Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage

Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS) resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA), which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.     

Discovery and SAR of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based HTS assay

A series of indole-2-carboxylic acid benzylidene-hydrazides has been identified as a new class of potent apoptosis inducers through a novel cell-based caspase HTS assay. The screening hit, 5-chloro-3-methyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (3a), was found to arrest T47D cells in G(2)/M and to induce apoptosis as measured by the flow cytometric analysis assay. A SAR study was carried out by modification of the substitutions on the indole and benzene rings. Substitution at the 3-position of the indole ring was found to be important for apoptotic activity. A 20-fold increase of apoptotic activity was achieved from screening hit 3a to 5-methyl-3-phenyl-indole-2-carboxylic acid (4-methylbenzylidene)-hydrazide (9a) and 5-chloro-3-phenyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (9b), with EC(50) value of 0.1microM in the caspase activation assay in T47D breast cancer cells. Compound 9b also was found to be highly active in a standard growth inhibition assay with a GI(50) value of 0.9microM in T47D cells. Compound 3a and its analogs were found to inhibit tubulin polymerization, which is the most probable primary mechanism of action of these compounds.     

The cGMP-binding, cGMP-specific phosphodiesterase (PDE5): intestinal cell expression, regulation and role in fluid secretion

The expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5, was studied in intestinal cells. Both PDE5A1 and PDE5A2 splice forms were cloned from the cDNA prepared from human colonic T84 cells, and PDE5 activity was dependent on increases in intracellular cGMP levels which correlated with increased phosphorylation of the enzyme. PDE5 expression was monitored in different regions of the gastrointestinal tract and nearly 50% of the phosphodiesterase activity in the duodenum, jejunum, ileum and colon was inhibited by sildenafil citrate. Administration of the stable toxin to intestinal loops resulted in activation of PDE5. Inhibition of PDE5 by sildenafil citrate led to fluid accumulation in loops, suggesting a possible explanation for the side effect of diarrhoea observed in individuals administered sildenafil citrate. Our results therefore represent the first study on the expression and regulation of PDE5 in intestinal tissue, and indicate that mechanisms to control its activity may have important consequences in intestinal physiology.     

Cloning of Protocatechuate 3,4-Dioxygenase Genes from Bradyrhizobium japonicum USDA110

A heterologous gene probe encoding the α and β subunits of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase (PCD) was used to detect its homolog in the genome of Bradyrhizobium japonicum USDA110. Three cosmid clones carrying a 2.2-kb BamHI insert showed high levels of PCD activity. SacI digestion of one of the genomic clones, pBjG17, produced a 2.5-kb insert DNA that complemented a PCD mutant of P. cepacia.     

CD3- T cells with cis- or trans-acting mutations affecting expression of T cell receptor beta-chain mRNA.

Mutants of an untransformed T cell clone that no longer respond to TCR/CD3 stimulation have been derived using a selection procedure based on the loss of functional response to Ag. This functional selection gives rise to clones of several different phenotypes. We have previously described mutants with a TCR/CD3+ cell surface phenotype whose TCR are uncoupled from cellular responses. We describe six additional mutants that do not express TCR/CD3 at the cell surface. One of the CD3- clones contains a deletion in the successfully rearranged TCR-alpha gene, whereas another carries a deletion in the successfully rearranged TCR-beta gene. TCR/CD3 expression in these deletion mutants can be restored by transfection of TCR-alpha or TCR-beta DNA. Four other clones do not express TCR-beta mRNA, yet contain no obvious deletions or rearrangements in the TCR-beta genes. One of these clones does not transcribe TCR-beta chain mRNA. The mutation in this clone does not reside in the TCR-beta gene itself, but may instead reside in a trans-acting regulatory element affecting TCR-beta gene expression, because the TCR-beta mRNA-phenotype is complemented by fusion with a TCR-alpha-beta- cell line. TCR-beta chain regulatory mutants will be valuable in contributing to our understanding of how TCR expression is regulated.     

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca₂₊ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca₂₊ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.