ABSTRACT
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary enzyme defect that affects over 400 million people worldwide. Deficiency in G6PD can cause hemolytic anemia, which can be fatal if it is not treated properly. Mass qualitative G6PD-deficiency screening methods have been available for some time; however, in this study we developed the first high-throughput quantitative method for measuring G6PD activity. A patient's blood can be analyzed directly from the original collection tubes on a Hitachi P modular serum work area (SWA), which dramatically reduces the analysis time and the cost of the assay. The results presented here may enable the development of other improved, automated assays of whole-blood samples.
Subject(s)
Clinical Laboratory Techniques/methods , Glucosephosphate Dehydrogenase Deficiency/diagnosis , Glucosephosphate Dehydrogenase/blood , Mass Screening/methods , Clinical Laboratory Techniques/economics , Glucosephosphate Dehydrogenase Deficiency/enzymology , Hemoglobins/analysis , Humans , Mass Screening/economics , Reference Values , Reproducibility of ResultsABSTRACT
Structure-activity relationship studies of substituted arylsulfoanilides as antiproliferatives, which are mediated by the partial depletion of intracellular Ca(2+) stores, resulted in the identification of compounds with micromolar activity against lung cancer cells in a growth inhibition assay. Incorporating the substitution pattern of the best arylsulfoanilides onto the 3-phenyloxindole scaffold resulted in a potent arylsulfoanilide-oxindole hybrid, 27. Compound 27 inhibits cancer cell growth by partial depletion of intracellular Ca(2+) stores and phosphorylation of eIF2alpha.
Subject(s)
Aniline Compounds/chemical synthesis , Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Sulfonamides/chemical synthesis , Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Calcium/metabolism , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Indoles/chemistry , Indoles/pharmacology , Mice , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
In an effort to generate novel translation initiation inhibitors for cancer therapy, a series of 2'-benzyloxy-5'-substituted-5-benzylidene-thiazolidine-2,4-thione and dione derivatives was synthesized and evaluated for activity in translation initiation specific assays. Several candidates of the 5-benzylidene-thiazolidine-2,4-diones (3c, 3d, and 3f) and -thiones (2b, 2e, and 2j), inhibit cell growth with low microM GI(50) mediated by inhibition of translation initiation, which involves partial depletion of intracellular Ca(2+) stores and strong phosphorylation of eIF2alpha.
Subject(s)
Antineoplastic Agents/chemical synthesis , Thiazoles/chemical synthesis , Thiones/chemical synthesis , Antineoplastic Agents/pharmacology , Calcium/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Eukaryotic Initiation Factor-2/metabolism , Humans , Phosphorylation , Protein Biosynthesis/drug effects , Structure-Activity Relationship , Thiazoles/pharmacology , Thiazolidinediones , Thiones/pharmacology , Time Factors , Tumor Cells, CulturedABSTRACT
By an analysis of PDB crystal structures, the mean conformations of protein strands bound in serine protease active sites are shown to contain extensively aligned atomic orbitals. The active-serine-bearing segment of each enzyme (subtilisin BPN' and beta-trypsin) also contains such alignments. The participating orbitals are almost identical in each system. All of the alignments converge on the targeted linkage. They suggest that a kind of through-strand polarizability is being optimized by evolution, presumably due to corresponding benefits in proteolysis rate. Such polarizability would help to explain the high values of kcat seen for long oligopeptide substrates. The idea predicts long substrates to be relatively reactive even under non-enzymatic conditions, which in fact they are.
Subject(s)
Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemistry , Binding Sites , Databases, Protein , Protein Binding , Protein Conformation , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Stereoisomerism , Structure-Activity Relationship , Subtilisin/chemistry , Subtilisin/physiology , Trypsin/chemistry , Trypsin/physiologyABSTRACT
Depletion of calcium from the endoplasmic reticulum has shown to affect protein synthesis and cell proliferation. The anticancer effect of troglitazone was reported to be mediated by depletion of intracellular calcium stores resulting in inhibition of translation initiation. The unsaturated form of troglitazone displays similar anticancer properties in vitro. In this letter, we report our findings on the minimum structural requirements for both compounds to retain their calcium release and antiproliferative activities.
Subject(s)
Antineoplastic Agents/chemical synthesis , Calcium/metabolism , Chromans/pharmacology , Thiazolidinediones/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Chromans/chemical synthesis , Eukaryotic Initiation Factor-2/metabolism , Humans , Phosphorylation/drug effects , Protein Biosynthesis/drug effects , Structure-Activity Relationship , Thiazolidinediones/chemical synthesis , TroglitazoneABSTRACT
Binding of eIF4E to the cap structure (m(7)GpppN) plays a critical role in mRNA translation. To study the interaction between eIF4E and cap, and to identify small molecule inhibitors of their binding, we synthesized a fluorescent-labeled cap analogue and used it to develop a fluorescence-polarization assay. This preliminary communication describes the synthesis of a fluorescein labeled 7-methylguanosinemonophosphate, and its dose dependent binding to purified human eIF4E as demonstrated by the fluorescence polarization assay.
Subject(s)
Fluorescein , RNA Cap Analogs/chemical synthesis , Eukaryotic Initiation Factor-4E/chemistry , Fluorescence Polarization , Fluorescent Dyes/chemical synthesis , Humans , Protein Binding , Protein Biosynthesis , RNA Cap Analogs/chemistryABSTRACT
A series of substituted 3,3-diphenyl-1,3-dihydro-indol-2-ones was synthesized from the corresponding isatins. The compounds were studied for cell growth inhibition mediated by partial depletion of intracellular Ca2+ stores that leads to phosphorylation of eIF2alpha. The diphenyloxindole (1) showed mechanism-specific antiproliferative activity that was comparable to known translation initiation inhibitors such as clotrimazole or troglitazone. SAR studies identified m'-tert-butyl and o-hydroxy substituted diphenyloxindole (25) as a lead compound for Ca2+-depletion-mediated inhibition of translation initiation.
Subject(s)
Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Calcium/metabolism , Cell Line , Cell Line, Tumor , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Eukaryotic Initiation Factor-2/metabolism , Fluorescence Resonance Energy Transfer , Humans , Indoles/chemistry , Indoles/pharmacology , Phosphorylation , Protein Biosynthesis/drug effects , Structure-Activity RelationshipABSTRACT
The modeling, synthesis, and characterization of oligomers containing 2-aminoquinazolin-5-yl 2'-deoxynucleotide residues are reported. The 2-aminoquinazoline residues sequence specifically bind via Hoogsteen base pairing as a third strand in the center of the major groove at T:A base pair Watson-Crick duplex sequences. Evidence for the formation of a sequence specific three-stranded structure is based on thermal denaturation UV-vis and fluorescence studies. The novel 2-aminoquinazoline C-nucleotide is a component of a system designed to overcome the homopurine requirement for triple helix structures.
