Resorufin Product Research Articles

Rapid detection of cefiderocol susceptibility/resistance in Acinetobacter baumannii

PurposeDue to its ability to disseminate worldwide and its multiple resistance trait, Acinetobacter baumannii is becoming a threat for public health worldwide. Cefiderocol (FDC) is a promising broad-spectrum cephalosporin recently approved for treating Gram-negative infection. The aim of this study was to develop a rapid test, namely the rapid FDC Acinetobacter baumannii NP test, for the detection of FDC susceptibility/resistance in A. baumannii since the current FDC susceptibility tests are rather time-consuming (at least 24 h).Materials and methodsThe rapid test is based on the reduction of resazurin to resorufin product by bacterial viable cells, thus detecting bacterial growth in the presence of FDC (38.4 mg/L). A color change from blue (resazurin) to violet or pink (resorufin) represents visual detection of bacterial growth. 95 randomly selected A. baumannii isolates were used to evaluate the performance of the rapid FDC Acinetobacter baumannii NP test.ResultsThe test showed 95.5% (95% CI 78.2–99.2%) and 100.0% (95% CI 95.0–100.0%) of sensitivity and specificity, respectively. All the results were obtained within 4 h30–4 h45 incubation time at 35 °C ± 2 °C, saving virtually one day when compared with currently-used antimicrobial susceptibility tests. The test showed only a single very major error, an isolate with a MIC of 8 mg/L.ConclusionsThe rapid FDC Acinetobacter baumannii NP test can be a valuable method which is easier and faster to interpret when compared with the gold standard broth microdilution method. The test showed remarkable performances; hence, it may be suitable for implementation in clinical microbiology routine laboratories.

Chemical Cascading Between Polymersomal Nanoreactor Populations

AbstractHarnessing interactions of functional nano‐compartments to generate larger particle assemblies allows studying diverse biological behaviors based on their population states and can lead to the development of smart materials. Herein, thiol‐functionalized polymersome nanoreactors are utilized as responsive organelle‐like nano‐compartments—with inherent capacity to associate into larger aggregates in response to change in the redox state of their environment—to study the kinetics of cascade reactions and explore functions of their collective under different population states. Two nanoreactor populations, glucose oxidase‐ and horseradish peroxidase‐loaded polymersomes, are prepared, and the results of their cascading upon addition of glucose are investigated. The kinetics of resorufin production in associated polymersomes and non‐associated polymersome populations are compared, observing a decreased rate upon association. For the associated populations, faster chemical cascading is found when the two types of nanoreactors are associated in a concerted step, as compared to sequential association. The addition of competing agents such as catalase impacts the communication between non‐associated polymersomes, whereas such an effect is less pronounced for the associated ones. Altogether, the results showcase the impact of collective associations on enzymatic cascading between organelle‐like nanoreactors.

Reduction in CYP1A1 and 2B2 activity at low oxygen tension

The Cytochrome P450 (CYP) enzyme family comprises a wide array of monooxygenases involved in the oxidation of endobiotic and xenobiotic molecules. The active site of a CYP enzyme contains an iron protoporphyrin center coordinated to a cysteine thiolate, and then, molecular oxygen is associated with the iron to be converted into dioxygen complex plus substrate. Reduction by CYP reductase expedites hydroxylation of the compound. In this oxidation reaction, insufficient oxygen molecules would affect enzyme catalysis. Nevertheless, biochemical data about CYP kinetics at low oxygen concentrations are not available. In this work, we present the results on the variation in rat liver microsomal CYP Vmax app and Km app under normal and hypoxic conditions. Using alkoxyresorufin molecules as substrates, the Vmax/Km ratios for resorufin production decreased from 426 to 393 for CYP1A1 and from 343 to 202 for CYP2B1 at a low oxygen concentration (4.1 ppm) compared to the ratios observed at a normal oxygen concentration (6.5 ppm). Additionally, the bacterial mutagenicity of 2-aminoanthracene and cyclophosphamide, decreased by 32% and 42%, respectively, at low oxygen concentrations. These results support the hypothesis that low oxygen availability is implicated in the low efficiency of substrate oxidation by CYP.

UPLC‐MS/MS Analysis of Ethoxyresorufin O‐Deethylation (EROD) Kinetics in Rat Kidney Microsomes

PurposeFormation of resorufin from ethoxyresorufin has been widely used to assess cytochrome P450 1A1 (CYP1A1) activity. Additionally, the kinetics of CYP1A1 activity has been well characterized in the liver microsomes by spectrophotometric/chromatographic methods. However, studies in kidney microsomes are scarce, most likely due to the lower microsomal content of CYP1A1 and lower production of the resorufin in the kidneys relative to those in the liver. In the present study, we aimed to develop a microscale enzymatic incubation method, coupled with a sensitive UPLC‐MS/MS assay for the characterization of kinetic parameters of 7‐ethoxyresorufin O‐deethylation (EROD) in the kidney microsomes.MethodsKidney microsomes were prepared from adult male Sprague‐Dawley rats (n = 3) by differential centrifugation. Protein (0.1, 0.25, 0.5 mg/mL) and time (2.5, 5.0, 7.5, and 10 min) linearity experiments with regard to the rate of production of resorufin from the substrate were determined (n = 3) using a substrate concentration of 0.5 μM. For determination of the Michaelis‐Menten (MM) kinetic parameters of EROD, the reaction mixtures (50 μL), containing microsomal proteins (0.25 mg/mL) were incubated with different concentrations of ethoxyresorufin (0.1–5.0 μM) in the presence of NADPH (1 mM) for 10 min at 37°C. The reaction was terminated by the addition of 100 μL of acetonitrile, containing internal standard (d6‐resorufin). After centrifugation, the supernatant (5 μL) was injected onto a UPLC column (Phenomenex C18), and sample components were separated using a binary gradient mobile phase (A: 5 mM ammonium acetate in water and B: acetonitrile/methanol, 95:5) at a flow rate of 0.3 mL/min. The UPLC–MS/MS system used the electrospray ionization technique in positive ion mode, and the transitions of m/z 213.9→185.9 and 220→192 were used to quantitate resorufin and the IS, respectively. Analytical method validation was performed as per regulatory guidelines.ResultsThe analytical method met the acceptance criteria for method validation in the linear range of 0.5 nM to 75 nM resorufin, with a lower limit of quantitation of 0.5 nM. The intra‐ and inter‐day precision was in the range of 4.55–12.4% and 6.17–9.82%, respectively. The overall recoveries of resorufin (90%–99%) and IS (85%–103%) were relatively high, suggesting an almost complete recovery from the protein precipitation method and confirming the lack of matrix effect on the samples. Using the optimized incubation and quantitation methods, we were able to reduce the incubation volume (50 μL), microsomal protein amount (12.5 μg), and incubation time (10 min), compared with reported methods. The method was successfully applied to the estimation of the MM kinetic parameters of 7‐ethoxyresorufin O‐deethylase activity in the rat kidney microsomes (Fig 1), which showed a maximum velocity of 1.92 ± 0.25 pmol/min/mg and a MM constant of 1.24 ± 0.2 μM (mean ± SD).ConclusionThe use of a sensitive and validated UPLC‐MS/MS method allows determination of the MM kinetic parameters of CYP1A1 activity in the rat kidney microsomes.Support or Funding InformationFunding: Chapman University School of PharmacyFormation rate of resorufin from ethoxyresorufin in rat kidney microsomal incubations (n = 3)Figure 1

Survival Tests for Leptospira spp.

Measuring viability is an important and necessary assessment in studying microorganisms. Several methods can be applied to Leptospira spp., each with advantages and inconveniencies. Here, we describe the traditional colony-forming unit method, together with two other methods based, respectively, on the reducing capacity of live cells (Alamar Blue® Assay) and differential staining of live and dead cells (LIVE/DEAD BacLight®). The Alamar Blue® Assay uses the blue reagent resazurin, which can be reduced into the pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance or fluorescence reading. The LIVE/DEAD BacLight® assay uses a mixture of two nucleic acid dyes (Syto9 and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The two other methods are not laborious and well-adapted to high-throughput studies, but the range of detection and linearity arelimited.

In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC50, CC50 and SI Values

Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols for the in vitro screening of a natural product-derived compound required for the determination of its antileishmanial potency. For this purpose, the Total Phenolic Fraction (TPF) derived from extra virgin olive oil is evaluated through the in vitro cell culture method against extracellular promastigote and intracellular amastigote Leishmania spp. forms. The aim of this article is to describe a step-by-step procedure that can be easily applied to accurately estimate the 50% inhibitory concentration (IC50), the 50% cytotoxic concentration (CC50) and the selectivity index (SI) via the resazurin reduction assay. These protocols are based on the ability of resazurin (oxidized blue form) to be irreversibly reduced by enzymes in viable cells and generate a red fluorescent resorufin product and can be easily expanded to the investigation of the antimicrobial activity in other microorganisms.

Triggered Reversible Reconfiguration of G-Quadruplex-Bridged "Domino"-Type Origami Dimers: Application of the Systems for Programmed Catalysis.

The reversible and switchable reconfiguration of the two-origami-dimer mixture AB plus CD into the dimer mixture DA plus BC and back using the triggered formation of K+-ion-stabilized G-quadruplexes and subsequent treatment with 18-crown-6-ether is presented. The reconfiguration processes are followed by atomic force microscopy imaging of the dimer structures that include tiles marked with 0, 1, 2, and 3 4× hairpin labels. By the functionalization of AB and CD dimers with the Mg2+-ion-dependent DNAzyme subunits, the AB plus CD mixture leads to the cleavage of the fluorophore- and quencher-modified substrate of the DNAzyme and to the activation of the fluorescence of the fluorophore (fluorescein)-modified fragment product. The K+-ion-induced isomerization of the mixture of AB plus CD into the mixture DA plus BC separates the Mg2+-ion-dependent DNAzyme subunits and concomitantly reconfigures the K+-ion-stabilized G-quadruplex associated with the two dimers. After the binding of hemin to the G-quadruplexes, the hemin/G-quadruplex DNAzyme is generated, leading to the catalyzed oxidation of AmplexRed by H2O2 to yield the fluorescent resorufin product. By the cyclic treatment of the AB plus CD mixture with K+ ions to yield the DA plus BC mixture and the subsequent recovery of the AB plus CD mixture by subjecting the DA plus BC mixture to 18-crown-6-ether, the fluorescence output signals of the system are switched on and off between the fluorescence of fluorescein and resorufin, respectively.

TRPV1 mediates capsaicin-stimulated metabolic activity but not cell death or inhibition of interleukin-1β release in human THP-1 monocytes

Human monocytes and dendritic cells express transient receptor potential vanilloid 1 (TRPV1) which may play a role in mediating the inflammatory, immune and cancer surveillance responses of these cells. The aim of the present study was to investigate TRPV1 expression and function in THP-1 monocytic cells. RT-PCR and Western blot were used to detect TRPV1. The metabolic activity and viability of THP-1 cells following exposure to vanilloids was assessed using resorufin production from rezazurin. Cytokine release was measured using ELISA. TRPV1 was expressed in cultured THP-1 monocytic cells and naïve monocytes. Lower concentrations (<250 μM) of capsaicin, but not other putative TRPV1 agonists, were shown to stimulate cell metabolic activity, whereas at concentrations >250 μM, all agonists decreased metabolic activity. Capsaicin-stimulated THP-1 metabolic activity was blocked by the TRPV1 antagonist, 5-iodo-resiniferatoxin (5′-IRTX), whereas the decline in resorufin production by THP-1 cells at higher capsaicin concentrations (due to cell death), was not affected by 5′-IRTX. Finally, capsaicin (≤125 μM) significantly increased lipopolysaccharide-stimulated IL-6 and TNF-α release from THP-1 cells, whereas phytohaemagglutinin-stimulated IL-1β, TNF-α, MCP-1 and IL-6 release were concentration-dependently inhibited by capsaicin. Modulation of IL-1β release was not TRPV1 mediated. Overall, these results show that functional TRPV1 channels are present in naïve monocytes and THP-1 cells, and when activated, increase cell metabolic activity. In addition, capsaicin modifies cytokine release from THP-1 cells and induces cell death, most likely by a mechanism that is independent of TRPV1 activation.

MicroRNA Sensor Based on Magnetic Beads and Enzymatic Probes

MicroRNAs are associated with multiple cellular processes and diseases. Here, we designed a highly sensitive, magnetically retrievable biosensor using magnetic beads (MBs) as a model RNA sensor. The assay utilized two biotinylated probes, which were hybridized to the complementary target miRNA in a sandwich assay format. One of the biotinylated ends of the hybridization complex was immobilized onto the surface of a NeutrAvidin (NAV) coated MB and the other biotinylated end was conjugated to HRP via NAV-biotin interaction. The results were presented by colorimetric absorbance of the resorufin product from amplex red oxidation. We show that by combining the use of MBs as well as bio-specific immobilization, the sensitivity of miRNA detection is down to 100 pM. This model HRP-MBs system can be used for simple, rapid colorimetric quantification of low level DNA/RNA or other small molecules.

Logic Control of Enzyme-Like Gold Nanoparticles for Selective Detection of Lead and Mercury Ions

Functional logic gates based on lead ions (Pb(2+)) and mercury ions (Hg(2+)) that induce peroxidase-like activities in gold nanoparticles (Au NPs) in the presence of platinum (Pt(4+)) and bismuth ions (Bi(3+)) are presented. The "AND" logic gate is constructed using Pt(4+)/Pb(2+) as the input and the peroxidase-like activity of the Au NPs as the output; this logic gate is denoted as "Pt(4+)/Pb(2+)(AND)-Au NPPOX". When Pt(4+) and Pb(2+) coexist, strong metallophilic interactions (between Pt and Pb atoms/ions) and aurophilic interactions (between Au and Pb/Pt atoms/ions) result in significant increases in the deposition of Pt and Pb atoms/ions onto the Au NPs, leading to enhanced peroxidase-like activity. The "INHIBIT" logic gate is fabricated by using Bi(3+) and Hg(2+) as the input and the peroxidase-like activity of the Au NPs as the output; this logic gate is denoted as "Bi(3+)/Hg(2+)(INHIBIT)-Au NPPOX". High peroxidase-like activity of Au NPs in the presence of Bi(3+) is a result of the various valence (oxidation) states of Bi(3+) and Au (Au(+)/Au(0)) atoms on the nanoparticle's surface. When Bi(3+) and Hg(2+) coexist, strong Hg-Au amalgamation results in a large decrease in the peroxidase-like activity of the Au NPs. These two probes (Pt(4+)/Pb(2+)(AND)-Au NPPOX and Bi(3+)/Hg(2+)(INHIBIT)-Au NPPOX) allow selective detection of Pb(2+) and Hg(2+) down to nanomolar quantities. The practicality of these two probes has been validated by analysis of Pb(2+) and Hg(2+) in environmental water samples (tap water, river water, and lake water). In addition, an integrated logic circuit based on the color change (formation of reddish resorufin product) and generation of O2 bubbles from these two probes has been constructed, allowing visual detection of Pb(2+) and Hg(2+) in aqueous solution.

Investigation of expression and activity levels of primary rat hepatocyte detoxication genes under various flow rates and cell densities in microfluidic biochips

We investigated the behavior of primary rat hepatocytes in biochips using a microfluidic platform (the integrated dynamic cell culture microchip). We studied the effects of cell inoculation densities (0.2-0.5 × 10(6) cells/biochip) and perfusion flow rates (10, 25, and 40 µL/min) during 72 h of perfusion. No effects were observed on hepatocyte morphology, but the levels of mRNA and CYP1A2 activity were found to be dependent on the initial cell densities and flow rates. The dataset made it possible to extract a best estimated range of parameters in which the rat hepatocytes appeared the most functional in the biochips. Namely, at 0.25 × 10(6) inoculated cells cultivated at 25 µL/min for 72 h, we demonstrated better induction of the expression of all the genes analyzed in comparison with other cell densities and flow rates. More precisely, when primary rat hepatocytes were cultivated at these conditions, the time-lapse analysis demonstrated an over expression of CYP3A1, CYP2B1, ABCC1b and ABCC2 in the biochips when compared to the postextraction levels. Furthermore, the AHR, CYP1A2, GSTA2, SULT1A1, and UGT1A6 levels remained higher than 50% of the postextraction values whereas values of HNF4α, CEBP, and PXR remained higher than 20% during the duration of the culture process. Nevertheless, an important reduction in mRNA levels was found for the xenosensors CAR and FXR, and the related CYP (CYP2E1, CYP7A1, CYP3A2, and CYP2D2). CYP1A2 functionality was illustrated by 700 ± 100 pmol/h/10(6) cells resorufin production. This study highlighted the functionality in optimized conditions of primary rat hepatocytes in parallelized microfluidic cultures and their potential for drug screening applications.

Hemin/G-Quadruplex-Catalyzed Aerobic Oxidation of Thiols to Disulfides: Application of the Process for the Development of Sensors and Aptasensors and for Probing Acetylcholine Esterase Activity

This study describes the novel hemin/G-quadruplex DNAzyme-catalyzed aerobic oxidation of thiols to disulfides and the respective mechanism. The mechanism of the reaction involves the DNAzyme-catalyzed oxidation of thiols to disulfides and the thiol-mediated autocatalytic generation of H2O2 from oxygen. The coupling of a concomitant H2O2-mediated hemin/G-quadruplex-catalyzed oxidation of Amplex Red to the fluorescent resorufin as a transduction module provides a fluorescent signal for probing the catalyzed oxidation of the thiol to disulfides and for probing sensing processes that yield the hemin/G-quadruplex as a functional label. Accordingly, a versatile sensing method for analyzing thiols (L-cysteine, glutathione) using the H2O2-mediated DNAzyme-catalyzed oxidation of Amplex Red to the resorufin was developed. Also, the L-cysteine and Amplex Red system was implemented as an auxiliary fluorescent transduction module for probing recognition events that form the catalytic hemin/G-quadruplex structures. This is exemplified with the development of thrombin aptasensor. The thrombin/thrombin binding aptamer recognition complex binds hemin, and the resulting catalytic complex activates the auxiliary transduction module, involving the aerobic oxidation of l-cysteine and the concomitant formation of the fluorescent resorufin. Finally, the hemin/G-quadruplex DNAzyme/Amplex Red system was used to follow the activity of acetylcholine esterase, AChE, and to probe its inhibition. The AChE-catalyzed hydrolysis of acetylthiocholine to the thiol-functionalized thiocholine enabled the probing of the enzymatic activity of AChE through the hemin/G-quadruplex-catalyzed aerobic oxidation of thiocholine to the respective disulfide and the concomitant generation of the fluorescent resorufin product.

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg−1, with 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.

Abstract 3673: Identifying the target of an orphan compound: CB30865 is a Nampt inhibitor

Abstract Background: Drug discovery based on cellular phenotypes is impeded by the challenge of identifying the molecular target. To alleviate this problem, Myriad Pharmaceuticals has developed a chemical proteomics process to identify cellular proteins that bind to small molecules. CB30865 is a potent and selective cytotoxic compound of previously unknown mechanism of action (1). Using our technology, we have unequivocally demonstrated that CB30865 cytotoxicity is due to inhibition of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first step in the synthesis of NAD from nicotinamide. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases. Materials and Methods: Direct target affinity purification (DTAP) was performed using analogs of CB30865 (MPI-0479883, MPI-0482594) coupled to beads. Purified proteins were identified by nano-flow reversed-phase liquid chromatography and tandem mass spectrometry using an LTQ-Orbitrap. In vitro Nampt activity was measured in a coupled biochemical assay based on the production of fluorescent resorufin. Nampt cellular activity was assayed by measuring levels of NAD and NAD-dependent polyADP-ribosylation. Results: As reported (1), 3-pyridyl, but not 2-pyridyl, analogs of CB30865 were potently cytotoxic. A 55 kD protein was purified efficiently using DTAP with MPI-0479883 (a 3-pyridyl CB30856 analog) but not MPI-0482594 (a 2-pyridyl analog). Preincubation of cellular lysates with uncoupled 3-pyridyl but not 2-pyridyl analogs potently blocked binding of the 55 kD protein. Based on LC-MS/MS analyses, the 55 kD protein was identified as Nampt. Immunoblotting of DTAPs using a Nampt specific antibody confirmed the MS identification. Furthermore, biochemical and cellular assays of Nampt activity demonstrated pyridyl-isomer specific enzyme inhibition. Finally, cellular Nampt inhibition and cytotoxicity were completely rescued by treating cells with 10 µM nicotinic acid as an alternate NAD source. Conclusions: The chemical proteomics DTAP approach is a powerful tool to identify molecular targets of orphan compounds. We demonstrate this here through identification of Nampt as the molecular target of CB30865 and further implicate NAD metabolism as a functional target for cancer. (1) Skelton LA, Ormerod MG, Titley J, Kimbell R, Brunton LA, Jackman AL. A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus. Br J Cancer. 1999 Apr;79(11-12):1692-701. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3673.

Abstract LB-288: MPI-0486348: A novel inhibitor of nicotinamide phosphoribosyltransferase (Nampt) induces tumor regression in a preclinical model

Abstract Background: Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the recycling of nicotinamide into NAD. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases (PARPs). Identification of Nampt as the target of an orphan cytotoxic compound using chemical proteomics spawned a Nampt drug discovery program at Myriad. We have developed a potent and selective inhibitor of Nampt that inhibits the growth of cancer cells in culture and induces tumor regression in a mouse xenograft model. Materials and Methods: In vitro Nampt activity was measured in a coupled biochemical assay based on the production of fluorescent resorufin. Viability was determined by measuring cellular ATP levels at 72 hrs following addition of compound. Nampt cellular activity was assayed by measuring levels of NAD and NAD-dependent poly (ADP)-ribosylation (PAR). Pharmacokinetic studies were conducted in female CD-1 mice. Anti-tumor activity was evaluated in an HCT116 human colon carcinoma xenograft in nu/nu mice. Results: MPI-0486348 inhibited Nampt activity in vitro with an average IC50 value of 40 pM. Depending upon the time of compound exposure, MPI-0486348 showed TC50 values of 180 pM to 20 nM in HCT116 cells. MPI-0486348 treatment also reduced cellular NAD levels and nuclear PAR levels, with IC50 values of 170 pM and 120 pM, respectively. These cellular effects of MPI-0486348 were prevented by coadministration of nicotinic acid, demonstrating on target mechanism of action. In mouse pharmacokinetic studies, absolute oral bioavailability of 47% was determined using 40% Captisol as the vehicle at an oral dose of 10 mg/kg. The same dose in rats gave an absolute oral bioavailability of 46%. Finally, in HCT116 tumor-bearing mice, a single oral dose of MPI-0486348 (50 or 75 mg/kg) resulted in 31% and 67% regression, respectively. Conclusions: MPI-0486348 is an orally bioavailable small molecule inhibitor of Nampt and shows promising preclinical results in a human xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-288.

Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.

Plasma Membrane Cholesterol Content Affects Nitric Oxide Diffusion Dynamics and Signaling

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

Light‐Induced Triggering of Peroxidase Activity Using Quantum Dots

Light‐Induced Triggering of Peroxidase Activity Using Quantum Dots

A new technique for assaying cytochrome P450 enzyme activity in a single cell

A new microspectrofluorometric technique for measuring the ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 (CYP)1A1 in single living cells is described. The system, which uses a perfusion chamber and an HPLC pump, allowed cells to be stained, fixed, blocked, and washed by injecting each treatment solution into the on-line carrier stream of buffer from the sampling block of the HPLC pump. After addition of the substrate 7-ethoxyresorufin, the fluorescence intensity of the metabolite resorufin was measured in individual cells. Fluorescence intensity steeply increased to a unique peak for each cell and then decreased to the basal level. Furthermore, CYP1A1 in each cell was stained with its antibody and quantified using the fluorescence intensity of an FITC-conjugated secondary antibody. EROD activity was normalized using the FITC fluorescence. The results show that the initial slopes and peak values of resorufin production by the cells were dependent on the CYP1A1 level. Treatment of hepatocytes with two nonspecific P450 inhibitors, cimetidine and SKF-525A, suppressed EROD activity.

Mechanism to study 1:1 stoichiometry of NADPH and alkoxyphenoxazones metabolism spectrophotometrically in subcellular biological preparations

Our prior studies have shown that pentoxyresorufin-O-dealkylation (PROD) can be measured spectrophotometrically with simultaneous monitoring of stoichiometry of NADPH/substrate and NADP/product as 10:1:10:1 [Rastogi et al. FEBS Letters 512 (2002) 121-124]. In the present investigation, mechanism of action of other enzymes in modulating the stoichiometry of alkoxyphenoxazones metabolism to 1:1 for electron donor/substrate and oxidized electron donor/product in the same incubation mixture was studied. The spectrophotometric analysis reveals 10:1 ratio between NADPH and pentoxyresorufin (PRF)-ethoxyresorufin (ERF) in microsomal system. The high ratio of electron donor to substrate is due to the presence of the other forms of P-450, which may participate in endogenous metabolism of compounds, thereby reducing the ratio to 4:1 and 7:1 for NADPH/PRF-ERF. Incubation of dicumarol in the microsomal PROD or ethoxyresorufin-O-dealkylase (EROD) assay led to significant decrease in the consumption of NADPH with a ratio of 4:1 and 7:1 for NADPH/PRF-ERF which is due to inhibition of NADPH cytochrome c (P-450) reductase. In post mitochondrial fraction (S-9), the ratio of 11:1 and 15:1 is seen for NADPH/PRF-ERF. The addition of dicumarol in S-9 fraction showed enhanced rate of alkoxyphenoxazone utilization, suggesting the possibility of reduced resorufin product as a feedback inhibitor. Equating the ratio of NADPH/substrate(s) derived after endogenous utilization of NADPH with the ratio after accounting for NADPH consumption following dicumarol addition in either S-9 or microsomal fraction, a 1:1 mol of NADPH/substrate(s) and oxidized electron donor/product is obtained. The results further suggest that cytosolic fraction may interfere in monitoring the formation of resorufin during dealkylation of alkoxyphenoxazones making dicumarol a mandatory cofactor.