Peptide Fusion Inhibitors Research Articles

Inhibition of Measles Viral Fusion Is Enhanced by Targeting Multiple Domains of the Fusion Protein.

Measles virus (MeV) infection remains a significant public health threat despite ongoing global efforts to increase vaccine coverage. As eradication of MeV stalls, and vulnerable populations expand, effective antivirals against MeV are in high demand. Here, we describe the development of an antiviral peptide that targets the MeV fusion (F) protein. This antiviral peptide construct is composed of a carbobenzoxy-d-Phe-l-Phe-Gly (fusion inhibitor peptide; FIP) conjugated to a lipidated MeV F C-terminal heptad repeat (HRC) domain derivative. Initial in vitro testing showed high antiviral potency and specific targeting of MeV F-associated cell plasma membranes, with minimal cytotoxicity. The FIP and HRC-derived peptide conjugates showed synergistic antiviral activities when administered individually. However, their chemical conjugation resulted in markedly increased antiviral potency. In vitro mechanistic experiments revealed that the FIP-HRC lipid conjugate exerted its antiviral activity predominantly through stabilization of the prefusion F, while HRC-derived peptides alone act predominantly on the F protein after its activation. Coupled with in vivo experiments showing effective prevention of MeV infection in cotton rats, FIP-HRC lipid conjugates show promise as potential MeV antivirals via specific targeting and stabilization of the prefusion MeV F structure.

A "Two-Birds-One-Stone" Approach toward the Design of Bifunctional Human Immunodeficiency Virus Type 1 Entry Inhibitors Targeting the CCR5 Coreceptor and gp41 N-Terminal Heptad Repeat Region.

Previous studies have reported the stepwise nature of human immunodeficiency virus type 1 (HIV-1) entry and the pivotal role of coreceptor CCR5 and the gp41 N-terminal heptad repeat (NHR) region in this event. With this in mind, we herein report a dual-targeted drug compound featuring bifunctional entry inhibitors, consisting of a piperidine-4-carboxamide-based CCR5 antagonist, TAK-220, and a gp41 NHR-targeting fusion-inhibitory peptide, C34. The resultant chimeras were constructed by linking both pharmacophores with a polyethylene glycol spacer. One chimera, CP12TAK, exhibited exceptionally potent antiviral activity, about 40- and 306-fold over that of its parent inhibitors, C34 and TAK-220, respectively. In addition to R5-tropic viruses, CP12TAK also strongly inhibited infection of X4-tropic HIV-1 strains. These data are promising for the further development of CP12TAK as a new anti-HIV-1 drug. Results show that this strategy could be extended to the design of therapies against infection of other enveloped viruses.

Peptide Amphiphilic-Based Supramolecular Structures with Anti-HIV-1 Activity.

In a previous work, we defined a novel HIV-1 fusion inhibitor peptide (E1P47) with a broad spectrum of activity against viruses from different clades, subtypes, and tropisms. With the aim to enhance its efficacy, in the present work we address the design and synthesis of several peptide amphiphiles (PAs) based on the E1P47 peptide sequence to target the lipid rafts of the cell membrane where the cell–cell fusion process takes place. We report the synthesis of novel PAs having a hydrophobic moiety covalently attached to the peptide sequence through a hydrophilic spacer of polyethylene glycol. Characterization of self-assembly in condensed phase and aqueous solution as well as their interaction with model membranes was analyzed by several biophysical methods. Our results demonstrated that the length of the spacer of polyethylene glycol, the position of the peptide conjugation as well as the type of the hydrophobic residue determine the antiviral activity of the construct. Peptide amphiphiles with one alkyl tail either in C-terminus (C-PAmonoalkyl) or in N-terminus (N-PAmonoalkyl) showed the highest anti-HIV-1 activities in the cellular model of TZM-bl cells or in a preclinical model of the human mucosal tissue explants.

A bivalent protein targeting glycans and HR1 domain in spike protein potently inhibited infection of SARS-CoV-2 and other human coronaviruses

BackgroundOur previous studies have shown that combining the antiviral lectin GRFT and the pan-CoV fusion inhibitory peptide EK1 results in highly potent inhibitory activity against SARS-CoV-2 infection. In this study, we aimed to design and construct a bivalent protein consisting of GRFT and EK1 components and evaluate its inhibitory activity and mechanism of action against infection by SARS-CoV-2 and its mutants, as well as other human coronaviruses (HCoVs).MethodsThe bivalent proteins were expressed in E. coli and purified with Ni-NTA column. HIV backbone-based pseudovirus (PsV) infection and HCoV S-mediated cell–cell fusion assays were performed to test their inhibitory activity. ELISA and Native-PAGE were conducted to illustrate the mechanism of action of these bivalent proteins. Five-day-old newborn mice were intranasally administrated with a selected bivalent protein before or after HCoV-OC43 challenge, and its protective effect was monitored for 14 days.ResultsAmong the three bivalent proteins purified, GL25E exhibited the most potent inhibitory activity against infection of SARS-CoV-2 PsVs expressing wild-type and mutated S protein. GL25E was significantly more effective than GRFT and EK1 alone in inhibiting HCoV S-mediated cell–cell fusion, as well as infection by SARS-CoV-2 and other HCoVs, including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and HCoV-OC43. GL25E could inhibit authentic SASR-CoV-2, HCoV-OC43 and HCoV-229E infection in vitro and prevent newborn mice from authentic HCoV-OC43 infection in vivo. GL25E could bind to glycans in the S1 subunit and HR1 in the S2 subunit in S protein, showing a mechanism of action similar to that of GRFT and EK1 alone.ConclusionsSince GL25E showed highly potent and broad-spectrum inhibitory activity against infection of SARS-CoV-2 and its mutants, as well as other HCoVs, it is a promising candidate for further development as a broad-spectrum anti-HCoV therapeutic and prophylactic to treat and prevent COVID-19 and other emerging HCoV diseases.

Exploring SARS-CoV-2 Spikes Glycoproteins for Designing Potential Antiviral Targets.

Till today, the globe is still struggling with the newly emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and known as coronavirus disease 2019 (COVID-19). It has resulted in multiple fatalities from SARSs all around the world. A year after the global pandemic, the World Health Organization (WHO) has reported more than 79 million confirmed cases of COVID-19 and over 1.7 million deaths, making it one of the worst and most difficult pandemics encompassed in the modern history. The ongoing triad of escalating infections, mortality, and economic loss has urgently called for recognizing SARS-CoV-2 cell entry mechanisms as a crucial step in the initial stages of infection and to which possible interventional strategies should be targeted. To mediate host cell infections, Coronaviruses utilize the immunogenic studded spikes glycoproteins on its surface as a key factor for attachment, fusion, and entrance to host cells. Herein, we shed the light on a potential strategy involving disruption of SARS-CoV-2 S protein interaction with host cell receptors through design of neutralizing antibodies targeting receptor binding domain in S1 subunit, small peptide inhibitors, peptide fusion inhibitors against S2, host cell angiotensin converting enzymes 2 (ACE2), and protease inhibitors, aiming to pave the way for controlling viral cell entrance. In this review, we also highlight the recent research advances in the antiviral drugs that target the highly exposed spike protein, aiming to stem the COVID-19 pandemic.

Discovery of New Fusion Inhibitor Peptides against SARS-CoV-2 by Targeting the Spike S2 Subunit.

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

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

Cholesterol: A key player in membrane fusion that modulates the efficacy of fusion inhibitor peptides.

The interaction of cholesterol with the neighboring lipids modulates several physical properties of the membrane. Mostly, it affects membrane fluidity, membrane permeability, lateral diffusion of lipids, bilayer thickness, and water penetration into the lipid bilayer. Due to the smaller head group to hydrophobic cross-sectional area of the tail, cholesterol induces intrinsic negative curvature to the membrane. The interaction of cholesterol with sphingolipids forms lipid rafts; generates phase separation in the membrane. The cholesterol-dependent modifications of membrane physical properties modulate viral infections by affecting the fusion between viral and host cell membranes. Cholesterol demonstrates a strong impact on the structure, depth of penetration, conformation, and organization of fusion peptides in membrane milieu. Further, cholesterol has been implicated to modify the fusion inhibitory efficiency of peptide-based membrane fusion inhibitors.

Structure-based design and characterization of novel fusion-inhibitory lipopeptides against SARS-CoV-2 and emerging variants

ABSTRACT The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has severely impacted the global public health and socio-economic stability, calling for effective vaccines and therapeutics. In this study, we continued our efforts to develop more efficient SARS-CoV-2 fusion inhibitors and achieved significant findings. First, we found that the membrane-proximal external region (MPER) sequence of SARS-CoV-2 spike fusion protein plays a critical role in viral infectivity and can serve as an ideal template for design of fusion-inhibitory peptides. Second, a panel of novel lipopeptides was generated with greatly improved activity in inhibiting SARS-CoV-2 fusion and infection. Third, we showed that the new inhibitors maintained the potent inhibitory activity against emerging SARS-CoV-2 variants, including those with the major mutations of the B.1.1.7 and B.1.351 strains circulating in the United Kingdom and South Africa, respectively. Fourth, the new inhibitors also cross-inhibited other human CoVs, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Fifth, the structural properties of the new inhibitors were characterized by circular dichroism (CD) spectroscopy and crystallographic approach, which revealed the mechanisms underlying the high binding and inhibition. Combined, our studies provide important information for understanding the mechanism of SARS-CoV-2 fusion and a framework for the development of peptide therapeutics for the treatment of SARS-CoV-2 and other CoVs.

Importance of structure-based studies for the design of a novel HIV-1 inhibitor peptide

Based on the structure of an HIV-1 entry inhibitor peptide two stapled- and a retro-enantio peptides have been designed to provide novel prevention interventions against HIV transmission. The three peptides show greater inhibitory potencies in cellular and mucosal tissue pre-clinical models than the parent sequence and the retro-enantio shows a strengthened proteolytic stability. Since HIV-1 fusion inhibitor peptides need to be embedded in the membrane to properly interact with their viral target, the structural features were determined by NMR spectroscopy in micelles and solved by using restrained molecular dynamics calculations. Both parent and retro-enantio peptides demonstrate a topology compatible with a shared helix–turn–helix conformation and assemble similarly in the membrane maintaining the active conformation needed for its interaction with the viral target site. This study represents a straightforward approach to design new targeted peptides as HIV-1 fusion inhibitors and lead us to define a retro-enantio peptide as a good candidate for pre-exposure prophylaxis against HIV-1.

Long-Acting Efavirenz and HIV-1 Fusion Inhibitor Peptide Co-loaded Polymer-Lipid Hybrid Nanoparticles: Statistical Optimization, Cellular Uptake, and In Vivo Biodistribution.

The objective of the present study was to develop long-acting efavirenz (Efa)-enfuvirtide (Enf) Co-loaded polymer-lipid hybrid nanoparticles (PLN) with improved intracellular delivery to target T-cells and macrophage cells located in multiple human immunodeficiency virus sanctuaries. The Box-Behnken design was utilized to optimize three high-risk factors, namely, Efa amount, sonication time for primary emulsion, and sonication time for aqueous nanodispersion obtained from preliminary studies. Lyophilized Efa-Enf Co-loaded PLN using trehalose elicited spherical morphology, drug amorphization on incorporation, and absence of drug-excipient interaction. In vitro release studies revealed an sustained release of both the drugs from PLN with the differential release profile. Efa-Enf Co-loaded PLN exhibited low hemolytic, platelet and leukocyte aggregation as well as low cytotoxicity in Jurkat E6.1 T-cells and U937 macrophage cells. Circular dichroism spectra confirmed the presence of an α-helix form of Enf after encapsulation in PLN. Coumarin-6-loaded PLN exhibited enhanced cellular uptake in Jurkat E6.1 T-cells and U937 macrophage cells in comparison to free coumarin-6, as evidenced by fluorescence microscopy and flow cytometry. In vivo biodistribution studies after intravenous administration of near-infrared dye-loaded PLN (surrogate for Efa-Enf PLN) revealed non-uniform distribution within 2 h in the order of spleen ≥ liver > lymph node > thymus > lungs > female reproductive tract (FRT) > heart > kidneys > brain. However, subcutaneous administration caused non-uniform biodistribution after 3 days, eliciting a long-acting slow release from the injection site depot until day 5 in the infection-spread site (lymph nodes and FRT), reservoir sites (liver and spleen) and the difficult-to-access site (brain). Furthermore, it presents a vital illustration of the available tissue-specific drug concentration prediction from simulated surrogate PLN.

Lipid Vesicles Loaded with an HIV-1 Fusion Inhibitor Peptide as a Potential Microbicide.

The effective use of fusion inhibitor peptides against cervical and colorectal infections requires the development of sustained release formulations. In this work we comparatively study two different formulations based on polymeric nanoparticles and lipid vesicles to propose a suitable delivery nanosystem for releasing an HIV-1 fusion inhibitor peptide in vaginal mucosa. Polymeric nanoparticles of poly-d,l-lactic-co-glycolic acid (PLGA) and lipid large unilamellar vesicles loaded with the inhibitor peptide were prepared. Both formulations showed average sizes and polydispersity index values corresponding to monodisperse systems appropriate for vaginal permeation. High entrapment efficiency of the inhibitor peptide was achieved in lipid vesicles, which was probably due to the peptide’s hydrophobic nature. In addition, both nanocarriers remained stable after two weeks stored at 4 °C. While PLGA nanoparticles (NPs) did not show any delay in peptide release, lipid vesicles demonstrated favorably prolonged release of the peptide. Lipid vesicles were shown to improve the retention of the peptide on ex vivo vaginal tissue in a concentration sufficient to exert its pharmacological effect. Thus, the small size of lipid vesicles, their lipid-based composition as well as their ability to enhance peptide penetration on vaginal tissue led us to consider this formulation as a better nanosystem than polymeric nanoparticles for the sustained delivery of the HIV-1 fusion inhibitor peptide in vaginal tissues.

Broad-Spectrum Coronavirus Fusion Inhibitors to Combat COVID-19 and Other Emerging Coronavirus Diseases.

In the past 17 years, three novel coronaviruses have caused severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the coronavirus disease 2019 (COVID-19). As emerging infectious diseases, they were characterized by their novel pathogens and transmissibility without available clinical drugs or vaccines. This is especially true for the newly identified COVID-19 caused by SARS coronavirus 2 (SARS-CoV-2) for which, to date, no specific antiviral drugs or vaccines have been approved. Similar to SARS and MERS, the lag time in the development of therapeutics is likely to take months to years. These facts call for the development of broad-spectrum anti-coronavirus drugs targeting a conserved target site. This review will systematically describe potential broad-spectrum coronavirus fusion inhibitors, including antibodies, protease inhibitors, and peptide fusion inhibitors, along with a discussion of their advantages and disadvantages.

The Efficacy of Designed Anti-Measles Virus Peptides Depends on the Stability of Self-Assembled Clusters

The resurgence of several infectious diseases, like measles, has driven the search for new chemotherapeutics to prevent and treat viral infections. Self-assembling antiviral peptides are a promising class of entry inhibitors capable of meeting this need. Fusion inhibitory peptides derived from the heptad repeat of the C-terminal (HRC) of the measles fusion protein, dimerized and conjugated with lipophilic groups, were found to be efficacious against measles virus. The structures of the self-assembled nanoparticles formed by these peptides modulated their activity. Based on the analysis of a L454W mutation in the fusion protein of a naturally occurring measles viral isolate, HRC peptides bearing the tryptophan residue at position 454 (HRC-L454W) were synthesized with the goal of improving membrane anchoring and manipulating self-assembly. Monomeric and dimeric peptides, whether conjugated or not to a single lipophilic group, reduced infection in vivo. Bis-conjugation with lipophilic groups, in contrast, abrogated activity. Based on the physicochemical properties of self-assembly and membrane insertion kinetics of the HRC-L454W peptides we show that bis-conjugation increases the stability and order of the inner core of the spontaneously self-assembled nanoparticles, resulting in their compaction. The presence of the tryptophan residue also increases steric hindrance effects in the nanoparticle of the dimeric peptides, contributing to inter-peptide cluster meshing, but the same level of compaction is not achieved. We propose that the highly ordered packing and stability of molecular clusters forming the inner core of self-assembled nanoparticles prevent efficient dissociation of the peptides in vivo, hindering their release and therefore eliminating their antiviral efficacy.

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.

IgG Fc-binding motif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies.

The clinical application of conventional peptide drugs, such as the HIV-1 fusion inhibitor enfuvirtide, is limited by their short half-life in vivo. To overcome this limitation, we developed a new strategy to extend the in vivo half-life of a short HIV-1 fusion inhibitory peptide, CP24, by fusing it with the human IgG Fc-binding peptide (IBP). The newly engineered peptide IBP-CP24 exhibited potent and broad anti-HIV-1 activity with IC50 values ranging from 0.2 to 173.7 nM for inhibiting a broad spectrum of HIV-1 strains with different subtypes and tropisms, including those resistant to enfuvirtide. Most importantly, its half-life in the plasma of rhesus monkeys was 46.1 h, about 26- and 14-fold longer than that of CP24 (t1/2 = 1.7 h) and enfuvirtide (t1/2 = 3 h), respectively. IBP-CP24 intravenously administered in rhesus monkeys could not induce significant IBP-CP24-specific antibody response and it showed no obvious in vitro or in vivo toxicity. In the prophylactic study, humanized mice pretreated with IBP-CP24 were protected from HIV-1 infection. As a therapeutic treatment, coadministration of IBP-CP24 and normal human IgG to humanized mice with chronic HIV-1 infection resulted in a significant decrease of plasma viremia. Combining IBP-CP24 with a broad neutralizing antibody (bNAb) targeting CD4-binding site (CD4bs) in gp120 or a membrane proximal external region (MPER) in gp41 exhibited synergistic effect, resulting in significant dose-reduction of the bNAb and IBP-CP24. These results suggest that IBP-CP24 has the potential to be further developed as a new HIV-1 fusion inhibitor-based, long-acting anti-HIV drug that can be used alone or in combination with a bNAb for treatment and prevention of HIV-1 infection.

Suitable fusion of N-terminal heptad repeats to achieve covalently stabilized potent N-peptide inhibitors of HIV-1 infection

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.

Engineering a Novel Antibody-Peptide Bispecific Fusion Protein Against MERS-CoV.

In recent years, tremendous efforts have been made in the engineering of bispecific or multi-specific antibody-based therapeutics by combining two or more functional antigen-recognizing elements into a single construct. However, to the best of our knowledge there has been no reported cases of effective antiviral antibody-peptide bispecific fusion proteins. We previously developed potent fully human monoclonal antibodies and inhibitory peptides against Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a novel coronavirus that causes severe acute respiratory illness with high mortality. Here, we describe the generation of antibody-peptide bispecific fusion proteins, each of which contains an anti-MERS-CoV single-chain antibody m336 (or normal human IgG1 CH3 domain as a control) linked with, or without, a MERS-CoV fusion inhibitory peptide HR2P. We found that one of these fusion proteins, designated as m336 diabody-pep, exhibited more potent inhibitory activity than the antibody or the peptide alone against pseudotyped MERS-CoV infection and MERS-CoV S protein-mediated cell-cell fusion, suggesting its potential to be developed as an effective bispecific immunotherapeutic for clinical use.

Trimeric heptad repeat synthetic peptides HR1 and HR2 efficiently inhibit HIV-1 entry.

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.

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.