This study is the first to examine combinations of CCR5 drugs that are currently in development for HIV-1 therapy. Surprisingly, we observed potent antiviral synergy between the CCR5 MAb PRO 140 and each of three structurally distinct small-molecule CCR5 antagonists. Consistent, high-level synergy was observed across various assay systems, viral isolates, target cells, and inhibition levels. PRO 140 and small-molecule CCR5 antagonists were more potently synergistic when used together rather than in combination with inhibitors that block other stages of HIV-1 entry. In contrast, additive effects were observed for combinations of two small-molecule CCR5 antagonists. Competition binding studies revealed complex and nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors and suggest that the synergistic interactions occur at the level of receptor binding. Our findings have implications for the potential use of novel dual-CCR5 regimens for HIV-1 therapy.
Robust synergy between MAb and small-molecule CCR5 inhibitors was observed in this study, and this finding is consistent with that of a recent report (36
). Potent synergy was observed for both cell-cell and virus-cell fusion, and there was a good concordance of findings in these two well-established assay systems. Comparable levels of synergy were observed for PRO 140 in combination with each of three small-molecule CCR5 antagonists from unrelated chemical series. In addition, consistent synergy was observed for each of two CCR5 target cells and two well-characterized HIV-1 envelopes. HIV-1JR-FL
was isolated from the brain of an AIDS patient at autopsy (31
). Like other late-stage neurovirulent viruses (17
encodes an envelope with high binding affinity for CCR5 (36
). Lastly, similar levels of synergy were observed when PRO 140 and maraviroc were tested against HIV-1BaL
) in preliminary studies (data not shown).
Synergy increased with increasing levels of viral inhibition and translated into in vitro dose reductions of up to 14-fold. Viewed alternatively, this degree of synergy provides a corresponding increase in antiviral pressure at a given concentration of drugs, thereby improving viral suppression and potentially delaying the emergence of drug-resistant virus.
We also observed potent synergy for RANTES used in combination with either maraviroc or PRO 140. Endogenous levels of RANTES may afford some protection against HIV-1 disease progression during natural infection (16
), and therefore, our finding of synergy has important and positive implications for CCR5-targeted therapies of HIV-1. Antiviral synergy between RANTES and PRO 140 is not surprising based on our prior observation that RANTES signaling is not blocked by antiviral concentrations of murine PRO 140 (PA14) (33
). Synergy between RANTES and maraviroc is less easily explained given that maraviroc is a potent CCR5 antagonist. However, our findings are consistent with prior observations of synergy between the small-molecule CCR5 antagonist SCH-C and aminooxypentane-RANTES (44
), a RANTES derivative that has been evaluated as a potential topical microbicide (19
In contrast to the robust synergy observed between MAb and small-molecule CCR5 antagonists, additive effects were observed for combinations of small-molecule CCR5 antagonists. A lack of cooperativity is consistent with the view that these molecules compete for binding to a common pocket on CCR5 (13
). However, synergy is not required to derive clinical benefit from combination therapy. No interference was observed between small-molecule CCR5 antagonists, and this finding has relevance when considering combining such agents in the clinic.
Similarly, we did not observe potent synergy between PRO 140 and inhibitors of HIV-1 attachment (PRO 542 and BMS-378806), fusion (enfuvirtide), or reverse transcriptase (zidovudine and nevirapine), and these findings underscore the significance of the synergy observed for PRO 140 and small-molecule CCR5 antagonists. A number of prior studies have examined interactions between various small-molecule CCR5 antagonists (maraviroc, SCH-C, TAK-220, TAK-652, and E913) and drugs from each of the existing HIV-1 treatment classes. Most (42
) but not all (11
) studies have reported broad synergy between CCR5 inhibitors and the other HIV-1 treatment classes, and the divergent results may reflect differences in the compounds and methods used for antiviral testing as well as differences in the methods used for data analysis. When maraviroc was tested against 20 licensed antiretroviral agents, additive effects were observed in all but three cases, in which modest synergy was reported (11
). This result is consistent with our findings for combinations of PRO 140 and HIV-1 inhibitors that do not target CCR5.
Synergy between anti-HIV-1 drugs may stem from a variety of mechanisms. In mixed virus cultures, one compound may inhibit virus resistant to a second compound (18
), and NRTI/NNRTI combinations may overcome specific reverse transcriptase-mediated resistance mechanisms (4
). Metabolic interactions between inhibitors may increase their effective intracellular drug concentrations (28
), and synergistic entry inhibitors may disrupt interdependent steps in the entry cascade (29
). The present study examined clonal viral envelopes rather than mixed populations, and the extracellular nature of the target argues against metabolic interactions. Multiple domains of gp120 contribute to CCR5 binding (8
), but it is unclear at present whether these interactions represent separate or discrete events during infection.
Our findings indicate that antiviral synergy between MAb and small-molecule CCR5 inhibitors may occur at the level of the receptor, although the mechanism remains poorly defined. As discussed above, MAbs and small molecules bind distinct loci on CCR5 (13
). When preincubated with CCR5 cells in the present study, PRO 140 completely blocked subsequent binding of maraviroc to the receptor, although the PRO 140 concentrations were higher than those needed to block HIV-1 entry into the same cells. In contrast, preincubation of CCR5 cells with supersaturating concentrations of maraviroc or vicriviroc reduced PRO 140 binding by 50% or less. As one possible explanation, PRO 140 could recognize CCR5 conformers that are not bound by maraviroc or vicriviroc. Although cell surface CCR5 exists in multiple conformations (22
), it seems unlikely that the small-molecule antagonists could demonstrate potent antiviral activity while failing to bind a significant fraction of cell surface CCR5. In this regard, it is important to note that a common cellular background (CEM.NKR-CCR5 cells) was used for competition binding and antiviral studies, and therefore, the findings are not related to cell-specific differences in CCR5 expression.
In our view, a more plausible explanation for our findings is that PRO 140 is capable of forming a ternary complex with maraviroc-bound CCR5, and this ternary complex provides an increased barrier to HIV-1 entry. Within the context of this model, PRO 140 may bind maraviroc-bound CCR5 somewhat less efficiently than free CCR5, as evidenced by the modest reduction in PRO 140 binding in the presence of maraviroc. However, we note that our studies do not demonstrate the existence of a ternary complex, and additional studies will be required to test this model and to further define the mechanisms of synergy between MAb and small-molecule CCR5 inhibitors. To this end, time-of-addition studies have indicated that murine PRO 140 (PA14) acts at a later stage of the fusion cascade than does the small-molecule CCR5 antagonist SCH-C (36
). This report proposed that SCH-C blocks gp120 binding to CCR5, while PA14 prevents ill-defined postbinding events.
The combination index method is widely used to assess drug-drug interactions. In this method, cooperativity is often defined on the basis of empirical CI values (e.g., <0.9 for synergy and >1.1 for antagonism) irrespective of interassay variability. Statistical analyses are performed infrequently, and even more rarely are adjustments made for multiple comparisons. In the absence of such analyses, there is increased potential to overestimate the number of synergistic combinations.
We adopted a rigorous approach for identifying synergistic effects. CI values were tested for statistical significance against the null hypothesis of additivity (CI = 1). In addition, our studies determined 20 to 30 different CI values per experiment (Tables and ), as is common in synergy studies. In order to reduce the potential for spurious positive results, we reduced the significance level using the Bonferroni correction. We also evaluated a mock combination as a control. We conclude that numerous apparent synergies (CI < 0.9) could not be distinguished from interassay variation based on the available data. Nevertheless, PRO 140 and small-molecule inhibitors demonstrated significant synergy under every test condition, lending credence to this finding. Combinations with CI values that trended towards significance in the present survey, such as the PRO 140/enfuvirtide combination, could be explored in future studies.
A growing body of data indicates that MAb and small-molecule CCR5 antagonists represent distinct subclasses of CCR5 inhibitors, and a number of important parallels can be drawn between NRTI and NNRTI on the one hand and between MAb and small-molecule CCR5 antagonists on the other. In each instance, there are distinct binding loci for the inhibitors on the target protein (reverse transcriptase or CCR5). One set of inhibitors (NNRTI or small-molecule CCR5 antagonists) acts via allosteric mechanisms, while the other set (NRTI or CCR5 MAbs) acts as a competitive inhibitor. Like NRTI and NNRTI, MAb and small-molecule CCR5 inhibitors are synergistic and possess complementary patterns of viral resistance in vitro in preliminary testing (20
). NRTI and NNRTI represent important and distinct treatment classes even though they target the same protein, and MAb and small-molecule CCR5 inhibitors similarly may offer distinct HIV-1 treatment modalities.