Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
Biochem Pharmacol ; 61(2): 179-89, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11163332

ABSTRACT

The in vivo administration of enzyme-inhibiting drugs for cancer and infectious disease often results in overexpression of the targeted enzyme. We have developed an enzyme-catalyzed therapeutic agent (ECTA) approach in which an enzyme overexpressed within the resistant cells is recruited as an intracellular catalyst for converting a relatively non-toxic substrate to a toxic product. We have investigated the potential of the ECTA approach to circumvent the thymidylate synthase (TS) overexpression-based resistance of tumor cells to conventional fluoropyrimidine [i.e. 5-fluorouracil (5-FU)] cancer chemotherapy. (E)-5-(2-Bromovinyl)-2'-deoxy-5'-uridyl phenyl L-methoxyalaninylphosphoramidate (NB1011) is a pronucleotide analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), an antiviral agent known to be a substrate for TS when in the 5'-monophosphorylated form. NB1011 was synthesized and found to be at least 10-fold more cytotoxic to 5-FU-resistant, TS-overexpressing colorectal tumor cells than to normal cells. This finding demonstrates that the ECTA approach to the design of novel chemotherapeutics results in compounds that are selectively cytotoxic to tumor cell lines that overexpress the target enzyme, TS, and therefore may be useful in the treatment of fluoropyrimidine-resistant cancer.


Subject(s)
Antineoplastic Agents/metabolism , Bromodeoxyuridine/analogs & derivatives , Bromodeoxyuridine/metabolism , Drug Design , Prodrugs/metabolism , Thymidylate Synthase/metabolism , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Bromodeoxyuridine/administration & dosage , Bromodeoxyuridine/pharmacology , Deoxyuracil Nucleotides/chemistry , Deoxyuracil Nucleotides/metabolism , Disease Models, Animal , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Nude , Prodrugs/administration & dosage , Prodrugs/pharmacology , Thymidylate Synthase/administration & dosage , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Anal Biochem ; 263(1): 57-61, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9750143

ABSTRACT

I have designed an unamplified assay for human telomerase based on the hybridization protection assay. This assay measures in vitro synthesis of the human telomeric repeat DNA sequence (TTAGGG)N by solution hybridization with a chemiluminescent acridinium ester oligonucleotide probe. The assay is capable of detecting less than 0. 1 fmol of the telomerase repeat DNA sequence, and has a linear range extending to 3.5 fmol/reaction. Using this assay, telomerase activity can be measured in less than 60 min. The hybridization protection assay can be used to determine the amount of telomerase activity in a cell-free extract obtained from as few as 10(5) cells, and is three to four orders of magnitude less sensitive than PCR-based telomerase assays. However, the precision, accuracy, and rapidity of this telomerase assay make it suitable for enzyme isolation, enzyme characterization, and high-throughput screening applications.


Subject(s)
In Situ Hybridization/methods , Telomerase/analysis , Acridines/chemistry , DNA Primers , DNA Probes , Endopeptidase K/chemistry , Endopeptidase K/metabolism , Humans , Luminescent Measurements , Repetitive Sequences, Nucleic Acid , Ribonucleases/chemistry , Ribonucleases/metabolism , Telomerase/antagonists & inhibitors , Telomerase/metabolism
3.
J Biol Chem ; 267(25): 17508-11, 1992 Sep 05.
Article in English | MEDLINE | ID: mdl-1517203

ABSTRACT

The tripeptide pyroGlu-Tyr-Pro amide was isolated from an aqueous extract prepared from dried alfalfa pellets. The tripeptide was quantitated using a competitive radioimmunoassay in which 125I-labeled thyrotropin-releasing hormone (TRH), is displaced from antibody specific to TRH (pyroGlu-His-Pro amide). The pyroGlu-Tyr-Pro amide was purified by passing the filtered extract through QAE-Sephadex A25 at pH 5, followed by open bed chromatography on C18 silica using an H2O/methanol gradient, then preparative high performance liquid chromatography (HPLC) on microbondapak C18 using a 10 mM HCl/methanol gradient, followed by G-10-Sephadex chromatography, SP-C25-Sephadex chromatography, QAE-Sephadex chromatography at pH 10.1, analytical HPLC on a microbondapak C18 column eluted with 10 mm HCl/acetonitrile, and analytical HPLC reverse phase chromatography on an APEX phenyl column eluted with H2O/acetonitrile. The tripeptide was essentially homogeneous after the final chromatography step, as judged by correspondence of immunoreactivity with A280. The sequence of the alfalfa tripeptide was determined to be Glu-Tyr-Pro by gas phase sequencing, after hydrolysis of pyroglutamic acid by mild acid hydrolysis. The mass of the alfalfa tripeptide was 389.1, as determined by fast ion bombardment mass spectroscopy, and was found to be identical to the mass of synthetic pyroGlu-Tyr-Pro amide. The sequence of the alfalfa tripeptide was also verified using B/E-linked scanning. I conclude that the tripeptide isolated from alfalfa differs from human thyrotropin-releasing hormone only by the substitution of tyrosine for histidine at position 2. The role of pyroGlu-Tyr-Pro amide in alfalfa is not known, but the existence of a family of thyrotropin-related peptides occurring in both the animal and the plant kingdoms indicates that the thyrotropin related peptides have a wide phylogenetic distribution.


Subject(s)
Medicago sativa/chemistry , Oligopeptides/chemistry , Thyrotropin-Releasing Hormone/chemistry , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Mass Spectrometry , Molecular Structure , Oligopeptides/isolation & purification , Pyrrolidonecarboxylic Acid/analogs & derivatives , Sequence Homology, Nucleic Acid
4.
Eur J Biochem ; 195(1): 65-70, 1991 Jan 01.
Article in English | MEDLINE | ID: mdl-1991477

ABSTRACT

The purification is reported of an endopeptidase, XSCEP1 (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z-Phe-Arg-Amc (Z, benzyloxycarbonyl; Amc, 7-amidomethylcoumarin) as substrate, was fractionated by gradient ion-exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury-agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possessed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase; it was activated by dithiothreitol and EDTA and inhibited by the mechanism-based inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P1 position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val-Arg-Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.


Subject(s)
Cysteine Endopeptidases/isolation & purification , Sebaceous Glands/enzymology , Amino Acid Sequence , Animals , Chromatography, Affinity/methods , Chromatography, Ion Exchange/methods , Cysteine Endopeptidases/metabolism , Electrophoresis, Polyacrylamide Gel/methods , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Peptides/metabolism , Sebaceous Glands/metabolism , Substrate Specificity , Xenopus laevis
SELECTION OF CITATIONS
SEARCH DETAIL
...