HIV-1协同受体及其抑制剂研究进展

协同受体CCR5和CXCR4分别是嗜巨噬细胞性HIV-1和嗜T细胞性TIV-1侵入靶细胞的主要受体。CCR5抑制剂如TAK-779、SCH—C等，和CXCR4抑制剂如AMD3100、T22等，能分别与CCR5和CXCR4结合，从而阻断HIV-1侵入靶细胞。本文综述了HIV-1与CCR5和CXCR4的结合机制及其抑制剂的研究进展。     

Peptide T inhibits HIV-1 infection mediated by the chemokine receptor-5 (CCR5)

Peptide T, which is derived from the V2 region of HIV-1, inhibits replication of R5 and dual-tropic (R5/X4) HIV-1 strains in monocyte-derived macrophages (MDMs), microglia, and primary CD4(+)T cells. Little to no inhibition by peptide T was observed with lab adapted X4 viruses such as IIIB, MN, or NL4-3 propagated in CD4(+) T cells or in the MAGI entry assay. The more clinically relevant R5/X4 early passage patient isolates were inhibited via either the X4 or R5 chemokine receptors, although inhibition was greater with R5 compared to X4 receptors. Virus inhibition ranged from 60 to 99%, depending on the assay, receptor target, viral isolate and amount of added virus. Peak inhibitory effects were detected at concentrations from 10(-12) to 10(-9) M. Peptide T acted to block viral entry as it inhibited in the MAGI cell assay and blocked infection in the luciferase reporter assay using HIV virions pseudotyped with ADA envelope. These results using early passage virus grown in primary cells, together with two different entry reporter assays, show that peptide T selectively inhibits HIV replication using chemokine receptor CCR5 compared to CXC4, explaining past inconsistencies of in vitro antiviral effects.     

Variation of Macrophage Tropism among HIV-1 R5 Envelopes in Brain and Other Tissues

Human immunodeficiency virus (HIV)-positive individuals frequently suffer from progressive encephelopathy, which is characterized by sensory neuropathy, sensory myelopathy, and dementia. Our group and others have reported the presence of highly macrophage-tropic R5 variants of HIV-1 in brain tissue of patients with neurological complications. These variants are able to exploit low amounts of CD4 and/or CCR5 for infection and potentially confer an expanded tropism for any cell types that express low CD4 and/or CCR5. In contrast to the brain-derived envelopes, we found that envelopes from lymph node tissue, blood, or semen were predominantly non-macrophage-tropic and required high amounts of CD4 for infection. Nevertheless, where tested, the non-macrophage-tropic envelopes conferred efficient replication in primary CD4⁺ T-cell cultures. Determinants of R5 macrophage tropism appear to involve changes in the CD4 binding site, although further unknown determinants are also involved. The variation of R5 envelopes also affects their sensitivity to inhibition by ligands and entry inhibitors that target CD4 and CCR5. In summary, HIV-1 R5 viruses vary extensively in macrophage tropism. In the brain, highly macrophage-tropic variants may represent neurotropic or neurovirulent viruses. In addition, variation in R5 macrophage tropism may also have implications (1) for transmission, depending on what role macrophages or cells that express low CD4 and/or CCR5 play in the establishment of infection in a new host, and (2) for pathogenesis and depletion of CD4⁺ T cells (i.e., do highly macrophage-tropic variants confer a broader tropism among CD4⁺ T-cell populations late in disease and contribute to their depletion?).     

Genetic Knockouts Suggest a Critical Role for HIV Co-Receptors in Models of HIV gp120-Induced Brain Injury

Infection with HIV-1 frequently affects the brain and causes NeuroAIDS prior to the development of overt AIDS. The HIV-1 envelope protein gp120 interacts with host CD4 and chemokine co-receptors to initiate infection of macrophages and lymphocytes. In addition, the virus or fragments of it, such as gp120, cause macrophages to produce neurotoxins and trigger neuronal injury and apoptosis. Moreover, the two major HIV co-receptors, the chemokine receptors CCR5 and CXCR4, serve numerous physiological functions and are widely expressed beyond immune cells, including cells in the brain. Therefore, HIV co-receptors are poised to play a direct and indirect part in the development of NeuroAIDS. Although rodents are not permissive to infection with wild type HIV-1, viral co-receptors - more than CD4 - are highly conserved between species, suggesting the animals can be suitable models for mechanistic studies addressing effects of receptor-ligand interaction other than infection. Of note, transgenic mice expressing HIV gp120 in the brain share several pathological hallmarks with NeuroAIDS brains. Against this background, we will discuss recently completed or initiated, ongoing studies that utilize HIV co-receptor knockout and viral gp120-transgenic mice as models for in vitro and in vivo experimentation in order to address the potential roles of HIV gp120 and its co-receptors in the development of NeuroAIDS.     

A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains

The V3-loop region in the envelope protein gp120 of HIV is critical for viral infection, but its interaction with the target cells is not clear. Using synthetic peptides, representing linear V3 sequences as reagents, we obtained evidence to show inhibition of infection by both T-cell- and macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) (X4 and R5, respectively), without interfering with gp120-CD4 interaction, by the V3 peptides through binding to host cell membrane glycosphingolipids (GSL). Synthetic peptides mimicking the central 15-21 amino acid sequence of the V3-loop region in both X4 and R5 strains of HIV-1 competed with and blocked the entry of both types of HIV isolates. These HIV-inhibitory V3 peptides exhibited specific binding to target cells that was not competed by antibodies to either the primary receptor CD4 or the co-receptors CXCR-4 and CCR5. However, R15K, the V3 peptide from HIV-1 IIIB gp120 exhibited specific binding to three distinct cell surface GSL: GM3, Gb3, and GalCer. Further, R15K inhibited GSL binding of gp120 from both HIV-1 IIIB (X4, Gb3-binding strain) and HIV-1 89.6 (X4R5, GM3-binding strain). Together, these results suggest a critical V3-mediated post-CD4-binding event involving cell surface GSL binding represented by the HIV-inhibitory V3 peptides, that is common for the entry of diverse HIV-1 strains and may be targeted for the development of novel HIV therapeutics aimed at blocking viral entry.     

Conformational properties of HIV-1 gp120/V3 immunogenic domains

Infection of target host cells by the human immunodeficiency virus-1 (HIV-1) is a multi-step process involving a series of conformational changes in the viral gp120 and gp41 proteins. Gp120 binding to the main cell receptor, CD4, on the surface of cells expressing this molecule, and interaction with the cell chemokine receptors CCR5 and CXCR4, are among the key events for HIV-1 infection. These steps are crucial for the virus and offer potential therapeutic targets. For this reason, understanding the structure and the physicochemical characteristics of the gp120 in relation to these interactions has drawn much attention. This review article focuses on the biologically important V3 region of the gp120 and summarizes the functional role, the sequence variation and the conformational features of V3 peptides, which are important for co-receptor selectivity, specificity and interaction. Synthetic V3 peptides have been extensively studied by NMR spectroscopy and X-ray crystallography, in solution or in solid state, in their free or bound form, and valuable information was generated with the aim to be exploited in the design of new, effective inhibitors of HIV-1 infection. The features of the potential gp120 interacting sites on the two chemokine co-receptors, CCR5 and CXCR4, are also discussed, and co-receptor blocking molecules under clinical trial are also reported.     

Genetic polymorphisms in the chemokine and chemokine receptors: impact on clinical course and therapy of the human immunodeficiency virus type 1 infection (HIV-1)

The natural history and pathogenic processes of infection by the human immunodeficiency virus type 1 (HIV-1) are complex, variable, and dependent upon a multitude of viral and host factors and their interactions. The CCR5-Delta32 allele remains the most important genetic factor known to be associated with host resistance to the HIV-1 infection. However, other mutations in the CCR5, CCR2, CX(3)CR1, CXCL12 (SDF1), and CCL5 (RANTES) genes have been identified and associated with host resistance and/or susceptibility to HIV-1 infection and disease progression. Some studies have also suggested that chemokine receptor gene polymorphisms may affect response to potent antiretroviral therapy. This article reviews the polymorphisms already described in the mutant chemokine receptors or ligands and their impact on the host susceptibility to HIV-1 infection and on the clinical course of the disease, as well as the development of new anti-HIV therapies that takes into account these potential targets in the host. These genetic polymorphisms could be used as genetic markers to detect individuals at higher risk of developing either a faster disease progression or therapeutic failure. Once these individuals are identified, therapeutic strategies based on either different, more aggressive drugs or combinations of drugs can be used, either alone or in combination with shorter intervals for therapeutic monitoring. Pharmacogenetics is very likely to underlie future therapies for HIV-1 infection, and current patients with multi-resistance to the existing antiretroviral agents could also benefit from this approach. These developments also underscore the importance of continuing the investigation of new therapies targeted to the host in order to inhibit the HIV-1 entry into the host cells.     

Immunoreactive cycloimmunogen design based on conformational epitopes derived from human immunodeficiency virus type 1 coreceptors: cyclic dodecapeptides mimic undecapeptidyl arches of extracellular loop-2 in chemokine receptor and inhibit human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) requires a chemokine receptor (CCR5 or CXCR4) as a coreceptor not only for initiate viral entry but also protecting highly conserved neutralization epitopes from the attack of neutralizing antibodies. Over the past decade, many studies have provided new insights into the HIV entry mechanism and have focused on developing an effective vaccine strategy. However, to date, no vaccine that can provide protection from HIV-1 infection has been developed. One reason for the disappointing results has been the inability of current vaccine candidates to elicit a broadly reactive immunity to viral proteins such as the envelope (env) protein. Here, we propose that chemokine receptors are attractive targets of vaccine development because their structures are highly conserved and that our synthetic cycloimmunogens can mimic conformational-specific epitopes of undecapeptidyl arches (UPAs: R(168)-C(178) in CCR5, N(176)-C(186) in CXCR4) and be useful for HIV-1 novel vaccine development.     

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.     

Chemokine receptors in HIV-1 and SIV infection

Seven transmembrane segment (7TMS) receptors for chemokines and related molecules have been demonstrated to be essential, in addition to CD4, for HIV and SIV infection. The β-chemokine receptor CCR5 is the primary, perhaps sole, coreceptor for HIV-1 during the early and chronic phases of infection, and supports infection by most primary HIV-1 and many SIV isolates. Late-stage primary and laboratory-adapted HIV-1, HIV-2, and SIV isolates can use other 7TMS receptors. CXCR4 appears especially important in late-stage HIV infection; several related receptors can also be used. The specificity of SIV viruses is similar. Commonalities among these receptors, combined with analyses of mutated molecules, indicate that discrete, conformationally-dependent sites on the chemokine receptors determine their association with the third variable and conserved regions of viral envelope glycoproteins. These studies are useful for elucidating the mechanism and molecular determinants of HIV-1 entry, and of inhibitors to that entry.     

Anti-HIV activity and conformational studies of peptides derived from the C-terminal sequence of SDF-1

The entry of the human immunodeficiency virus type 1 (HIV-1) into target cells requires the interaction of viral envelope glycoprotein, gp120, with the human CD4 glycoprotein and a chemokine receptor, usually CCR5 or CXCR4. The natural ligand for CXCR4 is the chemokine SDF-1 that inhibits entry and replication of X4 HIV-1 strains. SDF-1 is produced in two forms, SDF-1alpha (68 residues) and SDF-1beta (72 residues); the difference between them lies in the additional four C-terminal amino acids in the SDF-1beta sequence. Despite the relevance of the N-terminal site in determining the SDF anti HIV-1 activity, SDF-1beta has a stronger activity than SDF-1alpha. Here we demonstrate that a synthetic peptide mapped on the C-terminus of SDF-1beta presents inhibitory activity, whereas an analogue reproducing the C-terminal trait of SDF-1alpha does not show any activity. The opposite biological effect of the two peptides correlates with the type of interaction they each have with heparin and chondroitin sulfate.     

Cytokine and chemokine based control of HIV infection and replication

HIV infects and propagates into CD4+ T lymphocytes and macrophages, although many other cell types play an important role in virus spreading and pathogenesis. In addition to regulatory viral proteins, the cytokine network has early been implicated as a major controller of the plastic capacity of HIV to spread productively or rather remain silently integrated in the chromosomes of infected cells. The recent discovery of CCR5 and CXCR4 as essential entry co-receptors together with CD4 has highlighted a novel and potentially important step in the pharmacological hunt for more effective antiviral agents. In addition to regulate HIV expression and replication, several cytokines have demonstrated the capacity of up- or down-modulating chemokine receptors including CCR5 and CXCR4 with the consequence of influencing the susceptibility of T cells and macrophages to HIV infection. Pharmacological agents such as pertussis toxin B-oligomer have demonstrated HIV suppressive effects via non competitive binding of CCR5, whereas interferons or interleukin-16 (IL-16) can prevent post-entry steps in HIV expression. At the clinical level, several cytokines or their receptors are useful markers for monitoring disease progression and its consequence on the immune system. Cytokine-based therapy represents a realistic complementary approach to traditional antiretroviral therapy potentially capable of restoring important adaptive or innate immune functions ultimately curtailing HIV spreading and its consequences on the immune system, as exemplified by the experimental clinical use of IL-2.     

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.     

Current drug design of anti-HIV agents through the inhibition of C-C chemokine receptor type 5

Human immunodeficiency virus (HIV) is the responsible causal agent of acquired immunodeficiency syndrome (AIDS), a condition in humans in which the immune system begins to fail, allowing the entry of opportunistic infections. HIV infection in humans is considered pandemic by the World Health Organization (WHO). HIV needs to use a protein as a co-receptor to enter its target cells. Several chemokine receptors can in principle act as viral co-receptors, but the chemokine (C-C motif) receptor 5 (CCR5) is likely the most physiologically important co-receptor during natural infection. For this reason the development of new CCR5 inhibitors like anti-HIV agents, constitutes a challenge for the scientific community. The present review will focus on the current state of the design of novel anti-HIV drugs, and how the existing computer aided-drug design methodologies, have been effective in the search of new anti-HIV agents. In addition, a QSAR model based on substructural descirptors is presented as a rapid, rational and promising alternative for the discovery of anti-HIV agents through the inhibition of the CCR5.     

Effect of CCR5 receptor antagonists on endocytosis of the human CCR5 receptor in CHO-K1 cells

Background and purpose:

The CCR5 chemokine receptor is a member of the G protein-coupled receptor (GPCR) family that is expressed by macrophages, memory T-lymphocytes and dendritic cells and is activated by chemotactic proteins (e.g. MIP-1α [CCL3], MIP-1β [CCL4] and RANTES [CCL5]). CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus-1 (HIV-1) and some chemokines can inhibit HIV-1 infection by stimulating CCR5 receptor endocytosis. The aim of this study was to evaluate the effect of CCR5 antagonists on CCR5 endocytosis.

Experimental approach:

The effects of CCR5 agonists and antagonists on receptor internalization in CHO cells, expressing a C-terminal green fluorescent protein-tagged human CCR5 receptor (CCR5-GFP), were quantified using a confocal imaging plate reader.

Key results:

MIP-1α [CCL3], MIP-1β [CCL4] and RANTES [CCL5] were all able to stimulate potently the internalization of CCR5-GFP. This effect was inhibited by the non-peptide antagonist TAK 779. The CCR5 peptide antagonist met-RANTES antagonized MIP-1α-mediated increases in intracellular free calcium but was also able to stimulate a substantial internalization of the human CCR5-GFP receptor. However, CHO cells exhibited an aminopeptidase activity that was able to metabolize sufficient met-RANTES into an agonist metabolite capable of stimulating calcium mobilization via CCR5 receptors in naïve cells.

Conclusions and implications:

These data suggest that there is an endogenous aminopeptidase activity on the surface of CHO cells, that produces a slow internalization of the receptor following a time-dependent conversion of receptor-bound met-RANTES from a CCR5 receptor antagonist into a CCR5 agonist molecule.