• drug design;
  • HIV-1 entry inhibitors;
  • poly arginine-aminoglycoside conjugates;
  • structure–function relationship


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. References
  7. Supporting Information

We present the design, synthesis, anti-HIV-1 and mode of action of neomycin 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 1α (SDF-1α)/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 better 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.


aminoglycoside-arginine conjugates


N-α-acetyl-nona-d-arginine amide


aminoglycosides poly d- and l-arginine conjugates


CC chemokine receptor 5


CXC chemokine receptor type 4




N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride


fluorescein isothiocyanate


glycoprotein 120


human erythrocytes




median fluorescent intensity




hexa-arginine-neomycin conjugate




stromal cell-derived factor 1


Tat responsive element.

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 target 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. Blocking 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 inhibition of HIV-1 proliferation has been observed in in vitro studies with several entry inhibitor combinations, 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 target 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 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-1α 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-α-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 efficacious 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 translocational 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 conjugated 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 neomycin (or other members of this aminoglycoside group, e.g. neamine and paromomycin) with poly arginine (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-aminoglycoside conjugates (APACs) was designed and synthesized, and their cell uptake and antiviral activities were determined. We further investigated how APACs block HIV-1 gp120 interaction with CXCR4 and compete with its natural ligand SDF-1α to CXCR4.


Figure 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.

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  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. References
  7. Supporting Information

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 reactivity 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 synthesis was undertaken for conjugation of the peptides with the aminoglycosides (Fig. 2).


Figure 2.  Schematic representation of the synthesis of aminoglycoside-arginine peptide conjugates.

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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-butoxycarbonyloxy)-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 methylene carbon(s)] of neamine, paromomycin and neomycin 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 presence 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 confirmed by mass spectrometry. The coupling of arginine peptides (6- and 9-mers) with 1a, 2a and 3a was performed using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) as a coupling reagent in the presence of 1-hydroxybenzotriazole (HOBT) and diisopropylethylamine (DIEA). The tertiary amine DIEA, used in the reaction mixture, is not sufficiently basic to deprotonate the guanidinium headgroup. 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, paromomycin 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).

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 abbreviationMS (m/z)

As expected, d- and l-arginine (6- and 9-mers) peptide aminoglycoside conjugates displayed mirror-image CD spectra and random conformation (see supplementary 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 temperature with some modifications, as previously reported, for NeoR and other aminoglycoside arginine conjugates [18,20]. As previously reported for FITC-aminoglycosides [21] FITC is bound to the free aminomethylene (-CH2NH2) group of neomycin. After removal of the solvents under reduced pressure, the FITC derivatives were purified by extraction 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 neamine, 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 µm of the neomycin B was noticed (Table 2). However, a short chain of two l-arginines already conjugated to neamine (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 µm. R6 presented significantly lower antiviral activity (EC50 of 110 µm) in comparison to its aminoglycoside conjugates Neam-R6, Paramo-R6 and Neo-R6 (EC50 of 70, 31, and 40 µm, respectively). By contrast, the antiviral activity of the free nonamer arginine R9 (EC50 of 33 µm) was similar to that of its aminoglycoside conjugates. The EC50 of d/l-9-mer-arginine neamine conjugate (Neam-R/r9, Neam-RRrRrRrRR; Table 1) showed a somewhat lower value of EC50 (28 µm) compared to Neam-R9 (37.5 µm; Table 2), but significantly higher than Neam-r9 (EC50 of 4.2 µm; Table 3).

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


EC50m)110 ± 2070 ± 1031 ± 1040 ± 1233 ± 337.5 ± 2.531 ± 930 ± 728 ± 231> 200
CC50m)ND210160200120175140150160ND> 300
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 cytotoxicity 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.
CompoundEC50m) Cytotoxicity CC50m)
IIIBBa-LaAZTbProteasebNeoRbClade AcClade CcLAIELI
  • a

    M-tropic HIV-1 viral isolate;

  • b

    b resistant isolate;

  • c

    c clinical isolate.

r65.8 ± 1.7> 5015.2 ± 6.24.9 ± 1.06.8 ± 0.78.0 ± 1.05.3 ± 0.3NDND170
Neam-r65.9 ± 4.2> 507.0 ± 4.02.2 ± 0.46.2 ± 5.83.2 ± 1.31.7 ±
Neo-r63.1 ± 3.7> 5010.4 ± 4.14.0 ± 1.04.8 ± 2.25.7 ± 2.83.0 ±
r91.5 ± 0.6> 509.7 ± 5.91.4 ± 0.62.0 ± 1.22.2 ± 0.31.6 ± 0.4NDND150
Neam-r94.2 ± 4.8> 5010.4 ± 2.72.4 ± 0.46.0 ± 5.05.1 ± 2.92.7 ±
Neo-r91.6 ± 0.7> 506.6 ± 5.91.5 ± 0.42.8 ± 2.52.1 ± 0.41.2 ±

d-APACs inhibited a variety of T-tropic HIV-1 isolates, both laboratory adapted and clinical isolates, as well as resistant strains, including NeoR resistant (NeoRres) virus, in the EC50 range of 1.2–6.2 µm, with the exception of AZT resistant virus, in which the EC50 range was 6.6–10.4 µm (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 µm (against Proteaser virus) and 1.6 ± 0.7 µm (against HIV-1IIIB) and 1.6 ± 0.4 µm and 1.2 ± 0.5 µm against clade C virus (a clinical isolate) for r9 peptide and Neo-r9 conjugate, respectively (Table 3). A similar relative ratio of the EC50 for the NeoRres 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.


Figure 3.  Inhibitory effect of Neam-r9 and Neo-r9 on HIV-1IIIB replication. cMAGI cells were infected for 2 h at 37 °C in the absence or presence of 0.78–50 µm 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 compounds. Cell infectivity was then determined. bsl00066, APACs were present during the infection step and after the cells were washed; ▪, APACs were present only during the first 2 h, before the cells were washed.

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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 analogue 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.


Figure 4.  Confocal microscopy images of cMAGI cells stained with the APACs–FITC conjugates. The cells were incubated for 30 min with 5 and 15 µm 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 panels comprise the same fields as upper panels, but with confocal fluorescent microscopy.

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APACs inhibit monoclonal anti-CXCR4 mAb binding to cells

We have previously found that a variety of AACs (aminoglycosides neamine, paromomycin, neomycin and gentamicin conjugated via each one of the free amino groups of the aminoglycoside to arginines; e.g. six arginines 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 binding of the phycoerythrin (PE) labeled 12G5 mAb to CXCR4 in MT2 cells was examined. The main 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 concentrations for the l-APACs (20 and 80 µm) and two concentrations for the d-APACs (2 and 10 µm) 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 µm 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% inhibition in the presence of 2 µm of Neo-r9. Similar measurements 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 µm 9-mer-d-arginine-neomycin conjugate (Neo-r9) achieved 95.3% inhibition of mAb 12G5 binding 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 neomycin B, neamine and paromomycin, at concentrations of up to 20 µm, 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 degradation of l-arginine conjugates is likely to occur.


Figure 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/Pi and analyzed by flow cytometry. The MFI are shown in parenthesis. PE-conjugated isotype matched antibodies served as negative control. Data are representative of at least two experiments.

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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.
CompoundInhibition (%)
l-APACs20 µm90 µm
d-APACs2 µm10 µm

APACs affect cell migration induced by SDF-1α

Next, we investigated whether APACs cause cell migration, similar to the natural interaction between SDF-1α and CXCR4, or affect the cell migration induced by SDF-1α. We used G2 cells (human T-tropic cell) in the present study because we could not attain SDF-1α 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 concentrations (0–10 µm), on cell migration in the absence or presence of 6.3 nm SDF-1α is shown in Fig. 6. The total number of cells that migrated in the presence of 6.3 nm SDF-1α 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-1α induced migration was noticed by APACs. 0.5 and 1 µm Neo-r9 reduced SDF-1α induced cell migration by 25% and 100%, respectively. In comparison to Neo-r9, Neo-r6 showed reduced inhibition of SDF-1α 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-1α. Thus, not only the length of the d-Arg peptide, but also the aminoglycoside residue core may play a role in competing with SDF-1α binding to CXCR4. The l-Arg-aminoglycosides revealed lower migration inhibition activities compared to the d-analogues (Fig. 6B). By contrast to d-Arg-9-mer (r9), R9 (0.5 µm) did not inhibit cell migration induced by SDF-1α. Neamine l-Arg conjugates also exhibited lower inhibition compared to their d-analogues.


Figure 6.  G2 cell migration induced by SDF-1α in the presence and absence of APACs. (A) The effect of d-APACs at different concentrations on SDF-1α (6.3 nm) induced cell migration. Cell migration induced by SDF-1α data are considered as 100%. (B) The effect of l-APACs on cell migration induced by 6.3 nm SDF-1α. Data are representative of three independent experiments.

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APACs do not cause hemolysis

To investigate the possibility of intravenal administration of APACs, the hemolytic activity of the APACs was studied as described in Experimental procedures. No hemolysis was noted up to concentrations of 100 µm for several l- and d-derivative APACs (data not shown).


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. References
  7. Supporting Information

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 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 demonstrate that d-APACs inhibit SDF-1α-induced cell migration. It is well known that the SDF-1α competes with monoclobal anti-CXCR4 serum 12G5, and inhibits HIV-1 infection mediated by the CXCR4 coreceptor [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 demonstrated in competition experiments using monoclonal 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 abilities 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 accordance 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 interface, found that the d-amino acid peptides compete with 125I-SDF-1α and monoclonal antibody 12G5 binding to CXCR4 with a potency and selectivity comparable 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 functions 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-1α 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)/EC50 ratio, 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 TI50 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. However, 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 multisite 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 degradation by proteolytic enzymes found in the cell cytoplasm. 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 configuration to the l- than the d-arginine peptide configuration. 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 during 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 significantly lower compared to that of the d-peptide aminoglycoside 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 conjugates 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), indicating 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 further elucidate their mode of antiviral action. However, although the cells could be grown for several days in the presence of > 100 µm APACs without any signs of cytotoxicity in the absence of HIV-1, during the development of resistance in the presence of HIV-1, even at relatively low concentrations of APACs (approximately 25 µm), 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 antagonists, such as AMD3100, T140 and ALX40-4C [11], which also affect the normal natural cascade of effects caused by the SDF-1α–CXCR4 interaction, are now being actively pursued as stem cell mobilizers for transplantation 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

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. References
  7. Supporting Information

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 methanolysis 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-methylmorpholine, 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 performed with 0.2 mm silica-coated aluminum sheets, visualization 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 mL·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 mL·min−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 (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 mixture (ethanol/acetone/NH4OH, 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 solvents 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 acetone/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 (CH2Cl2/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, concentrated and the residue was treated with ether to obtain a white solid, which was again made slurry in ether and decanted. 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 residue was resuspended in ether; the ether was decanted and a white material was obtained. Column chromatography (silica gel, CH2Cl2/MeOH/NH4OH, 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 1H NMR (500 MHz, D2O).

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 provided in the Supplementary material. Mass spectra of all APACs confirmed their purity and the the relative molecular masses of their free base (for the d-APACs, see Table 1).

Fluorescent probes – APACs-FITC

The fluorescent probes had been prepared as published previously [18,41] with some modifications. The acetate counter 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 ethanol. 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 U·mL−1 penicillin; 100 µg·mL−1 streptomycin; 0.25 µg·mL−1 fungizone; 200 µg·mL−1 G418; 1 µg·mL−1 puromycin). The cMAGI assay is based on the ability of HIV-1 Tat to transactivate the expression of an integrated β-galactosidase reporter gene driven by the HIV-long-terminal repeats [42,43]. The β-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-indoyl-β-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 inhibition 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 syncytium 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 concentrations of APACs. After 3 days of incubation at 37 °C, live cells were counted in each well by trypan blue exclusion assay and the CC50 determined by plotting the number of live cells versus compound concentration.

Cellular uptake using APACs and poly arginine peptide-fluorescent derivatives: assay by confocal microscopy

cMAGI cells were incubated in a eight-well plate (2000 cells·well−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 µm. After incubation, cells were washed with phosphate buffered saline three times prior to confocal microscopy measurements (Olympus IX70 FV500 or Axiovert 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 Dickinson, San Jose, CA, USA) as previously described [18]. Briefly, 0.5 × 106 MT2 cells were washed with ice-cold NaCl/Pi 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 different concentrations of APACs and arginine peptides. After 30 min of incubation, the cells were washed with ice-cold wash buffer and fixed in NaCl/Pi containing 1% paraformaldehyde. 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 CellQuestTM software (Becton Dickinson).

Chemotaxis migration assay

G2 cells, from a human precursor-B acute lymphoblastic leukemia cell line (kindly provided by T. Lapidot, the Weizmann Institute), were used for the chemotaxis migration assay. Cell migration was assessed in 24-well chemotaxis chambers (6.5 mm diameter, 5 µm pore size, tissue culture treated polycarbonate membrane polystyrene plates; Costar, Corning Incorporated, Corning, NY, USA). Six hundred µl RPMI media with or without 50 ng·mL−1 (6.3 nm) of SDF-1α 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/Pi. The tested compounds (APACs) in NaCl/Pi solution were serially diluted in a 96-well round bottom plate and then 50 µL of hRBC suspension were added to reach a final volume of 100 µL (final erythrocyte concentration 4% v/v and final compounds concentrations 1.56–100 µm). The resulting suspensions 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 monitored by measuring the absorbance at 540 nm. Controls for zero hemolysis and 100% hemolysis consisted of hRBC suspended in NaCl/Pi and Triton 1%, respectively.


  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. References
  7. Supporting Information
ejb_6169_sm_DocS1_FigS1.pdfMissing — Supporting info item

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