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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.
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 . Several peptide-derived and other small molecule inhibitors of CXCR4- and CCR5-mediated HIV-1 infection have been reviewed . 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) . ALX40-4C was the first CXCR4 antagonist to be tested in HIV-1 infected individuals .
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 . 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 . Poly arginine-containing peptides are also known as potent furin inhibitors, with the 9-mer d-poly arginine being the most active inhibitor . 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) . 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 . 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  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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- Experimental procedures
- 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  (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.  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 .
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, 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 .
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 .
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 , 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 , 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)].