Guattegaumerine

Effects of Verapamil and Two Bisbenzylisoquinolines, Curine and Guattegaumerine Extracted from Isolona hexaloba, on the Inhibition of ABC Transporters from Pseudomonas aeruginosa.[Pubmed:35625344]

Antibiotics (Basel). 2022 May 21;11(5):700.

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.[Pubmed:35566380]

Molecules. 2022 May 9;27(9):3030.

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.

Complete biosynthesis of the bisbenzylisoquinoline alkaloids guattegaumerine and berbamunine in yeast.[Pubmed:34903659]

Proc Natl Acad Sci U S A. 2021 Dec 21;118(51):e2112520118.

Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs Guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of Guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of Guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.

Guattegaumerine protects primary cultured cortical neurons against oxidative stress injury induced by hydrogen peroxide concomitant with serum deprivation.[Pubmed:19015976]

Cell Mol Neurobiol. 2009 May;29(3):355-64.

Guattegaumerine is a natural product with characteristics of being lipophilic and reaching high concentration in the brain, but its function in the central nervous system has not yet been observed. This study was designed to evaluate the neuroprotective effects of Guattegaumerine on rat primary cultured cortical neurons. Following a 24-h exposure of the cells to combined serum-starvation and hydrogen peroxide, a significant augment in neuron damage as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release were observed. Preincubation of Guattegaumerine dramatically improved the cell viability and inhibited LDH release. Preincubation of Guattegaumerine also dramatically inhibited malondialhehyde (MDA) production and elevated the decreased total antioxidative capacity in cells caused by the combined injury. Results of flow cytometry and immunohistochemistry showed that pre-addition of Guattegaumerine interrupted the apoptosis of the neurons, reversed the up regulation of the pro-apoptotic gene (Bax) and the down regulation of the anti-apoptotic gene (Bcl-2). Furthermore, Guattegaumerine suppressed the increase of intracellular calcium ([Ca(2+)](i)) stimulated by either H(2)O(2) or KCl in Ca(2+)-containing extracellular solutions, and high concentration of 2.5 microM Guattegaumerine also suppressed the increase of [Ca(2+)](i) induced by H(2)O(2) in Ca(2+)-free solution. These observations suggested that Guattegaumerine may possess potential protection against oxidative stress injury, which might be beneficial for neurons.

Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera.[Pubmed:7892226]

Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2071-5.

A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer Guattegaumerine or one molecule each of (R)- and (S)-N-methylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.

The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures.[Pubmed:8380416]

J Biol Chem. 1993 Jan 15;268(2):823-31.

A new cytochrome P-450 enzyme, isolated from Berberis stolonifera plant cell suspension cultures, has been purified to electrophoretic homogeneity. The purified hemoprotein migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a minimal M(r) = 46,000. The enzyme could be purified to a high specific content of P-450 (18.2 nmol/mg protein) after fast protein liquid chromatofocusing, displaying an isoelectric point of 6.05. Spectral analysis of the homogeneous enzyme showed that it is predominantly low spin in the oxidized state, with a slight red-shifted ferrous carbonyl complex that exhibits a maximum at 452 nm. The purified cytochrome P-450, successfully reconstituted with NADPH-cytochrome P-450 reductase, displayed a maximal turnover rate of 50 nmol of substrate/nmol of P-450/min. In the purified and reconstituted form, the enzyme catalyzed the oxidation of three different chiral benzyltetrahydroisoquinoline substrates, namely (S)-coclaurine, (R)-N-methylcoclaurine, and (S)-N-methylcoclaurine, leading to the formation of three distinct dimeric products, (R,S)-berbamunine, (R,S)-2'-norberbamunine, and (R,R)-Guattegaumerine, that are also present in the plant cell cultures in vivo. This is the first report of a P-450 enzyme that mediates regio- and stereoselective intermolecular oxidative phenol coupling to furnish natural dimeric compounds. In this catalytic cycle cytochrome P-450 functions as an oxidant in a bisubstrate reaction without transfer of the activated oxygen atom to either of the two chiral substrates.

[Guattegaumerine, New Bisbenzylisoquinoline Alkaloid from Guatteria gaumeri.].[Pubmed:17405005]

Planta Med. 1983 Sep;49(9):25-7.

The aqueous-alcoholic extract of the bark of GUATTERIA GAUMERI (Mexican Annonaceae) is used for the treatment of cholelithiasis and hypercholesterolemia. Therefore it seems very interesting to perform a phytochemical study of this drug in order to know its composition and to determine, if possible, all the active principles. We describe now the isolation of the main alkaloid named Guattegaumerine. This new bisbenzylisoquinoline alkaloid has been assigned structure 1, on the basis of spectroscopic, chemical and chromatographic evidence.