Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.

BACKGROUND: The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors. OBJECTIVE: The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction. METHODS: The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software. RESULTS: The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation. CONCLUSION: We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

Improving Anti-HIV activity and pharmacokinetics of enfuvirtide (T20) by modification with oligomannose.

Dendritic cells (DCs) play a pivotal role in controlling HIV-1 infections of CD4+ T cells. DC-SIGN, which is expressed on the surface of DCs, efficiently captures HIV-1 virions by binding to the highly mannosylated membrane protein, gp120, and then the DCs transport the virus to target T cells in lymphoid organs. This study explored the modification of T20, a peptide inhibitor of HIV-1 fusion, by conjugation of the N-terminus with varying sizes of oligomannose, which are DC-SIGN-specific carbohydrates, aiming to create dual-targeting HIV inhibitors. Mechanistic studies indicated the dual-target binding of the conjugates. Antiviral assays demonstrated that N-terminal mannosylation of T20 resulted in increased inhibition of the viral infection of TZM-b1 cells (EC50 = 0.3-0.8 vs. 1.4 nM). Pentamannosylated T20 (M5-T20) exhibited a stronger inhibitory effect on virus entry into DC-SIGN+ 293T cells compared with T20 (67% vs. 50% inhibition at 500 µM). M5-T20 displayed an extended half-life in rats relative to T20 (T1/2: 8.56 vs. 1.64 h, respectively). These conjugates represent a potential new treatment for HIV infections with improved antiviral activity and pharmacokinetics, and this strategy may prove useful in developing dual-target inhibitors for other pathogens that require DC-SIGN involvement for infection.

Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities.

Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 µM while the standard one had 0.0180 µM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.

Isopeptide Bond Bundling Superhelix for Designing Antivirals against Enveloped Viruses with Class I Fusion Proteins: A Review.

Viral infection has become one of the worst human lethal diseases. In recent years, major gains have been made in the research of peptide-based antiviral agents on account of the mechanism of viral membrane fusion, among which the peptide Enfuvirtide has been listed for the treatment of AIDS. This paper reviewed a new way to design peptide-based antiviral agents by "bundling" superhelix with isopeptide bonds to construct the active advanced structure. It can solve the problem that peptide precursor compounds derived from the natural sequence of viral envelope protein tend to aggregate and precipitate under physiological conditions and low activity and endow the peptide agents with the feature of thermal stability, protease stability and in vitro metabolic stability. This approach is also providing a new way of thinking for the research and development of broad-spectrum peptide-based antiviral agents.

Action of enfuvirtide on the pregnancy of albino rats ( Rattus norvegicus albinus, Rodentia, Mammalia ): biological assay and functional and histological analyses of exposed maternal-fetal organs.

OBJECTIVE: • No drug related fetal malformations. • No adverse effects on vital organ functions. To assess the effects of enfuvirtide on pregnancy in albino rats and their fetuses. METHODS: Forty pregnant EPM 1 Wistar rats were randomly allocated into four groups: control (E) (distilled water twice/day), G1 (4mg/kg/day enfuvirtide), G2 (12mg/kg/day enfuvirtide), and G3 (36mg/kg/day enfuvirtide) groups. On the 20th day of gestation, the rats were anesthetized and subjected to cesarean section. Their blood was collected for laboratory analysis, and they were sacrificed. The offspring's fragments of their kidneys, liver, and placentas and the maternal rats' fragments of their lungs, kidneys, and liver were separated in the immediate postpartum period for light microscopy analysis. RESULTS: No maternal deaths occurred. In the second week at the end of pregnancy, the mean weight of the G3 Group was significantly lower than that of the G2 Group (p=0.029 and p=0.028, respectively). Analyzing blood laboratory parameters, the G1 Group had the lowest mean amylase level, and the G2 Group had the lowest mean hemoglobin level and the highest mean platelet count. In the morphological analysis, there were no changes in organs, such as the kidneys and liver, in both the maternal rats and offspring. Three maternal rats in the G3 Group had pulmonary inflammation in the lungs. CONCLUSION: Enfuvirtide has no significant adverse effects on pregnancy, conceptual products, or functional alterations in maternal rats.

Development of a HPLC fluorometric method for the quantification of enfuvirtide following in vitro releasing studies on thermosensitive in situ forming gel.

Due to the presence of peptidase and protease in the gastrointestinal tract, peptides are subjected to digestion and inactivation when administrated orally. To avoid degradation and maintain the desired efficacy of peptide drugs, there is a demand to develop transdermal and intradermal delivery systems. This requires efficient and specific analytical methods to separate and quantify the peptide drugs from the formulation and the skin matrix in the early stages of pharmaceutical development. A high-performance liquid chromatography (HPLC) system equipped with a fluorometric detector was used to quantify enfuvirtide, which is the first fusion inhibitor for HIV treatment. The HPLC method was developed and validated according to the ICH Q2(R1) guidelines. The viability of the method was demonstrated during in vitro studies, where samples were analysed following intradermal administration of a thermosensitive in situ forming gel. Compared with previously reported methods, this assay proved efficient, sensitive and accurate, with a detection limit of 0.74 µg/mL and a run time of 9 min, mitigating the use of any internal standards and detergents. The addition of an organic solvent to the samples successfully solved the problem of low recovery caused by the adsorption of the drug to the plastic consumables in the sample treatment process. The amount of enfuvirtide releasing from the in situ gel through skin after 7 hours was 16.25 ± 7.08 µg, which was significantly lower than the reconstituted FUZEON® itself (26.68 ± 10.45 µg), showing a longer release profile. The results may be beneficial as a constructive input for future enfuvirtide quantification within a preclinical setting through in vitro release studies across the skin.

Enzyme Responsive Delivery of Anti-Retroviral Peptide via Smart Hydrogel.

In response to an urgent need for advanced formulations for the delivery of anti-retrovirals, a stimuli-sensitive hydrogel formulation that intravaginally delivers HIV-1 entry inhibitor upon being exposed to a specific protease was developed. The hydrogel formulation consists of PEG-azide and PEG-DBCO covalently linked to the entry inhibitor peptide, enfuvirtide, via substrate linker that is designed to undergo proteolysis by prostate specific antigen (PSA) present in seminal fluid and release innate enfuvirtide. Of the tested PSA substrate linkers (HSSKLQYY, GISSFYSSK, AYLMYY, and AYLMGRR), HSSKLQ was found to be an optimal candidate for PEG-based hydrogel with kcat/KM of 2.2 M-1 s-1. The PEG-based hydrogel displayed a pseudoplastic, thixotropic behavior with overall viscosity varying between 1516 and 2.2 Pa.s, within the biologically relevant shear rates of 0.01-100 s-1. It also exhibited viscoelastic properties appropriate for uniform spreading and being retained in vagina. PEG-based hydrogels were loaded with N3-HSSKLQ-enfuvirtide (HF42) that is customarily synthesized enfuvirtide prodrug with its N-terminus connected to HSSKLQ linker. The stimuli-sensitive PEG-based hydrogel formulations upon being exposed to PSA released 36.5 ± 4.8% of enfuvirtide over 24 h in human ejaculate mimic of vaginal simulant fluid and seminal simulant fluid mixed in 1:3 ratio, which is significantly greater than its IC50. The PEG-based hydrogel was non-cytotoxic to both vaginal epithelial cells (VK2/E6E7) and murine macrophages (RAW 264.7) and did not significantly induce the production of nitric oxide, an inflammatory mediator. The PEG-based hydrogel is found to have suitable physicochemical properties for an intravaginal formulation of the PSA substrate-linked anti-retrovirals and is safe towards vaginal epithelium. It is capable of delivering enfuvirtide with effective concentrations to prevent women from HIV-1 infection.

The Glu143 Residue Might Play a Significant Role in T20 Peptide Binding to HIV-1 Receptor gp41: An In Silico Study.

Despite the enormous efforts made to develop other fusion inhibitors for HIV, the enfuvirtide (known as T20) peptide is the only approved HIV-1 inhibitory drug so far. Investigating the role of potential residues of the T20 peptide's conformational dynamics could help us to understand the role of potential residues of the T20 peptide. We investigated T20 peptide conformation and binding interactions with the HIV-1 receptor (i.e., gp41) using MD simulations and docking techniques, respectively. Although the mutation of E143 into alanine decreased the flexibility of the E143A mutant, the conformational compactness of the mutant was increased. This suggests a potential role of E143 in the T20 peptide's conformation. Interestingly, the free energy landscape showed a significant change in the wild-type T20 minimum, as the E143A mutant produced two observed minima. Finally, the docking results of T20 to the gp41 receptor showed a different binding interaction in comparison to the E143A mutant. This suggests that E143 residue can influence the binding interaction with the gp41 receptor. Overall, the E143 residue showed a significant role in conformation and binding to the HIV-1 receptor. These findings can be helpful in optimizing and developing HIV-1 inhibitor peptides.

In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to LP-40, Enfuvirtide-Based Lipopeptide Inhibitor.

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-1NL4-3 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.

Virus Entry Inhibitors: Past, Present, and Future.

The approval of enfuvirtide marked a milestone for the development of virus entry inhibitor-based antiviral therapeutics. Since then, more peptide-, small-molecule-, and protein-based entry inhibitors have been identified and approved for viral diseases. Here we reviewed the development of virus entry inhibitors and the advantages and disadvantages of peptide-, small-molecule-, and protein-based entry inhibitors, herein summarizing the future trend of these antivirals. Virus entry inhibitors take effect outside the host cell, making them good candidates for development as pre- and post-exposure prophylaxis, microbicides, and therapeutics. This chapter, as well as this book, provides more information on the development and modification of peptide-, small-molecule-, and protein-based virus entry inhibitors.

Enfuvirtide, an HIV-1 fusion inhibitor peptide, can act as a potent SARS-CoV-2 fusion inhibitor: an in silico drug repurposing study.

Regarding the urgency of therapeutic measures for coronavirus disease 2019 (COVID-19) pandemic, the use of available drugs with FDA approval is preferred because of the less time and cost required for their development. In silico drug repurposing is an accurate way to speed up the screening of the existing FDA-approved drugs to find a therapeutic option for COVID-19. The similarity in SARS-CoV-2 and HIV-1 fusion mechanism to host cells can be a key point for Inhibit SARS-CoV-2 entry into host cells by HIV fusion inhibitors. Accordingly, in this study, an HIV-1 fusion inhibitor called Enfuvirtide (Enf) was selected. The affinity and essential residues involving in the Enf binding to the S2 protein of SARS-CoV-2, HIV-1 gp41 protein and angiotensin-converting enzyme 2 (ACE-2) as a negative control, was evaluated using molecular docking. Eventually, Enf-S2 and Enf-gp41 protein complexes were simulated by molecular dynamics (MD) in terms of binding affinity and stability. Based on the most important criteria such as docking score, cluster size, energy and dissociation constant, the strongest interaction was observed between Enf with the S2 protein. In addition, MD results confirmed that Enf-S2 protein interaction was remarkably stable and caused the S2 protein residues to undergo the fewest fluctuations. In conclusion, it can be stated that Enf can act as a strong SARS-CoV-2 fusion inhibitor and demonstrates the potential to enter the clinical trial phase of COVID-19. Communicated by Ramaswamy H. Sarma.

A Derivative of the D5 Monoclonal Antibody That Targets the gp41 N-Heptad Repeat of HIV-1 with Broad Tier-2-Neutralizing Activity.

HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the prehairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date, these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5's six antibody complementarity-determining regions. One variant, D5_AR, demonstrated 6-fold enhancement in the 50% inhibitory dose (ID50) against lentivirus pseudotyped with HXB2 Env. D5_AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5_AR IgG was greatly enhanced in target cells expressing FcγRI, with ID50 values of <0.1 µg/ml; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5_AR have equivalent neutralization potency in IgG, Fab, and single-chain variable-fragment (scFv) formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR and support investigation of anti-NHR mAbs in nonhuman primate passive immunization studies. IMPORTANCE Despite advances in antiretroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective HIV vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 prehairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date, anti-NHR antibodies have not been potent. Here, we engineered D5_AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5_AR was greatly potentiated in cells expressing FcγRI; FcγRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.

HIV-1 gp41 genetic diversity and enfuvirtide resistance-associated mutations among enfuvirtide-naïve patients in southern China.

BACKGROUND: Human immunodeficiency virus (HIV) increasing molecular diversity and emergence of drug resistant mutants remain a major concern in China. Enfuvirtide (ENF/T-20) is the first entry inhibitor used in patients failing highly active antiretroviral therapy (HAART). However, data on HIV-1 gp41genetic diversity and primary ENF resistance-associated mutations among treatment-naïve patients in China is limited. The objective was to identify molecular diversity and ENF resistance patterns of HIV-1 in southern China, using envelope (env) gp41 sequences and bioinformatics tools, which may help optimize antiretroviral therapy. METHODS: From November 2018 to January 2019, 439 blood plasma samples from ENF-naïve patients were collected from Shenzhen, Wuhan and Chongqing, of which 396 HIV env regions were sequenced and subtyped, and were performed the analysis of drug resistance-associated mutations (DRMs). RESULTS: The main subtypes were circulating recombinant form (CRF) 01_AE (30.6 %) and CRF07_BC (48.7 %). CRF55_01B had been the fourth subtype in the study, and many rare CRFs were observed. Notably, CRF02_AG and CRF_BF strains typically found in Africa and US respectively were identified amongst Chinese patients. Known DRMs were detected in 27.5 % (109/396) of ENF treatment-naïve patients. One major DRM (L44 M), many secondary DRMs (including N126 K, E137 K, S138A), and lots of polymorphisms were found in the study, which have been proved to elevate resistance to ENF. CONCLUSIONS: HIV-1 molecular diversity was observed in the study, which indicating that HIV-1 variability is becoming increasingly complex in southern China. A diverse set of primary DRMs discovered in this study described the serious threat to ART, which reminds us the urgent need of timely surveillance of HIV-1 viral diversity and drug resistance in China.

Immunostimulatory effects on THP-1 cells by peptide or protein pharmaceuticals associated with injection site reactions.

Injection site reaction (ISR) is a common side-effect associated with the use of peptide or protein pharmaceuticals. These types of pharmaceuticals-induced activation of antigen-presenting cells is assumed to be a key step in the pathogenesis of immune-mediated ISR. The present study was designed to evaluate the immunostimulatory properties of peptide or protein pharmaceuticals using human monocytic THP-1 cells. Here, THP-1 cells, with or without phorbol-12-myristate-13-acetate (PMA) pretreatment, were exposed to enfuvirtide and glatiramer acetate (positive controls) or evolocumab (negative control) for 6 or 24 h. PMA treatment differentiated non-adherent monocytic THP-1 (nTHP-1) cells into adherent macrophagic THP-1 (pTHP-1) cells that highly express CD11b and CD36. Enfuvirtide increased the release of cytokines, e.g. TNFα, MIP-1ß, and MCP-1, and expression of CD86 and CD54 on nTHP-1 cells at 24 h. Similar immunostimulatory properties of glatiramer acetate were observed both in the nTHP-1 and pTHP-1 cells at 6 h, but the responses were very weak in the pTHP-1 cells. Evolocumab did not affect cytokine secretion or cell surface marker expression in either cell type. Taken together, these in vitro THP-1 cell assays revealed the immunostimulatory properties of enfuvirtide and glatiramer acetate. This assay platform thus could serve as a powerful tool in evaluating potential immune-related ISR risks of peptide or protein pharmaceuticals in humans.

Enfuvirtide-Protoporphyrin IX Dual-Loaded Liposomes: In Vitro Evidence of Synergy against HIV-1 Entry into Cells.

We have developed a nanocarrier consisting of large unilamellar vesicles (LUVs) for combined delivery of two human immunodeficiency virus type 1 (HIV-1) entry inhibitors, enfuvirtide (ENF) and protoporphyrin IX (PPIX). The intrinsic lipophilicity of ENF and PPIX, a fusion inhibitor and an attachment inhibitor, respectively, leads to their spontaneous incorporation into the lipid bilayer of the LUVs nanocarrier. Both entry inhibitors partition significantly toward LUVs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a 9:1 mixture of POPC:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), representative of conventional and immune-evasive drug delivery formulations, respectively. These colocalize in the core of lipid membranes. Dual-loaded nanocarriers are monodispersed and retain the size distribution, thermotropic behavior, and surface charge of the unloaded form. Combination of the two entry inhibitors in the nanocarrier resulted in improved synergy against HIV-1 entry compared to combination in free form, strongly when immune-evasive formulations are used. We propose that the improved action of the entry inhibitors when loaded into the nanocarriers results from their slow release at the site of viral entry. Overall, liposomes remain largely unexplored platforms for combination of viral entry inhibitors, with potential for improvement of current antiretroviral therapy drug safety and application. Our work calls for a reappraisal of the potential of entry inhibitor combinations and delivery for clinical use in antiretroviral therapy.

Mutations of Glu560 within HIV-1 Envelope Glycoprotein N-terminal heptad repeat region contribute to resistance to peptide inhibitors of virus entry.

BACKGROUND: Peptides corresponding to N- and C-terminal heptad repeat regions (HR1 and HR2, respectively) of gp41 can inhibit HIV-1 infection in a dominant negative manner by interfering with refolding of the viral HR1 and HR2 to form a six-helix bundle (6HB) that induces fusion between viral and host cell membranes. Previously, we found that HIV-1 acquired the mutations of Glu560 (E560) in HR1 of envelope (Env) to escape peptide inhibitors. The present study aimed to elucidate the critical role of position 560 in the virus entry and potential resistance mechanisms. RESULTS: The Glu560Lys/Asp/Gly (E560K/D/G) mutations in HR1 of gp41 that are selected under the pressure of N- and C-peptide inhibitors modified its molecular interactions with HR2 to change 6HB stability and peptide inhibitor binding. E560K mutation increased 6HB thermostability and resulted in resistance to N peptide inhibitors, but E560G or E560D as compensatory mutations destabilized the 6HB to reduce inhibitor binding and resulted in increased resistance to C peptide inhibitor, T20. Significantly, the neutralizing activities of all mutants to soluble CD4 and broadly neutralizing antibodies targeting membrane proximal external region, 2F5 and 4E10 were improved, indicating the mutations of E560 could regulate Env conformations through cross interactions with gp120 or gp41. The molecular modeling analysis of E560K/D/G mutants suggested that position 560 might interact with the residues within two potentially flexible topological layer 1 and layer 2 in the gp120 inner domain to apparently affect the CD4 utilization. The E560K/D/G mutations changed its interactions with Gln650 (Q650) in HR2 to contribute to the resistance of peptide inhibitors. CONCLUSIONS: These findings identify the contributions of mutations of E560K/D/G in the highly conserved gp41 and highlight Env's high degree of plasticity for virus entry and inhibitor design.

The Tryptophan-Rich Motif of HIV-1 gp41 Can Interact with the N-Terminal Deep Pocket Site: New Insights into the Structure and Function of gp41 and Its Inhibitors.

Refolding of the HIV-1 gp41 N- and C-terminal heptad repeats (NHR and CHR, respectively) into a six-helix bundle (6-HB) juxtaposes viral and cellular membranes for fusion. The CHR-derived peptide T20 is the only clinically approved viral fusion inhibitor and has potent anti-HIV activity; however, its mechanism of action is not fully understood. In this study, we surprisingly found that T20 disrupted the α-helical conformation of the NHR-derived peptide N54 through its C-terminal tryptophan-rich motif (TRM) and that synthetic short peptides containing the TRM sequence, TRM8 and TRM12, disrupted the N54 helix in a dose-dependent manner. Interestingly, TRM8 efficiently interfered with the secondary structures of three overlapping NHR peptides (N44, N38, and N28) and interacted with N28, which contains mainly the deep NHR pocket-forming sequence, with high affinity, suggesting that TRM targeted the NHR pocket site to mediate the disruption. Unlike TRM8, the short peptide corresponding to the pocket-binding domain (PBD) of the CHR helix had no such disruptive effect, and the CHR peptide C34 could form a stable 6-HB with the NHR helix; however, addition of the TRM to the C terminus of C34 resulted in a peptide (C46) that destroyed the NHR helix. Although the TRM peptides alone had no anti-HIV activity and could not block the formation of 6-HB conformation, substitution of the TRM for the PBD in C34 resulted in a mutant inhibitor (C34TRM) with high binding and inhibitory capacities. Combined, the present data inform a new mode of action of T20 and the structure-function relationship of gp41.IMPORTANCE The HIV-1 Env glycoprotein mediates membrane fusion and is conformationally labile. Despite extensive efforts, the structural property of the native fusion protein gp41 is largely unknown, and the mechanism of action of the gp41-derived fusion inhibitor T20 remains elusive. Here, we report that T20 and its C-terminal tryptophan-rich motif (TRM) can efficiently impair the conformation of the gp41 N-terminal heptad repeat (NHR) coiled coil by interacting with the deep NHR pocket site. The TRM sequence has been verified to possess the ability to replace the pocket-binding domain of C34, a fusion inhibitor peptide with high anti-HIV potency. Therefore, our studies have not only facilitated understanding of the mechanism of action of T20 and developed novel HIV-1 fusion inhibitors but also provided new insights into the structural property of the prefusion state of gp41.

Long-Acting HIV-1 Fusion Inhibitory Peptides and their Mechanisms of Action.

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 (EC50) of about 36 nM. They also inhibited infection of the laboratory-adapted HIV-1 strain NL4-3 with EC50 of about 4-5 nM, and against 47 HIV-1 clinical isolates circulating in China with mean EC50 of PEG2kC34 and PEG5kC34 of about 26 nM and 32 nM, respectively. The plasma half-life (t1/2) of PEG2kC34 and PEG5kC34 was 2.6 h and 5.1 h, respectively, and the t1/2 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.

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins.

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR-NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.