RESUMO
A new dual-functional implant based on gellan-xanthan hydrogel with calcium-magnesium silicate ceramic diopside and recombinant lysostaphin and bone morphogenetic protein 2 (BMP-2)-ray is developed. In this composite, BMP-2 is immobilized on microparticles of diopside while lysostaphin is mixed directly into the hydrogel, providing sustained release of BMP-2 to allow gradual bone formation and rapid release of lysostaphin to eliminate infection immediately after implantation. Introduction of diopside of up to 3% (w/v) has a negligible effect on the mechanical properties of the hydrogel but provides a high sorption capacity for BMP-2. The hydrogels show good biocompatibility and antibacterial activity. Lysostaphin released from the implants over a 3 h period efficiently kills planktonic cells and completely destroys 24 h pre-formed biofilms of Staphylococcus aureus. Furthermore, in vivo experiments in a mouse model of critically-sized cranial defects infected with S. aureus show a complete lack of osteogenesis when implants contain only BMP-2, whereas, in the presence of lysostaphin, complete closure of the defect with newly formed mineralized bone tissue is observed. Thus, the new implantable gellan-xanthan hydrogel with diopside and recombinant lysostaphin and BMP-2 shows both osteogenic and antibacterial properties and represents a promising material for the treatment and/or prevention of osteomyelitis after bone trauma.
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The development of new and effective antibacterials for pharmaceutical or cosmetic skin care that have a low potential for the emergence and expansion of bacterial resistance is of high demand in scientific and applied research. Great hopes are placed on alternative agents such as bactericidal peptidoglycan hydrolases, depolymerases, etc. Enzybiotic-based preparations are being studied for the treatment of various infections and, among others, can be used as topical formulations and dressings with protein-polysaccharide complexes. Here, we investigate the antibiofilm properties of a novel enzybiotic cocktail of phage endolysin LysSi3 and bacteriocin lysostaphin, formulated in the alginate gel matrix and its ability to control the opportunistic skin-colonizing bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as mixed-species biofilms. Our results propose that the application of SiL-gel affects different components of biofilm extracellular polymeric substances, disrupts the matrix, and eliminates the bacteria embedded in it. This composition is highly effective against biofilms composed of Gram-negative and Gram-positive species and does not possess significant cytotoxic effects. Our data form the basis for the development of antibacterial skin care products with a gentle but effective mode of action.
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Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.
Assuntos
Endopeptidases , Insulisina , Humanos , Animais , Cavalos , Endopeptidases/genética , Sequência de Aminoácidos , Biotecnologia , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , ImunossupressoresRESUMO
Lysostaphin is a zinc-dependent endopeptidase that is effective against both antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus aureus. Lysostaphin is typically purified on cation-exchange or metal-chelate affinity resins, and there are data indicating potential influence of the chromatographic resin on the lysostaphin activity. In this study, we systematically investigated the impact of the resin used to purify the recombinant lysostaphin on its activity. To this end, recombinant lysostaphin with an additional histidine tag at the C-terminus was purified using a cation-exchange resin, three types of nickel-chelate resins with different strength of metal ion binding, or a zinc-chelate resin. Lysostaphin samples purified on the cation-exchange resin (WorkBeads 40S), the nickel-chelate resin with a strong nickel ion binding (WorkBeads NiMAC), and the zinc-chelate resin (WorkBeads NTA with immobilized zinc ions) had equal activity. On the contrary, the activity of lysostaphin preparations purified on nickel-chelate resins with medium (WorkBeads Ni-NTA) and relatively weak (WorkBeads Ni-IDA) nickel ion binding was significantly reduced. The decrease in activity can be explained by the interaction of lysostaphin with the nickel ions leached from the resin and is caused by either the exchange of the zinc ion in the lysostaphin active center with a nickel ion from the resin, or binding of an additional ion that inhibits the enzymatic activity. Removal of the metal ions from the active site of lysostaphin and subsequent incorporation of the native zinc ions lead to complete restoration of the activity of the enzyme.
Assuntos
Lisostafina , Níquel , Níquel/química , Metais/química , Quelantes/química , Zinco/química , Cromatografia de Afinidade/métodos , AntibacterianosRESUMO
Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Dissulfetos , Epitopos , Humanos , Imunização , Camundongos , Proteínas Recombinantes/genética , SARS-CoV-2RESUMO
A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Epitopos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , COVID-19/genética , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/isolamento & purificação , Vacinas contra COVID-19/farmacologia , Epitopos/genética , Epitopos/imunologia , Epitopos/isolamento & purificação , Epitopos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/farmacologiaRESUMO
Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.
RESUMO
The spread of bacterial strains resistant to commonly used antibiotics urges the development of novel antibacterial compounds. Ideally, these novel antimicrobials should be less prone to the development of resistance. Peptidoglycan-degrading enzymes are a promising class of compounds with a fundamentally different mode of action compared to traditionally used antibiotics. The difference in the mechanism of action implies differences both in the mechanisms of resistance and the chances of its emergence. To critically assess the potential of resistance development to peptidoglycan-degrading enzymes, we review the available evidence for the development of resistance to these enzymes in vitro, along with the known mechanisms of resistance to lysozyme, bacteriocins, autolysins, and phage endolysins. We conclude that genetic determinants of resistance to peptidoglycan-degrading enzymes are unlikely to readily emerge de novo. However, resistance to these enzymes would probably spread by the horizontal transfer between intrinsically resistant and susceptible species. Finally, we speculate that the higher cost of the therapeutics based on peptidoglycan degrading enzymes compared to classical antibiotics might result in less misuse, which in turn would lead to lower selective pressure, making these antibacterials less prone to resistance development.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Farmacorresistência Bacteriana , Enzimas/farmacologia , Peptidoglicano/química , Animais , Bactérias/metabolismo , Bactérias/virologia , Infecções Bacterianas/microbiologia , Bacteriófagos/enzimologia , Bacteriófagos/fisiologia , Humanos , Peptidoglicano/metabolismoRESUMO
Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.
Assuntos
Proteínas de Bactérias , Lisostafina , Proteínas Recombinantes de Fusão , Albumina Sérica/química , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacocinética , Proteínas de Bactérias/farmacologia , Feminino , Lisostafina/química , Lisostafina/genética , Lisostafina/farmacocinética , Lisostafina/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.
Assuntos
Antibacterianos/química , Antibacterianos/farmacocinética , Lisostafina/química , Lisostafina/farmacocinética , Multimerização Proteica , Sequência de Aminoácidos , Área Sob a Curva , Catálise , Ativação Enzimática , Lisostafina/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Proteica , Staphylococcus/efeitos dos fármacosRESUMO
BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Ilhas de CpG , Dextranos/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genéticaRESUMO
BACKGROUND: The control of genome stability is relevant for the worldwide BCG vaccine preventing the acute forms of childhood tuberculosis. BCG sub-strains whole genome comparative analysis and revealing the triggers of sub-strains transition were the purpose of our investigation. RESULTS: Whole genome sequencing of three BCG Russia seed lots (1963, 1982, 2006 years) confirmed the stability of vaccine sub-strain genome. Comparative analysis of three Mycobacteruim bovis and nine M. bovis BCG genomes shown that differences between "early" and "late" sub-strains BCG genomes were associated with specific prophage profiles. Several prophages common to all BCG genomes included ORFs which were homologues to Caudovirales. Surprisingly very different prophage profiles characterized BCG Tice and BCG Montreal genomes. These prophages contained ORFs which were homologues to Herpesviruses. Phylogeny of strains cohort based on genome maps restriction analysis and whole genomes sequence data were in agreement with prophage profiles. Pair-wise alignment of unique BCG Tice and BCG Montreal prophage sequences and BCG Russia 368 genome demonstrated only similarity of fragmetary sequences that suggested the contribution of prophages in genome mosaic structure formation. CONCLUSIONS: Control of the extended sequences is important for genome with mosaic structure. Prophage search tools are effective instruments in this analysis.
Assuntos
Bacteriófagos/genética , Elementos de DNA Transponíveis , Genoma Bacteriano , Mycobacterium bovis/genética , Sequência de Aminoácidos , Vacina BCG/genética , Vacina BCG/imunologia , Biologia Computacional/métodos , DNA Bacteriano , Ordem dos Genes , Rearranjo Gênico , Genoma Viral , Instabilidade Genômica , Genômica/métodos , Anotação de Sequência Molecular , Mycobacterium bovis/classificação , Mycobacterium bovis/imunologia , Mycobacterium bovis/virologia , FilogeniaRESUMO
BACKGROUND AND AIM: The order Burkholderiales became more abundant in the healthcare units since the late 1970s; it is especially dangerous for intensive care unit patients and patients with chronic lung diseases. The goal of this investigation was to reveal the real variability of the order Burkholderiales representatives and to estimate their phylogenetic relationships. METHODS: 16S rDNA and genes of the Burkholderia cenocepacia complex (Bcc) Multi Locus Sequence Typing (MLST) scheme were used for the bacteria detection. RESULTS: . A huge diversity of genome size and organization was revealed in the order Burkholderiales that may prove the adaptability of this taxon's representatives. The following variability of the Burkholderiales in Russian healthcare units has been revealed: Burkholderiaceae (Burkholderia, Pandoraea, and Lautropia), Alcaligenaceae (Achromobacter), and Comamonadaceae (Variovorax). The Burkholderia genus was the most diverse and was represented by 5 species and 16 sequence types (ST). ST709 and 728 were transmissible and often encountered in cystic fibrosis patients and in hospitals. A. xylosoxidans was estimated by 15 genotypes. The strains of first and second ones were the most numerous. CONCLUSIONS: Phylogenetic position of the genus Lautropia with smaller genome is ambiguous. The Bcc MLST scheme is applicable for all Burkholderiales representatives for resolving the epidemiological problems.
Assuntos
Infecções por Burkholderia/genética , Burkholderiaceae/genética , Filogenia , RNA Ribossômico 16S/genética , Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/microbiologia , Burkholderiaceae/patogenicidade , Variação Genética , Genótipo , Humanos , Especificidade da EspécieRESUMO
BACKGROUND AND AIM: Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. METHODS: The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). RESULTS: The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. CONCLUSIONS: Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions.
Assuntos
Leptospira/genética , Leptospirose/microbiologia , Loci Gênicos , Genoma Bacteriano/genética , Genótipo , Leptospira/ultraestrutura , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Fenótipo , Filogenia , Federação Russa , Análise de Sequência de DNARESUMO
Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 µm) and activated factor XI (Ki = 94 ± 11 µm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 µm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.
Assuntos
Fator XIa/antagonistas & inibidores , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Zea mays , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/metabolismo , Tripsina/metabolismoRESUMO
Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.
Assuntos
Enzimas de Restrição do DNA/genética , Genoma Arqueal , Genoma Bacteriano , Metilases de Modificação do DNA/genética , Enzimas de Restrição do DNA/classificação , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , GenômicaRESUMO
PURPOSE: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). METHODS AND MATERIALS: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. RESULTS: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines. CONCLUSION: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.
Assuntos
Astato/uso terapêutico , Núcleo Celular/efeitos da radiação , Portadores de Fármacos/uso terapêutico , Receptores ErbB/metabolismo , Partículas alfa/uso terapêutico , Antígenos Transformantes de Poliomavirus/metabolismo , Astato/farmacocinética , Benzoatos/farmacocinética , Benzoatos/uso terapêutico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Intervalos de Confiança , Di-Hidropteridina Redutase/farmacocinética , Toxina Diftérica/farmacocinética , Portadores de Fármacos/farmacocinética , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Proteínas de Escherichia coli/farmacocinética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Guanidina/análogos & derivados , Guanidina/farmacocinética , Guanidina/uso terapêutico , Guanidinas/farmacocinética , Guanidinas/uso terapêutico , Hemeproteínas/farmacocinética , Humanos , NADH NADPH Oxirredutases/farmacocinética , Radioimunoterapia/métodosRESUMO
A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed high-affinity interactions with ErbB1 receptors and alpha/beta-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and (*)OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications.
Assuntos
Núcleo Celular/metabolismo , Di-Hidropteridina Redutase/administração & dosagem , Toxina Diftérica/administração & dosagem , Fator de Crescimento Epidérmico/administração & dosagem , Receptores ErbB/metabolismo , Proteínas de Escherichia coli/administração & dosagem , Hemeproteínas/administração & dosagem , NADH NADPH Oxirredutases/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Células NIH 3T3 , Sinais de Localização Nuclear , Espécies Reativas de Oxigênio , Proteínas Recombinantes/administração & dosagemRESUMO
The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities. Thus, photosensitizers (PSs) used in anti-cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced. Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) alpha-melanocyte-stimulating hormone as an internalizable, cell-specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T-antigen; 3) an Escherichia coli hemoglobin-like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin. These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non-MSH receptor-overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs. The modular fusion proteins described here for the first time, capable of cell-specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.