One of the most interesting aspects of BA derivatives is that the locations of side chains can orchestrate their mechanisms of action. A side chain in the C-3 position is needed for anti-HIV-1 maturation activity (18
). On the other hand, a C-28 side chain on the BA molecule is required for anti-HIV-1 entry activity (12
). The most well-characterized anti-HIV-1 maturation BA derivative is DSB (PA-457). DSB does not inhibit HIV-1 entry, but it is a potent anti-HIV-1 maturation inhibitor (18
). DSB was shown to be active against many HIV-1 isolates and is currently in phase II clinical trials. In contrast to the BA maturation inhibitors, the molecular mechanism of BA entry inhibitors was elusive. Although BA entry inhibitors have been shown to potently inhibit T-cell-adapted viruses such as HIV-1IIIB
or NL4-3, their spectra of anti-HIV-1 activity were poorly characterized. The data presented in this study clearly indicated that IC9564 and A43-D exhibited broad-spectrum anti-HIV activity at submicromolar concentrations. It is worth noting that clade C viruses, which account for approximately 50% of all HIV-1 isolates in the world, are relatively more sensitive to the BA entry inhibitors than are clade A and clade B viruses.
All the viruses listed in Table are R5 viruses, except the X4 viruses HXB2 and NL4-3. One BA entry inhibitor, RPR103611, was shown to have antientry activity against some X4 viruses, with little activity against R5 viruses (17
). Since RPR103611 and IC9564 are steroisomers, their mechanisms of anti-HIV action are likely to be the same. However, it remains a possibility that a minor structural difference in BA derivatives could affect their mechanisms of action. For example, PA-457 is a BA derivative that inhibits HIV-1 maturation without anti-HIV-1 entry activity.
The purpose of studying the interaction between IC9564 and other HIV-1 entry inhibitors was to gain insights into where the drug binding site resides. The strong influence of IC9564 on the antifusion activity of TAK-779 suggests that the IC9564 binding site might be intimately related to chemokine receptor binding sites in gp120. The binding of IC9564 might have resulted in an HIV-1 Env structure that retains a minimal function for chemokine receptor binding but that has reached a threshold that is susceptible to the CCR5 inhibitor TAK-779. It will be interesting to determine whether IC9564 can also strongly affect the anti-HIV-1 entry activity of other classes of CCR5 inhibitors. Since the experiments were designed to study the mechanism of action of IC9564 rather than the synergy between IC9564 and other entry inhibitors, the degree of synergy between the BA derivatives and other HIV-1 entry inhibitors remains to be determined.
The ability of the BA derivatives to inhibit primary HIV-1 isolates across the most prevalent genetic subtypes (clades A, B, and C) suggests that these compounds target a conserved functional motif in gp120. However, results obtained with drug-resistant viruses suggested that the V3 loop of gp120 is the main target. The strong competition of IC9564 with V3 monoclonal antibody binding to gp120 also supports the notion that V3 is the likely target of the compounds. The V3 region, by definition, contains a highly variable sequence. Thus, the broad anti-HIV activity of uncharged, HIV-1-specific A43-D was unexpected. It is likely that the drugs interact with a conserved functional motif in V3. Despite considerable sequence variation, a conserved V3 structural motif is likely needed to interact with chemokine receptors for virus-cell fusion to take place. Such a conserved V3 feature was implicated in previous studies on the structure of the V3 loop (13
). Some V3 monoclonal antibodies, such as 447-52D, were shown to neutralize many primary isolates (41
). Although IC9564 can compete with 447-52D for V3 binding, their mechanisms of action might not be identical. For example, all the clade B viruses used in this study are sensitive to the BA entry inhibitors, whereas only two of the clade B viruses tested, SS1196.1 and 6535.3, were sensitive to 447-52D (Table ). SS1196.1 and another clade B virus, WITO4160.33, exhibited similar sensitivities to the BA derivatives. On the other hand, WITO4160.33 was not sensitive to 447-52D. Therefore, the BA entry inhibitors might have targeted a structural motif different from that of the 447-52D epitope. A similar argument against the notion that the G306R mutation in the IC9564-resistant virus is where the drug binding site resides could also be made. For example, HXB2 virus, which possesses an Arg (R) at amino acid residue 306, is very sensitive to the BA derivatives (Table ). The G306R mutation in gp120 of M2-NLDH, which occurred at a site known to be a key determinant for coreceptor tropism, might result in a V3 conformation that is unfavorable for interactions with the BA entry inhibitors.
The V3 loop is one of the key structural motifs that are critical for chemokine receptor binding. It appears that the binding of BA derivatives to the V3 loop interfered with interactions between gp120 and chemokine receptors. The interaction of gp120 with chemokine receptors is thought to trigger subsequent conformational changes in gp41. This is in agreement with our previous observations that IC9564 induced an aberrant conformational change in gp120 and inhibited the conformational changes in gp41 (15
). Thus, the BA derivatives could inhibit HIV-1 entry processes by inducing a fusion-incompetent gp120 conformation that is no longer capable of interacting with chemokine receptors and triggering the subsequent conformational changes in gp41 needed for fusion.
The computer docking model for the interaction between the BA derivatives and the V3 loop (Fig. ) appears to agree with our previous structure-activity relationship study. For example, a bulky C-3 side chain of the A ring in the BA scaffold compromised the antientry activity of BA derivatives (14
). It is possible that a bulky C-3 side chain negatively impacts the ability of the A ring to fit into the ligand binding pocket in the V3 base. Furthermore, the C-28 side chain is where the anti-HIV-1-entry activity resides. Alignment of the C-28 side chain with the V3 stem toward the N-terminal half of the V3 loop might impact the ability of V3 to interact with other structural motifs in gp120 and chemokine receptors.
In summary, the BA derivatives studied here were shown to possess potent broad-spectrum anti-HIV-1 activity. The most prevalent clade C viruses were relatively sensitive to this activity. In addition to broad anti-HIV-1 activity, IC9564 was able to affect the antifusion activity of other entry inhibitors. The greater-than-2-log10 alteration in the anti-HIV-1 entry activity of the R5 inhibitor TAK-779 is remarkable. Although the ability of IC9564 to sensitize HIV-1 to TAK-779 was unexpected, the most surprising result was that the V3 region of gp120 is a critical determinant for the antientry activity of the BA derivatives. The inhibition of a variety of primary HIV-1 isolates by targeting V3 supports the notion that a conserved structural motif exists in the V3 region. Such a conserved V3 structure could be an interesting target for both drug and vaccine development against HIV-1 infection.