PERSPECTIVE
For reprint orders, please contact:
[email protected]
Resistance against inhibitors of HIV-1 entry into target cells Victor G Kramer1,2 & Mark A Wainberg*,1,2
ABSTRACT HIV resistance against currently approved entry inhibitors, the chemokine receptor-5 (CCR5) antagonist maraviroc and the fusion inhibitor enfuvirtide (T-20), manifests in a complex manner that is distinct from the resistance patterns against other classes of antiretroviral drugs. Several attachment and fusion inhibitors are currently under various stages of development. Whereas CCR5 co-receptor antagonists have been widely studied until now, because patients who lack CCR5 are healthy and protected to some extent from HIV-infection, CXCR4-antagonist development has been slower, due to limited antiviral activity and potential toxicity given that CXCR4 may have essential cellular functions. Novel fusion inhibitor development is focusing on orally available small-molecule inhibitors that might replace T-20, which needs to be administered by subcutaneous injection. HAART for HIV has led to a marked decrease in morbidity and mortality. Drug candidates for HAART regimens come from the following classes: reverse transcriptase inhibitors, protease inhibitors, integrase strand-transfer inhibitors and entry inhibitors. A typical regimen includes two nucleoside/nucleotide analog reverse transcriptase inhibitors and either a non-nucleoside reverse transcription inhibitor or a protease inhibitor. A number of concerns arise as a result of lifelong HAART, including development of drug resistance, long-term toxicity and unfavorable drug–drug reactions. It is therefore vital to continue to develop and refine new drug classes in an effort to broaden the treatment options that are available. This review focuses on currently approved and candidate entry inhibitors and the development of resistance to agents in this class. HIV enters host target cells through a complex, step-wise process that begins with virus envelope subunit gp120 binding to the CD4 receptor on the cell surface (reviewed in [1,2]). The gp41 and gp120 subunits are the result of furin cleavage of gp160, which is the major product of the HIV env gene, in the Golgi apparatus [3] . After cleavage, noncovalently associated trimers that are the result of their cleavage are transported to the cell membrane where they are incorporated into budding virions [4] . CD4 D1 domain binding to a conserved site on gp120 causes the latter to undergo a conformational shift that allows it to bind to one of two co-receptors at the cell surface, CCR5 or CXCR4. These co-receptors are G-protein coupled receptors that possess an extracellular N-terminus, intracellular C-terminus and six loops – equally divided into intracellular and extracellular loops. These receptors are responsible for lymphocyte chemotaxis and activation and, in the context of the immune system, are expressed on T cells, monocytes, macrophages and dendritic cells. The conformational shift involves V1/V2 loop changes as well as exposure of the co-receptor binding site [5–8] . The specific co-receptor that gp120 interacts with
KEYWORDS
• Entry inhibitors • HIV • resistance
McGill AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada Department of Experimental Medicine, McGill University, Montreal, QC, Canada *Author for correspondence: Tel.: +1 514 340 8222; Fax: +1 514 340 7537;
[email protected] 1 2
10.2217/FVL.14.104 © 2015 Future Medicine Ltd
Future Virol. (2015) 10(2), 97–112
part of
ISSN 1746-0794
97
Perspective Kramer & Wainberg is determined by the amino acid composition of the V3 loop [9–11] . Viruses that interact with CXCR4 are positively charged at amino acids 11, 24 and 25 on the V3 loop. Once gp120 binds to a co-receptor, it induces further conformational change that allows the gp41 fusion peptide to insert itself into the cell membrane. This precipitates the formation of a six-helix bundle comprised of six heptad repeat domains, three of which are termed HR1 and three others that are termed HR2 [12–14] . The bundle is formed when the C-terminal HR2 region binds to the N-terminal HR1 region in an opposing orientation. The formation of this bundle brings the virus and cell membrane into close proximity and culminates in fusion, allowing the viral capsid to enter the cytoplasm and begin reverse transcription (Figures 1 & 2) . Each step of the entry cascade has potential targets for inhibition. Thus, unlike other antiretroviral classes, the entry inhibitor class consists of a number of agents with diverse antiviral mechanisms that act at disparate points in the viral entry cascade. These steps include: CD4 binding; co-receptor binding;
and membrane fusion. The first step in the cascade, CD4 binding, is not targeted by any currently approved agent. Although previous candidates were shown to inhibit HIV entry in tissue culture, this was not reflected in patients [15,16] . The blockage of the CD4 receptor carries an inherent risk of immunological disruption, as this receptor is essential in processes involving antigen recognition. Soluble CD4 (sCD4) was developed as a promising early candidate following identification of the role of CD4 in entry, Despite promising results in vitro, sCD4 was ineffective in patients in clinical trials and subinhibitory concentrations of sCD4 were found to enhance infection [17] . Candidate compounds still under development in this subclass include BMS-663068 [18] , a drug that binds to gp120 and prevents attachment to CD4. Earlier generations of this drug (BMS-488043) required high doses for antiviral activity [19] . BMS-663068 has now been shown to lower HIV-1 viral load by at least 1 log10 during 8 days of monotherapy [18] . These compounds induce a conformational change in gp120 that render it unable to bind to CD4 [20] . A Phase IIb trial – Viral capsid – gp41 – gp120 – CCR5 or CXCR4 – CD4
Figure 1. Diagram of HIV-1 entry. (A) gp120/gp41 trimer on the virus surface attaches to CD4 on the surface of the cell membrane. (B) Native gp120/gp41 trimer, unbound to CD4. (C) gp120 conformation bound to CD4. (D) Interaction between gp120 and co-receptor, post CD4-attachment. gp120 binds to the N-terminus and extracellular loop 2 of the co-receptor. This allows the gp41 fusion peptide to insert into the cell membrane. (E) Formation of the six-helix bundle following interaction of the HR1 and HR2 domains of gp41, postfusion peptide insertion. This brings together viral and cellular membranes for fusion. (F) Fusion of viral and cellular membranes creates a pore that viral capsid uses to enter the target cell.
98
Future Virol. (2015) 10(2)
future science group
Resistance against inhibitors of HIV-1 entry into target cells
Perspective
gp120 N–
C1
V1
V2
C2
C3
V3
V4
C4
V5
C5
–C
gp41 N–
FP
HR1
Hinge
HR2
MPER TM
–C
Figure 2. Schematic representation of HIV gp120 and gp41 envelope proteins on the virion surface. (A) gp120 contains five conserved domains (C1–C5) and five variable domains (V1–V5). (B) gp41 contains an FP, HR1, HR2, the hinge region between both, the MPER and the TM. FP: Fusion peptide; HR1: Heptad repeat region 1; HR2: Heptad repeat region 2; MPER: Membrane proximal external region; TM: transmembrane anchor.
with this drug as part of combination therapy has recently completed. A postattachment inhibitor, the monoclonal antibody ibalizumab, has been developed that targets the D2 domain of the CD4 receptor [21] . It blocks gp120 conformational change following the gp120-CD4 interaction. Used as a single dose, it has shown antiviral activity with HIV viral load reductions up to 1.5 log10 when given together with optimized background therapy (OBT) [22] . Phase IIb trials demonstrated that weekly or biweekly doses led to viral-load reductions of up to 1.7 log10 together with OBT [23] . Ibalizumab is currently in Phase I subcutaneous administration dose-escalation trials for subcutaneous administration in HIV-negative individuals [24] . CD4 inhibitors are currently the only subclass of entry inhibition without drugs for HIV therapy. CCR5 co-receptor inhibitors are the bestresearched entry inhibitor subclass. This is due to a variety of factors such as the existence of individuals who do not express CCR5 due to a homozygous gene variant (CCR5 delta32/delta32). These individuals possess a high-level of resistance to HIV-1 CCR5-tropic infection [25–27] . Heterozygotes for this deletion experienced slower disease progression [25,27–30] . A lack of CCR5 has not led to major detrimental effects. Interestingly, CCR5 homozygotes show increased susceptibility to West Nile virus and tickborne encephalitis infections [31,32] . Recent reports described several potential benefits of CCR5 antagonists that extend beyond antiviral effect such as a reduction in inflammation [33,34] , although not on progression of rheumatoid arthritis [35] . CCR5-blockade has also led to an increase in levels of CD4 T cells, though
future science group
it is unknown whether these regenerated T cells are extant cells that have gone uninfected or a de novo population of other replicative cells [33] . Finally, one HIV-positive patient who had his viremia controlled by HAART subsequently developed acute myeloid leukemia and received a hematopoietic stem cell transplant from a homozygous CCR5 delta32/delta32 donor [36] . HAART was stopped before transplantation and, after 5 years, his viremia has not returned [37,38] . This has been attributed to the fact that the donor was a homozygous CCR5 delta32/delta32 donor. Similar cases where the donors had normal CCR5 expression were evaluated; the recipients received HAART posttransplant and exhibited undetectable viremia up to 32 weeks [39–41] . The recipients’ viral load rebounded following planned antiretroviral therapy interruption [41] . The differences in treatment outcomes of these patients illustrate the importance of the continued presence of the viral reservoir and continued focus on therapeutic CCR5 blockade. CXCR4 is the alternative co-receptor that a virus may use to enter cells in conjunction with CD4. It is unknown what the long-term effects of CXCR4-blockade are, though there are concerns that it may have serious ramifications due to the fact that cases of CXCR4-receptor augmentation in humans often results in serious immunodeficiency (wart, hypogammaglobulinemia, infection, myelokathexis [WHIM] syndrome) [42,43] and CXCR4 knockout in mice led to abnormalities in embryonic development [44–47] . CXCR4 functions as a receptor of CXCL12 and does not appear to be as redundant as CCR5. Homozygotes possessing mutant ligand SDF1–3′A displayed delayed
www.futuremedicine.com
99
Perspective Kramer & Wainberg progression to AIDS, possibly due to increased production of CXCL12 which competes with CXCR4 tropic strains and prevents entry [48,49] . Promising CXCR4 inhibitors such as AMD3100 have demonstrated antiviral activity in vitro but did not exhibit the same antiviral efficacy in vivo [50,51] . This may be attributed to the fact that CXCR4-tropic strains of HIV are rarely found on their own in vivo; they generally appear in the presence of CCR5-tropic virus, even in late-stage disease (Figure 3) [52–55] . Although CXCR4 blockade may completely inhibit the CXCR4-tropic component of the viral quasispecies, CCR5-tropic replication may continue unabated in the presence of CXCR4 antagonists. AMD3100, initially developed as an HIV antiretroviral, is no longer being developed for HIV therapy but is now being used as a hematopoietic stem cell mobilizer (Pleraxifor) in order to increase stem cell harvests before transplantation. Additionally, AMD3100 is now undergoing clinical trials for treatment of WHIM syndrome as it ablates hyper-CXCR4 signaling following binding to CXCL12 [43,56–57] .
●●Maraviroc
Maraviroc (MVC) is a CCR5 entry inhibitor that prevents CCR5-tropic virus from infecting a target cell. It is currently the only CCR5inhibitor approved for HIV-1 treatment by the US FDA and other regulatory agencies for both first-line and second-line therapy [58] . It is not yet licensed for first-line therapy in Europe [59] . Since a majority of new transmissions are CCR5-tropic [60] , it is reasonable that MVC could have its greatest benefit early in treatment (Figure 3) . CXCR4-tropic variants are more likely to develop over time, rendering the use of MVC inappropriate [54–55,61–62] . MVC is unique among currently approved antiretrovirals in that it exerts its antiviral properties by binding to a cellular target rather than a viral one. It binds at a cavity formed by the 7 transmembrane helices that is distinct from the gp120 binding site, and induces a conformational change that renders the receptor unusable for entry [63–65] . This allosteric inhibition prevents CCR5 ligand (CCL3, CCL4
R5-tropic: no tropism switch
B
R5-tropic to X4-tropic switch
Viral load
A
Entry inhibitors currently approved for treatment
Recent infection
Chronic infection
Late-stage infection
Time
Recent infection
Chronic infection
Late-stage infection
Time
CD4 inhibitor CCR5 inhibitor CXCR4 inhibitor Fusion inhibitor Treatment effective
Treatment ineffective
Figure 3. Relative viral load through three stages of HIV-1 disease course and corresponding efficacy of entry inhibitor subclasses. (A) patients infected with a CCR5-tropic virus that does not undergo tropism switch. CD4, CCR5 and fusion inhibitors are effective for treatment; CXCR4 inhibitors are not. (B) patients infected with a CCR5-tropic virus that switches tropism to CXCR4, characterized by a pronounced increase in viral load at late-stage disease. CD4 and fusion inhibitors are effective throughout disease course. The efficacy of CCR5 inhibitors decreases with a concomitant rise in CXCR4 tropism during chronic and late-stage infection; CXCR4 inhibitors are more effective during these stages.
100
Future Virol. (2015) 10(2)
future science group
Resistance against inhibitors of HIV-1 entry into target cells and CCL5) signaling, although there does not appear to be any sequelae associated with such interference [63] . MVC, as a cellular inhibitor, may have effects beyond its ostensible antiviral activity. The results of the MOTIVATE-1 and MOTIVATE-2 trials showed that treatmentexperienced patients who received MVC with OBT had increased CD4 + T-cell counts versus placebo groups [66,67] . This increase in CD4 + T-cell count was observed in both patients with solely CCR5-tropic virus, and also in patients harboring CXCR4 and dual/mixed population virus at failure. It is possible that the increase in CD4 + T cells results from anti-inflammatory effects that are a natural consequence of CCR5blockade, since naive cells may no longer be recruited to sites of inflammation. The precise mechanisms responsible for the increased CD4 + cell count are still under investigation. MVC appears to limit graft versus host disease (GVHD) in patients receiving allogeneic stemcell transplants. Phase I and II trials revealed decreased incidence of GVHD in patients taking a 33-day course of combination of maraviroc, tacrolimus and methotrexate (23.6% grades II–IV disease; 5.9% grade III or IV) compared with patients taking only tacrolimus and methotrexate (38.5% grades II–IV disease; 21.9% grade III or IV) [68] . This is attributed to MVCmediated inhibition of CCR5 internalization and lymphocyte recruitment. CCR5 was recently implicated as the receptor required for Staphylococcus aureus-produced leukotoxin ED toxicity [69] . Leukotoxin ED is one of four pore-forming toxins produced by the bacteria and is responsible for targeted killing of macrophages, dendritic cells and effector memory T cells, and can serve as an immune evasion strategy by this bacterial pathogen. MVC, along with natural ligands and a monoclonal antibody to ECL-2, reduced the interaction between leukotoxin and CCR5, whereas antibody 3A9, specific for the CCR5 N-terminus, did not affect the interaction. Although MVC treatment had no effect on leukotoxin ED-mediated killing of neutrophils and monocytes, CCR5 antagonists may potentially represent complementary therapeutic strategies in the treatment of S. aureus infections. CCR5 delta32/delta32 homozygotes appear to have more favorable outcomes in rheumatoid arthritis [70,71] . Indeed, it had been hypothesized that CCR5 blockade might mediate rheumatoid
future science group
Perspective
arthritis and maraviroc was originally developed with antiarthritic use in mind. However, maraviroc did not demonstrate any beneficial effects when used to treat rheumatoid arthritis in conjunction with methotrexate [35] . ●●Enfuvirtide (T-20)
Fusion inhibitors exert their antiviral effect during the last stage of viral entry through inhibition of virus and cell membrane fusion. Enfuvirtide (T-20) is the only approved representative of the fusion inhibitor subclass of HIV entry inhibitors. It was the first entry inhibitor approved for therapy and is indicated for inclusion in salvage regimens in treatment-experienced patients despite requiring subcutaneous administration. Two randomized Phase III trials (TORO 1 and 2) showed reduced viral loads to
