ABSTRACT
Two new classes of selenazolidine-4(R)-carboxylic acids (2-oxo and 2-methyl-SCAs) were synthesized and characterized. Both were designed as latent forms of selenocysteine, intended to provide a chemically superior delivery form for selenium. The prodrugs may be clinically useful when selenium supplementation at supranutritional levels is indicated, such as in cancer chemoprevention.
Subject(s)
Organoselenium Compounds/chemistry , Prodrugs/chemical synthesis , Proline/analogs & derivatives , Proline/chemistry , Selenium/chemistry , Selenocysteine/chemistry , Organoselenium Compounds/metabolism , Proline/metabolism , Selenocysteine/metabolismABSTRACT
A method for the separation and quantitation of several important biological thiolamines is described. The procedure employs a C18 reversed-phase HPLC system to separate the dinitrophenyl derivatives of reduced and oxidized glutathione and cysteine and relies on an internal standard, Nepsilon-methyllysine, to minimize experimental error. The method was validated in three matrices (water, HepG2 cell lysates, and mouse liver homogenates) using several criteria. The detector response was linear for the dinitrophenyl derivatives of glutathione, glutathione disulfide, cysteine, and cystine in the concentrations ranging from 10 to 50 nmol/ml. Inter- and intra-day variation, percent recovery in the biological matrices, and limits of detection and quantitation were determined. For the most accurate determination, it is essential that standard curves be produced daily and in the same matrix as that being analyzed.
Subject(s)
Amines/analysis , Chromatography, High Pressure Liquid/methods , Sulfhydryl Compounds/chemistry , Amines/chemistry , Animals , Humans , Mice , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Representatives of two classes of thiazolidine prodrug forms of the well-known radioprotective agents L-cysteine, cysteamine, and 2-[(aminopropyl)amino]ethanethiol (WR-1065) were synthesized by condensing the parent thiolamine with an appropriate carbonyl donor. Inherent toxicity of the prodrugs was assessed in V79 cells using a clonogenic survival assay. Protection against radiation-induced cell death was measured similarly after exposure to 0--8 Gy gamma ((137)Cs) radiation. Antimutagenic activity was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. All thiazolidine prodrugs exhibited less toxicity than their parent thiolamines, sometimes dramatically so. Protection against radiation-induced cell death was observed for the 2-alkylthiazolidine, 2(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)thiazolidine (RibCyst), which produced a protection factor at 8 Gy of 1.8; the cysteine analogue, 2(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid (RibCys), was less active. RibCyst also exhibited excellent antimutational activity, rivaling that of WR-1065. The 2-oxothiazolidine analogues showed little activity in either determination under the conditions tested, perhaps due to their enhanced chemical and biochemical stability.
Subject(s)
Antimutagenic Agents/chemical synthesis , Cysteamine/chemistry , Cysteine/chemistry , Mercaptoethylamines/chemistry , Prodrugs/chemical synthesis , Radiation-Protective Agents/chemical synthesis , Sugar Alcohols/chemical synthesis , Thiazoles/chemical synthesis , Animals , Antimutagenic Agents/chemistry , Antimutagenic Agents/pharmacology , Cell Death/drug effects , Cell Line , Cricetinae , Cricetulus , Gamma Rays , Mutagenicity Tests , Prodrugs/chemistry , Prodrugs/pharmacology , Radiation-Protective Agents/chemistry , Radiation-Protective Agents/pharmacology , Structure-Activity Relationship , Sugar Alcohols/chemistry , Sugar Alcohols/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , ThiazolidinesABSTRACT
Protein farnesyltransferase (PFTase) catalyzes the alkylation of cysteine in C-terminal CaaX sequences of a variety of proteins, including Ras, nuclear lamins, large G-proteins, and phosphodiesterases, by farnesyl diphosphate (FPP). These modifications enhance the ability of the proteins to associate with membranes and are essential for their respective functions. The binding mechanism for yeast PFTase was deduced from a combination of steady-state kinetic and equilibrium studies. Rates for prenylation were measured by a continuous assay based on an enhancement in the fluorescence of the dansyl moiety in pentapeptide dansyl-GCVIA upon farnesylation by FPP. Unreactive substrate analogs for FPP and dansyl-GCVIA gave steady-state inhibition patterns for the dead-end inhibitors typical of an ordered sequential mechanism in which FPP adds to the enzyme before the peptide. The kinetic analysis was complicated by substrate inhibition for dansyl-GCVIA. The substrate inhibition was reversed at high concentrations of FPP, indicating that formation of the nonproductive enzyme--peptide complex is competitive with respect to FPP. Progress curves were fitted to an integrated form of the rate expression to determine the catalytic constant, kcat = 4.5 +/- 1.9 s-1, and the Michaelis constant for dansyl-GCVIA, KMD = 0.9 +/- 0.1 microM. The dissociation constant for FPP, KD = 75 +/- 15 nM, was measured using a membrane retention assay.
