Emerging drug targets for antiretroviral therapy.

Current targets for antiretroviral therapy (ART) include the viral enzymes reverse transcriptase and protease. The use of a combination of inhibitors targeting these enzymes can reduce viral load for a prolonged period and delay disease progression. However, complications of ART, including the emergence of viruses resistant to current drugs, are driving the development of new antiretroviral agents targeting not only the reverse transcriptase and protease enzymes but novel targets as well. Indeed, enfuvirtide, an inhibitor targeting the viral envelope protein (Env) was recently approved for use in combination therapy in individuals not responding to current antiretroviral regimens. Emerging drug targets for ART include: (i) inhibitors that directly or indirectly target Env; (ii) the HIV enzyme integrase; and (iii) inhibitors of maturation that target the substrate of the protease enzyme. Env mediates entry of HIV into target cells via a multistep process that presents three distinct targets for inhibition by viral and cellular-specific agents. First, attachment of virions to the cell surface via nonspecific interactions and CD4 binding can be blocked by inhibitors that include cyanovirin-N, cyclotriazadisulfonamide analogues, PRO 2000, TNX 355 and PRO 542. In addition, BMS 806 can block CD4-induced conformational changes. Secondly, Env interactions with the co-receptor molecules can be targeted by CCR5 antagonists including SCH-D, maraviroc (UK 427857) and aplaviroc (GW 873140), and the CXCR4 antagonist AMD 070. Thirdly, fusion of viral and cellular membranes can be inhibited by peptides such as enfuvirtide and tifuvirtide (T 1249). The development of entry inhibitors has been rapid, with an increasing number entering clinical trials. Moreover, some entry inhibitors are also being evaluated as candidate microbicides to prevent mucosal transmission of HIV. The integrase enzyme facilitates the integration of viral DNA into the host cell genome. The uniqueness and specificity of this reaction makes integrase an attractive drug target. However, integrase inhibitors have been slow to reach clinical development, although recent contenders, including L 870810, show promise. Inhibitors that target viral maturation via a unique mode of action, such as PA 457, also have potential. In addition, recent advances in our understanding of cellular pathways involved in the life cycle of HIV have also identified novel targets that may have potential for future antiretroviral intervention, including interactions between the cellular proteins APOBEC3G and TSG101, and the viral proteins Vif and p6, respectively. In summary, a number of antiretroviral agents in development make HIV entry, integration and maturation emerging drug targets. A multifaceted approach to ART, using combinations of inhibitors that target different steps of the viral life cycle, has the best potential for long-term control of HIV infection. Furthermore, the development of microbicides targeting HIV holds promise for reducing HIV transmission events.

Macrophages and lymphocytes differentially modulate the ability of RANTES to inhibit HIV-1 infection.

The beta-chemokines MIP-1alpha, MIP-1beta, and RANTES inhibit HIV-1 infection of CD4+ T cells by inhibiting interactions between the virus and CCR5 receptors. However, while beta-chemokine-mediated inhibition of HIV-1 infection of primary lymphocytes is well documented, conflicting results have been obtained using primary macrophages as the virus target. Here, we show that the beta-chemokine RANTES inhibits virus entry into both cellular targets of the virus, lymphocytes and macrophages. However, while virus entry is inhibited at the moment of infection in both cell types, the amount of virus progeny is lowered only in lymphocytes. In macrophages, early-entry restriction is lost during long-term cultivation, and the amount of virus produced by RANTES-treated macrophages is similar to the untreated cultures, suggesting an enhanced virus replication. We further show that at least two distinct cellular responses to RANTES treatment in primary lymphocytes and macrophages contribute to this phenomenon. In lymphocytes, exposure to RANTES significantly increases the pool of inhibitory beta-chemokines through intracellular signals that result in increased production of MIP-1alpha and MIP-1beta, thereby amplifying the antiviral effects of RANTES. In macrophages this amplification step does not occur. In fact, RANTES added to the macrophages is efficiently cleared from the culture, without inducing synthesis of beta-chemokines. Our results demonstrate dichotomous effects of RANTES on HIV-1 entry at the moment of infection, and on production and spread of virus progeny in primary macrophages. Since macrophages serve as a reservoir of HIV-1, this may contribute to the failure of endogenous chemokines to successfully eradicate the virus.

Functional HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by HIV strain X4.

HIV can cross the intact epithelium of genital mucosae via Langerhans cells. Fresh Langerhans cells are known to express CD4 and CCR5. The presence of CXCR4 on the surface of cultured but not freshly isolated Langerhans cells has been described. In the present study, we demonstrate that CXCR4 was expressed by fresh Langerhans cells isolated and purified from epidermis. However, the percentage of Langerhans cells expressing CXCR4 or CCR5 increased during maturation of the cells in culture, especially in the presence of exogenous granulocyte-macrophage colony-stimulating factor. To determine whether CXCR4 was functional, freshly isolated Langerhans cells were infected with HIV LAI, a T-cell-tropic strain, and p24 protein production was measured in culture supernatants. p24 production was observed when infected Langerhans cells were cocultured with SupT1 cells. However, the presence of HIV provirus DNA was evidenced within the infected Langerhans cells by nested PCR. Ultrastructural studies confirmed the formation of syncytia when Langerhans cells were cocultured with SupT1 cells. Preincubation of Langerhans cells with azidothymidine or SDF-1-alpha, a natural ligand for CXCR4, prevented infection. These data demonstrated that CXCR4 is present on the surface of Langerhans cells freshly isolated from human skin epidermis and that this expression is functional.

The future of HIV infection: gene therapy and RNA interference.

The description of the mechanism of RNA interference (RNAi) has generated enormous interest in the biomedical field. A previously unrecognized pathway in which small interfering, 21 to 23 mer, double-stranded RNA (siRNA) mediates sequence-specific degradation of mRNA is becoming one the most useful techniques in cell biology and genetics research. Based on the potency, specificity and physiology of RNAi to silence gene expression, much is expected from its use as a therapeutic tool. The first evidence of RNAi as a suppressor of HIV replication has already been reported, thus providing a new impetus to the development of molecular or gene therapy approaches to HIV infection.

CD4 modulation and inhibition of HIV-1 infectivity induced by monosialoganglioside GM1 in vitro.

The addition of monosialoganglioside GM1 to serum-free culture medium efficiently and specifically inhibited CD4 antigen expression on normal T lymphocytes from peripheral blood or thymus as well as on cells from H9 and Molt-3 lines; other molecules such as CD3, CD2 and CD8 were not affected. Subsequent addition of fetal calf serum or bovine and human serum albumin blocked GM1 action on CD4 expression, most likely through the formation of ganglioside-albumin complexes. Removal of GM1 from the medium was followed by the prompt reappearance of CD4 on the cell surface. GM1 treatment of H9 and Molt-3 cells greatly reduced HIV-1 infectivity, which was evaluated by reverse transcriptase activity levels in culture supernatants and p24 detection on target cells. GM1 also inhibited syncytial formation in Molt-3 cells even when treatment was initiated 24h after infection. The GM1 effect on HIV-1 infectivity, however, was not long-lasting since removal of the compound was followed by a rapid increase in viral replication, probably due to CD4 re-expression and HIV-1 propagation from a few initially infected cells.

Recent advances in understanding the molecular mechanisms of HIV-1 entry and fusion: revisiting current targets and considering new options for therapeutic intervention.

Recent advances in our understanding of the cellular and molecular mechanisms of HIV-1 entry provide the basis for novel therapeutic strategies that prevent viral penetration of the target cell-membrane, while reducing detrimental virus and treatment effects on cells and prolonging virion exposure to immune defenses. A number of potential sites for therapeutic intervention become accessible during the narrow window between virus attachment and the subsequent fusion of viral envelope with the cell membrane. Initial approaches considered for prevention of HIV-1 entry included the use of natural ligands, small-molecule inhibitors and/or monoclonal antibodies, which could interfere with gp120-CD4 and/or gp120-CXCR4/CCR5 interaction. Others avenues pursued were the use of agents that interfere with the conformational changes of gp120 or gp41 leading to subsequent fusion of viral and cellular membranes. More recently, strategies have emerged that involve inhibition of thiol/disulfide oxidoreductases (factors which facilitate Env transition from an inactive to a fusion-competent conformation) to block redox exchange, thereby impeding the entry process. This review provides a summary of the cellular and viral factors mediating the HIV-1 entry process, with an emphasis on novel therapeutics targeting Env-receptor/coreceptor interaction, Env conformational change and the membrane fusion process.

Unwelcome guests with master keys: how HIV enters cells and how it can be stopped.

HIV entry to host cells begins with binding of the viral envelope protein to CD4 molecules on the host cell surface. This binding initiates conformational changes in the envelope protein that result in binding to a coreceptor (CCR5 or CXCR4), exposure of a previously hidden domain in the viral protein, insertion of a viral fusion peptide into the host-cell membrane and fusing the viral and cell membranes. Each of these steps provides an opportunity for intervention to prevent viral entry, and a number of agents targeting these steps are in development. Studies of coreceptor inhibitors and fusion inhibitors have indicated the presence of host and viral factors that can result in variability of antiretroviral effect. Improved understanding of these factors will help to guide clinical use of these new agents. This article summarizes a presentation by Robert W. Doms, MD, PhD, at the International AIDS Society-USA course in Chicago in May 2004.

Virus entry as a target for anti-HIV intervention.

The replicative cycle of the human immunodeficiency virus (HIV) can be interrupted at several stages. Until recently only the viral reverse transcriptase and protease were the only enzymes targeted by antiretroviral agents. However, the first HIV entry inhibitor (T-20, Enfuvirtide, Fuseon) to be used in humans has been approved by the Food and Drug Administration (FDA). The HIV entry process is considered as an attractive target for chemotherapeutic intervention, as blocking HIV entry into its target cell leads to suppression of viral infectivity, replication and the cytotoxicity induced by virus-cell contacts. HIV-1 entry into target cells is a multistep process: virus attachment is initiated by the binding of trimeric envelope glycoprotein gp120 complexes on the virions to glycosylated T-cell surface receptor (CD4) and HIV GPCR coreceptors (CCR5 or CXCR4) leading to envelope glycoprotein gp41-dependent fusion-pore formation and membrane fusion. A number of compounds are being developed to specifically target each of these steps leading to virus entry and some compounds have reached early clinical development. Conversely, agents such as the CCR5 antagonist Tak-779 and the CXCR4 antagonist AMD3100 are not longer being thought as relevant anti-HIV agents but have given way to new analogues with improved properties. This review summarizes the current state of HIV entry inhibitors, their mechanisms of action and their therapeutic value against HIV infection and AIDS.

Prostratin induces HIV activation and downregulates HIV receptors in peripheral blood lymphocytes.

Induction of HIV expression through lymphocyte activation has been proposed as a strategy to purge latent reservoirs. Prostratin is a non-tumourogenic phorbol ester that delays HIV replication in vitro, but paradoxically activates HIV expression in latently infected cells. To get a better insight into the mechanisms of action of prostratin, we have analysed the effect of prostratin on HIV activation and HIV receptor and coreceptors' surface expression in human lymphocytes. Peripheral blood mononuclear cells (PBMCs) were transfected with luciferase expression constructs under the control of wild type HIV-long terminal repeat (LTR) and consensus sequences for transcription factors involved in HIV-LTR transactivation (NF-kappaB, SP1, NFAT). Prostratin stimulates transactivation of LTR vectors, kappaB- and SP-1-driven luciferase constructs. In another set of experiments, PBMCs were transfected with a full-length infectious viral clone. Prostratin induced HIV transcription and viral expression as detected by luciferase activity in cellular extracts and p24 levels in culture supernatants, respectively. Expression of the HIV coreceptors CCR5 and CXCR4 was decreased by prostratin and, concomitantly, prostratin inhibited the infection of PBMCs with R5 and X4 strains. However, prostratin did not inhibit infection with a pseudotyped viral clone that enters into the cells independently of HIV receptors. These results help to explain the paradoxical effects of prostratin. On one hand, prostratin induces HIV activation in latently infected cells through the induction of NF-kappaB and Sp1. On the other hand, strong and persistent downregulation of HIV receptors decreases infection of new targets and delays HIV propagation. These data support the potential use of prostratin to activate HIV from latency and purge viral reservoirs.

Inhibitors of the entry of HIV into host cells.

The development of mechanistic insight into the process by which HIV enters host cells has revealed a panoply of targets that offer considerable potential as sites for pharmacological intervention. The gp120/gp41 protein complex, expressed on the virion surface, mediates HIV entry by a process initiated by the engagement of the host cell receptor CD4. Subtle conformational changes triggered by this interaction expose elements of gp120 to the seven-transmembrane, G protein-coupled chemokine receptors CCR5 or CXCR4 expressed on host cells, a contact that relieves constraints imposed on gp41 by gp120. This leads to a major conformational rearrangement of gp41, which results in the insertion of the fusion peptide into the host cell membrane and the assembly of the amino terminus heptad repeat into a trimeric form that is subsequently recognized by the carboxy terminal heptad repeat. The latter process leads to juxtaposition of the viral and host cell membranes, a prelude to fusion. The most prominent strategies and targets that are actively being exploited as drug discovery opportunities are inhibition of the attachment of HIV to host cells, blockade of chemokine receptors and interference with the function of gp41. Inhibitors of each of these steps in the HIV entry process with potential clinical relevance are reviewed in the context of their status in the drug development process. The most significant entity to emerge from this area of research to date is enfuvirtide, a 36-amino acid derivative that interferes with the function of gp41. Enfuvirtide is the first HIV entry inhibitor to be granted a license for marketing (it was approved in the US and Europe in March 2003), and its introduction portends the beginning of what promises to be an exciting new era of HIV therapy.

Inhibition of entry of HIV into cells by poly(A).poly(U).

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U), is a synthetic double-stranded RNA that has been shown to manifest both antitumoral and immunodulatory activities. Previously, we have reported that poly(A).poly(U) inhibits HIV infection in cell cultures. Here we provide direct evidence to demonstrate that the inhibitory action of poly(A).poly(U) is through its capacity to prevent entry of HIV particles into CD4-positive T lymphocytes. Such inhibition of HIV entry is also observed in the case of other polyanions such as heparin, dextran sulfate, and poly(I).poly(C). The mechanism of inhibition appears to occur postbinding of HIV particles to the CD4 receptor molecules, because the binding of the external envelope glycoprotein of HIV-1 (gp120) is not affected significantly in the presence of poly(A).poly(U) or other polyanions. These results confirm the potential of poly(A).poly(U) as an antiviral drug against HIV infection.

Unexploited viral and host targets for the treatment of human immunodeficiency virus type 1 infection.

To date, all approved drugs for the treatment of infection by human immunodeficiency virus type 1 (HIV-1) target either of two viral enzymes, reverse transcriptase or protease. Drugs targeting different macromolecules could improve upon current shortcomings (ex, drug resistance, metabolism, toxicity, formulation) and provide foundations for novel combination therapies. This review will focus on the two key challenges for any new target--target validation (demonstrating the role in the disease), and target tractability (the likelihood of identifying modulators of that target that have drug-like properties). For this discussion, drug-like molecules are orally active, relatively small organic molecules. All of the virally-encoded proteins (other than reverse transcriptase and protease) and the host targets that have been postulated to be critical for HIV-1 proliferation will be reviewed.

Bicyclams, a class of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/CXCR-4.

Bicyclams are a novel class of antiviral compounds which are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. The prototype compound, AMD3100, has an IC50 of 1-10 ng/ml, which is a least 100,000 fold lower than the cytotoxic concentration. AMD3100 does not inhibit virus binding to the CD4 receptor and based on time-of-addition experiments, has been assumed to interact with the HIV fusion-uncoating process. Resistance of HIV-1 strains to AMD3100 is associated with the accumulation of several mutations in the viral envelope glycoprotein gp120. Here, we demonstrate that AMD3100 interacts with fusin (CXCR-4), the coreceptor used by T-tropic viruses to infect the target cells. The replication of NL4-3 wild type virus and NL4-3 dextran sulfate-resistant virus was inhibited by the CXC-chemokine, stromal cell-derived factor 1 (SDF-1), the natural ligand for CXCR-4. In contrast, the replication of the HIV-1 NL4-3 AMD3100-resistant virus was no longer inhibited by SDF-1. The bicyclams are the first low-molecular-weight anti-HIV agents shown to interact with the coreceptor for T-tropic viruses.

Thalidomide and the immune system. 4. Down-regulation of the CD26 receptor, probably involved in the binding of HIV components to T cells in primates.

Thalidomide (Thd) is capable of down-regulating the CD26 receptor on CD4+ lymphocytes after treatment of healthy volunteers. Similar effects are observed when marmosets (Callithrix jacchus) are treated with Thd. The Ta1 epitope of the CD26 receptor has recently been shown to bind the HIV-1 Tat trans-activating protein, and CD26 has also been suggested to be a coreceptor for the binding of the V3 loop of the gp120 HIV envelope protein. This might provide a hint for possible therapeutic interventions.

The influence of D-Ala1-peptide T amide, an analogue of HIV glycoprotein 120 fragment, on the CD4--anti CD4 lymphocyte interaction.

D-Ala1-peptide T amide (DAPTA), synthetic analogue of the HIV glycoprotein 120 sequence, was used to study its binding to specific cellular receptor of HIV, CD4. The analogue contains eight aminoacid residues including 4 threonine residues; its molecular weight being 992. We have shown the temperature- and dose-dependent inhibitory effect of peptide T on the CD4 - anti CD4 binding. The strongest effect was noted at 37 degrees C with the peptide dose of 150 nM: 62% inhibition of binding. Other means of possible blocking of HIV attachment to the host cellular receptor are discussed.

A pathophysiological approach to antiretroviral therapy.

A discussion of the pathophysiology of HIV infection is important not only to understand both routine and novel therapeutic approaches, but also to appreciate the challenges of long-term control and viral eradication. This article will first briefly review certain pathophysiologic principles of HIV infection that have particular therapeutic implications, and then discuss general tenets of antiretroviral therapy, followed by new developments in the field.

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Drug resistance and coreceptor usage in HIV type 1 subtype C-infected children initiating or failing highly active antiretroviral therapy in South Africa.

HIV-1 drug resistance monitoring in resource-poor settings is crucial due to limited drug alternatives. Recent reports of the increased prevalence of CXCR4 usage in subtype C infections may have implications for CCR5 antagonists in therapy. We investigated the prevalence of drug resistance mutations and CXCR4 coreceptor utilization of viruses from HIV-1 subtype C-infected children. Fifty-one children with virological failure during highly active antiretroviral therapy (HAART) and 43 HAART-naive children were recruited. Drug resistance genotyping and coreceptor utilization assessment by phenotypic and genotypic methods were performed. At least one significant drug resistance mutation was present in 85.4% of HAART-failing children. Thymidine analogue mutations (TAMs) were detected in 58.5% of HAART-failing children and 39.0% had ≥3 TAMs. CXCR4 (X4) or dual (R5X4)/mixed (R5, X4) (D/M)-tropic viruses were found in 54.3% of HAART-failing and 9.4% of HAART-naive children (p<0.0001); however, the HAART-failing children were significantly older (p<0.0001). In multivariate logistic regression, significant predictors of CXCR4 usage included antiretroviral treatment, older age, and lower percent CD4(+) T cell counts. The majority of genotypic prediction tools had low sensitivity (≤65.0%) and high specificity (≥87.5%) for predicting CXCR4 usage. Extensive drug resistance, including the high percentage of TAMs found, may compromise future drug choices for children, highlighting the need for improved treatment monitoring and adherence counseling. Additionally, the increased prevalence of X4/D/M viruses in HAART-failing children suggests limited use of CCR5 antagonists in salvage therapy. Enhanced genotypic prediction tools are needed as current tools are not sensitive enough for predicting CXCR4 usage.