HIV entry inhibitors targeting gp41: from polypeptides to small-molecule compounds

HIV envelope glycoprotein transmembrane subunit gp41 plays a critical role in the fusion between viral and target cell membranes. Upon gp120 binding to CD4 and a coreceptor (CCR5 or CXCR4), gp41 changes its conformation by forming N-helix trimer between N-heptad repeats (NHRs) and then six-helix bundle between the N-trimer and the C-heptad repeats (CHRs). Peptides derived from the NHR and CHR of gp41 extracellular region have demonstrated potent inhibitory activity on the HIV mediated cell fusion. One of these peptides, T-20, became the first success of a new class of anti-HIV agents, named HIV entry inhibitors. However, a relatively long peptide such as T-20 suffers from several limitations including lack of oral bioavailability and high cost of production. Great efforts have been made to develop alternative peptides and proteins with improved anti-HIV-1 activity, increased bioavailability and reduced cost of production. The most promising approach is the development of small molecule HIV entry inhibitors targeting gp41. Any molecule that blocks the process of NHR homotrimerization and the six-helix bundle formation by targeting the gp41 NHR, NHR trimer and CHR may inhibit HIV-mediated membrane fusion. The progress in development of those anti-HIV agents targeting gp41, from polypeptides to small-molecule compounds, is reviewed.     

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。     

The emerging role of fusion inhibitors in HIV infection

Fusion of HIV with its host cell requires the interaction of the viral envelope glycoprotein 120 (gp120) with the chemokine receptor CXCR4 [T cell-tropic (T-tropic) or X4 HIV strains] or CCR5 [macrophage-tropic (M-tropic) or R5 HIV strains] followed by a 'spring-loaded' action of the glycoprotein 41 (gp41) that ensures fusion of the viral and cellular lipid membranes and permits the viral nucleocapsid to enter the cell. The overall fusion process can be blocked by a number of compounds. These include siamycin analogues, SPC 3 (a synthetic peptide derived from the V3 domain of gp120), pentafuside (T 20, DP 178) [a synthetic peptide corresponding to amino acid residues 127 to 162 of gp41], the betulinic acid derivative RPR 103611, TAK 779 (a low molecular weight non-peptide CCR5 antagonist) and a number of compounds (T 22, T 134, ALX40-4C, CGP64222 and AMD 3100) that are targeted at the CXCR4 receptor. In particular, the bicyclam AMD 3100 has proved highly potent and selective as a CXCR4 antagonist that blocks the infectivity of X4 HIV strains in the nanomolar concentration range. The proof-of-concept that fusion inhibitors should be able to suppress viral replication in vivo has been demonstrated with pentafuside. Pentafuside and AMD 3100 have now proceeded to phase II clinical trials.     

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.     

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.     

HIV包膜蛋白的结构及其相应的病毒进入抑制剂

HIV-1病毒包膜蛋白gp120和gp41在病毒感染中起着重要的作用。在病毒进入细胞的过程中,gp120先和CD4分子结合,发生构象改变,进而导致gp41构象的变化,使病毒包膜和细胞膜融合而感染细胞。与gp120或者gp41相结合的多肽、大分子和小分子化合物,都可能影响HIV-1病毒包膜和靶细胞膜结合的过程,从而起到抗HIV-1病毒的作用。该文对gp120和gp41的结构及其相互作用,以及以HIV-1包膜糖蛋白为靶点的病毒进入抑制剂类抗艾滋病药物进行综述。     

作用于HIV包膜蛋白亚基gp41的多肽类融合抑制剂

HIVgp4 1是病毒包膜上介导HIV与靶细胞膜融合的跨膜糖蛋白 ,其包膜外区包括位于N末端的融合多肽和下游的N末端重复序列 (NHR) ,以及C末端的重复序列(CHR)。衍生于gp4 1NHR和CHR的N 多肽和C 多肽具有抑制HIV与靶细胞融合的活性 ,其中C 多肽T 2 0已获得美国FDA批准 ,成为继逆转录酶抑制剂和蛋白酶抑制剂后的第三类抗艾滋病药物 ,即HIV融合抑制剂。由于T 2 0等为多肽类药物 ,容易被体内蛋白酶降解 ,临床剂量大 ,用基因工程手段难以满足需要 ,因此只能采用多肽合成技术进行生产 ,成本高昂。为此 ,人们希望寻找到具有相似机制的活性短肽 ,或通过多肽设计使活性多肽适合用基因工程进行大规模生产。近年来 ,对N 多肽和C 多肽进行结构改造已成为研究的热点 ,出现了许多相对分子质量较小 ,不容易被内源性蛋白酶降解 ,或者能用基因工程手段生产的活性多肽 ,同时多肽药物抑制HIV感染的作用机制也因此而逐渐清晰起来     

Inhibiting HIV-1 entry with fusion inhibitors

In recent years, tremendous progress has been made in understanding the HIV-1 entry process in which the viral and cellular membranes are fused, resulting in the subsequent delivery of the viral genome into the host cell. The mechanistic insight gained from these studies has led to the formulation of exciting new approaches for therapeutic intervention. One of the first and clinically most advanced drugs to emerge from this effort is the fusion inhibitor T20. T20 acts by freezing a transient structural intermediate of the HIV-1 fusion process, thus blocking an essential step in viral entry. With phase III clinical trials already well underway, the success of T20 indicates that targeting of the viral entry process will soon be an important component of antiretroviral therapy. This review addresses this rapidly developing area of HIV research, with a focus on the mechanistic role of fusion inhibitors targeted to the HIV-1 gp41 transmembrane glycoprotein. We will review the results of recent clinical trials with T20 and discuss possible mechanisms of viral escape through the evolution of drug-resistant HIV-1 variants. We will also discuss ongoing research on fusion inhibitor susceptibility testing and the development of new improved fusion inhibitors.     

Enfuvirtide

Despite the overall success of antiretroviral medications in reducing the morbidity and mortality associated with HIV infection, many patients on treatment suffer progressive disease due to intolerance or the development of resistant viral strains. Consequently, considerable research focuses on the development of new classes of antiretroviral agents with mechanisms of action different to the current classes. Enfuvirtide (T-20, pentafuside, Fuzeon), the first drug of a new class of antiretroviral medications known as fusion inhibitors, blocks the fusion of the virus particle with the host target cell. The viral entry process begins with the attachment of viral surface glycoprotein gp120 to the host cell CD4 and chemokine receptor sites. Viral gp41 then undergoes a conformational change enabling fusion of both membranes, a critical step in the viral life cycle. Enfuvirtide is a synthetic peptide that binds to gp41, preventing the conformational change required for membrane fusion. Based on potent in vitro activity, a Phase I clinical trial of intravenous enfuvirtide was conducted that demonstrated a substantial decline in HIV plasma viral load in the highest dose group and no serious adverse effects. Phase II trials evaluated regimens of both continuous subcutaneous infusions and intermittent subcutaneous injections. Intermittent injections were pharmacokinetically superior to continuous infusions and were associated with fewer administration difficulties. For some subjects who added enfuvirtide monotherapy to an already failing regimen, the beneficial effect on viral load reduction appeared short-lived, suggesting the development of resistance. Two large randomised clinical trials comparing "optimised background" (best available, individualised regimens based on patient history and resistance assays) versus optimised background plus enfuvirtide have recently shown a significant virological advantage (approximately 1 log(10) difference from controls) at 24 weeks. In all trials to date, very few significant adverse effects have been seen--minor injection site reactions are frequent, but rarely treatment limiting. Based on these studies, enfuvirtide will likely play a significant role in the treatment of patients with limited treatment options.     

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。     

HIV-1融合抑制剂研究现状及发展趋势

HIV-1融合抑制剂是继逆转录酶和蛋白酶抑制剂后的新一类抗HIV感染药物, 通过阻断病毒与靶细胞膜的融合从而抑制病毒进入靶细胞, 在感染的初始环节切断HIV-1的传播, 其中多肽类融合抑制剂T-20已于2003年上市。HIV-1融合抑制剂以HIV-1跨膜糖蛋白gp41为作用靶标, 它们是一些天然或合成的多肽以及小分子化合物, 通过与gp41功能区结合从而抑制其促融合功能的发挥。近年来, 随着对膜融合过程分子机制以及gp41功能研究的不断深入, 新的以gp41不同功能区为靶点的融合抑制剂分子不断被发现, 成为倍受关注的研究热点之一。本文着重对近年来HIV-1融合抑制剂的研究现状及发展趋势进行综述。     

Enfuvirtide

Despite the overall success of antiretroviral medications in reducing the morbidity and mortality associated with HIV infection, many patients on treatment suffer progressive disease due to intolerance or the development of resistant viral strains. Consequently, considerable research focuses on the development of new classes of antiretroviral agents with mechanisms of action different to the current classes. Enfuvirtide (T-20, pentafuside, Fuzeon^?), the first drug of a new class of antiretroviral medications known as fusion inhibitors, blocks the fusion of the virus particle with the host target cell. The viral entry process begins with the attachment of viral surface glycoprotein gp120 to the host cell CD4 and chemokine receptor sites. Viral gp41 then undergoes a conformational change enabling fusion of both membranes, a critical step in the viral life cycle. Enfuvirtide is a synthetic peptide that binds to gp41, preventing the conformational change required for membrane fusion. Based on potent in vitro activity, a Phase I clinical trial of intravenous enfuvirtide was conducted that demonstrated a substantial decline in HIV plasma viral load in the highest dose group and no serious adverse effects. Phase II trials evaluated regimens of both continuous subcutaneous infusions and intermittent subcutaneous injections. Intermittent injections were pharmacokinetically superior to continuous infusions and were associated with fewer administration difficulties. For some subjects who added enfuvirtide monotherapy to an already failing regimen, the beneficial effect on viral load reduction appeared short-lived, suggesting the development of resistance. Two large randomised clinical trials comparing ‘optimised background’ (best available, individualised regimens based on patient history and resistance assays) versus optimised background plus enfuvirtide have recently shown a significant virological advantage (~ 1 log₁₀ difference from controls) at 24 weeks. In all trials to date, very few significant adverse effects have been seen – minor injection site reactions are frequent, but rarely treatment limiting. Based on these studies, enfuvirtide will likely play a significant role in the treatment of patients with limited treatment options.     

A simple screening system for anti-HIV drugs: syncytium formation assay using T-cell line tropic and macrophage tropic HIV env expressing cell lines--establishment and validation.

The first step in cellular entry of HIV involves binding of the viral envelope glycoprotein complex (gp120/gp41) to specific receptor molecules on the target cells. The cell-cell fusion (syncytium formation) between env expressing cells and CD4+ cells mimics the viral infection of the host cells. To search for anti-HIV substances preventing this process, we constructed the recombinant cell lines, HeLa/CD4/Lac-Z and HeLa/T-env/Tat for T-cell tropic (HIV-1(NL4-3)) system, and HOS/CD4/CCR5/Lac-Z and HeLa/M-env/Tat for macrophage tropic (HIV-1(SF162)) system. When each pair of cells were co-incubated for 20 hours, the multinuclear giant cells (syncytia) were formed and beta-galactosidase was expressed. These systems are less biohazardous because no infectious virus particles are used. Their validity in screening for anti-HIV substances which inhibit syncytium formation was confirmed using various known HIV entry inhibitors.     

HIV-1 gp41 as a target for viral entry inhibition

The recent success of the fusion inhibitor T-20 (enfuvirtide) in clinical studies has ushered in a new chapter in the development of anti-HIV-1 therapeutics. T-20 is the first FDA-approved drug that targets the viral transmembrane protein gp41. This protein, along with gp120, promotes viral entry through a coordinated cascade of conformational transitions that lead to the fusion of the HIV-1 and target cell membranes. The interaction of gp120 with CD4 and a chemokine receptor stimulates gp41 to extend and bridge the space between the virus and cell. Subsequently, gp41 collapses into a trimer-of-hairpins structure that brings the viral and cellular membranes into close proximity necessary for fusion. Enfuvirtide targets the gp41 amino-terminal region exposed in the transient extended state, blocking the ultimate collapse into the trimer-of hairpins and inhibiting membrane fusion. The vulnerability of this transient extended state has stimulated the development of new agents, ranging from small molecules to large proteins, that bind to gp41 and inhibit its structural transformations. The discovery and characterization of these inhibitors have not only led to new antiviral strategies, but have also shed light on the accessibility of gp41 epitopes that might play a role in HIV-1 vaccine development.     

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.