Effects of Verapamil and Two Bisbenzylisoquinolines, Curine and Guattegaumerine Extracted from Isolona hexaloba, on the Inhibition of ABC Transporters from Pseudomonas aeruginosa.

The biological effects of alkaloids, curine, guattegaumerine, and verapamil, on Pseudomonas aeruginosa were investigated. These molecules did not inhibit P. aeruginosa growth but increased the sensitivity of this bacterium to carbenicillin, novobiocin, and erythromycin. The results of another study indicate that curine and guattegaumerine were competitors of verapamil and acted as inhibitors of eukaryotic ABCB1 efflux pump. A BLAST-P carried out between a bacterial MDR transporter LmrA from Lactococcus lactis, a human MDR1/P-glycoprotein (ABCB1), and ABC proteins of P.aeruginosa highlighted five potential candidates that have this bacterium. A study on the sensitivity to carbenicillin in the presence of verapamil allowed us to identify the product of gene PA1113 as the ABC transporter involved in the influx of carbenicillin. Similarly, novobiocin transport performed in the presence of verapamil and a docking analysis highlighted protein MsbA (Lipid A flippase, gene PA4997) as a potential candidate in novobiocin efflux. MsbA has previously been identified as a multidrug transporter in E. coli, and as P. aeruginosa MsbA presented 76% identity with E. coli MsbA, it is possible that novobiocin efflux involves this ABC transporter, accounting for about 30% of the bacterium resistance to this antibiotic.

Effects of Two Natural Bisbenzylisoquinolines, Curine and Guattegaumerine, Extracted from Isolona hexaloba on Rhodamine Efflux by Abcb1b from Rat Glycocholic-Acid-Resistant Hepatocarcinoma Cells.

To develop new therapeutic molecules, it is essential to understand the biological effects and targets of clinically relevant compounds. In this article, we describe the extraction and characterization of two alkaloids from the roots of Isolona hexaloba-curine and guattegaumerine. The effect of these alkaloids on the multidrug efflux pump ABCB1 (MDR1/P-Glycoprotein) and their antiproliferative properties were studied. Compared to verapamil, a widely used inhibitor of P-gp, curine and guattegaumerine were found to be weak inhibitors of MDR1/P-Glycoprotein. The highest inhibition of efflux produced by verapamil disappeared in the presence of curine or guattegaumerine as competitors, and the most pronounced effect was achieved with curine. Altogether, this work has provided new insights into the biological effects of these alkaloids on the rat Mdr1b P-gp efflux mechanism and would be beneficial in the design of potent P-gp inhibitors.

Identification of the PA1113 Gene Product as an ABC Transporter Involved in the Uptake of Carbenicillin in Pseudomonas aeruginosa PAO1.

The resistance of Pseudomonas aeruginosa to antibiotics is multi factorial and complex. Whereas efflux pumps such as MexAB-OprM have been thought to predominate, here we show that a novel ATP Binding Cassette (ABC) transporter that mediates influx of carbenicillin from the periplasm to the cytoplasm and away from its cell wall target plays an important role in the resistance of P. aeruginosa to this antibiotic. Treatment of P. aeruginosa with verapamil, an inhibitor of ABC transporters in eukaryotic cells, increases its sensitivity to carbenicillin. Using amino acid sequence homology with known verapamil protein targets as a probe, we determined that the PA1113 gene product, an ABC transporter, mediates carbenicillin uptake into the bacterial cytoplasm. Docking and pharmacological analyses showed that verapamil and carbenicillin compete for the same site on the PA1113 gene protein, explaining the inhibitory effect of verapamil on carbenicillin uptake, and furthermore suggest that the PA1113 ABC transporter accounts for about 30% of P. aeruginosa carbenicillin resistance. Our findings demonstrate that the PA1113 gene product helps mediate carbenicillin resistance by transporting it away from its cell wall target and represents a promising new therapeutic target.

Altered hepatobiliary gene expressions in PFIC1: ATP8B1 gene defect is associated with CFTR downregulation.

Recent reports in patients with PFIC1 have indicated that a gene defect in ATP8B1 could cause deregulations in bile salt transporters through decreased expression and/or activity of FXR. This study aimed to: (1) define ATP8B1 expression in human hepatobiliary cell types, and (2) determine whether ATP8B1 defect affects gene expressions related to bile secretion in these cells. ATP8B1 expression was detected by RT-PCR in hepatocytes and cholangiocytes isolated from normal human liver and gallbladder. ATP8B1 mRNA levels were 20- and 200-fold higher in bile duct and gallbladder epithelial cells, respectively, than in hepatocytes. RT-PCR analyses of the liver from two patients with PFIC1, one with PFIC2, one with biliary atresia, showed that, compared to normal liver, hepatic expressions of FXR, SHP, CYP7A1, ASBT were decreased at least by 90% in all cholestatic disorders. In contrast, NTCP transcripts were less decreased (by < or = 30% vs. 97%) in PFIC1 as compared with other cholestatic disorders, while BSEP transcripts, in agreement with BSEP immunohistochemical signals, were normal or less decreased (by 50% vs. 97%). CFTR hepatic expression was decreased (by 80%), exclusively in PFIC1, while bile duct mass was not reduced, as ascertained by cytokeratin-19 immunolabeling. In Mz-ChA-2 human biliary epithelial cells, a significant decrease in CFTR expression was associated with ATP8B1 invalidation by siRNA. In conclusion, cholangiocytes are a major site ofATP8B1 hepatobiliary expression. A defect of ATP8B1 along with CFTR downregulation can impair the contribution of these cells to bile secretion, and potentially explain the extrahepatic cystic fibrosis-like manifestations that occur in PFIC1.