New methods based on capillary electrophoresis for in vitro evaluation of protein tau phosphorylation by glycogen synthase kinase 3-ß.

The hyperphosphorylation of tau protein is associated with the development of the neuronal pathology of Alzheimer's disease. As most conventional methods study only particular phosphorylation sites of tau, it is necessary to develop a simple and reliable assay to determine the phosphorylation of tau at multiple sites. Capillary electrophoresis (CE)-based enzymatic assays are not yet used to monitor tau phosphorylation. The present work aims to develop CE-based assays to evaluate tau phosphorylation by the glycogen synthase kinase 3-ß (GSK3ß). A novel pre-capillary CE assay was first developed. An in-capillary CE-based enzymatic assay was also used since this approach is known to be time- and cost- effective. The enzymatic reaction was monitored by quantifying the product adenosine 5'- diphosphate (ADP). The influence of two classes of glycosaminoglycan (GAG), namely heparin and heparan sulfate, on the phosphorylation reaction was also assessed. Results obtained by both CE approaches were comparable and in excellent agreement with those reported in the literature using conventional radiometric and immunoblotting methods. In fact, CE results confirmed the inductory effect of the sulfated sugars heparin and heparan sulfate on tau hyperphosphorylation, probably because of the exposition of new sites phosphorylatable by GSK3ß. This study shows that simple (no-labeling), rapid (less than 30 min per assay), and eco-friendly (no-radioactivity) CE-based kinase assays can give insight into the abnormal phosphorylation of tau. They can be extended to screen different modulators of tau phosphorylation to highlight their function and to develop effective drugs for neurodegenerative disease treatments.

New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway.

Human kinases are one of the most promising targets for cancer therapy. Methods able to measure the effects of drugs on these cell agents remain crucial for biologists and medicinal chemists. The current work therefore sought to develop an in-capillary enzymatic assay based on capillary electrophoresis (CE) to evaluate the inhibition of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and the mammalian target of rapamycin (mTOR). These kinases belong to the same signaling pathway PI3K/Akt/mTOR. For this proposal, the capillary was used as a nanoreactor in which a few nanoliters of the kinase, its substrate, adenosine triphosphate (ATP), and the potent inhibitor were separately injected. A transverse diffusion of laminar flow profiles (TDLFP) approach was employed to mix the reactants. Adenosine diphosphate (ADP ) was detected online at 254 nm. The CE assay was first developed on the α isoform of PI3K. It was compared to five commercial kits frequently used to assess kinase inhibition, based on time-resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence. Each assay was evaluated in terms of sensitivity (S/B), reproducibility (Z'), and variability (r (2)). This CE method was easily extended to assay the inhibition of the ß, γ, and δ isoforms of PI3K, and of the other kinases of the pathway, Akt1 and mTOR, since it is based on in-capillary mixing by TDLFP and on ADP quantification by simple UV absorption. This work shows for the first time the evaluation of inhibitors of the kinases of the PI3K/Akt/mTOR pathway using a common in-capillary CE assay. Several inhibitors with a wide range of affinity toward these enzymes were tested.

Electrophoretically mediated microanalysis for in-capillary electrical cell lysis and fast enzyme quantification by capillary electrophoresis.

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. ß-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-ß-D-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the ß-galactosidase--released from the lysed cells--by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

In-capillary reactant mixing for monitoring glycerol kinase kinetics by CE.

CE was used for the first time to study the two-substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine-5'-triphosphate were in-capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine-5'-triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero-potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine-5'-diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose-1,6-bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in-capillary mixed using an electrophoretically mediated microanalysis approach.

Potential Inhibitory Effect of Apis mellifera's Venom and of Its Two Main Components-Melittin and PLA2-on Escherichia coli F1F0-ATPase.

Bacterial resistance has become a worrying problem for human health, especially since certain bacterial strains of Escherichia coli (E. coli) can cause very serious infections. Thus, the search for novel natural inhibitors with new bacterial targets would be crucial to overcome resistance to antibiotics. Here, we evaluate the inhibitory effects of Apis mellifera bee venom (BV-Am) and of its two main components -melittin and phospholipase A2 (PLA2)- on E. coli F1F0-ATPase enzyme, a crucial molecular target for the survival of these bacteria. Thus, we optimized a spectrophotometric method to evaluate the enzymatic activity by quantifying the released phosphate from ATP hydrolysis catalyzed by E. coli F1F0-ATPase. The protocol developed for inhibition assays of this enzyme was validated by two reference inhibitors, thymoquinone (IC50 = 57.5 µM) and quercetin (IC50 = 30 µM). Results showed that BV-Am has a dose-dependent inhibitory effect on E. coli F1F0-ATPase with 50% inhibition at 18.43 ± 0.92 µg/mL. Melittin inhibits this enzyme with IC50 = 9.03 ± 0.27 µM, emphasizing a more inhibitory effect than the two previous reference inhibitors adopted. Likewise, PLA2 inhibits E. coli F1F0-ATPase with a dose-dependent effect (50% inhibition at 2.11 ± 0.11 µg/mL) and its combination with melittin enhanced the inhibition extent of this enzyme. Crude venom and mainly melittin and PLA2, inhibit E. coli F1F0-ATPase and could be considered as important candidates for combating resistant bacteria.

Screening of Some Essential Oil Constituents as Potential Inhibitors of the ATP Synthase of Escherichia coli.

Many novel bacterial targets and natural inhibitors of enzymes are currently being considered to overcome antibiotic resistance of Escherichia coli. Hence, in this study, 20 essential oil constituents were screened for their potential inhibitory effect on E. coli ATP synthase. This enzyme is involved in the hydrolysis of ATP into ADP and inorganic phosphate (Pi). First, E. coli membrane ATP synthase was isolated via cell lysis. A spectrophotometric method was optimized to quantify the released phosphate from ATP hydrolysis in order to follow the enzymatic activity. The method was validated by determining the kinetic parameters of this reaction (Km = 144.66 µM and Vmax = 270.27 µM/min), and through the inhibition assays of ATP synthase using three reference inhibitors, thymoquinone (half maximal inhibitory concentration [IC50 ] = 50.93 µM), resveratrol (maximum inhibition of 40%), and quercetin (IC50 = 29.01 µM). Among the studied essential oil components, α-terpinene was the most potent inhibitor (IC50 = 19.74 µM) followed by ß-pinene, isoeugenol, eugenol, and estragole.

Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis.

Myrosinase is a unique enzyme that catalyzes the hydrolysis of glucosinolates (GLS) to isothiocyanate (ITC), glucose and sulfate. Isothiocyanates display a diversified very interesting biological activity. In this study, capillary electrophoresis (CE) was used for the first time for evaluating myrosinase kinetics (maximum velocity Vmax and Michaelis-Menten constant Km) and to assess the affinity of a variety of substrates toward this enzyme. The pre-capillary approach was chosen since it is very simple to conduct. For this, the enzymatic reaction was performed in a micro-vial. The reaction mixture volume was of only 100 µL and the incubation lasted only 5 min at 37±1°C. Short-end injection of few tens of nanoliters (~25 nL) of the reaction mixture was performed which decreased analysis time without using any electroosmotic modifier. The sulfate produced was detected and quantified with a contactless capacitively coupled conductivity detector (C(4)D) allowing the evaluation of myrosinase kinetics. This study shows, that capillary electrophoresis with contactless conductivity detection can be very useful for monitoring myrosinase activity. Comparing to the conventional spectrophotometric method (1982), the CE method developed here is simple, automated, economic, rapid (incubation for few minutes) and robust. Results compared very well with those reported in literature using the conventional method. Moreover, the affinity of a variety of natural and synthetic glucosinolates toward this enzyme has been assessed for the first time.

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing.

A capillary electrophoresis (CE)-based enzyme assay method has been developed to screen protein kinase inhibitors. Four human kinases GSK3ß, DYRK1A, CDK5/p25 and CDK1/cyclin B were chosen to test this novel method. These enzymes have been identified as very promising targets to develop treatments against cancer and neurodegenerative diseases. The efficiency of drugs against these relevant biological targets has never been carried out by CE. For this proposal, the capillary was used as a nanoreactor in which four reactants (the enzyme, its two substrates and its potential inhibitor) were successively injected, mixed by using transverse diffusion of laminar flow profiles and incubated. The adenosine 5'-diphosphate (ADP) formed during the enzymatic reaction was detected by UV and quantified. The efficiency of the developed CE method was validated by determining the IC50 values of a wide variety of inhibitors covering a large domain of affinity toward kinases and containing representative and chemically divergent skeletons. Excellent agreement was found between the results obtained by CE and those reported in the literature when using conventional radiometric enzyme assays. Moreover, CE was successfully used to determine the inhibitory effect of several potential inhibitors that was not yet assessed by conventional methods and is crucial for structure activity relation studies. This novel CE method is simple, rapid, very economic (few tens of nanoliters per IC50) and eco-friendly since no radioactivity was required. It could be extended to high-throughput screening of kinase inhibitors, which is of great interest for biomedical and pharmaceutical research fields.

Capillary electrophoresis as a novel technique for screening natural flavonoids as kinase inhibitors.

Capillary electrophoresis (CE) was used for the first time to evaluate the inhibition activity of aglycone flavonoids (such as quercetin and isorhamnetin) and some of their glycosylated derivatives toward human kinases. The cyclin-dependant kinase 5 (CDK5/p25) and the glycogen synthase kinase 3ß (GSK3ß) were chosen since they are very promising biological targets for developing treatments against neurodegenerative diseases and cancer. In a previous work, we developed an in-capillary kinase CE assay where the capillary was used as an enzymatic nanoreactor in which the kinase, its substrate, adenosine 5'-triphosphate (ATP) and its potential inhibitor were mixed by using transverse diffusion of laminar flow profiles (TDLFP). The product adenosine 5'-diphosphate (ADP) was then detected at 254nm and quantified. In this work, this assay was improved to reduce, for the first time, the dilution effect commonly observed with the TDLFP approach. Under the new conditions established herein, IC50 values for quercetin, kaempferol and flavopiridol were successfully obtained and were in the same order of magnitude of those reported in the literature using the conventional assay using radioactive (33)P-ATP. It was shown that aglycone flavonoids have an inhibition activity more important than their glycosylated derivatives. CE was also proved to be very efficient for evaluating inhibition activity of complex samples such as crude extracts of sea buckthorn (SBT) berries obtained by solvent-free microwave extraction (SFME). This novel approach to combine SFME technique to a CE-based enzymatic assay is very interesting for evaluating the biological activity of natural material in a fast, simple, economic (no use of neither fluorescent nor radiometric labels) and green (no organic solvents) manner.

New development in in-capillary electrophoresis techniques for kinetic and inhibition study of enzymes.

Enzymes are often quantified by measuring their biological activity. Capillary electrophoresis is gaining its position in this field due to the ongoing trend to miniaturize biochemical assays. The aim of this work was to compare pre-capillary (off-line) and in-capillary electrophoresis techniques for studying enzymatic activity. The ß-galactosidase (ß-Gal) was chosen as a model enzyme. Each technique was optimized independently in order to decrease analyte consumption (to few tens of nanoliters), incubation time (to few seconds) and analysis time (below 1 min). Several experimental parameters (ionic strength of the background electrolyte (BGE) and of the incubation buffer, incubation time, injected volumes, …) were optimized by following peak efficiencies, resolution and repeatability. To monitor the performance of each technique, the catalytic constants (V(max) and K(m)) of 4-nitro-phenyl-d-galactopyranoside (PNPG) hydrolysis by ß-Gal as well as the inhibition constants (K(i) and IC(50)) by a competitive inhibitor 2-nitrophenyl-1-thio-ß-d-thiogalactopyranoside (ONPTG) were determined. The results obtained were cross compared and were also evaluated by comparison to a standard spectrophotometric method. EMMA proved to be the best technique in terms of sample consumption and speed. The short-end injection was successfully used which speeded-up electrophoretic analysis (<0.8 min). It is a very powerful tool for studying enzymatic inhibition. Usually, the inhibitor is injected in the capillary mixed to the substrate especially when both have similar mobilities. We show in this work, for the first time, that combining at-inlet reaction with EMMA-CE allows enzyme inhibition to be realized without any prior mixing of the substrate and the inhibitor. This approach is very interesting for screening inhibitors, rapidly and without excessive substrate consumption.