<i>In vitro</i> hepatic metabolism of the natural product quebecol

Quebecol (2,3,3-tri-(3-methoxy-4-hydroxyphenyl)-1-propanol) is a polyphenolic compound, which is formed during maple syrup production from Acer spp. Quebecol bears structural similarities to the chemotherapy drug tamoxifen, which has led to synthesis of structural analogues and investigations into their pharmacological properties, however there are no reports on the hepatic metabolism of quebecol.This interest in therapeutic properties spurred us to investigate the in vitro microsomal Phase I and II metabolism of quebecol. We were unable to detect any P450 metabolites for quebecol in either human liver microsomes (HLM) or rat liver microsomes (RLM). In contrast we observed marked formation of three glucuronide metabolites in both RLM and HLM, suggesting that clearance via Phase II pathways is likely to predominate.To further understand the hepatic contribution to first-pass glucuronidation we have validated an HPLC method following FDA and EMA guidelines (selectivity, linearity, accuracy, and precision) to quantify quebecol in microsomes. In vitro enzyme kinetics were performed for quebecol glucuronidation by HLM including 8 concentrations from 5–30 µM. We determined a Michaelis-Menten constant (KM) of 5.1 µM, intrinsic clearance (Clint,u) of 0.038 ± 0.001 mL/min/mg, and maximum velocity (Vmax) of 0.22 ± 0.01 µmol/min/mg. Quebecol (2,3,3-tri-(3-methoxy-4-hydroxyphenyl)-1-propanol) is a polyphenolic compound, which is formed during maple syrup production from Acer spp. Quebecol bears structural similarities to the chemotherapy drug tamoxifen, which has led to synthesis of structural analogues and investigations into their pharmacological properties, however there are no reports on the hepatic metabolism of quebecol. This interest in therapeutic properties spurred us to investigate the in vitro microsomal Phase I and II metabolism of quebecol. We were unable to detect any P450 metabolites for quebecol in either human liver microsomes (HLM) or rat liver microsomes (RLM). In contrast we observed marked formation of three glucuronide metabolites in both RLM and HLM, suggesting that clearance via Phase II pathways is likely to predominate. To further understand the hepatic contribution to first-pass glucuronidation we have validated an HPLC method following FDA and EMA guidelines (selectivity, linearity, accuracy, and precision) to quantify quebecol in microsomes. In vitro enzyme kinetics were performed for quebecol glucuronidation by HLM including 8 concentrations from 5–30 µM. We determined a Michaelis-Menten constant (KM) of 5.1 µM, intrinsic clearance (Clint,u) of 0.038 ± 0.001 mL/min/mg, and maximum velocity (Vmax) of 0.22 ± 0.01 µmol/min/mg.</p

The UVA and Aqueous Stability of Flavonoids Is Dependent on B-Ring Substitution

Flavonols such as kaempferol and quercetin are believed to provide protection against ultraviolet (UV)-induced damage to plants. Recent in vitro studies have examined the ability of flavonols to protect against UV-induced damage to mammalian cells. Stability of flavonols in cell culture media, however, has been problematic, especially for quercetin, one of the most widely studied flavonols. As part of our investigations into the potential for flavonols to protect skin against UV-induced damage, we have determined the stability of a series of flavonols that differ only in the number of substituents on the B-ring. We measured the stability of these flavonols over time to UVA radiation, Dulbecco’s modified Eagle’s medium (DMEM), and Dulbecco’s phosphate-buffered saline (DPBS) using high performance liquid chromatography with UV detection (HPLC–UV). The identification of the breakdown products of flavonols was accomplished by using a hybrid quadrupole linear ion trap mass spectrometer coupled with liquid chromatography. Tandem mass spectrometric analysis (MS/MS) of flavonol photoproducts was confirmed by comparing with the known standard samples. We have determined that flavonol stability decreases with increasing B-ring substitution, suggesting that future investigation of potential photoprotective flavonols will need to be cognizant of this trend

Novel Dimer Compounds That Bind α‑Synuclein Can Rescue Cell Growth in a Yeast Model Overexpressing α‑Synuclein. A Possible Prevention Strategy for Parkinson’s Disease

The misfolding of α-synuclein is a critical event in the death of dopaminergic neurons and the progression of Parkinson’s disease. Previously, it was suggested that drugs, which bind to α-synuclein and form a loop structure between the N- and C-termini, tend to be neuroprotective, whereas others, which cause a more compact structure, tend to be neurotoxic. To improve the binding to α-synuclein, eight novel compounds were synthesized from a caffeine scaffold attached to (<i>R</i>,<i>S</i>)-1-aminoindan, (<i>R</i>,<i>S</i>)-nicotine, and metformin, and their binding to α-synuclein determined through nanopore analysis and isothermal titration calorimetry. The ability of the dimers to interact with α-synuclein in a cell system was assayed in a yeast model of PD which expresses an AS-GFP (α-synuclein-Green Fluorescent Protein) construct under the control of a galactose promoter. In 5 mM galactose this yeast strain will not grow and large cytoplasmic foci are observed by fluorescent microscopy. Two of the dimers, C₈-6-I and C₈-6-N, at a concentration of 0.1 μM allowed the yeast to grow normally in 5 mM galactose and the AS-GFP became localized to the periphery of the cell. Both dimers were superior when compared to the monomeric compounds. The presence of the dimers also caused the disappearance of preformed cytoplasmic foci. Nanopore analysis of C₈-6-I and C₈-6-N were consistent with simultaneous binding to both the N- and C-terminus of α-synuclein but the binding constants were only 10⁵ M^–1

A Previously Unrecognized Kanosamine Biosynthesis Pathway in <i>Bacillus subtilis</i>

The <i>ntd</i> operon in <i>Bacillus subtilis</i> is essential for biosynthesis of 3,3′-neotrehalosadiamine (NTD), an unusual nonreducing disaccharide reported to have antibiotic properties. It has been proposed that the three enzymes encoded within this operon, NtdA, NtdB, and NtdC, constitute a complete set of enzymes required for NTD synthesis, although their functions have never been demonstrated <i>in vitro</i>. We now report that these enzymes catalyze the biosynthesis of kanosamine from glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-dehydrogenase, NtdA is a pyridoxal phosphate-dependent 3-oxo-glucose-6-phosphate:glutamate aminotransferase, and NtdB is a kanosamine-6-phosphate phosphatase. None of these enzymatic reactions have been reported before. This pathway represents an alternate route to the previously reported pathway from <i>Amycolatopsis mediterranei</i> which derives kanosamine from UDP-glucose