Synthesis and structure-activity relationship of potent, selective and orally active anthranilamide-based factor Xa inhibitors: application of weakly basic sulfoximine group as novel S4 binding element.

A novel series of potent and efficacious factor Xa inhibitors which possesses sulfoximine moiety as novel S4 binding element in anthranilamide chemotype has been identified. Lead optimization at this novel P4 group led to many potent factor Xa inhibitors with excellent anticoagulant activity in human plasma. Selected compounds were dosed orally in rats and checked for their ex vivo prothrombin time prolonging activity, which resulted in identification of compound 5-chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(diethylamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide (18f). The detailed pharmacokinetic evaluation and subsequent metabolism study of 18f suggested the presence of an active metabolite. The compound 18f and its active metabolite 18b demonstrated excellent in vivo efficacy in both arterial and venous thrombosis model in rats and were found to be highly selective against related serine proteases. Based on this promising profile, compound 18f was selected for further evaluation.

Discovery of inhibitors of plasminogen activator inhibitor-1: structure-activity study of 5-nitro-2-phenoxybenzoic acid derivatives.

Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral bioavailability in the rodents.

Innate immune defense in visceral leishmaniasis: cytokine mediated protective role by allogeneic effector cell.

Antileishmanial role of mouse splenic natural killer (NK) cell was studied in allogeneic condition. In vitro data indicates that NK cells of allogeneic (C57BL/6, H2(b)) non-leishmania exposed mouse have strong antileishmanial effect against Leishmania donovani infected BALB/c (H2(d)) macrophages. Physical contact between the effector (NK cell) and the target cells (infected macrophages) is essential in this system since; cell free supernatant generated after coculturing of effector cells with infected target cells fails to elicit any antileishmanial effect. Although NK cells from allogeneic mouse are strongly attached to the infected macrophages but unable to kill it in such interaction. The antileishmanial effect of allogeneic NK cells is mediated by TNF-alpha and not by IFN-gamma. In vivo cellular therapy of established infection with NK cells from non-leishmania exposed allogeneic mouse significantly reduces the total parasite burden in the spleen of infected animal.

In vitro PAI-1 inhibitory activity of oxalamide derivatives.

A number of oxalamide derivatives have been synthesized and evaluated for PAI-1 inhibitory activity. In vitro PAI-1 inhibitory activities of oxalamide derivatives are evaluated by chromogenic assay. Few compounds have shown significant PAI-1 inhibitory activity.

Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants.

Clostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of approximately 155 or approximately 200 kDa. Formation of the approximately 155 kDa complex causes a Ca(2+) influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the approximately 200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the approximately 155 and approximately 200 kDa complexes as approximately 425-500 kDa and approximately 550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPE-binding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and approximately 425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the approximately 425 kDa complex, indicating claudin-induced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for approximately 425-500 kDa CPE complex formation. These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability.

The importance of calcium influx, calpain and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin.

CaCo-2 cells exhibit apoptosis when treated with low doses of Clostridium perfringens enterotoxin (CPE), but develop oncosis when treated with high CPE doses. This study reports that the presence of extracellular Ca(2+) in treatment buffers is important for normal activation of both those cell death pathways in CPE-treated CaCo-2 cells. Normal development of CPE-induced cell death pathway effects, such as morphologic damage, DNA fragmentation, caspase activation, mitochondrial membrane depolarization and cytochrome c release, was strongly inhibited when CaCo-2 cells were CPE-treated in Ca(2+)-free buffers. When treatment buffers contained Ca(2+), CPE caused a rapid increase in CaCo-2 cell Ca(2+) levels, apparently because of increased Ca(2+) influx through a CPE pore. High CPE doses caused massive changes in cellular Ca(2+) levels that appear responsible for activating oncosis, whereas low CPE doses caused less perturbations in cellular Ca(2+) levels that appear responsible for activating apoptosis. Both CPE-induced apoptosis and oncosis were found to be calmodulin- and calpain-dependent processes. As Ca(2+) levels present in the intestinal lumen resemble those of Ca(2+)-containing treatment buffers used in this study, perturbations in cellular Ca(2+) levels and calpain/calmodulin-dependent processes are also probably important for inducing enterocyte cell death during CPE-mediated gastrointestinal disease.

New insights into the cytotoxic mechanisms of Clostridium perfringens enterotoxin.

Clostridium perfringens type A isolates producing the 35 kDa enterotoxin (CPE) are an important cause of food poisoning, human non-foodborne gastrointestinal (GI) disease, and some veterinary GI diseases. Studies using CPE knock-out mutants confirmed the importance of enterotoxin expression for the enteric virulence of CPE-positive type A isolates. CPE action involves formation of a series of complexes in mammalian plasma membranes. One such CPE-containing complex (of approximately 155 kDa) is important for the induction of plasma membrane permeability alterations, which are responsible for killing enterotoxin-treated mammalian cells. Those membrane permeability changes damage the epithelium, allowing the enterotoxin to interact with the tight junction (TJ) protein occludin. CPE:occludin interactions result in formation of an approximately 200 kDa CPE complex and internalization of occludin into the cytoplasm. That removal of occludin (and possibly other proteins) damages TJs and disrupts the normal paracellular permeability barrier of the intestinal epithelium, which may contribute to CPE-induced diarrhea. Recent studies demonstrated that low CPE doses kill mammalian cells by inducing a classic apoptotic pathway involving mitochondrial membrane depolarization, cytochrome C release, and caspase 3/7 activation. In contrast, high enterotoxin doses induce oncosis, a proinflammatory event. Thus, inflammation may also contribute to the GI symptoms of patients whose intestines contain high CPE levels. In summary, CPE is a unique, multifunctional toxin with cytotoxic, TJ-damaging, and (probably) proinflammatory activities.

Death pathways activated in CaCo-2 cells by Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE), a 35-kDa polypeptide, induces cytotoxic effects in the enterocyte-like CaCo-2 cell culture model. To identify the mammalian cell death pathway(s) mediating CPE-induced cell death, CaCo-2 cultures were treated with either 1 or 10 micro g of CPE per ml. Both CPE doses were found to induce morphological damage and DNA cleavage in CaCo-2 cells. The oncosis inhibitor glycine, but not a broad-spectrum caspase inhibitor, was able to transiently block both of those pathological effects in CaCo-2 cells treated with the higher, but not the lower, CPE dose. Conversely, a caspase 3/7 inhibitor (but not glycine or a caspase 1 inhibitor) blocked morphological damage and DNA cleavage in CaCo-2 cells treated with the lower, but not the higher, CPE dose. Collectively, these results indicate that lower CPE doses cause caspase 3/7-dependent apoptosis, while higher CPE doses induce oncosis. Apoptosis caused by the lower CPE dose was shown to proceed via a classical pathway involving mitochondrial membrane depolarization and cytochrome c release. As the CPE concentrations used in this study for demonstrating apoptosis and oncosis have pathophysiologic relevance, these results suggest that both oncosis and apoptosis may occur in the intestines during CPE-associated gastrointestinal disease.

Organization of the plasmid cpe Locus in Clostridium perfringens type A isolates.

Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.