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1.  Small-Molecule Inhibitors of Dengue-Virus Entry 
PLoS Pathogens  2012;8(4):e1002627.
Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal “stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC90s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a “pocket-open" state, which has lost affinity for the stem peptide and cannot support the final “zipping up" of the stem.
Author Summary
Fusion of viral and cellular membranes is necessary to establish infection by an enveloped virus. This process is facilitated by rearrangement of protein(s) present on the virion surface in response to molecular cues from the compartment from which fusion occurs, such as low pH of an endosome. Dengue virus is an enveloped virus in the flavivirus family; its “E" (for envelope) protein is the fusion mediator. We previously showed that peptides derived from the membrane proximal “stem" of the E protein bind a form of E that represents a late-stage fusion intermediate. We used this assay to screen for small-molecule inhibitors that compete for stem-peptide association with E. We describe one such inhibitor and its analogs that block viral fusion. These inhibitors also block infectivity if added to dengue virus before infection. Withdrawing the inhibitor before fusion reverses the blockage. We propose that these small molecules bind a hydrophobic pocket on the virion surface and that the virus carries them into the endosome, where they prevent viral fusion by stabilizing an intermediate conformation of the E protein that cannot complete the fusion-promoting conformational change. Identification of these fusion inhibitors shows that viral entry is a possible target for anti-flavivirus drugs.
PMCID: PMC3320583  PMID: 22496653
2.  ADS-J1 Inhibits Human Immunodeficiency Virus Type 1 Entry by Interacting with the gp41 Pocket Region and Blocking Fusion-Active gp41 Core Formation▿  
Antimicrobial Agents and Chemotherapy  2009;53(12):4987-4998.
We previously identified a small-molecule anti-human immunodeficiency virus type 1 (anti-HIV-1) compound, ADS-J1, using a computer-aided molecular docking technique for primary screening and a sandwich enzyme-linked immunosorbent assay (ELISA) as a secondary screening method. In the present study, we demonstrated that ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay. Further mechanism studies confirmed that ADS-J1 does not block gp120-CD4 binding and exhibits a marginal interaction with the HIV-1 coreceptor CXCR4. However, ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D peptide, PIE7. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding of ADS-J1 to NHR. These results suggest that ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry.
PMCID: PMC2786362  PMID: 19786602
3.  N-Substituted Pyrrole Derivatives as Novel Human Immunodeficiency Virus Type 1 Entry Inhibitors That Interfere with the gp41 Six-Helix Bundle Formation and Block Virus Fusion 
Antimicrobial Agents and Chemotherapy  2004;48(11):4349-4359.
A recently approved peptidic human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, T-20 (Fuzeon; Trimeris Inc.), has shown significant promise in clinical application for treating HIV-1-infected individuals who have failed to respond to the currently available antiretroviral drugs. However, T-20 must be injected twice daily and is too expensive. Therefore, it is essential to develop orally available small molecule HIV-1 fusion inhibitors. By screening a chemical library consisting of “drug-like” compounds, we identified two N-substituted pyrroles, designated NB-2 and NB-64, that inhibited HIV-1 replication at a low micromolar range. The absence of the COOH group in NB-2 and NB-64 resulted in a loss of anti-HIV-1 activity, suggesting that this acid group plays an important role in mediating the antiviral activity. NB-2 and NB-64 inhibited HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation and disrupting the α-helical conformation. They blocked a d-peptide binding to the hydrophobic pocket on surface of the gp41 internal trimeric coiled-coil domain. Computer-aided molecular docking analysis has shown that they fit inside the hydrophobic pocket and that their COOH group interacts with a positively charged residue (K574) around the pocket to form a salt bridge. These results suggest that NB-2 and NB-64 may bind to the gp41 hydrophobic pocket through hydrophobic and ionic interactions and block the formation of the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry. Therefore, NB-2 and NB-64 can be used as lead compounds toward designing and developing more potent small molecule HIV-1 fusion inhibitors targeting gp41.
PMCID: PMC525433  PMID: 15504864
4.  Paramagnetic relaxation assisted docking of a small indole compound in the HIV-1 gp41 hydrophobic pocket 
ACS chemical biology  2011;6(3):267-274.
The hydrophobic pocket contained within the gp41 coiled coil is an important target for small molecules designed to inhibit HIV-1 fusion. While various screening experiments have identified molecules purported to bind in this pocket, few have confirmed details of the interaction, instead relying on computational docking to predict the binding mode. This is made more challenging by the fact that residues lining the hydrophobic pocket are highly flexible, as is typical for a protein - protein interaction site, limiting the predictive power of computational tools. In this study, we report on an NMR method to define the binding mode of 1-5i, a compound in a series of newly developed indole inhibitors. We show that paramagnetic relaxation enhancement of ligand protons due to an MTSL group positioned close to the binding pocket could be applied quantitatively to distinguish between more than 30 different computational poses, selecting a single pose that agreed with the NMR data. In this pose, important hydrophobic and polar contacts occur with pocket lysine, tryptophan and glutamine residues, including putative hydrogen bonds between the ligand carboxylate and the lysine ε-amino group. A study of the ligand orientation suggests directions for optimization.
PMCID: PMC3060958  PMID: 21155611
5.  A Novel Fluorescence Intensity Screening Assay Identifies New Low-Molecular-Weight Inhibitors of the gp41 Coiled-Coil Domain of Human Immunodeficiency Virus Type 1▿  
A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophore-labeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z′) of 0.88 and can rank order inhibitors at 10 μM concentration with Kis in the range of 0.2 μM to 30 μM, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors.
PMCID: PMC1913228  PMID: 17452484
6.  Approaches for Identification of HIV-1 Entry Inhibitors Targeting gp41 Pocket 
Viruses  2013;5(1):127-149.
The hydrophobic pocket in the HIV-1 gp41 N-terminal heptad repeat (NHR) domain plays an important role in viral fusion and entry into the host cell, and serves as an attractive target for development of HIV-1 fusion/entry inhibitors. The peptide anti-HIV drug targeting gp41 NHR, T-20 (generic name: enfuvirtide; brand name: Fuzeon), was approved by the U.S. FDA in 2003 as the first HIV fusion/entry inhibitor for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, because T20 lacks the pocket-binding domain (PBD), it exhibits low anti-HIV-1 activity and short half-life. Therefore, several next-generation HIV fusion inhibitory peptides with PBD have been developed. They possess longer half-life and more potent antiviral activity against a broad spectrum of HIV-1 strains, including the T-20-resistant variants. Nonetheless, the clinical application of these peptides is still limited by the lack of oral availability and the high cost of production. Thus, development of small molecule compounds targeting the gp41 pocket with oral availability has been promoted. This review describes the main approaches for identification of HIV fusion/entry inhibitors targeting the gp41 pocket and summarizes the latest progress in developing these inhibitors as a new class of anti-HIV drugs.
PMCID: PMC3564113  PMID: 23344560
HIV-1; gp41; HIV fusion/entry inhibitors; small molecule compounds; hydrophobic pocket
7.  Charge-Surrounded Pockets and Electrostatic Interactions with Small Ions Modulate the Activity of Retroviral Fusion Proteins 
PLoS Pathogens  2011;7(2):e1001268.
Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry.
Author Summary
Human T-cell leukaemia virus types-1 (HTLV-1) and bovine leukaemia virus (BLV) are divergent blood borne viruses that cause hematological malignancies in humans and cattle respectively. In common with other enveloped viruses, infection of cells by HTLV-1 and BLV is dependent on the membrane fusion properties of the viral envelope glycoproteins. Here we have solved the crystal structure of the BLV transmembrane glycoprotein, and, through a functional and comparative analysis with HTLV-1, we have identified features that are critical to fusion protein function. In particular, we demonstrate that electrostatic interactions with small ions dramatically stabilize the assembly and fusion-associated forms of the BLV TM, but are not required for the cell surface display of native pre-fusogenic envelope. Moreover, we show that charged residues that border a deep hydrophobic pocket contribute directly to appropriate folding of fusion-active envelope and are critical to membrane fusion. Importantly, the charged residues that border the pocket are key features that determine the specificity and activity of peptide inhibitors of envelope function. Our study demonstrates that charge-surrounded pockets and electrostatic interactions with small ions are significant leitmotifs of diverse class-1 fusion proteins and that these elements represent ideal targets for novel small-molecule inhibitors of viral entry.
PMCID: PMC3033372  PMID: 21304939
8.  Stable Extended Human Immunodeficiency Virus Type 1 gp41 Coiled Coil as an Effective Target in an Assay for High-Affinity Fusion Inhibitors▿  
The human immunodeficiency virus type 1 (HIV-1) gp41 coiled-coil domain is an important target for fusion inhibitors, including the peptide T20, which has been approved as a drug against HIV-1. Research into nonpeptide fusion inhibitors has focused primarily on a hydrophobic pocket located within the coiled coil and has so far yielded compounds with relatively weak fusion inhibitory activity. Here, we describe metal ion-assisted stabilization of an extended 39-residue construct of gp41, which includes residues of the hydrophobic pocket and also of an extended groove N terminal to the hydrophobic pocket. We show that the presence of a metal ion and the high-affinity interaction between the receptor construct and cognate C-peptides result in a simple and highly selective assay for fusion inhibitors that may be used to scan large compound libraries. The long construct presents multiple potential binding sites along the extended coiled-coil groove. We demonstrate the modular use of assay probes to detect whether compounds bind in the hydrophobic pocket or elsewhere along the groove. Rapid detection and quantitation of hits can lead to the discovery of compounds binding to different sites along the groove and provide structure-activity relationship data for optimization. Compounds binding to adjacent sites could be linked to form more potent fusion inhibitors.
PMCID: PMC2687196  PMID: 19364877
9.  Design, Synthesis, and Structure-Activity Relationship of a Novel Series of 2-Aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidin-ylidenemethyl)furans as HIV-1 entry inhibitors* 
Journal of medicinal chemistry  2009;52(23):7631-7639.
We previously identified two small molecules targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole 12 (NB-2) and N-(3-carboxy-4-chloro) phenylpyrrole 13 (NB-64) that inhibit HIV-1 infection at low μM level. Based on molecular docking analysis, we designed a series of 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans. Compared with 12 and 13, these compounds have bigger molecular size (437–515 Da) and could occupy more space in the deep hydrophobic pocket on the gp41 NHR-trimer. Fifteen 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans (11a–o) were synthesized by Suzuki-Miyaura cross coupling, followed by a Knoevenagel condensation and tested for their anti-HIV-1activity and cytotoxicity on MT-2 cells. We found that all 15 compounds had improved anti-HIV-1 activity and 3 of them (11a, 11b, and 11d) exhibited inhibitory activity against replication of HIV-1 IIIB and 94UG103 at <100 nM range, more than 20-fold more potent than 12 and 13, suggesting that these compounds can serve as leads for development of novel small molecule HIV fusion inhibitors.
PMCID: PMC2802534  PMID: 19746983
10.  Computational analyses of curcuminoid analogs against kinase domain of HER2 
BMC Bioinformatics  2014;15(1):261.
Human epidermal growth factor receptor 2 (HER2) has an important role in cancer aggressiveness and poor prognosis. HER2 has been used as a drug target for cancers. In particular, to effectively treat HER2-positive cancer, small molecule inhibitors were developed to target HER2 kinase. Knowing that curcumin has been used as food to inhibit cancer activity, this study evaluated the efficacy of natural curcumins and curcumin analogs as HER2 inhibitors using in vitro and in silico studies. The curcumin analogs considered in this study composed of 4 groups classified by their core structure, β-diketone, monoketone, pyrazole, and isoxazole.
In the present study, both computational and experimental studies were performed. The specificity of curcumin analogs selected from the docked results was examined against human breast cancer cell lines. The screened curcumin compounds were then subjected to molecular dynamics simulation study. By modifying curcumin analogs, we found that protein-ligand affinity increases. The benzene ring with a hydroxyl group could enhance affinity by forming hydrophobic interactions and the hydrogen bond with the hydrophobic pocket. Hydroxyl, carbonyl or methoxy group also formed hydrogen bonds with residues in the adenine pocket and sugar pocket of HER2-TK. These modifications could suggest the new drug design for potentially effective HER2-TK inhibitors. Two outstanding compounds, bisdemethylcurcumin (AS-KTC006) and 3,5-bis((E)-3,4-dimethoxystyryl)isoxazole (AS-KTC021 ),were well oriented in the binding pocket almost in the simulation time, 30 ns. This evidence confirmed the results of cell-based assays and the docking studies. They possessed more distinguished interactions than known HER2-TK inhibitors, considering them as a promising drug in the near future.
The series of curcumin compounds were screened using a computational molecular docking and followed by human breast cancer cell lines assay. Both AS-KTC006 and AS-KTC021 could inhibit breast cancer cell lines though inhibiting of HER2-TK. The intermolecular interactions were confirmed by molecular dynamics simulation studies. This information would explore more understanding of curcuminoid structures and HER2-TK.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2105-15-261) contains supplementary material, which is available to authorized users.
PMCID: PMC4143557  PMID: 25089037
HER2; Tyrosine kinase; Curcuminoid analogs; Docking; Molecular dynamics simulation
11.  Design, Synthesis and Evaluation of Indole Compounds as Novel Inhibitors targeting Gp41 
A series of indole ring containing compounds were designed based on the structure of the gp41 complex in the region of the hydrophobic pocket. These compounds were synthesized using a Suzuki Coupling reaction, and evaluated using a fluorescence binding assay and cell-cell fusion assay. The observed inhibition constant of compound 7 was 2.1µM, and the IC50 for cell-cell fusion inhibition was 1.1µM. Assay data indicated that 7 is a promising lead compound for optimization into an effective low molecular weight fusion inhibitor.
PMCID: PMC2833348  PMID: 20153190
HIV; gp41; small molecule inhibitor; lead optimization; indole rings
12.  Structural and Functional Insights into the HIV-1 Maturation Inhibitor Binding Pocket 
PLoS Pathogens  2012;8(11):e1002997.
Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for in vitro. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased structural understanding of HIV-1 maturation inhibitor activity.
Author Summary
Maturation of HIV-1 particles, which occurs as they bud off from the infected cell, is triggered by the step-wise cleavage of the major viral structural polyprotein, Pr55Gag, to individual, mature Gag proteins. The viral protease is the enzyme responsible for Gag polyprotein cleavage. Maturation inhibitors prevent the viral protease from processing Gag at one particular cleavage site, but how they accomplish this is not understood. In this study, the ability of HIV-1 to become resistant to the two structurally distinct maturation inhibitors that have thus far been reported was examined. We found that one of these compounds, PF-46396, gives rise to resistance mutations that map to three domains in Gag, including a region known as the major homology region (MHR). The MHR is highly conserved among retroviruses and is known to be very important for virus assembly and maturation. These MHR mutants were observed to replicate much better in the presence of PF-46396 than in its absence; i.e., these mutants are compound-dependent. We were also able to select for second-site mutations in Gag that reversed the replication defects imposed by the MHR mutations. These results define residues in Gag that comprise the maturation inhibitor-binding pocket and also identify regions of Gag that structurally and functionally interact with the MHR.
PMCID: PMC3493477  PMID: 23144615
13.  NMR-assisted computational studies of peptidomimetic inhibitors bound in the hydrophobic pocket of HIV-1 glycoprotein 41 
Due to the inherently flexible nature of a protein – protein interaction surface, it is difficult both to inhibit the association with a small molecule, and to predict how it might bind to the surface. In this study, we have examined small molecules that mediate the interaction between a WWI motif on the C-helix of HIV-1 glycoprotein-41 and a deep hydrophobic pocket contained in the interior N-helical trimer. Association between these two components of gp41 leads to virus–cell and cell–cell fusion, which could be abrogated in the presence of an inhibitor that binds tightly in the pocket. We have studied a comprehensive combinatorial library of α-helical peptidomimetics, and found that compounds with strongly hydrophobic side chains had the highest affinity. Computational docking studies produced multiple possible binding modes due to the flexibility of both the binding site and the peptidomimetic compounds. We applied a transferred paramagnetic relaxation enhancement (PRE) experiment to two selected members of the library, and showed that addition of a few experimental constraints enabled definitive identification of unique binding poses. Computational docking results were extremely sensitive to side chain conformations, and slight variations could preclude observation of the experimentally validated poses. Different receptor structures were required for docking simulations to sample the correct pose for the two compounds. The study demonstrated the sensitivity of predicted poses to receptor structure and indicated the importance of experimental verification when docking to a malleable protein – protein interaction surface.
PMCID: PMC3777804  PMID: 23893342
14.  Design, Synthesis, and Biological Evaluation of N-Carboxyphenylpyrrole Derivatives as Potent HIV Fusion Inhibitors Targeting gp41 
Journal of medicinal chemistry  2008;51(24):7843-7854.
Based on the structures of small-molecule hits targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole (2, NB-2) and N-(3-carboxy-4-chloro)phenylpyrrole (A1, NB-64), 42 N-carboxyphenylpyrrole derivatives in two categories (A and B series) were designed and synthesized. We found that 11 compounds exhibited promising anti-HIV-1 activity at micromolar level and their antiviral activity was correlated with their inhibitory activity on gp41 six-helix bundle formation, suggesting that these compounds block HIV fusion and entry by disrupting gp41 core formation. The structure-activity relationship and molecular docking analysis revealed that the carboxyl group could interact with either Arg579 or Lys574 to form salt bridges and two methyl groups on the pyrrole ring were favorable for interaction with the residues in gp41 pocket. The most active compound, N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (A12), partially occupied the deep hydrophobic pocket, suggesting that enlarging the molecular size of A12 could improve its binding affinity and anti-HIV-1 activity for further development as a small-molecule HIV fusion and entry inhibitor.
PMCID: PMC2656571  PMID: 19053778
HIV-1 gp41; HIV entry inhibitor; six-helix bundle
15.  Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition ▿  
Journal of Virology  2008;82(22):11129-11139.
The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp632 located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys574 in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys574 plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp632 has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp632 substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys574 mutations, nonconserved substitutions of Asp632 also severely impaired the α-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp632 significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.
PMCID: PMC2573276  PMID: 18768964
16.  Amphipathic properties of HIV-1 gp41 fusion inhibitors 
Current topics in medicinal chemistry  2011;11(24):3022-3032.
Small molecule inhibition of HIV fusion has been an elusive goal, despite years of effort by both pharmaceutical and academic laboratories. In this review, we will discuss the amphipathic properties of both peptide and small molecule inhibitors of gp41-mediated fusion. Many of the peptides and small molecules that have been developed target a large hydrophobic pocket situated within the grooves of the coiled coil, a potential hotspot for inhibiting the trimer of hairpin formation that accompanies fusion. Peptide studies reveal molecular properties required for effective inhibition, including elongated structure and lipophilic or amphiphilic nature. The characteristics of peptides that bind in this pocket provide features that should be considered in small molecule development. Additionally, a novel site for small molecule inhibition of fusion has recently been suggested, involving residues of the loop and fusion peptide. We will review the small molecule structures that have been developed, evidence pointing to their mechanism of action and strategies towards improving their affinity. The data points to the need for a strongly amphiphilic character of the inhibitors, possibly as a means to mediate the membrane - protein interaction that occurs in gp41 in addition to the protein – protein interaction that accompanies the fusion-activating conformational transition.
PMCID: PMC3219813  PMID: 22044226
17.  Design of a Potent d-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance▿  
Journal of Virology  2010;84(21):11235-11244.
The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific d-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. d-Peptides (peptides composed of d-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first d-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.
PMCID: PMC2953169  PMID: 20719956
18.  The role of amphiphilicity and negative charge in glycoprotein 41 interactions in the hydrophobic pocket 
Journal of medicinal chemistry  2009;52(14):4338-4344.
The hydrophobic pocket within the coiled coil domain of HIV-1 gp41 is considered to be a hotspot suitable for small molecule intervention of fusion, although so far it has yielded only μM inhibitors. Previous peptide studies have identified specific hydrophobic interactions and a Lys-Asp salt bridge as contributing to binding affinity in the pocket. Negative charge appears to be critical for activity of small molecules. We have examined the role of charge and amphiphilic character in the interaction, by studying a series of short pocket binding peptides differing in charge, helical content and in the presence or absence of the Lys-Asp salt bridge, and a series of fatty acid salts with varying charge and hydrocarbon length. Quantitative binding analysis revealed that long range electrostatic forces and a greasy non-specific hydrophobic interaction were sufficient for μM potency. The results suggest that an extended interaction site may be necessary for higher potency. We examined a region of the coiled coil immediately C-terminal to the pocket, and found that specific salt bridge and hydrogen bond networks may reside in this region. Negatively charged groups extended towards or beyond the C-terminus of the pocket could therefore result in improved low molecular weight fusion inhibitors.
PMCID: PMC2724993  PMID: 19534533
HIV-1 gp41; hydrophobic pocket; electrostatics; peptide binding; fatty acid salts
19.  Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage 
Retrovirology  2013;10:144.
LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction.
We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket.
Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.
PMCID: PMC4222603  PMID: 24261564
HIV; Integrase; LEDGF; Antiretroviral activity; Drug discovery; Allosteric inhibition; Protein-protein interaction inhibitor; Integrase inhibitor; Co-crystallization
20.  HIV Capsid is a Tractable Target for Small Molecule Therapeutic Intervention 
PLoS Pathogens  2010;6(12):e1001220.
Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.
Author Summary
Although the current standard of care for Human Immunodeficiency Virus (HIV) is high, viral resistance has emerged to every drug currently in the clinic, in some cases rendering the entire class ineffective for patients. A new class of antiretroviral drugs would be effective against strains of HIV-1 that are resistant to any existing drug and would expand the therapeutic options available to patients. Capsid is the primary structural protein of HIV and a critical part of the viral replication cycle, both in the assembly of viral particles and in the infection of host cells. We report a new class of antiretrovirals that targets HIV-1 capsid and demonstrate that it is active at two critical stages in the viral replication cycle. These compounds were consistently effective against a range of clinical strains of HIV-1, from various sub-types, as well as HIV-2. Finally, the compounds bind in a unique pocket on capsid that has not previously been highlighted as a drug binding site. We believe this new class of antiretrovirals can serve as a starting point for the development of a new generation of HIV-1 therapeutics and, more generally, underscores the potential of capsid as a therapeutic target.
PMCID: PMC3000358  PMID: 21170360
21.  Peptide inhibitors of dengue virus and West Nile virus infectivity 
Virology Journal  2005;2:49.
Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) [1] in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 μM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 μM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 μM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.
PMCID: PMC1177995  PMID: 15927084
22.  Identification of a Small-Molecule Entry Inhibitor for Filoviruses▿ †  
Journal of Virology  2011;85(7):3106-3119.
Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC50s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.
PMCID: PMC3067866  PMID: 21270170
23.  Distinct Effects of Two HIV-1 Capsid Assembly Inhibitor Families That Bind the Same Site within the N-Terminal Domain of the Viral CA Protein 
Journal of Virology  2012;86(12):6643-6655.
The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.
PMCID: PMC3393593  PMID: 22496222
24.  Discovery of Small-Molecule HIV-1 Fusion and Integrase Inhibitors Oleuropein and Hydroxytyrosol: I. Fusion 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 EC50s of 66–58 nM, with no detectable toxicity. Our findings on effects on HIV-1 integrase are reported separately.
PMCID: PMC2790717  PMID: 17275783
HIV-1; AIDS; natural product; small molecule HIV-1 inhibitors; HIV-1 entry inhibitor; Olive Leaf Extract (OLE); Oleuropein (Ole); Hydroxytyrosol (HT); structure-function; molecular modeling
25.  Human Immunodeficiency Virus Type 1 Variants Resistant to First- and Second-Version Fusion Inhibitors and Cytopathic in Ex Vivo Human Lymphoid Tissue▿  
Journal of Virology  2007;81(12):6563-6572.
Human immunodeficiency virus type 1 (HIV-1) fusion inhibitors blocking viral entry by binding the gp41 heptad repeat 1 (HR1) region offer great promise for antiretroviral therapy, and the first of these inhibitors, T20 (Fuzeon; enfuvirtide), is successfully used in the clinic. It has been reported previously that changes in the 3-amino-acid GIV motif at positions 36 to 38 of gp41 HR1 mediate resistance to T20 but usually not to second-version fusion inhibitors, such as T1249, which target an overlapping but distinct region in HR1 including a conserved hydrophobic pocket (HP). Based on the common lack of cross-resistance and the difficulty of selecting T1249-resistant HIV-1 variants, it has been suggested that the determinants of resistance to first- and second-version fusion inhibitors may be different. To further assess HIV-1 resistance to fusion inhibitors and to analyze where changes in HR1 are tolerated, we randomized 16 codons in the HR1 region, including those making contact with HR2 codons and/or encoding residues in the GIV motif and the HP. We found that changes only at positions 37I, 38V, and 40Q near the N terminus of HR1 were tolerated. The propagation of randomly gp41-mutated HIV-1 variants in the presence of T1249 allowed the effective selection of highly resistant forms, all containing changes in the IV residues. Overall, the extent of T1249 resistance was inversely correlated to viral fitness and cytopathicity. Notably, one HIV-1 mutant showing ∼10-fold-reduced susceptibility to T1249 inhibition replicated with wild type-like kinetics and caused substantial CD4+-T-cell depletion in ex vivo-infected human lymphoid tissue in the presence and absence of an inhibitor. Taken together, our results show that the GIV motif also plays a key role in resistance to second-version fusion inhibitors and suggest that some resistant HIV-1 variants may be pathogenic in vivo.
PMCID: PMC1900115  PMID: 17428857

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