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 = 37.81 µM), 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.     

Computational study of bindings of HL9, a nonapeptide fragment of human lysozyme, to HIV-1 fusion protein gp41

HL9 is a nonapeptide fragment of human lysozyme which has been shown to have anti-HIV-1 activity in nanomolar concentration. This study aims to explain this inhibitory activity by using molecular dynamics (MD) simulation, focusing on the ectodomain of gp41, the envelope glycoprotein of HIV-1 crucial to membrane fusion. It was found that in HL9, two Trp residues separated by two others occupy the conserved hydrophobic pocket on gp41 and thus inhibit fusion in dominant-negative manner. Detailed HL9-gp41 binding interactions and free energies of binding were obtained through MD simulation and solvated interaction energies (SIE) calculation, giving a binding free energy of −8.25 kcal/mol which is in close agreement with the experimental value of −9.96 kcal/mol. Since C-helical region (C34) of gp41 also has two Trp residues separated by two others, this arrangement may be generalised and used to scan peptide library and to find those having similar manner of inhibition.     

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.     

An In Silico Analysis of the Binding Modes and Binding Affinities of Small Molecule Modulators of PDZ-Peptide Interactions

Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson’s disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K_i or K_d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K_i or K_d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

Computational study of bindings of olive leaf extract (OLE) to HIV-1 fusion protein gp41

Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41. Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors.     

Computational studies and peptidomimetic design for the human p53-MDM2 complex

The interaction between human p53 and MDM2 is a key event in controlling cell growth. Many studies have suggested that a p53 mimic would be sufficient to inhibit MDM2 to reduce cell growth in cancerous tissue. In order to design a potent p53 mimic, molecular dynamics (MD) simulations were used to examine the binding interface and the effect of mutating key residues in the human p53-MDM2 complex. The Generalized Born surface area (GBSA) method was used to estimate free energies of binding, and a computational alanine-scanning approach was used to calculate the relative effects in the free energy of binding for key mutations. Our calculations determine the free energy of binding for a model p53-MDM2 complex to be -7.4 kcal/mol, which is in very good agreement with the experimentally determined values (-6.6--8.8 kcal/mol). The alanine-scanning results are in good agreement with experimental data and calculations by other groups. We have used the information from our studies of human p53-MDM2 to design a beta-peptide mimic of p53. MD simulations of the mimic bound to MDM2 estimate a free energy of binding of -8.8 kcal/mol. We have also applied alanine scanning to the mimic-MDM2 complex and reveal which mutations are most likely to alter the binding affinity, possibly giving rise to escape mutants. The mimic was compared to nutlins, a new class of inhibitors that block the formation of the p53-MDM2 complex. There are interesting similarities between the nutlins and our mimic, and the differences point to ways that both inhibitors may be improved. Finally, an additional hydrophobic pocket is noted in the interior of MDM2. It may be possible to design new inhibitors to take advantage of that pocket.     

Toward full-sequence de novo protein design with flexible templates for human beta-defensin-2

In this article, we introduce and apply our de novo protein design framework, which observes true backbone flexibility, to the redesign of human β-defensin-2, a 41-residue cationic antimicrobial peptide of the innate immune system. The flexible design templates are generated using molecular dynamics simulations with both Generalized Born implicit solvation and explicit water molecules. These backbone templates were employed in addition to the x-ray crystal structure for designing human β-defensin-2. The computational efficiency of our framework was demonstrated with the full-sequence design of the peptide with flexible backbone templates, corresponding to the mutation of all positions except the native cysteines.     

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. fusion [corrected] inhibition

We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC(50)s of 66-58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article.     

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.     

An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.     

Footprint-based identification of viral entry inhibitors targeting HIVgp41

A targeted virtual screen to the N-helix hydrophobic pocket on HIVgp41 was performed using DOCK followed by re-ranking with a new footprint-based scoring function which employed native gp41 C-helix residues as a reference. Of ca. 500,000 small molecules screened, 115 were purchased, and 7 hits were identified with favorable binding (K(i)), cell-cell fusion (IC(50)), and cytotoxicity (CC(50)) profiles. Three of the seven active compounds would not have been discovered without the use of the footprints, demonstrating the utility of the method for structure-based design when a known reference compound or substrate is available.     

Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody

Elicitation of potent and broadly neutralizing antibodies is an important goal in designing an effective human immunodeficiency virus-1 (HIV-1) vaccine. The HIV-1 gp41 inner-core trimer represents a functionally and structurally conserved target for therapeutics. Here we report the 2.0-A-resolution crystal structure of the complex between the antigen-binding fragment of D5, an HIV-1 cross-neutralizing antibody, and 5-helix, a gp41 inner-core mimetic. Both binding and neutralization depend on residues in the D5 CDR H2 loop protruding into the conserved gp41 hydrophobic pocket, as well as a large pocket in D5 surrounding core gp41 residues. Kinetic analysis of D5 mutants with perturbed D5-gp41 interactions suggests that D5 persistence at the fusion intermediate is crucial for neutralization. Thus, our data validate the gp41 N-peptide trimer fusion intermediate as a target for neutralizing antibodies and provide a template for identification of more potent and broadly neutralizing molecules.     

The PP2A‐interactor TIP41 modulates ABA responses in Arabidopsis thaliana

Modulation of growth in response to environmental cues is a fundamental aspect of plant adaptation to abiotic stresses. TIP41 (TAP42 INTERACTING PROTEIN OF 41 kDa) is the Arabidopsis thaliana orthologue of proteins isolated in mammals and yeast that participate in the Target‐of‐Rapamycin (TOR) pathway, which modifies cell growth in response to nutrient status and environmental conditions. Here, we characterized the function of TIP41 in Arabidopsis. Expression analyses showed that TIP41 is constitutively expressed in vascular tissues, and is induced following long‐term exposure to NaCl, polyethylene glycol and abscisic acid (ABA), suggesting a role of TIP41 in adaptation to abiotic stress. Visualization of a fusion protein with yellow fluorescent protein indicated that TIP41 is localized in the cytoplasm and the nucleus. Abolished expression of TIP41 results in smaller plants with a lower number of rosette leaves and lateral roots, and an increased sensitivity to treatments with chemical TOR inhibitors, indicating that TOR signalling is affected in these mutants. In addition, tip41 mutants are hypersensitive to ABA at germination and seedling stage, whereas over‐expressing plants show higher tolerance. Several TOR‐ and ABA‐responsive genes are differentially expressed in tip41, including iron homeostasis, senescence and ethylene‐associated genes. In yeast and mammals, TIP41 provides a link between the TOR pathway and the protein phosphatase 2A (PP2A), which in plants participates in several ABA‐mediated mechanisms. Here, we showed an interaction of TIP41 with the catalytic subunit of PP2A. Taken together, these results offer important insights into the function of Arabidopsis TIP41 in the modulation of plant growth and ABA responses.     

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.     

Computational identification of self-inhibitory peptides from envelope proteins

Fusion process is known to be the initial step of viral infection and hence targeting the entry process is a promising strategy to design antiviral therapy. The self-inhibitory peptides derived from the enveloped (E) proteins function to inhibit the protein-protein interactions in the membrane fusion step mediated by the viral E protein. Thus, they have the potential to be developed into effective antiviral therapy. Herein, we have developed a Monte Carlo-based computational method with the aim to identify and optimize potential peptide hits from the E proteins. The stability of the peptides, which indicates their potential to bind in situ to the E proteins, was evaluated by two different scoring functions, dipolar distance-scaled, finite, ideal-gas reference state and residue-specific all-atom probability discriminatory function. The method was applied to α-helical Class I HIV-1 gp41, β-sheet Class II Dengue virus (DENV) type 2 E proteins, as well as Class III Herpes Simplex virus-1 (HSV-1) glycoprotein, a E protein with a mixture of α-helix and β-sheet structural fold. The peptide hits identified are in line with the druggable regions where the self-inhibitory peptide inhibitors for the three classes of viral fusion proteins were derived. Several novel peptides were identified from either the hydrophobic regions or the functionally important regions on Class II DENV-2 E protein and Class III HSV-1 gB. They have potential to disrupt the protein-protein interaction in the fusion process and may serve as starting points for the development of novel inhibitors for viral E proteins.     

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.