Discovery of tetrahydroindazoles as a novel class of potent and in vivo efficacious gamma secretase modulators

The identification and optimization of a novel series of centrally efficacious gamma secretase modulators (GSMs) offering an alternative to the privileged aryl imidazole motif is described. Chiral bicyclic tetrahydroindazolyl amine substituted triazolopyridines were identified as structurally distinct novel series of GSMs. Representative compound BI-1408 ((R)-42) was demonstrated to be centrally efficacious in rats at a 30?mg/kg oral dose.     

Ablating both Fabp1 and Scp2/Scpx (TKO) induces hepatic phospholipid and cholesterol accumulation in high fat-fed mice

Although singly ablating Fabp1 or Scp2/Scpx genes may exacerbate the impact of high fat diet (HFD) on whole body phenotype and non-alcoholic fatty liver disease (NAFLD), concomitant upregulation of the non-ablated gene, preference for ad libitum fed HFD, and sex differences complicate interpretation. Therefore, these issues were addressed in male and female mice ablated in both genes (Fabp1/Scp2/Scpx null or TKO) and pair-fed HFD. Wild-type (WT) males gained more body weight as fat tissue mass (FTM) and exhibited higher hepatic lipid accumulation than WT females. The greater hepatic lipid accumulation in WT males was associated with higher hepatic expression of enzymes in glyceride synthesis, higher hepatic bile acids, and upregulation of transporters involved in hepatic reuptake of serum bile acids. While TKO had little effect on whole body phenotype and hepatic bile acid accumulation in either sex, TKO increased hepatic accumulation of lipids in both, specifically phospholipid and cholesteryl esters in males and females and free cholesterol in females. TKO-induced increases in glycerides were attributed not only to complete loss of FABP1, SCP2 and SCPx, but also in part to sex-dependent upregulation of hepatic lipogenic enzymes. These data with WT and TKO mice pair-fed HFD indicate that: i) Sex significantly impacted the ability of HFD to increase body weight, induce hepatic lipid accumulation and increase hepatic bile acids; and ii) TKO exacerbated the HFD ability to induce hepatic lipid accumulation, regardless of sex, but did not significantly alter whole body phenotype in either sex.     

多药抗药基因Mdrl探针的克隆及初步应用

多药抗药基因Mdrl的表达水平与细胞的耐药性直接相关,检测Mdrl的表达水平可预测化疗的效果以及预后,用分子原位杂交的方法可检测单个细胞中Mdrl的表达水平.用PCR扩增方法获得了一段特异的DNA片段,并将其克隆到pUC18载体中,经DNA序列分析证明与文献报道一致,此探针可用于临床标本的分子杂交检测.     

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effe     

Biologic activity of extracts of delayed hypersensitivity skin reaction sites

Extracts obtained from skin sites of delayed hypersensitivity reactions show chemotactic activity for monocytes and lymphocytes but not neutrophils. The soluble extractable factors present at these sites have in vivo activity as well; they promote the accumulation of monocytes in peritoneal exudates and cause inflammatory reactions in the skin of nonimmunized animals. The skin inflammatory infiltrates are predominantly mononuclear and are similar to those of delayed hypersensitivity reactions in actively immunized guinea pigs. The extracts which produced these effects had no detectable MIF activity, nor permeability inducing activity in excess of that obtainable from normal skin.These monocyte and lymphocyte chemotactic factors were analyzed by sucrose density ultracentrifugation. By this technique the distribution of monocyte factors corresponded rather closely with that of the monocyte chemotactic factors obtained from an antigen-activated lymphocyte culture. Similar correspondence was obtained for the bulk of the lymphocyte chemotactic activity present in skin extracts and in culture supernatants. This suggests the possibility that the lymphokine-like substances in the skin extracts might in fact represent lymphokines. Further documentation of this point will provide a link between in vitro and in vivo manifestations of delayed hypersensitivity.     

Design,synthesis and molecular modeling studies of new series of s-triazine derivatives as antimicrobial agents against multi-drug resistant clinical isolates

Three novel series of s-triazine derivatives, including thirty-five new compounds 2a-d, 3a-3p, 4b-d, 5b-d, 6d-6d, and 7a-7f were synthesized comprising a diversity of substituents based on the structure of Astrazeneca arylaminotriazine DNA gyrase B inhibitor. The antimicrobial activity was determined for all compounds against Staphylococcus aureus, Escherichia coli and Candida albicans using the two-fold serial dilution technique and against reference standards Ampicillin for the antibacterial screening and Clotrimazole regarding the antifungal evaluation. The tested compounds showed strong to moderate antibacterial inhibitory action and weak antifungal activity. Compounds 3j and 6b were the most potent antibacterial agents against the tested strains and multi-drug resistant (MDR) clinical isolates of Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus (MRSA1) with minimal toxicity in comparison to the reference drugs. In silico molecular properties calculations and molecular docking study for 3j and 6b revealed that both compounds could be considered as promising antibacterial DNA gyrase B inhibitors.     

Caged-xanthone from Cratoxylum formosum ssp. pruniflorum inhibits malignant cancer phenotypes in multidrug-resistant human A549 lung cancer cells through down-regulation of NF-κB

Our recent study reported that multidrug-resistant (MDR) human A549 lung cancer cells (A549RT-eto) with the elevated expression of NF-κB showed epithelial–mesenchymal transition (EMT), increasing spheroid formation and elevating the expression levels of stemness-related factors, including Oct4, Nanog, Sox2, Bmi1, and Klf4. Therefore, when new therapeutic agents targeting these malignant cancer cells were explored, we found that caged-xanthone (CX) isolated from the roots of Cratoxylum formosum ssp. pruniflorum diminished the expression of NF-κB, P-glycoprotein (P-gp) protein levels, cell migration and invasion, and sphere-forming ability of A549RT-eto cells. To address the role of NF-κB in these malignant cancer features, we treated A549RT-eto cells with NF-κB siRNAs in the present work. We found that the knockdown of NF-κB inhibited EMT and sphere formation. Furthermore, co-treatment with CX and NF-κB siRNA accelerated the death of apoptotic cells through the decrease of P-gp protein levels. These results suggest that NF-κB was involved in malignant cancer phenotypes and MDR in A549RT-eto cells. Taken together, our findings suggest that CX can be a potential therapeutic agent for the treatment of malignant tumor cells.     

Phosphonodiamidate prodrugs of N-alkoxy analogs of a fosmidomycin surrogate as antimalarial and antitubercular agents

A series of N-alkoxy analogs of a l-leucine ethyl ester phosphonodiamidate prodrug of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. These compounds originate by merging a previously reported successful phosphonate derivatisation with favorable modifications of the hydroxamate moiety. None of the synthesized compounds showed enhanced activity against either P. falciparum or M. tuberculosis in comparison with the parent free hydroxamate analog.     

Ascorbate promotes the cellular accumulation of doxorubicin and reverses the multidrug resistance in breast cancer cells by inducing ROS-dependent ATP depletion

Chemotherapy is the most effective strategy for the treatment of metastatic breast cancer. However, P-glycoprotein (P-gp)-mediated multidrug resistance severely limits the efficacy of chemotherapy and is a major cause of the failure during chemotherapeutic treatment. In this study, we investigated the anticancer effects of combining chemotherapeutic drugs with ascorbate (AA) in human breast cancer cells. We found that combined administration of AA can improve the sensitivity of both MCF-7 and doxorubicin (Dox)-resistant MCF-7/Adr cells to Dox in vitro and in vivo by a reactive oxygen species (ROS)-dependent mechanism. Further studies proved that AA can promote the accumulation of Dox in MCF-7/Adr cells when combined with doxorubicin. AA had no effect on the expression of P-gp at the mRNA and protein levels, but could decrease its activity as demonstrated by an obvious inhibition of efflux of P-gp substrate Rh 123. AA reduced ATP levels in both MCF-7 and MCF-7/Adr cells, and pretreating AA-stimulating cells with catalase completely rescued ATP levels. With ATP reduction, we observed an increased cellular calcium and the appearance of vacuoles and micropores on the cell surface, indicating the increased cell membrane permeability in AA-treated MCF-7/Adr cells. The above results suggest that AA could promote the cellular accumulation of doxorubicin by inducing ROS-dependent ATP depletion. Clinically, a combination of AA with doxorubicin would be a novel strategy for reversal of the multidrug resistance in human breast cancer cells during chemotherapy.