RESUMO
The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. In the current study, we present a method to isolate individual antigen-specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR-specific B-cells from 1189 antibody-secreting cells (ASCs) and isolated 94 by CellCellector. We identified a diverse set of heavy chain complementarity-determining region sequences and cloned and expressed 20 into a standard human immunoglobulin G1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400 000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1196 unique EGFR-specific antibodies could be discovered. CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.
RESUMO
Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Linfócitos B/imunologia , Imunoglobulina G/imunologia , Animais , Linfócitos B/citologia , Células CHO , Cricetulus , Ensaio de Imunoadsorção Enzimática , Humanos , CamundongosRESUMO
HIV-1 Env protein is essential for host cell entry, and targeting Env remains an important antiretroviral strategy. We previously found that a peptide triazole thiol KR13 and its gold nanoparticle conjugate AuNP-KR13 directly and irreversibly inactivate the virus by targeting the Env protein, leading to virus gp120 shedding, membrane disruption and p24 capsid protein release. Here, we examined the consequences of targeting cell-surface Env with the virus inactivators. We found that both agents led to formation of non-infectious virus from transiently transfected HEK293T cells. The budded non-infectious viruses lacked Env gp120 but contained gp41. Importantly, budded virions also retained the capsid protein p24, in stark contrast to p24 leakage from viruses directly treated by these agents and arguing that the agents led to deformed viruses by transforming the cells at a stage before virus budding. We found that the Env inactivators caused gp120 shedding from the transiently transfected HEK293T cells as well as non-producer CHO-K1-gp160 cells. Additionally, AuNP-KR13 was cytotoxic against the virus-producing HEK293T and CHO-K1-gp160 cells, but not untransfected HEK293T or unmodified CHO-K1 cells. The results obtained reinforce the argument that cell-surface HIV-1 Env is metastable, as on virus particles, and provides a conformationally vulnerable target for virus suppression and infectious cell inactivation.
Assuntos
Antivirais/farmacologia , HIV-1/fisiologia , Peptídeos/farmacologia , Triazóis/farmacologia , Montagem de Vírus/efeitos dos fármacos , Inativação de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Animais , Células CHO , Cricetulus , Células HEK293 , HumanosRESUMO
Epstein-Barr virus (EBV) is an oncogenic herpesvirus that has been causally linked to the development of B-cell and epithelial malignancies. Early after infection, EBV induces a transient period of hyperproliferation that is suppressed by the activation of the DNA damage response and a G1/S-phase growth arrest. This growth arrest prevents long-term outgrowth of the majority of infected cells. We developed a method to isolate and characterize infected cells that arrest after this early burst of proliferation and integrated gene expression and metabolic profiling to gain a better understanding of the pathways that attenuate immortalization. We found that the arrested cells have a reduced level of mitochondrial respiration and a decrease in the expression of genes involved in the TCA cycle and oxidative phosphorylation. Indeed, the growth arrest in early infected cells could be rescued by supplementing the TCA cycle. Arrested cells were characterized by an increase in the expression of p53 pathway gene targets, including sestrins leading to activation of AMPK, a reduction in mTOR signaling, and, consequently, elevated autophagy that was important for cell survival. Autophagy was also critical to maintain early hyperproliferation during metabolic stress. Finally, in assessing the metabolic changes from early infection to long-term outgrowth, we found concomitant increases in glucose import and surface glucose transporter 1 (GLUT1) levels, leading to elevated glycolysis, oxidative phosphorylation, and suppression of basal autophagy. Our study demonstrates that oncogene-induced senescence triggered by a combination of metabolic and genotoxic stress acts as an intrinsic barrier to EBV-mediated transformation.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , Estresse Fisiológico , Autofagia/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linfócitos B/ultraestrutura , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Transformação Celular Viral/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Desoxiglucose/farmacologia , Dimetilformamida/farmacologia , Herpesvirus Humano 4/efeitos dos fármacos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Oncogenes , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
B-cell activation and proliferation can be induced by a variety of extracellular stimuli. The fate of an activated B cell following mitogen stimulation can be dictated by the strength or duration of the signal, the expression of downstream signaling components necessary to promote proliferation, and the cell intrinsic sensors and regulators of the proliferative program. Previously we have identified the DNA damage response (DDR) signaling pathway as a cell intrinsic sensor that is activated upon latent infection of primary human B cells by Epstein-Barr virus (EBV). Here we have assessed the role of the DDR as a limiting factor in the proliferative response to non-viral B-cell mitogens. We report that TLR9 activation through CpG-rich oligonucleotides induced B-cell hyper-proliferation and an ATM/Chk2 downstream signaling pathway. However, B-cell activation through the CD40 pathway coupled with interleukin-4 (IL-4) promoted proliferation less robustly and only a modest DDR. These two mitogens, but not EBV, modestly induced intrinsic apoptosis that was independent from the DDR. However, all three mitogens triggered a DDR-dependent G1/S phase cell cycle arrest preventing B-cell proliferation. The extent of G1/S arrest, as evidenced by release through Chk2 inhibition, correlated with B-cell proliferation rates. These findings have implications for the regulation of extra-follicular B-cell activation as it may pertain to the development of auto-immune diseases or lymphoma.
Assuntos
Linfócitos B/metabolismo , Quinase do Ponto de Checagem 2/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Mitógenos/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Proliferação de Células , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA/genética , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Ativação Linfocitária/genética , Mitógenos/metabolismo , Transdução de Sinais/genéticaRESUMO
BACKGROUND: We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. RESULTS: KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. CONCLUSIONS: The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Peptídeos/metabolismo , Compostos de Sulfidrila/metabolismo , Triazóis/metabolismo , Vírion/efeitos dos fármacos , Inativação de Vírus , Antivirais/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Conformação ProteicaRESUMO
Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , HIV-1/ultraestrutura , Microscopia Eletrônica de Transmissão , Plasmídeos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genéticaRESUMO
Viruses encounter many challenges within host cells in order to replicate their nucleic acid. In the case of DNA viruses, one challenge that must be overcome is recognition of viral DNA structures by the host DNA damage response (DDR) machinery. This is accomplished in elegant and unique ways by different viruses as each has specific needs and sensitivities dependent on its life cycle. In this review, we focus on three DNA tumor viruses and their interactions with the DDR. The viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV) account for nearly all of the virus-associated human cancers worldwide. These viruses have also been excellent models for the study of oncogenic virus-mediated cell transformation. In this review, we will discuss how each of these viruses engage and subvert aspects of the host DDR. The first level of DDR engagement is a result of the genetic linkage between the oncogenic potential of these viruses and their ability to replicate. Namely, the promotion of cells from quiescence into the cell cycle to facilitate virus replication can be sensed through aberrant cellular DNA replication structures which activate the DDR and hinder cell transformation. DNA tumor viruses subvert this growth-suppressive DDR through changes in viral oncoprotein expression which ultimately facilitate virus replication. An additional level of DDR engagement is through direct detection of replicating viral DNA. These interactions parallel those observed in other DNA virus systems in that the need to subvert these intrinsic sensors of aberrant DNA structure in order to replicate must be in place. DNA tumor viruses are no exception. This review will cover the molecular features of DNA tumor virus interactions with the host DDR and the consequences for virus replication.
Assuntos
Dano ao DNA , Vírus de DNA Tumorais/genética , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologia , Replicação Viral/genética , Animais , Dano ao DNA/fisiologia , HumanosRESUMO
Despite advances in HIV therapy, viral resistance and side-effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV-1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co-receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide's affinity and its ability to inhibit binding of CD4 and the co-receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single-round cell infection assays. Two mutations, D474A and T257A, caused large-scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co-receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development.
Assuntos
Fármacos Anti-HIV/química , Proteína gp120 do Envelope de HIV/química , Peptídeos/química , Triazóis/química , Fármacos Anti-HIV/farmacologia , Antígenos CD4/química , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação/genética , Peptídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Triazóis/metabolismoRESUMO
We investigated the derivation of non-natural peptide triazole dual receptor site antagonists of HIV-1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG-156 [R-I-N-N-I-X-W-S-E-A-M-M-CONH(2), in which X=ferrocenyltriazole-Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co-receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG-156, typified by UM-24 (Cit-N-N-I-X-W-S-CONH(2)) and containing the critical central stereospecific (L)X-(L)W cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM-24 to the greatest extent possible. The analogue with the critical "hot spot" residue Trp 6 replaced with L-3-benzothienylalanine (Bta) (KR-41), as well as a completely non-natural analogue containing D-amino acid substitutions outside the central cluster (KR-42, (D)Cit-(D)N-(D)N-(D)I-X-Bta-(D)S-CONH(2)), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV-1 Env gp120.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Calorimetria , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Ligação Proteica , Triazóis/química , Triazóis/farmacologiaRESUMO
The first stage of human immunodeficiency virus type 1 (HIV-1) infection involves the fusion of viral and host cellular membranes mediated by viral envelope glycoprotein gp120. Inhibitors that specifically target gp120 are gaining increased attention as therapeutics or preventatives to prevent the spread of HIV-1. One promising new group of inhibitors is the peptide triazoles, which bind to gp120 and simultaneously block its interaction with both CD4 and the coreceptor. In this study, we assessed the most potent peptide triazole, HNG-156, for inhibitory breadth, cytotoxicity, and efficacy, both alone and in combination with other antiviral compounds, against HIV-1. HNG-156 inhibited a panel of 16 subtype B and C isolates of HIV-1 in a single-round infection assay. Inhibition of cell infection by replication-competent clinical isolates of HIV-1 was also observed with HNG-156. We found that HNG-156 had a greater than predicted effect when combined with several other entry inhibitors or the reverse transcriptase inhibitor tenofovir. Overall, we find that HNG-156 is noncytotoxic, has a broad inhibition profile, and provides a positive combination with several inhibitors of the HIV-1 life cycle. These results support the pursuit of efficacy and toxicity analyses in more advanced cell and animal models to develop peptide triazole family inhibitors of HIV-1 into antagonists of HIV-1 infection.
Assuntos
Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Triazóis/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/toxicidade , Linhagem Celular , Quimioterapia Combinada , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/metabolismo , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/toxicidade , HIV-1/classificação , HIV-1/patogenicidade , Humanos , Organofosfonatos/farmacologia , Peptídeos/química , Peptídeos/toxicidade , Inibidores da Transcriptase Reversa/farmacologia , Tenofovir , Triazóis/química , Triazóis/toxicidade , Replicação Viral/efeitos dos fármacosRESUMO
Human interleukin-5 receptor α (IL5Rα) is a glycoprotein that contains four N-glycosylation sites in the extracellular region. Previously, we found that enzymatic deglycosylation of IL5Rα resulted in complete loss of IL5 binding. To localize the functionally important carbohydrate moieties, we employed site-directed mutagenesis at the N-glycosylation sites (Asn(15), Asn(111), Asn(196), and Asn(224)). Because Asn-to-Gln mutagenesis caused a significant loss of structural integrity, we used diverse mutations to identify stability-preserving changes. We also rationally designed mutations at and around the N-glycosylation sites based on sequence alignment with mouse IL5Rα and other cytokine receptors. These approaches were most successful at Asn(15), Asn(111), and Asn(224). In contrast, any replacement at Asn(196) severely reduced stability, with the N196T mutant having a reduced binding affinity for IL5 and diminished biological activity because of the lack of cell surface expression. Lectin inhibition analysis suggested that the carbohydrate at Asn(196) is unlikely involved in direct ligand binding. Taking this into account, we constructed a stable variant, with triple mutational deglycosylation (N15D, I109V/V110T/N111D, and L223R/N224Q). The re-engineered protein retained Asn(196) while the other three glycosylation sites were eliminated. This mostly deglycosylated variant had the same ligand binding affinity and biological activity as fully glycosylated IL5Rα, thus demonstrating a unique role for Asn(196) glycosylation in IL5Rα function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.
Assuntos
Asparagina/química , Asparagina/genética , Subunidade alfa de Receptor de Interleucina-5/química , Subunidade alfa de Receptor de Interleucina-5/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Asparagina/fisiologia , Configuração de Carboidratos , Linhagem Celular , Drosophila melanogaster , Variação Genética , Glicosilação , Humanos , Subunidade alfa de Receptor de Interleucina-5/fisiologia , Ligantes , Camundongos , Dados de Sequência Molecular , Ligação Proteica/genéticaAssuntos
Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Peptídeos/química , Triazóis/química , Triazóis/farmacologia , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral , Ouro/química , Proteína do Núcleo p24 do HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Nanopartículas Metálicas/químicaRESUMO
We sought to identify sequences in the monoclonal antibody m18 complementarity determining regions (CDRs) that are responsible for its interaction with HIV-1 gp120 and inhibition of the envelope receptor binding sites. In the accompanying paper (DOI 10.1021/bi101160r), we reported that m18 inhibits CD4 binding through a nonactivating mechanism that, at the same time, induces conformational effects leading to inhibition of the coreceptor site. Here, we sought to define the structural elements in m18 responsible for these actions. Direct binding and competition analyses using surface plasmon resonance showed that YU-2 gp120 binding is stabilized by a broad paratope of residues in the m18 CDRs. Additionally, several m18 residues were identified for which mutants retained high affinity for gp120 but had suppressed CD4 and 17b inhibition activities. A subset of these mutants did, however, neutralize HXBc2 viral infection. The results obtained in this work demonstrate that the combined m18 paratope contains subsets of residues that are differentially important for the binding and inhibition functions of the m18 neutralizing antibody. The data also add to prior observations that high-affinity antibodies that do not inhibit monomeric gp120 receptor site interactions may still exhibit significant antiviral activity.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD4/metabolismo , Epitopos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva , Antígenos CD4/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Epitopos/química , Epitopos/genética , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação ProteicaRESUMO
We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (KD≈5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.
Assuntos
Anticorpos Neutralizantes/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Conformação Proteica , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Sítios de Ligação/genética , Ligação Competitiva , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Calorimetria , Epitopos/imunologia , Epitopos/metabolismo , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for the viral surface protein gp120. This peptide class exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of the viral envelope protein with both CD4 and co-receptor. In this investigation, we minimized the structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxy- and amino-terminal residues from the parent 12-residue peptide 1 were found to have minimal effects on both affinity and antiviral activity. In contrast, the central triazole(Pro)-Trp cluster at residues 6 and 7 with ferrocenyl-triazole(Pro) (Ftp) was found to be critical for bioactivity. Amino-terminal residues distal to the central triazole(Pro)-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism, and induction of gp120 structuring, all properties that define the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole(Pro)-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen bonding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, thereby enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Peptídeos/metabolismo , Triazóis/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Antígenos CD4/metabolismo , Domínio Catalítico , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Dados de Sequência Molecular , Peptídeos/química , Ressonância de Plasmônio de Superfície , Termodinâmica , Triazóis/químicaRESUMO
In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2+3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Compostos Organometálicos/química , Fragmentos de Peptídeos/farmacologia , Triazóis/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Sítios de Ligação , Antígenos CD4/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Compostos Ferrosos/química , Anticorpos Anti-HIV/metabolismo , HIV-1/imunologia , Humanos , Metalocenos , Mimetismo Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/metabolismo , Triazóis/síntese química , Triazóis/química , Internalização do Vírus/efeitos dos fármacosRESUMO
The first, critical stage of HIV-1 infection is fusion of viral and host cellular membranes initiated by a viral envelope glycoprotein gp120. We evaluated the potential to form a chimeric protein entry inhibitor that combines the action of two gp120-targeting molecules, an allosteric peptide inhibitor 12p1 and a higher affinity carbohydrate-binding protein cyanovirin (CVN). In initial mixing experiments, we demonstrated that the inhibitors do not interfere with each other and instead show functional synergy in inhibiting viral cell infection. Based on this, we created a chimera, termed L5, with 12p1 fused to the C-terminal domain of CVN through a linker of five penta-peptide repeats. L5 revealed the same broad specificity as CVN for gp120 from a variety of clades and tropisms. By comparison to CVN, the L5 chimera exhibited substantially increased inhibition of gp120 binding to receptor CD4, coreceptor surrogate mAb 17b and gp120 antibody F105. These binding inhibition effects by the chimera reflected both the high affinity of the CVN domain and the allosteric action of the 12p1 domain. The results open up the possibility to form high potency chimeras, as well as noncovalent mixtures, as leads for HIV-1 envelope antagonism that can overcome potency limits and potential virus mutational resistance for either 12p1 or CVN alone.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteína gp120 do Envelope de HIV/química , Lectinas/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antígenos CD4/metabolismo , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Lectinas/química , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development.
Assuntos
Fármacos Anti-HIV/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vagina/efeitos dos fármacos , Animais , Western Blotting , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Feminino , Expressão Gênica , Vetores Genéticos , Proteína gp120 do Envelope de HIV/metabolismo , Células HeLa , Humanos , Corpos de Inclusão Viral/metabolismo , Concentração Inibidora 50 , Proteínas Recombinantes/genética , Análise de Sequência de Proteína , Vagina/microbiologiaRESUMO
Hormones, estrogen and prolactin, transforming growth factor-beta (TGF-beta), and matrixmetalloproteinases (MMPs) may modulate breast cancer progression. The goal of this research was to examine the regulation of expression of TGF-beta and MMPs (MMP-1, 2, 9) by estrogen and prolactin, independently and in combination, at physiological doses, and at doses stimulating cancer cell (T47D) proliferation in vitro. Prolactin, and estrogen synergistically, and similarly, inhibited the expression of TGF-beta and MMPs at physiological concentrations without altering cell proliferation, indicating a beneficial role of the hormones. The growth stimulating concentration of prolactin, but not estrogen, also inhibited the TGF-beta and MMP expression.