Intracellular Concentrations Of Substrates Research Articles

Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In Vitro/In Vivo Catalytic Performance Correlation.

Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.

Engineering of a Substrate Affinity Reduced S-Adenosyl-methionine Synthetase as a Novel Biosensor for Growth-Coupling Selection of L-Methionine Overproducers.

Biosensors are powerful tools for monitoring specific metabolites or controlling metabolic flux towards the products in a single cell, which play important roles in microbial cell factory construction. Despite their potential role in metabolic flux monitoring, the development of biosensors for small molecules is still limited. Reported biosensors often exhibit bottlenecks of poor specificity and a narrow dynamic range. Moreover, fine-tuning the substrate binding affinity of a crucial enzyme can decrease its catalytic activity, which ultimately results in the repression of the corresponding essential metabolite biosynthesis and impairs cell growth. However, increasing intracellular substrate concentration can elevate the availability of the essential metabolite and may lead to restore cellular growth. Herein, a new strategy was proposed for constructing whole-cell biosensors based on enzyme encoded by essential gene that offer inherent specificity and universality. Specifically, S-adenosyl-methionine synthetase (MetK) in E. coli was chosen as the crucial enzyme, and a series of MetK variants were identified that were sensitive to L-methionine concentration. This occurrence enabled the engineered cell to sense L-methionine and exhibit L-methionine dose-dependent cell growth. To improve the biosensor's dynamic range, an S-adenosyl-methionine catabolic enzyme was overexpressed to reduce the intracellular availability of S-adenosyl-methionine. The resulting whole-cell biosensor effectively coupled the intracellular concentration of L-methionine with growth and was successfully applied to select strains with enhanced L-methionine biosynthesis from random mutagenesis libraries. Overall, our study presents a universal strategy for designing and constructing growth-coupled biosensors based on crucial enzyme, which can be applied to select strains overproducing high value-added metabolites in cellular metabolism.

Lysine Crotonylation: An Emerging Player in DNA Damage Response.

The DNA damage response (DDR) system plays an important role in maintaining genome stability and preventing related diseases. The DDR network comprises many proteins and posttranslational modifications (PTMs) to proteins, which work in a coordinated manner to counteract various genotoxic stresses. Lysine crotonylation (Kcr) is a newly identified PTM occurring in both core histone and non-histone proteins in various organisms. This novel PTM is classified as a reversible acylation modification, which is regulated by a variety of acylases and deacylases and the intracellular crotonyl-CoA substrate concentration. Recent studies suggest that Kcr links cellular metabolism with gene regulation and is involved in numerous cellular processes. In this review, we summarize the regulatory mechanisms of Kcr and its functions in DDR, including its involvement in double-strand break (DSB)-induced transcriptional repression, DSB repair, and the DNA replication stress response.

Dynamic control of toxic natural product biosynthesis by an artificial regulatory circuit

To mimic the delicately regulated metabolism in nature for improved efficiency, artificial and customized regulatory components for dynamically controlling metabolic networks in multiple layers are essential in laboratory engineering. For this purpose, a novel regulatory component for controlling vanillin biosynthetic pathway was developed through directed evolution, which was responsive to both the product vanillin and substrate ferulic acid, with different capacities. This regulatory component facilitated pathway expression via dynamic control of the intracellular substrate and product concentrations. As vanillin is an antimicrobial compound, low pathway expression and vanillin formation levels enabled better cell growth at an early stage, and the product feedback-activated pathway expression at later stages significantly improved biosynthesis efficiency. This novel multiple-layer dynamic control was demonstrated effective in managing the trade-off between cell growth and production, leading to improved cell growth and vanillin production compared to the conventional or quorum-sensing promoter-controlled pathway. The multiple-layer dynamic control enabled by designed regulatory components responsive to multiple signals shows potential for wide applications in addition to the dynamic controls based on biosynthetic intermediate sensing and quorum sensing reported to date.

Metabolic control analysis of L-tryptophan production with Escherichia coli based on data from short-term perturbation experiments

E. coli strain NT1259 /pF112aroFBLkan was able to produce 14.3 g L−1 L-tryptophan within 68 h in a fed-batch process from glycerol on a 15 L scale. To gain detailed insight into metabolism of this E. coli strain in the fed-batch process, a sample of L-tryptophan producing cells was withdrawn after 47 h, was separated rapidly and then resuspended in four parallel stirred-tank bioreactors with fresh media. Four different carbon sources (glucose, glycerol, succinate, pyruvate) were supplied individually with varying feeding rates within 19 min and the metabolic reactions of the cells in the four parallel reactors were analyzed by quantification of extracellular and intracellular substrate, product and metabolite concentrations. Data analysis allowed the estimation of intracellular carbon fluxes and of thermodynamic limitations concerning intracellular concentrations and reaction energies. Carbon fluxes and intracellular metabolite concentrations enabled the estimation of elasticities and flux control coefficients by applying metabolic control analysis making use of a metabolic model considering 48 enzymatic reactions and 56 metabolites. As the flux control coefficients describe connections between enzyme activities and metabolic fluxes, they reveal genetic targets for strain improvement. Metabolic control analysis of the recombinant E. coli cells withdrawn from the fed-batch production process clearly indicated that (i) the supply of two precursors for L-tryptophan biosynthesis, L-serine and phosphoribosyl-pyrophosphate, as well as (ii) the formation of aromatic byproducts and (iii) the enzymatic steps of igps and trps2 within the L-tryptophan biosynthesis pathway have major impact on fed-batch production of L-tryptophan from glycerol and should be the targets for further strain improvements.

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate.

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Thermodynamic first law efficiency of membrane proteins

Proteins are nature’s biomolecular machines. Proteins, such as transporters, pumps and motors, have complex function/operating-machinery/mechanisms, comparable to the macro-scaled machines that we encounter in our daily life. These proteins, as it is for their macro-scaled counterparts, convert (part of) other/various forms of energy into work. In this study, we are performing the first law analysis on a set of proteins, including the dopamine transporter, glycine transporters I and II, glutamate transporter, sodium–potassium pump and Ca2+ ATPase. Each of these proteins operates on a thermodynamic/mechanic cycle to perform their function. In each of these cycles, they receive energy from a source, convert part of this energy into work and reject the remaining part of the energy to the environment. Conservation of energy principle was applied to the thermodynamic/mechanic cycle of each protein, and thermodynamic first law efficiency was evaluated for each cycle, which shows how much of the energy input per cycle was converted into useful work. Interestingly, calculations based on experimental data indicate that proteins can operate under a range of efficiencies, which vary based on the extracellular and intracellular ion and substrate concentrations. The lowest observed first law efficiency was 50%, which is a very high value if compared to the efficiency of the macro-scaled heat engines we encounter in our daily lives.Communicated by Ramaswamy H. Sarma

Hydroxylated and sulfated metabolites of commonly occurring airborne polychlorinated biphenyls inhibit human steroid sulfotransferases SULT1E1 and SULT2A1

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that are associated with varied adverse health effects. Lower chlorinated PCBs are prevalent in indoor and outdoor air and can be metabolized to their hydroxylated derivatives (OH-PCBs) followed by sulfation to form PCB sulfates. Sulfation is also a means of signal termination for steroid hormones. The human estrogen sulfotransferase (SULT1E1) and alcohol/hydroxysteroid sulfotransferase (SULT2A1) catalyze the formation of steroid sulfates that are inactive at steroid hormone receptors. We investigated the inhibition of SULT1E1 (IC50s ranging from 7.2 nM to greater than 10 μM) and SULT2A1 (IC50s from 1.3 μM to over 100 μM) by five lower-chlorinated OH-PCBs and their corresponding PCB sulfates relevant to airborne PCB-exposure. Several congeners of lower chlorinated OH-PCBs relevant to airborne PCB exposures were potent inhibitors of SULT1E1 and SULT2A1 and thus have the potential to disrupt regulation of intracellular concentrations of the receptor-active steroid substrates for these enzymes.

Extracts of Immature Orange (Aurantii fructus immaturus) and Citrus Unshiu Peel (Citri unshiu pericarpium) Induce P-Glycoprotein and Cytochrome P450 3A4 Expression via Upregulation of Pregnane X Receptor.

P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) are expressed in the intestine and are associated with drug absorption and metabolism. Pregnane X receptor (PXR) is the key molecule that regulates the expression of P-gp and CYP3A4. Given that PXR activity is regulated by a variety of compounds, it is possible that unknown PXR activators exist among known medicines. Kampo is a Japanese traditional medicine composed of various natural compounds. In particular, immature orange [Aurantii fructus immaturus (IO)] and citrus unshiu peel [Citri unshiu pericarpium (CP)] are common ingredients of kampo. A previous study reported that kampo containing IO or CP decreased the blood concentration of concomitant drugs via upregulation of CYP3A4 although the mechanism was unclear. Some flavonoids are indicated to alter P-gp and CYP3A4 activity via changes in PXR activity. Because IO and CP include various flavonoids, we speculated that the activity of P-gp and CYP3A4 in the intestine may be altered via changes in PXR activity when IO or CP is administered. We tested this hypothesis by using LS180 intestinal epithelial cells. The ethanol extract of IO contained narirutin and naringin, and that of CP contained narirutin and hesperidin. Ethanol extracts of IO and CP induced P-gp, CYP3A4, and PXR expression. The increase of P-gp and CYP3A4 expression by the IO and CP ethanol extracts was inhibited by ketoconazole, an inhibitor of PXR activation. The ethanol extract of IO and CP decreased the intracellular concentration of digoxin, a P-gp substrate, and this decrease was inhibited by cyclosporine A, a P-gp inhibitor. In contrast, CP, but not IO, stimulated the metabolism of testosterone, a CYP3A4 substrate, and this was inhibited by a CYP3A4 inhibitor. These findings indicate that the ethanol extract of IO and CP increased P-gp and CYP3A4 expression via induction of PXR protein. Moreover, this induction decreased the intracellular substrate concentration.

Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter

The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na+ concentrations because intracellular Na+ release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K+ and, notably, also H+. In addition, our data suggest that Cl− is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K+ or of H+ to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na+ release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na+ release and K+ (or H+) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form.

Bacillus subtilis 5'-nucleotidases with various functions and substrate specificities.

BackgroundIn Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5′-nucleotidases that hydrolyze the phosphate group of 5′-nucleotides. In Bacillus subtilis, genes encoding 5′-nucleotidase have remained to be identified.ResultsWe found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His6-tagged proteins. YcsE exhibited significant 5′-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5′-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not.ConclusionsAccordingly, in B. subtilis, at least YcsE and YktC acted as major 5′-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0866-5) contains supplementary material, which is available to authorized users.

Robust Luenberger observers for microalgal cultures

The advanced control of microalgal cultures usually requires the knowledge of several component concentrations, which are however not always measurable on-line. In this context, state estimation plays an important role, and software sensors should be robust to model uncertainties and measurement noise. In this study, two software sensors are designed in the form of extended Luenberger observers, using Lyapunov arguments and linear matrix inequalities (LMI). These observers are based on Droop model and a few available on-line sensors. The first observer design estimates the intracellular quota and substrate concentrations considering a linear differential inclusion modeling technique and a constant observer gain. On the other hand, the second one estimates only the intracellular quota concentration assuming uncertainties in the model parameters and a quasi-Linear Parameter Varying (quasi-LPV) representation of the nonlinear system. The results are presented considering simulated and experimental data from Dunaliella tertiolecta culture.

Role of Hepatic Efflux Transporters in Regulating Systemic and Hepatocyte Exposure to Xenobiotics

Hepatic efflux transporters include numerous well-known and emerging proteins localized to the canalicular or basolateral membrane of the hepatocyte that are responsible for the excretion of drugs into the bile or blood, respectively. Altered function of hepatic efflux transporters due to drug-drug interactions, genetic variation, and/or disease states may lead to changes in xenobiotic exposure in the hepatocyte and/or systemic circulation. This review focuses on transport proteins involved in the hepatocellular efflux of drugs and metabolites, discusses mechanisms of altered transporter function as well as the interplay between multiple transport pathways, and highlights the importance of considering intracellular unbound concentrations of transporter substrates and/or inhibitors. Methods to evaluate hepatic efflux transport and predict the effects of impaired transporter function on systemic and hepatocyte exposure are discussed, and the sandwich-cultured hepatocyte model to evaluate comprehensively the role of hepatic efflux in the hepatobiliary disposition of xenobiotics is characterized.

Tumor-Specific Expression of Organic Anion-Transporting Polypeptides: Transporters as Novel Targets for Cancer Therapy

Members of the organic anion transporter family (OATP) mediate the transmembrane uptake of clinical important drugs and hormones thereby affecting drug disposition and tissue penetration. Particularly OATP subfamily 1 is known to mediate the cellular uptake of anticancer drugs (e.g., methotrexate, derivatives of taxol and camptothecin, flavopiridol, and imatinib). Tissue-specific expression was shown for OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis, while other OATPs, for example, OATP4A1, are expressed in multiple cells and organs. Many different tumor entities show an altered expression of OATPs. OATP1B1/OATP1B3 are downregulated in liver tumors, but highly expressed in cancers in the gastrointestinal tract, breast, prostate, and lung. Similarly, testis-specific OATP6A1 is expressed in cancers in the lung, brain, and bladder. Due to their presence in various cancer tissues and their limited expression in normal tissues, OATP1B1, OATP1B3, and OATP6A1 could be a target for tumor immunotherapy. Otherwise, high levels of ubiquitous expressed OATP4A1 are found in colorectal cancers and their metastases. Therefore, this OATP might serve as biomarkers for these tumors. Expression of OATP is regulated by nuclear receptors, inflammatory cytokines, tissue factors, and also posttranslational modifications of the proteins. Through these processes, the distribution of the transporter in the tissue will be altered, and a shift from the plasma membrane to cytoplasmic compartments is possible. It will modify OATP uptake properties and, subsequently, change intracellular concentrations of drugs, hormones, and various other OATP substrates. Therefore, screening tumors for OATP expression before therapy should lead to an OATP-targeted therapy with higher efficacy and decreased side effects.

A template model for studying anticancer drug efflux transporter inhibitors in vitro

Efflux transporters play an important role in drug absorption and also in multidrug resistance. ABCG2 (BCRP) is an efflux transporter conferring cross-resistance to mitoxantrone (Mit), irinotecan (CPT11), and its active metabolite SN38. MBLI87, a new ABCG2 inhibitor has proven its efficacy against ABCG2-mediated efflux in vitro and in vivo. This work aimed at modeling and quantifying the cellular interaction between MBLI87 and different substrates using a mechanistic template model. An in vitro competition experiment study was carried out with HEK293 cells overexpressing ABCG2 exposed to fixed concentrations of substrates (Mit, CPT11, SN38) and to MBLI87 at several concentration levels. A nonlinear mixed-effects transport inhibition model was developed to fit intracellular drug concentrations. In this model, drugs cross the cell membrane through passive diffusion, active drug efflux is ABCG2 mediated, interaction between substrates and inhibitor occurs within the transporter. The interaction was found to be noncompetitive. The MBLI87 Ki was estimated to 141 nm for Mit, 289 nm for CPT11, and 1160 nm for SN38. The ratio of intrinsic transport clearance divided by diffusion clearance was estimated to 2.5 for Mit, 1.01 for CPT11, and 5.4 for SN38. The maximal increase in the intracellular substrate concentration that is possible to achieve by inhibition of the transporter was estimated to 1.5 for Mit, 0.1 for CPT11, and 4.4 for SN38. This mechanistic template model describes both drug accumulation and cellular transport, and the mixed-effects approach allows an estimation of intra- and interassay variability. This model is of great interest to study cytotoxic cellular pharmacokinetics.

The Anthelmintic Triclabendazole and Its Metabolites Inhibit the Membrane Transporter ABCG2/BCRP

ABCG2/BCRP is an ATP-binding cassette transporter that extrudes compounds from cells in the intestine, liver, kidney, and other organs, such as the mammary gland, affecting pharmacokinetics and milk secretion of antibiotics, anticancer drugs, and other compounds and mediating drug-drug interactions. In addition, ABCG2 expression in cancer cells may directly cause resistance by active efflux of anticancer drugs. The development of ABCG2 modulators is critical in order to improve drug pharmacokinetic properties, reduce milk secretion of xenotoxins, and/or increase the effective intracellular concentrations of substrates. Our purpose was to determine whether the anthelmintic triclabendazole (TCBZ) and its main plasma metabolites triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO(2)) inhibit ABCG2 activity. ATPase assays using human ABCG2-enriched membranes demonstrated a clear ABCG2 inhibition exerted by these compounds. Mitoxantrone accumulation assays using murine Abcg2- and human ABCG2-transduced MDCK-II cells confirmed that TCBZSO and TCBZSO(2) are ABCG2 inhibitors, reaching inhibitory potencies between 40 and 55% for a concentration range from 5 to 25 μM. Transepithelial transport assays of ABCG2 substrates in the presence of both TCBZ metabolites at 15 μM showed very efficient inhibition of the Abcg2/ABCG2-mediated transport of the antibacterial agents nitrofurantoin and danofloxacin. TCBZSO administration also inhibited nitrofurantoin Abcg2-mediated secretion into milk by more than 2-fold and increased plasma levels of the sulfonamide sulfasalazine by more than 1.5-fold in mice. These results support the potential role of TCBZSO and TCBZSO(2) as ABCG2 inhibitors to participate in drug interactions and modulate ABCG2-mediated pharmacokinetic processes.

In Silico Quantitative Structure-Activity Relationship Studies on P-gp Modulators of Tetrahydroisoquinoline-Ethyl-Phenylamine Series

BackgroundMultidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The drug efflux by a transport protein is the main reason for MDR. In humans, MDR mainly occurs when the ATP-binding cassette (ABC) family of proteins is overexpressed simultaneously. P-glycoprotein (P-gp) is most commonly associated with human MDR; it utilizes energy from adenosine triphosphate (ATP) to transport a number of substrates out of cells against concentration gradients. By the active transport of substrates against concentration gradients, intracellular concentrations of substrates are decreased. This leads to the cause of failure in cancer chemotherapy.ResultsHerein, we report Topomer CoMFA (Comparative Molecular Field Analysis) and HQSAR (Hologram Quantitative Structure Activity Relationship) models for third generation MDR modulators. The Topomer CoMFA model showed good correlation between the actual and predicted values for training set molecules. The developed model showed cross validated correlation coefficient (q2) = 0.536 and non-cross validated correlation coefficient (r2) = 0.975 with eight components. The best HQSAR model (q2 = 0.777, r2 = 0.956) with 5-8 atom counts was used to predict the activity of test set compounds. Both models were validated using test set compounds, and gave a good predictive values of 0.604 and 0.730.ConclusionsThe contour map near R1 indicates that substitution of a bulkier and polar group to the ortho position of the benzene ring enhances the inhibitory effect. This explains why compounds with a nitro group have good inhibitory potency. Molecular fragment analyses shed light on some essential structural and topological features of third generation MDR modulators. Fragments analysis showed that the presence of tertiary nitrogen, a central phenyl ring and an aromatic dimethoxy group contributed to the inhibitory effect. Based on contour map information and fragment information, five new molecules with variable R1 substituents were designed. The activity of these designed molecules was predicted by the Topomer CoMFA and HQSAR models. The novel compounds showed higher potency than existing compounds.

The effects of long‐term storage of human red blood cells on the glutathione synthesis rate and steady‐state concentration

Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage-dependent changes that may affect GSH production. RBC units (n = 6) in saline-adenine-glucose-mannitol (SAGM) solution were each divided into four transfusion bags and separate treatments were applied: 1) SAGM (control), 2) GSH precursor amino acids, 3) aminoguanidine, and 4) glyoxal. RBCs were sampled during 6 weeks of storage. Rejuvenated RBCs were also analyzed. After 6 weeks, the ATP concentration declined to 50 ± 5.5% (p < 0.05) of that in the fresh RBCs. For control RBCs, the GSH concentration decreased by 27 ± 6.5% (p < 0.05) and the rate of GSH synthesis by 45 ± 8% (p < 0.05). The rate of GSH synthesis in rejuvenated and amino acid-treated RBCs was unchanged after 6 weeks. Modeling identified that the decline in GSH synthesis was due to decreased intracellular substrate concentrations and reduced amino acid transport, secondary to decreased ATP concentration. This study has uniquely shown that the glutathione synthesis rate decreased significantly after 6 weeks in stored RBCs. Our results have identified potential opportunities for improvement of banked blood storage.

Improved Extrapolation of Hepatobiliary Clearance from in Vitro Sandwich Cultured Rat Hepatocytes through Absolute Quantification of Hepatobiliary Transporters

Previously we have reported that hepatobiliary transporter expressions in sandwich cultured hepatocytes (SCH) are altered 2- to 5-fold. This change could limit the model's predictive power for in vivo biliary clearance. The present study was designed to better establish in vitro to in vivo correlation (IVIVC) of biliary clearance. Eleven compounds representing the substrates of Mrp2/Abcc2, Bcrp/Abcg2 and Bsep/Abcb11 were tested in the sandwich cultured rat hepatocyte (SCRH) model. Simultaneously, the absolute difference of hepatobiliary transporters between rat livers and SCRH at day 5 post culture was determined by LC-MS/MS. This difference was integrated into the well-stirred hepatic prediction model. A correction factor named "g_factor" was mathematically defined to reflect the difference in hepatobiliary transporter expressions between the SCRH model and in vivo models, as well as the contribution of multiple transporters. When the g_factor correction was applied, the in vivo biliary clearance prediction was significantly improved. In addition, for those compounds which are poorly permeable and/or undergo transporter-dependent active uptake, the known intracellular concentrations of substrates were used to estimate intrinsic bile clearance. This led to further improvement in the prediction of in vivo bile secretion. While the rate-limiting processes of uptake transporters in the SCRH model remain to be further determined, we showed that integration of the absolute difference of hepatobiliary transporter proteins and transport contributions could improve the predictability of SCRH model. This integration is fundamental for increased confidence in the IVIVC of human biliary clearance.

Model Analysis of the Concentration-Dependent Permeability of P-gp Substrates

Recently, it was reported that the apparent Michaelis-Menten constant (Km(app)) of a P-glycoprotein (P-gp) substrate, defined for the extracellular substrate concentration, increases as the P-gp expression level in the cell increases. By its nature, the Km value should not depend on the level of P-gp expression. The purpose of this study is to establish a model which can estimate the Km value independent of the P-gp expression level in cells. The previously reported concentration-dependent permeability of verapamil, quinidine, and vinblastine in MDR1-MDCKII, P-gp-highly induced Caco-2, P-gp-induced Caco-2, normal Caco-2, and MDR1-knockdown Caco-2 cells data were analyzed using a model in which the Km value was defined for the intracellular substrate concentration. The estimated Km values defined for the substrate concentration inside the cells were almost the same among various cells with different levels of P-gp expression. The estimated Vmax values were approximately proportional to the P-gp expression level. The established kinetic model was found to be rational based on the results that the Km values of P-gp substrates were about the same for cells expressing various levels of P-gp, while the Vmax values were proportional to the expression levels of P-gp.