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Enveloped viruses infect host cells via protein-mediated membrane fusion. However, insights into the microscopic rearrangement induced by the viral proteins and peptides have not yet emerged. Here, we report a new methodology to extract viral fusion peptide (FP)-mediated biomembrane dynamical nanodomain fusion parameter, λ, based on stimulated emission depletion microscopy coupled with fluorescence correlation spectroscopy. We also define another dynamical parameter membrane gradient, defined in terms of the ratio of average lipid diffusion coefficients across dynamic crossover length scales, ξ. Significantly, we observe that λ as well as these mobility gradients are larger in the stiffer liquid-ordered (L o ) phase compared to the liquid-disordered phase and are more effective at the smaller nanodomain interfaces, which are only present in the L o phase. The results could possibly help to resolve a long-standing puzzle about the enhanced fusogenicity of FP in the L o phase. Results obtained from the diffusion results have been correlated with the human immunodeficiency virus gp41 FP-induced membrane fusion.

In our previous work, we replaced the TRM (tryptophan-rich motif) of T20 (Enfuvirtide) with fatty acid (C16) to obtain the novel lipopeptide LP-40, and LP-40 displayed enhanced antiviral activity. In this study, we investigated whether the C16 modification could enhance the high-resistance barrier of the inhibitor LP-40. To address this question, we performed an in vitro simultaneous screening of HIV-1 NL4-3 resistance to T20 and LP-40. The mechanism of drug resistance for HIV-1 Env was further studied using the expression and processing of the Env glycoprotein, the effect of the Env mutation on the entry and fusion ability of the virus, and an analysis of changes to the gp41 core structure. The results indicate that the LP-40 activity is enhanced and that it has a high resistance barrier. In a detailed analysis of the resistance sites, we found that mutations in L33S conferred a stronger resistance, except for the well-recognized mutations in amino acids 36-45 of gp41 NHR, which reduced the inhibitory activity of the CHR-derived peptides. The compensatory mutation of eight amino acids in the CHR region (NDQEEDYN) plays an important role in drug resistance. LP-40 and T20 have similar resistance mutation sites, and we speculate that the same resistance profile may arise if LP-40 is used in a clinical setting.

The development of peptide-based HIV entry inhibitors has made an important contribution to the stock of anti-HIV drugs. In particular, the peptide-based anti-HIV drugs enfuvirtide and albuvirtide were approved for clinical use by the U.S. FDA and CFDA in 2003 and 2018, respectively. Peptide-based HIV entry inhibitors exert antiviral activity by targeting the early stage of viral infection, i.e., binding of a viral surface protein to the receptor(s) on the host cell and the subsequent fusion between the viral and host cell membranes. Therefore, they are particularly useful for HIV-infected patients who have failed to respond to the highly active antiretroviral drugs (ARD) targeting the late stage of HIV replication, such as reverse transcriptase inhibitors and protease inhibitors. In this chapter, we will focus on the past, current, and future trends in research and development of peptide-based HIV entry inhibitors., (© 2022. Springer Nature Singapore Pte Ltd.)

The continued HIV/AIDS epidemic worldwide and the battle against emerging infectious diseases caused by coronaviruses underscore the need for the development of an ever-expanding repertoire of antiviral drugs. Entry inhibitors are of particular interest because of their potential to be used as therapeutic or prophylactic treatments for blocking viral invasion. HIV and coronaviruses utilize class I fusion proteins to facilitate their entry and membrane fusion. Discovery of a common hexameric coiled-coil fusion complex resulting from the packing of three C-terminal heptad repeat region from the fusion-mediating subunit of viral fusion proteins against trimeric coiled-coil made up by their N-terminal heptad repeat prompted the search for peptides mimicking the heptad repeat regions that could potentially inhibit viral entry. This has led to the development of effective peptides that are specific to the virus that is developed for. In this review, we focus on peptide-based entry dual inhibitors that block fusion process not only of HIV but also coronaviruses through interrupting their fusogenic six-helical bundle core and which hopefully will help to gain insight into the α-helical secondary structure- and coiled-coil superstructure-based strategies to design entry inhibitors with broad-spectrum antiviral activity against enveloped viruses with class I fusion proteins., (© 2022. Springer Nature Singapore Pte Ltd.)

Interrupting early events in the virus life cycle, such as those prior to the formation of provirus, could effectively prevent HIV-1 infection. HIV-1 entry into host cells is mediated by the envelope glycoprotein (Env) trimer, which is composed of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Hence, the development of novel inhibitors targeting these two glycoproteins could hold the key to early inhibition of HIV-1 infection. Small-molecule entry inhibitors targeting early events in the virus life cycle comprise a well-established class of useful drugs. Many libraries of small-molecule inhibitors have been established to screen potential drug candidates for a variety of targets based on computer docking, FRET, or peptide-linked assay. This chapter reviews the mechanisms of some small-molecule inhibitors targeting HIV-1 gp120 and gp41 and corresponding high-efficiency screening strategies for potential small-molecule inhibitors., (© 2022. Springer Nature Singapore Pte Ltd.)

Anti-human immunodeficiency virus type 1 (anti-HIV-1) fusion peptides have been studied for nearly 2 decades, but few candidates have found useful clinical applications. One factor underlying the failure of such agents to reach the clinic is their poor pharmacokinetic properties, and many efforts have been made to overcome this problem. In this study, we modified C34, a peptide inhibitor of HIV-1 fusion, at its conserved glycosylation site using polyethylene glycols (PEGs) of different molecular weights. PEG40-NC, a conjugate of C34 and branched PEG 40 kDa (PEG40), which has been previously shown to improve the pharmacokinetic profiles of proteins, showed a significantly extended half-life ( t 1/2 ; 10.39 h in rats), which compensated for decreased in vitro activity (50% effective concentration [EC 50 ] of 18.51 nM). PEG40-NC also showed a mechanism of action similar to that of C34. PEG40-NC monotherapy in acutely simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys significantly suppressed viral load compared with a control treatment. Efficacy was linked to the extended half-life and lymphatic exposure conferred by attached PEG40. These results highlight the potential of further clinical investigations of PEG40-NC in combination with antiretroviral therapy or other anti-HIV agents. IMPORTANCE Poor pharmacokinetics have severely hindered the clinical use of anti-HIV peptides. Different small molecules, such as lipid, cholesterol, and small PEG, were designed to modify peptides to improve their pharmacokinetics. In this study, we incorporated large branched PEG to anti-HIV peptide and obtained a conjugate with extended half-life and improved in vivo efficacy. The strategy we developed in this study can also be applicable for the development of other peptide candidates., (Copyright © 2020 American Society for Microbiology.)

N-terminal heptad repeat (NHR)-derived peptide (N-peptide) fusion inhibitors, which are derived from human immunodeficiency virus (HIV) envelope glycoprotein 41 (gp41), are limited by aggregation and unstable trimer conformation. However, they could function as potent inhibitors of viral infection by forming a coiled-coil structure covalently stabilized by interchain disulfide bonds. We previously synthesized N-peptides with potent anti-HIV-1 activity and high stability by coiled-coil fusion and covalent stabilization. Here, we attempted to study the effects of NHRs of chimeric N-peptides by fusing de novo coiled-coil isopeptide bridge-tethered T21 peptides of different NHR lengths. Peptides (T21N23) 3 and (T21N36) 3 was a more potent HIV-1 fusion inhibitor than (T21N17) 3 . The site of isopeptide bond formation was precisely controlled and had little influence on N-peptide properties. The N-peptide (T21N36) 3 , which had a similar conformation as the NHR trimer and interacted well with the C34 peptide, may be useful for screening other C-peptides and small-molecule fusion inhibitors, and for studying the interactions between the NHR trimer and C-terminal heptad repeats., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Inducing immune responses protecting from HIV infection or at least controlling replication poses a huge challenge to modern vaccinology. An increasingly discussed strategy to elicit a potent and broad neutralizing antibody response is the immobilization of HIV's trimeric envelope (Env) surface receptor on a nanoparticulate carrier. As a conceptual proof, we attached an Env variant (BG505 SOSIP.664) to highly stable and biocompatible silica nanoparticles (SiNPs) via site-specific covalent conjugation or nonspecific adsorption to SiNPs. First, we demonstrated the feasibility of SiNPs as platform for Env presentation by a thorough characterization process during which Env density, attachment stability, and antigenicity were evaluated for both formulations. Binding affinities to selected antibodies were in the low nanomolar range for both formulations confirming that the structural integrity of Env is retained after attachment. Second, we explored the recognition of SiNP conjugates by antigen presenting cells. Here, the uptake of Env attached to SiNPs via a site-specific covalent conjugation was 4.5-fold enhanced, whereas adsorbed Env resulted only in a moderate 1.4-fold increase compared with Env in its soluble form. Thus, we propose SiNPs with site-specifically and covalently conjugated Env preferably in a high density as a promising candidate for further investigations as vaccine platform., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: There is no effective and safe preventive/therapeutics vaccine against HIV-1 worldwide. Different viral proteins such as Nef, and two regions of Env including; variable loop of gp120 (V3) and membrane proximal external region of gp41 (MPER) are particularly important for vaccine development in different strategies and they are also the primary targets of cellular and humoral immune responses. On the other side, LDP12 is a new cell-penetrating peptide (CPP) which is capable of therapeutic application and cargoes delivery across the cellular membrane., Objective: In current study, we designed and produced Nef-MPER-V3 fusion protein harboring LDP12 that has the capability of being used in future vaccine studies., Methods: The CPP-protein was expressed in E. coli Rosseta (DE3) strain and purified through Ni-NTA column. Characterization of cellular delivery and toxicity of the recombinant protein were evaluated by western blotting and MTT assay., Results: Our results showed that the CPP-protein was successfully expressed and purified with high yield of 5 mg/L. Furthermore, non-cytotoxic effect was observed and specific band (~ 37 KDa) in western blotting indicated the capability of LDP12 to improve the rate of penetration into HEK-293T cells in comparison with a control sample., Conclusion: Altogether, the data indicated that LDP12 CPP could be utilized to internalize HIV-1 Nef-MPER-V3 protein into eukaryotic cell lines without any toxicity and represented a valuable potential vaccine candidate and this guarantees the further evaluation towards the assessment of its immunogenicity in mice, which is currently under process., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The trimeric heptad repeat domains HR1 and HR2 of the human immunodeficiency virus 1 (HIV-1) gp41 play a key role in HIV-1-entry by membrane fusion. To develop efficient inhibitors against this step, the corresponding trimeric-N36 and C34 peptides were designed and synthesized. Analysis by circular dichroism of monomeric and trimeric N36 and C34 peptides showed their capacities to adopt α-helical structures and to establish physical interactions. At the virological level, while trimeric-C34 conserves the same high anti-fusion activity as monomeric-C34, trimerization of N36-peptide induced a significant increase, reaching 500-times higher in anti-fusion activity, against R5-tropic virus-mediated fusion. This result was associated with increased stability of the N36 trimer peptide with respect to the monomeric form, as demonstrated by the comparative kinetics of their antiviral activities during 6-day incubation in a physiological medium. Collectively, our findings demonstrate that while the trimerization of C34 peptide had no beneficial effect on its stability and antiviral activity, the trimerization of N36 peptide strengthened both stability and antiviral activity. This approach, promotes trimers as new promising HIV-1 inhibitors and point to future development aimed toward innovative peptide fusion inhibitors, microbicides or as immunogens., (© 2019 The Author(s).)

The clinical application of HIV fusion inhibitor, enfuvirtide (T20), was limited mainly because of its short half-life. Here we designed and synthesized two PEGylated C34 peptides, PEG2kC34 and PEG5kC34, with the PEG chain length of 2 and 5 kDa, respectively, and evaluated their anti-HIV-1 activity and mechanisms of action. We found that these two PEGylated peptides could bind to the HIV-1 peptide N36 to form high affinity complexes with high α-helicity. The peptides PEG2kC34 and PEG5kC34 effectively inhibited HIV-1 Env-mediated cell-cell fusion with an effective concentration for 50% inhibition (EC 50 ) of about 36 nM. They also inhibited infection of the laboratory-adapted HIV-1 strain NL4-3 with EC 50 of about 4-5 nM, and against 47 HIV-1 clinical isolates circulating in China with mean EC 50 of PEG2kC34 and PEG5kC34 of about 26 nM and 32 nM, respectively. The plasma half-life (t 1/2 ) of PEG2kC34 and PEG5kC34 was 2.6 h and 5.1 h, respectively, and the t 1/2 of PEGylated C34 was about 2.4-fold and 4.6-fold longer than C34 (~1.1 h), respectively. These findings suggest that PEGylated C34 with broad-spectrum anti-HIV-1 activity and prolonged half-life can be further developed as a peptide fusion inhibitor-based long-acting anti-HIV drug for clinical use to treat HIV-infected patients who have failed to respond to current anti-retrovirus drugs.

C34, a 34-mer fragment peptide, is contained in the HIV-1 envelope protein gp41. A dimeric derivative of C34 linked through a disulfide bridge at its C terminus was synthesized and found to display potent anti-HIV activity, comparable with that of a previously reported PEGylated dimer of C34REG. The reduction in the size of the linker moiety for dimerization was thus successful, and this result might shed some light on the mechanism of the suppression of six-helix bundle formation by these C34 dimeric derivatives. Addition of a Gly-Cys(CH 2 CONH 2 )-Gly-Gly motif at the N-terminal position of a C34 monomeric derivative significantly increased the anti-HIV-1 activity. This moiety functions as a new pharmacophore, and this might provide a useful insight into the design of potent HIV-1 fusion inhibitors., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lung cancer is the leading cause of cancer-related deaths worldwide; hence novel treatments for this malignancy are eagerly needed. Since natural-based compounds represent a rich source of novel chemical entities in drug discovery, we have focused our attention on tambjamines, natural compounds isolated from marine invertebrates that have shown diverse pharmacological activities. Based on these structures, we have recently identified the novel indole-based tambjamine analog 21 (T21) as a promising antitumor agent, which modulates the expression of apoptotic proteins such as survivin. This antiapoptotic protein plays an important role in carcinogenesis and chemoresistance. In this work, we have elucidated the molecular mechanism by which the anticancer compound T21 exerts survivin inhibition and have validated this protein as a therapeutic target in different lung cancer models. T21 was able to reduce survivin protein levels in vitro by repressing its gene expression through the blockade of Janus kinase/Signal Transducer and Activator of Transcription-3 (JAK/STAT3)/survivin signaling pathway. Interestingly, this occurred even when the pathway was overstimulated with its ligand interleukin 6 (IL-6), which is frequently overexpressed in lung cancer patients who show poor clinical outcomes. Altogether, these results show T21 as a potent anticancer compound that effectively decreases survivin levels through STAT3 inhibition in lung cancer, appearing as a promising therapeutic drug for cancer treatment.

A short sequence on the gp41 envelope protein of HIV-1 is integral to infection by the virus. Without this sequence, termed the fusion peptide (FP), the virus is far less effective at fusing with the cellular membrane. One of the interesting features of the isolated FP is that it transitions between an α-helical conformation and a β-sheet conformation in lipid bilayer membranes as a function of lipid composition and concentration, and the transition correlates with fusion. To better understand how the conformations of the FP impact lipid bilayer membranes, a variant of the FP that does not strongly promote fusion, termed gp41rk, was studied. Circular dichroism spectroscopy, dynamic light scattering, small-angle neutron scattering (SANS) and neutron spin echo spectroscopy (NSE) were used to relate the conformation of gp41rk to the structure and mechanical properties of lipid bilayer membrane vesicles composed of a 7:3 molar ratio mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol). At a peptide-to-lipid ratio (P/L) of 1/200, it adopts an α-helical conformation, while gp41rk is a β-sheet at a P/L of 1/50 in the unilamellar vesicles. SANS reveals that the lipid bilayer membrane becomes thicker when gp41rk adopts a β-sheet conformation, which indicates that the high-concentration state of the peptide increases the order of the lipid acyl chains. At the same time, NSE demonstrates that the bilayer becomes more rigid, demonstrating that the β-sheet conformation, which correlates with fusion for the native FP sequence, stiffens the bilayer. The results have implications for the function of the FP., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The deep hydrophobic pocket of HIV-1 gp41 has been considered a drug target, but short-peptides targeting this site usually lack potent antiviral activity. By applying the M-T hook structure, we previously generated highly potent short-peptide fusion inhibitors that specifically targeted the pocket site, such as MT-SC22EK, HP23L, and LP-11. Here, the crystal structures of HP23L and LP-11 bound to the target mimic peptide N36 demonstrated the critical intrahelical and interhelical interactions, especially verifying that the hook-like conformation was finely adopted while the methionine residue was replaced by the oxidation-less prone residue leucine, and that addition of an extra glutamic acid significantly enhanced the binding and inhibitory activities. The structure of HP23L bound to N36 with two mutations (E49K and L57R) revealed the critical residues and motifs mediating drug resistance and provided new insights into the mechanism of action of inhibitors. Therefore, the present data help our understanding for the structure-activity relationship (SAR) of HIV-1 fusion inhibitors and facilitate the development of novel antiviral drugs.

Most peptide drugs have short half-lives, necessitating frequent injections that may induce skin sensitivity reactions; therefore, versatile prolonged-release delivery platforms are urgently needed. Here, we focused on an oxidatively and thermally responsive recombinant elastin-like polypeptide with periodic cysteine residues (cELP), which can rapidly and reversibly form a disulfide cross-linked network in which peptide can be physically incorporated. As a model for proof of concept, we used enfuvirtide, an antiretroviral fusion-inhibitor peptide approved for treatment of human immunodeficiency virus (HIV) infection. cELP was mixed with enfuvirtide and a small amount of hydrogen peroxide (to promote cross-linking), and the soluble mixture was injected subcutaneously. The oxidative cross-linking generates a network structure, causing the mixture to form a hydrogel in situ that serves as an enfuvirtide depot. We fabricated a series of enfuvirtide-containing hydrogels and examined their stability, enfuvirtide-releasing profile and anti-HIV potency in vitro. Among them, hydrophobic cELP hydrogel provided effective concentrations of enfuvirtide in blood of rats for up to 8 h, and the initial concentration peak was suppressed compared with that after injection of enfuvirtide alone. cELP hydrogels should be readily adaptable as platforms to provide effective depot systems for delivery of other anti-HIV peptides besides enfuvirtide., Statement of Significance: In this paper, we present an anti-HIV peptide delivery system using oxidatively and thermally responsive polypeptides that contain multiple periodic cysteine residues as an injectable biomaterial capable of in situ self-gelation, and we demonstrate its utility as an injectable depot capable of sustained release of anti-HIV peptides. The novelty of this work stems from the platform employed to provide the depot encapsulating the peptide drugs (without chemical conjugation), which consists of rationally designed, genetically engineered polypeptides that enable the release rate of the peptide drugs to be precisely controlled., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition. IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40., (Copyright © 2017 American Society for Microbiology.)

Protein- or peptide-based viral inactivators are being developed as novel antiviral drugs with improved efficacy, pharmacokinetics and toxicity profiles because they actively inactivate cell-free human immunodeficiency virus type 1 (HIV-1) virions before attachment to host cells. By contrast, most clinically used antiviral drugs must penetrate host cells to inhibit viral replication. In this study, we pre-treated HIV-1 particles with a gp120-targeting bispecific multivalent protein, 2Dm2m or 4Dm2m, in the presence or absence of the gp41-targeting HIV-1 fusion inhibitory peptides enfuvirtide (T20), T2635, or sifuvirtide (SFT). HIV-1 virions were separated from the inhibitors using PEG-6000, followed by testing of the residual infectivity of the HIV-1 virions. 2Dm2m and 4Dm2m exhibited significant inactivation activity against all HIV-1 strains tested with EC 50 values at the low nanomolar level, whereas none of the gp41-targeting peptides showed inactivation activity at concentrations up to 250 nM. Notably, these three peptides significantly enhanced protein-mediated inactivation against cell-free HIV-1 virions, including HIV-1 laboratory-adapted and primary HIV-1 strains, as well as those resistant to T20 or T2635 and virions released from reactivated latently HIV-1-infected cells. These results indicate that the gp120-targeting bispecific multivalent proteins 2Dm2m and 4Dm2m have potential for further development as HIV-1 inactivator-based antiviral drugs for use in the clinic, either alone or in combination with a gp41-targeting HIV-1 fusion inhibitor such as T20, to treat patients with HIV-1 infection and AIDS.

Peptides derived from the C-terminal heptad repeat (CHR) region of the human immunodeficiency virus type 1 (HIV-1) fusogenic protein gp41 are potent viral entry inhibitors, and currently, enfuvirtide (T-20) is the only one approved for clinical use; however, emerging drug resistance largely limits its efficacy. In this study, we generated a novel lipopeptide inhibitor, named LP-19, by integrating multiple design strategies, including an N-terminal M-T hook structure, an HIV-2 sequence, intrahelical salt bridges, and a membrane-anchoring lipid tail. LP-19 showed stable binding affinity and highly potent, broad, and long-lasting antiviral activity. In in vitro studies, LP-19 efficiently inhibited HIV-1-, HIV-2-, and simian immunodeficiency virus (SIV)-mediated cell fusion, viral entry, and infection, and it was highly active against diverse subtypes of primary HIV-1 isolates and inhibitor-resistant mutants. Ex vivo studies demonstrated that LP-19 exhibited dramatically increased anti-HIV activity and an extended half-life in rhesus macaques. In short-term monotherapy, LP-19 reduced viral loads to undetectable levels in acutely and chronically simian-human immunodeficiency virus (SHIV)-infected monkeys. Therefore, this study offers an ideal HIV-1/2 fusion inhibitor for clinical development and emphasizes the importance of the viral fusion step as a drug target. IMPORTANCE The peptide drug T-20 is the only viral fusion inhibitor in the clinic, which is used for combination therapy of HIV-1 infection; however, it requires a high dosage and easily induces drug resistance, calling for a new drug with significantly improved pharmaceutical profiles. Here, we have developed a short-lipopeptide-based fusion inhibitor, termed LP-19, which mainly targets the conserved gp41 pocket site and shows highly potent inhibitory activity against HIV-1, HIV-2, and even SIV isolates. LP-19 exhibits dramatically increased antiviral activity and an extended half-life in rhesus macaques, and it has potent therapeutic efficacy in SHIV-infected monkeys, highlighting its high potential as a new viral fusion inhibitor for clinical use., (Copyright © 2017 American Society for Microbiology.)

The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through modulation of the cytokine release and active inhibition of immune responses.

Most mechanistic studies on human immunodeficiency virus (HIV) peptide fusion inhibitors have focused on the interactions between fusion inhibitors and viral envelope proteins. However, the interactions of fusion inhibitors with viral membranes are also essential for the efficacy of these drugs. Here, we utilized surface plasmon resonance (SPR) technology to study the interactions between the HIV fusion inhibitor peptides sifuvirtide and enfuvirtide and biomembrane models. Sifuvirtide presented selectivity toward biomembrane models composed of saturated dipalmitoylphosphatidylcholine (DPPC) (32-fold higher compared with unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [POPC]) and sphingomyelin (SM) (31-fold higher compared with POPC), which are rigid compositions enriched in the HIV viral membrane. In contrast, enfuvirtide showed no significant selectively toward these rigid membrane models. Furthermore, the bindings of sifuvirtide and enfuvirtide to SM bilayers were markedly higher than those to monolayers (14-fold and 23-fold, respectively), indicating that the inner leaflet influences the binding of these drugs to SM bilayers. No obvious differences were noted in the bindings of either peptide to the other mono- and bilayer models tested, illustrating that both peptides interact with these membranes through surface-binding. The bindings of the inhibitor peptides to biomembranes were found to be driven predominantly by hydrophobic interactions rather than electrostatic interactions, as determined by comparing their affinities to those of positively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPC) to zwitterionic membrane models. The improved efficiency of sifuvirtide relative to enfuvirtide might be related to its ability to adsorb on rigid lipidic areas, such as the viral envelope and lipid rafts, which results in an increased sifuvirtide concentration at the fusion site.

20 (enfuvirtide) and other peptides derived from the human immunodeficiency virus type 1 (HIV-1) gp41 C-terminal heptad repeat (CHR) region inhibit HIV fusion by binding to the hydrophobic grooves on the N-terminal heptad repeat (NHR) trimer and blocking six-helix-bundle (6-HB) formation. Several strategies focusing on the binding grooves of the NHR trimer have been adopted to increase the antiviral activity of the CHR peptides. Here, we developed a novel and simple strategy to greatly enhance the potency of the existing peptide-based HIV fusion inhibitors. First, we identified a shallow pocket adjacent to the groove in the N-terminal region of NHR trimer as a new drug target, and then we designed several short artificial peptides to fit this target. After the addition of IDL (Ile-Asp-Leu) to the C terminus of CHR peptide WQ or MT-WQ, the conjugated peptides, WQ-IDL and MT-WQ-IDL, showed much more potent activities than WQ and T20, respectively, in inhibiting HIV-1 IIIB infection. WQ-IDL and MT-WQ-IDL were also more effective than WQ in blocking HIV-1 Env-mediated membrane fusion and had higher levels of binding affinity with NHR peptide N46. We solved the crystal structure of the 6-HB formed by MT-WQ-IDL and N46 and found that, besides the N-terminal MT hook tail, the IDL tail anchor of MT-WQ-IDL also binds with the shallow hydrophobic pocket outside the groove of the NHR trimer, resulting in enhanced inhibition of HIV-1 fusion with the target cell. It is expected that this novel approach can be widely used to improve the potency of peptidic fusion inhibitors against other enveloped viruses with class I fusion proteins., Importance: The hydrophobic groove of the human immunodeficiency virus type 1 (HIV-1) gp41 NHR trimer has been known as the classic drug target to develop fusion inhibitors derived from the gp41 CHR. Here, we developed a novel and simple strategy to improve the existing peptide-based HIV fusion inhibitors. We identified a shallow pocket adjacent to the groove in the NHR trimer and added a short artificial peptide consisting of three amino acids (IDL) to the C terminus of a fusion inhibitor to fit this new target. The inhibition activity of this new conjugated peptide was significantly enhanced, by 77-fold, making it much more potent than T20 (enfuvirtide) and suggesting that the IDL tail can be adopted for optimizing existing HIV-1 CHR peptide fusion inhibitors. This new approach of identifying a potential binding pocket outside the traditional target and creating an artificial tail anchor can be widely applied to design novel fusion inhibitors against other class I enveloped viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV)., (Copyright © 2016 American Society for Microbiology.)

Human immunodeficiency virus type 2 (HIV-2) has already spread to different regions worldwide, and currently about 1 to 2 million people have been infected, calling for new antiviral agents that are effective on both HIV-1 and HIV-2 isolates. T20 (enfuvirtide), a 36-mer peptide derived from the C-terminal heptad repeat region (CHR) of gp41, is the only clinically approved HIV-1 fusion inhibitor, but it easily induces drug resistance and is not active on HIV-2. In this study, we first demonstrated that the M-T hook structure was also vital to enhancing the binding stability and inhibitory activity of diverse CHR-based peptide inhibitors. We then designed a novel short peptide (23-mer), termed 2P23, by introducing the M-T hook structure, HIV-2 sequences, and salt bridge-forming residues. Promisingly, 2P23 was a highly stable helical peptide with high binding to the surrogate targets derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV). Consistent with this, 2P23 exhibited potent activity in inhibiting diverse subtypes of HIV-1 isolates, T20-resistant HIV-1 mutants, and a panel of primary HIV-2 isolates, HIV-2 mutants, and SIV isolates. Therefore, we conclude that 2P23 has high potential to be further developed for clinical use, and it is also an ideal tool for exploring the mechanisms of HIV-1/2- and SIV-mediated membrane fusion., Importance: The peptide drug T20 is the only approved HIV-1 fusion inhibitor, but it is not active on HIV-2 isolates, which have currently infected 1 to 2 million people and continue to spread worldwide. Recent studies have demonstrated that the M-T hook structure can greatly enhance the binding and antiviral activities of gp41 CHR-derived inhibitors, especially for short peptides that are otherwise inactive. By combining the hook structure, HIV-2 sequence, and salt bridge-based strategies, the short peptide 2P23 has been successfully designed. 2P23 exhibits prominent advantages over many other peptide fusion inhibitors, including its potent and broad activity on HIV-1, HIV-2, and even SIV isolates, its stability as a helical, oligomeric peptide, and its high binding to diverse targets. The small size of 2P23 would benefit its synthesis and significantly reduce production cost. Therefore, 2P23 is an ideal candidate for further development, and it also provides a novel tool for studying HIV-1/2- and SIV-mediated cell fusion., (Copyright © 2016 American Society for Microbiology.)

Peptides derived from HIV-1 transmembrane proteins have been extensively studied for antimicrobial activities, and they are known as antimicrobial peptides (AMPs). These AMPs have also been reported to potently combat the drug-resistant microbes. In this study, we demonstrated that peptide #6383 originated from HIV-1 MN strain membrane-spanning domain of gp41 was active (2-log reductions) at 100βg/mL (56.5βM) against methicillin-resistant Staphylococcus aureus (MRSA) in 10% and 50% human plasma-supplemented phosphate buffered saline (PBS). The activity was further enhanced (3-log reductions) in the presence of 5% human serum albumin (HSA) alone. All bactericidal activities were achieved within 6 hours. At 100μg/mL, the peptide showed only 13% toxicity against human erythrocytes. This peptide can serve as an attractive template for a design of a novel peptide antibiotic against drug-resistant bacteria. By sequence-specific engineering or modifications, we anticipated that the bactericidal activity and the reduced toxicity against human erythrocytes will be improved.

Lipid rafts of the plasma membrane have been shown to be gateways for HIV-1 budding and entry. In nature, many glycosyl-phosphatidylinositol (GPI) anchored proteins are targeted to the lipid rafts. In the present study we constructed two fusion genes, in which C34 peptide or AVF peptide control was genetically linked with a GPI-attachment signal. Recombinant lentiviruses expressing the fusion genes were used to transduce TZM.bl and CEMss-CCR5 cells. Here, we show that with a GPI attachment signal both C34 and AVF are targeted to the lipid rafts through a GPI anchor. GPI-C34, but not GPI-AVF, in transduced TZM.bl cells efficiently blocks the infection of diverse HIV-1 strains of various subtypes. GPI-C34-transduced CEMss-CCR5 cells are totally resistant to HIV-1 infection. Importantly, maximum percentage of inhibition (MPI) by GPI-C34 is comparable to, if not higher than, a very high concentration of soluble C34. Potent blocking by GPI-C34 is likely due to its high local concentration, which allows GPI-C34 to efficiently bind to the prehairpin intermediate and prevent its transition to six helical bundle, thereby interfering with membrane fusion and virus entry. Our findings should have important implications in GPI-anchor-based therapy against HIV-1.

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection.

The synthesis of a β-thiol asparagine derivative bearing a novel (2,4,6-trimethoxyphenyl)thiazolidine protecting group is described. The efficient incorporation of the amino acid into the N-termini of peptides is demonstrated as well as the utility of the β-thiol asparagine moiety for rapid ligation reactions with peptide thioesters. The streamlined synthesis of native peptide products could be accomplished using a one-pot radical desulfurization of the β-thiol auxiliary following the ligation event. The utility of the amino acid is highlighted in the efficient one-pot assembly of the HIV entry inhibitor enfuvirtide.

The highlighted next-generation HIV-1 fusion inhibitor peptide 1 is capped by two threonines. Here, we generated peptide 2 by deleting the T-T motif and compared their structural and antiviral properties. Significantly, two peptides showed similar helical and oligomeric states in solution, comparable binding affinities to the target, and no significant difference to inhibit HIV-1 fusion and infection. Also, the T-T motif was not associated with peptide 1 resistant mutations and its deletion did not affect peptide 1 against enfuvirtide-resistant HIV-1 mutants. The redundancy of the T-T motif was further verified by the model peptide C34 and short peptide inhibitors that mainly target the gp41 pocket, suggesting that the N-terminal T-T motif of peptide 1 could be removed or modified toward the development of new anti-HIV-1 drugs. Consistently, our data have verified that the M-T hook structure rather than the T-T motif is an efficient strategy for short peptide fusion inhibitors.

Background: Cryptococcal meningitis is the most common fungal infection of the central nervous system (CNS) in HIV/AIDS. HIV-1 virotoxins (e.g., gp41) are able to induce disorders of the blood-brain barrier (BBB), which mainly consists of BMEC. Our recent study suggests that α7 nAChR is an essential regulator of inflammation, which contributes to regulation of NF-κB signaling, neuroinflammation and BBB disorders caused by microbial (e.g., HIV-1 gp120) and non-microbial [e.g., methamphetamine (METH)] factors. However, the underlying mechanisms for multiple comorbidities are unclear., Methods: In this report, an aggravating role of α7 nAChR in host defense against CNS disorders caused by these comorbidities was demonstrated by chemical [inhibitor: methyllycaconitine (MLA)] and genetic (α7(-/-) mice) blockages of α7 nAChR., Results: As shown in our in vivo studies, BBB injury was significantly reduced in α7(-/-) mice infected with C. neoformans. Stimulation by the gp41 ectodomain peptide (gp41-I90) and METH was abolished in the α7(-/-) animals. C. neoformans and gp41-I90 could activate NF-κB. Gp41-I90- and METH-induced monocyte transmigration and senescence were significantly inhibited by MLA and CAPE (caffeic acid phenethyl ester, an NF-κB inhibitor)., Conclusions: Collectively, our data suggest that α7 nAChR plays a detrimental role in the host defense against C. neoformans- and HIV-1 associated comorbidity factors-induced BBB injury and CNS disorders.

Enfuvirtide (T20), is the first HIV fusion inhibitor approved for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, its clinical application is limited because of short half-life, drug resistance and cross-reactivity with the preexisting antibodies in HIV-infected patients. Using an artificial peptide strategy, we designed a peptide with non-native protein sequence, AP3, which exhibited potent antiviral activity against a broad spectrum of HIV-1 strains, including those resistant to T20, and had remarkably longer in vivo half-life than T20. While the preexisting antibodies in HIV-infected patients significantly suppressed T20's antiviral activity, these antibodies neither recognized AP3, nor attenuated its anti-HIV-1 activity. Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses.

A key barrier against developing preventive and therapeutic human immunodeficiency virus (HIV) vaccines is the inability of viral envelope glycoproteins to elicit broad and potent neutralizing antibodies. However, in the presence of fusion inhibitor enfuvirtide, we show that the nonneutralizing antibodies induced by the HIV type 1 (HIV-1) gp41 N-terminal heptad repeat (NHR) domain (N63) exhibit potent and broad neutralizing activity against laboratory-adapted HIV-1 strains, including the drug-resistant variants, and primary HIV-1 isolates with different subtypes, suggesting the potential of developing gp41-targeted HIV therapeutic vaccines., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The deep hydrophobic pocket on the N trimer of HIV-1 gp41 has been considered an ideal drug target. On the basis of the M-T hook structure, we recently developed short-peptide-based HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the pocket site and possess highly potent antiviral activity. In this study, we focused on investigating their resistance pathways and mechanisms by escape HIV-1 mutants to SC22EK, a template peptide for MTSC22 and HP23. Two substitutions, E49K and N126K, located, respectively, at the N- and C-heptad repeat regions of gp41, were identified as conferring high resistance to the inhibitors targeting the pocket and cross-resistance to enfuvirtide (T20) and sifuvirtide (SFT). The underlying mechanisms of SC22EK-induced resistance include the following: (i) significantly reduced binding affinity of the inhibitors, (ii) dramatically enhanced interaction of the viral six-helix bundle, and (iii)severely damaged functionality of the viral Env complex. Our data have provided important information for the structure-function relationship of gp41 and the structure-activity relationship of viral fusion inhibitors., Importance: Enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but the problem of resistance significantly limits its use, calling for new strategies or concepts to develop next-generation drugs. On the basis of the M-T hook structure, short-peptide HIV-1 fusion inhibitors specifically targeting the gp41 pocket site exhibit high binding and antiviral activities. Here, we investigated the molecular pathway of HIV-1 resistance to the short inhibitors by selecting and mapping the escape mutants. The key substitutions for resistance and the underlying mechanisms have been finely characterized. The data provide important information for the structure-function relationship of gp41 and its inhibitors and will definitely help our future development of novel drugs that block gp41-dependent fusion., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8+ T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4+ T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.

Many peptide-based therapeutics have short circulatory half-lives. We report here that the pharmacokinetics of an anti-HIV peptide drug enfuvirtide (ENF) can be dramatically improved by a chemical glycosylation approach. A set of glycosylated ENFs with varying glycosylation sites and glycan structures were synthesized. Among these, a sialic acid-introduced peptide (SL-ENF) demonstrated a 15-fold extended half-life in rats relative to ENF (T1/2: 23.1 vs 1.5 h), and its antiviral potency was comparable to that of ENF (EC50: 2 vs 3 nM). SL-ENF bound to a functional fragment of the HIV fusogenic protein gp41 and formed complexes with high affinity and α-helicity, revealing the mechanism behind its potent antiviral activity. Because it is widely accepted in biology that glycosylation protects proteins from denaturation and proteases, our approach may be useful for the development of novel protein and peptide drugs with enhanced pharmaceutical properties.

Triterpene saponins are a major group of active components in natural products with nonspecific antiviral activities, while T20 peptide (enfuvirtide), which contains a helix zone-binding domain (HBD), is a gp41-specific HIV-1 fusion inhibitor. In this paper, we report the design, synthesis, and structure-activity relationship (SAR) of a group of hybrid molecules in which bioactive triterpene sapogenins were covalently attached to the HBD-containing peptides via click chemistry. We found that either the triterpenes or peptide part alone showed weak activity against HIV-1 Env-mediated cell-cell fusion, while the hybrids generated a strong cooperative effect. Among them, P26-BApc exhibited anti-HIV-1 activity against both T20-sensitive and -resistant HIV-1 strains and improved pharmacokinetic properties. These results suggest that this scaffold design is a promising strategy for developing new HIV-1 fusion inhibitors and possibly novel antiviral therapeutics against other viruses with class I fusion proteins.

Fuzeon (Enfuvirtide, T20) is the first fusion inhibitor approved by the FDA of the USA for the treatment of HIV/AIDS in combination with other anti-retroviral drugs. Enfuvirtide is a synthetic peptide that blocks the entry of HIV into healthy host CD4 cells, which requires very high (90 mg twice daily) therapeutic doses. To increase the yield of Enfuvirtide, a gene polymerization strategy was introduced and recombinant T20 (rT20) was expressed in Escherichia coli as a five copy repeat polypeptide with a histidine-tag. The five copy rT20 was purified by Ni-affinity chromatography and cleaved to single rT20 units by cyanogen bromide. Finally, single rT20 units were purified by reversed phase chromatography giving a yield (400 mg/l) with a purity >95 %, which exhibited specific biological activity similar to Fuzeon.

Background: The membrane-proximal external region (MPER) of HIV-1 gp41 is particularly conserved and target for the potent broadly neutralizing monoclonal antibodies (bnMAbs) 2F5, 4E10 and 10E8. Epitope focusing and stabilization present promising strategies to enhance the quality of immune responses to specific epitopes., Results: The aim of this work was to design and evaluate novel immunogens based on the gp41 MPER with the potential to elicit cross-clade neutralizing antibodies. For that purpose, gp41 was truncated N-terminally in order to dispose immunodominant, non-neutralizing sites and enhance the exposure of conserved regions. To stabilize a trimeric conformation, heterologous GCN4 and HA2 zipper domains were fused based on an in silico "best-fit" model to the protein's amino terminus. Cell surface exposure of resulting proteins and their selective binding to bnMAbs 2F5 and 4E10 could be shown by cytometric analyses. Incorporation into VLPs and preservation of antigenic structures were verified by electron microscopy, and the oligomeric state was successfully stabilized by zipper domains. These gp41 immunogens were evaluated for antigenicity in an immunization study in rabbits primed with homologous DNA expression plasmids and boosted with virus-like particle (VLP) proteins. Low titers of anti-MPER antibodies were measured by IgG ELISA, and low neutralizing activity could be detected against a clade C and B viral isolate in sera., Conclusions: Thus, although neutralizing titers were very moderate, induction of cross-clade neutralizing antibodies seems possible following immunization with MPER-focusing immunogens. However, further refinement of MPER presentation and immunogenicity is clearly needed to induce substantial neutralization responses to these epitopes.

We previously described indole-containing compounds with the potential to inhibit HIV-1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here we report optimization and structure-activity relationship studies on the basic scaffold, defining the role of shape, contact surface area, and molecular properties. Thirty new compounds were evaluated in binding, cell-cell fusion, and viral replication assays. Below a 1 μM threshold, correlation between binding and biological activity was diminished, indicating an amphipathic requirement for activity in cells. The most active inhibitor 6j exhibited 0.6 μM binding affinity and 0.2 μM EC50 against cell-cell fusion and live virus replication and was active against T20 resistant strains. Twenty-two compounds with the same connectivity displayed a consensus pose in docking calculations, with rank order matching the biological activity. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion and demonstrates a potent low molecular weight fusion inhibitor.

Objectives: To develop new HIV-1 fusion inhibitors with improved antiviral activities and resistance profiles, we designed two categories of artificial peptides, each containing four heptad repeats (m4HR) conjugated with a pocket-specific small molecule (pssm) or pssm and cholesterol (chol), designated pssm-m4HR or pssm-m4HR-chol, respectively, and tested their anti-HIV-1 activity., Methods: We synthesized the artificial peptides and conjugated these peptides with pssm and chol using a standard solid-phase Fmoc protocol and a chemoselective thioether conjugation method, respectively. We tested the inhibitory activities of the peptide conjugates against HIV-1 Env-mediated cell-cell fusion and infection by laboratory-adapted and primary HIV-1 isolates, and enfuvirtide-resistant HIV-1 strains using cell-cell fusion and p24 production assays, respectively. We assessed their cytotoxicity towards MT-2 cells using the XTT assay., Results: We found that pssm-m4HR conjugates exhibited promising inhibitory activity against HIV-1 Env-mediated cell-cell fusion and laboratory-adapted HIV-1 replication with IC50 values at the low micromolar level, whereas the pssm-m4HR-chol conjugates exhibited dramatically increased anti-HIV-1 activities with IC50 values at the low nanomolar level. Some of the pssm-m4HR-chol conjugates (e.g. 5a and 5b) showed highly potent antiviral activity against infection by primary HIV-1 isolates and enfuvirtide-resistant HIV-1 strains. All the conjugates displayed no or low cytotoxicity towards MT-2 cells. The result of a prime/wash assay indicated pssm-m4HR-chol conjugates were strongly anchored to the membrane and sustained a potent inhibitory effect after washing., Conclusions: These results suggest this scaffold design is a promising strategy for developing novel peptide conjugates with improved antiviral activity against a broad spectrum of HIV-1 strains, including those highly resistant to enfuvirtide., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34-polyethylene glycol (PEG)₄-cholesterol] and HIVP4 [(C34-PEG₄)₂-cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy., Methods: Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays., Results: Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34-cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol., Conclusions: The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.

Unlabelled: HIV-1 membranes contain gp120-gp41 trimers. Binding of gp120 to CD4 and a coreceptor (CCR5 or CXCR4) reduces the constraint on metastable gp41, enabling a series of conformational changes that cause membrane fusion. An analytic difficulty occurs because these steps occur slowly and asynchronously within cohorts of adsorbed virions. We previously isolated HIV-1JRCSF variants that efficiently use CCR5 mutants severely damaged in the tyrosine-sulfated amino terminus or extracellular loop 2. Surprisingly, both independent adaptations included gp120 mutations S298N, F313L, and N403S, supporting other evidence that they function by weakening gp120's grip on gp41 rather than by altering gp120 binding to specific CCR5 sites. Although several natural HIV-1 isolates reportedly use CCR5(Δ18) (CCR5 with a deletion of 18 N-terminal amino acids, including the tyrosine-sulfated region) when the soluble tyrosine-sulfated peptide is present, we show that HIV-1JRCSF with the adaptive mutations [HIV-1JRCSF(Ad)] functions approximately 100 times more efficiently and that coreceptor activation is reversible, enabling synchronous efficient entry control under physiological conditions. This system revealed that three-stranded gp41 folding intermediates susceptible to the inhibitor enfuvirtide form slowly and asynchronously on cell surface virions but resolve rapidly, with virions generally forming only one target. Adsorbed virions asynchronously and transiently become competent for entry at 37°C but are inactivated if the CCR5 peptide is absent during their window of opportunity. This competency is conferred by endocytosis, which results in inactivation if the peptide is absent. For both wild-type and adapted HIV-1 isolates, early gp41 refolding steps obligatorily occur on cell surfaces, whereas the final step(s) is endosomal. This system powerfully dissects HIV-1 entry and inhibitor mechanisms., Importance: We present a powerful means to reversibly and efficiently activate or terminate HIV-1 entry by adding or removing a tyrosine-sulfated CCR5 peptide from the culture medium. This system uses stable cell clones and a variant of HIV-1JRCSF with three adaptive mutations. It enabled us to show that CCR5 coreceptor activation is rapidly reversible and to dissect aspects of entry that had previously been relatively intractable. Our analyses elucidate enfuvirtide (T-20) function and suggest that HIV-1 virions form only one nonredundant membrane fusion complex on cell surfaces. Additionally, we obtained novel and conclusive evidence that HIV-1 entry occurs in an assembly line manner, with some steps obligatorily occurring on cell surfaces and with final membrane fusion occurring in endosomes. Our results were confirmed for wild-type HIV-1. Thus, our paper provides major methodological and mechanistic insights about HIV-1 infection.

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naïve Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

Previous studies demonstrate that soluble HIV proteins impact both hepatocyte function and HCV replication in vitro. It has also been reported that HIV can productively infect hepatocytes. We therefore investigated the impact of HIV infection of hepatocytes on HCV expression. The Huh7.5JFH1 cell line that constitutively expresses infectious HCV was infected with the lab-adapted strains HIVNL4-3 or HIVYK-JRCSF. HCV expression was quantified via HCV core antigen ELISA, Western blot, and strand-specific real-time PCR for positive-sense and negative-sense HCV RNA. After HIVNL4-3 infection of Huh7.5JFH1 cells, positive-sense and negative-sense HCV RNA levels were elevated compared to HIV uninfected cells. Increased HCV RNA synthesis was also observed after infection of Huh7.5JFH1 cells with HIVYK-JRCSF. HIV-induced HCV core production was decreased in the presence of the anti-HIV drugs AZT, T20, and raltegravir, although these medications had a minimal effect on HCV expression in the absence of HIV. HCV core, NS3, and NS5A protein expression were increased after HIV infection of Huh7.5JFH1 cells. Chemically inactivated HIV had a minimal effect on HCV expression in Huh7.5JFH1 cells suggesting that ongoing viral replication was critical. These data demonstrate that HIV induces HCV RNA synthesis and protein production in vitro and complement previous in vivo reports that HCV RNA levels are elevated in individuals with HIV/HCV co-infection compared to those with HCV mono-infection. These findings suggest that HIV suppression may be a critical factor in controlling liver disease, particularly if the underlying liver disease is not treated.

The selective pressure of antiretroviral drugs (ARVs) targeting HIV-1 pol can promote drug resistance mutations in other genomic regions, such as env. Drug resistance among women should be monitored to avoid horizontal and mother-to-child transmission. To describe natural resistance to T-20 (enfuvirtide), gp41 env polymorphisms, mutations in pol and HIV-1 subtypes, 124 pregnant women were recruited. For 98 patients, the gp41 env, protease (PR) and reverse transcriptase (RT) fragments were sequenced. The patients were ARV naïve (n = 30), taking mother-to-child transmission prophylaxis (n = 50), or being treated with highly active ARV therapy/HAART (n = 18). The Stanford and IAS/USA databases and other sources were used to analyze PR/RT, gp41 env resistance mutations. The HIV-1 genetic diversity was analyzed by REGA/phylogenetic analyses. The patients' median age was 25 years (range, 16-42), 18.4% had AIDS. The frequency of natural resistance to T-20 (N42D, L44M, and R46M-low-impact mutations) was 6.1% (6/98); 20.4% (20/98) had compensatory mutations in HR2. The prevalence of transmitted drug resistance in the pol was 13.3% (4/30), and the prevalence of secondary drug resistance was 33.3% (6/18). Two patients were infected with multidrug resistant/MDR viruses. The analysis of HIV-1 subtypes (PR/RT/gp41) revealed that 61.2% (60/98) were subtype B, 12.2% (12/98) were subtype C, 4.1% (4/98) were subtype F1, and 22.4% (22/98) were possible recombinants (BF1 = 20.4%; BC = 2%). Natural resistance to T-20 was not associated with pol resistance or previous ARV use. The high rate of secondary resistance, including MDR, indicates that the number of women that may need T-20 salvage therapy may be higher than anticipated., (© 2013 Wiley Periodicals, Inc.)

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between D- and L-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a D-enantiomer of ISLAD CM (termed ISLAD D-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35-55)-specific line T-cells to the same extent as the l-motif form. Moreover, the D- and L-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD D-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35-55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the D-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.

Objectives: We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy., Methods: Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay., Results: At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity., Conclusions: The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.

BMS-626529 is a novel small-molecule HIV-1 attachment inhibitor active against both CCR5- and CXCR4-tropic viruses. BMS-626529 functions by preventing gp120 from binding to CD4. A prodrug of this compound, BMS-663068, is currently in clinical development. As a theoretical resistance pathway to BMS-663068 could be the development of a CD4-independent phenotype, we examined the activity of BMS-626529 against CD4-independent viruses and investigated whether resistance to BMS-626529 could be associated with a CD4-independent phenotype. Finally, we evaluated whether cross-resistance exists between BMS-626529 and other HIV-1 entry inhibitors. Two laboratory-derived envelopes with a CD4-independent phenotype (one CXCR4 tropic and one CCR5 tropic), five envelopes from clinical isolates with preexisting BMS-626529 resistance, and several site-specific mutant BMS-626529-resistant envelopes were examined for their dependence on CD4 for infectivity or susceptibility to BMS-626529. Viruses resistant to other entry inhibitors (enfuvirtide, maraviroc, and ibalizumab) were also examined for susceptibility to BMS-626529. Both CD4-independent laboratory isolates retained sensitivity to BMS-626529 in CD4(-) cells, while HIV-1 envelopes from viruses resistant to BMS-626529 exhibited no evidence of a CD4-independent phenotype. BMS-626529 also exhibited inhibitory activity against ibalizumab- and enfuvirtide-resistant envelopes. While there appeared to be some association between maraviroc resistance and reduced susceptibility to BMS-626529, an absolute correlation cannot be presumed, since some CCR5-tropic maraviroc-resistant envelopes remained sensitive to BMS-626529. Clinical use of the prodrug BMS-663068 is unlikely to promote resistance via generation of CD4-independent virus. No cross-resistance between BMS-626529 and other HIV entry inhibitors was observed, which could allow for sequential or concurrent use with different classes of entry inhibitors.

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15μM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides., (Copyright © 2013 Elsevier B.V. All rights reserved.)