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The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro.
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Acinetobacter baumannii , Antibacterianos , Péptidos Catiónicos Antimicrobianos , Proteínas Bacterianas , Proteínas Portadoras , Péptidos Catiónicos Antimicrobianos/química , Unión Proteica , Proteínas Bacterianas/química , Proteínas Portadoras/química , Antibacterianos/química , Conformación Proteica , Secuencia de Aminoácidos , Secuencia ConservadaRESUMEN
The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.
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Proteínas de Escherichia coli , Peptidomiméticos , Enterobacteriaceae , Lipopolisacáridos , Peptidomiméticos/farmacología , Escherichia coli , Antibacterianos/farmacología , Proteínas PortadorasRESUMEN
PURPOSE: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. METHODS: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. RESULTS: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. CONCLUSIONS: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.
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Descubrimiento de Drogas , Neoplasias Ováricas , Animales , Femenino , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Proteoma/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Descubrimiento de Drogas/métodosRESUMEN
Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ρ-TIA to the human G-protein coupled α1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics.
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Péptidos , Receptores Adrenérgicos alfa 1 , HumanosRESUMEN
Respiratory syncytial virus (RSV) can cause severe respiratory disease in humans, particularly in infants and the elderly. However, attempts to develop a safe and effective vaccine have so far been unsuccessful. Atomic-level structures of epitopes targeted by RSV-neutralizing antibodies are now known, including that bound by Motavizumab and its clinically used progenitor Palivizumab. We developed a chemically defined approach to RSV vaccine design, that allows control of both immunogenicity and safety features of the vaccine. Structure-guided antigen design and a synthetic nanoparticle delivery platform led to a vaccine candidate that elicits high titers of palivizumab-like, epitope-specific neutralizing antibodies. The vaccine protects preclinical animal models from RSV infection and lung pathology typical of vaccine-derived disease enhancement. The results suggest that the development of a safe and effective synthetic epitope-specific RSV vaccine may be feasible by combining this conformationally stabilized peptide and synthetic nanoparticle delivery system.
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An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.
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Antibacterianos/química , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Bacterias Gramnegativas/efectos de los fármacos , Peptidomiméticos/química , Peptidomiméticos/farmacología , Animales , Antibacterianos/efectos adversos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Productos Biológicos/química , Descubrimiento de Drogas , Farmacorresistencia Microbiana/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fluorescencia , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/patogenicidad , Humanos , Lipopolisacáridos/química , Compuestos Macrocíclicos/efectos adversos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutación , Peptidomiméticos/efectos adversos , Etiquetas de FotoafinidadRESUMEN
With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5'-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis in Escherichia coli.
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Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico Activo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformación ProteicaRESUMEN
The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral ß-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Because of its essential function in many Gram-negative bacteria, the LPS transport pathway is an interesting target for the development of new antibiotics. A family of macrocyclic peptidomimetics was discovered recently that target LptD and inhibit LPS transport specifically in Pseudomonas spp. The related molecule Murepavadin is in clinical development for the treatment of life-threatening infections caused by P. aeruginosa. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we characterized the binding site by cross-linking to a photolabeling probe. We used a hypothesis-free mass spectrometry-based proteomic approach to provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a ß-jellyroll domain and an N-terminal insert domain characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.
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Antibacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Peptidomiméticos/metabolismo , Pseudomonas aeruginosa/química , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Bacterias Gramnegativas/efectos de los fármacos , Lipopolisacáridos/metabolismo , Periplasma , Dominios Proteicos , Transporte de ProteínasRESUMEN
Protein epitope mimetics provide powerful tools to study biomolecular recognition in many areas of chemical biology. They may also provide access to new biologically active molecules and potentially to new classes of drug and vaccine candidates. Here we highlight approaches for the design of folded, structurally defined epitope mimetics, by incorporating backbone and side chains of hot residues onto a stable constrained scaffold. Using robust synthetic methods, the structural, biological, and physical properties of epitope mimetics can be optimized, by variation of both side chain and backbone chemistry. To illustrate the potential of protein epitope mimetics in medicinal chemistry and biotechnology, we present studies in two areas of infectology; the discovery of new antibiotics targeting essential outer membrane (OM) proteins in Gram-negative bacteria and the design of supramolecular synthetic vaccines. The discovery of new antibiotics with novel mechanisms of action, in particular to combat infections caused by Gram-negative pathogens, represents a major challenge in medicinal chemistry. We were inspired by naturally occurring cationic antimicrobial peptides to design structurally related peptidomimetics and to optimize their antimicrobial properties through library synthesis and screening. Through these efforts, we could show that antimicrobial ß-hairpin mimetics may have structures and properties that facilitate interactions with essential bacterial ß-barrel OM proteins. One recently discovered family of antimicrobial peptidomimetics targets the ß-barrel protein LptD in Pseudomonas spp. This protein plays a key role in lipopolysaccaride (LPS) transport to the cell surface during OM biogenesis. Through a highly selective interaction with LptD, the peptidomimetic blocks LPS transport, resulting in nanomolar antimicrobial activity against the important human pathogen P. aeruginosa. Epitope mimetics may also have great potential in the field of vaccinology, where structural information on complexes between neutralizing antibodies and their cognate epitopes can be taken as a starting point for B cell epitope mimetic design. In order to generate potent immune responses, an effective method of delivering epitope mimetics to relevant cells and tissues in the immune system is also required. For this, engineered synthetic nanoparticles (synthetic virus-like particles, SVLPs) prepared using supramolecular chemistry can be designed with optimal surface properties for efficient dendritic cell-mediated delivery of folded B-cell and linear T-cell epitopes, along with ligands for pattern recognition receptors, into lymphoid tissues. In this way, multivalent display of the epitope mimetics occurs over the surface of the nanoparticle, suitable for cross-linking B cell receptors. In this highly immunogenic format, strong epitope-specific humoral immune responses can be elicited that target infections caused by pathogenic microorganisms. Other potential applications of epitope mimetics in next-generation therapeutics are also discussed.
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Antibacterianos/química , Epítopos/química , Proteínas/química , Vacunas Sintéticas/química , Humanos , Sustancias Macromoleculares/química , Dominios ProteicosRESUMEN
Fully synthetic medium-sized macrocyclic peptides mimicking the key ß-hairpin and α-helical protein epitopes relevant in many protein-protein interactions have emerged as a novel class of drugs with the potential to fill an important gap between small molecules and proteins. Conformationally stabilized macrocyclic scaffolds represent ideal templates for medicinal chemists to incorporate bioactive peptide and protein pharmacophores in order to generate novel drugs to treat diseases with high unmet medical need. This review describes recent approaches to design and generate large libraries of such macrocycles, for hit identification, and for their efficient optimization. Finally, this review describes some of the most advanced protein epitope mimetic (PEM) macrocycles in clinical development.
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Materiales Biomiméticos/química , Descubrimiento de Drogas/métodos , Epítopos , Compuestos Macrocíclicos/química , Proteínas/química , Materiales Biomiméticos/farmacología , Humanos , Compuestos Macrocíclicos/farmacologíaRESUMEN
Antimicrobial resistance among Gram-negative bacteria is a growing problem, fueled by the paucity of new antibiotics that target these microorganisms. One novel family of macrocyclic ß-hairpin-shaped peptidomimetics was recently shown to act specifically against Pseudomonas spp. by a novel mechanism of action, targeting the outer membrane protein LptD, which mediates lipopolysaccharide transport to the cell surface during outer membrane biogenesis. Here we explore the mode of binding of one of these ß-hairpin peptidomimetics to LptD in Pseudomonas aeruginosa, by examining the effects on antimicrobial activity following N-methylation of individual peptide bonds. An N-methyl scan of the cyclic peptide revealed that residues on both sides of the ß-hairpin structure at a non-hydrogen bonding position likely mediate hydrogen-bonding interactions with the target LptD. Structural analyses by NMR spectroscopy further reinforce the conclusion that the folded ß-hairpin structure of the peptidomimetic is critical for binding to the target LptD. Finally, new NMe analogues with potent activity have been identified, which opens new avenues for optimization in this family of antimicrobial peptides.
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Aminoácidos/farmacología , Antibacterianos/síntesis química , Antibacterianos/farmacología , Compuestos Macrocíclicos/farmacología , Péptidos/farmacología , Peptidomiméticos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Aminoácidos/química , Aminoácidos/metabolismo , Antibacterianos/química , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Metilación , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Péptidos/química , Peptidomiméticos/síntesis química , Peptidomiméticos/químicaRESUMEN
LptE is an outer membrane (OM) lipoprotein found in Gram-negative bacteria, where it forms a complex with the ß-barrel lipopolysaccharide (LPS) transporter LptD. The LptD/E complex plays a key role in OM biogenesis, by translocating newly synthesized LPS molecules from the periplasm into the external leaflet of the asymmetric OM during cell growth. The LptD/E complex in Pseudomonas aeruginosa (Pa) is a target for macrocyclic ß-hairpin-shaped peptidomimetic antibiotics, which inhibit the transport of LPS to the cell surface. So far, the three-dimensional structure of the Pa LptD/E complex and the mode of interaction with these antibiotics are unknown. Here, we report the solution structure of a Pa LptE derivative lacking the N-terminal lipid membrane anchor, determined by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. The structure reveals a central five-stranded ß-sheet against which pack a long C-terminal and a short N-terminal α-helix, as found in homologues of LptE from other Gram-negative bacteria. One unique feature is an extended C-terminal helix in Pa LptE, which in a model of the Pa LptD/E complex appears to be long enough to contact the periplasmic domain of LptD. Chemical shift mapping experiments suggest only weak interactions occur between LptE and the oligosaccharide chains of LPS. The NMR structure of Pa LptE will be valuable for more detailed structural studies of the LptD/E complex from P. aeruginosa.
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Proteínas de la Membrana Bacteriana Externa/química , Membrana Celular/química , Lipopolisacáridos/metabolismo , Pseudomonas aeruginosa/metabolismo , Transporte Biológico , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Periplasma/metabolismo , Unión Proteica , Conformación Proteica , SolucionesRESUMEN
Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel ß-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected ß-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many ß-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of ß-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target ß-barrel proteins and the integrity of the Gram-negative OM.
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Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Péptidos/farmacología , Peptidomiméticos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
We report structural studies in aqueous solution on backbone cyclic peptides that possess potent antimicrobial activity specifically against Pseudomonas sp. The peptides target the ß-barrel outer membrane protein LptD, which plays an essential role in lipopolysaccharide transport to the outer membrane. The peptide L27-11 contains a 12-residue loop (T(1)W(2)L(3)K(4)K(5)R(6)R(7)W(8)K(9)K(10)A(11)K(12)) linked to a DPro-LPro template. Two related peptides were also studied, one with various Lys to ornithine or diaminobutyric acid substitutions as well as a DLys(6) (called LB-01), and another containing the same loop sequence, but linked to an LPro-DPro template (called LB-02). NMR studies and MD simulations show that L27-11 and LB-01 adopt ß-hairpin structures in solution. In contrast, LB-02 is more flexible and importantly, adopts a wide variety of different backbone conformations, but not ß-hairpin conformations. L27-11 and LB-01 show antimicrobial activity in the nanomolar range against Pseudomonas aeruginosa, whereas LB-02 is essentially inactive. Thus the ß-hairpin structure of the peptide is important for antimicrobial activity. An alanine scan of L27-11 revealed that tryptophan side chains (W(2)/W(8)) displayed on opposite faces of the ß-hairpin represent key groups contributing to antimicrobial activity.
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Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Péptidos/química , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Dicroismo Circular , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Péptidos/farmacología , Peptidomiméticos , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.
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Repetición de Anquirina , Proteína gp120 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/metabolismo , Proteínas/metabolismo , Inhibidores de Fusión de VIH/aislamiento & purificación , Humanos , Unión Proteica , Conformación Proteica , Proteínas/aislamiento & purificaciónRESUMEN
Spiropins for SPPS: The rigid structure of an anomerically stabilised spiroketal motif enables the appendage of substituents in a fixed conformation. To assess the ability of a spiroketal motif to induce a turn structure and participate in solid-phase peptide synthesis (SPPS), an Fmoc-spiroketal amino acid was synthesised and incorporated into a spiroketal-containing cyclic peptide.
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Aminoácidos/síntesis química , Furanos/síntesis química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Compuestos de Espiro/síntesis química , Secuencia de Aminoácidos , Aminoácidos/química , Furanos/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Compuestos de Espiro/químicaRESUMEN
Conformationally constrained peptidomimetics could be of great value in the design of vaccines targeting protective epitopes on viral and bacterial pathogens. But the poor immunogenicity of small synthetic molecules represents a serious obstacle for their use in vaccine development. Here, we show how a constrained epitope mimetic can be rendered highly immunogenic through multivalent display on the surface of synthetic virus-like nanoparticles. The target epitope is the V3 loop from the gp120 glycoprotein of HIV-1 bound to the neutralizing antibody F425-B4e8. The antibody-bound V3 loop adopts a ß-hairpin conformation, which is effectively stabilized by transplantation onto a D-Pro-L-Pro template. The resulting mimetic after coupling to synthetic virus-like particles elicited antibodies in rabbits that recognized recombinant gp120. The elicited antibodies also blocked infection by the neutralization sensitive tier-1 strain MN of HIV-1, as well as engineered viruses with the V1V2 loop deleted; this result is consistent with screening of V3 by the V1V2 loop in intact trimeric viral gp120 spikes. The results provide new insights into HIV-1 vaccine design based on the V3 loop, and illustrate how knowledge from structural biology can be exploited for the design of constrained epitope mimetics, which can be delivered to the immune system by using a highly immunogenic synthetic nanoparticle delivery system.
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Imitación Molecular , Péptidos/química , Vacunas Sintéticas , Virión , Vacunas contra el SIDA , Secuencia de Aminoácidos , Ensayo de Inmunoadsorción Enzimática , Proteína gp120 de Envoltorio del VIH/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The insect sex peptide (SP) elicits a variety of biological responses upon transfer to the mated female. SP contains 36 amino acids, including a tryptophan-rich N-terminal region, a central region containing five hydroxyproline (Hyp) residues, and a C-terminal region enclosed by a disulfide bridge. The solution structure of SP, studied here using NMR spectroscopy, includes a motif WPWN that adopts a type I ß-turn in the N-terminal Trp-rich region. This turn region is connected to the central Hyp-rich region, which adopts extended and/or PPII-like conformations. The C-terminal disulfide-bonded loop populates helical turns or nascent helical structure. Overall, the results reveal a rather flexible peptide that lacks a compact folded structure in solution.