References of "Farcas, Elena"
     in
Bookmark and Share    
See detailFlow-through partial-filling affinity capillary electrophoresis for the detection of weak ligand-target interactions: application to coagulation factor XIIa
Davoine, Clara ULiege; Farcas, Elena ULiege; Bouckaert, Charlotte et al

Poster (2019, December 11)

Coagulation factor XIIa (FXIIa) is a S1A serine protease implicated in several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. In ... [more ▼]

Coagulation factor XIIa (FXIIa) is a S1A serine protease implicated in several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. In the field of thrombosis, anti-FXIIa therapies could answer unmet medical needs such as the safe prevention of thrombosis in patients exposed to blood-contacting devices. The FXIIa inhibition also rises as a therapeutic strategy in patients suffering from hereditary angioedema and as an emerging research field in neuro-inflammatory and neurodegenerative disorders. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, and RNA-based technologies. In contrast, only a few data regarding the design of synthetic small molecular-weight inhibitors of FXIIa are available. Our team previously developed 3-carboxamido-benzopyrans and highlighted that aromatic guanidine is an attractive starting point. To facilitate chemical exploration, we decided to apply a fragment-based lead discovery approach (FBLD). However, the success of this approach rests on the ability to develop bioassays that are able to detect affinity in the µM-mM range. For this purpose, we develop a flow-through partial-filling affinity capillary electrophoresis designed to screen positively-charged molecules against FXIIa at physiological pH. [less ▲]

Detailed reference viewed: 44 (4 ULiège)
See detailAffinity capillary electrophoresis as a powerful tool for fragment-based screening: application to the inhibition of S1A serine proteases implicated in the coagulation cascade
Davoine, Clara ULiege; Farcas, Elena ULiege; Simon, François et al

Poster (2019, November 22)

Fragment-based drug discovery (FBDD) proved its efficacy and gained an increasing interest in the pharmaceutical industry and academia.[1] In contrast to the traditional medicinal chemistry approach, FBDD ... [more ▼]

Fragment-based drug discovery (FBDD) proved its efficacy and gained an increasing interest in the pharmaceutical industry and academia.[1] In contrast to the traditional medicinal chemistry approach, FBDD leads to small chemical templates that should be easier to tune into compounds displaying a high inhibitory potency associated with adequate selectivity and pharmacokinetic properties. However, the use of fragments during the screening implies that the affinity with the target is very weak. In consequence, the success of the FBDD approach strongly depends on the ability to develop bioassays that are sensitive enough to detect and gauge affinity in the µM-mM range. NMR methods, X-ray screening and surface plasmon resonance (SPR) are generally the most popular approaches reported in the literature. Nevertheless, these valuable techniques are rather expensive, time-consuming or unable to reflect the physiological environment. Generally, they also require highly purified reagents as well as large sample amounts. In this context, we investigated the ability of affinity capillary electrophoresis (ACE) for FBDD screening. ACE has the advantages to do not need highly purified, modified or immobilized species.[2] In this communication, we will report the development of the ACE approach for the screening of fragments towards two S1A serine proteases namely thrombin and FXIIa. We performed a direct and competitive ACE approach for the screening. The direct ACE method was designed to screen positively charged fragments. Indeed, in S1A serine proteases, the specificity pocket S1 is characterized by an aspartate (Asp189) at its bottom and a cationic group is expected to guide the fragment inside the active site [3]. Competitive ACE-binding is suitable for any fragments regardless of their charge, but the assay requires a higher concentration of ligands. [4] [1] D.A. Erlanson, W. Jahnke, Wiley-VCH2016; b) G. Siegal, E. Ab, J. Schultz, Drug Discov Today (2007),12 (23/24), 1032; c) CW. Murray, ML. Verdonk, DC. Rees, Trends Pharmacol Sci (2012), 33(5), 224. [2] E. Farcas, L. Pochet, J. Crommen, A.C. Servais, M. Fillet, JPBA (2017), 144, 195–212 [3] E. Farças, C. Bouckaert, A.C. Servais, J. Hanson, L. Pochet, M. Fillet, Anal Chim Acta (2017), 211-222. [4] E. Farças, J. Hanson, L. Pochet, M. Fillet, Anal Chim Acta (2018), 214-222 [less ▲]

Detailed reference viewed: 52 (4 ULiège)
See detailCapillary electrophoresis and microscale thermophoresis in fragment-based drug discovery : development of screening bioassays towards FXIIa
Davoine, Clara ULiege; Farcas, Elena ULiege; Thabault, Léopold et al

Poster (2019, March 24)

Tackling thrombotic disorders without affecting the hemostatic capacity remains a challenge in medicine. One of the strategies in the search for safer antithrombotic therapies is to target coagulation ... [more ▼]

Tackling thrombotic disorders without affecting the hemostatic capacity remains a challenge in medicine. One of the strategies in the search for safer antithrombotic therapies is to target coagulation factor XIIa (FXIIa).[1] This strategy have two substantial advantages : 1. No bleeding risk 2. Additional anti-inflammatory effects After limited results with the traditional medicinal chemistry approach (inhibitors in the μM range) [2], we decided to apply fragment-based drug discovery approach (FBDD). In its first step, FBDD strongly depends on the ability to detect and gauge weak affinity binders. In the scope of validating the fragment hits, the most robust strategy is to apply orthogonal techniques. In this work, we investigate the suitability of microscale thermophoresis and capillary electrophoresis for the discovery of new fragments that bind FXIIa. [1] a) J.I.Weitz, Thromb Res, 141 Suppl 2 (2016) S40-45; b) J.C. Fredenburgh, P.L. Gross, J.I. Weitz, Blood, 129 (2017) 147-154; c) A.T. Long, E. Kenne, R. Jung, T.A. Fuchs, T. Renne, J Thromb Haemost, 14 (2016) 427-437. [2] a) S. Robert, C. Bertolla, B. Masereel, J.M. Dogné, L. Pochet, Journal of Medicinal Chemistry, 51 (2008) 3077-3080; b) C. Bouckaert, S. Serra, G. Rondelet, E. Dolušić, J. Wouters, J.M. Dogné, R. Frédérick, L. Pochet, Eur J of Med Chem, 110 (2016) 181-194 [less ▲]

Detailed reference viewed: 97 (7 ULiège)
See detailDevelopment of fragments screening bioassays towards thrombin and FXIIa using capillary electrophoresis
Davoine, Clara ULiege; Farcas, Elena ULiege; Bouckaert, Charlotte et al

Poster (2018, December 19)

In this communication, we will report the development of a capillary electrophoresis approach for the screening of fragments towards thrombin and FXIIa. We performed direct and competitive ACE-binding ... [more ▼]

In this communication, we will report the development of a capillary electrophoresis approach for the screening of fragments towards thrombin and FXIIa. We performed direct and competitive ACE-binding assays for the screening of molecules at physiological pH. [less ▲]

Detailed reference viewed: 14 (2 ULiège)
See detailDevelopment of capillary electrophoresis bioassays for the screening of fragments : application to thrombin and FXIIa
Davoine, Clara ULiege; Farcas, Elena ULiege; Bouckaert, Charlotte et al

Poster (2018, November 23)

In this communication, we will report the development of a capillary electrophoresis approach for the screening of fragments towards thrombin and FXIIa. We performed direct and competitive ACE-binding ... [more ▼]

In this communication, we will report the development of a capillary electrophoresis approach for the screening of fragments towards thrombin and FXIIa. We performed direct and competitive ACE-binding assays for the screening of molecules at physiological pH. [less ▲]

Detailed reference viewed: 15 (2 ULiège)
Peer Reviewed
See detailPartial filling capillary electrophoretic mobility shift competition assay: a versatile and reliable tool for the assessment of weak biomolecular interactions
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Hanson, Julien ULiege et al

Conference (2018, September 10)

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both ... [more ▼]

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both academia and pharmaceutical industry. FBDD detects low molecular-weight (MW) ligands (fragments) that bind to biologically important targets, then a structure-guided fragment growing or merging approach is performed giving rise to potent molecules with drug-like properties. However, the analytical arsenal able to point out weak interactions is rather expensive, time consuming or unable to reflect the physiological environment. In this framework, we developed a generic, fully automated, microscale electrophoretic mobility shift competition assay that can be used for primary screening of weak biomolecular interactions between fragments and the target of interest. The affinity capillary electrophoresis (ACE) competitive approach is based on the monitoring of the competition of fragments with a known target inhibitor (PL) for the same active site. The consequence of the competition is a modification of PL electrophoretic mobility, modification that can be measured and used for ligand screening and/or IC50 determination. To achieve our goal, particular attention has been paid to the optimization of the binding environment parameters: an optimal buffer was used for the binding measurements, a partial filling approach was considered to gain sensitivity and to reduce protein consumption and a neutral dynamic coating was performed to reduce protein adsorption to the capillary wall. Moreover, the binding partners concentrations and the electrophoretic conditions were carefully optimized. It is noteworthy that the interactions occur in solution, using the protein in its native form, thus mimicking the physiological environment.The accuracy and reliability of the proposed method was demonstrated by monitoring the competition of two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat with a known thrombin inhibitor, pefabloc (PEFA). The measured IC50 were found to be in good accordance with the previously reported ones. Additionally, a small chemical library was built to evaluate the performance of the newly developed screening-bioassay. The optimized method proved to be remarkably reproducible (migration time RSDs < 1.2%) and selective. The results prove the high discriminatory potency of the method and its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, significantly expanding the applicability of the assay compared to a direct approach [1]. Finally, the ability of this approach to discriminate between competitive and irreversible thrombin binders was also demonstrated.References [1] E. Farcas, C. Bouckaert, A.-C. Servais, J. Hanson, L. Pochet, M. Fillet, Analytica Chimica Acta, 2017, 984, 211-222 [less ▲]

Detailed reference viewed: 88 (17 ULiège)
Full Text
Peer Reviewed
See detailTransverse diffusion of laminar flow profiles as a generic capillary electrophoresis method for in-line nanoreactor mixing: application to the investigation of antithrombotic activity
Farcas, Elena ULiege; Pochet, Lionel; Fillet, Marianne ULiege

in Talanta (2018), 188

Capillary electrophoresis (CE)instrumentwas used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened ... [more ▼]

Capillary electrophoresis (CE)instrumentwas used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened molecules was monitored in a quick and fully automated fashion using only few nanoliters of reactants. To achieve this goal, the targeted enzyme (thrombin) and the chromogenic substrate with or without the screened inhibitor were injected as separate plugs. The mixing of the reactants was then realized using electrophoretically mediated microanalysis (EMMA) or fast transverse diffusion of laminar flow profiles (TDLFP) procedure. The latest provided better mixing performance and was chosen to investigate the inhibitory potency of a fragment library. This very straightforward and fast CE activity assay showed results in good accordance with a previously developed CE affinity assay that confirms the potential of CE at the early stages of drug discovery, providing not only an efficient nanoscale bioreactor but also a selective and integrated separation device. [less ▲]

Detailed reference viewed: 27 (5 ULiège)
See detailContribution of capillary electrophoresis at the early stage of drug discovery
Farcas, Elena ULiege

Doctoral thesis (2018)

With the emergence of novel pharmaceutical targets, there is an imperative need for fast, reliable and cost-effective analytical techniques able to shorten the long and expensive process of drug ... [more ▼]

With the emergence of novel pharmaceutical targets, there is an imperative need for fast, reliable and cost-effective analytical techniques able to shorten the long and expensive process of drug development. In this context, we demonstrated that capillary electrophoresis (CE) could be a robust and efficient analytical tool, notably for the quantification of weak ligand-target interactions, for the investigation of ligands impact on targeted enzyme activity and for drug metabolism monitoring. To perform our proof of concept studies, the pathology targeted was thrombosis, since it remains one of the most important causes of morbidity and mortality worldwide. Two affinity CE approaches were designed to quantitate the interaction between thrombin (Thr) and low-affinity ligands in near physiological conditions. The direct binding approach consisted of monitoring the ligand electrophoretic mobility (µep) modification upon binding, while the indirect binding approach was based on the competition of the ligand with a known Thr inhibitor (PL). The modifications of PL µep as a consequence of the competition was in this case monitored. The developed affinity CE approaches allowed not only the accurate determination of complex dissociation constants (Kd)/half maximal inhibitory concentration (IC50) values, but they additionally allowed the conduction of highly discriminant and robust screening campaigns. It should be mentioned that the direct binding approach was suitable for the affinity determination only for cationic molecules that absorb in UV-VIS, while the indirect binding assay was extended to all kind of molecules, no matter their UV-VIS absorbance, or charging state. The information obtained during the affinity CE analyses was completed with the results generated by fast activity CE assays (analysis time less than 3 min). In this case, the capillary was employed as a multifunction nanoreactor on which the reagents were injected, mixed, incubated and the reaction product (the reporter molecule) was electrophoresed and detected. Two different mixing procedures, namely electrophoretically mediated microanalysis (EMMA) and transverse diffusion of laminar flow profiles (TDLFP) were investigated and the reaction parameters were optimized. In the context of molecules screening TDLFP approach proved to be the mixing procedure of choice, especially when the assayed molecules were charged species. The screening of our small library was performed in the optimized conditions and the results obtained were in good concordance with the affinity screening results. Additionally, a monolithic-functionalized GFP capillary column was investigated for method sensitivity enhancement. The usefulness of CE for metabolism studies realization was also demonstrated. A fully automated in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, underwent an in-line bioreaction in the presence of CYP1A1 giving rise to hydroxycoumarin. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The developed approaches demonstrate that CE is a powerful and robust tool to be considered in the early stages of drug discovery, due to its minimal sample requirements, ease of automation, its ability to detect and quantify weak interactions and to study drug metabolism in a fully-automated fashion and in near-physiological conditions. [less ▲]

Detailed reference viewed: 77 (11 ULiège)
Full Text
Peer Reviewed
See detailCapillary electrophoretic mobility shift displacement assay for the assessment of weak drug-protein interactions
Farcas, Elena ULiege; Hanson, Julien ULiege; Pochet, Lionel et al

in Analytica Chimica Acta (2018)

Only few reports describe the use of capillary electrophoresis in the context of Fragment Based Drug Discovery (FBDD). In this paper, we will present a generic, fully automated, microscale electrophoretic ... [more ▼]

Only few reports describe the use of capillary electrophoresis in the context of Fragment Based Drug Discovery (FBDD). In this paper, we will present a generic, fully automated, microscale electrophoretic mobility shift competition assay that can be used in FBDD for primary screening of weak biomolecular interactions between fragments and target protein. The accuracy and reliability of the present method was demonstrated by measuring the interaction between two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat. The measured IC50 were found to be in good accordance with the previously reported ones. Furthermore, we built a small chemical library to evaluate the performance and the advantage of our newly developed screening-bioassay compared to the direct affinity capillary electrophoresis-binding assay. The results demonstrate the high discriminatory power of the method and above all its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, greatly expanding the scope of the assay. Finally, we proved that this approach is able to discriminate between competitive binders and irreversible binders. Altogether, this work demonstrates that capillary electrophoresis could constitute an important added value in the arsenal used to screen fragments in drug discovery. [less ▲]

Detailed reference viewed: 30 (7 ULiège)
Peer Reviewed
See detailOptimization of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena ULiege; Bouckaert, Charlotte; Servais, Anne-Catherine ULiege et al

Conference (2017, June 20)

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations ... [more ▼]

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR. [less ▲]

Detailed reference viewed: 50 (17 ULiège)
Peer Reviewed
See detailOptimiaztion of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena ULiege; Bouckaert, Charlotte; Servais, Anne-Catherine ULiege et al

Poster (2017, June 19)

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations ... [more ▼]

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR. [less ▲]

Detailed reference viewed: 86 (8 ULiège)
Full Text
Peer Reviewed
See detailPartial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.
Farcas, Elena ULiege; Bouckaert, C.; Servais, Anne-Catherine ULiege et al

in Analytica Chimica Acta (2017), 984

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD ... [more ▼]

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the muM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD. [less ▲]

Detailed reference viewed: 36 (6 ULiège)
Full Text
Peer Reviewed
See detailCapillary electrophoresis in the context of drug discovery.
Farcas, Elena ULiege; Pochet, Lionel; Crommen, Jacques ULiege et al

in Journal of Pharmaceutical and Biomedical Analysis (2017)

Capillary Electrophoresis is a very efficient and resolutive separation technique used for many years in the analytical field. Despite all its assets, CE remains poorly used in drug discovery. This can be ... [more ▼]

Capillary Electrophoresis is a very efficient and resolutive separation technique used for many years in the analytical field. Despite all its assets, CE remains poorly used in drug discovery. This can be explained by the relatively low number of experienced CE practitioners, the maturity of HPLC in the pharmaceutical industry and some intrinsic limitations of the technique. The objective of this review is to focus our attention on recent developments of this technique in three different drug discovery areas: bioassays, drug-plasma interactions and drug metabolism studies. These developments were based on two important abilities of CE: the capacity to measure non-covalent interactions in solution and the ability to use a portion of the capillary as a reactor while the rest of the capillary is used for the separation of the product of the reaction. [less ▲]

Detailed reference viewed: 47 (8 ULiège)
Peer Reviewed
See detailOPTIMIZATION OF A FULLY AUTOMATED ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM FOR CYP1A1 ACTIVITY MONITORING
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline et al

Conference (2016, October 01)

Objective of the study Since CYP1A1, a member of cytochrome P450 superfamily, has been described to be over expressed in various types of cancer, our study was focused on the optimization of a fully ... [more ▼]

Objective of the study Since CYP1A1, a member of cytochrome P450 superfamily, has been described to be over expressed in various types of cancer, our study was focused on the optimization of a fully automated system for the monitoring of this particularly interesting enzyme. Moreover, the potentiality of this approach to screen CYP1A1 inhibitors was investigated. Materials and methods The experiments were carried out on a HP3DCE system using an on-column DAD. The EMMA procedure was performed by injecting a plug containing the co-factor(NADPH) and the substrate(7-ethoxycoumarin) between two plugs of CYP1A1 supersomes. The reaction was triggered by the application of a voltage switch. The voltage was then turned off to allow the metabolic reaction to occur. The separation of the components was then performed. Results Satisfying results were obtained using CYP1A1 at a concentration of 200 pmol/mL, while the incubation time was settled to 15 min. A DoE was performed to find the best mixing conditions. The amount of metabolite obtained was comparable to the one detected after conventional off-line metabolization. The ability of our system to monitor CYP1A1 inhibition was then proven with apigenin, a well-known CYP1A1 inhibitor. Conclusions The present study describes the development of a fully automatized in-capillary method for CYP1A1 activity monitoring and proves the potentialy of our system to be used for the screening of CYP1A1 inhibitors. The advantages of performing inline metabolization assays are mainly the miniaturization and the automatization of the process. Besides, the reagents consumption is drastically reduced due to the injection of few tens of nanoliters. [less ▲]

Detailed reference viewed: 30 (13 ULiège)
Full Text
Peer Reviewed
See detailCapillary electrophoresis method to determine siRNA complexation with cationic liposomes
Furst, Tania ULiege; Bettonville, Virginie ULiege; Farcas, Elena ULiege et al

in Electrophoresis (2016)

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally ... [more ▼]

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation are gel electrophoresis and RiboGreen® fluorescence-based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this work is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this research, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short-end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared to the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated to CE approach since no carcinogenic product is used. Finally, this methodology can also be extended to the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles. [less ▲]

Detailed reference viewed: 103 (30 ULiège)
Full Text
Peer Reviewed
See detailFully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline ULiege et al

in Electrophoresis (2016), 37(2), 248-255

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction ... [more ▼]

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction in the presence of CYP1A1 supersomes and NADPH as co-factor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after off-line metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our in-line system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors. [less ▲]

Detailed reference viewed: 65 (38 ULiège)
Peer Reviewed
See detailFULLY AUTOMATED ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM FOR CYP1A1 ACTIVITY MONITORING
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline ULiege et al

Poster (2015, June 23)

Introduction Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The ... [more ▼]

Introduction Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The identification of the enzymes involved in drug metabolism is thus of critical importance for the design of further clinical studies. The availability of specifically expressed human CYPs, namely supersomes, allows the investigation of the contribution of a single metabolic enzyme to the biotransformation pathway of the compound under investigation. CYP1A1, a member of the cytochrome P450 superfamily, was studied in this project. Interestingly, it has been described to be over expressed in various types of cancer. Consequently, CYP1A1 has emerged as a particularly interesting target for cancer therapy. Methods All the experiments were carried out on a HP3DCE system equipped with an on-column DAD. The EMMA procedure was performed by injecting a plug containing CYP1A1 supersomes, followed by a plug that contained the co-factor and the substrate, then another plug of CYP1A1 supersomes. The reaction was triggered by the application of a voltage switch. The voltage was then turned off to allow the metabolic reaction to occur. The separation of the components was then performed at -25 kV. Results The present study describes the development of a fully automatized in-capillary method to follow metabolization of 7-hydroxycoumarin and screen CYP1A1 inhibitors. After preliminary studies, satisfying results were obtained using CYP1A1 at a concentration of 200 pmol/mL, while the incubation time was settled to 15 min. Equal reactant plugs were injected at -50 mbar for 6 sec. The short-end injection performed gave rise to a baseline separation of the molecules (substrate, product, CYP1A1 and NADPH) in less than 2 minutes. Adequate plugs overlap was obtained using electrophoretic mixing. The DoE performed highlighted that the voltage switch has a great impact on the metabolite formation. The amount of product obtained in the optimal conditions was found to be comparable to the one detected after conventional off-line metabolization. Besides the interest of developing an automatized CE approach for metabolisation studies, we also wanted to investigate the potentiality of this approach to screen CYP1A1 inhibitors. The ability of our system to monitor CYP1A1 inhibition was undertaken with apigenin, a well-known inhibitor. It is noteworthy that the compatibility of our system with MEKC ensures its applicability to a large variety of molecules. Novel aspect Monitoring CYP1A1 activity using a rapid and fully automated EMMA method that could be used for new anticancer agents screening. [less ▲]

Detailed reference viewed: 71 (14 ULiège)
Peer Reviewed
See detailFully automated electrophoretically mediated microanalysis system for CYP1A1 activity monitoring
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline ULiege et al

Conference (2015, May 28)

In order to evaluate the potentiality of capillary electrophoresis for CYP1A1 activity monitoring, an in-line method was developed with the well-known 7-ethoxycoumarin substrate. The electrophoretically ... [more ▼]

In order to evaluate the potentiality of capillary electrophoresis for CYP1A1 activity monitoring, an in-line method was developed with the well-known 7-ethoxycoumarin substrate. The electrophoretically mediated microanalysis approach was used with CYP1A1 supersomes to provide a rapid and fully automated method. The in-line homogenous enzyme assay was performed in physiological conditions (pH 7.4), whereas a MEKC buffer was used as background electrolyte. In order to reduce the analysis time, the short end injection was performed. Firstly a plug containing CYP1A1 supersomes was hydrodynamically injected into a fused silica capillary, followed by a plug of co-factor (NADPH) and substrate (7-ethoxycoumarin) and finally another plug of CYP1A1 (sandwich mode). The experimental conditions were finely investigated and tuned by design of experiment methodology. The metabolization rate measured in the optimized conditions was comparable with the one obtained after off-line metabolization. Finally, inhibition studies were conducted and a significant decrease of 7-hydroxycoumarin formation was observed using apigenin as CYP1A1 potent inhibitor. [less ▲]

Detailed reference viewed: 38 (18 ULiège)