The Experts below are selected from a list of 3174 Experts worldwide ranked by ideXlab platform
Shibo Jiang - One of the best experts on this subject based on the ideXlab platform.
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a two birds one stone approach toward the design of bifunctional human immunodeficiency virus type 1 entry inhibitors targeting the ccr5 coreceptor and gp41 n terminal Heptad Repeat region
Journal of Medicinal Chemistry, 2021Co-Authors: Chao Wang, Xinling Wang, Huan Wang, Yang Liu, Shibo JiangAbstract:Previous studies have reported the stepwise nature of human immunodeficiency virus type 1 (HIV-1) entry and the pivotal role of coreceptor CCR5 and the gp41 N-terminal Heptad Repeat (NHR) region in this event. With this in mind, we herein report a dual-targeted drug compound featuring bifunctional entry inhibitors, consisting of a piperidine-4-carboxamide-based CCR5 antagonist, TAK-220, and a gp41 NHR-targeting fusion-inhibitory peptide, C34. The resultant chimeras were constructed by linking both pharmacophores with a polyethylene glycol spacer. One chimera, CP12TAK, exhibited exceptionally potent antiviral activity, about 40- and 306-fold over that of its parent inhibitors, C34 and TAK-220, respectively. In addition to R5-tropic viruses, CP12TAK also strongly inhibited infection of X4-tropic HIV-1 strains. These data are promising for the further development of CP12TAK as a new anti-HIV-1 drug. Results show that this strategy could be extended to the design of therapies against infection of other enveloped viruses.
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site specific isopeptide bridge tethering of chimeric gp41 n terminal Heptad Repeat helical trimers for the treatment of hiv 1 infection
Scientific Reports, 2016Co-Authors: Chao Wang, Shibo Jiang, Wenqing Lai, Xifeng Jiang, Tianhong Zhang, Zhenqing Zhang, Huixin Wang, Keliang LiuAbstract:Peptides derived from the N-terminal Heptad Repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection.
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an effective strategy for recapitulating n terminal Heptad Repeat trimers in enveloped virus surface glycoproteins for therapeutic applications
Chemical Science, 2016Co-Authors: Wenqing Lai, Shibo Jiang, Chao Wang, Qian Wang, Xifeng Jiang, Tianhong Zhang, Xi Zheng, Zhenqing Zhang, Fangting DongAbstract:Sequestering peptides derived from the N-terminal Heptad Repeat (NHR) of class I viral fusion proteins into a non-aggregating trimeric coiled-coil conformation remains a major challenge. Here, we implemented a synthetic strategy to stabilize NHR-helical trimers, with the human immunodeficiency virus type 1 (HIV-1) gp41 fusion protein as the initial focus. A set of trimeric scaffolds was realized in a synthetic gp41 NHR-derived peptide sequence by relying on the tractability of coiled-coil structures and an additional isopeptide bridge-tethering strategy. Among them, (N36M)3 folded as a highly stable helical trimer and exhibited promising inhibitory activity against HIV-1 infection, exceptional resistance to proteolysis, and effective native ligand-binding capability. We anticipate that the trimeric coiled-coil recapitulation methodology described herein may have broader applicability to yield NHR trimers of other class I enveloped viruses and to prepare helical tertiary structure mimetics of certain natural protein-protein interactions for biomedical applications.
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hydrophobic mutations in buried polar residues enhance hiv 1 gp41 n terminal Heptad Repeat c terminal Heptad Repeat interactions and c peptides anti hiv activity
AIDS, 2014Co-Authors: Baohua Zheng, Shibo Jiang, Keliang Liu, Kun Wang, Maosheng Cheng, Lifeng CaiAbstract:OBJECTIVE To investigate the effect of mutations in a highly conserved buried polar area on the function of HIV-1 gp41. DESIGN During HIV-1 entry, a six helical bundle (6-HB) formation between the C-terminal and N-terminal Heptad Repeat (CHR and NHR) of gp41 provides energy for virus cell membrane fusion. In 6-HB, residues at a and d (a-d) positions of CHR directly interact with NHR and are buried. They are considered critical residues for 6-HB stability and for anti-HIV-1 activity of CHR-derived peptides (C-peptides). Most of a-d residues in CHR are hydrophobic, as buried hydrophobic residues facilitate protein stability. However, HIV-1 gp41 CHR contains a highly conserved polar area with four successive buried a-d polar residues: S649/Q652/N656/E659. We mutated these buried polar residues to hydrophobic residues, either Leu or Ile, and studied its effect on the gp41 NHR-CHR interactions and anti-HIV activities of the C-peptides. METHODS We measured the C-peptide mutants' ability to form 6-HB with NHR, thermal stability of the 6-HBs and C-peptides' inhibitory activity against both T20-sensitive and resistant HIV-1 strains. RESULTS All the mutated C-peptides retained their ability to form stable 6-HB with NHR and strongly inhibited HIV-1 replication. Strikingly, S649L and E659I mutations endow C-peptide with a significantly enhanced activity against T20-resistant HIV-1 strains. CONCLUSION The highly conserved buried a-d polar residues in HIV-1 gp41 CHR can be mutated as a means of developing new fusion inhibitors against drug-resistant HIV-1 strains. The concept can also be utilized to design fusion inhibitors against other viruses with similar mechanisms.
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the conserved residue arg46 in the n terminal Heptad Repeat domain of hiv 1 gp41 is critical for viral fusion and entry
PLOS ONE, 2012Co-Authors: Xiaoyi Wang, Asim K Debnath, Shibo Jiang, Weiliang Xiong, Meili Wei, Yanxia Chen, Chungen PanAbstract:During the process of HIV-1 fusion with the target cell, the N-terminal Heptad Repeat (NHR) of gp41 interacts with the C-terminal Heptad Repeat (CHR) to form fusogenic six-helix bundle (6-HB) core. We previously identified a crucial residue for 6-HB formation and virus entry - Lys63 (K63) in the C-terminal region of NHR (aa 54–70), which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121) in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46), in the N-terminal region of NHR (aa 35–53), which forms a hydrogen bond with a polar residue, Asn43 (N43), in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB’s stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.
Trudy G Morrison - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of the membrane proximal Heptad Repeat of the newcastle disease virus fusion protein
Virology, 2001Co-Authors: Lori W Mcginnes, Theresa Sergel, Hong Chen, Ludwig Hamo, Steve Schwertz, Trudy G MorrisonAbstract:Paramyxovirus fusion proteins have two Heptad Repeat domains, HR1 and HR2, that have been implicated in the fusion activity of the protein. Peptides from these two domains form a six-stranded, coiled-coil with the HR1 sequences forming a central trimer and three molecules of the HR2 helix located within the grooves in the central trimer (Baker et al., 1999, Mol. Cell 3, 309; Zhao et al. 2000, Proc. Natl. Acad. Sci. USA 97, 14172). Nonconservative mutations were made in the HR2 domain of the Newcastle disease virus fusion protein in residues that are likely to form contacts with the HR1 core trimer. These residues form the hydrophobic face of the helix and adjacent residues ("a" and "g" positions in the HR2 helical wheel structure). Mutant proteins were characterized for effects on synthesis, steady-state levels, proteolytic cleavage, and surface expression as well as fusion activity as measured by syncytia formation, content mixing, and lipid mixing. While all mutant proteins were transport competent and proteolytically cleaved, these mutations did variously affect fusion activity of the protein. Nonconservative mutations in the "g" position had no effect on fusion. In contrast, single changes in the middle "a" position of HR2 inhibited lipid mixing, content mixing, and syncytia formation. A single mutation in the more carboxyl-terminal "a" position had minimal effects on lipid mixing but did inhibit content mixing and syncytia formation. These results are consistent with the idea that the HR2 domain is involved in posttranslational interactions with HR1 that mediate the close approach of membranes. These results also suggest that the HR2 domain, particularly the carboxyl-terminal region, plays an additional role in fusion, a role related to content mixing and syncytia formation.
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carbohydrate modifications of the ndv fusion protein Heptad Repeat domains influence maturation and fusion activity
Virology, 2001Co-Authors: Lori W Mcginnes, Theresa Sergel, Julie N Reitter, Trudy G MorrisonAbstract:Abstract The amino acid sequence of the fusion protein (F) of Newcastle disease virus (NDV) has six potential N-linked glycosylation addition sites, five in the ectodomain (at amino acids 85, 191, 366, 447, and 471) and one in the cytoplasmic domain at amino acid 542. Two of these sites, at positions 191 and 471, are within Heptad Repeat (HR) domains implicated in fusion activity of the protein. To determine glycosylation site usage as well as the function of added carbohydrate, each site was mutated by substituting alanine for the serine or threonine in the addition signal. The sizes of the resulting mutant proteins, expressed in Cos cells, showed that sites at amino acids 85, 191, 366, and 471 are used. This conclusion was verified by comparing sizes of mutant proteins missing all four used sites with that of unglycosylated F protein. The role of each added oligosaccharide in the structure and function of the F protein was determined by characterizing stability, proteolytic cleavage, surface expression, and fusion activity of the mutant proteins. Elimination of the site in F 2 at amino acid 85 had the most detrimental effect, decreasing cleavage, stability, and surface expression as well as fusion activity. The protein missing the site at 191, at the carboxyl terminus of the HR1 domain, also showed modestly reduced surface expression and negligible fusion activity. Proteins missing sites at 366 and 471 (within HR2) were expressed at nearly wild-type levels but had decreased fusion activity. These results suggest that all carbohydrate side chains, individually, influence the folding or activity of the NDV F protein. Importantly, carbohydrate modifications of the HR domains impact fusion activity of the protein.
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interaction of peptides with sequences from the newcastle disease virus fusion protein Heptad Repeat regions
Journal of Virology, 1999Co-Authors: John K Young, Matthew C Abramowitz, Trudy G MorrisonAbstract:Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several Heptad Repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix.
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mutations in the fusion peptide and Heptad Repeat regions of the newcastle disease virus fusion protein block fusion
Journal of Virology, 1994Co-Authors: T Sergelgermano, Cathy Mcquain, Trudy G MorrisonAbstract: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.
Yechiel Shai - One of the best experts on this subject based on the ideXlab platform.
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the hiv gp41 pocket binding domain enables c terminal Heptad Repeat transition from mediating membrane fusion to immune modulation
Biochemical Journal, 2016Co-Authors: Yoel A Klug, Gal Kapach, Etai Rotem, Benjamin Dubreuil, Yechiel ShaiAbstract:For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. The human immunodeficiency virus (HIV), utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled-coil is formed from three N and three C terminal Heptad Repeats, termed the six helix bundle, which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor signaling cascade. One of these regions encompasses the conserved pocket binding domain that is situated in the C terminal Heptad Repeat and stabilizes six-helix bundle formation. This could indicate that the pocket binding domain plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the pocket binding domain mediates T-cell suppression by stabilizing a T-cell receptor binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast to the potent fusion inhibitor C34. In addition, by focusing on six helix bundle conformation post its assembly, we shed light on a mechanism by which gp41’s function alternates from membrane fusion facilitation to suppression of T-cell receptor activation.
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membrane anchored hiv 1 n Heptad Repeat peptides are highly potent cell fusion inhibitors via an altered mode of action
PLOS Pathogens, 2009Co-Authors: Yael Wexlercohen, Yechiel ShaiAbstract:Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-Heptad Repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-Heptad Repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC50 increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors.
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structurally altered peptides reveal an important role for n terminal Heptad Repeat binding and stability in the inhibitory action of hiv 1 peptide dp178
Journal of Biological Chemistry, 2006Co-Authors: Yael Wexlercohen, Benitra T Johnson, Anu Puri, Robert Blumenthal, Yechiel ShaiAbstract:Human immunodeficiency virus 1 gp41 folds into a six-helix bundle whereby three C-terminal Heptad Repeat regions pack in an anti-parallel manner against the coiled-coil formed by three N-terminal Heptad Repeats (NHR). Peptides that inhibit bundle formation contributed significantly to the understanding of the entry mechanism of the virus. DP178, which partially overlaps C-terminal Heptad Repeats, prevents bundle formation through an undefined mechanism; additionally it has been suggested to bind other ENV regions and arrest fusion in an unknown manner. We used two structurally altered DP178 peptides; in each, two sequential amino acids were substituted into their d configuration, d-SQ in the hydrophilic N-terminal region and d-LW in the hydrophobic C-terminal. Importantly, we generated an elongated NHR peptide, N54, obtaining the full N-helix docking site for DP178. Interestingly, d-LW retained wild type fusion inhibitory activity, whereas d-SQ exhibited significantly reduced activity. In correlation with the inhibitory data, CD spectroscopy and fluorescence studies revealed that all the DP178 peptides interact with N54, albeit with different stabilities of the bundles. We conclude that strong binding of DP178 N-terminal region to the endogenous NHR, without significant contribution of the C-terminal sequence of DP178 to core formation, is vital for DP178 inhibition. The finding that d-amino acid incorporation in the C terminus did not affect activity or membrane binding as revealed by surface plasmon resonance correlates with an additional membrane binding site, or membrane anchoring role, for the C terminus, which works synergistically with the N terminus to inhibit fusion.
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the hiv 1 gp41 n terminal Heptad Repeat plays an essential role in membrane fusion
Biochemistry, 2002Co-Authors: Kelly Sackett, Yechiel ShaiAbstract: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.
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direct evidence that the n terminal Heptad Repeat of sendai virus fusion protein participates in membrane fusion
Journal of Molecular Biology, 1999Co-Authors: Jimut Kanti Ghosh, Yechiel ShaiAbstract:Recent studies have demonstrated the importance of Heptad Repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if Heptad Repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal Heptad Repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and Heptad Repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the Heptad Repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the Heptad Repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal Heptad Repeat in assisting the fusion peptide in mediating membrane fusion.
Jimut Kanti Ghosh - One of the best experts on this subject based on the ideXlab platform.
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selective phenylalanine to proline substitution for improved antimicrobial and anticancer activities of peptides designed on phenylalanine Heptad Repeat
Acta Biomaterialia, 2017Co-Authors: Amit Kumar Tripathi, Praveen K. Shukla, Tripti Kumari, Anshika Tandon, Mohd Sayeed, Tayyaba Afshan, Manoj Kathuria, Kalyan Mitra, Jimut Kanti GhoshAbstract:Abstract Introducing cell-selectivity in antimicrobial peptides (AMPs) without compromising the antimicrobial and anti-endotoxin properties is a crucial step towards the development of new antimicrobial agents. A peptide designed on phenylalanine Heptad Repeat possesses significant cytotoxicity along with desired antimicrobial and anti-endotoxin properties. Amino acid substitutions at ‘a’ and/or ‘d’ positions of Heptad Repeats of AMPs could alter their helical structure in mammalian membrane-mimetic environments and cytotoxicity towards mammalian cells. Since proline is a helix breaker, effects of selective proline substitution(s) at ‘a’ and/or ‘d’ positions of a 15-residue peptide designed on phenylalanine Heptad Repeat (FR-15) were investigated. Proline-substituted FR-15 variants were highly selective toward bacteria and fungi over hRBCs and murine 3T3 cells and also retained their antibacterial activities at high salt, serum and elevated temperatures. These non-cytotoxic variants also inhibited LPS-induced production of pro-inflammatory cytokines/chemokines in human monocytes, THP-1, RAW 264.7 and in BALB/c mice. The two non-cytotoxic variants (FR8P and FR11P) showed potent anti-cancer activity against highly metastatic human breast cancer cell line MDA-MB-231 with IC 50 values less than 10 μM. At sub-IC 50 concentrations, FR8P and FR11P also showed anti-migratory and anti-invasive effects against MDA-MB-231 cells. FR8P and FR11P induced cellular apoptosis by triggering intrinsic apoptotic pathway through depolarization of mitochondrial membrane potential and activation of caspases. Overall the results demonstrated the utilization of selective phenylalanine to proline substitution in a Heptad Repeat of phenylalanine residues for the design of cell-selective, broad-spectrum AMPs with significant anti-cancer properties. Statement of Significance We have demonstrated a methodology to design cell-selective potent antimicrobial and anti-endotoxin peptides by utilizing phenylalanine zipper as a template and replacement of phenylalanine residue(s) from “a” and/or “d” position(s) with proline residue(s) produced non-cytotoxic AMPs with improved antibacterial properties against the drug-resistant strains of bacteria. The work showed that the ‘a’ and ‘d’ positions of the phenylalanine Heptad Repeat could be replaced by an appropriate amino acid to control cytotoxicity of the peptide without compromising its potency in antimicrobial and anti-endotoxin properties. The direct bacterial membrane targeting mechanism of proline substituted analogs of parent peptide makes difficult for bacteria to grow resistance against them. The peptides designed could be lead molecules in the area of sepsis as they possess significant anti-LPS activities for in vitro and in vivo . Interestingly since cancer cells and bacterial cell membranes possess the structural resemblances, the cancer cells are also targets for these peptides making them lead molecules in this field. However, unlike in bacteria where the peptides showed membrane permeabilization property to lyse them, the peptides induced apoptosis in MDA-MB-231 breast cancer cells to inhibit their proliferation and growth. The results are significant because it reveals that “a” and “d” positions of a phenylalanine zipper can be utilized as switches to design cell-selective, antimicrobial, anti - endotoxin and anticancer peptides.
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single amino acid substitutions at specific positions of the Heptad Repeat sequence of piscidin 1 yielded novel analogs that show low cytotoxicity and in vitro and in vivo antiendotoxin activity
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Amit Kumar, Jimut Kanti Ghosh, Amit Kumar Tripathi, Manoj Kathuria, Kalyan Mitra, Sonal Shree, Jitendra Kumar Tripathi, R K Purshottam, Ravishankar RamachandranAbstract:Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long Heptad Repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the Heptad Repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 Heptad Repeat.
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Characterization of Antimicrobial, Cytotoxic, and Antiendotoxin Properties of Short Peptides with Different Hydrophobic Amino Acids at “a” and “d” Positions of a Heptad Repeat Sequence
2016Co-Authors: Sarfuddin Azmi, Saurabh Srivastava, Nripendra N. Mishra, Jitendra K. Tripathi, Praveen K. Shukla, Jimut Kanti GhoshAbstract:To understand the influence of different hydrophobic amino acids at “a” and “d” positions of a Heptad Repeat sequence on antimicrobial, cytotoxic, and antiendotoxin properties, four 15-residue peptides with leucine (LRP), phenylalanine (FRP), valine (VRP), and alanine (ARP) residues at these positions were designed, synthesized, and characterized. Although valine is similarly hydrophobic to leucine and phenylalanine, VRP showed significantly lesser cytotoxicity than LRP and FRP; further, the replacement of leucines with valines at “a” and “d” positions of melittin-Heptads drastically reduced its cytotoxicity. However, all four peptides exhibited significant antimicrobial activities that correlate well with their interactions with mammalian and bacterial cell membranes and the corresponding lipid vesicles. LRP most efficiently neutralized the LPS-induced pro-inflammatory mediators like NO, TNF-α, and IL-6 in macrophages followed by FRP, VRP, and ARP. The results could be useful for designing short antimicrobial and antiendotoxin peptides with understanding the basis of their activity
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characterization of antimicrobial cytotoxic and antiendotoxin properties of short peptides with different hydrophobic amino acids at a and d positions of a Heptad Repeat sequence
Journal of Medicinal Chemistry, 2013Co-Authors: Sarfuddin Azmi, Saurabh Srivastava, Nripendra N. Mishra, Praveen K. Shukla, Jitendra Kumar Tripathi, Jimut Kanti GhoshAbstract:To understand the influence of different hydrophobic amino acids at “a” and “d” positions of a Heptad Repeat sequence on antimicrobial, cytotoxic, and antiendotoxin properties, four 15-residue peptides with leucine (LRP), phenylalanine (FRP), valine (VRP), and alanine (ARP) residues at these positions were designed, synthesized, and characterized. Although valine is similarly hydrophobic to leucine and phenylalanine, VRP showed significantly lesser cytotoxicity than LRP and FRP; further, the replacement of leucines with valines at “a” and “d” positions of melittin-Heptads drastically reduced its cytotoxicity. However, all four peptides exhibited significant antimicrobial activities that correlate well with their interactions with mammalian and bacterial cell membranes and the corresponding lipid vesicles. LRP most efficiently neutralized the LPS-induced pro-inflammatory mediators like NO, TNF-α, and IL-6 in macrophages followed by FRP, VRP, and ARP. The results could be useful for designing short antimicr...
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direct evidence that the n terminal Heptad Repeat of sendai virus fusion protein participates in membrane fusion
Journal of Molecular Biology, 1999Co-Authors: Jimut Kanti Ghosh, Yechiel ShaiAbstract:Recent studies have demonstrated the importance of Heptad Repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if Heptad Repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal Heptad Repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and Heptad Repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the Heptad Repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the Heptad Repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal Heptad Repeat in assisting the fusion peptide in mediating membrane fusion.
Nobutaka Fujii - One of the best experts on this subject based on the ideXlab platform.
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a novel peptide derived from the fusion protein Heptad Repeat inhibits replication of subacute sclerosing panencephalitis virus in vitro and in vivo
PLOS ONE, 2016Co-Authors: Masahiro Watanabe, Koichi Hashimoto, Yusaku Abe, Eiichi Kodama, Ryota Nabika, Shinya Oishi, Shinichiro Ohara, Masatoki Sato, Yukihiko Kawasaki, Nobutaka FujiiAbstract:Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical Heptad Repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the Heptad Repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The Heptad Repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein Heptad Repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.
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characterization of hiv 1 resistance to a fusion inhibitor n36 derived from the gp41 amino terminal Heptad Repeat
Antiviral Research, 2010Co-Authors: Kazuki Izumi, Shinya Oishi, Shota Nakamura, Hiroaki Nakano, Kazuya Shimura, Yasuko Sakagami, Susumu Uchiyama, Tadayasu Ohkubo, Yuji Kobayashi, Nobutaka FujiiAbstract:A transmembrane glycoprotein of HIV-1, gp41, plays a central role in membrane fusion of HIV-1 and host cells. Peptides derived from the amino- and carboxyl-terminal Heptad Repeat (N-HR and C-HR, respectively) of gp41 inhibit this fusion. The mechanism of resistance to enfuvirtide, a C-HR-derived peptide, is well defined; however the mechanism of resistance to N-HR-derived peptides remains unclear. We characterized an HIV-1 isolate resistant to the N-HR-derived peptide, N36. This HIV-1 acquired a total of four amino acid substitutions, D36G, N126K and E137Q in gp41, and P183Q in gp120. Among these substitutions, N126K and/or E137Q conferred resistance to not only N36, but also C34, which is the corresponding C-HR-derived peptide fusion inhibitor. We performed crystallographic and biochemical analysis of the 6-helix bundle formed by synthetic gp41-derived peptides containing the N126K/E137Q substitutions. The structure of the 6-helix bundle with N126K/E137Q was identical to that in wild-type HIV-1 except for the presence of a new hydrogen bond. Denaturing experiments revealed that the stability of the 6-helix bundle of N126K/E137Q is greater than in the wild-type. These results suggest that the stabilizing effect of N126K/E137Q provides resistance to N36 and C34.
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Heptad Repeat derived peptides block protease mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway
Journal of Virology, 2008Co-Authors: Makoto Ujike, Eiichi Kodama, Nobutaka Fujii, Hiroki Nishikawa, Akira Otaka, Naoki Yamamoto, Norio Yamamoto, Masao Matsuoka, Fumihiro TaguchiAbstract:The peptides derived from the Heptad Repeat (HRP) of severe acute respiratory syndrome coronavirus (SCoV) spike protein (sHRPs) are known to inhibit SCoV infection, yet their efficacies are fairly low. Recently our research showed that some proteases facilitated SCoV's direct entry from the cell surface, resulting in a more efficient infection than the previously known infection via endosomal entry. To compare the inhibitory effect of the sHRP in each pathway, we selected two sHRPs, which showed a strong inhibitory effect on the interaction of two Heptad Repeats in a rapid and virus-free in vitro assay system. We found that they efficiently inhibited SCoV infection of the protease-mediated cell surface pathway but had little effect on the endosomal pathway. This finding suggests that sHRPs may effectively prevent infection in the lungs, where SCoV infection could be enhanced by proteases produced in this organ. This is the first observation that HRP exhibits different effects on virus that takes the endosomal pathway and virus that enters directly from the cell surface.