could cause an impressive decrease in the binding
affinity of compound 1 to IN, thus suggesting that residue
Lys103 of IN also plays an important role for compound 1
binding to IN.
In conclusion, we identified a novel compound
(compound 1) that could competitively inhibit IN binding
to viral DNA. The antivirus assay indicated that compound
1 showed good antiretroviral activity on HIV-induced CPE
in MT-4 cell culture. The SPR assay indicated that it bound
to the HIV-1 IN CCD domain. Molecular docking provides
a possible binding mode for compound 1/HIV-1 IN
interaction at the atomic level. Site-directed mutagenesis
analysis further identified that 3 key amino acid residues
(Lys103, Lys173, and Trp174) were involved in this
interaction. Our studies are expected to provide further
information in the identification of new drug-binding sites
and the elucidation of a potential IN inhibition mechanism,
thereby facilitating antiviral agent discovery.
Prof Xu SHEN, Prof Hua-liang JIANG and Prof Yun
TANG designed research; Li DU performed research,
analyzed data and wrote the paper; Ya-xue ZHAO
performed the molecular docking; Liu-meng YANG and
Yong-tang ZHENG determined the antiviral activity of the
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