RESUMO
Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from EnvSF162 into the EnvHXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the EnvHXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312-315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.
Assuntos
Infecções por HIV , HIV-1 , Anticorpos Neutralizantes , Epitopos , Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Humanos , Vírion/metabolismo , Internalização do VírusRESUMO
BACKGROUND: We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. RESULTS: HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. CONCLUSIONS: Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/química , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Peptídeos/farmacologia , Triazóis/farmacologia , Fármacos Anti-HIV/química , Sítios de Ligação , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Simulação de Acoplamento Molecular , Mutação , Peptídeos/química , Conformação Proteica , Triazóis/química , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket. RESULTS: Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data. CONCLUSIONS: These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular , Infecções por HIV/virologia , HIV-1/genética , Humanos , MutaçãoRESUMO
The homotrimeric HIV-1 envelope glycoprotein (Env) undergoes receptor-triggered structural changes that mediate viral entry through membrane fusion. This process is inhibited by chemokine receptor antagonists (CoRAs) that block Env-receptor interactions and by fusion inhibitors (FIs) that disrupt Env conformational transitions. Synergy between CoRAs and FIs has been attributed to a CoRA-dependent decrease in the rate of viral membrane fusion that extends the lifetime of the intermediate state targeted by FIs. Here, we demonstrated that the magnitude of CoRA/FI synergy unexpectedly depends on FI-binding affinity and the stoichiometry of chemokine receptor binding to trimeric Env. For C-peptide FIs (clinically represented by enfuvirtide), synergy waned as binding strength decreased until inhibitor combinations behaved additively. Curiously, this affinity dependence on synergy was absent for 5-Helix-type FIs. We linked this complex behavior to the CoRA dependence of Env deactivation following FI binding. For both FI classes, reducing chemokine receptor levels on target cells or eliminating competent chemokine receptor-binding sites on Env trimers resulted in a loss of synergistic activity. These data imply that the stoichiometry required for CoRA/FI synergy exceeds that required for HIV-1 entry. Our analysis suggests two distinct roles for chemokine receptor binding, one to trigger formation of the FI-sensitive intermediate state and another to facilitate subsequent conformational transitions. Together, our results could explain the wide variety of previously reported activities for CoRA/FI combinations. These findings also have implications for the combined use of CoRAs and FIs in antiviral therapies and point to a multifaceted role for chemokine receptor binding in promoting HIV-1 entry.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Internalização do Vírus/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Humanos , CinéticaRESUMO
A popular strategy for overcoming the limited plasma half-life of peptide heptad repeat 2 (HR2) fusion inhibitors against HIV-1 is conjugation with biocompatible polymers such as poly(ethylene glycol) (PEG). However, despite improved resistance to proteolysis and reduced renal elimination, covalent attachment of polymers often causes a loss in therapeutic potency. In this study, we investigated the molecular origins of the loss in potency upon conjugation of linear, midfunctional, and hyperbranched PEG-like polymers to peptides that inhibit HIV-1-host cell membrane fusion. Fluorescence binding assays revealed that polymer conjugation imparted mass transport limitations that manifested as coexistent slower association and dissociation rates from the gp41 target on HIV-1. Furthermore, reduced association kinetics rather than affinity disruption was responsible for the loss in antiviral potency. Finally, the binding assays indicated that the unmodified HR2-derived peptide demonstrated diffusion-limited binding. The observed high potency of the unmodified peptide in HIV-1 inhibition assays was therefore attributed to rapid peptide conformational changes upon binding to the gp41 prehairpin structure. This study emphasizes that the view in which polymer ligation to therapeutic peptides inadvertently leads to loss in potency due to a loss in binding affinity requires scientific verification on a case-by-case basis and that high peptide potency may be due to rapid target-binding events.
Assuntos
Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Humanos , Modelos Moleculares , Internalização do Vírus/efeitos dos fármacosRESUMO
Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI.
Assuntos
Proteína gp41 do Envelope de HIV/química , HIV-1/fisiologia , Internalização do Vírus , Linhagem Celular , HIV-1/química , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans.
Assuntos
Linfócitos T CD4-Positivos/virologia , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores CXCR4/metabolismo , Internalização do Vírus , Animais , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NODRESUMO
Two of the primary virulence regulators of Vibrio cholerae, ToxR and TcpP, function together with cognate effector proteins. ToxR undergoes regulated intramembrane proteolysis (RIP) during late stationary phase in response to nutrient limitation at alkaline pH; however, the specific function of its cognate ToxS remains unresolved. In this work, we found that ToxR rapidly becomes undetectable in a ΔtoxS mutant when cultures are exposed to either starvation conditions or after alkaline pH shock individually. A ΔtoxS mutant enters into a dormant state associated with the proteolysis of ToxR at a faster rate than wild-type, closely resembling a ΔtoxR mutant. Using a mutant with a periplasmic substitution in ToxS, we found that the proteases DegS and DegP function additively with VesC and a novel protease, TapA, to degrade ToxR in the mutant. Overall, the results shown here reveal a role for ToxS in the stabilization of ToxR by protecting the virulence regulator from premature proteolysis.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Endopeptidases/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , Mutação , Periplasma/metabolismo , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Proteólise , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fatores de Transcrição/genética , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , Fatores de Virulência/genéticaRESUMO
Severely immunodeficient mice such as the NOD/SCID/IL2rγ(null) (NSG) strain can be engrafted with human hematopoietic stem cells (HSCs), resulting in chimeric mice containing many components of the human immune system (Human Immune System mice or HIS mice). HIS mice can both support the replication of and recapitulate much of the immunological response to a variety of pathogens, including ones with strict human tropism, such as HIV-1. In an effort to develop a better mouse model for human infectious pathogen infection and possible immune resolution, we compared the human immune system reconstitution of NSG mice following injection with human CD34(+) HSCs purified from either fetal liver (FL) or umbilical cord blood (UCB). We analyzed reconstitution in standard NSG mice as well as a derivative of these mice containing an HLA.A2 encoding transgene (NSG.A2). HSCs from both sources effectively reconstituted hematopoietic lineages when injected into NSG mice. In marked contrast, total CD45(+) human hematopoietic cells in NSG.A2 mice were well reconstituted by HSCs from UCB but very poorly by HSCs purified from FL. Moreover, the reconstitution of T cell lineages in NSG.A2 mice by HSCs from UCB was inferior to that obtained using NSG mice. We also found that FL CD34(+) HSCs contain a much higher percentage of cells with a phenotype consistent with primitive progenitors than UCB HSCs. We discuss possible explanations for the influence of the HLA.A2 transgene on hematopoietic reconstitution using the two sources of HSCs.
Assuntos
Sangue Fetal/citologia , Antígeno HLA-A2/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Fígado/citologia , Animais , Antígenos CD34/metabolismo , Quimera/imunologia , Sangue Fetal/imunologia , Antígeno HLA-A2/imunologia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Fígado/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante HeterólogoRESUMO
Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Nanoconjugados/toxicidade , Peptídeos/farmacologia , Triazóis/farmacologia , Fármacos Anti-HIV/síntese química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ouro/química , Proteína gp120 do Envelope de HIV/química , HIV-1/crescimento & desenvolvimento , Humanos , Nanoconjugados/ultraestrutura , Tamanho da Partícula , Peptídeos/síntese química , Ligação Proteica , Triazóis/síntese química , Inativação de Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., "de-fatted"), and, as a result, their involvement in allergy has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. CONCLUSIONS/SIGNIFICANCE: Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is warranted and could potentially lead to the development of improved diagnostic and therapeutic tools.
Assuntos
Cynodon/química , Cisteína Proteases/farmacologia , Endo-1,4-beta-Xilanases/farmacologia , Células Epiteliais/efeitos dos fármacos , Imunoglobulina E/metabolismo , Proteínas de Plantas/farmacologia , Pólen/química , Mucosa Respiratória/efeitos dos fármacos , Sequência de Bases , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Fracionamento Químico , Cynodon/imunologia , Cynodon/ultraestrutura , Cisteína Proteases/isolamento & purificação , Cisteína Proteases/metabolismo , Relação Dose-Resposta a Droga , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Imunoglobulina E/química , Imunoglobulina E/imunologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Pólen/imunologia , Pólen/ultraestrutura , Ligação Proteica , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Rinite Alérgica Sazonal/imunologia , SolventesRESUMO
The prevalence of obesity among children and adults is increasing worldwide. There are substantial health risks and financial costs associated with the obesity epidemic that impact the practice of orthopaedic surgery. Patients with increased body mass index are more prone to sustaining distal extremity injuries than are those with a normal body mass index. Obese individuals are more likely than nonobese individuals to seek treatment for osteoarthritis of the knee.
Assuntos
Doenças Musculoesqueléticas/epidemiologia , Obesidade/epidemiologia , Procedimentos Ortopédicos/efeitos adversos , Ferimentos e Lesões/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Índice de Massa Corporal , Criança , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Doenças Musculoesqueléticas/diagnóstico , Doenças Musculoesqueléticas/cirurgia , Obesidade/diagnóstico , Obesidade/cirurgia , Procedimentos Ortopédicos/métodos , Osteoartrite do Quadril/diagnóstico , Osteoartrite do Quadril/epidemiologia , Osteoartrite do Quadril/cirurgia , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/epidemiologia , Osteoartrite do Joelho/cirurgia , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/fisiopatologia , Prognóstico , Valores de Referência , Medição de Risco , Distribuição por Sexo , Estados Unidos/epidemiologia , Ferimentos e Lesões/diagnóstico , Ferimentos e Lesões/cirurgiaRESUMO
This report describes the synthesis and properties of a series of polyvalent side chain peptide-synthetic polymer conjugates designed to block the CD4 binding site on gp120 and inhibit HIV-1 entry into a host cell. The peptide sequences in the conjugates are based on the CDR H3 region of the neutralizing anti-HIV-1 antibody IgG1 b12. Using a consecutive ester-amide/thiol-ene postpolymerization modification strategy, a library of polymer conjugates was prepared. Evaluation of the HIV-1 inhibitory properties revealed that midsized polymer conjugates displayed the highest antiviral activity, while shorter and longer conjugates proved to be less efficacious inhibitors. The lower molecular weight conjugates may not have sufficient length to span the distance between two neighboring gp120 containing spikes, while the higher molecular weight conjugates may be compromised due to a higher entropic penalty that would accompany their binding to the viral envelope. Although the IC(50) values for these polymer conjugates are higher than that of the parent IgG1 b12 antibody, the strategy presented here may represent an interesting antiviral approach due to the attractive properties of such polymer therapeutics (relatively inexpensive production and purification costs, high thermal and chemical stability in storage conditions, long half-life in biological tissues, low immunogenicity, and protection from proteolytic degradation).
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Peptídeos/química , Polímeros/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Antígenos CD4/química , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/química , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos/síntese química , Polímeros/síntese química , Polímeros/químicaRESUMO
The human genome encodes six isoforms of importin α that show greater than 60% sequence similarity and remarkable substrate specificity. The isoform importin α5 can bind phosphorylated cargos such as STAT1 and Epstein-Barr Virus Nuclear Antigen 1, as well as the influenza virus polymerase subunit PB2. In this work, we have studied the interaction of the nucleoporin Nup50 with importin α5. We show that the first 47 residues of Nup50 bind to the C terminus of importin α5 like a "clip," stabilizing the closed conformation of ARM 10. In vitro, Nup50 binds with high affinity either to empty importin α5 or to a preassembled complex of importin α5 bound to the C-terminal domain of the import cargo PB2, resulting in a trimeric complex. By contrast, PB2 can only bind with high affinity to importin α5 in the absence of Nup50. This suggests that Nup50 primary function may not be to actively displace the import cargo from importin α5 but rather to prevent cargo rebinding in preparation for recycling. This is the first evidence for a nucleoporin modulating the import reaction by directly altering the three-dimensional structure of an import adaptor.
Assuntos
Núcleo Celular/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas Nucleares/química , alfa Carioferinas/química , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMO
The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific D-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. D-peptides (peptides composed of D-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first D-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.
Assuntos
Desenho de Fármacos , Farmacorresistência Viral , Inibidores da Fusão de HIV/química , Peptídeos/farmacologia , Sítios de Ligação , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Peptídeos/uso terapêuticoRESUMO
Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.
Assuntos
Proteína gp41 do Envelope de HIV/efeitos dos fármacos , Inibidores da Fusão de HIV/farmacocinética , Proteínas de Transporte/farmacologia , Fusão Celular , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Cinética , Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes , Internalização do Vírus/efeitos dos fármacosRESUMO
Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K(D) = 30 nm), close to its IC50 concentration (approximately 3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (K(D) = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF --> ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012-21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (K(D) = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20 variant was significantly more potent against T20-resistant virus. The findings suggest that other factors in addition to binding affinity play a role in limiting T20 potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.
Assuntos
Materiais Biomiméticos/farmacologia , Proteína gp41 do Envelope de HIV/antagonistas & inibidores , Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/metabolismo , Fragmentos de Peptídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Materiais Biomiméticos/química , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Enfuvirtida , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/metabolismo , Células HeLa , Humanos , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Secundária de Proteína/genéticaRESUMO
With the continuing march of the AIDS epidemic and little hope for an effective vaccine in the near future, work to develop a topical strategy to prevent HIV infection is increasingly important. This stated, the track record of large scale "microbicide" trials has been disappointing with nonspecific inhibitors either failing to protect women from infection or even increasing HIV acquisition. Newer strategies that target directly the elements needed for viral entry into cells have shown promise in non-human primate models of HIV transmission and as these agents have not yet been broadly introduced in regions of highest HIV prevalence, they are particularly attractive for prophylaxis. We review here the agents that can block HIV cellular entry and that show promise as topical strategies or "virustats" to prevent mucosal transmission of HIV infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , HIV/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , HIV/fisiologia , Infecções por HIV/virologia , Humanos , Receptores de HIV/antagonistas & inibidores , Receptores de HIV/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.