Inhibition of human UDP-glucuronosyltransferase enzyme by Dabrafenib: Implications for drug-drug interactions.

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.

Effect of downscaling on the linearity range of a calibration curve in spectrofluorimetry.

Interest in the microfluidic environment, owing to its unique physical properties, is increasing in much innovative chemical, biological, or medicinal research. The possibility of exploiting and using new phenomena makes the microscale a powerful tool to improve currently used macroscopic methods and approaches. Previously, we reported that an increase in the surface area to volume ratio of a measuring cell could provide a wider linear range for fluorescein (Kwapiszewski et al., Anal. Bioanal. Chem. 403:151-155, 2012). Here, we present a broader study in this field to confirm the assumptions we presented before. We studied fluorophores with a large and a small Stokes shift using a standard cuvette and fabricated microfluidic detection cells having different surface area to volume ratios. We analyzed the effect of different configurations of the detection cell on the measured fluorescence signal. We also took into consideration the effect of concentration on the emission spectrum, and the effect of the surface area to volume ratio on the limit of linearity of the response of the selected fluorophores. We observed that downscaling, leading to an increase in the probability of collisions between molecules and cell walls with no energy transfer, results in an increase in the limit of linearity of the calibration curve of fluorophores. The results obtained suggest that microfluidic systems can be an alternative to the currently used approaches for widening the linearity of a calibration curve. Therefore, microsystems can be useful for studies of optically dense samples and samples that should not be diluted.

Improvement in the sensitivity of microfluidic ELISA through field amplified stacking of the enzyme reaction product.

In this article, we demonstrate a novel approach to enhancing the sensitivity of enzyme-linked immunosorbent assays (ELISA) through pre-concentration of the enzyme reaction product (resorufin/4-methylumbelliferone) in free solution. The reported pre-concentration was accomplished by transporting the resorufin/4-methylumbelliferone molecules produced in the ELISA process towards a high ionic-strength buffer stream in a microfluidic channel while applying a voltage drop across this merging region. A sharp change in the electric field around the junction of the two liquid streams was observed to abruptly slow down the negatively charged resorufin/4-methylumbelliferone species leading to the reported pre-concentration effect based on the field amplified stacking (FAS) technique. It has been shown that the resulting enhancement in the detectability of the enzyme reaction product significantly improves the signal-to-noise ratio in the system thereby reducing the smallest detectable analyte concentration in the ELISA method. Applying the above-described approach, we were able to detect mouse anti-BSA and human TNF-α at concentrations nearly 60-fold smaller than that possible on commercial microwell plates. For the human TNF-α sample, this improvement in assay sensitivity corresponded to a limit of detection (LOD) of 0.102pg mL(-1) using the FAS based microfluidic ELISA method as compared to 7.03pg mL(-1) obtained with the traditional microwell plate based approach. Moreover, because our ELISAs were performed in micrometer sized channels, they required sample volumes about two orders of magnitude smaller than that consumed in the latter case (1µL versus 100µL).

The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays.

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

Evaluation of quinazoline analogues as glucocerebrosidase inhibitors with chaperone activity.

Gaucher disease is a lysosomal storage disorder (LSD) caused by deficiency in the enzyme glucocerebrosidase (GC). Small molecule chaperones of protein folding and translocation have been proposed as a promising therapeutic approach to this LSD. Most small molecule chaperones described in the literature contain an iminosugar scaffold. Here we present the discovery and evaluation of a new series of GC inhibitors with a quinazoline core. We demonstrate that this series can improve the translocation of GC to the lysosome in patient-derived cells. To optimize this chemical series, systematic synthetic modifications were performed and the SAR was evaluated and compared using three different readouts of compound activity: enzymatic inhibition, enzyme thermostabilization, and lysosomal translocation of GC.

Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus.

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed beta-propeller structure with the model's highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl alpha-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred alpha2-3-linked sialic acids over the alpha2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.

Light emitting diode induced chemiluminescence and its application as a detector for high performance liquid chromatography.

Some categories of compounds, including quinones, coumarins, flavins, and xanthene dyes, were found to produce strong chemiluminescence (CL) signals with luminol in sample solution under the irradiation of light emitting diodes (LED) with proper wavelengths. Based on this phenomenon, a compact photochemical reactor was constructed to develop a novel LED induced CL detector for high performance liquid chromatography (HPLC). The effects of related parameters including LED wavelength, luminol concentration, flow rate, pH, and eluents of HPLC were investigated in detail. Under the optimized conditions, the limits of detections (LODs) were in the range of 0.2-80 ng mL(-1). The applications and accuracy of the proposed method were validated by analyzing food samples such as milk powder, beer, candy and beverage with satisfactory results.

Detection of degradation products of chemical warfare agents by highly porous molecularly imprinted microspheres.

The multiplication of terrorist actions in the recent events is alarming and the detection of chemical warfare agents (CWAs) has become one of the highest research priorities in the fields of security and public health. The biomimetic properties of molecularly imprinted polymers (MIPs) render them attractive for molecular recognition as well as sensing purposes. The degradation products of easily hydrolysable organophosphorus nerve agents such as pinacolyl methylphosphonate (PMP), a hydrolysis by-product of soman, are often used as templates in MIP synthesis. In this study, we describe the first example of PMP-imprinted polymer microspheres synthesized by precipitation polymerization. This one-step process involves methacrylic acid (MAA) as the monomer and divinylbenzene (DVB) as the cross-linker, in a toluene/acetonitrile mixture. Subsequent morphological characterizations of the PMP-imprinted particles show that they have diameters between 1 and 10 mum (as opposed to 4-5 mum for the non-imprinted microspheres), surface areas of up to 680 m(2) g(-1) and high porosities with pore sizes smaller than 2 nm. The present investigation also evidences the imprinting effect via batch binding experiments and reports on the use of a novel fluorescence-based methodology, where 4-methylumbelliferone (4MU) is utilised as a sensing agent to determine the PMP concentration in solution.

Development of an assay using 4-trifluoromethylumbelliferyl as a marker substrate for assessment of drug-drug interactions to multiple isoforms of UDP-glucuronosyltransferases.

Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs) and is one of the most important pathways for elimination of xenobiotics. The aim of the present study was to develop an in vitro assay for assessment of drug-drug interactions related to UGTs applicable to early drug discovery. 4-Trifluoromethylumbelliferyl was tested as a marker substrate for six human recombinant expressed UGT isoforms: 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7. It was shown that 4-trifluoromethylumbelliferyl was glucuronidated by all UGTs tested, except UGT1A4. By using a short HPLC gradient (7 min) and fluorescence detection, the enzyme kinetic parameters for these reactions were obtained. All reactions were found to follow classical Michaelis-Menten kinetics, with K(m) values between 29 microM (UGT1A9) and 80 microM (UGT1A3). The method was validated by using several known competitive inhibitors of UGTs. The most potent inhibition was observed for the reaction between 17alpha-ethynylestradiol and UGT1A1 (K(i) = 10.5 microM), and the weakest interaction was detected for acetaminophen and UGT1A9 (IC(50) > 1 mM). Taken together, we report the development of an assay using 4-trifluoromethylumbelliferyl as a marker substrate for five different human UGT isoforms suitable for the assessment of drug-drug interactions related to UGTs during early drug discovery.

A nonradioactive 96-well plate assay for screening of trans-sialidase activity.

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.

Inhibition of influenza A virus sialidase activity by sulfatide.

Sulfatide, which binds to influenza A viruses and prevents the viral infection, was found to inhibit the sialidase activities of influenza A viruses in a pH-dependent manner. The kinetic parameters of the effect of sulfatide on the sialidase activities of human influenza A viruses using fluorometric assay indicated that sulfatide was a powerful and non-competitive type inhibitor in low-pH conditions.

Optimisation of an enzymatic method for beta-galactosidase.

BACKGROUND: The enzyme beta-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity. METHODS: Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its utility. beta-galactosidase acts on the substrate 4-methylumbelliferyl-galactoside (MUG) to produce 4-methylumbelliferone (4-MU), detected fluorimetrically with excitation wavelength 355 nm and emission wavelength 460 nm. RESULTS: Reaction conditions in a citrate-phosphate buffer were optimised to give maximal enzyme activity: pH was optimal at 4.4 (range investigated 3.6-5.0) and substrate concentration at 3.33 mmol/l. A small specimen volume (10 microl) in 80 microl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 degrees C. Reaction was terminated by addition of 200 microl of glycine-NaOH, pH 12.8. The assay is linear to 3,000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2,012 U/ml was 4.7, 3.1, and 3.4, respectively (n=10). Inter-assay CV% at 51, 496, and 1,986 U/ml was 7.0, 4.0, and 3.9, respectively (n=10). CONCLUSIONS: The assay has greater practical utility and demonstrated significant differences in the perfusate beta-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.

Hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide in differing sample fractions of river waters and its implication for the detection of fecal pollution.

The hydrolysis rate of 4-methylumbelliferyl-beta-D-glucuronide (MUG-HR) was determined in unamended samples, filtered samples, and in corresponding buffer resuspended filter residues of various river waters of slight to excessive fecal pollution covering a four orders of magnitude range. Regression analysis of the log MUG-HR of the unamended water samples versus the log MUG-HR of the filter residues revealed a highly significant linear relationship (R2 = 0.94; p<0.001). The median of the MUG-HR of the filtrated water samples was about 10% the MUG-HR of the corresponding unamended water samples. If MUG-HR determinations were used as a surrogate for estimating fecal coliform contamination, both the MUG-HR of the unamended water samples and the MUG-HR of the filter residues would have been equally adequate techniques at river sites of higher fecal pollution levels. However, at river locations of decreased fecal pollution, MUG-HR determination of filter residues appeared to be the more sensitive technique in order to estimate fecal coliform concentrations.

A fluorogenic assay using pressure-driven flow on a microchip.

A fluorogenic assay for human T-cell phosphatase (TCPTP) was conducted on an etched glass microchip using pressure driven flow. The TCPTP enzyme catalyzes the removal of a phosphate group from 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP) to produce the fluorogenic product 6,8-difluoro-4-methylumbelliferone (DiFMU). Enzyme assays with real-time on-chip dilution were performed in both low-viscosity (1 cP) buffer and an enzyme solution containing 50% glycerol (6 cP). Single side channels connect a series of reagent wells to a main channel where the fluorescent product of the enzyme reaction passes the detector region. Flow regulation of mixed viscosity fluids requires a pressure control on each arm of the chip contributing to the overall flow. An 8-channel pressure controller was built to regulate the air pressure above all wells feeding channels of the chip, thereby controlling the dilution ratios of buffer, substrate and enzyme. Well pressures maintained a constant concentration of enzyme in the detector channel while adjusting the flow contribution of substrate and buffer. The substrate concentration was stepped over two orders of magnitude while verifying fluid dilutions using marker dyes. The kinetic parameters, Km, Vmax, and Kcat, showed good agreement with the values determined using a standard well plate and fluorometer.

Screening of non-Saccharomyces wine yeasts for the production of beta-D-xylosidase activity.

Fifty-four yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, were screened for the production of beta-D-xylosidase activity. Beta-D-xylosidase activity was only detected in eight yeast strains belonging to the genera Hanseniaspora (H. osmophila and H. uvarum) and Pichia (P. anomala). Beta-D-xylosidase preparations active against p-nitrophenyl-beta-D-xyloside were characterised with respect to their optimal pH and temperature conditions. H. uvarum 11105 and 11107 and P. anomala 10320 beta-D-xylosidase preparations were active at pH and temperature ranges and at concentrations of glucose and ethanol usually found during winemaking processes.

Analysis of urinary N-acetyl-beta-glucosaminidase by capillary zone electrophoresis.

N-Acetyl-beta-glucosaminidase (NAG), a glycosidase enzyme, present in serum, urine and the renal lysosomes is utilized clinically as an early marker for renal damage preceding the elevation of both blood urea nitrogen and creatinine. NAG is analyzed by CE after incubation of urine samples with the synthetic substrate methylumbelliferyl-beta-D-glucosaminide. The reaction mixture is introduced directly into the instrument without further treatment. The released reaction product, 4-methyl-umbelliferone, is separated at 13.2 kV in a 400 mM borate buffer, pH 8.1. Detection was achieved with either ultraviolet absorption or with fluorescence. The fluorescence detection was more sensitive and gave cleaner electropherograms. The CZE method correlated well with an automated kinetic fluorescent assay. 4-Methyl-umbelliferone conjugated to different substrates is used in the analysis of many enzymes involved in the inborn errors of metabolism.

Identification of a novel glycosaminoglycan core-like molecule. I. 500 MHz 1H NMR analysis using a nano-NMR probe indicates the presence of a terminal alpha-GalNAc residue capping 4-methylumbelliferyl-beta-D-xylosides.

beta-Xylosides compete with endogenous proteoglycan core proteins and act as alternate acceptors for synthesizing protein-free glycosaminoglycan chains. Their assembly on these alternate acceptors utilizes the same glycosyltransferases that make the protein-bound chains. Most studies using alternate acceptors focus on the production of sulfated glycosaminoglycan chains that are thought to be the major products. However, we previously showed that labeling melanoma cells with [6-3H]galactose in the presence of 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) beta-xylosides led to the synthesis of mostly di- to tetrasaccharide products including incomplete core structures. We have solved the structure of one of the previously unidentified products as, GalNAc alpha(1,4)GlcA beta(1,3)Gal beta(1,3)Gal beta(1,4)Xyl beta MU, based on compositional analysis by high performance liquid chromatography, fast atom bombardment, electrospray mass spectrometry, and one-dimensional and two-dimensional 1H NMR spectroscopy. The novel aspect of this molecule is the presence of a terminal alpha-Gal-NAc residue at a position that is normally occupied by beta-GalNAc in chondroitin/dermatan sulfate or by alpha-Glc-NAc in heparin or heparan sulfate chains. An alpha-GalNAc residue at this critical location may prevent further chain extension or influence the type of chain subsequently added to the common tetrasaccharide core.

Fluorimetric determination of alkaline phosphatase activity in human serum by use of a flow-through biosensor.

A fluorimetric method for the determination of alkaline phosphatase activity based on the use of a flow-through biosensor is reported. The biochemical basis of the method is the hydrolysis of 4-methyl-umbelliferone-phosphate catalyzed by the analyte with fluorimetric monitoring of the 4-methyl-umbelliferone formed (lambda ex = 365 nm, lambda em = 445 nm). The enhancement of sensitivity achieved when the reaction product is retained on the support packed in the flow-cell makes the method suitable for determination of the analytes in serum samples after 1:50 dilution. The linear range is found to be between 0.1 and 20 U l-1, with relative standard deviation less than 2.2%. The use of this biosensor was tested by the determination of the analyte in human serum from healthy and sick individuals with excellent recoveries (94-103%).