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See detailEvaluation of the analytical performances of two Raman handheld spectrophotometers for pharmaceutical solid dosage form quantitation
Coic, Laureen ULiege; Sacre, Pierre-Yves ULiege; Dispas, Amandine ULiege et al

in Talanta (2020), 214

This paper addresses the issue of pharmaceutical solid dosage form quantitation using handheld Raman spectrophotometers. The two spectrophotometers used are designed with different technologies: one ... [more ▼]

This paper addresses the issue of pharmaceutical solid dosage form quantitation using handheld Raman spectrophotometers. The two spectrophotometers used are designed with different technologies: one allows getting a more representative sampling with the Orbital Raster Scanning technology and the other one allows setting acquisition parameters. The goal was to evaluate which technology could provide the best analytical results. Several parameters were optimized to get the lowest prediction error in the end. The main objective of this study was to evaluate if this kind of instrument would be able to identify substandard medicines. For that purpose, two case-study were explored. At first, a full ICH Q2 (R1) compliant validation was performed for moderate Raman scatterer active pharmaceutical ingredient (API) in a specific formulation. It was successfully validated in the ±15% relative total error acceptance limits, with a RMSEP of 0.85% (w/w). Subsequently, it was interesting to evaluate the influence of excipients when the API is a high Raman scatterer. For that purpose, a multi-formulation model was developed and successfully validated with a RMSEP of 2.98% (w/w) in the best case. These two studies showed that thanks to the optimization of acquisition parameters, Raman handheld spectrophotometers methods were validated for two different case-study and could be applied to identify substandard medicines. [less ▲]

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See detailHeparin-Coated Liposomes Improve Antiplasmodial Activity and Reduce the Toxicity of Poupartone B
Ledoux, Allison ULiege; Mamede, Lucia; Palazzo, Claudio et al

in Planta Medica International Open (2020), 7(e), 7380

Poupartone B is an alkyl cyclohexenone derivative isolated from Poupartia borbonica. This compound demonstrated promising antimalarial activity (IC50 < 1 μg/mL), however, it was not de- void of toxicity ... [more ▼]

Poupartone B is an alkyl cyclohexenone derivative isolated from Poupartia borbonica. This compound demonstrated promising antimalarial activity (IC50 < 1 μg/mL), however, it was not de- void of toxicity. Thus, to reduce the adverse side effects of this natural bioactive molecule, a delivery strategy involving a na- nostructure was formulated. Additionally, poupartone B-load- ed liposomes were coated with heparin, a glycosaminoglycan that is known to target proteins on the surface of Plasmodium falciparum-infected red blood cells. The quantification of the compound in the formulation was performed by HPLC-DAD, while heparin was quantitated by 1H NMR spectroscopy. The liposomes’ antiplasmodial activity was tested on artemisinin- resistant P. falciparum isolate, and toxicity was evaluated on human HeLa cells and zebrafish embryos. Throughout this re- search, the formulation demonstrated higher antiplasmodial activities against both P. falciparum strains and a significant decrease of in vitro toxicity. The formulation improved the se- lectivity index 2 times in vitro and proved to be 3 times less toxic than the compound alone in the zebrafish embryo acute toxicity test. Hence, the use of this strategy to deliver natural products in Plasmodium-infected cells, particularly those with a narrow therapeutic margin, is proposed. [less ▲]

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See detailHow to analyse big data in hyperspectral imaging? Application for the elucidation of medicine composition
Coic, Laureen ULiege; Sacre, Pierre-Yves ULiege; Dispas, Amandine ULiege et al

Poster (2020, January)

Nowadays, there is an increase of interest regarding hyperspectral imaging in the pharmaceutical field because of the highly valuable information provided. It is a powerful tool to get the composition of ... [more ▼]

Nowadays, there is an increase of interest regarding hyperspectral imaging in the pharmaceutical field because of the highly valuable information provided. It is a powerful tool to get the composition of samples and to give the spatial distribution and/or homogeneity of each compound without destructing the sample. However, in the case of tablets, the surface to analyse can be wide (> 1 cm²) which provides huge file size (approximately 50 GB). The analysis of such kind of data is a challenge either on a common computer or on an affordable workstation because of “out of memory” issues. Those can sometimes be overcome by parallelizing functions but some interesting algorithms are not parallelizable. Moreover, central processing unit (CPU) computing is quickly limited. For example, spectral unmixing algorithms are rarely applicable on such amount of data without reducing it and, when possible, results are very long to obtain. Some strategies have to be developed to be able to analyse such data with a usual software, such as MATLAB®, on an affordable workstation. The objective of the study was to evaluate several spectral unmixing algorithms in combination with data reduction strategies to allow the elucidation of tablet composition. Two tablets of artemether/lumefantrine formulation were used for this study. One was a genuine Combiart® (manufacturer: Strides Arco Labs, batch 7227669, expiry date: June 2018) further called G1. The other one was a suspected falsified medicine Combiart® (manufacturer: Strides Arco Labs, batch 7225500, expiry date: August 2019) further called S1. The samples were gathered from a previous study [1]. The pharmaceutical tablets were analyzed with a FT-IR Cary 670/620 Agilent series microscope (Agilent Technologies) equipped with a 15x infrared objective, with a numerical aperture (NA) of 0.62 and a FPA (64x64) detector. The chemometric tools used for the study and that will be compared for this application are MCR-ALS, Vertex Component Analysis (VCA) and Pixel Purity Index (PPI). Moreover, two data reduction approaches will be evaluated: Kennard-Stone sub-sampling together with cube splitting and cube splitting only. Each pure spectrum was then matched with a homemade database using the hit quality index (HQI). The proposed approaches allowed analyzing data without “out of memory” issue and without losing any relevant information. Indeed, a preliminary test on the G1 sample with the different algorithms shown that the VCA algorithm elucidated the composition with an analysis time much smaller compared to other algorithms [1]. Moreover, the Kennard-Stone subsampling provided interesting results, probably because the information analyzed was much targeted and the spectral redundancy reduced. The workflow was then applied on the S1 sample and showed that the results were also improved compared to conventional protocols. Thanks to the development of the proposed workflow, it was possible to analyze big data to elucidate the composition of pharmaceutical tablets. It showed that easy-to-develop functions and cube splitting significantly decreased the memory consumption and analysis time (from 2 days to 3 hours) to obtain the same result for G1. In addition, it can be envisaged to use MCR-ALS algorithm in a second step after removing the pure spectra from VCA or PPI, to unmix the pixel information and thus, obtain the complete medicine composition elucidation. [less ▲]

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See detailUsing fragment-based lead discovery to generate new scaffolds for the development of FXIIa inhibitors
Davoine, Clara ULiege; Simon, François; Bouckaert, Charlotte et al

Poster (2020, January)

Tackling thrombotic disorders without affecting the hemostatic capacity remains a challenge in medicine. Up to now, the direct oral anticoagulants (DOACs) on the market induce severe bleeding side effects ... [more ▼]

Tackling thrombotic disorders without affecting the hemostatic capacity remains a challenge in medicine. Up to now, the direct oral anticoagulants (DOACs) on the market induce severe bleeding side effects. One of the strategies in the search for safer antithrombotic therapies is to target coagulation factor XIIa (FXIIa). Studies with different animal models suggest that the inhibition of FXII or FXIIa is an opportunity to develop anticoagulants devoid of a bleeding risk associated with anti-inflammatory properties. In addition, anti-FXII directed therapies could answer unmet medical needs such as the safe prevention of thrombosis in patients exposed to blood-contacting medical devices [1]. Besides this advantage in the field of thrombosis, the FXIIa inhibition also rises as a therapeutic strategy to interfere with excessive vascular leakage in patients suffering from hereditary angioedema [2] and as an emerging research field in neuro-inflammatory and neurodegenerative disorders [3]. 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 [4]. Encouraging results demonstrate that the compounds are anticoagulants and are quite selective for the contact phase pathway [4c]. Importantly, this study showed that aromatic guanidine is an attractive starting point in the design of FXIIa inhibitors. Besides the modulations of the 3-carboxamide coumarins, the search for new chemical scaffolds has been started. To facilitate chemical exploration, we decided to apply a fragment-based lead discovery approach (FBLD). With this aim in view, we set up a high concentration bioassay as primary screening and we elaborate an initial library of fragments bearing an amidine or a guanidine moiety. The library was further enlarged with available in-house compounds and with structures close to potent serine protease inhibitors described in the literature. For the constitution of this library, computational studies were also undertaken. [1] a) A.H. Schmaier, E.X. Stavrou, Res Pract Thromb Haemost. (2019), 1–8.; b) B. Tillman, D. Gailani, Semin Thromb Hemost. (2018), 44(1), 60–9. [2] J. Bjorkqvist, S. de Maat, U. Lewandrowski, A. Di Gennaro, C. Oschatz, K. Schonig, M.M. Nothen, C. Drouet, H. Braley, M.W. Nolte, A. Sickmann, C. Panousis, C. Maas, T. Renne, J Clin Invest, 125 (2015) 3132-3146. [3] S. Lorenzano, M. Inglese, T. Koudriavtseva, Editorial: Role of Coagulation Pathways in Neurological Diseases. Front Neurol (2019), 10, 1–3. [4] 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; c) C. Bouckaert , S. Zhu, J. W. P. Govers-Riemslag, M. Depoorter, S. L. Diamond, L. Pochet, Thromb Res, 157 (2017) 126-133. [less ▲]

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See detailDexfenfluramine and Pergolide Cause Heart Valve Disease via Valve Metabolic Reprogramming and Ongoing Matrix Remodeling.
Oury, Cécile ULiege; MARECHAL, Patrick ULiege; Donis, Nathalie ULiege et al

in International journal of molecular sciences (2020), 21(11),

Several clinical reports indicate that the use of amphetaminic anorectic drugs or ergot derivatives could cause valvular heart disease (VHD). We sought to investigate whether valvular lesions develop in ... [more ▼]

Several clinical reports indicate that the use of amphetaminic anorectic drugs or ergot derivatives could cause valvular heart disease (VHD). We sought to investigate whether valvular lesions develop in response to long-term oral administration of these drugs and to identify drug-targeted biological processes that may lead to VHD. Treatment of New Zealand White rabbits with pergolide, dexfenfluramine, or high-dose serotonin for 16 weeks induced valvular alterations characterized by extracellular matrix remodeling. Transcriptome profiling of tricuspid valves using RNA sequencing revealed distinct patterns of differentially expressed genes (DEGs) that clustered according to the different treatments. Genes that were affected by the three treatments were functionally enriched for reduced cell metabolism processes. The two drugs yielded more changes in gene expression than serotonin and shared most of the DEGs. These DEGs were mostly enriched for decreased biosynthetic processes, increased cell-matrix interaction, and cell response to growth factors, including TGF-β, which was associated with p38 MAPK activation. Treatment with pergolide specifically affected genes involved in homeostasis, which was corroborated by the activation of the master regulator of cell energy homeostasis, AMPK-α, as well as decreased levels of metabolism-related miR-107. Thus, both pergolide and dexfenfluramine may cause VHD through valve metabolic reprogramming and matrix remodeling. [less ▲]

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See detailHyphenation of capillary zone electrophoresis with mass spectrometry for proteomic analysis: Optimization and comparison of two coupling interfaces
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege et al

in Journal of Chromatography A (2020)

Capillary electrophoresis tandem mass spectrometry (CE–MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry ... [more ▼]

Capillary electrophoresis tandem mass spectrometry (CE–MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC–MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE–MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest. © 2020 [less ▲]

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See detailIon mobility high resolution mass spectrometry coupled to HILIC and CZE for the characterization of phosphodiester and phosphorothioate oligonucleotides
Demelenne, Alice ULiege; Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege et al

Conference (2019, December 11)

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically ... [more ▼]

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. With the emergence of oligonucleotides on the market, it is of increasing importance to develop analytical methods to study those modified oligonucleotides and their impurities. Chromatographic and electrophoretic approaches may be used for that purpose. In this work, poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS) were studied. For the chromatographic approach, Hydrophilic Interaction Liquid Chromatography (HILIC) was chosen as it represents good alternative to commonly used ion-pair reversed-phase liquid chromatography for the analysis of polar compounds. Moreover, it avoids the use of ion pairing agents, which makes it more compatible with mass spectrometric detection. For the electrophoretic approaches, a capillary zone electrophoresis (CZE) with a MS-compatible background electrolyte was employed. Both techniques were coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides. [less ▲]

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See detailProteomic analysis using capillary electrophoresis hyphenated with high resolution mass spectrometry: comparison of two coupling interfaces
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege et al

Poster (2019, December 11)

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC ... [more ▼]

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. The use of dynamic pH junction allowed the identification of more peptides and proteins compared to conventional injections. The sheath liquid composition was also optimized in order to enhance signal intensity. A nanoflow sheath liquid interface (EMASS-II) was compared to the traditional coaxial sheath liquid interface (Triple tube) in terms of number of identified peptides and proteins as well as in terms of sensitivity (peak area and peak height). The use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by a factor of 4 and 6-fold, respectively, compared to the Triple tube. [less ▲]

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See detailDevelopment of a sensitive CE-LIF method for the analysis of synthetic cathinones.
Emonts, Paul ULiege; Dispas, Amandine ULiege; Servais, Anne-Catherine ULiege et al

Poster (2019, December 11)

Synthetic cathinones (SCs) are phenylalkylamine compounds related to natural cathinone from Catha Edulis leaves. Given their structural similarities with amphetamines, these compounds are mainly drugs of ... [more ▼]

Synthetic cathinones (SCs) are phenylalkylamine compounds related to natural cathinone from Catha Edulis leaves. Given their structural similarities with amphetamines, these compounds are mainly drugs of abuse. Indeed these substances constitute the second most frequently seized group of new psychoactive substances (NPS) and counted more than 130 compounds in Europe (EMCDDA 2016). In this context, reliable analytical tools are required to track these substances. In this study, our goal was to develop a capillary electrophoresis separation method coupled to laser induced fluorescence detection (CE-LIF) to analyze SCs. CE separation was optimized by means of BGE composition study. Various additives have been investigated to achieve separation of these closely related compounds. Due to their lack of native fluorescence, analytes were derivatized using fluorescein isothiocyanate isomer I (FITC). A protocol adapted to small basic compounds was previously optimized using DoE strategy. The CE-LIF optimized method could be proposed as a generic method for the screening of SCs. [less ▲]

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See detailLC-UV as tool for nanovectorized anticancer peptide quality control: evaluation of encapsulation efficiency
Ilangala Booka, Ange ULiege; COBRAIVILLE, Gaël ULiege; EVRARD, Aude ULiege et al

Poster (2019, December 11)

Many peptides have today a recognized and growing therapeutic interest. However, their administration is not an easy task, since systemically administrated peptides may suffer from several issues such as ... [more ▼]

Many peptides have today a recognized and growing therapeutic interest. However, their administration is not an easy task, since systemically administrated peptides may suffer from several issues such as rapid elimination or unspecific biodistribution. Moreover, many compounds of this emerging class of therapeutics have their targets at the intracellular level, adding another challenge to their therapeutic success. LB19 is a peptidic selective inhibitors of LDHB that catalyzes the conversion of lactate + NAD to pyruvate + NADH + H+, a new therapeutic approach for cancer therapy. The necessity of intravenous administration of LB19 lead to the development of liposomes as drug carriers, combining the protective properties of PEG (stealth liposomes) with the transfection properties of pH-sensitive lipids. This general concept raises the need to develop analytical tools enabling the determination of the encapsulation efficiency of LB19 into these nanocarriers and quantitatively demonstrate their ability to achieve intracellular delivery of their payload. In this project, a LC-UV method was developed to selectively quantify this new anticancer peptide in liposomes. More particularly, we generated stability data of LB19 to support its formulation development. The LC-UV method was employed to study the adsorption of the peptide and demonstrate the impact of the adsorption behaviour on quantitative analysis during the evaluation of the encapsulation efficiency of liposomes. Altogether, this analytical part of our project has the potential to control the quality of the formulated LB19 loaded liposomes, and has also a goal to provide a LC-MS/MS method for the intracellular assay of the peptide on long run. [less ▲]

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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 ▲]

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See detailWhich tools can help to thwart “out of memory” issues during analysis of big data?
Coic, Laureen ULiege; Sacre, Pierre-Yves ULiege; Dispas, Amandine ULiege et al

Poster (2019, December 11)

Nowadays, data management and data analysis are more and more present in the scientist’s life. Indeed, there is an increase of cutting-edge technologies in the analytical field, which provide high quality ... [more ▼]

Nowadays, data management and data analysis are more and more present in the scientist’s life. Indeed, there is an increase of cutting-edge technologies in the analytical field, which provide high quality data but in high amount. In some cases, it is possible increase the data analysis power of a basic computer by parallelizing functions. However, central processing unit (CPU) computing is quickly limited. Another option is to perform analyses with the Graphics Processing Unit (GPU). However, it requires a high knowledge of coding and most usual toolboxes do not support GPU computing. That is why some chemometric strategies have to be developed to be able to analyse such amount of data with an accessible software, such as the MATLAB® environment, on an affordable workstation. In this study, several chemometrics algorithms will be evaluated for the data analysis of an infrared chemical image of a pharmaceutical tablet. The image (3.5 million of spectra of 767 wavenumbers) has been acquired on a FT-IR Cary 670/620 Agilent series microscope (Agilent Technologies). The file size of the raw image data is ~40GB and is impossible to analyse even on a workstation due to “Out of Memory” error. The goal of this work is to show that specific chemometrics strategies coupled to a little bit of coding enables the analysis keeping only the relevant information by reducing the size of the matrix in a smart way. [less ▲]

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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 ▲]

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See detailTargeted proteomics reveals serum amyloid A variants and alarmins S100A8-S100A9 as key plasma biomarkers of rheumatoid arthritis
Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege; Servais, Anne-Catherine ULiege et al

in Talanta (2019)

Serum amyloid A (SAA) and S100 (S100A8, S100A9 and S100A12) proteins were previously identified as biomarkers of interest for rheumatoid arthritis (RA). Among SAA family, two closely related isoforms (SAA ... [more ▼]

Serum amyloid A (SAA) and S100 (S100A8, S100A9 and S100A12) proteins were previously identified as biomarkers of interest for rheumatoid arthritis (RA). Among SAA family, two closely related isoforms (SAA-1 and SAA-2) are linked to the acute-phase of inflammation. They respectively exist under the form of three (α, β, and γ) and two (α and β) allelic variants. We developed a single run quantitative method for these protein variants and investigated their clinical relevance in the context of RA. The method was developed and validated according to regulations before being applied on plasma coming from RA patients (n = 46), other related inflammatory pathologies (n = 116) and controls (n = 62). Depending on the activity score of RA, SAA1 isoforms (mainly of SAA1α and SAA1β subtypes) were found to be differentially present in plasma revealing their dual role during the development of RA. In addition, the weight of SAA1α in the total SAA response varied from 32 to 80% depending on the pathology studied. A negative correlation between SAA1α and SAA1β was also highlighted for RA early-onset (r = −0.41). SAA2 and S100A8/S100A9 proteins were significantly overexpressed compared to control samples regardless of RA stage. The pathophysiological relevance of these quantitative and qualitative characteristics of the SAA response remains unknown. However, the significant negative correlation observed between SAA1α and SAA1β levels in RA early-onset suggests the existence of still unknown regulatory mechanisms in these diseases. [less ▲]

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See detailSeparation of Intact Parathyroid Hormone and Variants Using a Highly Sensitive Sheathless CE-ESI-MS/MS Method
Nyssen, Laurent ULiege; Fillet, Marianne ULiege; CAVALIER, Etienne ULiege et al

Poster (2019, September 26)

INTRODUCTION Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Moreover, immunoassays ... [more ▼]

INTRODUCTION Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Moreover, immunoassays may cross-react with PTH variants such as C-terminal fragments stemming from PTH catabolism. These issues make it difficult to compare results obtained in different laboratories. A reference quantification method is necessary to harmonize PTH assays, both sensitive and selective enough to detect PTH at low concentrations among a variety of closely related compounds. OBJECTIVES In this study, our main goal was to reach a very high sensitivity (pg/mL range) for the analysis of PTH and its variants. Two variants were selected, namely 7-84 PTH as C-terminal fragment and 1-34 PTH as related peptide, but also as potential internal standard for future works. METHODS To achieve our goal, we developed a sheathless CE-ESI-MS method for the separation of 1-34 PTH, 7-84 PTH, and 1-84 PTH. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis (t-ITP), field-amplified sample injection (FASI) and electrokinetic supercharging (EKS). RESULTS The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. 1-84 PTH (full length), 7-84 PTH and 1-34 PTH were separated using an acidic background electrolyte containing acetonitrile to reduce peptide adsorption onto the capillary wall. Ammonium acetate was used as sample medium to improve sensitivity through t-ITP. The method was then transferred to a sheathless CE-ESI-MS instrument. CE-MS on fused silica capillary was limited to µg/mL levels. Indeed, despite the MS detection, only samples containing at least 10 µg/mL of 1-84 PTH, 7-84 PTH, and 1-34 PTH could be analyzed. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH and 1-34 PTH were detected at 100 ng/mL using FASI while 1-84 PTH and 1-34 PTH were detected at 100 pg/mL using EKS. The estimated LODs (S/N = 3) for the EKS method were 25 pg/mL for 1-84 PTH and 10 pg/mL for 1-34 PTH, while there was no signal anymore for 7-84 PTH at these levels. CONCLUSION The developed sheathless CE-ESI-MS method has the potential to reach the low pg/mL range in biological samples after the optimization of the sample preparation method. [less ▲]

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See detailComparison of two capillary electrophoresis-mass spectrometry interfaces for proteomic analysis
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; Demelenne, Alice ULiege et al

Poster (2019, September 17)

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new ... [more ▼]

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new proteic entities of biological interest. As the samples are of high complexity and that some proteins could be present at very low concentrations, efficient and sensitive instruments have to be used in order to maximize peptide identification. Nowadays, capillary electrophoresis tandem mass spectrometry (CE-MS/MS) has gained interest in proteomic analysis as it is considered as complementary to the gold standard method, namely reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS). However, the coupling of CE with MS is not straightforward. Indeed, robust interface is needed in order to conserve the high-resolution in-capillary separation while ensuring a stable spray. For this purpose, optimization of basic parameters such as BGE composition was first carried out using a simple peptide mix. Several parameters were then optimized in order to maximize the sensitivity, such as the composition of the sheath liquid, the interface position and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). Finally, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. In our study, two commercialized interfaces were compared by analysing E. coli proteome digest. The coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) were both coupled with an IMS-qTOF-MS. Eventually, spray stability was found to be the main strength of the triple tube interface, whereas the EMASS-II interface was found to provide higher sensitivity thanks to the reduced flow rate of the sheath liquid. [less ▲]

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See detailCapillary electrophoresis tandem mass spectrometry for proteomic analysis: optimization and comparison of two coupling interfaces
Gou, Marie-Jia ULiege; Nys, Gwenaël ULiege; Demelenne, Alice ULiege et al

Poster (2019, September 01)

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new ... [more ▼]

Untargeted bottom-up proteomic analysis aims to identify the highest number of peptides from complex protein digests. The application of this strategy to real sample might lead to the discovery of new proteic entities. As the samples are of high complexity and that some proteins could be present at very low concentrations, efficient and sensitive instruments have to be used in order to maximize peptide identification. Nowadays, capillary electrophoresis tandem mass spectrometry (CE-MS/MS) has gained interest in proteomic analysis as it is considered as complementary to the gold standard method, namely reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS). However, the coupling of CE with MS is not straightforward. Indeed, robust interface is needed in order to conserve the high-resolution in-capillary separation while ensuring a stable spray. For this purpose, optimization of basic parameters such as BGE composition was first carried out using a simple peptide mix. Several parameters were then optimized in order to maximize the sensitivity, such as the composition of the sheath liquid, the interface position and different pre-concentration approaches (stacking, dynamic pH junction and transient isotachophoresis). Finally, transient isotachophoresis (tITP) was selected among other techniques and allowed the injection of large sample volumes without sacrificing separation efficiency. In our study, two commercialized interfaces were compared by analysing E. coli proteome digest. The coaxial sheath liquid interface (« Triple tube », Agilent Technologies) and the nanoflow sheath liquid interface (« EMASS-II », CMP Scientific) were both coupled with an IMS-qTOF-MS. Eventually, spray stability was found to be the main strength of the triple tube interface, whereas the EMASS-II interface was found to provide higher sensitivity thanks to the reduced flow rate of the sheath liquid. [less ▲]

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See detailDetermination of iohexol by capillary blood microsampling and UHPLC-MS/MS
Ion, Valentin ULiege; Le Goff, Caroline ULiege; Cavalier, Etienne ULiege et al

in Journal of Pharmaceutical Analysis (2019), 9(4), 259-265

One of the most important tools used to evaluate kidney function in the context of chronic kidney disease or other renal function related pathologies is the exploration of glomerular filtration rate (GFR ... [more ▼]

One of the most important tools used to evaluate kidney function in the context of chronic kidney disease or other renal function related pathologies is the exploration of glomerular filtration rate (GFR). Iohexol is up to this moment a good candidate molecule for the GFR assessment since it exhibits minimum protein binding rates and minimum extra-renal clearance, being neither secreted nor reabsorbed at the tubular level. This study proposes and evaluates a new LC-MS/MS method for the iohexol determination from capillary blood, prelevated using volumetric absorbative microsampling (VAMS) systems. As an alternative to VAMS, a brand new HemaPEN® device for micro-prelevation was also tested. A new high throughput sample preparation protocol adapted for iohexol quantification from whole blood VAMS samples was developed. The medium term stability study of iohexol in dried whole blood VAMS samples that was conducted showed a good stability of this molecule for up to 12 days. By collecting only 10 μL of blood, iohexol can be analyzed from dried whole blood VAMS samples for concentration ranges between 1 and 250 μg/mL. Due to the analyte stability in VAMS for up to 12 days, this approach might be successfully applied for GFR assessment for clinical cases allowing minimum invasiveness and even delayed analysis. © 2019 Xi'an Jiaotong University [less ▲]

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