The alpha-glucosidase inhibitor 1-deoxynojirimycin blocks human immunodeficiency virus envelope glycoprotein-mediated membrane fusion at the CXCR4 binding step.

1-Deoxynojirimycin (DNM) is a saccharide decoy that inhibits cellular alpha-glucosidase I-II activity. Treatment by DNM of human immunodeficiency virus (HIV)-infected lymphocyte cultures inhibits virus spread. The functional properties of the membrane-associated Env glycoprotein (Env) modified in the presence of DNM remain unclear because previous reports on this subject have essentially used recombinant soluble Envs whose properties differ notably from those of Env anchored on the surface of the virus. To model virus-associated Env synthesized in the presence of DNM, native Env was expressed at the surface of mammalian cells treated with DNM. As expected, its glycosylation pattern was altered in the presence of the inhibitor. Env was found able to bind CD4, whereas its ability to induce membrane fusion was abolished. The immunoreactivity of regions involved in interactions of Env with CXCR4 (V1, V2, C2, and V3) was modified and Env displayed altered interaction with this coreceptor. These results are consistent with the inhibition by DNM of virus entry at the Env/coreceptor interaction step. Finally, preliminary data indicate that suboptimal concentrations of DNM and natural or synthetic CXCR4 ligands used in combination potently inhibit the Env-mediated membrane fusion process. Altogether, our results suggest that DNM and its analogs deserve further investigation as anti-HIV agents in combination with experimental compounds targeting CXCR4 to inhibit each partner of this crucial step of HIV entry.     

Inhibition of HIV-1 by fusion inhibitors

The envelope glycoprotein complex (Env) is responsible for entry of the human immunodeficiency virus type 1 (HIV-1) into cells by mediating attachment to target cells and subsequent membrane fusion. Env consists of three gp120 subunits that mediate receptor and co-receptor attachment and three gp41 subunits responsible for membrane fusion. Several steps of the entry process can serve as drug targets. Receptor antagonists prevent attachment of gp120 to the receptor or co-receptor and conformational changes within gp41 required for membrane fusion can be inhibited by fusion inhibitors. Enfuvirtide (T20, Fuzeon) is a peptide based on the gp41 sequence and is the only approved fusion inhibitor. It prevents membrane fusion by competitively binding to gp41 and blocking the formation of the post-fusion structure. New generations of T20-like peptides have been developed with improved potency and stability. Besides T20 and derivatives, other fusion inhibitors have been developed that target different domains of gp41. Here we discuss the development of fusion inhibitors, their mode of action and their potential for incorporation in future drug regimens.     

Glycoside analogs of beta-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry

The interaction between HIV gp120 and galactose-containing cell surface glycolipids such as GalCer or Gb(3) is known to facilitate HIV binding to both CD4(+) as well as CD4(-) cells. In an effort to develop small molecule HIV-1 entry inhibitors with improved solubility and efficacy, we have synthesized a series of C-glycoside analogs of GalCer and tested their anti HIV-1 activity. The analogs were tested for gp120 binding using a HIV-1 (IIIB) V3-loop specific peptide. Two of the six analogs that interfered with gp120 binding also inhibited HIV Env-mediated cell-to-cell fusion and viral entry in the absence of any significant cytotoxicity. Analogs with two side chains did not show inhibition of fusion and/or infection under identical conditions. The inhibition of virus infection seen by these compounds was not coreceptor dependent, as they inhibited CXCR4, CCR5 as well as dual tropic viruses. These compounds showed inhibition of HIV entry at early steps in viral infection since the compounds were inactive if added post viral entry. Temperature-arrested state experiments showed that the compounds act at the level of virus attachment to the cells likely at a pre-CD4 engagement step. These compounds also showed inhibition of VSV glycoprotein-pseudotyped virus. The results presented here show that the glycoside derivatives of GalCer with simple side chains may serve as a novel class of small molecule HIV-1 entry inhibitors that would be active against a number of HIV isolates as well as other enveloped viruses.     

Maraviroc,a CCR5 Coreceptor Antagonist That Blocks Entry of Human Immunodeficiency Virus Type 1

Inhibition of the human immunodeficiency virus type 1 (HIV-1) coreceptor is an encouraging new approach to pharmacotherapy against HIV. The HIV-1 strain makes use of either the CCR5 or the CXCR4 coreceptor to gain access into host CD4⁺ cells. Maraviroc, the first HIV-1 CCR5 coreceptor antagonist, blocks entry of HIV-1. This recently approved drug has demonstrated clinically significant decreases in plasma concentrations of HIV-1 RNA and increases in CD4⁺ cell counts; however, it is indicated only for use as salvage therapy. Drug resistance is a concern, as is selective pressure on viral coreceptor use, because viral coreceptor targets may switch as disease progresses. In addition, before maraviroc therapy can be started, costly assays are required to determine the host's viral coreceptor tropism. Emerging therapies targeting CXCR4, the other HIV coreceptor, have shown promise in decreasing plasma concentrations of HIV-1 RNA. Long-term studies with both targets are required to explore the critical issues of efficacy and immunologic safety, as the function of these coreceptors is linked to host chemokine pathways.     

HIV entry into cells by CD4-independent mechanisms

Summary All HIV and SIV strains can enter cells by binding to cell-surface CD4. Therapeutics designed to intervene in viral entry by blocking HIV attachment to CD4, may not work if entry mechanisms independent of CD4 occur frequently in vivo. A range of cell-surface molecules as well as CD4 can bind gp120, yet few act as receptors for HIV infection, indicating that passive attachment to the cell surface is not sufficient to confer virus entry. In vitro, HIV entry independent of CD4 has frequently been described, although this route to infection is usually inefficient. Variants of HIV-1 and HIV-2 that infect CD4-negative cell types more efficiently can be selected in vitro. However, there is currently no evidence that such variants evolve in vivo. Furthermore, present knowledge suggests that few CD4-negative cells types are productively infected in vivo. It is thus unlikely that CD4-independent infection significantly influences HIV induced pathogenesis in vivo.     

作用于HIV gp120的进入抑制剂研究进展

HIV-1病毒为包膜病毒,其感染靶细胞的第一步是由HIV包膜蛋白表面亚基gp120与靶细胞上的CD4分子和辅助受体（趋化因子受体CCR5或CXCR4等）结合,导致gp41的构型发生改变,启动病毒包膜与靶细胞膜的融合。与gp120相结合的一些抗体、蛋白、多糖、多肽和小分子化合物,都可能影响HIV-1病毒包膜和靶细胞膜融合的过程,从而起到抗HIV-1病毒的作用。该文对近年来以HIV gp120为靶点的HIV进入抑制剂的研究进展进行综述。     

Progress in targeting HIV-1 entry

Current HIV entry inhibitors target the binding of the viral envelope glycoprotein gp120 to cellular CD4 and co-receptors, or block a late stage of the fusogenic activation of adjacent gp41. New targets are suggested by the role of cell surface protein disulfide isomerase (PDI), which attaches to the primary receptor CD4 close to the gp120-binding site. This could enable PDI to reduce gp120 disulfide bonds, which triggers the major conformational changes in gp120 and gp41 required for virus entry. Inhibiting cell surface PDI prevents HIV-1 entry. The new potential targets outlined are PDI activity as well as the sites of PDI-CD4 and PDI-gp120 interaction.     

HIV-1 (co)receptors: implications for vaccine and therapy design

The ultimate aim of therapy or vaccine design against HIV is to eliminate ongoing virus replication or prevent HIV infection. The task at hand is daunting given the wide array of HIV variants circulating and the immense degree of variation found within the virus, especially in the envelope glycoprotein. HIV utilizes the CD4 receptor and a range of 7 transmembrane chemokine coreceptors for cell entry, specifically CCR5 and CXCR4. These receptors provide a number of targets for therapy design, however, the finding that multiple receptors allow for viral entry suggest that targeting one may cause the virus to swirch to using another receptor. The molecular interactions directing coreceptor usage are complex and can involve the same modifications associated with escape from the effect of neutralizing antibodies (NAbs), indicating that they are not unrelated and can in all liklihood impact on each other. Furthermore, a large array of other receptors, other than CD4, CCR5 and/or CXCR4 can interact with HIV with consequences for HIV tranmssion as well as disease progression.     

新型抗艾滋病药物——HIV进入抑制剂的研究进展

HIV与靶细胞融合的过程是药物干预的重要环节。融合过程主要由H IV包被蛋白表面亚基gp120和跨膜亚基gp41介导。H IV gp120与靶细胞上的CD4分子和辅助受体(趋化因子受体CCR5或CXCR4等)结合,导致gp41的构型发生改变,启动病毒包膜与靶细胞膜的融合。在融合过程中,病毒和靶细胞上的这些蛋白和受体均可作为药物的作用靶点,寻找抑制H IV进入靶细胞的药物用来治疗H IV感染和艾滋病。作用于gp41的肽类药物T-20已被美国FDA批准上市,表明继逆转录酶抑制剂和蛋白酶抑制剂后,H IV进入抑制剂作为第3类抗H IV药物开始在临床上应用。作为一种新机制的抗H IV药物,H IV进入抑制剂单独或与逆转录酶抑制剂和蛋白酶抑制剂联合应用,将有助于提高药物的疗效,降低毒副作用,并可望挽救对现有抗H IV药物耐药的艾滋病病人的生命。该文综述了近年来H IV进入抑制剂的研究进展。     

Targeting HIV-1 gp41-induced fusion and pathogenesis for anti-viral therapy

HIV gp41 is a metastable protein whose native conformation is maintained in the form of a heterodimer with gp120. The non-covalently associated gp41/gp120 complex forms a trimer on the virus surface. As gp120 engages with HIV's receptor, CD4, and coreceptor, CXCR4 or CCR5, gp41 undergoes several conformational changes resulting in fusion between the viral and cellular membranes. Several lipophilic and amphiphilic domains have been shown to be critical in that process. While the obvious function of gp41 in viral entry is well-established its role in cellular membrane fusion and the link with pathogenesis are only now beginning to appear. Recent targeting of gp41 via fusion inhibitors has revealed an important role of this protein not only in viral entry but also in bystander apoptosis and HIV pathogenesis. Studies by our group and others have shown that the phenomenon of gp41-mediated hemifusion initiates apoptosis in bystander cells and correlates with virus pathogenesis. More interestingly, recent clinical evidence suggests that gp41 mutants arising after Enfuvirtide therapy are associated with CD4 cell increase and immunological benefits. This has in turn been correlated to a decrease in bystander apoptosis in our in vitro as well as in vivo assays. Although a great deal of work has been done to unravel HIV-1 gp41-mediated fusion mechanisms, the factors that regulate gp41-mediated fusion versus hemifusion and the mechanism by which hemifusion initiates bystander apoptosis are not fully understood. Further insight into these issues will open new avenues for drug development making gp41 a critical anti-HIV target both for neutralization and virus attenuation.     

Virus attachment and entry offer numerous targets for antiviral therapy

In 2003 the first virus entry inhibitor, the anti-HIV peptide T20 (Fuzeon, enfuvirtide), was approved for treatment of advanced Human Immunodeficiency Virus Type 1 infection. T20 is an unconventional antiviral drug, as it does not target a viral replicase or protease but a conformational transition within the HIV1 fusion protein gp41 required for virus-cell membrane fusion. Beyond membrane fusion, numerous early drug targets have been identified that will allow for large scale screening or structure-based drug design. The first encounter of the virus with the host might be through binding to attachment receptors, such as the C-type lectins DC- and L-SIGN, which might play an important role of infection for a large number of enveloped viruses by capturing, concentrating and transmitting infectious virions. Once a virus reaches its target cell, a cascade of events generally starting with the interaction of viral envelope glycoproteins with specific entry receptors and co-receptors is necessary in order to trigger the virus-cell membrane fusion. The present review will highlight recent advances in the identification of new drugs and targets at the level of virus entry for three major human pathogens accounting for several hundred million infections worldwide: HIV, Dengue Virus and Hepatitis C virus.     

Properties of HIV envelope expressed in the presence of SPC3, an Env-derived peptide drug under phase II clinical trials

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4⁺ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4⁺ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.     

Potential drug targets on the HIV-1 envelope glycoproteins,gp120 and gp41

HIV-1 entry is an attractive target for anti-HIV-1 therapy. However, there are no entry inhibitors approved for the clinical treatment of HIV-1 infection. This is likely to be changed in the near future since promising HIV-1 entry inhibitors, such as T20 and some chemokine receptor antagonists, are in the pipeline to join the repertoire of anti-HIV-1 therapeutics. This review will focus on what might be potential targets on the key components of the viral entry machinery, gp120 and gp41. These two molecules are the viral proteins responsible for HIV-1 entry. Binding to CD4 induces a series of structural changes in gp120 and allows it to interact with chemokine receptors. The receptor binding eventually triggers conformational changes in gp41, which result in the formation of a fusion active molecule to attack the cell membrane. The structural and functional motifs that operate this delicate fusion machinery could become the Achilles' heel of the virus.     

The use of Envelope for HIV therapeutics: from vaccines to co-receptors

The Envelope protein (Env) of the human immunodeficiency virus (HIV) has been a primary target and tool for antiviral drug and vaccine development since the discovery of the virus. The study of Env has led to a knowledge of the virus life-cycle, structure and immunological response that has helped lead HIV-directed therapeutic strategies in new, unexpected directions. In particular, the discovery of chemokine receptors as the HIV co-receptors in 1996 is allowing a new generation of antiviral and vaccine candidates to be developed that are based on the ability to block the function of Env in mediating viral entry. This review discusses the primary roles of Env throughout its course of investigation as a tool for vaccine development and as a target for drug screening, emphasising the recent role of Env in the discovery and exploration of the co-receptors. The use of gp120/chemokine binding assays, chemokine receptor structure-function studies and co-receptor polymorphisms are discussed in the context of the development of high-throughput screening assays, creation of immunogens with enhanced vaccine potential and targeting of the co-receptors CCR5 and CXCR4 by small-molecule inhibitors of HIV entry.     

Small-molecule HIV-1 entry inhibitors targeting gp120 and gp41: a patent review (2010-2015)

Introduction: It is essential to discover and develop small-molecule HIV-1 entry inhibitors with suitable pharmaceutical properties.

Areas covered: We review the development of small-molecule HIV-1 entry inhibitors as evidenced in patents, patent applications, and related research articles published between 2010 and 2015.

Expert opinion: HIV-1 Env gp120 and gp41 are important targets for development of HIV-1 entry inhibitors. The Phe43 pocket in gp120 and the highly conserved hydrophobic pocket on gp41 NHR-trimer are important targets for identification of HIV-1 attachment and fusion inhibitors, respectively. Compounds that bind to Phe43 pocket can block viral gp120 binding to CD4 on T cells, thus inhibiting HIV-1 attachment. However, most compounds targeting Phe43 pocket identified so far are HIV-1 entry agonists with the ability to enhance infectivity of HIV-1 in CD4-negative cells. Therefore, it is essential to identify HIV-1 entry antagonist-based HIV-1 attachment/entry inhibitors. Compounds binding to the gp41 hydrophobic pocket may inhibit CHR binding to the gp41 NHR trimer, thus blocking six-helix bundle formation and gp41-mediated virus-cell fusion. However, most lead compounds targeting this pocket have low potency, possibly because the pocket is too big or too deep. Therefore, it is necessary to identify other pockets in gp41 for developing HIV-1 fusion/entry inhibitors.