RESUMO
Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as "TIGER") utilizes PCR with broad-range primers to amplify products from a wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses allows for calculations of base compositions for the broad-range PCR products, which can then be compared to a database for identification. PCR/ESI-MS has the benefits of PCR in sensitivity and high-throughput capacity, but also has the distinct advantage of being able to detect and identify organisms with no prior characterization or sequence data. Existing broad range PCR primers, designed with an emphasis on human pathogens, were tested for their ability to amplify DNA of well characterized phytobacterial strains, as well as to populate the existing PCR/ESI-MS bacterial database with base counts. In a blinded panel study, PCR/ESI-MS successfully identified 93% of unknown bacterial DNAs to the genus level and 73% to the species/subspecies level. Additionally, PCR/ESI-MS was capable of detecting and identifying multiple bacteria within the same sample. The sensitivity of PCR/ESI-MS was consistent with other PCR based assays, and the specificity varied depending on the bacterial species. Preliminary tests with real life samples demonstrate a high potential for using PCR/ESI-MS systems for agricultural diagnostic applications.
Assuntos
Bactérias/genética , Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Bactérias/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Reprodutibilidade dos TestesRESUMO
Cancer therapy based on the delivery of enzymes to tumour sites has advanced in several directions since antibody-directed enzyme/prodrug therapy was first described. It has been shown that methoxypolyethylene glycol (MPEG) can be used to deliver enzyme to a variety of solid tumours. MPEG-enzyme conjugates show reduced immunogenicity and may allow repeated treatment with enzymes of bacterial origin. Enzyme delivery to tumours by polymers can be used to convert a low toxicity prodrug to a potent cytotoxic agent. An example of such a prodrug is CB1954, which can be activated by a human enzyme in the presence of a cosubstrate. Tumour-located enzymes can also be used in conjunction with a combination of antimetabolites and rescue agents. The rescue agent protects normal tissue but is degraded at cancer sites by the enzyme, thus deprotecting the tumour and allowing prolonged antimetabolite action.
Assuntos
Antineoplásicos/uso terapêutico , Terapia Enzimática , Neoplasias/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Aziridinas/uso terapêutico , Portadores de Fármacos/uso terapêutico , Humanos , Imunoconjugados/uso terapêutico , Polietilenoglicóis/uso terapêutico , Pró-Fármacos/metabolismo , gama-Glutamil Hidrolase/uso terapêuticoRESUMO
BACKGROUND: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G2 (CPG2), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. RESULTS: We have determined the crystal structure of CPG2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. CONCLUSIONS: We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.
Assuntos
Antineoplásicos/química , Proteínas de Bactérias/química , Conformação Proteica , gama-Glutamil Hidrolase/química , Aminopeptidases/química , Antineoplásicos/uso terapêutico , Proteínas de Bactérias/uso terapêutico , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dimerização , Evolução Molecular , Exopeptidases , Leucil Aminopeptidase/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/classificação , Pseudomonas/enzimologia , Relação Estrutura-Atividade , Zinco/química , gama-Glutamil Hidrolase/uso terapêuticoRESUMO
The use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed "antibody-directed enzyme prodrug therapy" (ADEPT), has attracted considerable interest since the concept was first described in 1987. Prodrug forms of both clinically used anticancer agents and novel cytotoxic compounds have been developed to take advantage of potential prodrug-generating technology employing a variety of enzymes with widely differing substrate specificities. A particular advantage of the ADEPT approach is that it may allow the use of extremely potent agents such as nitrogen mustards and palytoxin, which are too toxic to be readily used in conventional chemotherapy. Preliminary studies using an antibody-enzyme conjugate constructed with a bacterial enzyme and a murine monoclonal antibody not only have established the value of the ADEPT technique, but also have highlighted the potential problem of immunogenicity of proteins of nonhuman origin. This problem has been tackled in the first instance by the use of immunosuppressive agents, but long-term solutions are being investigated in the development of second-generation ADEPT systems, including the development of human antibody-human enzyme fusion proteins and catalytic antibodies. Such improvements, coupled with further refinement of the prodrug-drug element of the system and the wide variety of antibody-enzyme-drug combinations available, should mean that ADEPT-based approaches will form an important element of the search for the anticancer drugs of the future.
Assuntos
Imunoconjugados/uso terapêutico , Pró-Fármacos/administração & dosagem , Fosfatase Alcalina/administração & dosagem , Amidoidrolases/administração & dosagem , Aminopeptidases/administração & dosagem , Carboxipeptidases/administração & dosagem , Glicosídeo Hidrolases/administração & dosagem , Humanos , Nitrorredutases/administração & dosagem , Nucleosídeo Desaminases/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , beta-Lactamases/administração & dosagemRESUMO
The specific activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase was measured in lymphocytes isolated from the blood of normal subjects, from patients with chronic lymphocytic leukemia, and from tonsil tissue. The mean specific activity of cyclic adenosine 3':5'-monophosphate phosphodiesterase in the lymphocytes from patients with untreated chronic lymphocytic leukemia was lower than that in lymphocytes from the blood of normal subjects or from tonsils. Cyclic adenosine 3':5'-monophosphate phosphodiesterase levels did not correlate with differences in B- and T-cell lymphocyte subpopulations or with peripheral blood lymphocyte counts.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/sangue , Leucemia Linfoide/enzimologia , Linfócitos/enzimologia , Diester Fosfórico Hidrolases/sangue , Linfócitos B/enzimologia , Humanos , Contagem de Leucócitos , Linfócitos T/enzimologiaRESUMO
A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.
Assuntos
Antineoplásicos/farmacocinética , Aziridinas/farmacocinética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Pró-Fármacos/farmacocinética , Animais , Antineoplásicos/toxicidade , Aziridinas/toxicidade , Biotransformação , Linhagem Celular , Cricetinae , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacologia , Compostos de Piridínio/metabolismo , Compostos de Piridínio/farmacologia , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
The enzyme carboxypeptidase G2 (CPG2) was conjugated to the rat IgG2a monoclonal antibody (mAb) ICR12, which recognizes the external domain of the human c-erbB2 protooncogene product. The conjugate retained antigen-binding and enzyme activity. Radiolabeled conjugate localized efficiently and stably to MDA MB 361 breast carcinoma xenografts, which overexpress the c-erbB2 gene product p185. Radiotracer determinations and plasma enzyme activity studies in nu/nu mice gave conjugate blood clearance rate half-lives of approximately 4 days. In separate antibody-directed enzyme prodrug therapy regimes, one dose of the 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid prodrug was administered to nu/nu mice bearing established MDA MB 361 tumors (mean volume, 170-260 mm3). In mice which had received ICR12-CPG2 12-14 days previously, sustained dose-dependent tumor stasis or regressions were effected, which in some cases persisted throughout observation periods of up to 90 days. In control mice which had received the isotype-matched irrelevant mAb ICR16-CPG2 conjugate, tumors grew progressively, as did those in mice treated with prodrug alone, or treated simultaneously with ICR12-CPG2 and prodrug at the maximum tolerated dose. Control chemotherapy with conventional drugs proved toxic and induced only minimal growth delays.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Receptor ErbB-2/imunologia , gama-Glutamil Hidrolase/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Transplante de Neoplasias , Ratos , Transplante HeterólogoRESUMO
ZD2767 represents an improved version of antibody-directed enzyme prodrug therapy. It consists of a conjugate of the F(ab')2 A5B7 antibody fragment and carboxypeptidase G2 (CPG2) and a prodrug, 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid. The IC50 of the prodrug against LoVo colorectal tumor cells was 47 microM, and cleavage by CPG2 released the potent bis-iodo phenol mustard drug (IC50 = 0.34 microM). The drug killed both proliferating and quiescent LoVo cells. Administration of the ZD2767 conjugate to nude mice bearing LoVo colorectal xenografts resulted in approximately 1% of injected ZD2767 conjugate localizing/g of tumor after 72 h, and blood and normal tissue levels of the conjugate were 10-50-fold lower. A single round of therapy involving the administration of the prodrug 72 h after the conjugate to athymic mice bearing established LoVo xenografts resulted in approximately 50% of the tumors undergoing complete regressions, tumor growth delays greater than 30 days, and little toxicity (as judged by body-weight loss). Similar studies using a control antibody-CPG2 conjugate that does not bind to LoVo tumor cells resulted in a growth delay of less than 5 days, confirming the tumor specificity of this approach. These studies demonstrate the potential of ZD2767 for the treatment of colorectal cancer.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Compostos de Mostarda Nitrogenada/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Feminino , Humanos , Imunotoxinas/administração & dosagem , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante Heterólogo , gama-Glutamil Hidrolase/metabolismoRESUMO
An FMN-dependent nitroreductase enzyme isolated from Escherichia coli B has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with cell parameters a = b = 57.74 A, c = 275.51 A and two molecules per asymmetric unit. Diffraction extends to beyond 1.9 A.
Assuntos
Escherichia coli/enzimologia , Nitrorredutases/química , Cristalização , Cristalografia por Raios X , Mononucleotídeo de Flavina , Estrutura MolecularRESUMO
The full three-dimensional structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent, sulfonamide hydroxamic acid inhibitor (CGS 27023) has been determined by NMR spectroscopy. The results reveal a core domain for the protein consisting of three alpha-helices and five beta-sheet strands with an overall tertiary fold similar to the catalytic domains of other matrix metalloproteinase family members. The S1' pocket, which is the major site of hydrophobic binding interaction, was found to be a wide cleft spanning the length of the protein and presenting facile opportunity for inhibitor extension deep into the pocket. Comparison with the reported X-ray structure of collagenase-3 showed evidence of flexibility for the loop region flanking the S1' pocket in both NMR and X-ray data. This flexibility was corroborated by NMR dynamics studies. Inhibitor binding placed the methoxy phenyl ring in the S1' pocket with the remainder of the molecule primarily solvent-exposed. The binding mode for this inhibitor was found to be similar with respect to stromelysin-1 and collagenase-1; however, subtle comparative differences in the interactions between inhibitor and enzyme were observed for the three MMPs that were consistent with their respective binding potencies.
Assuntos
Colagenases/química , Inibidores Enzimáticos/química , Ácidos Hidroxâmicos/química , Metaloproteinase 1 da Matriz/química , Metaloproteinase 3 da Matriz/química , Sulfonamidas/química , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Espectroscopia de Ressonância Magnética , Metaloproteinase 13 da Matriz , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Many plants produce constitutive antifungal molecules belonging to the saponin family of secondary metabolites, which have been implicated in plant defense. Successful pathogens of these plants must presumably have some means of combating the chemical defenses of their hosts. In the oat root pathogen Gaeumannomyces graminis, the saponin-detoxifying enzyme avenacinase has been shown to be essential for pathogenicity. A number of other phytopathogenic fungi also produce saponin-degrading enzymes, although the significance of these for saponin resistance and pathogenicity has not yet been established. The tomato leaf spot pathogen Septoria lycopersici secretes the enzyme tomatinase, which degrades the tomato steroidal glycoalkaloid alpha-tomatine. Here we report the isolation and characterization of tomatinase-deficient mutants of S. lycopersici following targeted gene disruption. Tomatinase-minus mutants were more sensitive to alpha-tomatine than the wild-type strain. They could, however, still grow in the presence of 1 mM alpha-tomatine, suggesting that nondegradative mechanisms of tolerance are also important. There were no obvious effects of loss of tomatinase on macroscopic lesion formation on tomato leaves, but trypan blue staining of infected tissue during the early stages of infection revealed more dying mesophyll cells in leaves that had been inoculated with tomatinase-minus mutants. Expression of a defense-related basic beta-1,3 glucanase gene was also enhanced in these leaves. These differences in plant response may be associated with subtle differences in the growth of the wild-type and mutant strains during infection. Alternatively, tomatinase may be involved in suppression of plant defense mechanisms.
Assuntos
Glicosídeo Hidrolases/genética , Fungos Mitospóricos/enzimologia , Fungos Mitospóricos/patogenicidade , Solanum lycopersicum/microbiologia , Solanum lycopersicum/fisiologia , Avena/microbiologia , Resistência a Medicamentos , Deleção de Genes , Magnaporthe/patogenicidade , Fungos Mitospóricos/genética , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , Tomatina/metabolismo , Tomatina/toxicidadeRESUMO
The anti-fungal, steroidal, glycoalkaloid saponin, alpha-tomatine, is present in uninfected tomato plants in substantial concentrations, and may contribute to the protection of tomato plants against attack by phytopathogenic fungi. In general, successful fungal pathogens of tomato are more resistant to alpha-tomatine in vitro than fungi that do not infect this plant. For a number of tomato pathogens, this resistance has been associated with the ability to detoxify alpha-tomatine through the action of enzymes known as tomatinases. In contrast, the biotrophic tomato pathogen Cladosporium fulvum is sensitive to alpha-tomatine and is unable to detoxify this saponin. This paper describes the effects of heterologous expression of the cDNA encoding tomatinase from the necrotroph Septoria lycopersici in two different physiological races of C. fulvum. Tomatinase-producing C. fulvum transformants showed increased sporulation on cotyledons of susceptible tomato lines. They also caused more extensive infection of seedlings of resistant tomato lines. Thus, alpha-tomatine may contribute to the ability of tomato to restrict the growth of C. fulvum in both compatible and incompatible interactions.
Assuntos
Cladosporium/fisiologia , Glicosídeo Hidrolases/biossíntese , Fungos Mitospóricos/fisiologia , Solanum lycopersicum/microbiologia , Solanum lycopersicum/fisiologia , Tomatina/metabolismo , Tomatina/farmacologia , Antifúngicos/farmacologia , Cladosporium/efeitos dos fármacos , Cladosporium/patogenicidade , Cotilédone , Fungos Mitospóricos/efeitos dos fármacos , Esporos FúngicosRESUMO
A plasmid vector, pIJ699, which provides positive selection for cloned DNA, was constructed using the replication functions of the Streptomyces wide-host-range multi-copy plasmid pIJ101. The selection for inserts is based on the principle that plasmids with long uninterrupted perfect palindromes (inverted repeats) are 'not viable' in bacteria. For cloning, pIJ699 is digested with BglII. This produces two fragments, one of which is the linearized vector, with two arms of the palindrome at its ends, and the other is a 'spacer' which is needed to keep the inverted repeat sequences apart. The vector fragment is separated from the 'spacer' fragment and ligated with the DNA to be cloned. Plasmids with a fragment of cloned DNA, but not the circularized vector, give rise to thiostrepton-resistant transformants in Streptomyces lividans. The inverted repeat sequences contain a strong transcription terminator which reduces transcriptional read-through both in and out of the cloned fragment. This improves the stability of many hybrid plasmids and facilitates the study of the regulation of cloned genes.
Assuntos
Vetores Genéticos , Plasmídeos , Streptomyces/genética , Clonagem Molecular , Enzimas de Restrição do DNA , Mapeamento de Nucleotídeos , Regiões Terminadoras GenéticasRESUMO
Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines. A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells. Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase. Significant bystander cell killing was dependent on a high cell density. In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.
Assuntos
Antineoplásicos/uso terapêutico , Aziridinas/uso terapêutico , Neoplasias Colorretais/terapia , Terapia Genética , Nitrorredutases/genética , Neoplasias Pancreáticas/terapia , Pró-Fármacos/uso terapêutico , Antineoplásicos/farmacologia , Aziridinas/farmacologia , Relação Dose-Resposta a Droga , Escherichia coli/genética , Vetores Genéticos , Humanos , Pró-Fármacos/farmacologia , Retroviridae , Células Tumorais CultivadasRESUMO
Increased tumour uptake of antibodies and antibody-drug conjugates has been demonstrated following pretreatment of animals with recombinant human tumour necrosis factor-alpha (rTNF-alpha) and interleukin 2 immunoconjugates. The experiments reported here were performed to determine whether improved tumour localisation of antibody-carboxypeptidase G2 conjugates could be achieved, with a view to applying this technology to antibody-directed enzyme-prodrug therapy (ADEPT). B6CF1 mice bearing the Ly-2.1+ murine thymoma E3 were simultaneously injected with 2.0 micrograms rTNF-alpha and 3.5 micrograms (74kBq) 125I-labelled murine anti-Ly-2.1-CPG2 conjugate. Mice in control groups received phosphate buffered saline in place of rTNF-alpha. The conjugate corresponded in molecular weight to a mixture of 1:1 and 2:1 (CPG2:IgG) conjugate and retained its antigen binding specificity and enzymic activity in vitro. A significant increase in tumour uptake was observed 24 h after administration when rTNF-alpha-treated animals were compared to controls (28.1 +/- 9.7%/g and 11.6 +/- 2.3%/g, respectively). Other tissues, most notably gut, skin and kidney also showed an increased localisation of conjugate. By 48 h, analysis of tissue:blood ratios demonstrated that although tumour:blood ratios were significantly higher in rTNF-alpha-treated animals (P < 0.05), all the other tissue:blood ratios were not significantly different between the two groups.
Assuntos
Carboxipeptidases/metabolismo , Imunoglobulina G/metabolismo , Imunotoxinas/metabolismo , Interleucina-2/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Eletroforese em Gel de Poliacrilamida , Camundongos , Camundongos Endogâmicos , Timoma/metabolismo , Timoma/terapia , Neoplasias do Timo/metabolismo , Neoplasias do Timo/terapia , Fatores de Tempo , Distribuição TecidualRESUMO
Three novel prodrugs have been designed for use as anticancer agents. Each is a bifunctional alkylating agent which has been protected to form a relatively inactive prodrug. They are designed to be activated to their corresponding alkylating agents at a tumour site by prior administration of an antitumour antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in a two-phase system called antibody-directed enzyme prodrug therapy (ADEPT). The Km and Vmax values for three different antibody-CPG2 conjugates were determined in relation to each prodrug. The Km values ranged from 4.5-12 mumol/l and the Vmax from 0.5-1.6 mumol/U/min. Athymic Nu/Nu mice with palpable transplanted human choriocarcinoma xenografts, which are resistant to conventional chemotherapy, were treated with anti-human chorionic gonadotropin antibodies conjugated to CPG2. This was followed by each of the three novel prodrugs. Significant increase in survival was obtained in three of the regimens tested using only one course of treatment. This demonstrates the potential of a tumour-localised bacterial enzyme to activate protected alkylating agents in order to eradicate an established human xenograft.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/uso terapêutico , Coriocarcinoma/tratamento farmacológico , Pró-Fármacos/uso terapêutico , gama-Glutamil Hidrolase/administração & dosagem , Alquilantes/uso terapêutico , Animais , Coriocarcinoma/mortalidade , Gonadotropina Coriônica/imunologia , Portadores de Fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante HeterólogoRESUMO
Conjugates of F(ab')2 fragment of the monoclonal antibody A5B7 coupled to carboxypeptidase G2 (CPG2) have been produced using the heterobifunctional reagents 2-mercapto-[S-acetyl]acetic acid, N-hydroxysuccinimide ester (SATA) and m-maleimidobenzoyl-N-hydroxysuccinimide ester (SMPB). The effect of various levels of modifying reagent on enzyme activity and antigen binding activity were determined, and it was shown that whilst CPG2 is relatively sensitive to modification, insertion of three maleimide groups per CPG2 resulted in the loss of 30% of enzyme activity; A5B7 F(ab')2 was insensitive to modification, little or no activity being lost. The coupling efficiency of the reaction was shown to be fairly constant over a wide range of substitution levels. There was thus no advantage to be gained in using high substitution levels, which may result in loss of enzyme activity. The formation of undesired high molecular weight aggregates could be controlled by adjustment of the protein concentration during the final coupling step.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/síntese química , Pró-Fármacos/química , gama-Glutamil Hidrolase/química , Antineoplásicos/uso terapêutico , Sítios de Ligação de Anticorpos , Desenho de Fármacos , Fragmentos Fab das Imunoglobulinas/química , Métodos , Pró-Fármacos/uso terapêutico , Succinimidas , Compostos de Sulfidrila/química , Sulfetos , Tioglicolatos , gama-Glutamil Hidrolase/imunologia , gama-Glutamil Hidrolase/metabolismoRESUMO
Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics.
Assuntos
Antineoplásicos Alquilantes/síntese química , Compostos de Mostarda Nitrogenada/síntese química , Pró-Fármacos/síntese química , Anticorpos Monoclonais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Células Tumorais Cultivadas , gama-Glutamil HidrolaseRESUMO
Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 microM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos/síntese química , Imunotoxinas , Pró-Fármacos/síntese química , gama-Glutamil Hidrolase/metabolismo , Mostarda de Anilina/síntese química , Mostarda de Anilina/metabolismo , Mostarda de Anilina/farmacologia , Morte Celular , Divisão Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Humanos , Estrutura Molecular , Células Tumorais CultivadasRESUMO
Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.