Covalent fusion inhibitors targeting HIV-1 gp41 deep pocket

Covalent inhibitors form covalent adducts with their target, thus permanently inhibiting a physiological process. Peptide fusion inhibitors, such as T20 (Fuzeon, enfuvirtide) and C34, interact with the N-terminal heptad repeat of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein to form an inactive hetero six-helix bundle (6-HB) to prevent HIV-1 infection of host cells. A covalent strategy was applied to peptide fusion inhibitor design by introducing a thioester group into C34-like peptide. The modified peptide maintains the specific interaction with its target N36. After the 6-HB formation, a covalent bond between C- and N-peptides was formed by an inter-helical acyl transfer reaction, as characterized by various biophysical and biochemical methods. The covalent reaction between the reactive C-peptide fusion inhibitor and its N-peptide target is highly selective, and the reaction greatly increases the thermostability of the 6-HB. The modified peptide maintains high potency against HIV-1-mediated cell–cell fusion and infection.     

The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.     

Selection with a peptide fusion inhibitor corresponding to the first heptad repeat of HIV-1 gp41 identifies two genetic pathways conferring cross-resistance to peptide fusion inhibitors corresponding to the first and second heptad repeats (HR1 and HR2) of gp41

We generated four HIV-1 cultures that are resistant to a peptide fusion inhibitor corresponding to the first heptad repeat of gp41 in order to study mechanisms of resistance and gain insights into envelope glycoprotein-mediated membrane fusion. Two genetic pathways emerged that were defined by acquisition of a specific mutation in either the first or second heptad repeat region of gp41 (HR1 or the HR2, respectively). Each pathway was enriched in mutations that clustered in either HR2 and V3 or in HR1 and residues in or near CD4 contact sites. The gp41 mutations in both pathways not only accounted for resistance to the selecting HR1 peptide but also conferred cross-resistance to HR2 peptide fusion inhibitors and enhanced the stability of the six-helix bundle formed by the self-assembly of HR1 and HR2. The gp120 mutations alone enhanced fusion but did not appear to directly contribute to resistance. The implications of these findings for resistance mechanisms and regulation of envelope-mediated fusion are discussed.     

Synergistic inhibition of HIV-1 envelope-mediated membrane fusion by inhibitors targeting the N and C-terminal heptad repeats of gp41

The human immunodeficiency virus type-1 (HIV-1) envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. Here, we show that N(CCG)-gp41, a class 2 inhibitor, and N36(Mut(e,g)), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudo-typed virus neutralization assay. The mechanistic, as well as potential therapeutic, implications of these observations for HIV-Env-mediated membrane fusion are discussed.     

Design,synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC₅₀ = 53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus–cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.     

Synthetic Fab fragments that bind the HIV-1 gp41 heptad repeat regions

Recent work has demonstrated that antibody phage display libraries containing restricted diversity in the complementarity determining regions (CDRs) can be used to target a wide variety of antigens with high affinity and specificity. In the most extreme case, antibodies whose combining sites are comprised of only two residues – tyrosine and serine – have been identified against several protein antigens. [F.A. Fellouse, B. Li, D.M. Compaan, A.A. Peden, S.G. Hymowitz, S.S. Sidhu, J. Mol. Biol. 348 (2005) 1153–1162.] Here, we report the isolation and characterization of antigen-binding fragments (Fabs) from such “minimalist” diversity synthetic antibody libraries that bind the heptad repeat regions of human immunodeficiency virus type 1 (HIV-1) gp41. We show that these Fabs are highly specific for the HIV-1 epitope and comparable in affinity to a single chain variable fragment (scFv) derived from a natural antibody repertoire that targets the same region. Since the heptad repeat regions of HIV-1 gp41 are required for viral entry, these Fabs have potential for use in therapeutic, research, or diagnostic applications.     

Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide

A variety of molecules in human blood have been implicated in the inhibition of HIV-1. However, it remained elusive which circulating natural compounds are most effective in controlling viral replication in vivo. To identify natural HIV-1 inhibitors we screened a comprehensive peptide library generated from human hemofiltrate. The most potent fraction contained a 20-residue peptide, designated VIRUS-INHIBITORY PEPTIDE (VIRIP), corresponding to the C-proximal region of alpha1-antitrypsin, the most abundant circulating serine protease inhibitor. We found that VIRIP inhibits a wide variety of HIV-1 strains including those resistant to current antiretroviral drugs. Further analysis demonstrated that VIRIP blocks HIV-1 entry by interacting with the gp41 fusion peptide and showed that a few amino acid changes increase its antiretroviral potency by two orders of magnitude. Thus, as a highly specific natural inhibitor of the HIV-1 gp41 fusion peptide, VIRIP may lead to the development of another class of antiretroviral drugs.     

ADS-J1 inhibits HIV-1 infection and membrane fusion by targeting the highly conserved pocket in the gp41 NHR-trimer

We previously identified a potent small-molecule human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, termed ADS-J1, and hypothesized that it mainly targeted the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR) trimer. However, this hypothesis has been challenged by the fact that ADS-J1 cannot induce drug-resistance mutation in the gp41 pocket region. Therefore, we show herein that HIV-1 mutants resistant to T2635, a peptide derived from the gp41 C-terminal heptad repeat (CHR) region with pocket-binding domain (PBD), were also resistant to ADS-J1. We also show that pseudoviruses with mutations at positions 64 and 67 in the gp41 pocket region were highly resistant to ADS-J1 and C34, another CHR-peptide with PBD, but relatively sensitive to T20, a CHR-peptide without PBD. ADS-J1 could effectively bind to N36Fd, a mimic of the gp41 NHR-trimer with pocket exposed, and block binding of C34 to N36Fd trimer to form six-helix bundle (6-HB). However, ADS-J1 was less effective in binding to N36Fd trimer with mutations in the gp41 pocket region, such as N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, and N36(Q66R)Fd, as well as less effective in blocking 6-HB formation between C34 and these mutant N36Fd trimers. These results confirm that ADS-J1 mainly targets the pocket region in the HIV-1 gp41 NHR trimer and suggest that it could be used as a lead for developing small-molecule HIV fusion inhibitors and as a molecule probe for studying the mechanisms of gp41-mediated membrane fusion.     

HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain

Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.     

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide¹ is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41 μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.     

Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41

Retrocyclin RC-101, a theta-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat 2 (HR2) region of glycoprotein 41 (gp41) and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101, and then analyzed the HIV-1 sequence database at Los Alamos National Laboratory (New Mexico, USA) for residue variations in the heptad repeat 1 (HR1) and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single-round replication assays in the presence of 1.25-10 microg x mL(-1) RC-101. Apart from one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide.     

Oligomerization of the hydrophobic heptad repeat of gp41.

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.     

Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor

T-20 is a synthetic peptide that corresponds to 36 amino acids within the C-terminal heptad repeat region (HR2) of human immunodeficiency virus type 1 (HIV-1) gp41. T-20 has been shown to potently inhibit viral replication of HIV-1 both in vitro and in vivo and is currently being evaluated in a Phase III clinical trial. T-649 is an inhibitory peptide that also corresponds to 36 amino acids within HR2. This sequence overlaps the T-20 sequence but is shifted 10 residues toward the N terminus of gp41. Both inhibitors are thought to exert their antiviral activity by interfering with the conformational changes that occur within gp41 to promote membrane fusion following gp120 interactions with CD4 and coreceptor molecules. We have shown previously that coreceptor specificity defined by the V3 loop of gp120 modulates sensitivity to T-20 and that a critical region within the N-terminal heptad repeat (HR1) of gp41 is the major determinant of sensitivity (C. A. Derdeyn et al., J. Virol. 74:8358-8367, 2000). This report shows that (i) regions within gp41 distinct from those associated with T-20 sensitivity govern the baseline sensitivity to T-649 and (ii) T-649 sensitivity of chimeric viruses that contain sequences derived from CXCR4- and CCR5-specific envelopes is also modulated by coreceptor specificity. Moreover, the pattern of sensitivity of CCR5-specific chimeras with only minor differences in their V3 loop was consistent for both inhibitors, suggesting that the individual affinity for coreceptor may influence accessibility of these inhibitors to their target sequence. Finally, an analysis of the sensitivity of 55 primary, inhibitor-naive HIV-1 isolates found that higher concentrations of T-20 (P < 0.001) and T-649 (P = 0.016) were required to inhibit CCR5-specific viruses compared to viruses that utilize CXCR4. The results presented here implicate gp120-coreceptor interactions in driving the complex conformational changes that occur in gp41 to promote fusion and entry and suggest that sensitivity to different HR1-directed fusion inhibitors is governed by distinct regions of gp41 but is consistently modulated by coreceptor specificity.     

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

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

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.     

The interactions of the N-terminal fusogenic peptide of HIV-1 gp41 with neutral phospholipids

We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of the HIV-1_LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the ¹³C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study. Received: 8 June 1998 / Revised version: 18 November 1998 / Accepted: 28 December 1998     

Mutations in the fusion peptide and heptad repeat regions of the Newcastle disease virus fusion protein block fusion.

Nonconservative mutations were introduced by site-specific mutagenesis into the fusion peptide and the adjacent heptad repeat region of the fusion protein of Newcastle disease virus in order to determine the role of both regions in the fusion activity of the protein. Mutations in both regions that allowed for proper folding and intracellular transport of the protein blocked the fusion activity of the protein when assayed in the presence of the hemagglutinin-neuraminidase protein.     

Thermodynamics of peptide inhibitor binding to HIV-1 gp41

The gp41 subunit of the human immunodeficiency virus type 1 envelope glycoprotein mediates fusion of the cellular and viral membranes. The gp41 ectodomain is a trimer of alpha-helical hairpins, where N-terminal helices form a parallel three-stranded coiled-coil core and C-terminal helices pack around the core. A deep hydrophobic pocket on the N-terminal core represents an attractive target for antiviral therapeutics. We have employed a soluble derivative of the gp41 core ectodomain and small cyclic disulfide D-peptide inhibitors to define the stoichiometry, affinity, and thermodynamics of ligand binding to this pocket using isothermal titration calorimetry. These inhibitors bind with micromolar affinity to the pocket with the expected stoichiometry of three peptides per gp41 core trimer. There are no cooperative interactions among the three binding sites. Linear eight- or nine-residue D-peptides derived from the pocket-binding domain of the cyclic molecules also bind specifically. A negative heat capacity change is observed and is consistent with burial of hydrophobic surface upon binding. Contrary to expectations for a reaction dominated by the classical hydrophobic effect, peptide binding is enthalpically driven and is opposed by an unfavorable negative entropy change. The calorimetry data support models whereby dominant negative inhibitors bind to a transiently exposed surface on the prefusion intermediate state of gp41 and disrupt subsequent resolution to the fusion-active six-stranded hairpin conformation.     

Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.