RESUMO
BACKGROUND/AIM: Breast cancer (BC) is the most common malignant disease worldwide. Localized stages of BC can be successfully treated by surgery. However, local recurrence occurs in about 4-10% of patients, requiring systemic treatments that impair the patients' quality of life and shortens life expectancy. Therefore, new therapeutic options are needed, which can be used intraoperatively and contribute to the complete removal of residual tumor cells in the surgical area. In the present study, we describe a cysteine-modified variant of the anti-HER2 antibody trastuzumab, that was coupled to the silicon phthalocyanine photosensitizer dye WB692-CB1 for the photoimmunotherapy (PIT) of BC. MATERIALS AND METHODS: The cysteine modified trastuzumab variant was cloned and expressed in Expi293F cells. After purification via immobilized affinity chromatography, the antibody was coupled to the dye. Cell binding of the antibody and the antibody dye conjugate was measured by flow cytometry. After incubation of BC cells with the conjugate and activation of the dye by irradiation with red light, cell viability was determined. RESULTS: The antibody and the conjugate showed specific binding to HER2-expressing BC cells. Treatment of the HER2high BC cell line SK-BR-3 with the conjugate followed by irradiation with a red light dose of 32 J/cm2 led to complete cell killing within 24 h. CONCLUSION: Our novel antibody dye conjugate represents a promising candidate for intraoperative treatment of localized BC, aiming to eliminate residual tumor cells in the surgical area and potentially reduce local recurrence, thereby improving recovery prospects for BC patients.
Assuntos
Neoplasias da Mama , Imunoterapia , Receptor ErbB-2 , Trastuzumab , Humanos , Neoplasias da Mama/terapia , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Receptor ErbB-2/metabolismo , Imunoterapia/métodos , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Fototerapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Linhagem Celular TumoralRESUMO
BACKGROUND/AIM: The standard treatment for localized prostate cancer involves surgical removal of the prostate with curative intent. However, when tumor cells persist in the operation site, there is high risk of local recurrence and tumor spread, leading to stressful follow-up treatments, impaired quality of life, and reduced overall survival. This study examined photoimmunotherapy (PIT) as a new treatment option for prostate cancer cells. MATERIALS AND METHODS: We generated conjugates consisting of either a humanized antibody or Fab fragments thereof targeting the prostate specific membrane antigen (PSMA), along with our silicon phthalocyanine photosensitizer dye WB692-CB1. PSMA-expressing prostate cancer cells were incubated with the antibody dye or Fab dye conjugates and cell binding was measured using flow cytometry. Cells were irradiated with varying doses of red light for dye activation, and cytotoxicity was determined by erythrosin B staining and subsequent analysis using a Neubauer counting chamber. RESULTS: Specific cytotoxicity was induced with the antibody dye conjugate in the prostate cancer cells in a light dose-dependent manner. Treatment of the cells with the Fab dye conjugate resulted in lower cytotoxicity, which could be attributed to a reduced binding affinity and a reduced dye uptake of the Fab fragment. CONCLUSION: Our new antibody dye and Fab dye conjugates offer potential for future intraoperative PIT in patients with localized prostate cancer, with the aim to ensure complete removal of tumor cells from the surgical area, to avoid local recurrence, and to improve clinical outcome.
Assuntos
Antígenos de Superfície , Fragmentos Fab das Imunoglobulinas , Imunoterapia , Neoplasias da Próstata , Humanos , Masculino , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Neoplasias da Próstata/terapia , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/patologia , Imunoterapia/métodos , Linhagem Celular Tumoral , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Glutamato Carboxipeptidase II/imunologia , Glutamato Carboxipeptidase II/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Fototerapia/métodos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêuticoRESUMO
BACKGROUND/AIM: Although there are curative treatment options for non-muscle-invasive bladder cancer, the recurrence of this tumor is high. Therefore, novel targeted therapies are needed for the complete removal of bladder cancer cells in stages of localized disease, in order to avoid local recurrence, to spare bladder cancer patients from stressful and expensive treatment procedures and to increase their quality of life and life expectancy. This study tested a new approach for the photoimmunotherapy (PIT) of bladder cancer. MATERIALS AND METHODS: We generated a cysteine modified recombinant version of the antibody cetuximab targeting the epidermal growth factor receptor (EGFR) on the surface of bladder cancer cells. Then, we coupled the novel photoactivatable phthalocyanine dye WB692-CB1 via a maleimide linker to the free cysteines of the antibody. PIT was performed by incubating bladder cancer cells with the antibody dye conjugate followed by irradiation with visible red light. RESULTS: The conjugate was able to induce specific cytotoxicity in EGFR-positive bladder cancer cells in a light dose-dependent manner. Enhanced cytotoxicity in RT112 bladder cancer cells was evoked by addition of a second antibody dye conjugate targeting HER2 or by repeated cycles of PIT. CONCLUSION: Our new antibody dye conjugate targeting EGFR-expressing bladder cancer cells is a promising candidate for the future PIT of bladder cancer patients.
Assuntos
Receptores ErbB , Imunoconjugados , Imunoterapia , Receptor ErbB-2 , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/terapia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Receptores ErbB/imunologia , Receptores ErbB/antagonistas & inibidores , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Imunoterapia/métodos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Linhagem Celular Tumoral , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Cetuximab/química , Fototerapia/métodosRESUMO
Many membrane proteins utilize dimerization to transmit signals across the cell membrane via regulation of the lateral binding affinity. The complexity of natural membrane proteins hampers the understanding of this regulation on a biophysical level. We designed simplified membrane proteins from well-defined soluble dimerization domains with tunable affinities, flexible linkers, and an inert membrane anchor. Live-cell single-molecule imaging demonstrates that their dimerization affinity indeed depends on the strength of their binding domains. We confirm that as predicted, the 2-dimensional affinity increases with the 3-dimensional binding affinity of the binding domains and decreases with linker lengths. Models of extended and coiled linkers delineate an expected range of 2-dimensional affinities, and our observations for proteins with medium binding strength agree well with the models. Our work helps in understanding the function of membrane proteins and has important implications for the design of synthetic receptors.
Assuntos
Proteínas de Membrana , Membrana Celular , Dimerização , MembranasRESUMO
A library of 40,000 compounds was screened for inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high-throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC(50) values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC(50) values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.
Assuntos
Antibacterianos/química , Antimaláricos/química , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , Plasmodium falciparum/enzimologia , Pirimidinas/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Antimaláricos/síntese química , Antimaláricos/farmacologia , Arabidopsis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Conformação Molecular , Fósforo-Oxigênio Liases/antagonistas & inibidores , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC(50) of 4 mum (K(i) = 1 mum). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-(13)C(6)]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [(13)C(3)]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC(-) mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1(-) and YscM2(-) mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a "load-and-shoot cycle" model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.
Assuntos
Proteínas de Bactérias/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Via Secretória/fisiologia , Fatores de Transcrição/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidade , Aminoácidos/biossíntese , Aminoácidos/genética , Proteínas de Bactérias/genética , Cálcio/metabolismo , Cálcio/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Ácido Pirúvico/metabolismo , Via Secretória/efeitos dos fármacos , Edulcorantes/metabolismo , Edulcorantes/farmacologia , Fatores de Transcrição/genética , Yersinia enterocolitica/genéticaRESUMO
Enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are therapeutic targets for the treatment of important infectious diseases. Whereas this pathway is absent in humans, it is used by plants, many eubacteria and apicomplexan protozoa, including major human pathogens such as Plasmodium falciparum and Mycobacterium tuberculosis. Herein, we report on the design, preparation and biological evaluation of a new series of ligands for IspE protein, a kinase from this pathway. These inhibitors were developed for the inhibition of IspE from Escherichia coli, using structure-based design approaches. Structure-activity relationships (SARs) and a co-crystal structure of Aquifex aeolicus IspE bound to a representative inhibitor validate the proposed binding mode. The crystal structure shows that the ligand binds in the substrate-rather than the adenosine 5'-triphosphate (ATP)-binding pocket. As predicted, a cyclopropyl substituent occupies a small cavity not used by the substrate. The optimal volume occupancy of this cavity is explored in detail. In the co-crystal structure, a diphosphate anion binds to the Gly-rich loop, which normally accepts the triphosphate moiety of ATP. This structure provides useful insights for future structure-based developments of inhibitors for the parasite enzymes.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Modelos Biológicos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/química , Concentração Inibidora 50 , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Relação Estrutura-AtividadeRESUMO
2C-Methyl-D-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1-deoxy-D-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-D-erythritol 4-phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol-aldol sequence. We report the preparation of (13)C-labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol-aldol reaction sequence. In reaction mixtures containing large amounts of 2C-methyl-D-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1-(13)C(1)]-2C-methyl-D-erythritol 4-phosphate and [3-(13)C(1)]2C-methyl-D-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-D-xylulose 5-phosphate to give 1-deoxy-L-ribulose 5-phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol-aldol mechanism cannot be ruled out conclusively, the data show that a retroldol-aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.
Assuntos
Aldose-Cetose Isomerases/química , Complexos Multienzimáticos/química , Oxirredutases/química , Terpenos/metabolismo , Aldose-Cetose Isomerases/classificação , Aldose-Cetose Isomerases/metabolismo , Arabidopsis/enzimologia , Catálise , Complexos Multienzimáticos/classificação , Complexos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/enzimologia , Ressonância Magnética Nuclear Biomolecular , Oxirredutases/classificação , Oxirredutases/metabolismo , Filogenia , EstereoisomerismoRESUMO
4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP alpha-phosphate not the binding site for the methyl-D-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Bioquímica/métodos , Química Farmacêutica/métodos , Desenho de Fármacos , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Ligação ProteicaRESUMO
The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co-crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co-crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well-conserved ATP-binding Gly-rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure-based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.
Assuntos
Citidina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Ácido Mevalônico/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Terpenos/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Citidina/análogos & derivados , Citidina/síntese química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fenilalanina/química , Fenilalanina/metabolismo , Plasmodium falciparum/crescimento & desenvolvimentoAssuntos
Antimaláricos/síntese química , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/síntese química , Proteínas de Escherichia coli/antagonistas & inibidores , Ácido Mevalônico/química , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Terpenos/química , Antimaláricos/uso terapêutico , Ligação Competitiva , Desenho de Fármacos , Inibidores Enzimáticos/uso terapêutico , Proteínas de Escherichia coli/química , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/química , Ácido Mevalônico/metabolismo , Fosfatos/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Terpenos/metabolismo , Thermus thermophilus/enzimologia , Transferases/antagonistas & inibidores , Transferases/químicaRESUMO
The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1).