Supercoiling Structure-Based Design of a Trimeric Coiled-Coil Peptide with High Potency against HIV-1 and Human ß-Coronavirus Infection.

Hexameric structure formation through packing of three C-terminal helices and an N-terminal trimeric coiled-coil core has been proposed as a general mechanism of class I enveloped virus entry. In this process, the C-terminal helical repeat (HR2) region of viral membrane fusion proteins becomes transiently exposed and accessible to N-terminal helical repeat (HR1) trimer-based fusion inhibitors. Herein, we describe a mimetic of the HIV-1 gp41 HR1 trimer, N3G, as a promising therapeutic against HIV-1 infection. Surprisingly, we found that in addition to protection against HIV-1 infection, N3G was also highly effective in inhibiting infection of human ß-coronaviruses, including MERS-CoV, HCoV-OC43, and SARS-CoV-2, possibly by binding the HR2 region in the spike protein of ß-coronaviruses to block their hexameric structure formation. These studies demonstrate the potential utility of anti-HIV-1 HR1 peptides in inhibiting human ß-coronavirus infection. Moreover, this strategy could be extended to the design of broad-spectrum antivirals based on the supercoiling structure of peptides.

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection.

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.

A targeted covalent small molecule inhibitor of HIV-1 fusion.

We describe a low molecular weight covalent inhibitor targeting a conserved lysine residue within the hydrophobic pocket of HIV-1 glycoprotein-41. The inhibitor bound selectively to the hydrophobic pocket and exhibited an order of magnitude enhancement of anti-fusion activity against HIV-1 compared to its non-covalent counterpart. The findings represent a significant advance in the quest to obtain non-peptide fusion inhibitors.

Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis in vivo.

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4+ T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4+ T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent d-peptide HIV entry inhibitor.

Cholesterol-PIE12-trimer (CPT31) is a potent d-peptide HIV entry inhibitor that targets the highly conserved gp41 N-peptide pocket region. CPT31 exhibited strong inhibitory breadth against diverse panels of primary virus isolates. In a simian-HIV chimeric virus AD8 (SHIVAD8) macaque model, CPT31 prevented infection from a single high-dose rectal challenge. In chronically infected animals, CPT31 monotherapy rapidly reduced viral load by ∼2 logs before rebound occurred due to the emergence of drug resistance. In chronically infected animals with viremia initially controlled by combination antiretroviral therapy (cART), CPT31 monotherapy prevented viral rebound after discontinuation of cART. These data establish CPT31 as a promising candidate for HIV prevention and treatment.

Design, Characterization, and Biopharmaceutical Behavior of Nanoparticles Loaded with an HIV-1 Fusion Inhibitor Peptide.

New therapeutic alternatives to fight against the spread of HIV-1 are based on peptides designed to inhibit the early steps of HIV-1 fusion in target cells. However, drawbacks, such as bioavailability, short half-life, rapid clearance, and poor ability to cross the physiological barriers, make such peptides unattractive for the pharmaceutical industry. Here we developed, optimized, and characterized polymeric nanoparticles (NPs) coated with glycol chitosan to incorporate and release an HIV-1 fusion inhibitor peptide (E1) inside the vaginal mucosa. The NPs were prepared by a modified double emulsion method, and optimization was carried out by a factorial design. In vitro, ex vivo, and in vivo studies were carried out to evaluate the optimized formulation. The results indicate that the physicochemical features of these NPs enable them to incorporate and release HIV fusion inhibitor peptides to the vaginal mucosa before the fusion step takes place.

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

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

A novel peptide shows excellent anti-HIV-1 potency as a gp41 fusion inhibitor.

Fusion inhibitors of HIV prevent the virus from entering into the target cell via the interaction with gp41, which stops the process of spatial rearrangement of the viral envelope protein. A series of peptides have been designed and screened to obtain a highly potent novel sequence. Among them, CT105 possesses the most potent anti-viral ability at low nanomolar IC50 values against a panel of HIV-1 pseudoviruses from A, B, C and A1/D subtypes, whereas T20 shows much weaker potency. CT105 also shows excellent inhibitory activity at 260 pico molar IC50 against HIV-1 replication. As a fusion inhibitor, CT105 has a strong ability to interrupt gp41 core formation. The terminal half-life of CT105 possesses 1.72-fold longer than that of T20 as determined by developing an indirect competitive ELISA method. The results suggest that this artificial peptide CT105 could be a favorable architype for further optimization and modification.

HIV-1 gp41-targeting fusion inhibitory peptides enhance the gp120-targeting protein-mediated inactivation of HIV-1 virions.

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 EC50 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.

Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer.

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett.2015, 25 2853-59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC50=37.81µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.

A Helical Short-Peptide Fusion Inhibitor with Highly Potent Activity against Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus.

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.

Helix-Grafted Pleckstrin Homology Domains Suppress HIV-1 Infection of CD4-Positive Cells.

The size, functional group diversity and three-dimensional structure of proteins often allow these biomolecules to bind disease-relevant structures that challenge or evade small-molecule discovery. Additionally, folded proteins are often much more stable in biologically relevant environments compared to their peptide counterparts. We recently showed that helix-grafted display-extensive resurfacing and elongation of an existing solvent-exposed helix in a pleckstrin homology (PH) domain-led to a new protein that binds a surrogate of HIV-1 gp41, a validated target for inhibition of HIV-1 entry. Expanding on this work, we prepared a number of human-derived helix-grafted-display PH domains of varied helix length and measured properties relevant to therapeutic and basic research applications. In particular, we showed that some of these new reagents expressed well as recombinant proteins in Escherichia coli, were relatively stable in human serum, bound a mimic of pre-fusogenic HIV-1 gp41 in vitro and in complex biological environments, and significantly lowered the incidence of HIV-1 infection of CD4-positive cells.

Inhibition of HIV Entry by Targeting the Envelope Transmembrane Subunit gp41.

BACKGROUND: The transmembrane subunit of the HIV envelope protein, gp41 is a vulnerable target to inhibit HIV entry. There is one fusion inhibitor T20 (brand name: Fuzeon, generic name: enfuvirtide) available by prescription. However, it has several drawbacks such as a high level of development of drug resistance, a short-half life in vivo, rapid renal clearance, low oral bioavailability, and it is only used as a salvage therapy. Therefore, investigators have been studying a variety of different modalities to attempt to overcome these limitations. METHODS: Comprehensive literature searches were performed on HIV gp41, inhibition mechanisms, and inhibitors. The latest structural information was collected, and multiple inhibition strategies targeting gp41 were reviewed. RESULTS: Many of the recent advances in inhibitors were peptide-based. Several creative modification strategies have also been performed to improve inhibitory efficacy of peptides and to overcome the drawbacks of T20 treatment. Small compounds have also been an area of intense research. There is a wide variety in development from those identified by virtual screens targeting specific regions of the protein to natural products. Finally, broadly neutralizing antibodies have also been important area of research. The inaccessible nature of the target regions for antibodies is a challenge, however, extensive efforts to develop better neutralizing antibodies are ongoing. CONCLUSION: The fusogenic protein, gp41 has been extensively studied as a promising target to inhibit membrane fusion between the virus and target cells. At the same time, it is a challenging target because the vulnerable conformations of the protein are exposed only transiently. However, advances in biochemical, biophysical, structural, and immunological studies are coming together to move the field closer to an understanding of gp41 structure and function that will lead to the development of novel drugs and vaccines.

Mechanism of HIV-1 Resistance to Short-Peptide Fusion Inhibitors Targeting the Gp41 Pocket.

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.

A simple contact mapping algorithm for identifying potential peptide mimetics in protein-protein interaction partners.

A simple, static contact mapping algorithm has been developed as a first step at identifying potential peptide biomimetics from protein interaction partner structure files. This rapid and simple mapping algorithm, "OpenContact" provides screened or parsed protein interaction files based on specified criteria for interatomic separation distances and interatomic potential interactions. The algorithm, which uses all-atom Amber03 force field models, was blindly tested on several unrelated cases from the literature where potential peptide mimetics have been experimentally developed to varying degrees of success. In all cases, the screening algorithm efficiently predicted proposed or potential peptide biomimetics, or close variations thereof, and provided complete atom-atom interaction data necessary for further detailed analysis and drug development. In addition, we used the static parsing/mapping method to develop a peptide mimetic to the cancer protein target, epidermal growth factor receptor. In this case, secondary, loop structure for the peptide was indicated from the intra-protein mapping, and the peptide was subsequently synthesized and shown to exhibit successful binding to the target protein. The case studies, which all involved experimental peptide drug advancement, illustrate many of the challenges associated with the development of peptide biomimetics, in general.

Singlet oxygen effects on lipid membranes: implications for the mechanism of action of broad-spectrum viral fusion inhibitors.

It was reported recently that a new aryl methyldiene rhodanine derivative, LJ001, and oxazolidine-2,4-dithione, JL103, act on the viral membrane, inhibiting its fusion with a target cell membrane. The aim of the present study was to investigate the interactions of these two active compounds and an inactive analogue used as a negative control, LJ025, with biological membrane models, in order to clarify the mechanism of action at the molecular level of these new broad-spectrum enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen (1O2). Changes in the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that 1O2 production by LJ001 and JL103 is able to induce several changes on membrane properties, specially related to a decrease in its fluidity, concomitant with an increase in the order of the polar headgroup region, resulting in an inhibition of the membrane fusion necessary for cell infection.

Development of HIV-1 fusion inhibitors targeting gp41.

The HIV-1 envelope protein glycoprotein 41 (gp41) is crucial in the HIV-1 infection process, therefore gp41 has emerged as an attractive target for drug design against AIDS. During the past few decades, tremendous efforts have been made on developing inhibitors that can prevent the HIV-1 entry process via suppressing functional gp41. In this review, the development of HIV-1 fusion inhibitors targeting gp41 including peptide inhibitors, small molecule inhibitors, vaccines and neutralized antibodies will be discussed.

Synthesized peptide inhibitors of HIV-1 gp41-dependent membrane fusion.

Fusion of viral and cellular membranes is an essential step for HIV-1 infection. This process offers an attractive target for developing antiviral agents. T20 (Enfuvirtide, Fuzeon), a 36-amino acid peptide derived from the C-terminal heptad repeat region of HIV-1 gp41, is the first and only clinically approved HIV-1 fusion inhibitor that being used for treatment of HIV/AIDS patients failed to respond to current antiretroviral drugs. However, T20-resistance can be acquired rather easily in vitro and in vivo. T1249 is considered as a representative of the second generation fusion inhibitors, but its clinical evaluation was halted due to the problem of drug formulation. To overcome these challenges, a number of strategies have been applied to develop the third or next-generation inhibitors with the significantly improved antiviral activity and pharmacokinetic profiles. Promisingly, several peptides are currently under clinical trials, such as Sifuvirtide and VIR-576. Recently, several high resolution crystal structures of HIV-1 fusion inhibitor peptides were reported, which reveal the key residues or motifs underlying their potency against diverse HIV-1 variants. This review highlights the development of the representative peptide inhibitors of HIV-1 fusion toward providing some insights into the future of this class of anti-HIV drugs.

Discovery of critical residues for viral entry and inhibition through structural Insight of HIV-1 fusion inhibitor CP621-652.

The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the (621)QIWNNMT(627) motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621-652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621-652 complexed by T21. We find that the (621)QIWNNMT(627) motif does not maintain the α-helical conformation. Instead, residues Met(626) and Thr(627) form a unique hook-like structure (denoted as M-T hook), in which Thr(627) redirects the peptide chain to position Met(626) above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met(626) caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621-652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.

Amino acid derivatives of the (-) enantiomer of gossypol are effective fusion inhibitors of human immunodeficiency virus type 1.

T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs. Gossypol is a polyphenolic aldehyde extracted from cotton plants. By modifying the (-) enantiomer of gossypol with a series of small molecules, we have found that neutral amino acids with aliphatic group derivatives of (-) gossypol show the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. Additionally, the selectivity index of the (-) gossypol-neutral amino acid conjugates is increased 100-fold when compared with (-) gossypol alone. It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1 replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and VSV-G pseudotyped virus assay, we demonstrate that the alanine-(-) gossypol derivative ((-)G-Ala) is an effective HIV-1 entry inhibitor. Further mechanistic analysis revealed that (-)G-Ala neither blocks gp120-CD4 binding nor interacts with the HIV-1 co-receptor CXCR4. Results from sandwich ELISA, native-PAGE and circular dichroism (CD) show that (-)G-Ala inhibits the cell fusion-activated gp41 core domain. Moreover, (-)G-Ala binds to the HIV-5-Helix protein and blocking D-peptide (PIE7) binding to the hydrophobic pocket on the surface of the gp41 internal trimeric coiled-coil domain. The contraceptive properties of (-) gossypol and amino acid derivatives of (-) gossypol are also discussed. Collectively, our results indicate that (-)G-Ala may bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core to inhibit HIV-1-mediated membrane fusion and subsequent viral entry.