gp120-CD4 Interaction Research Articles

Discovery of small molecular inhibitors targeting HIV-1 gp120–CD4 interaction drived from BMS-378806

The HIV-1 entry into host cells is a complex, multi-factors involved, and multi-step process. Especially, the attachment of HIV-1 envelope glycoprotein gp120 to the host cell receptor CD4 is the first key step during entry process, representing a promising antiviral therapeutic target. Among the HIV-1 attachment inhibitors blocking the interaction between gp120 and CD4 cells, BMS-378806 and NBD-556 are two representative small molecular chemical entities. Particularly, BMS-378806 and its derivatives are newly identified class of orally bioavailable HIV-1 inhibitors that interfere gp120–CD4 interaction. In this review, we focused on describing the structure–activity relationships (SARs), structural modifications, in vitro or even in vivo pharmacodynamics and pharmacokinetics of BMS-378806 and its analogues as HIV-1 gp120 attachment inhibitors. In addition, the brief SARs, structural modifications of NBD-556 and its derivatives targeting the “Phe-43 cavity” as CD4 mimics were also described.

Surfactant protein D inhibits HIV-1 infection of target cells via interference with gp120-CD4 interaction and modulates pro-inflammatory cytokine production.

Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection.

Exploration of the Binding Mode of Indole Derivatives as Potent HIV-1 Inhibitors Using Molecular Docking Simulations

The HIV-1 envelope glycoprotein gp120 plays a vital role in the entry of the virus into the host cells. The crucial role of the glycoprotein suggests gp120 as potential drug target for the future antiviral therapies. Identification of the binding mode of small drug like compounds has been an important goal in drug design. In the current study we attempt to propose binding mode of indole derivatives in the binding pocket of gp120. These derivatives are reported to inhibit HIV-1 by acting as attachment inhibitors that bind to gp120 and prevent the gp120-CD4 interaction and thus inhibit the infectivity of HIV-1. To elucidate the molecular basis of the small molecules interactions to inhibit the glycoprotein function we employed the molecular docking simulation approach. This study provides insights to elucidate the binding pattern of indole-based gp120 inhibitors and may help in the rational design of novel HIV-1 inhibitors with improved potency.

The Potent Human Immunodeficiency Virus Type 1 (HIV-1) Entry Inhibitor HR212 Blocks Formation of the Envelope Glycoprotein gp41 Six-Helix Bundle

HR212, a recombinant protein composed of the heptad repeat, is a rationally designed human immunodeficiency virus type 1 (HIV-1) fusion inhibitor. This protein can be easily produced by Escherichia coli at a low cost. Previously, studies indicated that HR212 can efficiently inhibit the entry and replication of both laboratory and clinical HIV-1 strains, and this protein is more stable and less sensitive to proteinases than T20. The procedure of HIV-1 entry into the host cells can be divided into three main steps: gp120-CD4 interactions, coreceptor binding, and gp41 six-helix bundle formation and subsequent membrane fusion. The present study demonstrates that HR212 does not block gp120-CD4 binding or interfere with binding to the coreceptors CXCR4 and CCR5. Instead, HR212 efficiently blocks the six-helix bundle formation between peptides derived from the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) region of gp41. Fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) indicated that HR212 could form a complex with peptide N36 to block gp41 fusogenic core formation. These results suggest that HR212 inhibits HIV-1 entry by targeting the NHR region of gp41. Therefore, HR212 can potentially be developed as a novel, high-efficiency, specific HIV-1 entry inhibitor.

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.

Association of Enhanced HIV-1 Neutralization by a Single Y681H Substitution in gp41 with Increased gp120-CD4 Interaction and Macrophage Infectivity

HIV-1 variants that show unusual sensitivity to autologous antibodies due to presence of critical neutralization signatures would likely contribute towards rational envelope based HIV-1 vaccine design. In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies. Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes. The increased gp120-CD4 interaction was further associated with relative exposure of CD4-induced epitopes and macrophage infectivity. In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.

MiniCD4 protein resistance mutations affect binding to the HIV-1 gp120 CD4 binding site and decrease entry efficiency

BackgroundBinding of the viral envelope protein (Env), and particularly of its gp120 subunit, to the cellular CD4 receptor is the first essential step of the HIV-1 entry process. The CD4 binding site (CD4bs) of gp120, and especially a recessed cavity occupied by the CD4 Phe43 residue, are known to be highly conserved among the different circulating subtypes and therefore constitute particularly interesting targets for vaccine and drug design. The miniCD4 proteins are a promising class of CD4bs inhibitors. Studying virus evolution under pressure of CD4bs inhibitors could provide insight on the gp120-CD4 interaction and viral entry.ResultsThe present study reports on the resistance induction of two subtype B HIV-1 against the most active miniCD4, M48U1, and its ancestor, M48, and how these mutated positions affect CD4bs recognition, entry efficiency, and sensitivity to other CD4bs inhibitors. Resistance against M48U1 was always associated with S375R/N substitution in both BaL and SF162; M48 resistance was associated with D474N substitution in SF162 and with H105Y substitution in BaL. In addition, some other mutations at position V255 and G471 were of importance for SF162 resistant viruses. Except for 474, all of these mutated positions are conserved, and introducing them into an SF162 Env expressing infectious molecular clone (pBRNL4.3 SF162) resulted in decreased entry efficiency. Furthermore, resistant mutants showed at least some cross-resistance towards other CD4bs inhibitors, the V3 monoclonal antibody 447-52D and some even against the monoclonal antibody 17b, of which the epitope overlaps the co-receptor binding site.ConclusionsThe mutations H105Y, V255M, S375R/N, G471R/E, and D474N are found to be involved in resistance towards M48 and M48U1. All mutated positions are part of, or in close proximity to, the CD4bs; most are highly conserved, and all have an impact on the entry efficiency, suggesting their importance for optimal virus infectivity.

Lignosulfonic acid exhibits broadly anti-HIV-1 activity--potential as a microbicide candidate for the prevention of HIV-1 sexual transmission.

Some secondary metabolites from plants show to have potent inhibitory activities against microbial pathogens, such as human immunodeficiency virus (HIV), herpes simplex virus (HSV), Treponema pallidum, Neisseria gonorrhoeae, etc. Here we report that lignosulfonic acid (LSA), a polymeric lignin derivative, exhibits potent and broad activity against HIV-1 isolates of diverse subtypes including two North America strains and a number of Chinese clinical isolates values ranging from 21.4 to 633 nM. Distinct from other polyanions, LSA functions as an entry inhibitor with multiple targets on viral gp120 as well as on host receptor CD4 and co-receptors CCR5/CXCR4. LSA blocks viral entry as determined by time-of-drug addiction and cell-cell fusion assays. Moreover, LSA inhibits CD4-gp120 interaction by blocking the binding of antibodies specific for CD4-binding sites (CD4bs) and for the V3 loop of gp120. Similarly, LSA interacts with CCR5 and CXCR4 via its inhibition of specific anti-CCR5 and anti-CXCR4 antibodies, respectively. Interestingly, the combination of LSA with AZT and Nevirapine exhibits synergism in viral inhibition. For the purpose of microbicide development, LSA displays low in vitro cytotoxicity to human genital tract epithelial cells, does not stimulate NF-κB activation and has no significant up-regulation of IL-1α/β and IL-8 as compared with N-9. Lastly, LSA shows no adverse effect on the epithelial integrity and the junctional protein expression. Taken together, our findings suggest that LSA can be a potential candidate for tropical microbicide.

Structure-based design, synthesis, and characterization of dual hotspot small-molecule HIV-1 entry inhibitors.

Cellular infection by HIV-1 is initiated with a binding event between the viral envelope glycoprotein gp120 and the cellular receptor protein CD4. The CD4-gp120 interface is dominated by two hotspots: a hydrophobic gp120 cavity capped by Phe43(CD4) and an electrostatic interaction between residues Arg59(CD4) and Asp368(gp120). The CD4 mimetic small-molecule NBD-556 (1) binds within the gp120 cavity; however, 1 and related congeners demonstrate limited viral neutralization breadth. Herein, we report the design, synthesis, characterization, and X-ray structures of gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm for inhibiting the CD4-gp120 protein-protein interaction.

A Molecular Dynamics Simulation of Peptide-Triazole HIV Entry Inhibitor Binding to gp120 Hydrophobic Core

The envelope spike on HIV surface is a non-covalent trimer of gp120 and gp41 dimers; with gp120 responsible for binding to the CD4 receptor and the mandatory coreceptor, CCR5 or CXCR4. Blocking the gp120-CD4 or gp120-coreceptor interaction can block viral entry and infection. The HNG family of peptides (sequence RINNIXWSEAMM, X= derivatized azidoproline) is a promising class of dual-site entry inhibitors thought to allosterically inhibit the binding process and trap the flexible gp120 molecule in an inactive state. To date, there have been no reports on the structure of the peptide or the peptide/gp120 complex. Here we have used Molecular Dynamics to develop a docking model in which the peptide binds in a tripartite fashion, preventing the formation of the bridging sheet in gp120, which is necessary for coreceptor attachment and initiation of entry. The protein undergoes significant induced fit conformational changes involving movement of large loops which allows the peptide to bind tightly. Our model is consistent with experimental observations on the peptide footprint on gp120, pointing to a hydrophobic core underneath the bridging sheet and close to the F43 pocket as the putative binding site. Our results provide an explanation for why the stereochemistry of the triazole moiety on the proline is important for peptide function, in addition to explaining the inactivity of D-tryptophan in the sequence. Furthermore, saturation transfer difference (STD) NMR experiments point to the I-X-P hydrophobic center on the peptide as the central contact point in the complex, which is consistent with the peptide pose proposed in MD studies. Our model may provide a unique basis for rational design of allosteric entry inhibitors of HIV and ultimately small molecule inhibitors of viral entry.

Host-Soluble Galectin-1 Promotes HIV-1 Replication through a Direct Interaction with Glycans of Viral gp120 and Host CD4

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4(+) T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.

Conformational alterations in the CD4 binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and fusogenicity of HIV-1 envelopes derived from brain and other tissues

BackgroundCD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81).FindingsThree-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop.ConclusionsUsing a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.

Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4). Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120.

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120–CD4 interactions and fusogenicity

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones ( n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120–CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120–CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120–CD4 interactions and Env fusogenicity.

CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist

Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block the gp120–CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-binding site. A structure–activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized and their utility evaluated.

Topological Layers in the HIV-1 gp120 Inner Domain Regulate gp41 Interaction and CD4-Triggered Conformational Transitions

The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (layers 1 and 2) in the gp120 inner domain. Both layers apparently contribute to the noncovalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, layer 1-layer 2 interactions strengthen gp120-CD4 binding by reducing the off rate. Layer 1-layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner-domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41.

P07-03. HIV gp120 interaction with CD4+ T cells induces local intracellular signaling and creates an F-actin depleted zone in the virological synapse

Results The data show that gp120 and ICAM-1 on the bilayers can induce the formation of a synaptic structure by noninfected CD4+ T cells with some characteristics of an immunological synapse (IS). There is a transient stop signal and the interface contains a central supramolecular activation cluster (cSMAC) of gp120-CD4 interactions surrounded by a peripheral supramolecular activation cluster (pSMAC) of ICAM-1-LFA-1 interactions. As with the IS, the cSMAC is also formed from microclusters that are enriched in active Src family kinases. However, while Src kinase signaling is suppressed in the cSMAC of the IS, the Src family kinases and other TCR signaling components remain active for a prolonged period in the cSMAC of the VS. Notably, the gp120-enriched cSMAC region of the VS is depleted of f-actin.

Discovery of a small molecule inhibitor through interference with the gp120–CD4 interaction

A series of piperazine derivatives were designed and synthesised as gp120–CD4 inhibitors. SAR studies led to the discovery of potent inhibitors in a cell based anti viral assay represented by compounds 9 and 28. The rat pharmacokinetic and antiviral profiles of selected compounds are also presented.

CD4 T Cells Treated with gp120 Acquire a CD45R0+/CD45RA+ Phenotype

HIV-infected patients exhibit quantitative and qualitative defects in CD4 T cells, including having increased numbers of CD4+CD45R0+/CD45RA+ T cells, although it remains unclear how these cells arise. Here we demonstrate that gp120 treatment of activated but not resting primary human CD4 T cells decreases number of cells with single positive CD45R0+/CD45RA- effector memory phenotype while proportionally increasing the subset of cells with double positive CD45R0+/CD45RA+ mixed phenotype. We found that double positive CD45R0+/CD45RA+CD4 T cells preferentially undergo apoptosis while single positive CD45R0+/CD45RA- and CD45R0-/CD45RA+ do not. Blocking gp120-CD4 interaction with sCD4 or inhibition Lck activity reverses gp120 induced increase in double positive CD45R0+/CD45RA+CD4 T cells and subsequently diminishes the apoptosis of double positive CD45R0+/CD45RA+ cells. Altogether these data indicate that gp120 ligation of the CD4 receptor increases the number of double positive CD45R0+/CD45RA+ CD4 T cells which subsequently undergo apoptosis in a CD4 dependent manner.

Engagement of the CD4 Receptor Affects the Redistribution of Lck to the Immunological Synapse in Primary T Cells: Implications for T-Cell Activation during Human Immunodeficiency Virus Type 1 Infection

Understanding the molecular mechanisms underlying dysregulated immune responses in human immunodeficiency virus type 1 (HIV-1) infection is crucial for the control of HIV/AIDS. Despite the postulate that HIV envelope glycoprotein gp120-CD4 interactions lead to impaired T-cell responses, the precise mechanisms underlying such association are not clear. To address this, we analyzed Lck and F-actin redistribution into the immunological synapse in stimulated human primary CD4(+) T cells from HIV-1-infected donors. Similar experiments were performed with CD4(+) T cells from HIV-uninfected donors, which were exposed to anti-CD4 domain 1 antibodies, as an in vitro model of gp120-CD4 interactions, or aldithriol-inactivated HIV-1 virions before stimulation. CD4(+) T cells from HIV-infected patients exhibited a two- to threefold inhibition of both Lck and F-actin recruitment into the synapse, compared to cells from uninfected donors. Interestingly, defective recruitment of Lck was ameliorated following suppressive highly active antiretroviral therapy. Engagement of the CD4 receptor on T cells from HIV-uninfected donors before anti-CD3/CD28 stimulation led to similar defects. Furthermore, the redistribution of Lck into lipid rafts was abrogated by CD4 preengagement. Our results suggest that the engagement of CD4 by HIV gp120 prior to T-cell receptor stimulation leads to dysregulation of early signaling events and could consequently play an important role in impaired CD4(+) T-cell function.