RESUMO
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.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Animais , Antibacterianos/efeitos adversos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Produtos Biológicos/química , Descoberta de Drogas , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fluorescência , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/patogenicidade , Humanos , Lipopolissacarídeos/química , Compostos Macrocíclicos/efeitos adversos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutação , Peptidomiméticos/efeitos adversos , Marcadores de FotoafinidadeRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.
Assuntos
Infecções por HIV/genética , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Antivirais/química , Antivirais/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Ligantes , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/genética , Fator B de Elongação Transcricional Positiva/química , Fator B de Elongação Transcricional Positiva/genética , Ligação Proteica , RNA Viral/genética , Transcrição Gênica/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Dendritic cells (DCs) play critical roles in developing immune defenses. One important aspect is interaction with pathogen-associated molecular patterns (PAMPs)/danger-associated molecular patterns, including di- and triacylated lipopeptides. Isolated or synthetic lipopeptides are potent vaccine adjuvants, interacting with cell surface TLR2 heterodimers. In contrast, deep embedment within bacteria cell walls would impair lipopeptide interaction with cell surface TLR2, requiring degradation for PAMP recognition. Accordingly, DC processing in the absence of surface TLR2 ligation was defined using synthetic virus-like particles (SVLPs) carrying hydrophobic TLR2 PAMPs within di- and triacylated lipopeptide cores (P2Cys-SVLPs and P3Cys-SVLPs) compared with SVLPs lacking immunomodulatory lipopeptides. DCs rapidly and efficiently internalized SVLPs, which was dominated by slow endocytic processing via macropinocytosis, although some caveolar endocytosis was implicated. This delivered SVLPs primarily into macropinosomes often interacting with EEA-1+ early endosomes. Although endoplasmic reticulum association was occasionally noted, association with recycling/sorting structures was not observed. Involvement of LysoTracker+ structures slowly increased with time, with SVLPs present in such structures ultimately dominating. Only SVLPs carrying di- and triacylated lipopeptide cores induced DC activation and maturation independently of surface TLR2 ligation. Intracellular recognition of SVLP TLR2 ligands was confirmed by observing SVLPs' association with internal TLR2, which had similar kinetics to SVLP association with LysoTracker. This related to inflammatory cytokine induction by SVLP+ DCs, with adaptive immune response activation ex vivo/in vivo. Importantly, particular DCs, not monocytes, recognized intracellular exposure of the TLR2 PAMPs carried by di- and triacylated SVLP cores, which indicates subset-distinct recognition of functional internal TLR2 ligands. Thus, vaccines carrying hydrophobic TLR2 ligands would interact with particular DCs for efficient induction of specific immunity in the absence of additional adjuvant.
Assuntos
Células Dendríticas/imunologia , Lipopeptídeos/química , Moléculas com Motivos Associados a Patógenos/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Imunidade Adaptativa , Adjuvantes Imunológicos , Animais , Diferenciação Celular , Citocinas/imunologia , Células Dendríticas/metabolismo , Endocitose , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/fisiologia , Endossomos/imunologia , Endossomos/metabolismo , Lipopeptídeos/imunologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Moléculas com Motivos Associados a Patógenos/química , Moléculas com Motivos Associados a Patógenos/metabolismo , Sus scrofa , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/químicaRESUMO
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.
Assuntos
Antibacterianos/química , Epitopos/química , Proteínas/química , Vacinas Sintéticas/química , Humanos , Substâncias Macromoleculares/química , Domínios ProteicosRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
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.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/química , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Transporte Biológico , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Periplasma/metabolismo , Ligação Proteica , Conformação Proteica , SoluçõesRESUMO
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.
Assuntos
Aminoácidos/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Compostos Macrocíclicos/farmacologia , Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Aminoácidos/química , Aminoácidos/metabolismo , Antibacterianos/química , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Metilação , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/química , Peptidomiméticos/síntese química , Peptidomiméticos/químicaRESUMO
The antimicrobial activity of polymyxins against Gram-negative bacteria has been known for several decades, but the mechanism of action leading to cell death has not been fully explored. A key step after binding of the antibiotic to lipopolysaccharide (LPS) exposed at the cell surface is 'self-promoted uptake' across the outer membrane (OM), in which the antibiotic traverses the asymmetric LPS-phospholipid bilayer before reaching the periplasm and finally targeting and disrupting the bacterial phospholipid inner membrane. The work described here was prompted by the hypothesis that polymyxins might interact with proteins in the OM, as part of their self-promoted uptake and permeabilizing effects. One way to test this is through photolabeling experiments. We describe the design and synthesis of a photoprobe based upon polymyxin B, containing photoleucine and an N-acyl group with a terminal alkyne suitable for coupling to a biotin tag using click chemistry. The resulting photoprobe retains potent antimicrobial activity, and in initial photolabeling experiments with Escherichia coli ATCC25922 is shown to photolabel several OM proteins. This photoprobe might be a valuable tool in more detailed studies on the mechanism of action of this family of antibiotics.
Assuntos
Antibacterianos/síntese química , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Sondas Moleculares/síntese química , Polimixina B/síntese química , Coloração e Rotulagem/métodos , Alcinos/química , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/química , Biotina/química , Membrana Celular/química , Química Click , Escherichia coli/química , Escherichia coli/fisiologia , Luz , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana , Técnicas de Sonda Molecular , Sondas Moleculares/farmacologia , Fosfolipídeos/química , Processos Fotoquímicos , Polimixina B/análogos & derivados , Polimixina B/farmacologia , Técnicas de Síntese em Fase Sólida/métodosRESUMO
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.
Assuntos
Repetição de Anquirina , Proteína gp120 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/metabolismo , Proteínas/metabolismo , Inibidores da Fusão de HIV/isolamento & purificação , Humanos , Ligação Proteica , Conformação Proteica , Proteínas/isolamento & purificaçãoRESUMO
Vancomycin is an important nosocomial antibiotic containing a glycosylated, cross-linked and doubly chlorinated heptapeptide backbone. During the biosynthesis of the vancomycin aglycone, two ß-hydroxytyrosine (Bht) residues are inserted at positions-2 and -6 into the heptapeptide backbone by a non-ribosomal peptide synthetase. A single flavin-dependent chlorinase (VhaA) is responsible for chlorinating both Bht residues at some ill-defined point in the assembly process. We show here using in vitro assays that VhaA is able to introduce a chlorine atom into each aromatic ring of both Bht residues at positions-2 and -6 of a peptide carrier protein-bound hexapeptide. The results suggest that VhaA can recognize and chlorinate two quite different sites within a linear hexapeptide intermediate during vancomycin biosynthesis.
Assuntos
Halogenação , Oligopeptídeos/metabolismo , Oxirredutases/metabolismo , Proteínas/metabolismo , Vancomicina/biossíntese , Cromatografia Líquida de Alta Pressão , Oligopeptídeos/química , Peptídeo Sintases/metabolismo , Vancomicina/químicaRESUMO
The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50) â¼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (-) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.
Assuntos
Fármacos Anti-HIV/farmacologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Transcrição Reversa/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Expressão Gênica , Regulação Viral da Expressão Gênica , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/virologia , Peptídeos Cíclicos/metabolismo , RNA Viral/genética , Ativação Transcricional/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
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.
Assuntos
Aminoácidos/síntese química , Furanos/síntese química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Compostos de Espiro/síntese química , Sequência de Aminoácidos , Aminoácidos/química , Furanos/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos de Espiro/químicaRESUMO
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.
Assuntos
Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/química , Peptídeos/química , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Dicroísmo Circular , Testes de Sensibilidade Microbiana , Peptídeos/síntese química , Peptídeos/farmacologia , Peptidomiméticos , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
This review highlights the growing importance of protein epitope mimetics in the discovery of new biologically active molecules and their potential applications in drug and vaccine research. The focus is on folded ß-hairpin mimetics, which are designed to mimic ß-hairpin motifs in biologically important peptides and proteins. An ever-growing number of protein crystal structures reveal how ß-hairpin motifs often play key roles in protein-protein and protein-nucleic acid interactions. This review illustrates how using protein structures as a starting point for small-molecule mimetic design can provide novel ligands as protein-protein interaction inhibitors, as protease inhibitors, and as ligands for chemokine receptors and folded RNA targets, as well as novel antibiotics to combat the growing health threat posed by the emergence of antibiotic-resistant bacteria. The ß-hairpin antibiotics are shown to target a ß-barrel outer membrane protein (LptD) in Pseudomonas sp., which is essential for the biogenesis of the outer cell membrane. Another exciting prospect is that protein epitope mimetics will be of increasing importance in synthetic vaccine design, in the emerging field of structural vaccinology. Crystal structures of protective antibodies bound to their pathogen-derived epitopes provide an ideal starting point for the design of synthetic epitope mimetics. The mimetics can be delivered to the immune system in a highly immunogenic format on the surface of synthetic virus-like particles. The scientific challenges in molecular design remain great, but the potential significance of success in this area is even greater.
Assuntos
Epitopos/química , Fragmentos de Peptídeos/química , Vacinas Sintéticas/química , Sequência de Aminoácidos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Epitopos/imunologia , Humanos , Modelos Moleculares , Mimetismo Molecular , Fragmentos de Peptídeos/imunologia , Estrutura Secundária de ProteínaRESUMO
The pharmacological disruption of the interaction between the HIV Tat protein and its cognate transactivation response RNA (TAR) would generate novel anti-viral drugs with a low susceptibility to drug resistance, but efforts to discover ligands with sufficient potency to warrant pharmaceutical development have been unsuccessful. We have previously described a family of structurally constrained ß-hairpin peptides that potently inhibits viral growth in HIV-infected cells. The nuclear magnetic resonance (NMR) structure of an inhibitory complex revealed that the peptide makes intimate contacts with the 3-nt bulge and the upper helix of the RNA hairpin, but that a single residue contacts the apical loop where recruitment of the essential cellular co-factor cyclin T1 occurs. Attempting to extend the peptide to form more interactions with the RNA loop, we examined a library of longer peptides and achieved > 6-fold improvement in affinity. The structure of TAR bound to one of the extended peptides reveals that the peptide slides down the major groove of the RNA, relative to our design, in order to maintain critical interactions with TAR. These conserved contacts involve three amino acid side chains and identify critical interaction points required for potent and specific binding to TAR RNA. They constitute a template of essential interactions required for inhibition of this RNA.
Assuntos
Fármacos Anti-HIV/química , Repetição Terminal Longa de HIV , Peptídeos/química , RNA Viral/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Sítios de Ligação , HIV-1/genética , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos Cíclicos/químicaRESUMO
Macromolecular structures represent an interesting starting point for the design and synthesis of small-molecule mimetics of surface epitopes that mediate protein-protein and protein-nucleic acid interactions. The resulting protein epitope mimetics (PEMs) provide a source of new biologically active molecules that are useful as biomolecular probes in chemical biology, as well as novel drug or vaccine candidates. This is illustrated here through studies on PEMs as synthetic vaccine candidates targeting the malaria parasite and the human immunodeficiency virus type-1 (HIV-1). In addition, various folded PEMs with ß-hairpin structures have been designed that target protein-protein and protein-nucleic acid interactions, as well as others that interact with cellular receptors such as CXCR4 and the bacterial outer membrane protein LptD. In this last example, the PEMs possess a novel antibiotic activity that has so far not been observed with traditional small synthetic molecules or natural products.
Assuntos
Epitopos/química , Mimetismo Molecular , Proteínas/química , Animais , Antibacterianos/química , Descoberta de Drogas , HIV-1/imunologia , Plasmodium/imunologia , Vacinas SintéticasRESUMO
The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹4C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface.
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Peptidomiméticos/farmacologia , Periplasma/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Radioisótopos de Carbono , Membrana Celular/metabolismo , Escherichia coli , Teste de Complementação Genética , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Microscopia Eletrônica , Estrutura Molecular , Mutação , Peptidomiméticos/química , Periplasma/metabolismo , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , TransfecçãoRESUMO
The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/genética , Mimetismo Molecular , Conformação de Ácido Nucleico , Peptídeos Cíclicos/metabolismo , RNA Viral/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , Arginina/metabolismo , Sequência de Bases , Interações Hidrofóbicas e Hidrofílicas , Vírus da Imunodeficiência Bovina/química , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Fosfatos/metabolismo , Ligação Proteica , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , Solventes , Eletricidade EstáticaRESUMO
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.