Identification of a Small-Molecule Inhibitor of HIV-1 Assembly that Targets the Phosphatidylinositol (4,5)-bisphosphate Binding Site of the HIV-1 Matrix Protein

Article abstract of DOI:10.1002/cmdc.201200577

The development of drug resistance remains a critical problem for current HIV-1 antiviral therapies, creating a need for new inhibitors of HIV-1 replication. We previously reported on a novel anti-HIV-1 compound, N²-(phenoxyacetyl)-N-[4-(1-piperidinylcarbonyl)benzyl]glycinamide (14), that binds to the highly conserved phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂) binding pocket of the HIV-1 matrix (MA) protein. In this study, we re-evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV-1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2-(4-{[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]methyl})-1-piperazinyl)-N-(4-methylphenyl)acetamide (7), 3-(2-ethoxyphenyl)-5-[[4-(4-nitrophenyl)piperazin-1-yl]methyl]-1,2,4-oxadiazole (17), and N-[4-ethoxy-3-(1-piperidinylsulfonyl)phenyl]-2-(imidazo[2,1-b][1,3]thiazol-6-yl)acetamide (18), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV-1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7. Additionally, compound 7 displays a broadly neutralizing anti-HIV activity, with IC₅₀ values of 7.5-15.6μM for the groupM isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti-HIV-1 therapeutics.

Full text of DOI:10.1002/cmdc.201200577

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DOI: 10.1002/cmdc.201200577
Identification of a Small-Molecule Inhibitor of HIV-1
Assembly that Targets the Phosphatidylinositol (4,5)-
bisphosphate Binding Site of the HIV-1 Matrix Protein
Isaac Zentner,[a] Luz-Jeannette Sierra,[b] Ayesha K. Fraser,[a] Lina Maciunas,[a]
Marie K. Mankowski,[c] Andrei Vinnik,[d] Peter Fedichev,[d] Roger G. Ptak,[c] Julio Martꢀn-
Garcꢀa,[b] and Simon Cocklin*[a]
The development of drug resistance remains a critical problem
for current HIV-1 antiviral therapies, creating a need for new in-
hibitors of HIV-1 replication. We previously reported on a novel
anti-HIV-1 compound, N2-(phenoxyacetyl)-N-[4-(1-piperidinyl-
carbonyl)benzyl]glycinamide (14), that binds to the highly con-
served phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) bind-
ing pocket of the HIV-1 matrix (MA) protein. In this study, we
re-evaluate the hits from the virtual screen used to identify
compound 14 and test them directly in an HIV-1 replication
assay using primary human peripheral blood mononuclear
cells. This study resulted in the identification of three new
compounds with antiviral activity; 2-(4-{[3-(4-fluorophenyl)-
1,2,4-oxadiazol-5-yl]methyl})-1-piperazinyl)-N-(4-methylphenyl)-
acetamide (7), 3-(2-ethoxyphenyl)-5-[[4-(4-nitrophenyl)pipera-
zin-1-yl]methyl]-1,2,4-oxadiazole (17), and N-[4-ethoxy-3-(1-pi-
peridinylsulfonyl)phenyl]-2-(imidazo[2,1-b][1,3]thiazol-6-yl)ace-
tamide (18), with compound 7 being the most potent of these
hits. Mechanistic studies on 7 demonstrated that it directly in-
teracts with and functions through HIV-1 MA. In accordance
with our drug target, compound 7 competes with PI(4,5)P2 for
MA binding and, as a result, diminishes the production of new
virus. Mutation of residues within the PI(4,5)P2 binding site of
MA decreased the antiviral effect of compound 7. Additionally,
compound 7 displays a broadly neutralizing anti-HIV activity,
with IC50 values of 7.5–15.6 mm for the group M isolates tested.
Taken together, these results point towards a novel chemical
probe that can be used to more closely study the biological
role of MA and could, through further optimization, lead to
a new class of anti-HIV-1 therapeutics.
Introduction
Owing to the emergence of drug-resistant strains and the cu-
mulative toxicities associated with current therapies, demand
remains for new inhibitors of HIV-1 replication. The HIV-
1 matrix (MA) protein, encoded as the N-terminal portion of
Gag, is critically involved in the late, assembly stages in the life
cycle of HIV-1. Firstly, HIV-1 MA is proposed to act as a scaffold
that brings together Env and Gag proteins in infected cells.
Several lines of evidence support an interaction of MA with
the gp41 cytoplasmic tail during assembly and a role for MA in
facilitating the incorporation of Env into virus particle.[1] Until
recently, this interaction was thought to be direct;[1e,2] however,
other studies have implicated the participation of a cellular
factor in both the interaction between Env and Gag, and the
process of envelope incorporation.[3]
Secondly, the HIV-1 MA protein directs the localization of
Gag to the plasma membrane. Specific interaction of MA with
cellular transport proteins has been implicated in the traffick-
ing of Gag to the plasma membrane. Once at the membrane,
membrane binding of HIV-1 Gag is mediated by two signals in
MA: the N-terminal myristic acid and the conserved basic
region between amino acids 17 and 31.[4] The myristate moiety
is considered to be regulated by a mechanism termed a myris-
toyl switch, whereby the N-terminal myristate is sequestered in
the MA globular domain, but a structural change exposes the
myristate and enhances Gag membrane binding.[5] The MA
basic domain is also involved in specific localization of Gag to
the plasma membrane, with mutations in this domain shifting
Gag localization from the plasma membrane to intracellular
vesicles in HeLa and Tcells.[6] Perhaps most critically, however,
the basic domain of MA has been demonstrated to interact
with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), a minor
[a] I. Zentner,+ A. K. Fraser, L. Maciunas, Dr. S. Cocklin
Department of Biochemistry & Molecular Biology
Drexel University College of Medicine
245 North 15th Street, Philadelphia, PA 19102 (USA)
E-mail: scocklin@drexelmed.edu
[b] L.-J. Sierra,+ Dr. J. Martꢀn-Garcꢀa
Department of Microbiology & Immunology
Drexel University College of Medicine
245 North 15th Street, Philadelphia, PA 19102 (USA)
[c] M. K. Mankowski, R. G. Ptak
Department of Infectious Disease Research
Southern Research Institute
431 Aviation Way, Frederick, MD 21701 (USA)
[d] Dr. A. Vinnik, Dr. P. Fedichev
QuantumLead, Quantum Pharmaceuticals
Kosmonavta Volkova 6A-606, Moscow 125171 (Russia)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200577.
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phospholipid that is concentrated primarily on the cytoplasmic
leaflet of the plasma membrane.[5f,7] PI(4,5)P2 binding to HIV-
1 MA is thought to serve two functions: inducing the confor-
mational change that triggers myristate exposure[5f] and acting
as a site-specific membrane anchor, allowing the targeting of
Gag to the plasma membrane.[8,7c]
Table 1. Therapeutic spectrum of compound 7 against HIV-1 subtypes.[a]
HIV-1 group M isolate
92UG031[b]
IC50 [mm]
Antiviral index
(TC50/IC50)
The structure of the PI(4,5)P2–MA complex has been solved
by NMR spectroscopy.[5f] The PI(4,5)P2 binding site resides in
a cleft in the HIV-1 MA globular head domain and is composed
of a large number of charged residues. The majority of these
residues are highly conserved across the HIV-1 subtypes, high-
lighting the functional importance of the PI(4,5)P2–MA inter-
action in the HIV-1 replication cycle. Given its functional impor-
tance and its high degree of conservation across isolates, we
propose that the PI(4,5)P2 binding site might represent a new,
attractive antiviral target. Using a combination of virtual and
surface plasmon resonance (SPR)-based screening, we have
previously identified a compound (Enamine ID T5872535; see
Table S1 in the Supporting Information) that interacts with HIV-
1 MA in the PI(4,5)P2 binding site and displays broad-range an-
tiviral activity.[9] In this study, we re-evaluated the compounds
identified from the initial virtual screening, this time, however,
using antiviral efficacy as the primary screen. This approach re-
sulted in the identification of three new compounds with anti-
viral activity. Mechanistic studies of the lead compound (7),
demonstrated that it interacts with the MA protein, disrupts
the interaction between MA and PI(4,5)P2, and possesses
broad-spectrum anti-HIV-1 activity.
(clade A)
(clade B)
(clade B)
(clade B)
(clade C)
(clade C)
(clade D)
(clade E)
(clade F)
(clade G)
11.7ꢀ0.6
7.5ꢀ1.7
12.3ꢀ2.9
15.6ꢀ3.8
7.8ꢀ2.1
11.0ꢀ2.8
8.5ꢀ0.8
11.7ꢀ6.2
13.0ꢀ0.1
12.5ꢀ6.8
>8.6
>13.3
>8.1
>6.4
>12.8
>9.1
>11.8
>8.6
>7.7
>8.0
92BR030[b]
89BZ167[c]
MDR769[e]
92BR025[b]
93IN101[b]
92UG024[c]
CMU08[c]
93BR020[d]
G3[b]
[a] TC50 values for all compounds were determined to be >100 mm in this
study. IC50 values are the mean ꢀ standard deviation (SD) of three repli-
cates. [b] CCR5-tropic.[c] CXCR4-tropic. [d] CCR5/CXCR4 dual-tropic.
[e] Clinical isolate presumed to be group M.
replication of genetically diverse isolates, especially isolates
from the most globally prevalent subtypes (A, B, and C). There-
fore, the antiviral efficacy of compound 7 was further evaluat-
ed in a standardized PBMC-based anti-HIV-1 assay with a panel
of HIV-1 clinical isolates from different geographic locations
that included HIV-1 group M subtypes A–G (Table 1). The panel
included CCR5-tropic (R5), CXCR4-tropic (X4), and dual-tropic
(R5X4) viruses. Compound 7 inhibited the replication of all the
group M viruses tested, further highlighting the potential of
targeting the MA protein.
Results and Discussion
Identification of compound 7
To identify small molecules capable of binding to and inhibit-
ing the functions of HIV-1 MA, we previously coupled a struc-
ture-based in silico screening with a surface plasmon reso-
nance (SPR)-based primary screen, which identified one active
hit compound (14).[9] Therefore, to identify other potential hits
that were not identified in the SPR-based screen, but still rep-
resent matrix-targeted inhibitors of HIV-1 replication, we evalu-
ated the 19 compounds identified from the initial virtual
screen (Table S1 in the Supporting Information) in a standar-
dized HIV-1 replication assay (Table S2 in the Supporting Infor-
mation). This led to the discovery of the novel antiviral com-
pounds 7, 17, and 18, as well as confirmation of compound 14
as a genuine hit. The lead compound from this analysis, com-
pound 7, inhibited the replication of the primary isolate HIV-
189BZ167 with the lowest IC50 value, showed minimal toxicity, and
did not inhibit the replication of a panel of non-retroviruses
(Table 1; see also Table S3 in the Supporting Information). In
Compound 7 exerts its action in the late, assembly stages of
the HIV-1 life cycle
To determine the stage at which compound 7 exerts its anti-
viral effect, we made use of a single-round infection assay. The
modular nature of this assay allows the determination of
whether a compound acts on the early, preintegration events,
or on the late, assembly events in the virus life cycle. Effects
on assembly were identified by incubating the viral producer
cells in the absence or presence of various concentrations of
compound 7. Supernatants containing HIV-1HxBc2 envelope-
pseudotyped virus (which encodes for firefly luciferase as a re-
porter gene) were then diluted tenfold and used to infect
U87–CD4–CXCR4 target cells. Compound-induced effects are
manifested as a decrease in infectivity in the target cells, com-
pared with those infected with virus from untreated cells. Ef-
fects on early-stage events were determined by using virus
produced in the absence of compound for infection of target
cells in the absence or presence of various concentrations of
compound 7. As can be seen in Figure 1, compound 7 displays
effects only on the late, assembly stages of the HIV-1 replication
cycle. Quantification of the virus produced in the presence of
100 mm of the compound indicates that it functions by
decreasing the amount of virus released from the cells, as
addition, compound 7 inhibited the multi-drug-resistant (MDR)
[10]
strain HIV-1MDR769
,
suggesting that it functions in a different
manner than drugs currently in use as part of the “highly
active antiretroviral therapy” (HAART) cocktail.
The genetic diversity of HIV can pose certain problems for
the development of new antiviral drugs. Therefore, in order to
be clinically viable, a compound must be able to inhibit the
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sought to investigate the proposed interaction of compound 7
with the HIV-1 MA protein. To explore the interaction of com-
pound 7 with HIV-1 MA, we employed SPR. Using SPR and
a novel HaloTag method of orientation, compound 14 was pre-
viously shown to be capable of interacting with HIV-1 MA in
the PI(4,5)P2 binding site.[9] However, the HaloTag ligand
(chloroalkane) can nonspecifically interact with compounds
that display hydrophobic characteristics.[9] Therefore, we decid-
ed to immobilize HIV-1 MA directly on the sensor chip surface.
In order to preserve the PI(4,5)P2 binding site and create
a more active protein surface, the immobilization of the MA
protein was performed in the presence of compound 14. As
can be seen in Figure 2a, compound 7 interacts with HIV-1 MA
with an equilibrium dissociation constant (KD) of 22.6(ꢀ
3.1) mm. This value is in remarkable agreement with the IC50
value derived from the single-round infection assay (IC50 =
21.2 mm).
Having demonstrated that compound 7 interacts with HIV-
1 MA, we next sought to establish whether compound 7 can
compete with PI(4,5)P2 for binding to MA. To accomplish this,
we established an SPR assay that allowed monitoring of the in-
teraction of HIV-1 MA with a soluble form of PI(4,5)P2. The solu-
ble, biotinylated PI(4,5)P2 was attached to a sensor chip, and
purified MA protein (1 mm) in the presence or absence of com-
pound 7 (75 mm) was passed over this surface. Using the affini-
ty of the compound 7–MA interaction, we established the frac-
tion of MA bound to compound 7 under the experimental
conditions and normalized the results to the unbound fraction
(for details, see the Experimental Section). As can be seen in
Figure 2b, the presence of compound 7 decreased the ability
of MA to interact with PI(4,5)P2 by 89% under the assay condi-
tions. This result, coupled with the decrease in the amount of
virus produced in the presence of compound 7, suggests
a mechanism of action whereby compound 7 disrupts the in-
teraction of Gag with PI(4,5)P2 at the plasma membrane, result-
ing in a failure of the new virions to bud correctly.
Figure 1. Compound 7 functions in the late stages of HIV-1 replication. a) Ef-
fects of compound 7 on early-stage events were determined by producing
virus via transfection of 293T cells (as described in the Experimental Section)
for infection of U87.CD4.CXCR4 target cells in the absence or presence of
various concentrations of compound 7. b) To assess the effect of compound
7 on late-stage events, recombinant virus was produced from 293T cells
either in the absence or presence of compound 7, and the culture super-
natants containing pseudotype stocks were diluted tenfold and then used
to infect target cells in the absence of test compound. Compound-induced
effects are manifested as a decrease in infectivity in the target cells (mea-
sured as luciferase activity), normalized against the infectivity of virus from
untreated cells. Data are the mean ꢀ standard error of the mean (SEM) from
at least four independent experiments (each performed in triplicate). Com-
pound 7 (early stages): IC50 =not applicable; Compound 7 (late stages):
IC50 =21.2 mm. The antiviral effect of compound 7 was determined to be
due to a specific effect on the virus as the compound was not cytotoxic to
293T cells up to a test concentration high of 1 mm (cf. Figure S2 in the
Supporting Information), giving a therapeutic index in this assay of >47.
Mutation of residues in the PI(4,5)P2-binding site of HIV-1 MA
reduces the efficacy of compound 7
Having demonstrated that compound 7 binds to HIV-1 MA
and competes with PI(4,5)P2 for binding, we next sought to
demonstrate that the antiviral effect is mediated through this
interaction. Changes to residues within the PI(4,5)P2 binding
site can render the virus nonfunctional. However, mutations in
L21 have been previously described to have no effect on virus
particle production,[6a] and a virus harboring the T81A mutation
is replication competent.[11] As both of these residues are in
proximity to compound 7 in the original docking model,[9] we
changed the codons in the gag gene present in the proviral
backbone that code for residues L21 and T81 to code for ala-
nine residues and produced the mutant viruses in the pres-
ence or absence of compound 7 (100 mm). The effect of com-
pound 7 on the production of both wild-type (WT) and the
L21A and T81A mutant viruses was assessed by p24 content in
the supernatants after 48 h. Figure 3a shows the results of this
analysis. Compound 7 decreased the amount of WT virus pro-
judged by p24 content (see the Supporting Information).
These observations are in line with the proposed action of
compound 7 through interaction with the HIV-1 MA protein.
Compound 7 binds to HIV-1 MA and competes with PI(4,5)P2
Having determined that compound 7 has antiviral activity and
exerts its effect in the late stages of the viral replication cycle
by decreasing the total amount of virus produced, we next
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Figure 2. a) Sensorgrams depicting the interaction of compound 7 with im-
mobilized HIV-1lAI MA. Compound concentrations of 0, 6.25, 12.5, 25, 50, and
100 mm were tested and are represented as orange, magenta, green, blue,
cyan and red lines, respectively. The insert shows the equilibrium plot used
to derive the equilibrium dissociation constant (KD). Compound 7–MA inter-
action: KD =22.6(ꢀ3.1) mm. b) Compound 7 competes with PI(4,5)P2 for
binding to HIV-1 MA. MA protein (1 mm) in the presence or absence of com-
pound 7 (75 mm) was passed over a PI(4,5)P2 surface, and the response was
recorded. To allow comparison, the responses were normalized with respect
to the degree of free MA.
Figure 3. a) Effect of compound 7 on the production of HIV-1 wild-type and
mutant pseudotypes as judged by supernatant p24 content. 293T cells were
transfected for production of luciferase-reporter pseudotypes (as described
in the Experimental Section) in the absence or in the presence of compound
7 (100 mm), and the culture supernatants containing pseudotype stocks
were then analyzed by p24 ELISA. Shown are the mean values of four inde-
pendent productions, with error bars depicting the standard error of the
mean (SEM). The indicated p value was derived from a two-tailed paired
t-test analysis. b) Refined predicted binding mode of compound 7 in the
PI(4,5)P2 binding site of HIV-1 MA. Binding site residues (L21, R22, W36, R76,
T81, and K98) are represented as dark sticks, whereas compound 7 is shown
in green sticks. Residues highlighted in blue are thought to be critical for
compound binding. Yellow dashed lines represent hydrogen bonds.
duced to approximately 39ꢀ2.5% of the control with no com-
pound (DMSO only). However, changing residues L21 and T81
to alanine completely abrogated the effects of compound 7.
We next sought to refine the docking model and to identify
the most probable binding mode of compound 7 to see
whether or not the model could offer any insight into the mu-
tational results. Exhaustive dockings to multiple conformations
of HIV-1 MA resulted in the binding pose shown in Figure 3b.
This docking model suggests plausible explanations of why
mutation of residues L21 and T81 residues abolishes the antivi-
ral activity compound 7. In this model, the carbonyl oxygen of
the acetamide linker of compound 7 points towards the hy-
droxy group of T81 in the base of the binding groove to form
a hydrogen bond, whereas L21 forms a weak hydrophobic
contact with the aliphatic carbons of the piperazine moiety of
compound 7. Disrupting either of these interactions would
result in decreased affinity of compound 7 for the target MA
protein and the concomitant decrease in potency observed in
the single round infection assay. Future structural studies on
compound 7 or higher affinity analogues in complex with MA
will ultimately determine the validity of this proposed model.
Conclusions
We have identified a novel compound (7) that binds to and
functions through the conserved PI(4,5)P2 binding site on HIV-
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topathic effect assays against a panel of viruses from different
classes were performed as described previously.[13]
1 MA, inhibits the production of new virus, and exhibits broad-
spectrum anti-HIV activity with IC50 values in the range of 7.5
to 15.6 mm for the group M isolates tested (Table 1). This com-
pound provides a novel chemical probe to investigate the
roles of MA in the HIV-1 replication cycle and could serve as
a good starting point for the development of a new class of
HIV therapeutics through optimization by medicinal chemistry
approaches.
Cells: Human embryonic kidney 293T cells were cultured in Dulbec-
co’s modified Eagle medium (DMEM) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), l-glutamine, and antibi-
otics (penicillin/streptomycin). Human astroglioma U87 cells stably
transfected for the expression of CD4 and CXCR4 (obtained from
Prof. Hongkui Deng (Peking University) and Prof. Dan Littman
(New York University, USA), through the AIDS Research and Refer-
ence Reagent Program, Division of AIDS, NIAID, NIH)[14] were cul-
tured in DMEM supplemented with 10% FBS, l-glutamine, penicil-
lin streptomycin, puromycin and G418 (Gibco BRL Life Technolo-
gies, Grand Island, NY, USA).
Experimental Section
Virtual screening
The in silico screening was previously reported.[9] Refinement of
the docking model of compound 7 was performed with QUANTUM
software utilities (Quantum Pharmaceuticals, Moscow, Russia)[12] on
a total of 40 different conformations of the MA protein derived
from the Protein Data Bank (PDB) and from molecular dynamic
simulations. In addition, several analogues of compound 7 that
have antiviral activity (Cocklin et al., unpublished data) were simi-
larly docked into the 40 protein conformers and consensus posi-
tions were used to establish the most probable binding mode of
compound 7.
Production of pseudotypes: Single-round infectious envelope-pseu-
dotyped luciferase-reporter viruses were produced in 293T cells co-
transfected by calcium phosphate precipitation (Profection Mam-
malian Transfection System) with the envelope-deficient HIV-1 NL4-
3 vector (pNL4-3-LucR+Eꢁ; a gift from Prof. Nathaniel R. Landau,
New York University, USA),[15] which carries the luciferase-reporter
gene, and the HxBc2 envelope-expressing vector (obtained from
Dr. Kathleen Page (University of Southern California, USA) and Prof.
Littman through the AIDS Research and Reference Reagent Pro-
gram, Division of AIDS, NIAID, NIH).[16] All MA mutations within the
pNL4-3-LucR+Eꢁ vector were synthesized and validated by Gene-
script (Piscataway, NJ, USA). After 6 h of incubation at 378C, the
DNA-containing medium was removed, cells were washed, and
fresh medium with or without compound 7 (at concentrations be-
tween 3.1 and 100 mm) was added. Supernatants containing the
envelope-pseudotyped viruses were collected two days later, clari-
fied by centrifugation, aliquoted, and stored at ꢁ808C until use.
Pseudotype stocks were quantified by p24gag content.
Chemistry
All chemicals were purchased from Enamine Ltd and were 95%
pure or greater. Compound 7 was resynthesized to 98% purity
using the route shown in Scheme 1. Briefly, diisopropylethylamine
(208 mg, 1.612 mmol) was added dropwise to a mixture of pipera-
zine
I
(342 mg, 1.465 mmol) and compound II (312 mg,
1.465 mmol) in dry DMF (1.5 mL). The reaction mixture was sonicat-
ed in an ultrasonic bath at 608C for 3 h. The mixture was then di-
luted with water (10 mL), and the resultant precipitate was isolated
by filtration and washed with EtOH (1 mL) to give compound III
(513 mg, 86%).
Single-round infection assays: To determine whether compound 7
affected early stages of the viral life cycle, 50 mL medium alone or
containing compound 7 at twice the desired final concentration
(between 3.1 and 100 mm) was added to the U87.CD4.CXCR4
target cells plated in 96-well plates, and 50 mL of pseu-
dotype stocks (not treated during production in 293T
cells) was added immediately. Alternatively, to study the
inhibitory effects of compound 7 during the late stages
of the viral life cycle, untreated or compound 7-treated
pseudotype stocks were diluted 1:10 with fresh
medium, and an aliquot of 100 mL was used for infection
of U87.CD4.CXCR4 target cells; this dilution ensured that
any effect observed was due to the concentration of the
compound present during viral production and not to
the residual amount of compound present during infec-
tion of target cells. Assessment of the effects of com-
pound 7 on the late stages of infection with pseudotype
stocks produced with a luciferase-reporter vector encod-
ing a mutant MA was performed in the same manner.
Infections were performed for two days and quantified
by measuring luciferase activity in cell lysates (Luciferase Assay
System, Promega) using a microplate luminometer (GloMax, Prom-
ega).
Scheme 1. Synthesis of compound III: Reagents and conditions: N,N-diisopropylethyl-
amine (1.1 equiv), DMF, 608C, 3 h, 86%.
Biology
Anti-HIV efficacy evaluation in human peripheral blood mononuclear
cells: All viral isolates were obtained from the AIDS Research and
Reference Reagent Program, Division of AIDS, National Institute of
Allergy and Infectious Diseases (NIAID), US National Institutes of
Health (NIH), and used to perform antiviral evaluations in human
PBMCs as described previously.[9,13]
Compound cytotoxicity: Supernatants from target cells treated with
or without compound 7 to test its effects at early stages, and pseu-
dotype stocks produced with or without compounds to evaluate
their effects at late stages, were used for analysis of cytotoxicity.
Using a
Determination of the antiviral spectrum of MA-targeted compounds:
To determine the spectrum of antiviral activity of compound 7, cy-
colorimetric assay (Takara Bio Inc., Japan), these effects were esti-
mated through the presence in culture supernatants of lactate de-
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hydrogenase (a stable cytoplasmic enzyme), which would indicate
plasma membrane damage.
sensor chip to high density (8000 RU) using standard amine cou-
pling. However, in order to preserve the PI(4,5)P2 binding site and
create a more active protein surface, the immobilization of the MA
protein was performed in the presence of 1.5 mm compound 14. A
reference surface was similarly created by immobilizing a nonspecif-
ic antibody ARC4033 (antimouse/rat interferon-g: BioSource; Invi-
trogen, Carlsbad, CA, USA).
p24 enzyme-linked immunosorbent assays (ELISAs): p24 ELISAs were
performed to determine the amount of virus produced in the pres-
ence or absence of compound 7. The ELISA kits used were ob-
tained from either Zeptometrix Corp. (Buffalo, NY, USA) or Ad-
vanced Bioscience Laboratories (Kensington, MD, USA).
Binding assays were performed with PBS-T with 3% DMSO as the
running buffer. Samples of compound 7 were prepared as previ-
ously described[9] and injected across the MA and antibody refer-
ence surfaces at a flow rate of 100 mLminꢁ1, for a 1 min association
phase, followed by a 5 min dissociation phase using the “one-shot
kinetics” capability of the Proteon instrument.[19] Specific regenera-
tion of the surfaces between injections was not needed owing to
the nature of the interaction. Data were analyzed using the Pro-
teOn Manager software version 3.0 (Bio-Rad). The responses of
a buffer injection and the responses from the reference flow cell
were subtracted to account for nonspecific binding and injection
artifacts. The experimental data could not be adequately fitted to
a simple 1:1 binding model as the individual on and off rates were
outside of the dynamic range of the instrument. Therefore, the
equilibrium dissociation constant (KD) was obtained by fitting equi-
librium binding data on the ProteOn Manager software, using the
four-parameter Equation (1), where Rhigh is the response value at
high analyte concentrations, Rlow is the response at an analyte con-
centration of zero, A1 is the midrange concentration and is equiva-
lent to the equilibrium constant (KD), and A2 is the slope factor.
Recombinant protein production and purification: The MA region of
the HIV-1 gag gene was amplified from plasmid pLAI (a generous
gift from Drs. Evelyn Kilareski and Brian Wigdahl, both Drexel Uni-
versity College of Medicine, Philadelphia, USA) using primers de-
signed to facilitate ligation-independent cloning into the vector
pETHSUL.[17] This vector is designed for the insertion of genes of in-
terest in frame with an N-terminal small ubiquitin-related modifier
(SUMO) tag.[17] The recombinant pETHSUL plasmid was verified for
the presence of MA insert by restriction digestion and sequence
analysis (Genewiz, Inc., South Plainfield, NJ, USA). The resultant
vector was designated pSUMO-MA. The purification of H6SUMO-
MA was achieved via immobilized metal affinity chromatography
using a TALON cobalt resin affinity column (ClonTech).
The Escherichia coli strain BL21 (DE3) Codon+-RIL (Stratagene, La
Jolla, CA, USA) was used for expression of H6SUMO-MA from
pSUMO-MA. Luria-Bertani broth (2 mL) containing 100 mgmLꢁ1 am-
picillin and 50 mgmLꢁ1 chloramphenicol was inoculated with
a single transformed colony, and the colony was allowed to grow
at 378C for 9 h. A 100 mL aliquot of the preculture was used to in-
oculate 100 mL of the autoinducing media ZYP-5052[18] containing
100 mgmLꢁ1 ampicillin and 34 mgmLꢁ1 chloramphenicol. The cul-
ture was grown at 308C for 16 h. Cells were harvested by centrifu-
gation at 1076ꢂg for 20 min at 48C, and the pellet was suspended
in 30 mL phosphate-buffered saline (PBS) (Roche, Nutley, NJ, USA)
containing 2.5 mm imidazole. Cells were lysed by sonication, and
the supernatant clarified by centrifugation at 11,952ꢂg (SS-34, Sor-
vall RC 5C Plus) for 20 min at 48C. The supernatant was removed
and applied to a TALON cobalt resin affinity column (ClonTech),
previously equilibrated with PBS.
À
Á
Rhigh ꢁ Rlow
Response ¼ Rhigh
þ
ꢀ
ꢁ
A2
ð1Þ
concn
1 þ
A1
For PI(4,5)P2 competition assays, biotin–PI(4,5)P2 (Echelon Biosci-
ences, Inc., Salt Lake City, UT, USA) was captured on the surface of
a GLC chip, to which streptavidin had been immobilized using
standard amine coupling.
A reference surface with captured
biotin–PI(3)P was also created. MA protein (1 mm) in the presence
or absence of compound 7 (75 mm) was passed over this surface
and the response recorded. The average responses from five data
sets were used. To normalize the competition data to the degree
of free MA, the concentration of free ligand and degree of satura-
tion of the HIV-1 MA were calculated directly using Equations (2)
and (3). The relation between the total ligand concentration [X]T
and free ligand concentration [X] is given by Equation (2), where KA
is the association constant, [P] is the concentration of macromole-
cule, and n is the stoichiometry. After determining the free concen-
tration of MA, the fraction bound (Fb) was calculated using Equa-
tion (3).
Loosely bound proteins were removed via seven-column volumes
of PBS containing 7.5 mm imidazole. Tightly associated proteins
were eluted in three-column volumes of PBS containing 250 mm
imidazole. The eluates were then pooled and made 1 mm with re-
spect to ethylenediamine tetraacetic acid (EDTA). To this pooled
sample, 10 mg of a recombinant His6-tagged form of the catalytic
domain (dtUD1) of the Saccharomyces cerevisiae SUMO hydrolase
was added.[17] Cleavage was allowed to proceed for 4 h at 188C.
Following cleavage, the sample was dialyzed at 48C overnight
against 2 L of PBS to remove any imidazole. After dialysis, the
dtUD1-catalyzed cleavage reaction was subjected to a second
cobalt affinity purification using the TALON cobalt resin affinity
column. In this purification step, however, the cleaved MA protein
passes straight through the column owing to removal of the His6
tag. Subsequently, the subtractively purified MA was dialyzed over-
night at 48C against 25 mm Tris-HCl, pH 8.0 containing 10% glycer-
ol. This dialyzed sample was then filtered and loaded onto a 5 mL
Hi-Trap Q HP column (GE Healthcare, Chalfont, UK). The flow-
through, containing the MA protein, was concentrated, flash
frozen in liquid nitrogen, and stored at ꢁ808C.
½Xꢂ ꢃ KA ꢃ n ꢃ ½Pꢂ
ð2Þ
ð3Þ
½XꢂT ¼ ½Xꢂ þ
1 þ KA ꢃ ½Xꢂ
KA½Xꢂ
Fb ¼
1 þ KA ꢃ ½Xꢂ
Acknowledgements
This work was supported in part by the US National Institutes of
Health (NIH)/US National Institute of Allergy and Infectious Dis-
eases (NIAID) (1R03AI078790-01A1 and 1R21AI087388-01A1 to
S.C.), and by the US NIH/US National Institute of Neurological
Disorders and Stroke (NINDS)(R01NS065727 to J.M.-G.). Andrei
Surface plasmon resonance (SPR): Interaction analyses were per-
formed on a ProteOn XPR36 SPR Protein Interaction Array System
(Bio-Rad Laboratories, Hercules, CA, USA). ProteOn GLH sensor
chips were preconditioned and equilibrated as previously de-
scribed.[9] HIV-1LAI MA was immobilized to the flow cells of a GLH
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 426 – 432 431

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Keywords: antiviral agents
· HIV-1 · matrix proteins ·
phosphatidylinositol 4,5-bisphosphates · virtual screening
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Published online on January 29, 2013
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ChemMedChem 2013, 8, 426 – 432 432