Oregano Oil and Its Principal Component, Carvacrol, Inhibit HIV-1 Fusion into Target Cells.

Oregano essential oil has long been known for its health-promoting benefits. Here, we report its activity against viral replication. Oregano oil was found to specifically inhibit lentiviruses, such as human and simian immunodeficiency viruses (HIV and SIV), irrespective of virus tropism, but not hepatitis C virus, adenovirus 5 (ADV5), Zika virus, and influenza (H1N1) virus. Oregano oil's most abundant components, carvacrol and its isomer, thymol, were shown to block virus-target cell fusion while not perturbing other stages of the virus life cycle. We detected changes in virus particle density, suggesting that cholesterol depletion from the HIV-1 envelope membrane reduces virus entry. Furthermore, infection was rescued by adding exogenous cholesterol. The evolution of viral resistance to carvacrol supported this mechanism of action with the identification of mutations in the viral gp41 fusion protein that counteracted cholesterol depletion. In addition, resistance to carvacrol emerged later than typically observed for other clinically used drugs, strengthening its antiviral potential. Structure-activity relationship studies revealed key motifs of carvacrol and thymol required for HIV neutralization and identified previously unknown active analogs. Carvacrol was also shown to additively cooperate with antiretroviral therapy. In sum, oregano oil and improved carvacrol and thymol analogs could be considered to supplement current HIV therapeutics.IMPORTANCE Oregano essential oil has multiple benefits in traditional medicine, cosmetics, and food industries. Carvacrol and its analog, thymol, are well-described components of oregano oil. Here, we show that these compounds inhibit HIV-target cell fusion independently of viral tropism. Our results suggest that carvacrol and thymol alter the cholesterol content of the viral membrane, blocking HIV-1 entry into the target cell. Resistance to carvacrol has selected for viruses with mutations in the viral envelope glycoprotein, gp41. This protein is known for its interaction with cholesterol present in membrane lipid rafts. Together, these results demonstrate the potential of therapies targeting the viral envelope membrane, and oregano oil is a safe supplement to antiretrovirals, potentially delaying disease progression and resistance development.

HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

Trilobatin as an HIV-1 entry inhibitor targeting the HIV-1 Gp41 envelope.

HIV-1 transmembrane protein gp41 plays a crucial role by forming a stable six-helix bundle during HIV entry. Due to highly conserved sequence of gp41, the development of an effective and safe small-molecule compound targeting gp41 is a good choice. Currently, natural polyanionic ingredients with anti-HIV activities have aroused concern. Here, we first discovered that a glycosylated dihydrochalcone, trilobatin, exhibited broad anti-HIV-1 activity and low cytotoxicity in vitro. Site-directed mutagenesis analysis suggested that the hydrophobic residue (I564) located in gp41 pocket-forming site is pivotal for anti-HIV activity of trilobatin. Furthermore, trilobatin displayed synergistic anti-HIV activities combined with other antiretroviral agents. Trilobatin has a good potential to be developed as a small-molecule HIV-1 entry inhibitor for clinical combination therapy.

Potential HIV-1 fusion inhibitors mimicking gp41-specific broadly neutralizing antibody 10E8: In silico discovery and prediction of antiviral potency.

An integrated computational approach to in silico drug design was used to identify novel HIV-1 fusion inhibitor scaffolds mimicking broadly neutralizing antibody (bNab) 10E8 targeting the membrane proximal external region (MPER) of the HIV-1 gp41 protein. This computer-based approach included (i) generation of pharmacophore models representing 3D-arrangements of chemical functionalities that make bNAb 10E8 active towards the gp41 MPER segment, (ii) shape and pharmacophore-based identification of the 10E8-mimetic candidates by a web-oriented virtual screening platform pepMMsMIMIC, (iii) high-throughput docking of the identified compounds with the gp41 MPER peptide, and (iv) molecular dynamics simulations of the docked structures followed by binding free energy calculations. As a result, eight hits-able to mimic pharmacophore properties of bNAb 10E8 by specific and effective interactions with the MPER region of the HIV-1 protein gp41 were selected as the most probable 10E8-mimetic candidates. Similar to 10E8, the predicted compounds target the critically important residues of a highly conserved hinge region of the MPER peptide that provides a conformational flexibility necessary for its functioning in cell-virus membrane fusion process. In light of the data obtained, the identified small molecules may present promising HIV-1 fusion inhibitor scaffolds for the design of novel potent antiviral drugs.

Synthesis, biophysical characterization and anti-HIV activity of d(TG3AG) Quadruplexes bearing hydrophobic tails at the 5'-end.

Novel conjugated G-quadruplex-forming d(TG3AG) oligonucleotides, linked to hydrophobic groups through phosphodiester bonds at 5'-end, have been synthesized as potential anti-HIV aptamers, via a fully automated, online phosphoramidite-based solid-phase strategy. Conjugated quadruplexes showed pronounced anti-HIV activity with some preference for HIV-1, with inhibitory activity invariably in the low micromolar range. The CD and DSC monitored thermal denaturation studies on the resulting quadruplexes, indicated the insertion of lipophilic residue at the 5'-end, conferring always improved stability to the quadruplex complex (20<ΔTm<40°C). The data suggest no direct functional relationship between the thermal stability and anti-HIV activity of the folded conjugated G-quartets. It would appear that the nature of the residue at 5' end of the d(TG3AG) quadruplexes plays an important role in the thermodynamic stabilization but a minor influence on the anti-HIV activity. Moreover, a detailed CD and DSC analyses indicate a monophasic behaviour for sequences I and V, while for ODNs (II-IV) clearly show that these quadruplex structures deviate from simple two-state melting, supporting the hypothesis that intermediate states along the dissociation pathway may exist.

Approaches for identification of HIV-1 entry inhibitors targeting gp41 pocket.

The hydrophobic pocket in the HIV-1 gp41 N-terminal heptad repeat (NHR) domain plays an important role in viral fusion and entry into the host cell, and serves as an attractive target for development of HIV-1 fusion/entry inhibitors. The peptide anti-HIV drug targeting gp41 NHR, T-20 (generic name: enfuvirtide; brand name: Fuzeon), was approved by the U.S. FDA in 2003 as the first HIV fusion/entry inhibitor for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, because T20 lacks the pocket-binding domain (PBD), it exhibits low anti-HIV-1 activity and short half-life. Therefore, several next-generation HIV fusion inhibitory peptides with PBD have been developed. They possess longer half-life and more potent antiviral activity against a broad spectrum of HIV-1 strains, including the T-20-resistant variants. Nonetheless, the clinical application of these peptides is still limited by the lack of oral availability and the high cost of production. Thus, development of small molecule compounds targeting the gp41 pocket with oral availability has been promoted. This review describes the main approaches for identification of HIV fusion/entry inhibitors targeting the gp41 pocket and summarizes the latest progress in developing these inhibitors as a new class of anti-HIV drugs.

Discovery of novel anti-HIV active G-quadruplex-forming oligonucleotides.

A series of d((5')TGGGAG(3')) sequences, 5'-conjugated with a variety of aromatic groups through phosphodiester linkages, were synthesized, showing CD spectra diagnostic of parallel-stranded, tetramolecular G-quadruplex structures. When tested for anti-HIV-1 and HIV-2 activity, potent inhibition of HIV-1 infection in CEM cell cultures was found, associated with high selectivity index values. Surface Plasmon Resonance assays revealed specific binding to HIV-1 gp120 and gp41.

A novel enzyme-linked immunosorbent assay for screening HIV-1 fusion inhibitors targeting HIV-1 Gp41 core structure.

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein mediates the fusion of viral and host cell membranes. As the HIV-1 enters the host cells, the 2 helical regions, HR1 and HR2, in the ectodomain of gp41 can form a 6-helix bundle, which brings the viral and target cell membranes to close proximity and serves as an attractive target for developing HIV-1 fusion inhibitors. Now, there are several cell- and molecule-based assays to identify potential HIV-1 fusion inhibitors targeting gp41. However, these assays cannot be used universally because they are time-consuming, inconvenient, and expensive. In the present study, the authors expressed and purified GST-HR121 and C43-30a proteins that were derived from the HIV-1 gp41 ectodomain region. GST-HR121 has a function similar to the HR1 peptide of gp41, whereas C43-30a is an HR2-derived peptide that added 50 amino acid residues (aa) in the N-terminal of C43. Further research found they could interact with each other, and a potential HIV-1 fusion inhibitor could inhibit this interaction. On the basis of this fact, a novel, rapid, and economic enzyme-linked immunosorbent assay was established, which can be developed for high-throughput screening of HIV-1 fusion inhibitors.

[A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins].

OBJECTIVE: To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors. METHODS: HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method. RESULTS: No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner. CONCLUSION: We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.

Computational study of bindings of olive leaf extract (OLE) to HIV-1 fusion protein gp41.

Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41. Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors.

Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation.

Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.

Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro.

Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

High-throughput screening method of inhibitors that block the interaction between 2 helical regions of HIV-1 gp41.

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors.

Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays an important role in the virus entry. During the process of fusion between the viral and target cell membranes, the N- and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.

High throughput screening for cyanovirin-N mimetics binding to HIV-1 gp41.

The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp41 is an important mediator of viral entry into host cells. Previous studies showed that the virucidal protein cyanovirin-N (CV-N) bound to both gp120 and gp41, and that this binding was associated with its antiviral activity. We constructed an HTS assay based on the interaction of europium-labeled CV-N with recombinant glycosylated gp41 ectodomain to support identification of small-molecule mimetics of CV-N that might be developed as antiviral drug leads. Primary screening of over 107,000 natural product extracts in the assay yielded 347 confirmed hits. Secondary assays eliminated extracts that bound directly to labeled CV-N or for which the simple sugars mannose and N-acetylglucosamine blocked the interaction with gp41 (lectin activity). Extracts were further prioritized based on anti-HIV activity and other biological, biochemical, and chemical criteria. The distribution of source organism taxonomy of active extracts was analyzed, as was the cross-correlation of activity between the CV-N-gp41 binding competition assay and the previously reported CV-N-gp120 binding competition assay. A limited set of extracts was selected for bioassay-guided fractionation.

Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1.

As one of the initial mucosal transmission pathways of HIV (HIV-1), epithelial cells translocate HIV-1 from apical to basolateral surface by nondegradative transcytosis. Transcytosis is initiated when HIV-1 envelope glycoproteins bind to the epithelial cell membrane. Here we show that the transmembrane gp41 subunit of the viral envelope binds to the epithelial glycosphingolipid galactosyl ceramide (Gal Cer), an alternative receptor for HIV-1, at a site involving the conserved ELDKWA epitope. Disrupting the raft organization of the Gal Cer-containing microdomains at the apical surface inhibited HIV-1 transcytosis. Immunological studies confirmed the critical role of the conserved ELDKWA hexapeptide in HIV-1 transcytosis. Mucosal IgA, but not IgG, from seropositive subjects targeted the conserved peptide, neutralized gp41 binding to Gal Cer, and blocked HIV-1 transcytosis. These results underscore the important role of secretory IgA in designing strategies for mucosal protection against HIV-1 infection.

A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure.

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.

Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements.

The trimeric, alpha-helical coiled-coil core of the HIV-1 gp41 ectodomain is thought to be part of a transient, receptor-triggered intermediate in the refolding of the envelope glycoprotein into a fusion-active conformation. In an effort to discover small organic inhibitors that block gp41 activation, we have generated a biased combinatorial chemical library of non-natural binding elements targeted to the gp41 core. From this library of 61,275 potential ligands, we have identified elements that, when covalently attached to a peptide derived from the gp41 outer-layer alpha-helix, contribute to the formation of a stable complex with the inner core and to inhibition of gp41-mediated cell fusion.

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.