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

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-

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: aviva.lapidot@weizmann.ac.il

(Received 29 August 2007, revised 18

Octo-ber 2007, accepted 29 OctoOcto-ber 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.

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-norbornene-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

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 NO2) 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 neomyparomomy-cin), 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.

free aminomethylene (-CH2NH2) 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 containingD-arginine peptides

(6- and 9-mers) display significantly higher

anti-HIV-1 activity then theirL-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-1IIIB activity, with the concentration that caused 50% inhibition of viral production (EC50) being 50 lm R6 presented significantly lower antivi-ral activity (EC50 of 110 lm) in comparison to its aminoglycoside conjugates Neam-R6, Paramo-R6 and Neo-R6 (EC50 of 70, 31, and 40 lm, respectively)

By contrast, the antiviral activity of the free non-amer arginine R9 (EC50 of 33 lm) was similar to that of its aminoglycoside conjugates The EC50 of Fig 2 Schematic representation of the synthesis of aminoglycoside-arginine peptide conjugates.

d⁄ l-9-mer-arginine neamine conjugate (Neam-R ⁄ r9,

Neam-RRrRrRrRR; Table 1) showed a somewhat

lower value of EC50 (28 lm) compared to Neam-R9

(37.5 lm; Table 2), but significantly higher than

Neam-r9 (EC50 of 4.2 lm; Table 3)

d-APACs inhibited a variety of T-tropic HIV-1

iso-lates, both laboratory adapted and clinical isoiso-lates, as

well as resistant strains, including NeoR resistant

(NeoRres) virus, in the EC50range of 1.2–6.2 lm, with

the exception of AZT resistant virus, in which the

EC50 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-1IIIB wild-type virus and its NeoRres variant (Table 3) In general, the d-9-mer-peptide and its aminoglycoside conjugates revealed significantly lower EC50 For example, 1.5 ± 0.4 lm (against Proteaser virus) and 1.6 ± 0.7 lm (against HIV-1IIIB)

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)

Table 2 Antiviral activity of L -APACs against HIV-1 IIIB virus ND, not determined.

EC50(l M ) 110 ± 20 70 ± 10 31 ± 10 40 ± 12 33 ± 3 37.5 ± 2.5 31 ± 9 30 ± 7 28 ± 2 31 > 200

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

EC50(l M )

Cytotoxicity

CC 50 (l M )

a M-tropic HIV-1 viral isolate; b resistant isolate; c clinical isolate.

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 EC50for the NeoRresvirus 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 37C 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-1IIIB

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 panpan-els, but with confocal fluorescent microscopy.

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 paromoneo-mycin, 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.

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)

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.

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 4C), 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.

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 125I-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- or l-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 (TI50), which

is the 50% cytotoxic concentration (CC50)⁄ EC50ratio,

of the compounds For example, the TI50 of Neo-r9 against HIV-1IIIB is 80 in MT2 cells compared to 94 for NeoR in MT2 against HIV-1IIIB [18], whereas the relevant TI50for 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

EC50 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 4C, 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

Interestingly, d-peptide conjugates are as effective

against NeoR resistant (NeoRres) HIV-1 isolate [20,30]

as against the wild-type virus HIV-1IIIB(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 NeoRres

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(NO2)-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)1at 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- andL⁄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