HIV microbicides: state-of-the-art and new perspectives on the development of entry inhibitors

1141 ISSN 1756-8919 Future Med. Chem. (2010) 2(7), 1141–1159 10.4155/FMC.10.203 © 2010 Future Science Ltd Review HIV infects more than 15,000 people every day. Most of these new infections are sexually transmitted and, so, compounds acting at a mucosal level and impeding infection would have a major epidemiological impact. Infections can be prevented by vaccines or by substances that block the penetration of pathogens in the body. In the first case we use adjuvanted antigens to elicit an immune response that will be able to avoid the insurgence of disease. It is important to notice that vaccines do not necessarily prevent infection (sterilizing immunity). Rather, these compounds impede disease: vaccine-stimulated immune responses modify the natural history of infection, avoiding the appearance of full-blown disease. Notably, with the exception of the results for the recent RV144 trial, the so-called ‘Thai trial’ [1,2], which showed a very modest possible preventative effect, no vaccine has worked to impede HIV infection, and no effective vaccine is foreseeable in the near future. The second way to prevent infection is to block the penetration of a pathogen in the body. This modality does not imply active participation of the immune response; rather, physical and/or chemical means are used to impede the contact between pathogens and the host cell, and the subsequent penetration of the pathogen into such cells. In the case of HIV, the simplest preventative device is a condom. If properly used, condoms are highly effective in impeding infection. Nevertheless, possible ethical considerations aside, cultural factors often keep men and women from using condoms, or women from asking their partners to use such devices, in many parts of the world. Finally, it has been repeatedly shown that reduced risk of HIV transmission is only associated with consistent but not with occasional condom use. These considerations, the evidence of the ever-growing spread of new infections despite the availability of condoms and the objective difficulty in designing effective vaccines, led to the exploration of the ability of microbicides to prevent sexual transmission of HIV [3–7]. Microbicides can be classified into five different groups: nSurfactants; nVaginal milieu protectors; nViral entry inhibitors; nAgents that target viral replication (reverse transcriptase inhibitors); nOther compounds, whose mechanisms are unknown [4]. Surfactants can disrupt membranes nonspecifically and have been evaluated as topical microbicides. The protectors of vaginal milieu either operate as direct acidifying agents or as enhancers of lactobacilli production. Inhibitors of viral entry are a broad class of molecules, which can target either host cells or viral targets, and can block different steps of the pathways HIV microbicides: state-of-the-art and new perspectives on the development of entry inhibitors Since the discovery of HIV at the beginning of the 1980s, numerous efforts have been devoted to the search of an efficient vaccine. There are at least 25 drugs available for HIV treatment, but no cure is available. The observation that therapy for HIV disease is life long and that these drugs are associated with a number of side effects underlines the need for approaches aimed at preventing rather than treating infection. Additionally, the economic burden of treatment for the HIV infection occupies an increasing share of healthcare expenditure, making current practices likely to become difficult to sustain in the long run. Unfortunately, no effective vaccine for this disease is foreseeable in the near future. Microbicides could be an alternate way to build preventative approaches to HIV infection. Strategies based on preventing the virus from reaching its target cells seem to have some room for development and application. In this review we explore the state-of-the-art of available microbicides, focusing on HIV entry inhibitors. In addition, we discuss new compounds that show anti-HIV activity, which could be effective candidates. Jose J Reina1, Anna Bernardi1, Mario Clerici2,3 & Javier Rojo†4 1Universita’ degli Studi di Milano, Dipartimento di Chimica Organica e Industriale and CISI, via Venezian 21, 20133 Milano, Italy 2Universita’ degli Studi di Milano, Dipartimento di Scienze e Tecnologie Biomediche,via Flli Cervi 93, 20090 Segrate, Italy 3Don C. Gnocchi ONLUS Foundation IRCCS, Via Capecelatro 66, 20148 Milano, Italy 4Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC – Universidad de Sevilla, Av. Américo Vespucio 49, 41092 Sevilla, Spain †Author for correspondence: Tel.: +34 954 489 568 Fax: +34 954 460 565 E-mail: [email protected] For reprint orders, please contact [email protected] Review | Reina, Bernardi, Clerici & Rojo Future Med. Chem. (2010) 2(7) 1142 future science group used by HIV to cross mucosal barriers and to infect CD4+ lymphocytes, (e.g., the attachment of HIV to host cells, the fusion of virus and host cell membranes, or the entry of HIV into host cells). More than 70 preclinical and 50 clinical trials have been performed using compounds that fall in each of the five classes. In the preclinical Phase, in vitro assays and cervical explants models are used; some clinical experimentation has proceeded to Phase III, the evaluation of possible efficacy. In brief, none of the compounds that reached Phase III has demonstrated any effect on the prevention of infection [8–10]. HIV entry process & potential molecular targets for antiviral therapy HIV entry in CD4+ cells is a very complex process that proceeds through a series of steps (FiguRe 1): ngp120, expressed on the viral surface, binds to the CD4 molecule present on host cells; nThis process induces a conformational change in gp120 that allows it to bind either CCR5 or CXCR4, the main co-receptors for HIV; nAt this point, a rearrangement in a second viral protein, gp41, exposes on the surface of HIV a ‘fusion peptide’; nThe fusion peptide activates the complex fusion between the virus and the host cell membranes. In 2000, it was suggested that gp120 recognition at the mucosal level by the dendritic cell membrane lectin, specific ICAM-3 grabbing nonintegrin (DC-SIGN) may contribute to infection by promoting viral transmission and/or by inducing a signaling cascade, which results in immunosuppression [11–17]. Based on previously described steps, molecules binding to the host cell receptors CD4, CCR5, CXCR4, or possibly to DC-SIGN, could act as inhibitors of viral entry and stop the initial events leading to productive infection. A similar effect can be obtained by targeting the viral envelope and blocking, for instance, gp120 or the ‘fusion peptide’. All these are, as a consequence, very interesting approaches for the prevention of infection [18–23]. Indeed, as will be discussed in the following sections, much research has been dedicated to the discovery of entry inhibitors and many candidates have emerged for drug development. Negatively charged polyanions capable of binding to the surface of HIV were found to prevent infection in vitro. The fact that these compounds, besides being effective in vitro, are also easy to produce and economical, made them perfect potential candidates. Nevertheless, clinical trials of polyanions, including dextran sulfate and cellulose sulfate, failed to demonstrate any protection in vivo and, rather disappointingly, even increased plasma HIV viral load of infected individuals. Similarly, recent results from a Phase III clinical trial of the anionic inhibitor PRO-2000 have been disappointing [24]. Lectins targeting viral glycoproteins and potentially able to inhibit the binding process between receptors at the target cell membranes and the virus at the initial steps of infection, have been proven to be efficient in vitro, but their ability to modulate HIV infection in vivo has not yet undergone any clinical trial. Human and/or humanized monoclonal antibodies endowed with the ability to bind the CD4 binding domain and neutralize viral infectivity have been tested in vitro and in macaque models. In the animal model, these antibodies (e.g., B12, 2F5, 2G12 and 4E10) showed a promising, although not total, ability to prevent infection (clinical trials have not yet been started). CCR5 inhibitors, the newest class of antivirals used in clinical practice, are potentially very important, as viruses that utilize this co-receptor are associated with almost the totality of mucosally transmitted HIV infections. CCR5 inhibitors were tested in the macaque model and were shown to effectively block vaginal infection (clinical trials based on the utilization of these molecules as mucosal tools to prevent infection have not yet started). Notably, infection can also be prevented in vitro by the use of high doses of RANTES, the chemo kine that binds CCR5. This observation led to the develop ment of a number of RANTES analogues that are endowed with potent anti viral activity. Some of these analogues have been shown to protect macaques against vaginal infection. Finally, peptides designed to interfere with the processes resulting in the exposure of the fusion peptide on the surface of HIV (e.g., C52L) can convincingly prevent infection in the macaque model. Nevertheless, the effect is only observed in the presence of extremely elevated (100 mM) concentrations of drug. The development of compounds with the ability to prevent mucosal infection has also been slowed down by a number of other practical HIV microbicides: state-of-the-art & new perspectives on the development of entry inhibitors | Review www.future-science.com 1143 future science group issues. To summarize, an effective compound, besides being able to prevent infection, will also have to be: nSafe and nontoxic; nDevoid of inflammatory activity; nResistant to seminal fluid, to vaginal pH and body temperature; nEasy and simple to use (e.g., non oily and nonleaking); nEconomical; nSimple to produce and store; nEndowed with durable activity. Finally, even if receptive anal intercourse (RAI) is less frequent than vaginal intercourse (RVI), the median estimate of transmission risk for RAI is approximately 20-fold higher than that observed for RVI. These considerations indicate that the design of compounds able to prevent sexual transmission of HIV altogether will need to take into account the necessity of protecting both the genital tract and the rectum. In the following sections we will discuss literature data concerning the discovery and development of HIV entry inhibitors. Molecules will be grouped according to the location of their target either on the host cell membrane or the viral envelope. Finally, recent reports exploring the potential for the combination of diverse entry inhibitors and of entry inhibitors with other microbicides will be discussed. Host cell targets n CD4 inhibitors CD4 is the main receptor for HIV-1 entry and, as a consequence, is a privileged target for strategies for preventing the infection of target cells. However, most antiviral compounds were targeted against the CD4 binding site of gp120 rather than against CD4 itself (gp120-targeting compounds are reviewed in the appropriate section). One of the few inhibitors targeting CD4 is an anti-CD4 humanized monoclonal antibody, known as ibalizumab or TNX-355 [25,26]. This molecule was developed as a potent in vitro inhibitor of HIV and has successfully completed Phase II clinical trials. Ibalizumab does not work by preventing gp120 binding to CD4, but appears to decrease the flexibility of CD4 and to hinder access of CD4-bound gp120 to the CCR5 and CXCR4 co-receptors. Recently, a bifunctional molecule comprising ibalizumab and two fusion inhibitor peptides has been described [27]. This novel bifunctional inhibitor was reported to display improved potency and favorable pharmacokinetic properties and may offer a novel approach to block both CCR5and CXCR4-using viral variants. n Co-receptor inhibitors The most important HIV-1 entry inhibitors targeting one of the cell membrane co-receptors (CXCR4 and CCR5) are shown in Table 1. Antagonists of the CCR5 co-receptor were CCR5 CXCR4 gp41 gp120 gp41 HIV Recognition and binding Fusion and penetration CD4 Core Future Med. Chem. © Future Science Group (2010) Figure 1. HIV-1 entry steps and some potential targets. Review | Reina, Bernardi, Clerici & Rojo Future Med. Chem. (2010) 2(7) 1144 future science group investigated more thoroughly because individuals carrying a natural deletion (Δ32) in the CCR5 gene were found to be highly resistant to infection by CCR5-tropic strains and apparently healthy [28]. Furthermore, with few well-documented exceptions, primary infection is only supported by CCR5-directed strains of the virus. Tropism switch was found to occur in a high number of HIV infections and CXCR4-tropic variants may emerge when the CCR5-tropic virus is suppressed. This phenomenon, is nevertheless seen in the chronic phase of HIV infection only. In addition, systemic use of CXCR4 antagonists is likely to lead to adverse effects by suppressing the physiological functions of this receptor, which is involved with the migration and development of hematopoietic cells. Hence, research on HIV entry inhibitors has focused mostly on CCR5 antagonists. Nonetheless, CXCR4 antagonists have also been investigated. In particular, two compounds have been described, AMD3100 (Plerixafor) [29] and KRH-1636 [30] (entries 1 & 2; Table 1, FiguRe 2), the most advanced of which, AMD3100, was found to possess little antiviral activity, but was approved by the US FDA (December 2008) for stem cell mobilization [31]. It was recently reported that the Cu2+ complex of AMD3100 shows improved potency relative to the starting compound, which was related to the increased residence time of the copper complex on CXCR4 [32]. Recent investigations focused on the optimization of a previous lead (AMD070; FiguRe 2) [33] have led to the identification of an orally bioavailable compound with subnanomolar anti-HIV-1activity (1; FiguRe 2) [34]. Many CCR5 small-molecule antagonists have been reported [22,23,35–37]. Early attempts included Takeda’s TAK-779 [38,39] and Merck’s CMPD-167 (Table 1; entry 3&4) [40,41]. Three compounds, maraviroc [42,43], vicriviroc [44,45] and aplaviroc [46] have reached clinical trials (entries 5–7; Table 1 & FiguRe 2). Aplaviroc belongs to a class of spirodiketopiperazine derivatives (FiguRe 2). Its development was halted in Table 1. HIV-1 entry inhibitors targeting co-receptors in CD4+ cells. Entry Generic name Experimental code Company Molecule type Target Notes Ref. 1 Plerixafor AMD3100 Small molecule CXCR4 Little antiviral activity shown in clinical trials evolved into a stem cell-mobilizing agent. [29,31] 2 KRH-1636 Kureha Chem Industry Small molecule CXCR4 [30] 3 TAK-779 TAK-220 TAK-652 Takeda Chemical Ind. Small molecule CCR5 TAK-779 currently not further developed. TAK-652 currently developed by Tobira Therapeutics as TBR-652. [31,38,39,140] 4 CMPD167 Merck Small molecule CCR5 [40,41] 5 Maraviroc UK-427,857 ViiV Healthcare Pfizer Small molecule CCR5 US FDA approved Aug 2007 [42,43] 6 Vicriviroc SCH-D; SCH-417690 Schering-Plough Corporation (now Merck) Small molecule CCR5 Experimental Phase II ongoing. Possible association with increased risk for malignancies. [31,44,45] 7 Aplaviroc GlaxoSmithKline Small molecule CCR5 Discontinued for liver toxicity. [46] 8 PRO 140 Progenics Pharmaceuticals Monoclonal antibody CCR5 Phase II trial concluded. [60] 9 Modified RANTES (AOP-RANTES) Modification of RANTES, a CCR5-binding chemokine CCR5 [59] HIV microbicides: state-of-the-art & new perspectives on the development of entry inhibitors | Review www.future-science.com 1145 future science group 2005 for dose-related liver toxicity problems. However, recent reports have concluded that coadministration of aplaviroc with the immunomodulatory drug rapamycin, which is able to reduce CCR5 density (receptors/cell), enhances the antiviral activity of aplaviroc, allowing a reduction of active concentration to nontoxic doses (as much as 25-fold in vitro) and could provide an effective means to control infection [47]. Optimization of aplaviroc’s spirodiketopiperazine framework with improved pharmacokinetic profiles has been reported very recently by Ono Pharmaceuticals [48]. Vicriviroc contains a piperazine scaffold (FiguRe 2) and was developed by ScheringPlough starting from screening data; it exhibited N N H N N N N 1 F F N HO N N N N N N N N N O F F F O N N H N O H O O O H O H N H N N N H N H N N H N H N N H O H N N H O N H N HH 2 N O NN N N O H O F N N H N N N N N H 2 Maraviroc (CCR5) Vicriviroc (CCR5) Aplaviroc (CCR5) CMPD 167 (CCR5) KRH-1636 (CXCR4) Plerixafor (AMD3100) (CXCR4) AMD070 or AMD11070 (CXCR4) (CXCR4) Figure 2. Small-molecule co-receptor antagonists as inhibitors of HIV entry in CD4+ cells. The receptor targeted is in brackets. Review | Reina, Bernardi, Clerici & Rojo Future Med. Chem. (2010) 2(7) 1146 future science group excellent selectivity for CCR5 receptors but clinical trials were recently interrupted, at least for the treatment of naive patients [201]. Maraviroc is the only CCR5 inhibitor currently approved by the FDA (Selzentry). It is based on a tropane scaffold (FiguRe 2) and its development, starting from high-throughput screening (HTS) leads, has been reviewed elsewhere [42,43]. The development and optimization of a new class of CCR5 antagonists replacing the tropane core of maraviroc by piperidine was recently described by Pfizer [49,50]. Other recent reports on the discovery and optimization of novel CCR5 antagonists as inhibitors of HIV-1 infection have uncovered selective and potent analogs, which include structural elements from the previous chemotypes [51–54] or novel, gem-disubstituted azacycles and one octahydro pyrrolo[3,4-c]pyrrole scaffold [55]. The different CCR5 antagonists, despite being rather diverse in shape and electronic properties, are all predicted to bind to a putative hydrophobic binding pocket located in the transmembrane domain of the receptor [56–58]. Macromolecules capable of antagonizing CCR5 include modifications of its natural ligand, the CCR5 chemokine also known as RANTES [59]. Notably, RANTES is not the only natural ligand of CCR5, as other chemokines, including MIP-1a, MIP-1b (its variant LD78b) and MCP2 can bind this receptor. Monoclonal antibodies have also been developed that recognize epitopes on CCR5 [60]. Their use in combination with small molecule antagonists has been recently reported as a promising new strategy for anti-HIV-1 therapy [61,62]. The success of some compounds such as maraviroc, approved by the FDA for clinical use, validates viral entry inhibitors as a new class of antiretroviral drugs for clinical use. However, some problems remain. The HIV envelope glyco protein is highly variable with diverse genotypes and, hence, the susceptibility of viral strains to different antagonists targeting the viral envelope glycoprotein may vary significantly. In addition, for the appropriate selection of treatment with co-receptor antagonists, HIV patients must initially be analyzed for the tropism of the virus they harbour. Moreover, because CCR5 and CXCR4 co-receptors are host cellular targets, the long-term effects and safety of drug use must also be closely monitored. With increasing knowledge of the HIV entry process, other targets involved in different stages of viral entry have emerged and will be discussed in the following sections. n DC-SIGN inhibitors A potential therapeutic target was recently identified in DC-SIGN, a receptor involved in the early stages of HIV infection [11]. DC-SIGN is a tetrameric calcium-dependent (C-type) lectin, expressed by dendritic cells (DC), which specifically recognizes highly glycosylated structures displayed at the surface of several pathogens [63–66]. Recognition by DC-SIGN was reported to play a key role in HIV transmission and is considered an interesting new target for the design of anti-viral agents [67–71]. DC-SIGN+ immature DCs, located in vaginal, cervical and rectal mucosae are among the first cell types to encounter HIV during sexual transmission. DC-SIGN expressed by DC at mucosal tissues captures HIV at low titre by binding the envelope glycoprotein gp120. The lectin appears to act as an attachment factor, rather than an entry receptor, binding and concentrating HIV on the cell surface. Initial reports described two different pathways that may be involved in the transmission of DC-SIGN-bound HIV to T lymphocytes [72,73]. The first one, responsible for short term HIV transfer (24 h after HIV exposure), involves virion internalization into intracellular compartments where the virus is protected from degradation and retains a high infective capacity during DC migration to lymphoid tissues (the ‘Trojan horse model’) [74]. The second, involved in long-term HIV transfer (72 h after exposure), follows DC infection in cis by transfer of DC-SIGN-bound virus to canonical HIV entry receptor (CD4 and CCR5) resulting in infected DC with a continuous production of virus for the T-cells [73,75]. In both cases, upon arrival at lymphoid tissues DC efficiently transmit HIV to CD4+ T lymphocytes, a process called infection in trans. These models have been challenged [76] and other reports have shown that the majority of virus internalized by DC is regularly processed by major histocompatibility complex presentation [77,78]. On the other hand, HIV sequestration by and stimulation of DC-SIGN was reported to help HIV evade immune responses and spread to cells by alternative pathways, such as the triggering of activities required for HIV replication [79] or induction of immunosuppressive signals[12–17]. A timely and comprehensive discussion of the role of DC-SIGN in HIV infection has recently been published [80]. Despite uncertainties about the mechanism of action of this lectin, various groups are working to design molecules capable of blocking the interaction between DC-SIGN and HIV, with HIV microbicides: state-of-the-art & new perspectives on the development of entry inhibitors | Review www.future-science.com 1147 future science group the goal of disclosing a new strategy to prevent HIV transmission and infection of the host. A handful of compounds have been described so far (Tables 2 & 3). The main natural ligands recognized by the carbohydrate recognition domain of DC-SIGN are the high mannose glycan, (Man)9(GlcNAc)2 also known as Man9, and a group of fucosylated oligosaccharides bearing Lewis-type epitopes [74]. Highly mannosylated polyvalent compounds, structural analogues of Man9 terminal dior trisaccharides, and mimics of the Lewis-× trisaccharide have been investigated and found to be capable of antagonizing binding of gp120 to DC-SIGN. Although most of the work was concerned with mannose or mannose analogues, a fucose-based antagonist (6) was recently reported (entry 8; Table 2) [70]. The ligand is based on a a-fucosylamide anchor and inhibits DC-SIGN binding to mannosylated bovine serum albumin (BSA) with potency Table 2. Known monovalent ligands of dendritic cell-specific ICAM-3 grabbing nonintegrin. Entry Compound IC50 (mM) Inhibition (%) Assay Ref. 1 O OH HO CH 2 OH OH HO H 2 NH 2 C 2 0.35†SPR (gp120 chip) [69] 2 Mana-1,2-Mana-1,2-Man 73‡Microarrays [141] 3 Mana-1,2-Mana-1,6-Mana-1,6-Man 73‡Microarrays [141] 4 N H N SN S H N N Me Me Me O 3 1.6 x 10-3§ Competition with immobilized DC-SIGN [68] 5 O O ONH2 HO HO HO OH MeOOC MeOOC 4 0.62¶Ebola cis-infection model [67] 0.6# 1.0†† SPR (inhibition of ManBSA binding) [86] 6 O O HO HO HO OH MeOOC MeOOC O O HO HO OH ONH 2 5 0.05# 0.125†† SPR (inhibition of ManBSA binding) [86] 7 Lewis-x trisaccharide 0.8#,‡‡ SPR (inhibition of ManBSA binding) [70] 8 O OH OH Me OH H N HH O HN O COOMe OH HO 6 0.35#,‡‡ SPR (inhibition of ManBSA binding) [70] †IC50 of mannose in the same assay 17 mM. ‡100% is Man9 in the same assay. §IC50 of ManNAc in the same assay 6.9 mM. ¶Mana-1,2-Mana-1-OCH2CH2NH2 is 1.9 mM in the same assay. #IC50 of mannose in the same assay 1.8 mM. ††IC50 of mannose in the same assay 2.5 mM. ‡‡IC50 of fucose in the same assay 1.2 mM. DC-SIGN: Dendritic cell-specific ICAM-3 grabbing nonintegrin; Man: Mannose; ManBSA: Mannose bovine serum albumin; SPR: Surface plasmon resonance. Review | Reina, Bernardi, Clerici & Rojo Future Med. Chem. (2010) 2(7) 1148 future science group similar to, or slightly better than, the natural ligand’s Lewis-x. This molecule represents an interesting lead for further optimization. Multivalent presentation of mannose using hyperbranched dendritic polymers of Boltorn type were shown to bind DC-SIGN and inhibit interaction with gp120-coated surface plasmon resonance (SPR) chips (entry 1; Table 3) [81–83]. The Wong group reported oligo mannose dendrons that display complex oligo mannoses in high density (entries 2 & 3; Table 3) and inhibit binding of gp120 to recombinant dimeric DC-SIGN with IC50 in the nanomolar range [84]. These systems are excellent models of DC-SIGN inhibition, but their practical application as therapeutic agents seems unlikely, given the complexity of the oligosaccharides used. Penadés, Alcami and their groups have reported that gold nanoparticles (GNP) displaying various linear and branched mannosyl oligosaccharides (Manno-GNPs, entries 4 & 5; Table 3) are potent inhibitors of DC-SIGN mediated HIV trans-infection of human activated peripheral blood mono-nuclear cells [85]. However, in vivo use of GNPs raises some concerns, mostly because of the potential toxicity produced by gold accumulation. Noncarbohydrate inhibitors with IC50 values in the low micromolar range, such as compound 3 (entry 4; Table 2), were identified by Kiessling and Borrok via HTS of approximately 36,000 compounds from commercial libraries [68]. Additional results from the Fleet group indicate that 2-C-substituted branched d-mannose analogues such as compound 2 (entry 1; Table 2) bind to DC-SIGN with significantly greater affinity than mannose [69]. We have described two glycomimetic compounds, 4 and 5 (entries 5 & 6 respectively; Table 2), which are structural mimics of linear diand tri-mannosides and exhibit moderate anti-infective action against DC-SIGN-mediated infections by HIV and Ebola [67,86]. A tetravalent dendron con-A tetravalent dendron containing four copies of the linear trimannoside mimic 5 (entry 7; Table 3) was found to inhibit DC-SIGN mediated HIV trans-infection of CD4+ T lymphocytes at low micromolar range. The anti-infective action is exerted upstream of Table 3. Affinity comparison for polyvalent ligands of dendritic cell-specific ICAM-3 grabbing nonintegrin. Entry Type of sugar Support Valency IC50 Rel. Pot. Assay Ref. 1 Man 3G Boltorn dendrimer 32 0.337 µM 118 Ebola cis-infection model [82] 0.1 µM SPR (high-density DC-SIGN ECD) [83] 50 µM SPR gp120 chip [81] 2 Man4 (Mana-1,2-Mana-1,2-Mana1,3-ManaR) 2G alkynyl dendrimer 9 0.020 µM†gp120 –Fc-DC-SIGN ELISA [84] 0.16 µM Competition with Man4-coated array 3 Man92G alkynyl dendrimer 9 0.008 µM†gp120 –Fc-DC-SIGN ELISA [84] 0.026 µM Competition with Man4-coated array 4 Mana-1,2-Man GNP 22 2‡–37§ nM HIV trans-infection model [85] 25 0.12 µM 100% inhibition 2.9 SPR gp120 chip [141] 5 Man4 (Mana-1,2-Mana-1,2-Mana1,3-ManaR) GNP 56 0.34‡– 0.83§ nM HIV trans-infection model [85] 6 Man Tetravalent boltorn dendron 4 50 µM HIV trans-infection model [86] 75 (mimic of Mana-1,2Mana-1,6-Mana) Tetravalent boltorn dendron 4 5 µM HIV trans-infection model [86] †Man is 8.5 mM in the same assay. ‡JR-Renilla viral strain (R5). §NL-Renilla viral strain (X4). DC-SIGN: Dendritic cell-specific ICAM-3 grabbing nonintegrin; ECD: Extracellular domain; GNP: Glyconanoparticle; Man: Mannose; SPR: Surface plasmon resonance. HIV microbicides: state-of-the-art & new perspectives on the development of entry inhibitors | Review www.future-science.com 1149 future science group cytokine involvement and, therefore, is independent of viral tropism, as shown by inhibition of a series of laboratory-adapted strains and primary isolates with different tropism. This tetravalent compound presents high solubility in physiologipresents high solubility in physiologi-presents high solubility in physiological media, a neglectable cytotoxicity and a longlasting effect and was found to be stable for at least 1 week at pH 5 (an acidic medium close to that found at the vaginal mucosa, pH 4). Timecourse studies showed that the antiviral effect of this compound persists for hours, even after the B-THP/ DC-SIGN+ cells used were exposed to HIV after washing out the antagonist. The mechanism of this inhibition after removal may be based on the persistency of the multivalent ligand on the receptor binding site (slow off-rate of the tetravalent compound from the protein). However, flow cytometry studies suggested that exposure of B-THP/DC-SIGN+ cells to this compound may also alter the observed cell surface concentration of DC-SIGN, possibly by induced endocytosis. Depletion of receptor membrane concentration is an interesting feature of this compound and it could at least partially account for its antiviral activity. Inhibition of DC-SIGN is not devoid of potential drawbacks. First and foremost, efficient inhibition of this lectin will prevent recognition of other pathogens besides HIV and, thus, may lead to a generalized reduction of the host immune response. This effect may be limited by topical, rather than systemic, use of inhibitors. Virus targets n gp120 binders Polyanions are one of the most popular groups of compounds to be formulated as microbicides due to their low cost and easy production. These compounds interact with the positively charged region of gp120, basically the conservative V3 loop, inhibiting the interaction with negative areas of the cell membrane surface. Different formulations of these compounds have reached clinical Phase trials (Table 4). PRO 2000/5 or PRO-2000, a formaldehyde-sodium 2-naphthalene sulfonate polymer, was recently demonstrated to be ineffective in Phase III clinical trials [24]. SPL7013, a lysine-based dendrimer with naphthalene disulfonic acid surface groups, also known as VivaGel™, is in Phase I/II clinical trials [87]. Dextrin sulfate (DxS) is also currently in clinical trials [88]. Some of these polyanions, in contrast to their very good in vitro activity, have failed to demonstrate efficacy in preventing HIV transmission in Phase III clinical trials, as was the case of cellulose sulfate (CS), terminated preterminated prematurely in early 2007 [89], and Carraguard [90]. Although some of these polyanion compounds enhance HIV infection, apparently this effect is not related to the nature of the polyanion, but to the assay conditions and therefore cannot be extrapolated [91]. CD4 is considered to be the main recepis considered to be the main recep-is considered to be the main receptor for the HIV-1. So, gp120-CD4 interaction has been one of the first targets against which anti-HIV drugs were developed. In 1990 it was reported that recombinant soluble CD4 (sCD4) protein was able to block HIV entry and subsequent infection in vitro [92]. However, Phase I/II clinical trials demonstrated that higher concentrations of this sCD4 were needed to inhibit infection in vivo using HIV primary isolates. Apparently, the binding of sCD4 to gp120 should produce conformational changes on the gp120 envelope protein, leading to the exposure of co-receptor binding sites. These changes could allow gp120 to bind the chemokine receptor at the cell surface, inducing virus–cell membrane fusion and virus entry [93]. This fact could explain the results found in the infection Table 4. Current status of polyanions as microbicides. Compound Administration Composition Company Current status Ref. PRO2000 Topical 0.5% gel Formaldehyde-sodium 2-naphthalenesulfonate polymer Indevus Pharmaceuticals Failed in Phase III trials [24] SPL7013 VivaGel (3% SPL7013 gel) Lysine dendrimer with naphtalene disulfonic acids Starpharma Phase I/II trials [87] Carraguard Gel from carrageenan Sulfated polysaccharide Ineffective in Phase III trials [90] Dextrin sulfate Oral or intravenous administration Polysulfate dextran Phase I/II trials [88] Cellulose sulfate (Ushecell) Topical 6% gel Polysulfate cellulose Polydex Pharmaceuticals Ineffective in Phase III trials [89] Review | Reina, Bernardi, Clerici & Rojo Future Med. Chem. (2010) 2(7) 1156 future science group 21 Esté JA, Telenti A. HIV entry inhibitors. Lancet 370(9581), 81–88 (2007). 22 Qian K, Morris-Natschke SL, Lee K-H. HIV entry inhibitors and their potential in HIV therapy. Med. Res. Rev. 29(2), 369–393 (2009). 23 Kuritzkes DR. HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS 4(2), 82–87 (2009). 24 Chisembele M, Crook A, Gafos M et al. PRO2000 is ineffective in preventing HIV infection: results of the MDP301 Phase III Microbicide trial (Abstract 87LB). Presented at: CROI. San Francisco, CA, USA, 16–19 February 2010. 25 Moore JP, Sattentau QJ, Klasse PJ, Burkly LC. A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4+ cells. J. Virol. 66(8), 4784–4793 (1992). 26 Kuritzkes DR, Jacobson J, Powderly WG et al. Antiretroviral activity of the anti-CD4 monoclonal antibody TNX-355 in patients infected with HIV type 1. J. Infect. Dis. 189(2), 286–291 (2004). n Important proof of concept. 27 Ji C, Kopetzki E, Jekle A et al. CD4anchoring HIV-1 fusion inhibitor with enhanced potency and in vivo stability. J. Biol. Chem. 284(8), 5175–5185 (2009). 28 Ford SL, Reddy YS, Anderson MT et al. Single-dose safety and pharmacokinetics of brecanavir, a novel human immunodeficiency virus protease inhibitor. Antimicrob. Agents Chemother. 50(6), 2201–2206 (2006). 29 Donzella GA, Schols D, Lin SW et al. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat. Med. 4(1), 72–77 (1998). 30 Ichiyama K, Yokoyama-Kumakura S, Tanaka Y et al. A duodenally absorbable CXC chemokine receptor 4 antagonist, KRH-1636, exhibits a potent and selective anti-HIV-1 activity Proc. Natl Acad. Sci. USA 100(7), 4185–4190 (2003). 31 De Clercq E. Antiviral drug discovery: ten more compounds, and ten more stories (Part B). Med. Res. Rev. 29(4), 571–610 (2009). 32 Khan A, Nicholson G, Greenman J et al. Binding optimization through coordination chemistry: CXCR4 chemokine receptor antagonists from ultrarigid metal complexes. J. Am. Chem. Soc. 131(10), 3416–3417 (2009). 33 Skerlj RT, Bridger GJ, Kaller A et al. Discovery of novel small molecule orally bioavailable C-X-C chemokine receptor 4 antagonists that are potent inhibitors of T-tropic (X4) HIV-1 replication. J. Med. Chem. 53(8), 3376–3388 (2010). 34 Gudmundsson KS, Sebahar PR, Richardson LD et al. Amine substituted N-(1H-benzimidazol-2ylmethyl)-5,6,7,8tetrahydro-8-quinolinamines as CXCR4 antagonists with potent activity against HIV-1. Bioorg. Med. Chem. Lett. 19(17), 5048–5052 (2009). 35 Maeda K, Nakata H, Ogata H et al. The current status of, and challenges in, the development of CCR5 inhibitors as therapeutics for HIV-1 infection. Curr. Opin. Pharmacol. 4(5), 447–452 (2004). 36 Wang T, Duan Y. HIV co-receptor CCR5: structure and interactions with inhibitors. Infect. Disord. Drug Targets 9(3), 279–288 (2009). 37 Pulley SR. CCR5 antagonists: from discovery to clinical efficacy. In: Chemokine Biology - Basic Research and Clinical Application (Volume. 2). Kuldeep N, Gordon LL, Bernhard M (Eds.). Birkhauser Basel, Switzerland, 145–163 (2007). 38 Baba M, Nishimura O, Kanzaki N et al. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc. Natl Acad. Sci. USA 96(10), 5698–5703 (1999). n Article that opens the road to the development of CCR5 inhibitors. 39 Dragic T, Trkola A, Thompson DA et al. A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5. Proc. Natl Acad. Sci. USA 97(10), 5639–5644 (2000). n Basis allowing the development of CCR5 inhibitors. 40 Veazey RS, Klasse PJ, Schader SM et al. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature 438(3), 99–102 (2005). 41 Schröder C, Pierson RN, Nguyen B-NH et al. CCR5 blockade modulates inflammation and alloimmunity in primates. J. Immunol. 179(4), 2289–2299 (2007). 42 Wood A, Armour D. The discovery of the CCR5 receptor antagonist, UK-427,857, a new agent for the treatment of HIV infection and AIDS. Prog. Med. Chem. 43, 239–271 (2005). n Describes the discovery of the only CCR5 antagonist currently approved by the US FDA. 43 Sayana S, Khanlou H. Maraviroc: a new CCR5 antagonist. Expert Rev. Anti Infect. Ther. 7(1), 9–19 (2009). 44 Taga JR, Steensma RW, McCombie SW et al. Piperazine-based CCR5 antagonists as HIV-1 inhibitors. II. Discovery of 1-[(2,4-dimethyl-3-pyridinyl) carbonyl]-4methyl-4-[3(S)-methyl-4- [1(S)-[4-(trifluoro-methyl)phenyl] ethyl]-1-piperazinyl]- piperidine N1-oxide (Sch-350634), an orally bioavailable, potent CCR5 antagonist. J. Med. Chem. 44(21), 3343–3346 (2001). 45 Strizki JM. Tremblay C, Xu S et al. Discovery and characterization of vicriviroc (SCH 417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 49(12), 4911–4919 (2005). 46 Maeda K, Nakata H, Koh Y et al. Spirodiketopiperazine-based CCR5 inhibitor which preserves CC-chemokine/CCR5 interactions and exerts potent activity against R5 human immunodeficiency virus type 1 in vitro. J. Virol. 78(16), 8654–8662 (2004). 47 Latinovic O, Heredia A, Gallo RC, Reitz M, Le N, Redfield RR. Rapamycin enhances aplaviroc anti-HIV activity: implications for the clinical development of novel CCR5 antagonists. Antiviral Res. 83(1), 86–89 (2009). 48 Nishizawa R, Nishiyama T, Hisaichi K et al. Spirodiketopiperazine-based CCR5 antagonists: improvement of their pharmacokinetic profiles. Bioorg. Med. Chem. Lett. 20(2), 763–766 (2010). 49 Barber CG, Blakemore DC, Chiva J-Y, Eastwood RL, Middleton DS, Paradowski KA. 1-amido-1-phenyl-3piperidinylbutanes – CCR5 antagonists for the treatment of HIV: Part 1. Bioorg. Med. Chem. Lett. 19(4), 1075–1079 (2009). 50 Barber CG, Blakemore DC, Chiva J-Y, Eastwood RL, Middleton DS, Paradowski KA. 1-amido-1-phenyl-3piperidinylbutanes - CCR5 antagonists for the treatment of HIV: Part 2. Bioorg. Med. Chem. Lett. 19(5), 1499–1503 (2009). 51 Pryde DC, Corless M, Fenwick DR et al. The design and discovery of novel amide CCR5 antagonists. Bioorg. Med. Chem. Lett. 19(4), 1084–1088 (2009). 52 Duan M, Aquino C, Ferris R et al. [2-(4-phenyl-4-piperidinyl)ethyl]amine based CCR5 antagonists: derivatizations at the N-terminal of the piperidine ring. Bioorg. Med. Chem. Lett., 19(6), 1610–1613 (2009). HIV microbicides: state-of-the-art & new perspectives on the development of entry inhibitors | Review www.future-science.com 1157 future science group 53 Duan M, Aquino C, Dorsey GF, Ferris R, Kazmierski WM. 4,4-disubstituted cyclohexylamine based CCR5 chemokine receptor antagonists as anti-HIV-1 agents. Bioorg. Med. Chem. Lett. 19(17), 4988–4992 (2009). 54 Rotstein DM, Gabriel SD, Makra F et al. Spiropiperidine CCR5 antagonists. Bioorg. Med. Chem. Lett. 19(18), 5401–5406 (2009). 55 Lemoine RC, Petersen AC, Setti L et al. Evaluation of secondary amide replacements in a series of CCR5 antagonists as a means to increase intrinsic membrane permeability. Part 1: optimization of gem-disubstituted azacycles. Bioorg. Med. Chem. Lett. 20(2), 704–708 (2010). 56 Maeda K, Das D, Ogata-Aoki H et al. Structural and molecular interactions of CCR5 inhibitors with CCR5. J. Biol. Chem. 281(18), 12688–12698 (2006). n First comprehensive structural interpretation of the activity of CCR5 antagonists. 57 Kondru R, Zhang J, Ji C et al. Molecular interactions of CCR5 with major classes of small-molecule anti-HIV CCR5 antagonists. Mol. Pharmacol. 73(3), 789–800 (2008). 58 Wang T, Duan Y. Binding modes of CCR5-targeting HIV entry inhibitors: partial and full antagonists. J. Mol. Graph. Model. 26(8), 287–295 (2008). 59 Hartley O, Gaertner H, Wilken J et al. Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors. Proc. Natl Acad. Sci. USA 101(47), 16460–16465 (2004). 60 Trkola A, Ketas TJ, Nagashima KA et al. Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140. J. Virol. 75(2), 579–588 (2001). 61 Murga JD, Franti M, Pevear DC et al. Potent antiviral synergy between monoclonal antibody and small-molecule CCR5 inhibitors of human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 50(10), 3289–3296 (2006). 62 Ji C, Zhang J, Dioszegi M et al. CCR5 Small-molecule antagonists and monoclonal antibodies exert potent synergistic antiviral effects by cobinding to the receptor. Mol. Pharmacol. 72(1), 18–28 (2007). 63 Geijtenbeek TBH, Torensma R, Van Vliet SJ et al. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100(5), 575–585 (2000). 64 Feinberg H, Mitchell DA, Drickamer K, Weis W. Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294(5549), 2163–2166 (2001). n Describes the high-resolution x-ray structures of DC-SIGN complexes with both fucosylated and mannosylated oligosaccharides. 65 Mitchell DA, Fadden AJ, Drickamer K. A novel mechanism of carbohydrate recognition by the C-type Lectins DC-SIGN and DC-SIGNR. J. Biol. Chem. 276(31), 28939–28945 (2001). 66 van Kooyk Y, Geijtenbeek TBH. DC-SIGN: escape mechanism for pathogens. Nat. Rev. Immunol. 3(9), 697–709 (2003). 67 Reina JJ, Sattin S, Invernizzi D et al. 1,2-mannobioside mimic: synthesis, DC-SIGN interaction by NMR and docking, and antiviral activity. ChemMedChem 2(7), 1030–1036 (2007). 68 Borrok MJ, Kiessling LL. Non-carbohydrate inhibitors of the lectin DC-SIGN. J. Am. Chem. Soc. 129(42), 12780–12785 (2007). 69 Mitchell DA, Jones NA, Hunter SJ et al. Synthesis of 2-C-branched derivatives of d-mannose: 2-C-aminomethyl-d-mannose binds to the human C-type lectin DC-SIGN with affinity greater than an order of magnitude compared with that of d-mannose. Tetrahedron: Asymmetry 18(12), 1502–1510 (2007). 70 Timpano G, Tabarani G, Anderluh M et al. Synthesis of novel DC-SIGN ligands with an a-fucosylamide anchor. ChemBioChem 9(12), 1921–1930 (2008). 71 Ernst B, Magnani JL. From carbohydrate leads to glycomimetic drugs. Nat. Rev. Drug Discovery 8(8), 661–677 (2009). 72 Turville SG, Santos JJ, Frank I et al. Immunodeficiency virus uptake, turnover, and 2-phase transfer in human dendritic cells. Blood 103(6), 2170–2179 (2004). 73 de Witte L, Nabatov A, Geijtenbeek TBH. Distinct roles for DC-SIGN+-dendritic cells and langerhans cells in HIV-1 transmission. Trends Mol. Med. 14(1), 12–19 (2008). 74 Kwon DS, Gregorio G, Bitton N et al. DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity 16(1), 135–144 (2002). n Important work that clarifies the role of DC-SIGN in HIV infection. 75 Burleigh L, Lozach P-Y, Schiffer C et al. Infection of dendritic cells (DCs), not DC-SIGN-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to T cells. J. Virol. 80(6), 2949–2957 (2006). n Clarifies the role of DC-SIGN in HIV infection. 76 Boggiano C, Manel N, Littman DR. Dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of DC-SIGN. J. Virol. 81(5), 2519–2523 (2007). 77 Moris A, Nobile C, Buseyne F, Porrot F, Abastado JP, Schwartz O. DC-SIGN promotes exogenous MHC-I-restricted HIV-1 antigen presentation. Blood 103(7), 2648–2654 (2004). 78 Moris A, Pajot A, Blanchet F, Guivel-Benhassine F, Salcedo M, Schwartz O. Dendritic cells and HIV-specific CD4+ T cells: HIV antigen presentation, T-cell activation, and viral transfer. Blood 108(5), 1643–1651(2006). 79 Hodges A, Sharrocks K, Edelmann M et al. Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication. Nat. Immunol. 8(6), 569–577 (2007). 80 Steffen I, Tsegaye TS, Poehlmann S. Lectin-like interactions in virus-cell recognition: human immunodeficiency virus and C-type lectin interactions. In: Microbial Glycobiology, Structures, Relevance and Applications. Moran A, Holst O, Brennan P, von Itzstein M (Eds). Elsevier, NY, USA, 567–584 (2009). 81 Tabarani G, Reina JJ, Ebel C et al. Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding. FEBS Lett. 580(10), 2402–2408 (2006). 82 Rojo J, Delgado R. Glycodendritic structures: promising new antiviral drugs. J. Antimicrob. Chemother. 54(3), 579–581 (2004). 83 Lasala F, Arce E, Otero JR et al. Mannosyl glycodendritic structure inhibits DC-SIGNmediated Ebola virus infection in cis and in trans. Antimicrob. Agents Chemother. 47(12), 3970–3972 (2003). 84 Wang S-K, Liang P-H, Astronomo RD et al. Targeting the carbohydrates on HIV-1: interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc. Natl Acad. Sci. USA 105(10), 3690–3695 (2008). 85 Martinez-Avila O, Bedoya LM, Marradi M et al. Multivalent manno-glyconanoparticles inhibit DC-SIGN-mediated HIV-1 transinfection of human T cells. ChemBioChem 10(11), 1806–1809 (2009). 86 Sattin S, Daghetti A, Thépaut M et al. Inhibition of DC-SIGN-mediated HIV infection by a linear trimannoside mimic in a tetravalent presentation. ACS Chem. Biol. 5(3), 301–312 (2010). Review | Reina, Bernardi, Clerici & Rojo Future Med. Chem. (2010) 2(7) 1158 future science group 87 O’Loughlin J, Millwood IY, McDonald HM, Price CF, Kaldor JM, Paull JR. Safety, tolerability, and pharmacokinetics of SPL7013 gel (VivaGel): a dose ranging, Phase I study. Sex. Transm. Dis. 37(2),100–104 (2010). 88 Fletcher PS, Wallace GS, Mesquita PMM, Shattock RJ. Candidate polyanion microbicides inhibit HIV-1 infection and dissemination pathways in human cervical explants. Retrovirology 3, 46 (2006). 89 Van Damme L, Govinden R, Mirembe FM et al. Lack of effectiveness of cellulose 21 sulfate gel for the prevention of vaginal HIV transmission. N. Engl. J. Med. 359(5), 463–472 (2008). n Disappointing results of a well-conducted clinical trial. 90 Skoler-Karpoff S, Ramjee G, Ahmed K et al. Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomized, double-blind, placebo-controlled trial. Lancet 372(9654), 1977–1987 (2008). n First report to suggest possible efficacy of microbicides in preventing HIV infection. 91 Sonza S, Johnson A, Tyssen D et al. Enhancement of human immunodeficiency virus type 1 replication is not intrinsic to all polyanion-based microbicides. Antimicrob. Agents Chemother. 53(8), 3565–3568 (2009). 92 Daar ES, Li XL, Moudgil T, Ho DD. High concentrations of recombinant soluble CD4 are required to neutralized primary immunodeficiency virus type 1 isolates. Proc. Natl Acad. Sci. USA 87(17), 6574–6578 (1990). 93 Haim H, Si Z, Madani N et al. Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state. PLOS Pathogen 5(4), e1000360 (2009). 94 Fouts T, Godfrey K, Bobb K et al. Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques. Proc. Natl Acad. Sci. USA 99(18), 11842–11847 (2002). 95 Martin L, Stricher F, Missé D et al. Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes. Nat. Biotech. 21(1), 71–76 (2003). 96 Baleux F, Loureiro-Morais L, Hersant Y et al. A synthetic CD4-heparan sulfate glycoconjugate inhibits CCR5 and CXCR4 HIV-1 attachment and entry. Nat. Chem. Biol. 5(10), 743–748 (2009). 97 Pantophlet R, Burton DR. Gp120: target for neutralizing HIV-1 antibodies. Annu. Rev. Immunol. 24, 739–769 (2006). 98 Phogat S, Wyatt RT, Hedestam GBK. Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets. J. Intern. Med. 262(1), 26–43 (2007). 99 Buton DR, Pyati J, Koduri R et al. Efficient neutralization of primary isolates HIV-1 by a recombinant human monoclonal antibody. Science 266(5187), 1024–1027 (1994). 100 Trkola A, Purtscher M, Muster T et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70(2), 1100–1108 (1996). 101 Muster T, Steindl F, Purtscher M et al. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67(11), 6642–6647 (1993). 102 Kwong PD, Wilson IA. HIV-1 and influenza antibodies: seeing antigens in new ways. Nat. Immunol. 10(6), 573–578 (2009). 103 Huber M, Olson WC, Trkola A. Antibodies for HIV treatment and prevention: window of opportunity? In: Human Antibody Therapeutics for Viral Disease. Current Topics 39 in Microbiology and Immunology (volume 317). Dessain SK (Ed.) Springer-Verlag, Berlin, Heidelberg, 39–66 (2008). 104 Balzarini J. Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral Res. 71(2–3), 237–247 (2006). 105 Balzarini J. Targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy. Nat. Rev. Microbiol. 5(8), 583–597 (2007). 106 Boyd MR, Gustafson KR, McMahon JB et al. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelop glycoprotein gp120: potential applications to microbicide development. Antimicrob. Agents Chemother. 41(7), 1521–1530 (1997). 107 Mori T, O’Keefe BR, Sowder II RC et al. Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp. J. Biol. Chem. 280(10), 9345–9353 (2005). 108 Tsai CC, Emau P, Jiang Y et al. Cyanovirin-N inhibits AIDS virus infection in vaginal transmission models. AIDS Res. Hum. Retroviruses 20(1), 11–18 (2004). 109 Tsai CC, Emau P, Jiang Y et al. Cyanovirin-N inhibits AIDS virus infection in vaginal transmission models. AIDS Res. Hum. Retroviruses 19(7), 535–541 (2003). 110 Buffa V, Stieh D, Mamhood N, Hu Q, Fletcher P, Shattock RJ. Cyanovirin-N potently inhibits human immunodeficiency virus type 1 infection in cellular and cervical explants models. J. Gen. Virol. 90(1), 234–243 (2009). 111 Hunskens D, Vermeire K, Vandemeulebroucke E, Balzarini J, Schols D. Safety concerns for the potential use of cyanovirin-N as a microbicidal anti-HIV agent. Int. J. Biochem. Cell Biol. 40(12), 2802–2814 (2008). 112 O’Keefe BR, Vojdani F, Buffa V et al. Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component. Proc. Natl Acad. Sci. USA 106(15), 6099–6104 (2009). 113 Jay JI, Lai BE, Myszka DG et al. Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors. Mol. Pharmaceutics 7(1), 116–129 (2010). 114 Balzarini J, Van Laethem K, Daelemans D et al. Pramicidin A, a carbohydrate-binding nonpeptidic lead compound for treatment of infections with viruses with highly glycosylated envelopes, such as human immunodeficiency virus. J. Virol. 81(1), 362–373 (2007). 115 Balzarini J, Van Laethem K, François K et al. Pradimicin S, a highly-soluble non-peptidic small-size carbohydrate-binding antibiotic, is an anti-HIV drug lead for both microbicidal and systemic use. Antimicrob. Agents Chemother. 54(4), 1425–1435 (2010). 116 Wang T, Zhang Z, Wallace OB et al. Discovery of 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-c]- pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806): a novel HIV-1 attachment inhibitor that interferes with CD4-gp120 interactions. J. Med. Chem. 46(20), 4236–4239 (2003). 117 Lin PF, Blair W, Wang T et al. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding. Proc. Natl Acad. Sci. USA 100(19), 11013–11018 (2003). 118 Wang T, Yin Z, Zhang Z et al. Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment. 5. An evolution from indole to azaindoles leading to the discovery of 1-(4ben zoyl piperazin-1-yl)-2-(4,7-dimethoxy1H-pyrrolo[2,3-c]-pyridin-3-yl)ethane-1,2dione (BMS-488043), a drug candidate that demonstrate antiviral activity in HIV-1-infected subjects. J. Med. Chem. 52(23), 7778–7787 (2009). 119 Qadir MI, Malik SA. HIV fusion inhibitors. Rev. Med. Virol. 20(1), 23–33 (2010). 120 Moore JP, Doms RW. The entry of entry inhibitors: a fusion science and medicine. Proc. Natl Acad. Sci. USA 100(19), 10598–10602 (2003). 121 Liu S, Jing W, Cgeung B et al. HIV gp41 C-terminal heptad repeat contains multifunctional domains. J. Chem. Biol. 282(13), 9612–9620 (2007). HIV microbicides: state-of-the-art & new perspectives on the development of entry inhibitors | Review www.future-science.com 1159 future science group 122 Pan C, Cai L, Lu H, Qi Z, Jiang S. Combinations of the first and next generations of human immunodeficiency virus (HIV) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtidesensitive and-resistant HIV type 1 strains. J. Virol. 83(16), 7862–7872 (2009). 123 Ingallinella P, Bianchi E, Ladwa NA et al. Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency. Proc. Natl Acad. Sci. USA 106(14), 5801–5806 (2009). 124 Eckert DM, Malashkevich VN, Hong LH, Carr PA, Kim PS. Inhibiting HIV-1 entry: discovery of d-peptide inhibitors that target the gp41 coiled-coil pocket. Cell 99(1), 103–115 (1999). 125 Welch DB, VanDemark AP, Heroux A, Hill CP, Kay MS. Potent d-peptide inhibitors of HIV-1 entry. Proc. Natl Acad. Sci. USA 104(43), 16828–16833 (2007). 126 Mayaux JF, Bousseau A, Pauwels R et al. Triterpene derivatives that block entry of human immunodeficiency virus type 1 into cells. Proc. Natl Acad. Sci. USA 91(9), 3564–3568 (1994). 127 Lai W, Huang L, Ho P, Li Z, Montefiori D, Chen CH. Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 52(1), 128–136 (2008). 128 Baell JB, Holloway GA. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 53(7), 2719–2740 (2010). 129 Qian K, Yu D, Chen CH et al. Anti-AIDS agents. 78. Design, synthesis, metabolic stability assessment, and antiviral evaluation of novel betulinic acid derivatives as potent anti-human immunodeficiency virus (HIV) agents. J. Med. Chem. 52(10), 3248–3258 (2009). 130 Jiang S, Lu H, Liu S, Zhao Q, He Y, Debnath AK. N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion. Antimicrob. Agents Chemother. 48(11), 4349–4359 (2004). 131 Liu K, Lu H, Hou L et al. Design, synthesis, and biological evaluation of N-carboxyphenylpyrrole derivatives as potent HIV fusion inhibitors targeting gp41. J. Med. Chem. 51(24), 7843–7854 (2008). 132 Wang Y, Lu H, Zhu Q, Jiang S, Liao Y. Structure-based design, synthesis amd biological evaluation of new N-carboxyphenylpyrrole derivatives as HIV fusion inhibitors targeting gp41. Bioorg. Med. Chem. Lett. 20(1), 189–192 (2010). 133 Smith PF, Ogundele A, Forrest A et al. Phase I and II study of the safety, virologic effect, and pharmacokinetics/ pharmacodynamics of single-dose 3-O-(3´,3´- dimethylsuccinyl)betulinic acid (bevirimat) against human immunodeficiency virus infection. Antimicrob. Agents Chemother. 51(10), 3574–3581 (2007). 134 Stewart KD, Huth JR, Ng TI et al. Nonpeptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket. Bioorg. Med. Chem. Lett. 20(2), 612–617 (2010). 135 Ketas Thomas J, Schader SM, Zurita J et al. Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes. Virology 364(2), 431–440 (2007). 136 Jenabian MA, Saidi H, Charpentier C et al. In vitro synergistic activity against CCR5tropic HIV-1 with combinations of potential candidate microbicide molecules HHA, KRV2110 and enfurvitide (T20). J. Antimicrob. Chemother. 64(6), 1192–1195 (2009). 137 Auwerx J, François KO, Vanstreels E et al. Capture and transmission of HIV-1 by the C-type lectin L-SIGN (DC-SIGNR) is inhibited by carbohydrate-binding agents and polyanions. Antiviral Res. 83(1), 61–70 (2009). 138 Gantlett KE, Weber JN, Sattentau QJ. Synergistic inhibition of HIV-1 infection by combinations of soluble polyanions with other potential microbicides. Antiviral Res. 75, 188–197 (2007). 139 Yeni PG, Hammer SM, Hirsch MS et al. Treatment for adult HIV infection: 2004 recommendations of the International AIDS Society-USA Panel, JAMA 292(2), 251–265 (2004). 140 Seto M, Aikawa K, Miyamoto N et al. Highly potent and orally active CCR5 antagonists as anti-HIV-1 agents: synthesis and biological activities of 1-benzazocine derivatives containing a sulfoxide moiety. J. Med. Chem. 49(6), 2037–2048 (2006). n Data suggesting a possible use of CCR5 inhibitors in non-HIVmediated pathologies. 141 Martínez-Ávila O, Hijazi K, Marradi M et al. Gold manno-glyconanoparticles: multivalent systems to block HIV-1 gp120 binding to the lectin DC-SIGN. Chem. Eur. J. 15(38), 9874–9888 (2009). n Website 201 AIDSMEDS www.aidsmeds.com/articles/hiv-vivrivirovccr5–2446–18031.html.