Báo cáo khoa học: Structure–function relationship of novel X4 HIV-1 entry inhibitors – L- and D-arginine peptide-aminoglycoside conjugates pptx

Structure–function relationship of novel X4 HIV-1 entry
inhibitors –
L- and D-arginine peptide-aminoglycoside
conjugates
Ravi Hegde, Gadi Borkow, Alexander Berchanski and Aviva Lapidot
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel
Significant advances in understanding the process by
which HIV-1 enters the host cells have been the focus
of considerable interest, owing to the possibility to tar-
get the HIV-1 receptors for therapeutic intervention.
The multistep nature of HIV-1 entry provides multisite
targeting at the entrance door of HIV-1 to cells. Block-
ing HIV-1 entry to its host cells has clear advantages
over blocking subsequent stages in the life cycle of the
virus. Indeed, potent cooperative and synergistic inhi-
bition of HIV-1 proliferation has been observed in
in vitro studies with several entry inhibitor combina-
tions, interacting with different steps of the HIV-1-cell
entry cascade. Targeting a compound to several steps
of the viral-cell entry, and also to subsequent steps in
the viral life cycle, promises an even more effective
therapeutic by reducing the probability of HIV-1 to
develop resistance [1–6]. Using one drug that can tar-
get multiple sites and ⁄ or steps in the viral life cycle will
have obvious advantages in clinical use.
The viral envelope protein plays a critical role in
HIV-1 entry to cells. HIV-1 entry is initiated by the
interaction of the viral envelope glycoprotein 120
(gp120) with the host cell receptor CD4, and mainly
with the CXC chemokine receptor type 4 (CXCR4)
and CC chemokine receptor 5 (CCR5). The CXCR4

receptor and its only natural chemokine ligand stromal
Keywords
drug design; HIV-1 entry inhibitors; poly
arginine-aminoglycoside conjugates;
structure–function relationship
Correspondence
A. Lapidot, Department of Organic
Chemistry, The Weizmann Institute of
Science, Rehovot 76100, Israel
Fax: +972 8 9344142
Tel: +972 8 9343413
E-mail:
(Received 29 August 2007, revised 18 Octo-
ber 2007, accepted 29 October 2007)
doi:10.1111/j.1742-4658.2007.06169.x
We present the design, synthesis, anti-HIV-1 and mode of action of neomy-
cin and neamine conjugated at specific sites to arginine 6- and 9-mers
d- and l-arginine peptides (APACs). The d-APACs inhibit the infectivity
of X4 HIV-1 strains by one or two orders of magnitude more potently than
their respective l-APACs. d-arginine conjugates exhibit significantly higher
affinity towards CXC chemokine receptor type 4 (CXCR4) than their
l-arginine analogs, as determined by their inhibition of monoclonal anti-
CXCR4 mAb 12G5 binding to cells and of stromal cell-derived factor 1a
(SDF-1a) ⁄ CXCL12 induced cell migration. These results indicate that
APACs inhibit X4 HIV-1 cell entry by interacting with CXCR4 residues
common to glycoprotein 120 and monoclonal anti-CXCR4 mAb 12G5
binding. d-APACs readily concentrate in the nucleus, whereas the
l-APACs do not. 9-mer-d-arginine analogues are more efficient inhibitors
than the 6-mer-d-arginine conjugates and the neomycin-d-polymers are bet-
ter inhibitors than their respective neamine conjugates. This and further

structure–function studies of APACs may provide new target(s) and lead
compound(s) of more potent HIV-1 cell entry inhibitors.
Abbreviations
AAC, aminoglycoside-arginine conjugates; ALX40-4C, N-a-acetyl-nona-
D-arginine amide; APACs, aminoglycosides poly D- and L-arginine
conjugates; CCR5, CC chemokine receptor 5; CXCR4, CXC chemokine receptor type 4; DIEA, diisopropylethylamine; EDC,
N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide hydrochloride; FITC, fluorescein isothiocyanate; gp120, glycoprotein 120; hRBC, human
erythrocytes; HOBT, 1-hydroxybenzotriazole; MFI, median fluorescent intensity; NBND, N-(tert-butoxycarbonyloxy)-5-norbornene-endo-2,3-
dicarboximide; NeoR, hexa-arginine-neomycin conjugate; PE, phycoerythrin; SDF-1, stromal cell-derived factor 1; TAR, Tat responsive
element.
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6523
cell-derived factor 1 (SDF-1) are crucial for embryonic
development, and have been implicated in various
pathological conditions, including HIV-1 infection and
cancer metastasis [7,8]. SDF-1a has been found to
inhibit X4-tropic HIV-1 isolates by blocking viral cell
entry [9]. Several peptide-derived and other small
molecule inhibitors of CXCR4- and CCR5-mediated
HIV-1 infection have been reviewed [5]. One example
of a CXCR4 antagonist that blocks infection by X4
strains of HIV-1 and SDF-1 binding is a N-a-acetyl-
nona-d-arginine amide (ALX40-4C) [10]. ALX40-4C
was the first CXCR4 antagonist to be tested in HIV-1
infected individuals [11].
An additional critical step in HIV-1 infection is effi-
cacious transactivation of the viral genes in the
infected host cell. Interestingly, an arginine rich basic
peptide, derived from HIV-1 transactivator protein
(Tat) (positions 48–60), has been reported to have the
ability to translocate through the cell membrane and

accumulate in the nucleus. It was also presented that
various arginine-rich peptides have a potent transloca-
tional activity very similar to Tat (48–60), including
such peptides in which l-arginines were substituted
with d-arginines [12]. Optimal cellular and nuclear
uptake was reported to be more effective for arginine
polymers that were 7–9 mers in length compared to
similar lengths of lysine polymers [13]. Poly arginine-
containing peptides are also known as potent furin
inhibitors, with the 9-mer d-poly arginine being the
most active inhibitor [14]. Cell penetrating peptides
such as l- and d-oligo-arginines have been recently
reported to enhance the cellular uptake of antisense
oligonucleotides, with the d-oligo-arginines having the
highest stability in cell culture compared to their
l-analogues [15,16].
Based on peptide models of HIV-1 Tat responsive
element (TAR) RNA binding, NMR structures of
TAR–ligand complexes and aminoglycoside–RNA
interactions, we have designed and synthesized a set of
conjugates of aminoglycoside antibiotics with arginine
(AACs) [1]. The AACs display high affinity to the
HIV-1 TAR RNA in HIV-1 long-terminal repeats and
to HIV-1 Rev responsive element [17,18].
Interestingly, we found that conjugates of AACs, in
addition to inhibiting viral gene transactivation, block
HIV-1 cell entry by interacting with CXCR4 [1]. The
finding that the hexa-arginine-neomycin conjugate
(NeoR; which contains six arginine moieties conju-
gated to the three pyranoside rings of neomycin B;

Fig. 1) is the most efficient anti-HIV-1 compound
among all the other aminoglycoside derivatives [1]
prompted us to question whether conjugation of neo-
mycin (or other members of this aminoglycoside
group, e.g. neamine and paromomycin) with poly argi-
nine (6- and 9-mers), would lead to more potent HIV-
1 inhibitors than a manifold of arginine conjugated via
the amino groups of the aminoglycosides. Thus, a new
set of poly arginine 6-mer and 9-mer d- and l-amino-
glycoside conjugates (APACs) was designed and syn-
thesized, and their cell uptake and antiviral activities
were determined. We further investigated how APACs
block HIV-1 gp120 interaction with CXCR4 and com-
pete with its natural ligand SDF-1a to CXCR4.
Results
Synthesis and chemical characterization
of APACs
The synthesis of the regioselectively functionalized
aminoglycosides (derivatives 1a, 2a and 3a; Fig. 2)
presented a challenge due to the presence of several
primary amines of approximately comparable reactiv-
ity in each of the aminoglycoside used in this study.
Within several primary amino groups, one amino
group of neamine (ring I) and paromomycin (ring IV)
and two amino groups of neomycin (rings I and IV)
are located at primary carbons. Thus, a multistep syn-
thesis was undertaken for conjugation of the peptides
with the aminoglycosides (Fig. 2).
Different approaches for selective protection of amino
groups of aminoglycosides have been reviewed [19]. A

procedure based on differences in reactivity of the
amino groups towards weak acylating agents appears
most attractive, particularly the reagent N-(tert -but-
oxycarbonyloxy)-5-norbo rnene-endo-2,3-dicarboximide
(NBND). The extent of selectivity shown by NBND is
unprecedented, which makes this reagent ideally suited
for application to aminoglycoside chemistry.
The unhindered amino groups [attached to methy-
lene carbon(s)] of neamine, paromomycin and neomy-
cin were blocked with tert-butoxycarbonyl groups by
the reaction of aminoglycoside with one equivalent of
NBND (in dioxane ⁄ water 1 : 1) medium. Under this
condition, only mono-Boc-neomycin derivative was
obtained as demonstrated by mass spectrometry. The
second step of the synthesis involved full protection of
the remaining amino groups with Cbz, achieved by the
reaction of benzylchloroformate (CbzCl) in the pres-
ence of sodium carbonate in high yield. Then, the
‘Boc’ group was removed by a classical trifluoroacetic
acid cleavage, affording free aminomethyl derivatives
1a, 2a and 3a (Fig. 2). The products were purified by
silica gel column chromatography before being con-
firmed by mass spectrometry. The coupling of arginine
peptides (6- and 9-mers) with 1a, 2a and 3a was
HIV inhibition by L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6524 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS
performed using N-(3-dimethylaminopropyl)-N¢-ethyl-
carbodiimide hydrochloride (EDC) as a coupling
reagent in the presence of 1-hydroxybenzotriazole
(HOBT) and diisopropylethylamine (DIEA). The ter-

tiary amine DIEA, used in the reaction mixture, is not
sufficiently basic to deprotonate the guanidinium head-
group. Finally, APACs were obtained by deprotecting
the remaining protecting groups (Cbz and NO
2
)by
hydrogenation using Pd ⁄ C (10%).
Of the three sets of compounds of 6- and 9-mers of
l-, d- and l ⁄ d-enantiomers of arginine chains and
their aminoglycoside conjugates (neamine, paromomy-
cin and neomycin), 17 compounds in total, only poly
d-arginines and their aminoglycoside conjugates, and
9-mer l-arginine, are represented in Table 1. The
purity of all compounds was approximately 95%, as
determined by HPLC analysis and proven by MALDI-
TOF, and confirmed by combustion analysis. In the
case of neomycin, conjugates might be a 1 : 1 mixture
of two neomycin derivatives, in which either ring I or
IV is conjugated to the arginine chain (Fig. 1).
As expected, d- and l-arginine (6- and 9-mers) pep-
tide aminoglycoside conjugates displayed mirror-image
CD spectra and random conformation (see supplemen-
tary Fig. S1).
Fluorescent probes: APACs-FITC
The acetate counter ions of Neo-r9 and Neo-R9
were removed by Amberlite IRA 400 (OH
–
) ion-
exchange resin prior to their reaction with fluorescein
isothiocyanate (FITC) in water ⁄ methanol ⁄ dioxane

(1:1:1, v⁄ v ⁄ v) medium in the presence of two
equivalents of triethyl amine for 2 h at room temper-
ature with some modifications, as previously
reported, for NeoR and other aminoglycoside argi-
nine conjugates [18,20]. As previously reported for
FITC-aminoglycosides [21] FITC is bound to the
A
B
Fig. 1. (A) Schematic representation of
APACs and aminoglycosides used. All
APACs were prepared as acetate salts. R,
L-arginine; r, D-arginine. (B) CXCR4-bound
conformations of NeoR, Neo-r9, and Neo-r6.
The aminoglycoside cores of compounds
are colored in gray, the arginine moieties
are shown in black.
R. Hegde et al. HIV inhibition by
L ⁄ D-poly arginine-aminoglycosides
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6525
free aminomethylene (-CH
2
NH
2
) group of neomycin.
After removal of the solvents under reduced pres-
sure, the FITC derivatives were purified by extrac-
tion with absolute ethanol. Finally, FITC conjugates
were converted into acetate salt and characterized by
mass spectrometry.
APACs containing

D-arginine peptides
(6- and 9-mers) display significantly higher
anti-HIV-1 activity then their
L-arginine
aminoglycoside analogues
APACs group A comprises the aminoglycosides ne-
amine, paromomycin and neomycin, conjugated to
6- or 9-mer l-arginine. As shown in Table 2, their
ability to inhibit HIV-1 infectivity is significantly
lower than their d-arginine aminoglycoside analogues
(group B). No antiviral activity up to 200 lm of the
neomycin B was noticed (Table 2). However, a short
chain of two l-arginines already conjugated to ne-
amine (data not included in Table 1) revealed a low
anti-HIV-1
IIIB
activity, with the concentration that
caused 50% inhibition of viral production (EC
50
)
being 50 lm. R6 presented significantly lower antivi-
ral activity (EC
50
of 110 lm) in comparison to its
aminoglycoside conjugates Neam-R6, Paramo-R6 and
Neo-R6 (EC
50
of 70, 31, and 40 lm, respectively).
By contrast, the antiviral activity of the free non-
amer arginine R9 (EC

50
of 33 lm) was similar to
that of its aminoglycoside conjugates. The EC
50
of
Fig. 2. Schematic representation of the synthesis of aminoglycoside-arginine peptide conjugates.
HIV inhibition by
L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6526 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS
d ⁄ l-9-mer-arginine neamine conjugate (Neam-R ⁄ r9,
Neam-RRrRrRrRR; Table 1) showed a somewhat
lower value of EC
50
(28 lm) compared to Neam-R9
(37.5 lm; Table 2), but significantly higher than
Neam-r9 (EC
50
of 4.2 lm; Table 3).
d-APACs inhibited a variety of T-tropic HIV-1 iso-
lates, both laboratory adapted and clinical isolates, as
well as resistant strains, including NeoR resistant
(NeoR
res
) virus, in the EC
50
range of 1.2–6.2 lm, with
the exception of AZT resistant virus, in which the
EC
50
range was 6.6–10.4 lm (Table 3). By contrast to

NeoR [18], the APACs did not inhibit HIV-1 Ba-L, an
M-tropic HIV-1 laboratory isolate that uses CCR5
and not CXCR4 for cell entry. Neo-r9 does not inhibit
the binding of 2D7 mAb against CCR5 (data not
shown). Taken together, this suggests that APACs
interfere with HIV-1 entry step by interacting with
CXCR4.
Significant differences were found between the
antiviral potency of APACs containing 6- and 9-mers
d-arginine, and the two aminoglycosides; neamine and
neomycin. In general, the 9-mer d-arginine conjugates
were approximately two- to three-fold more active than
the 6-mer d-arginine conjugates and the neomycin-
d-9-arginine conjugate was significantly more active
than the neamine respective arginine conjugate. There
were no significant differences between their capacities
to inhibit HIV-1
IIIB
wild-type virus and its NeoR
res
variant (Table 3). In general, the d-9-mer-peptide and
its aminoglycoside conjugates revealed significantly
lower EC
50
. For example, 1.5 ± 0.4 lm (against
Protease
r
virus) and 1.6 ± 0.7 lm (against HIV-1
IIIB
)

Table 1. D-peptides and their aminoglycoside conjugates. R, L-arginine; r, D-arginine; Amg, aminoglycoside as detailed in the third column;
–, no core.
Peptide ⁄ conjugate
Aminoglycoside
(Amg)
Compound
abbreviation
MS (m ⁄ z)
Calculated Observed
HO-rrrrrr-NHAc – r6 997.178 997.61
Amg-rrrrrr-NHAc Neamine Neam-r6 1301.523 1301.80
Amg-rrrrrr-NHAc Neomycin Neo-r6 1593.809 1594.14
HO-rrrrrrrrr-NHAc – r9 1465.742 1466.00
Amg-rrrrrrrrr-NHAc Neamine Neam-r9 1770.076 1770.43
Amg-rrrrrrrrr-NHAc Neomycin Neo-r9 2062.362 2063.00
HO-RRRRRRRRR-NHAc – R9 1465.710 1466.13
Table 2. Antiviral activity of L-APACs against HIV-1
IIIB
virus. ND, not determined.
R6 Neam-R6 Paramo-R6 Neo-R6 R9 Neam-R9 Paramo-R9 Neo-R9 Neam-R ⁄ r9
a
R9-Ac Neo-mycin
EC
50
(lM) 110 ± 20 70 ± 10 31 ± 10 40 ± 12 33 ± 3 37.5 ± 2.5 31 ± 9 30 ± 7 28 ± 2 31 > 200
CC
50
(lM) ND 210 160 200 120 175 140 150 160 ND > 300
a
Neam-RRrRrRrRR.

Table 3. Antiviral activity of D-APACs against HIV-1 clinical isolates and laboratory strains. The 50% effective concentration which inhibited
HIV-1 replication was determined as described in Experimental procedures. Cytotoxicity was measured by trypan blue exclusion assay for
MT2 cells. The data are the average of three independent experiments. The antiviral experiments were performed in triplicate and the cyto-
toxicity assays were performed in duplicate. All isolates tested are T-tropic HIV-1 isolates (isolates that use CXCR4 as its main coreceptor),
with the exception of HIV-1 Ba-L. ND, not determined.
Compound
EC
50
(lM)
Cytotoxicity
CC
50
(lM)
IIIB Ba-L
a
AZT
b
Protease
b
NeoR
b
Clade A
c
Clade C
c
LAI ELI
r6 5.8 ± 1.7 > 50 15.2 ± 6.2 4.9 ± 1.0 6.8 ± 0.7 8.0 ± 1.0 5.3 ± 0.3 ND ND 170
Neam-r6 5.9 ± 4.2 > 50 7.0 ± 4.0 2.2 ± 0.4 6.2 ± 5.8 3.2 ± 1.3 1.7 ± 0.1 13.9 2.9 150
Neo-r6 3.1 ± 3.7 > 50 10.4 ± 4.1 4.0 ± 1.0 4.8 ± 2.2 5.7 ± 2.8 3.0 ± 0.8 2.7 1.3 155
r9 1.5 ± 0.6 > 50 9.7 ± 5.9 1.4 ± 0.6 2.0 ± 1.2 2.2 ± 0.3 1.6 ± 0.4 ND ND 150

Neam-r9 4.2 ± 4.8 > 50 10.4 ± 2.7 2.4 ± 0.4 6.0 ± 5.0 5.1 ± 2.9 2.7 ± 0.5 9.7 4.3 150
Neo-r9 1.6 ± 0.7 > 50 6.6 ± 5.9 1.5 ± 0.4 2.8 ± 2.5 2.1 ± 0.4 1.2 ± 0.5 1.9 1.3 120
a
M-tropic HIV-1 viral isolate;
b
resistant isolate;
c
clinical isolate.
R. Hegde et al. HIV inhibition by
L ⁄ D-poly arginine-aminoglycosides
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6527
and 1.6 ± 0.4 lm and 1.2 ± 0.5 lm against clade C
virus (a clinical isolate) for r9 peptide and Neo-r9 con-
jugate, respectively (Table 3). A similar relative ratio of
the EC
50
for the NeoR
res
virus was observed (Table 3).
The finding that the presence of APACs only during
the first 2 h of cell infection was sufficient to inhibit
T-tropic HIV-1 isolates (Fig. 3) suggests that Neam-r9
and Neo-r9 may interfere with the binding of the viral
envelope to the cell.
D-arginine (6- and 9-mers) peptide-
aminoglycosides are readily internalized
and concentrated in the cell nucleus and
extra-nuclear organelles
FITC derivatives of d-arginine (6- and 9-mers) and
their aminoglycoside-neamine and -neomycin conjugate

FITC derivatives (for FITC derivatives preparation,
see Experimental procedures) display efficient cell
uptake and accumulate intracellularly and in the
nucleus. For example, Fig. 4 shows a representative
experiment in which cMAGI cells were incubated for
30 min at 37 °C with the fluorescent derivative (FITC)
of Neo-R9. As revealed by confocal microscopy, and
as indicated by the white full and dotted arrows, the
Neo-r9 FITC derivative is concentrated both in the
nucleus and in extra-nuclear organelle(s). By contrast,
the l-peptide aminoglycoside derivatives display lower
uptake efficiency, and do not concentrate in the
nucleus, but are widely dispersed throughout the cells
(Fig. 4). Of note, cellular uptake and ⁄ or cell membrane
interaction by d-arginine 9-mer aminoglycoside-FITC
derivative (Neo-r9-FITC) was reduced in the presence
of five-fold higher concentration of its l-peptide ana-
logue Neo-R9 (measured by fluorescent activated cell
sorting analysis, data not shown), indicating that the
d- and l-arginine aminoglycoside derivatives compete
for cell entry, and that the same cellular component(s)
is involved in their cell uptake.
APACs inhibit monoclonal anti-CXCR4 mAb
binding to cells
We have previously found that a variety of AACs (am-
inoglycosides neamine, paromomycin, neomycin and
gentamicin conjugated via each one of the free amino
groups of the aminoglycoside to arginines; e.g. six argi-
nines are conjugates to neomycin) interact with
CXCR4 (the main cellular coreceptor for T-tropic

HIV-1 isolates), but not with CCR5 [20,22,23]. Thus,
the capacity of the various APACs to block the bind-
ing of the phycoerythrin (PE) labeled 12G5 mAb
to CXCR4 in MT2 cells was examined. The main
Fig. 3. Inhibitory effect of Neam-r9 and Neo-r9 on HIV-1
IIIB
replica-
tion. cMAGI cells were infected for 2 h at 37 °C in the absence or
presence of 0.78–50 l
M Neam-r9 or Neo-r9 followed by a cell
wash. The cells were then incubated for a further 4 days in the
absence or presence of the appropriate concentrations of the com-
pounds. Cell infectivity was then determined. m, APACs were pres-
ent during the infection step and after the cells were washed; j,
APACs were present only during the first 2 h, before the cells were
washed.
Neo-R9
Neo-r9
Fig. 4. Confocal microscopy images of cMAGI cells stained with
the APACs–FITC conjugates. The cells were incubated for 30 min
with 5 and 15 l
M FITC–conjugates of Neo-r9 and Neo-R9. The
arrows indicate uptake of Neo-r9-FITC by the cell nucleus. The
upper panels show optical microscopy of the cells; the lower pan-
els comprise the same fields as upper panels, but with confocal
fluorescent microscopy.
HIV inhibition by
L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6528 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS
purpose of the study examining the inhibition of the

12G5 mAb binding to CXCR4 by the several APACs
was to distinguish between the capacities of the d- and
l-aminoglycoside conjugates to interact with CXCR4.
Due to the nature of the experiments, two concentra-
tions for the l-APACs (20 and 80 lm) and two con-
centrations for the d-APACs (2 and 10 lm) were
chosen. As shown for one representative experiment in
Fig. 5 for Neo-r9, the median fluorescent intensity
(MFI) of 12G5 mAb binding to MT2 cells was 55.56,
whereas that of the isotype control was 4.0. In the
presence of 2 and 10 lm of Neo-r9, the MFI of the
mAb binding to cells was reduced to 6.44 and 3.08,
respectively, thus already achieving almost 100% inhi-
bition in the presence of 2 lm of Neo-r9. Similar mea-
surements and data analysis were performed for all
APACs comprising 6- and 9-mers d- and l-arginines
conjugated to different aminoglycosides (Table 4). As
shown in Table 4, the d-arginine-neamine conjugates
inhibit 30–120-fold more potently than the l-peptide
conjugates the mAb interaction with CXCR4. The
9-mer-d-arginine activity was approximately 115-fold
higher than the corresponding l-peptide (Table 4). In
addition, 2 lm 9-mer-d-arginine-neomycin conjugate
(Neo-r9) achieved 95.3% inhibition of mAb 12G5 bind-
ing in comparison to 67.3% for the respective neamine
d-conjugate (Neam-r9) and 81% to the free 9-mer-
d-peptide (r9). Whereas, the free aminoglycosides neo-
mycin B, neamine and paromomycin, at concentrations
Table 4. Percent inhibition of 12G5 mAb binding to CXCR4 by
R-peptide and their conjugates, and r-peptides and their conjugates,

to neamine and neomycin. The percent of inhibition of 12G5
binding to the cells was calculated by the formula:
100 ) [(A ) B ⁄ C ) B) · 100]; where A is the MFI obtained in the
presence of APACs and 12G5 mAb, B is the MFI obtained with
cells exposed to the isotype match control Ab only, and C is the
MFI obtained with cells incubated with 12G5 mAb only. ND, not
determined.
Compound Inhibition (%)
L-APACs 20 lM 90 lM
Neam-R6 35.4 ND
R9 ND 52.8
Neam-R9 ND 30.1
D-APACs 2 lM 10 lM
r6 1.6 70.9
Neam-r6 91.1 100
Neo-r6 79 99.3
r9 81.3 99.6
Neam-r9 67.3 100
Neo-r9 95.3 100
a- Isotype control (4.0)
b- mAb only (55.56)
c- mAb + 2 µ
M
Neam-r9 (20.84)
d- mAb + 10 µ
M
Neam-r9 (2.52)
a- Isotype control (4.0)
b- mAb only (55.56)
c- mAb + 2 µ

M
Neam-r6 (9.6)
d- mAb + 10 µ
M
Neam-r6 (3.13)
a- Isotype control (4.0)
b- mAb only (55.56)
c- mAb + 2 µ
M
Neo-r9 (6.44)
d- mAb + 10 µ
M
Neo-r9 (3.08)
a- Isotype control (4.0)
b- mAb only (55.56)
c- mAb + 2 µ
M
Neo-r6 (14.8)
d- mAb + 10 µ
M
Neo-r6 (4.39)
b- mAb only (55.56)
a- Isotype control (4.0)
c- mAb + 2 µ
M
r9 (13.65)
d- mAb + 10 µ
M
r9 (4.44)
a- Isotype control (4.0)

b- mAb only (55.56)
c- mAb + 2 µ
M
r6 (54.72)
d- mAb + 10 µ
M
r6 (19)
a
b
c
d
a
b
c
d
a
d
c
b
a
a
d
d
c
c
b
b
a
d
c

b
Fig. 5. Competition of APACs (r6, Neam-r6, Neo-r6, r9, Neam-r9
and Neo-r9) and 12G5 mAb binding to CXCR4 on MT2 cells. Cells
were incubated with monoclonal PE-anti-CXCR4 conjugated mAb
(12G5) alone or in the presence of APACs for 30 min at 4 °C. The
cells were then washed twice with NaCl ⁄ P
i
and analyzed by flow
cytometry. The MFI are shown in parenthesis. PE-conjugated iso-
type matched antibodies served as negative control. Data are repre-
sentative of at least two experiments.
R. Hegde et al. HIV inhibition by
L ⁄ D-poly arginine-aminoglycosides
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6529
of up to 20 lm, did not exhibit any competition with
mAb 12G5 binding to CXCR4 [20]. Of note, under the
conditions used for APACs, inhibition of monoclonal
anti-CXCR4 mAb binding (30 min at 4 °C), no degra-
dation of l-arginine conjugates is likely to occur.
APACs affect cell migration induced by SDF-1a
Next, we investigated whether APACs cause cell
migration, similar to the natural interaction between
SDF-1a and CXCR4, or affect the cell migration
induced by SDF-1a. We used G2 cells (human T-tro-
pic cell) in the present study because we could not
attain SDF-1a induced migration of the MT-2 cells,
which was the cell line used in the antiviral studies.
The effect of all our new APACs, at increasing concen-
trations (0–10 lm), on cell migration in the absence or
presence of 6.3 nm SDF-1a is shown in Fig. 6. The

total number of cells that migrated in the presence of
6.3 nm SDF-1a served as the reference 100% cell
migration. No cell migration resulted in the presence
of APACs only, at all examined concentrations. By
contrast, a dose-dependent inhibition of SDF-1a
induced migration was noticed by APACs. 0.5 and
1 lm Neo-r9 reduced SDF-1a induced cell migration
by 25% and 100%, respectively. In comparison to
Neo-r9, Neo-r6 showed reduced inhibition of SDF-1a
induced cell migration (Fig. 6A). Similarly, Neam-r9,
in which the aminoglycoside residue was replaced from
neomycin to neamine, resulted in an approximately
two-fold lower inhibition of the cell migration induced
by SDF-1a. Thus, not only the length of the d-Arg
peptide, but also the aminoglycoside residue core may
play a role in competing with SDF-1a binding to
CXCR4. The l-Arg-aminoglycosides revealed lower
migration inhibition activities compared to the d-ana-
logues (Fig. 6B). By contrast to d-Arg-9-mer (r9), R9
(0.5 lm) did not inhibit cell migration induced by
SDF-1a. Neamine l-Arg conjugates also exhibited
lower inhibition compared to their d-analogues.
APACs do not cause hemolysis
To investigate the possibility of intravenal administra-
tion of APACs, the hemolytic activity of the APACs
was studied as described in Experimental procedures.
No hemolysis was noted up to concentrations of
100 lm for several l- and d-derivative APACs (data
not shown).
Discussion

Conjugates of aminoglycoside antibiotics with arginine
(AACs) target two critical steps of the HIV-1 life cycle:
HIV-1 cell entry and viral genes transactivation [1,17]
HIV-1 cell entry is inhibited by their interaction with
CXCR4 on the cell surface and HIV-1 viral genes
transactivation is inhibited by AACs interaction with
HIV-1 TAR RNA in the cell nucleus [1,18]. We
hypothesized that conjugating poly arginine (6- and
A B
Fig. 6. G2 cell migration induced by SDF-1a in the presence and absence of APACs. (A) The effect of D-APACs at different concentrations
on SDF-1a (6.3 n
M) induced cell migration. Cell migration induced by SDF-1a data are considered as 100%. (B) The effect of L-APACs on cell
migration induced by 6.3 n
M SDF-1a. Data are representative of three independent experiments.
HIV inhibition by
L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6530 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS
9-mers) to an aminoglycoside core could result in
potent multitarget HIV-1 inhibitors. The sphere-like
NeoR-CXCR4 binding conformer reveals a completely
different structure compared to the extended structure
of Neo-r9 and Neo-r6 in complex with CXCR4 [24]
(Fig. 1B). Indeed, in the present study, we found that
d-APACs, but not l-APACs, inhibit a wide range of
T-tropic HIV-1 isolates, interact with CXCR4 and
readily cross the cell membrane. Moreover, we demon-
strate that d-APACs inhibit SDF-1a-induced cell
migration. It is well known that the SDF-1a competes
with monoclobal anti-CXCR4 serum 12G5, and inhib-
its HIV-1 infection mediated by the CXCR4 corecep-

tor [25–27]. All the above suggest that our compounds
directly compete with HIV-1 on CXCR4 binding.
The d-APACs inhibit a wide range of T-tropic HIV-
1 viral isolates. The d-peptide conjugates interact with
CXCR4 with at least 30-fold higher affinity than their
respective l-peptide conjugates. This was clearly dem-
onstrated in competition experiments using monoclo-
nal anti-CXCR4 mAb 12G5. Interestingly, similar
positive charged arginine side chains, either of d- and
l-peptides conjugated to aminoglycosides with extra
+3 or +5 charged groups of neamine and neomycin,
respectively, revealed significant different binding abili-
ties to CXCR4 in the present study. The enhanced
interaction with the CXCR4 receptor of the d-peptide
conjugates over the l-peptide conjugates is in accor-
dance with their increased antiviral potency, indicating
that the conformational nature of the molecule, rather
than its overall charge, is critical for antiviral efficacy.
Zhou et al. [28] who synthesized d- and l-amino
acid peptides derived from natural chemokines and
tested the stereo specificity of the CXCR4–ligand inter-
face, found that the d-amino acid peptides compete
with
125
I-SDF-1a and monoclonal antibody 12G5
binding to CXCR4 with a potency and selectivity com-
parable with or higher than that of their l-peptide
counterparts. Acting as CXCR4 antagonists, the
d-peptides also showed significant activity in inhibiting
the replication of CXCR4-dependent HIV-1 strains.

Their result indicated that the peptide of opposite
chirality recognize similar or at least overlapping site(s)
of the CXCR4 receptor. The different stereochemical
requirements for CXCR4 binding and signaling func-
tions have been recently established [29].
The length of the poly arginine (6-mer versus 9-mer)
as well as the aminoglycoside core of the APACs,
exhibits differential effects on the capacity of the
APACs with respect to inhibiting SDF-1a induced cell
migration, supporting the notion that, in addition to
the d-orl-configuration, the core and the length of
the arginine chain affect the stereo-specificity of the
interaction of the APACs with CXCR4. This is further
manifested by the 50% therapeutic index (TI
50
), which
is the 50% cytotoxic concentration (CC
50
) ⁄ EC
50
ratio,
of the compounds. For example, the TI
50
of Neo-r9
against HIV-1
IIIB
is 80 in MT2 cells compared to 94
for NeoR in MT2 against HIV-1
IIIB
[18], whereas the

relevant TI
50
for Neo-r6 is only 50.
Another possible explanation to the higher antiviral
potency of the d- over the l-APACs may be due to
their cellular localization. The cell uptake of the
d- and l-APACs is comparable and cannot account
solely for the differences in antiviral potencies. How-
ever, as demonstrated by confocal microscopy (Fig. 4),
the d-APACs concentrate in the nucleus, whereas the
l-APACs do not, or at least nuclear localization of the
l-APACs takes significantly longer. The fast nuclear
localization of Neo-r9 may inhibit or compete with
HIV-1 Tat–TAR interaction similar to NeoR and
other aminoglycoside conjugates [17,18]. This possible
additional antiviral mechanism of APACs has to be
further elucidated. The possibility that NeoR and
other members of this group of compounds are multi-
site HIV-1 inhibitors has recently been reviewed [1].
It may, however, be that the prolonged retention of
the l-peptide aminoglycoside conjugates in the cell
cytosol results in their increased proteolytic degrada-
tion by proteolytic enzymes found in the cell cyto-
plasm. This is in accordance with recent findings that
d-configuration arginine-rich cell penetrating peptides
were completely stable, whereas their l-analogues were
degraded in HeLa cells [15,16]. Accordingly, the lower
EC
50
of the d ⁄ l-9-mer-arginine neamine conjugate

(Neam-R ⁄ r9, Neam-RRrRrRrRR; Table 2) compared
to Neam-R9, but significantly higher than Neam-r9,
may be due to a somewhat decreased proteolysis of
this compound as a result of its more similar configu-
ration to the l- than the d-arginine peptide configura-
tion. As previously reported, when there are two
adjacent arginine of l-configuration in a peptide,
proteolysis may occur more readily than when these
l-arginines are separated by d-arginine [16].
No degradation is likely to occur of the l-peptide dur-
ing 30 min of its incubation with cells at 4 °C, under the
conditions used in the competition reaction with mAb
12G5 binding to CXCR4, in which their efficacy was sig-
nificantly lower compared to that of the d-peptide ami-
noglycoside conjugates. Taken together, these results
reduce the likelihood that degradation of the l-peptides
aminoglycoside conjugates occurred extracellularly. But
in accordance with a recent report [16], only d-arginine
conjugates are resistant to intracellular degradation.
Thus, the l-arginine configuration and ⁄ or their conju-
gates are not suitable candidates as anti-HIV drugs.
R. Hegde et al. HIV inhibition by L ⁄ D-poly arginine-aminoglycosides
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6531
Interestingly, d-peptide conjugates are as effective
against NeoR resistant (NeoR
res
) HIV-1 isolate [20,30]
as against the wild-type virus HIV-1
IIIB
(Table 3), indi-

cating that obvious differences in the APACs mode of
HIV-1 viral infectivity inhibition exist from that of
NeoR. Analysis of mutations that arise in NeoR
res
viral isolates revealed the appearance of mutations in
the constant regions C3 and C4, and in the variable
region V4 of gp120, and in gp41, in the HR2 domain
[20,30], thus decreasing the capacity of NeoR to inhibit
the viral interaction with CXCR4. We intended to
develop resistance viral isolates in vitro against selected
APACs, as we did previously for NeoR [20,30], to fur-
ther elucidate their mode of antiviral action. However,
although the cells could be grown for several days in
the presence of > 100 lm APACs without any signs of
cytotoxicity in the absence of HIV-1, during the devel-
opment of resistance in the presence of HIV-1, even at
relatively low concentrations of APACs (approximately
25 lm), cytotoxicity occurred preventing the selection
of resistant viral isolates (data not shown). The reasons
for this phenomenon are still not clear to us and are
currently under investigation.
Altogether, the present study establishes that
d-APACs may serve as lead compounds to generate
potent multitarget X4 HIV-1 inhibitors. Although,
d-APACs did not inhibit R5 HIV-1 Ba-L, other R5
HIV-1 strains were not tested, but will be tested in
future studies.
CXCR4 plays an important role in cancer metastases
and other diseases [31,32]. Importantly, CXCR4 antago-
nists, such as AMD3100, T140 and ALX40-4C [11],

which also affect the normal natural cascade of effects
caused by the SDF-1a–CXCR4 interaction, are now
being actively pursued as stem cell mobilizers for trans-
plantation in patients with multiple myeloma and non-
Hodgkin’s lymphoma and as potential anti-metastatic
and anti-rheumatoid arthritis agents [33–36]. Because
APACs interact with CXCR4, such as AMD3100 and
T140, we are now also exploring their capacity to serve
as anti-metastatic agents. Aminoglycosides are known
as antibiotics; thus, exploring the efficacy of APACs
against microbial pathogens has been initiated [US
patent 10 ⁄ 831 224 (US 2006 ⁄ 0166867 A1)].
Experimental procedures
Materials and analytical procedures
Neomycin B and paromomycin were purchased from Sigma
(Rehovot, Israel) as sulfate salts and were used as free base
aminoglycosides. Neamine was synthesized by acidic meth-
anolysis of neomycin sulfate as described previously [1].
The obtained neamine hydrochloride was converted to a
free base using Amberlite IRA 400 (OH
–
) ion-exchange
resin. NBND was prepared as previously described using
N-hydroxy-5-norbornene-endo-2,3-dicarboximide (Aldrich,
Steinheim, Germany) and di-tert-butyl dicarbonate (Fluka,
Steinheim, Germany) in the presence of thallous ethoxide
(Aldrich) [1].Benzylchloroformate(CbzCl), HOBT,N-methyl-
morpholine, Cbz-Arg(NO
2
)-OH and palladium on charcoal

(10%) (Fluka), EDC (Aldrich), Fmoc-Arg(Pbf)-OH (d- and
l-enantiomers) and benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate (Orpegen Pharma,
Heidelberg, Germany) were reagent grade and were used
without further purification.
Column chromatography employed Merck silica gel
(Kieselgel 60; 0.063–0.200 mm). Analytical TLC was per-
formed with 0.2 mm silica-coated aluminum sheets, visuali-
zation by UV light or by spraying an aqueous solution of
ninhydrin (0.25%) and then heating the aluminum sheet.
Analytical RP-HPLC: E040720-5-1 Vydac (Deerfield, IL,
USA) C18 column (0.46 cm · 25.0 cm), flow rate of 1 mLÆ
min
)1
at 220, 230 and 280 nm, 5–65% linear acetonitrile
gradient in water with 0.1% trifluoroacetic acid over
30 min. Preparative RP-HPLC: E040519-4-4 Vydac C18
column (2.2 cm · 25.0 cm), flow rate of 8 mLÆmin
)1
at 220,
230 and 280 nm, 5–65% linear acetonitrile gradient in
water with 0.1% trifluoroacetic acid over 30 min. The
major HPLC peak was collected and further identified by
MALDI-TOF.
Peptide synthesis
Arginine peptides l-, d- and l ⁄ d (6- and 9-mers), and their
N-terminal acetylated derivatives were synthesized manually
by standard solid phase peptide synthesis technique (see
Supplementary material).
Synthesis of L-, D- and L ⁄ D-poly arginine

(6- and 9-mers) conjugates of neamine,
paromomycin and neomycin ) general
procedure for the synthesis of compounds
1a, 2a and 3a (Fig. 2)
Regioselective introduction of the tert-butoxycarbonyl
protective group at the unhindered amino group [attached
to primary carbon(s)] of neamine, paromomycin and
neomycin was performed as previously described [37–39].
Protection of the remaining amino groups was achieved
by a conventional method using benzylchloroformate and
sodium carbonate in acetone ⁄ water [40]. Deprotection of
the ‘tert-butoxycarbonyl’ group using trifluoroacetic acid
afforded the compounds 1a, 2a and 3a (Fig. 2).
Briefly, each one of the free base aminoglycosides
(neamine, paromomycin and neomycin) was dissolved in a
mixture of dioxane ⁄ water (1 : 1, v ⁄ v) and triethylamine
HIV inhibition by L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6532 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS
(1.5 equivalents) was added to the solution and stirred for
10 min. NBND (1 equivalent) was added (in one portion)
and the reaction mixture was stirred at room temperature
for approximately 15 h. TLC analysis of the reaction mix-
ture (ethanol ⁄ acetone ⁄ NH
4
OH, 1 : 1 : 1) demonstrated the
presence of one major product, which was revealed to be
the desired monocarbamoylated product, with traces of the
starting material and polycarbamoylated products. The sol-
vents were removed under reduced pressure; and the residue
was dissolved in water and washed with ethyl acetate

(3 · 25 mL). The aqueous layer was evaporated under
reduced pressure and the residue was dissolved in ace-
tone ⁄ water (7 : 3, v ⁄ v); sodium carbonate (1.5 equivalents,
for each free amino group) and the mixture cooled to 0 °C.
Benzylchloroformate (1.5 equivalents, for each free amino
group) in acetone was added drop wise to the mixture and
stirred at 0 °C for 2 h and then left at room temperature
for 15 h. TLC analysis (CH
2
Cl
2
⁄ MeOH, 8.5 : 1.5) of the
reaction mixture demonstrated complete conversion of the
starting material to the desired compound. Solvents were
removed under reduced pressure, and extracted three times
with warm ethyl acetate. The ethyl acetate layer was
washed twice with water, dried over sodium sulfate, concen-
trated and the residue was treated with ether to obtain a
white solid, which was again made slurry in ether and dec-
anted. The solid material was dissolved in dichloromethane
and treated with trifluoroacetic acid (10% v ⁄ v) at room
temperature for 3 h. Next, it was concentrated and the resi-
due was resuspended in ether; the ether was decanted and a
white material was obtained. Column chromatography (sil-
ica gel, CH
2
Cl
2
⁄ MeOH ⁄ NH
4

OH, 8.4 : 1.4 : 0.2) afforded
the pure compounds 1a, 2a and 3a.
MS: m ⁄ z MALDI-TOF; Compound 1a: 747.176
(M+Na), calcd., 747.586; Compound 2a: 1152.61(M
+
.),
calcd., 1152.163; Compound 3a: 1307.391(M+Na), calcd.,
1307.302. Also confirmed by
1
H NMR (500 MHz, D
2
O).
A mixture of DIEA (1.2 equivalents), arginine peptide
(trifluoroacetic acid salt, 1.1 equivalents), HOBT
(1.1 equivalents) and EDC (1.5 equivalents) was prepared
in dimethyformamide and left at room temperature for
10 min; then it was added to cooled dimethyformamide
solution of the protected aminoglycoside (1a, 2a or 3a).
The resulting mixture was stirred for approximately 15 h,
and was concentrated under reduced pressure. Detailed
procedures for hydrogenation and purification are pro-
vided in the Supplementary material. Mass spectra of all
APACs confirmed their purity and the the relative molecu-
lar masses of their free base (for the d-APACs, see
Table 1).
Fluorescent probes – APACs-FITC
The fluorescent probes had been prepared as published pre-
viously [18,41] with some modifications. The acetate coun-
ter ions of APACs and oligopeptides were removed by
Amberlite IRA 400 (OH

–
) ion-exchange resin prior to their
reaction with FITC (Sigma) in water ⁄ methanol ⁄ dioxane
(1:1:1,v⁄ v ⁄ v) medium in the presence of two equivalents
of triethyl amine for 2 h at room temperature. After
removal of the solvents under reduced pressure, the FITC
derivatives were purified by extraction with absolute etha-
nol. Finally, FITC conjugates were converted into acetate
salt and characterized by mass spectrometry.
Cell culture and inhibition of HIV-1 replication
MT2 (lymphocyte cell line permissive to T-tropic HIV-1
isolates) were cultured in RPMI 1640, containing 10% fetal
bovine serum and antibiotics. cMAGI HIV-1 reporter cells
were cultured in DMEM, containing 10% fetal bovine
serum and antibiotics (100 UÆmL
)1
penicillin; 100 lgÆmL
)1
streptomycin; 0.25 lgÆmL
)1
fungizone; 200 lgÆmL
)1
G418;
1 lgÆmL
)1
puromycin). The cMAGI assay is based on the
ability of HIV-1 Tat to transactivate the expression of an
integrated b-galactosidase reporter gene driven by the HIV-
long-terminal repeats [42,43]. The b-galactosidase reporter
has been modified to remain localized in the nucleus where

it can be detected with the X-gal (5-bromo-4-chloro-3-in-
doyl-b-d-galactopyranoside) substrate as an intense blue
nuclear stain within a few (3–4) days of infection. HIV-1
isolates were propagated by subculture in MT2 as described
previously [18]. Aliquots of cell-free culture supernatants
were used as viral inoculum. APACs and poly arginine
(6- and 9-mers) were dissolved in RPMI 1640 medium.
Viral inhibition was determined by incubating MT2 or
cMAGI HIV-1 reporter cells with 0.2–0.5 multiplicity of
infection of HIV-1 wild-type or resistant virus for 4 days at
37 °C in the presence or absence of various concentrations
of APACs and poly arginine peptides. HIV-1 infection of
cMAGI cells was determined by counting the number of
HIV-1-infected cells (stained blue). The percent of inhibi-
tion was determined according to the formula:
100 ) (A ⁄ B) · 100, where A is number of HIV-1 infected
cells in which the tested compounds were present, and B is
the number of HIV-1 infected cells in the presence of virus
alone. The cytopathic effects of the viral infection of MT2
cells were also analyzed by microscopic assessment of syn-
cytium formation.
Cytotoxicity measurements
Cytotoxicity of the compounds was determined by trypan
blue exclusion assay in MT2 cells. Briefly, MT2 cells were
grown in RPMI medium supplemented with 10% fetal
bovine serum in the presence or absence of various concen-
trations of APACs. After 3 days of incubation at 37 °C,
live cells were counted in each well by trypan blue exclusion
assay and the CC
50

determined by plotting the number of
live cells versus compound concentration.
R. Hegde et al. HIV inhibition by L ⁄ D-poly arginine-aminoglycosides
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6533
Cellular uptake using APACs and poly arginine
peptide-fluorescent derivatives: assay by confocal
microscopy
cMAGI cells were incubated in a eight-well plate (2000 cell-
sÆwell
)1
) for 30 min at 37 °C with each one of the APACs-
FITC and peptide-FITC derivatives at a final concentration
of 5 and 15 lm. After incubation, cells were washed with
phosphate buffered saline three times prior to confocal
microscopy measurements (Olympus IX70 FV500 or Axi-
overt 100M confocal laser scanning microscope; Oylmpus,
Tokyo, Japan).
Interaction of APACs with CXCR4 receptor
Interactions of APACs and arginine-peptides with CXCR4
were determined by flow cytometry (FACScan; Becton Dick-
inson, San Jose, CA, USA) as previously described [18].
Briefly, 0.5 · 10
6
MT2 cells were washed with ice-cold
NaCl ⁄ P
i
containing 0.1% sodium azide (wash buffer) and
incubated at 4 °C with monoclonal anti-CXCR4 mAb,
12G5, conjugated to PE, in the absence or presence of differ-
ent concentrations of APACs and arginine peptides. After

30 min of incubation, the cells were washed with ice-cold
wash buffer and fixed in NaCl ⁄ P
i
containing 1% paraformal-
dehyde. Non-specific fluorescence was assessed by using an
isotype control. For each sample 3000–10 000 events were
acquired. Data were analyzed and processed using Cell-
Quest
TM
software (Becton Dickinson).
Chemotaxis migration assay
G2 cells, from a human precursor-B acute lymphoblastic
leukemia cell line (kindly provided by T. Lapidot, the Weiz-
mann Institute), were used for the chemotaxis migration
assay. Cell migration was assessed in 24-well chemotaxis
chambers (6.5 mm diameter, 5 lm pore size, tissue culture
treated polycarbonate membrane polystyrene plates; Costar,
Corning Incorporated, Corning, NY, USA). Six hundred ll
RPMI media with or without 50 ngÆmL
)1
(6.3 nm) of SDF-
1a and with or without different concentrations of APACs
were added to the lower wells. After 3 h of incubation at
37 °C, the upper chambers were removed and the numbers
of migrated cells in the lower chambers were counted by
flow cytometry.
Hemolytic activity
Human erythrocytes (hRBC) were isolated from fresh
blood units by centrifugating them three times for 10 min
at 400 g and resuspending them in NaCl ⁄ P

i
. The tested
compounds (APACs) in NaCl ⁄ P
i
solution were serially
diluted in a 96-well round bottom plate and then 50 lLof
hRBC suspension were added to reach a final volume of
100 lL (final erythrocyte concentration 4% v⁄ v and final
compounds concentrations 1.56–100 lm). The resulting sus-
pensions were agitated for 60 min at 37 °C. The samples
were then centrifuged at 400 g for 10 min. Supernatant
from all the wells was then transferred to another 96-well
flat bottom plate and the release of hemoglobin was moni-
tored by measuring the absorbance at 540 nm. Controls for
zero hemolysis and 100% hemolysis consisted of hRBC sus-
pended in NaCl ⁄ P
i
and Triton 1%, respectively.
References
1 Borkow G & Lapidot A (2005) Multi-targeting the
entrance door to block HIV-1. Curr Drug Targets Infect
Disord 5, 3–15.
2 Cooley LA & Lewin SR (2003) HIV-1 cell entry and
advances in viral entry inhibitor therapy. J Clin Virol
26, 121–132.
3 Gallo SA, Finnegan CM, Viard M, Raviv Y, Dimitrov
A, Rawat SS, Puri A, Durell S & Blumenthal R (2003)
The HIV Env-mediated fusion reaction. Biochim Bio-
phys Acta 1614, 36–50.
4 Pierson TC & Doms RW (2003) HIV-1 entry and

its inhibition. Curr Top Microbiol Immunol 281,
1–27.
5 Pierson TC, Doms RW & Pohlmann S (2004) Prospects
of HIV-1 entry inhibitors as novel therapeutics. Rev
Med Virol 14, 255–270.
6 Zaitseva M, Peden K & Golding H (2003) HIV
coreceptors: role of structure, posttranslational
modifications, and internalization in viral-cell fusion
and as targets for entry inhibitors. Biochim Biophys Acta
1614, 51–61.
7 Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawaba-
ta K, Kataoka Y, Kitamura Y, Matsushima K, Yoshida
N, Nishikawa S et al. (1998) The chemokine receptor
CXCR4 is essential for vascularization of the gastro-
intestinal tract. Nature 393, 591–594.
8 Zou YR, Kottmann AH, Kuroda M, Taniuchi I & Litt-
man DR (1998) Function of the chemokine receptor
CXCR4 in haematopoiesis and in cerebellar develop-
ment. Nature 393, 595–599.
9 Bleul CC, Farzan M, Choe H, Parolin C, Clark-
Lewis I, Sodroski J & Springer TA (1996) The
lymphocyte chemoattractant SDF-1 is a ligand for
LESTR ⁄ fusin and blocks HIV-1 entry. Nature 382,
829–833.
10 Doranz BJ, Grovit-Ferbas K, Sharron MP, Mao SH,
Goetz MB, Daar ES, Doms RW & O’Brien WA (1997)
A small-molecule inhibitor directed against the
chemokine receptor CXCR4 prevents its use as an
HIV-1 coreceptor. J Exp Med 186, 1395–1400.
11 Doranz BJ, Filion LG, Diaz-Mitoma F, Sitar DS, Sahai

J, Baribaud F, Orsini MJ, Benovic JL, Cameron W &
Doms RW (2001) Safe use of the CXCR4 inhibitor
HIV inhibition by L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6534 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS
ALX40-4C in humans. AIDS Res Hum Retroviruses 17 ,
475–486.
12 Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S,
Ueda K & Sugiura Y (2001) Arginine-rich peptides. An
abundant source of membrane-permeable peptides
having potential as carriers for intracellular protein
delivery. J Biol Chem 276, 5836–5840.
13 Mitchell DJ, Kim DT, Steinman L, Fathman CG &
Rothbard JB (2000) Polyarginine enters cells more
efficiently than other polycationic homopolymers.
J Pept Res 56, 318–325.
14 Kacprzak MM, Peinado JR, Than ME, Appel J,
Henrich S, Lipkind G, Houghten RA, Bode W &
Lindberg I (2004) Inhibition of furin by polyarginine-
containing peptides: nanomolar inhibition by
nona-d-arginine. J Biol Chem 279, 36788–36794.
15 Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE,
Nelson MH, Iversen PL & Moulton HM (2007) Cell-
penetrating peptides as transporters for morpholino
oligomers: effects of amino acid composition on intra-
cellular delivery and cytotoxicity. Nucleic Acids Res 35,
5182–5191.
16 Youngblood DS, Hatlevig SA, Hassinger JN, Iversen
PL & Moulton HM (2007) Stability of cell-penetrating
peptide-morpholino oligomer conjugates in human
serum and in cells. Bioconjug Chem 18, 50–60.

17 Lapidot A, Vijayabaskar V, Litovchick A, YuJ & James
TL (2004) Structure–activity relationships of aminogly-
coside-arginine conjugates that bind HIV-1 RNAs as
determined by fluorescence and NMR spectroscopy.
FEBS Lett 577, 415–421.
18 Litovchick A, Lapidot A, Eisenstein M, Kalinkovich A
& Borkow G (2001) Neomycin B-arginine conjugate, a
novel HIV-1 Tat antagonist: synthesis and anti-HIV
activities. Biochemistry 40, 15612–15623.
19 Schepdael AV, Busson R & Claes P (1992) The selective
protection of amino functions in aminoglycosides: a
review of possible approaches. Bull Soc Chim Belg 101,
709–717.
20 Borkow G, Vijayabaskar V, Lara HH, Kalinkovich A
& Lapidot A (2003) Structure–activity relationship of
neomycin, paromomycin, and neamine–arginine
conjugates, targeting HIV-1 gp120-CXCR4 binding
step. Antiviral Res 60, 181–192.
21 Arbuzova A, Martushova K, Hangyas-Mihalyne G,
Morris AJ, Ozaki S, Prestwich GD & McLaughlin S
(2000) Fluorescently labeled neomycin as a probe of
phosphatidylinositol-4,5-bisphosphate in membranes.
Biochim Biophys Acta 1464, 35–48.
22 Cabrera C, Gutierrez A, Blanco J, Barretina J, Litov-
chick A, Lapidot A, Evdokimov AG, Clotet B & Este
JA (2000) Anti-human immunodeficiency virus activity
of novel aminoglycoside–arginine conjugates at early
stages of infection. AIDS Res Hum Retroviruses 16,
627–634.
23 Litovchick A, Evdokimov AG & Lapidot A (1999)

Arginine–aminoglycoside conjugates that bind to HIV
transactivation responsive element RNA in vitro. FEBS
Lett 445, 73–79.
24 Berchanski A & Lapidot A (2007) Prediction of HIV-1
entry inhibitors neomycin–arginine conjugates inter-
action with the CD4-gp120 binding site by molecular
modeling and multistep docking procedure. Biochim
Biophys Acta 1768, 2107–2119.
25 Amara A, Gall SL, Schwartz O, Salamero J, Montes
M, Loetscher P, Baggiolini M, Virelizier JL & Arenz-
ana-Seisdedos F (1997) HIV coreceptor downregulation
as antiviral principle: SDF-1alpha-dependent internali-
zation of the chemokine receptor CXCR4 contributes to
inhibition of HIV replication. J Exp Med 186, 139–146.
26 Brelot A, Heveker N, Montes M & Alizon M (2000)
Identification of residues of CXCR4 critical for human
immunodeficiency virus coreceptor and chemokine
receptor activities. J Biol Chem 275, 23736–23744.
27 Mondor I, Moulard M, Ugolini S, Klasse PJ, Hoxie J,
Amara A, Delaunay T, Wyatt R, Sodroski J & Satten-
tau QJ (1998) Interactions among HIV gp120, CD4,
and CXCR4: dependence on CD4 expression level,
gp120 viral origin, conservation of the gp120. Virology
248, 394–405.
28 Zhou N, Luo Z, Luo J, Fan X, Cayabyab M, Hiraoka
M, Liu D, Han X, Pesavento J, Dong CZ et al. (2002)
Exploring the stereochemistry of CXCR4-peptide recog-
nition and inhibiting HIV-1 entry with d-peptides
derived from chemokines. J Biol Chem 277, 17476–
17485.

29 Dong CZ, Kumar S, Choi WT, Madani N, Tian S, An
J, Sodroski JG & Huang Z (2005) Different stereochem-
ical requirements for CXCR4 binding and signaling
functions as revealed by an anti-HIV, d-amino acid-con-
taining SMM-chemokine ligand. J Med Chem 48, 7923–
7924.
30 Borkow G, Lara HH & Lapidot A (2003) Mutations in
gp41 and gp120 of HIV-1 isolates resistant to hexa-
arginine neomycin B conjugate. Biochem Biophys Res
Commun 312, 1047–1052.
31 Scala S, Giuliano P, Ascierto PA, Ierano C, Franco R,
Napolitano M, Ottaiano A, Lombardi ML, Luongo M,
Simeone E et al. (2006) Human melanoma metastases
express functional CXCR4. Clin Cancer Res 12, 2427–
2433.
32 Tamamura H, Tsutsumi H & Fujii N (2006) The
chemokine receptor CXCR4 as a therapeutic target for
several diseases. Mini Rev Med Chem 6, 989–995.
33 Tamamura H, Tsutsumi H, Masuno H & Fujii N (2007)
Development of low molecular weight CXCR4 antago-
nists by exploratory structural tuning of cyclic tetra-
and pentapeptide-scaffolds towards the treatment of
HIV infection, cancer metastasis and rheumatoid arthri-
tis. Curr Med Chem 14, 93–102.
R. Hegde et al. HIV inhibition by L ⁄ D-poly arginine-aminoglycosides
FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS 6535
34 Flomenberg N, Devine SM, Dipersio JF, Liesveld JL,
McCarty JM, Rowley SD, Vesole DH, Badel K &
Calandra G (2005) The use of AMD3100 plus G-CSF
for autologous hematopoietic progenitor cell mobiliza-

tion is superior to G-CSF alone. Blood 106, 1867–
1874.
35 Devine SM, Flomenberg N, Vesole DH, Liesveld J,
Weisdorf D, Badel K, Calandra G & Dipersio JF (2004)
Rapid mobilization of CD34+ cells following adminis-
tration of the CXCR4 antagonist AMD3100 to patients
with multiple myeloma and non-Hodgkin’s lymphoma.
J Clin Oncol 22, 1095–1102.
36 Smith MC, Luker KE, Garbow JR, Prior JL, Jackson
E, Piwnica-Worms D & Luker GD (2004) CXCR4
regulates growth of both primary and metastatic breast
cancer. Cancer Res 64, 8604–8612.
37 Grapsas I, Cho YJ & Mobashery S (1994) N-(tert-but-
oxycarbonyloxy)-5-norbornene-endr2,3-dicarboximide,
a reagent for the regio selective introduction of the tert-
butoxycarbonyl (BOC) protective group at unhindered
amines: application to aminoglycoside chemistry. J Org
Chem 59, 1918–1922.
38 Grapsas I, Lerner SA & Mobashery S (2001) Conjoint
molecules of cephalosporins and aminoglycosides. Arch
Pharm (Weinheim) 334, 295–301.
39 Sainlos M, Belmont P, Vigneron J-P, Lehn P & Lehn
J-M (2003) Aminoglycoside-derived cationic lipids for
gene transfection: synthesis of kanamycin A derivatives.
Eur J Org Chem 15, 2764–2774.
40 Kumar V & Remers WA (1978) Aminoglycoside
antibiotics. 1. Regiospecific partial syntheses of
ribostamycin and butirosin B. J Org Chem 43, 3327–
3331.
41 Borkow G, Lara HH, Ayash-Rashkovsky M, Tavor E,

Lapidot A, Bentwich Z & Honigman A (2004) Adenovi-
rus expressing a bioluminescence reporter gene and
cMAGI cell assay for the detection of HIV-1. Virus
Genes 29, 257–265.
42 Chackerian B, Long EM, Luciw PA & Overbaugh J
(1997) Human immunodeficiency virus type 1 corecep-
tors participate in postentry stages in the virus replica-
tion cycle and function in simian immunodeficiency
virus infection. J Virol 71, 3932–3939.
43 Zussman A, Lara L, Lara HH, Bentwich Z & Borkow
G (2003) Blocking of cell-free and cell-associated HIV-1
transmission through human cervix organ culture with
UC781. AIDS 17, 653–661.
Supplementary material
The following supplementary material is available
online:
Doc. S1. General procedure for arginine peptide-amino-
glycoside conjugates and chemotaxis migration assay.
Fig. S1. CD spectra. (A) Neo-r9 (j), Neo-R9 (m),
Neo-r6 (h) and Neo-R6 (n). (B) Neam-r9 (j), Neam-
R9 (m), Neam-r6 (h) and Neam-R6 (n).
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
HIV inhibition by L ⁄ D-poly arginine-aminoglycosides R. Hegde et al.
6536 FEBS Journal 274 (2007) 6523–6536 ª 2007 The Authors Journal compilation ª 2007 FEBS