RESUMO
We report on the use of nitric oxide-mediated transcriptional activation (NOMETA) as an innovative means to detect and access new classes of microbial natural products encoded within silent biosynthetic gene clusters. A small library of termite nest- and mangrove-derived fungi and actinomyces was subjected to cultivation profiling using a miniaturized 24-well format approach (MATRIX) in the presence and absence of nitric oxide, with the resulting metabolomes subjected to comparative chemical analysis using UPLC-DAD and GNPS molecular networking. This strategy prompted study of Talaromyces sp. CMB-TN6F and Coccidiodes sp. CMB-TN39F, leading to discovery of the triterpene glycoside pullenvalenes A-D (1-4), featuring an unprecedented triterpene carbon skeleton and rare 6-O-methyl-N-acetyl-d-glucosaminyl glycoside residues. Structure elucidation of 1-4 was achieved by a combination of detailed spectroscopic analysis, chemical degradation, derivatization and synthesis, and biosynthetic considerations.
Assuntos
Aminoglicosídeos , Isópteros , Óxido Nítrico , Triterpenos , Animais , Triterpenos/farmacologia , Triterpenos/química , Triterpenos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Estrutura Molecular , Isópteros/microbiologia , Aminoglicosídeos/farmacologia , Austrália , Ativação Transcricional/efeitos dos fármacos , Fungos/metabolismo , Talaromyces/química , Talaromyces/metabolismo , Actinomyces/metabolismo , Actinomyces/efeitos dos fármacosRESUMO
A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.
Assuntos
Dirofilaria immitis , Streptomyces , Animais , Streptomyces/química , Dirofilaria immitis/efeitos dos fármacos , Austrália , Ovinos , Fezes/parasitologia , Fezes/microbiologiaRESUMO
Chemical analysis of cultures of a Queensland mud dauber wasp nest-derived fungus, Talaromyces sp. CMB-MW102, yielded the known dimeric oxaphenalenone duclauxin (1) along with a family of new 1-deoxy-d-glucosamine adducts, glyclauxins A-E (2-6). Despite 1D NMR spectra of 2-6 being compromised by broadening of selected resonances, structures inclusive of absolute configuration were assigned on the basis of detailed spectroscopic analysis and biogenetic considerations, as well as biomimetic semisynthesis and chemical interconversion. For example, exposure of duclauxin (1) to synthetic 1-deoxy-d-glucosamine yielded glyclauxin B (3), while on handling and storage, glyclauxins C (4) and D (5) (bearing a 7-OMe moiety) proved chemically labile and underwent quantitative transformation to glyclauxins B (3) and A (2), respectively. These latter observations on chemical reactivity and stability informed a proposed biogenetic relationship linking all known members of the extended duclauxin family. Notwithstanding their potential status as artifacts, the detection of glyclauxins B (3) and A (2) in a fresh CMB-MW102 culture extract confirmed their natural product status.
Assuntos
Talaromyces , Vespas , Animais , Aminoglicosídeos , Talaromyces/química , Vespas/microbiologia , Austrália , Antibacterianos/química , Estrutura MolecularRESUMO
Application of a miniaturized 24-well plate system for cultivation profiling (MATRIX) permitted optimization of the cultivation conditions for the marine-derived fungus Talaromyces sp. CMB-TU011, facilitating access to the rare cycloheptapeptide talarolide A (1) along with three new analogues, B-D (2-4). Detailed spectroscopic analysis supported by Marfey's analysis methodology was refined to resolve N-Me-l-Ala from N-Me-d-Ala, l-allo-Ile from l-Ile and l-Leu, and partial and total syntheses of 2, and permitted unambiguous assignment of structures for 1 (revised) and 2-4. Consideration of diagnostic ROESY correlations for the hydroxamates 1 and 3-4, and a calculated solution structure for 1, revealed how cross-ring H-bonding to the hydroxamate moiety influences (defines/stabilizes) the cyclic peptide conformation. Such knowledge draws attention to the prospect that hydroxamates may be used as molecular bridges to access new cyclic peptide conformations, offering the prospect of new biological properties, including enhanced oral bioavailability.
RESUMO
BACKGROUND: Approximately 400 million individuals are infected with hookworms globally. Protective vaccines are needed to prevent reinfections, which often occur after drug treatment in endemic areas. Ideal vaccines are highly efficacious and well tolerated, and do not present risks to patient safety. Peptide vaccines can generate specific, highly protective responses because they focus on minimal antigenic target(s) with a specific immunoprotective mechanism. Necator americanus aspartyl protease 1 (Na-APR-1) is one of the most promising hookworm vaccine antigens. The neutralizing epitope p3 (TSLIAGPKAQVEAIQKYIGAEL), together with universal the TH epitope P25 (KLIPNASLIENCTKAEL), has been used previously to produce peptide vaccines and was found to protect BALB/c mice against rodent hookworm infections, resulting in worm burden reductions of up to 98%. However, because of extensive digestion in the gastrointestinal tract, large oral vaccination doses were necessary to achieve this level of efficacy. OBJECTIVE: We sought to overcome the limitations of oral vaccine delivery and to investigate protective efficacy and immune correlates of protection. Herein, we examined 5 different peptide vaccines following intraperitoneal injection, to compare their efficacy with that of the clinical protein antigen APR-1. METHODS: BALB/c mice were immunized with p3-P25-based antigen that was adjuvanted with (1) lipid core peptide, (2) polymethyl methacrylate, (3) linear polyleucine, and (4) branched polyleucine (BL10), or with (5) CpG/aluminum hydroxide adjuvant (alum)-adjuvanted control and protein-based (6) CpG/alum-adjuvanted Na-APR-1. The mice sera, saliva, and feces were sampled for immune response evaluation. The immunized mice were further challenged via hookworm larvae infection, and protection was evaluated by conducting intestinal hookworm counts. RESULTS: BL10 and lipid core peptide generated the highest serum anti-Na-APR-1 IgG and fecal anti-APR-1 IgG titers, but only BL10 generated significant fecal anti-Na-APR-1 IgA titers. Upon challenge, immunization with CpG/alum-adjuvanted p3-P25, BL10, and lipid core peptide provided the highest worm burden reductions of 75%, 77%, and 59%, respectively, whereas the group immunized with Na-APR-1 had only modest worm reduction of 26%. The relationships between serum anti-Na-APR-1 IgG, fecal anti-Na-APR-1 IgA and IgG, and worm burden reduction were established with R2 values greater than or equal to 0.9, and the crucial role of both anti-Na-APR-1 IgG and IgA responses was identified. CONCLUSIONS: We demonstrated for the first time that p3-based vaccine candidates are safer and can deliver higher protection against hookworm infection compared with the clinical vaccine candidate, Na-APR-1.
Assuntos
Infecções por Uncinaria , Vacinas de Subunidades Antigênicas , Adjuvantes Imunológicos , Hidróxido de Alumínio , Animais , Epitopos , Infecções por Uncinaria/prevenção & controle , Imunoglobulina A , Imunoglobulina G , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Necator americanus , Vacinas de Subunidades Antigênicas/efeitos adversosRESUMO
Hookworms are hematophagous nematode parasites that have infected a billion people worldwide. Anthelmintic drugs have limited efficacy and do not prevent reinfection. Therefore, prophylactic vaccines are in high demand. Whole parasite vaccines are allergic and unsafe; thus, research into subunit vaccines has been warranted. A comprehensive overview of protein or peptide subunit vaccines' safety, protective efficacy, and associated immune responses is provided herein. The differences between the immune responses against hookworm infection by patients from epidemic versus nonepidemic areas are discussed in detail. Moreover, the different immunologic mechanisms of protection are discussed, including those that rely on allergic and nonallergic humoral and antibody-dependent cellular responses. The allergic and autoimmune potential of hookworm antigens is also explored, as are the immunoregulatory responses induced by the hookworm secretome. The potential of oral mucosal immunizations has been overlooked. Oral immunity against hookworms is a long-lived and safer immune response that is associated with elimination of infection and protective against reinfections. However, the harsh conditions of the gastrointestinal environment necessitates special oral delivery systems to unlock vaccines' protective potential. The potential for development of safer and more effective peptide- and protein-based anthelmintic vaccines is explored herein.
Assuntos
Antígenos de Helmintos/imunologia , Infecções por Uncinaria/imunologia , Intestinos/imunologia , Necatoríase/imunologia , Vacinas/imunologia , Ancylostomatoidea/imunologia , Animais , Humanos , Imunidade nas Mucosas , Vacinas de Subunidades AntigênicasRESUMO
Peptide-based vaccines are composed of small, defined, antigenic peptide epitopes. They are designed to induce well-controlled immune responses. Multiple epitopes are often employed in these vaccines to cover strain variability of a pathogen. However, peptide epitopes cannot stimulate adequate immune responses on their own and require an adjuvant (immune stimulant) and/or delivery system. Here, we designed and synthesized a multiepitope vaccine candidate against Group A Streptococcus (GAS) composed of several B-cell epitopes (J8, PL1, and 88/30) derived from GAS M-protein, universal PADRE T-helper cell epitope, and a polyleucine self-adjuvanting unit. The vaccine components were conjugated together (using mercapto-maleimide and azide-alkyne Huisgen cycloaddition reactions) or delivered as a mixture. The conjugated multiepitope vaccine candidate self-assembled into small nanoparticles and chain-like aggregated nanoparticles (CLANs) that were able to induce the production of J8-, PL1-, and 88/30-specific antibodies in mice. The multiepitope conjugate and the physical mixture of conjugates bearing the individual epitopes produced similar nanoparticles and induced comparable immune responses. Hence, simple physical mixing can replace complex chemical conjugation to produce multiepitope nanoparticles with equivalent morphology and immunological efficacy. This greatly simplifies vaccine production.
Assuntos
Streptococcus pyogenesRESUMO
New drugs aimed at novel targets are urgently needed to combat the increasing rate of drug-resistant tuberculosis (TB). Herein, the National Cancer Institute Developmental Therapeutic Program (NCI-DTP) chemical library was screened against a promising new target, ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. From this library, 6-hydroxy-2-methylthiazolo[4,5-d]pyrimidine-5,7(4H,6H)-dione (NSC116565) was identified as a potent time-dependent inhibitor of Mycobacteriumâ tuberculosis (Mt) KARI with a Ki of 95.4â nm. Isothermal titration calorimetry studies showed that this inhibitor bound to MtKARI in the presence and absence of the cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), which was confirmed by crystal structures of the compound in complex with closely related Staphylococcusâ aureus KARI. It is also shown that NSC116565 inhibits the growth of H37Ra and H37Rv strains of Mt with MIC50 values of 2.93 and 6.06â µm, respectively. These results further validate KARI as a TB drug target and show that NSC116565 is a promising lead for anti-TB drug development.
Assuntos
Antituberculosos/farmacologia , Cetol-Ácido Redutoisomerase/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Pirimidinonas/farmacologia , Linhagem Celular , Humanos , Cetol-Ácido Redutoisomerase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , NADP/metabolismo , Staphylococcus aureus/enzimologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
We recently reported that polyethylenimine (PEI; molecular weight of 600 Da) acted as a vaccine adjuvant for liposomal group A Streptococcus (GAS) vaccines, eliciting immune responses in vivo with IgG antibodies giving opsonic activity against five Australian GAS clinical isolates. However, to date, no investigation comparing the structure-activity relationship between the molecular weight of PEI and its adjuvanting activity in vaccine development has been performed. We hypothesized that the molecular weight and quantity of PEI in a liposomal vaccine will impact its adjuvanting properties. In this study, we successfully formulated liposomes containing different molecular weights of PEI (600, 1800, 10k and 25k Da) and equivalents of PEI (0.5, 1 and 2) of branched PEI. Outbred mice were administrated the vaccine formulations intranasally, and the mice that received a high ratio of PEI 600 reported a stronger immune response than the mice that received a lower ratio of PEI 600. Interestingly, mice that received the same quantity of PEI 600, PEI 10k and PEI 25k showed similar immune responses in vivo and in vitro. This comparative study highlights the ratio of PEI present in the liposome vaccines impacts adjuvanting activity, however, PEI molecular weight did not significantly enhance its adjuvanting properties. We also report that the stability of PEI liposomes is critical for vaccines to elicit the desired immune response.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Lipossomos/química , Polietilenoimina/uso terapêutico , Infecções Estreptocócicas/terapia , Vacinas Estreptocócicas/uso terapêutico , Adjuvantes Imunológicos/química , Animais , Camundongos , Estrutura Molecular , Peso Molecular , Polietilenoimina/química , Vacinas Estreptocócicas/imunologia , Streptococcus/imunologia , Relação Estrutura-Atividade , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200â nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8â µm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72â Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data.
Assuntos
Cetol-Ácido Redutoisomerase/metabolismo , Magnésio/química , Mycobacterium tuberculosis/enzimologia , NADP/química , Staphylococcus aureus/metabolismo , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Humanos , Cetol-Ácido Redutoisomerase/química , Mycobacterium tuberculosis/química , NADP/metabolismo , Staphylococcus aureus/químicaRESUMO
The inhibition of tyrosinase is an established strategy for treating hyperpigmentation. Our previous findings demonstrated that cinnamic acid and benzoic acid scaffolds can be effective tyrosinase inhibitors with low toxicity. The hydroxyl substituted benzoic and cinnamic acid moieties of these precursors were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase. The most active compound, (2-(3-methoxyphenoxy)-2-oxoethyl (E)-3-(4-hydroxyphenyl) acrylate) 6c, inhibited tyrosinase with an IC50 of 5.7⯵M, while (2-(3-methoxyphenoxy)-2-oxoethyl 2, 4-dihydroxybenzoate) 4d had an IC50 of 23.8⯵M. In comparison, the positive control, kojic acid showed tyrosinase inhibition with an IC50â¯=â¯16.7⯵M. Analysis of enzyme kinetics revealed that 6c and 4d displayed noncompetitive reversible inhibition of the second tyrosinase enzymatic reaction with Ki values of 11⯵M and 130⯵M respectively. In silico docking studies with mushroom tyrosinase (PDB ID 2Y9X) predicted possible binding modes in the catalytic site for these active compounds. The phenolic para-hydroxy group of the most active compound 6c is predicted to interact with the catalytic site Cu++ ion. The methoxy part of this compound is predicted to form a hydrogen bond with Arg 268. Compound 6c had no observable toxic effects on cell morphology or cell viability at the highest tested concentration of 91.4⯵M. When dosed at 91.4⯵M onto B16F10 melanoma cells in vitro6c showed anti-melanogenic effects equivalent to kojic acid at 880⯵M. 6c displayed no PAINS (pan-assay interference compounds) alerts. Our results show that compound 6c is a more potent tyrosinase inhibitor than kojic acid and is a candidate for further development. Our exposition of the details of the interactions between 6c and the catalytic pocket of tyrosinase provides a basis for rational design of additional potent inhibitors of tyrosinase, built on the cinnamic acid scaffold.
Assuntos
Ácido Benzoico/uso terapêutico , Cinamatos/uso terapêutico , Melanoma/tratamento farmacológico , Simulação de Acoplamento Molecular/métodos , Ácido Benzoico/farmacologia , Cinamatos/farmacologia , Humanos , Relação Estrutura-AtividadeRESUMO
In order to improve the immunogenicity of peptide-based vaccines against group A Streptococcus (GAS), lipid moieties (C16 lipoamino acid and cholic acid) were conjugated with peptide antigen (P25-J8) and further modified with α-poly(glutamic acid) (α-PGA). Thus, positively charged lipopeptide vaccine candidates LCP-1 (P25-K(J8)-SS-C16-C16) and LCP-2 (P25-K(J8)-SS-K(cholic acid)) were synthesized. Negatively charged LCP-3 (P25-K(PGA-J8)-SS-K(cholic acid)) was also produced by attaching α-PGA to the J8 N-terminus of LCP-2. Polyelectrolyte complex (PEC) nanoparticles were formulated with heparin and/or trimethyl chitosan (TMC) for delivery of the lipopeptide vaccine candidates. The ability of the antigen-loaded nanoparticles to induce humoral immune responses was examined in outbred female Swiss mice following intranasal immunization. The antibodies produced were opsonic against all clinical GAS isolates tested.
Assuntos
Lipopeptídeos/imunologia , Streptococcus pyogenes/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos , Administração Intranasal , Animais , Anticorpos/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Homólogo 5 da Proteína Cromobox , Feminino , Humanos , Imunidade Humoral , Lipopeptídeos/administração & dosagem , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Camundongos , Nanopartículas/química , Polieletrólitos/química , Ácido Poliglutâmico/química , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/prevenção & controle , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/farmacologiaRESUMO
Based on a structure-guided approach, aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles were designed to target metallo-ß-lactamases (MBLs), using Klebsiella pneumoniaeNDM-1 as a model. The in vitro MBLs inhibition showed remarkable inhibition constant for most of the designed compounds at a low micromolar range (1.5-16.4 µM) against NDM-1, IMP-1 and AIM-1 MBLs. Furthermore, all compounds showed promising antibacterial activity against (K+, K1-K9) resistant clinical isolates of K. pneumoniae and were able to re-sensitize resistant K. pneumoniae (K5) strain towards meropenem and cefalexin. Besides, in vivo toxicity testing exhibited that the most active compound was non-toxic and well tolerated by the experimental animals orally up to 350 mg/kg and up to 125 mg/kg parenterally. The docking experiments on NDM-1 and IMP-1 rationalized the observed in vitro MBLs inhibition activity. Generally, this work presents a fruitful matrix to extend the chemical space for MBLs inhibition. This aids in tackling drug-resistance issues in antibacterial treatment.
Assuntos
Antibacterianos/farmacologia , Hidrazonas/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Pirazóis/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Hidrazonas/síntese química , Hidrazonas/química , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/química , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/síntese química , Inibidores de beta-Lactamases/químicaRESUMO
The development of polymer-based nanoparticulate delivery systems for siRNA is important for the clinical success of gene therapy. However, there are some major drawbacks that need to be overcome. Short interfering RNA (siRNA) has been investigated as a potential therapeutic drug to silence disease-associated genes, but its usage is limited due to the lack of effective and safe nanocarriers. In this study, DOPE-PEI, a nanoparticle consisting of the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) conjugated with low-molecular-weight, 600 Da, branched polyethylenimine (PEI) was produced and optimized for siRNA delivery. This delivery system was modified with other components such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DOPE-PEG2K), DOPE-PEG3.4K-bombesin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane (DOPE/DOTAP) and tested on PC-3 cells. The conjugation of DOPE to PEI polymer (DOPE-PEI) improved the efficiency of PEI to deliver siRNA into the cytosol and knockdown genes, but demonstrated high toxicity. The addition of DOPE-PEG2K reduced cellular toxicity by masking the surface positive charge of the DOPE-PEI/siRNA complex, with the incorporation of a gastrin-releasing peptide receptor (GRPR) targeting peptide and DOPE/DOTAP components improving the cellular uptake of siRNA into targeted cells and the siRNA knockdown efficiency.
Assuntos
Nanopartículas/química , Peptídeos/química , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Portadores de Fármacos/química , Ácidos Graxos Monoinsaturados/química , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Iminas/química , Lipídeos/química , Células PC-3 , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Polietilenos/química , Compostos de Amônio Quaternário/química , Receptores da Bombesina/metabolismoRESUMO
Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.
Assuntos
Aminoácidos/química , Quitosana/química , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , PeptídeosRESUMO
Four groupâ A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.
Assuntos
Adjuvantes Imunológicos/síntese química , Imunoglobulina G/sangue , Lipídeos/imunologia , Vacinas Estreptocócicas/síntese química , Vacinas Estreptocócicas/imunologia , Adjuvantes Imunológicos/química , Animais , Ensaio de Imunoadsorção Enzimática , Lipídeos/síntese química , Lipídeos/química , Camundongos , Tamanho da Partícula , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Vacinas Estreptocócicas/químicaRESUMO
Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure.
Assuntos
Vacinas Bacterianas/química , Vacinas Bacterianas/farmacologia , Lipídeos/química , Peptídeos/química , Streptococcus pyogenes/imunologia , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Camundongos , Microscopia Eletrônica de Transmissão , Relação Estrutura-AtividadeRESUMO
Immunotherapy is one of the most promising strategies for the treatment of cancer. Human papillomavirus (HPV) is responsible for virtually all cases of cervical cancer. The main purpose of a therapeutic HPV vaccine is to stimulate CD8(+) cytotoxic T lymphocytes (CTLs) that can eradicate HPV infected cells. HPV oncoproteins E6 and E7 are continuously expressed and are essential for maintaining the growth of HPV-associated tumor cells. We designed polymer-based multi-antigenic formulations/constructs that were comprised of the E6 and E7 peptide epitopes. We developed an N-terminus-based epitope conjugation to conjugate two unprotected peptides to poly tert-butyl acrylate. This method allowed for the incorporation of the two antigens into a polymeric dendrimer in a strictly equimolar ratio. The most effective formulations eliminated tumors in up to 50% of treated mice. Tumor recurrence was not observed up to 3months post initial challenge.
Assuntos
Antígenos/química , Vacinas contra Papillomavirus/uso terapêutico , Peptídeos/química , Polímeros/química , Neoplasias do Colo do Útero/prevenção & controle , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Epitopos/química , Epitopos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/imunologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Human papillomaviruses (HPVs) are associated with various cancers, with HPV16 linked to more than half of cervical cancer cases. Vaccines to prevent HPV infection and cancer development have proven effective, but are not useful in individuals with prior HPV exposure. Treatment vaccines to eradicate or control HPV-associated lesions are therefore desirable for these patients. Herein we describe the development of a process to enable the production of semisynthetic vaccines based on the site-specific attachment of synthetic bacterial lipid analogs (e.g., Pam2Cys) to a non-oncogenic mutant HPV16 E7 protein to generate molecularly defined vaccines. Many cytotoxic lymphocyte (CTL) epitopes from E7 are delivered by this approach; potentially ensuring that large numbers of immunized individuals can generate CTLs to clear HPV infected cells. Delivery of this construct reduced the growth of HPV16-associated tumors in a TC1 mouse model, the effects of which were better than the potent CTL epitope HPV16 E7(44-57) administered with Montanide ISA51 adjuvant.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Lipopeptídeos/química , Neoplasias/tratamento farmacológico , Proteínas E7 de Papillomavirus/efeitos dos fármacos , Infecções por Papillomavirus/terapia , Proteínas Recombinantes , Adjuvantes Imunológicos/síntese química , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/síntese química , Técnicas de Química Sintética , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Lipopeptídeos/síntese química , Lipopeptídeos/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.