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Multiple sequence alignment of homoserine-acetyltransferases, serine-acetyltransferases and homoserine-succinyltransferases show they all belong to MetX family, having evolved from a common ancestor by conserving the catalytic site and substrate binding residues. The discrimination in the substrate selection arises due to the presence of substrate-specific residues lining the substrate-binding pocket. Mutation of Ala59 and Gly62 to Gly and Pro respectively in homoserine-acetyltransferase from M. tuberculosis resulted in a serine-acetyltransferase like enzyme as it acetylated both l -homoserine and l -serine. Homoserine-acetyltransferase from M. tuberculosis when mutated at positon 322 where Leu was converted to Arg, resulted in succinylation over acetylation of l -homoserine. Our studies establish the importance of the substrate binding residues in determining the type of activity possessed by MetX family, despite all of them having the same catalytic triad Ser-Asp-His. Hence key residues at the substrate binding pocket dictate whether the given enzyme shows predominant transferase or hydrolase activity.

Introduction: Estimation of residual apixaban plasma concentrations may be requested in the management of emergencies. This study aims at assessing the performance of specific anti-Xa assays calibrated with apixaban on real-life samples with low apixaban plasma concentrations (®-Liquid Anti-Xa assay (STA ® LAX) and the low and normal procedures of the Biophen ®Direct Factor Xa Inhibitors (DiXaI) assay was tested on 134 blood samples, collected from patients on apixaban, wherefrom 74 patients received LMWH after apixaban cessation. The results were compared with the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Results: The Biophen ®DiXaI, Biophen ®DiXaI LOW, and STA ®LAX showed very good correlation with LC-MS/MS measurements in patients without LMWH administration (Spearman r.95,.99, and.98, respectively). Their limits of quantitation were defined at 48, 24, and 12 ng/mL, respectively. The Bland-Altman test measured mean bias (SD) at 5.6 (13.1), −2.5 (5.0), and −0.8 (6.1) ng/ml, respectively. The Spearman r of the Biophen ®DiXaI decreased to 0.64 in presence of low apixaban concentrations. The Spearman r of the Biophen ®DiXaI LOW and STA ®LAX decreased to 0.39 and 0.26, respectively, in presence of LMWH. Conclusions: The accuracy of the low methodologies (Biophen ®DiXaI LOW and STA ®LAX) is slightly improved for low apixaban plasma concentrations, compared with the normal procedure of Biophen ®DiXaI. The interference of LMWH on the low methodologies is measurable, however, less important than the previously reported interference of LMWH on rivaroxaban calibrated specific anti-Xa assays.

Fragment-based lead discovery is a usual strategy in drug discovery to identify innovative lead compounds. The success of this approach strongly relies on the capacity to detect weak binders and characterize their binding site. NMR and X-ray crystallography are the conventional technologies used to tackle this challenge. However, their large protein consumption and the cost of equipment reduce their accessibility. Here, an affinity capillary electrophoresis methodology was developed that enables the detection of mM binders, the determination of dissociation constants, and the characterization of the fragment binding site. On the basis of multiple equilibrium theory, dissociation constants in the μM-mM range were determined, and a new methodology is proposed to establish graphically if two fragments bind the same protein pocket. The applicability of this methodology was demonstrated experimentally on coagulation factor XIIa by evaluating pairs of fragments with expected behavior. This study reinforces the significance of using affinity capillary electrophoresis to gather valuable information for medicinal chemistry projects.

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.

The metA (Rv3341) gene from Mycobacterium tuberculosis H37Rv strain encodes a homoserine-acetyltransferase (HAT) enzyme, also called MetA. This enzyme plays a key role in the biosynthetic pathway of methionine and is a potential target for the development of antimicrobial drugs. Purified MetA showed 40 kDa molecular mass on SDS-PAGE. Manual docking was performed with substrates acetyl-CoA, l-homoserine, and p-nitrophenylacetate using crystal structure coordinates of MetA (PDB ID 6PUX) from M. tuberculosis. Multiple sequence alignment indicated that catalytic triad residues Ser157, Asp320, His350 were conserved across species in acetyltransferases, esterases, and hydrolases. As a conserved pentapeptide, GXSMG belongs to α/β hydrolase superfamily and it shares similarity with esterases and hydrolases from different sources. Hydrolase activity of MetA was tested using (PNPA), N-acetylglycine, N-acetylmethionine and Phe-Gly as substrate. LC-MS confirmed that MetA possessed HAT activity, but no homoserine-succinyltransferase (HST) and serine-acetyltransferase (SAT) activities. Replacing acetyl-CoA with PNPA as acetyl group donor showed a drastic reduction in transferase activity, arising due to the interaction of R227 of the enzyme with PNPA. This could prevent the binding of the second substrate in the right orientation and results in the preferential transfer of the acetyl group to water, thus exhibiting hydrolase rather than transferase activity. In this paper, we report that MetA has both transferase and hydrolase activity depending on the correct orientation of the second substrate and the availability of the amino acids involved in enzyme-substrate interaction.

Capillary electrophoresis (CE) instrument was 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.

Introduction: Availability of a rapid and reliable platelet activation assay avoiding limitations of current techniques would be valuable to diagnose heparin-induced thrombocytopenia and platelet secretion disorders. Objectives: The first aim was to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify in a single run TxB2 synthesized and serotonin released from platelets. The second aim was to use our method in association with light transmission aggregometry (LTA) to select good platelet responders for the diagnosis of HIT. Methods: Electrospray ionization and chromatographic separation were optimized for the simultaneous dosage of serotonin and TxB2. The method was validated according to the European Medicines Agency (EMA) guideline for bioanalytical method validation. LTA was performed with monoclonal anti-CD9 (clone ALB6) as platelet activator to select good responders. Results: Detection was performed using a tandem mass spectrometer with alternated positive and negative electrospray ionization. The total run time was 6 minutes. The method was validated for calibration curves, precision, accuracy, lower limit of quantification, carry-over, selectivity, and matrix effect. Platelet response to ALB6 was highly variable among donors. Conclusion: We developed and validated a UHPLC-MS/MS method for the simultaneous quantification of TxB2 and serotonin.

The present study describes the identification of highly potent dimeric 1,2,4-benzothiadiazine 1,1-dioxide (BTD)-type positive allosteric modulators of the AMPA receptors (AMPApams) obtained by linking two monomeric BTD scaffolds through their respective 6-positions. Using previous X-ray data from monomeric BTDs cocrystallized with the GluA2 ligand-binding domain (LBD), a molecular modeling approach was performed to predict the preferred dimeric combinations. Two 6,6-ethylene-linked dimeric BTD compounds (16 and 22) were prepared and evaluated as AMPApams on HEK293 cells expressing GluA2 o(Q) (calcium flux experiment). These compounds were found to be about 10,000 times more potent than their respective monomers, the most active dimeric compound being the bis-4-cyclopropyl-substituted compound 22 [6,6′-(ethane-1,2-diyl)bis(4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC 50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC 50 = 13 nM) was successfully cocrystallized with the GluA2 o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

Over the last decade, the coagulation factor XIIa (FXIIa) has seen renewed interest as a therapeutic target. Indeed, its inhibition could offer a protection against thrombosis without increasing the risk of bleeding. Moreover, it could answer the need for a safe prevention of blood-contacting medical devices-related thrombosis. Among the FXII and FXIIa inhibitors already described in literature, organic small-molecular-weight inhibitors are rather left behind. In this study, we were focused on the discovery and assessment of water soluble small molecules. First, a search within our library of compounds flagged two promising hits. Indeed, enzymes and plasma assays suggested they have a greater activity on the contact factors (FXIa, plasma kallikrein and FXIIa) than on the TF pathway. Then, simple pharmacomodulations were undertaken with the aim to design more selective FXIIa inhibitors. This afforded compounds having different degrees of selectivity. All compounds were finally screened in whole blood using an 8-channel microfluidic model and thromboelastometry measurements. Interestingly, all molecules interfered with the thrombus formation and one of them could be considered as a small organic contact inhibitor. (C) 2017 Elsevier Ltd. All rights reserved.

In this study, a partial-filling affinity capillary electrophoresis (pf-ACE) method was developed for the cross-validation of fragment hits revealed by chromogenic factor XIIa (FXIIa) assay. Chromogenic assay produces false positives, mainly due to spectrophotometric interferences and sample purity issues. pf-ACE was selected as counter-screening technology because of its separative character and the fact that the target does not have to be attached or tagged. The effects of protein plug length, applied voltage and composition of the running buffer were examined and optimized. Detection limit in terms of dissociation constant was estimated at 400 μM. The affinity evaluation was performed close to physiological conditions (pH 7.4, ionic strength 0.13 mol L−1) in a poly (ethylene oxide)-coated capillary of 75 μm internal diameter x 33 cm length with an applied voltage of 3 kV. This method uncovered chromogenic assay's false positives due to zinc contamination. Moreover, pf-ACE supported the evaluation of compounds absorbing at 405 nm.

Direct oral anticoagulants (DOACs) are now widely used in various thromboembolic diseases. Although routine therapeutic drug monitoring is not required, cumulative evidence suggests the usefulness of point measurement in some situations. Mass spectrometry is highly sensitive and specific and is considered the gold standard for the accurate measurement of DOAC plasma concentrations. However, lack of 24/7 availability, labour-intensive sample preparation and slow turn-around time of the analysis reduce its usefulness in emergency situations. Several coagulation or chromogenic assays have been introduced to estimate the plasma concentration of DOACs. In daily practice, these assays are recommended in most cases since they provide sufficient agreement with mass spectrometry over a broad range of concentrations. However, the assays are not perfect, and improvements are required to increase their sensitivity and/or specificity. This article focuses on the pros and the cons of mass spectrometry and specific coagulation/chromogenic assays.

Inhibitors of the coagulation factor XIIa (FXIIa) are attractive to detail the roles of this protease in hemostasis and thrombosis, to suppress artifact due to contact pathway activation in blood coagulation assays, and they are promising as antithrombotic therapy. The 3-carboxamide coumarins have been previously described as small-molecular-weight FXIIa inhibitors. In this study, we report a structure-activity relationship (SAR) study around this scaffold with the aim to discover new selective FXIIa inhibitors with an improved physico-chemical profile. To better understand these SAR, docking experiments were undertaken. For this purpose, we built an original hybrid model of FXIIa. This model has the advantage to gather the best features from the recently published crystal structure of FXIIa in its zymogen form and a more classical homology model. Results with the hybrid model are encouraging as they help understanding the activity and selectivity of our best compounds.

d -Alanyl- d -alanine ligase (Ddl) is a validated and attractive target among the bacterial enzymes involved in peptidoglycan biosynthesis. In the present work, we investigated the pharmacomodulations of the benzoylthiosemicarbazide scaffold to identify new Ddl inhibitors with antibacterial potency. Five novel series of thiosemicarbazide analogues, 1,2,4-thiotriazole-3-thiones, 1,3,4-thiadiazoles, phenylthiosemicarbazones, diacylthiosemicarbazides and thioureas were synthesized via straightforward procedures, then tested against Ddl and on susceptible or resistant bacterial strains. Among these, the thiosemicarbazone and thiotriazole were identified as the most promising scaffolds with Ddl inhibition potency in the micromolar range. Antimicrobial evaluation of salicylaldehyde-4(N)-(3,4-dichlorophenyl) thiosemicarbazone 33, one of the best compounds in our study, revealed interesting antimicrobial activities with values of 3.12–6.25 μM (1.06–2.12 μg/mL) against VRE strains and 12.5–25.0 μM (4.25–8.50 μg/mL) towards MRSA and VRSA strains. A detailed mechanistic study was conducted on the Ddl inhibitors 4-(3,4-dichlorophenyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 20 and compound 33, and revealed a bactericidal effect at 5 × MIC concentration after 7 h and 24 h, respectively, and a bacteriostatic effect at 1 × MIC or 2 × MIC without any sign of bacterial membrane disruption at these lower concentrations. Finally, 20 and 33 were proved to target Ddl in bacterio via intracellular LC-MS dosage of d -Ala, l -Ala and d -Ala- d -Ala. Although, at this stage, our results indicate that other mechanisms might be involved to explain the antimicrobial potency of our compounds, their ability to inhibit the growth of strains resistant to usual antibiotics, as well as strains that express alternative ligases, sets the stage for the development of new antimicrobial agents potentially less sensitive to resistance mechanisms.

Introduction: Chromogenic anti-Xa assays are the most appropriate tests to estimate the amount of betrixaban in plasma but the sensitivity of available tests is limited and improvements are needed to encompass the on-therapy range. Methods: Betrixaban was spiked at concentrations ranging from 0 to 500 ng/mL in plasma from healthy donors. Three commercial tests were used (Biophen®DiXaI®, STA®Liquid Anti-Xa, and HemosIL®Liquid Anti-Xa), and adaptation of their sample dilution scheme was performed. These new methodologies were also tested on plasma spiked with amounts of unfractionated heparin (UFH), low molecular weight heparins (LMWH), or fondaparinux covering the on-therapy ranges to evaluate their sensitivity to indirect factor Xa inhibitors. Results: Results showed concentration-dependent decreases in OD/min inversely proportional to the dilutions. While modifications improve the sensitivity of these tests to betrixaban (eg, ½*OD/min of 502 ng/mL [95% CI: 495-508 ng/mL] for Biophen®DiXaI® [1:50] is reduced to 51 ng/mL [95% CI: 50-52 ng/mL] for improved Biophen®DiXaI® [1:5]), results also showed an increased sensitivity to indirect factor Xa inhibitors, except for Biophen®DiXaI® which remains insensitive to UFH and LMWH. Conclusions: Results showed that the improvement of current chromogenic anti-Xa methodologies enhances the sensitivity of these assays to betrixaban but also to indirect factor Xa inhibitors. This lack of specificity may lead to overestimation of betrixaban concentrations in patients bridged with heparins. To avoid this cross-interference, the use of the Biophen®DiXaI® may be a solution except for fondaparinux which remains active even in the presence of the Biophen®DiXaI®’s specific buffer. For the other chromogenic assays, the conception and validation of specific buffer is required.

Introduction Betrixaban is a novel direct oral factor Xa inhibitor approved by the Food and Drug Administration for prophylaxis of venous thromboembolism in adult patients hospitalized for an acute illness at risk for thromboembolic complications. Assessment of the anti-coagulant effect of betrixaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced. Objective The aim of this study is to determine which coagulation assay(s) should be used to assess the impact of betrixaban on haemostasis and provide laboratory guidance for their interpretation. Materials and Methods Betrixaban was spiked at final concentrations ranging from 0 to 250 ng/mL in platelet-poor plasma. Different reagents from several manufacturers were tested and the impact of betrixaban on pro-thrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russel viper venom time (dRVV-T), chromogenic anti-Xa assays, thrombin generation assay (TGA), and a large panel of haemostasis diagnostic tests has been assessed. Results A concentration-dependent prolongation of aPTT, PT and dRVV-T is observed. The sensitivity mainly depends on the reagent. Chromogenic anti-Xa assays show high sensitivity depending on the reagent and/or the methodology. These assays applicable for other direct factor Xa inhibitors have to be adapted to obtain a relevant range of measurement. TGA may also be attractive to assess the anti-coagulant activity of betrixaban. Conclusion Adapted chromogenic anti-Xa assays are the most appropriate assays to estimate the concentration of betrixaban. Betrixaban significantly affects several haemostasis diagnostic tests and this needs to be taken into consideration when requesting and interpreting such tests.

Direct oral anticoagulants (DOAC) are substrates for the ABCB1 transporter (also called P-glycoprotein), an active efflux pump. ABCB1 polymorphisms have been previously reported to influence the pharmacokinetics of several drugs such as immunosuppressants and tyrosine kinase inhibitors. Recently, in vivo studies have suggested that genetic variants might contribute to the inter-individual variability in DOAC plasma concentrations. Therefore, we evaluated the in vitro effect of the most common coding ABCB1 single nucleotide polymorphisms (SNP), 1236 C > T-2677G > T-3435C > T, and the coding ABCB1 1199 G > A SNP on the transport activity towards rivaroxaban. HEK293 cells were transfected to overexpress the ABCB1 wild-type (1236C-2677G-3435C, 1199 G) or variant proteins (1236C-2677G-3435T, 1236T-2677T-3435T or 1199 A). ABCB1 expression decreased the intracellular accumulation of rivaroxaban, when compared to control cells. This confirms the involvement of ABCB1 in the active transport of rivaroxaban. However, the ABCB1 1236 C > T-2677G > T-3435C > T and 1199 G > A SNPs had no significant influence on the intracellular accumulation of rivaroxaban when compared to the wild-type protein. These results suggest that the ABCB1 coding SNPs investigated in the present study are unlikely to contribute to the inter-individual variability in rivaroxaban plasma concentrations.

In this study, a fully automated incapillary system was developed to monitor the activity of CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an inline bioreaction in the presence of CYP1A1 supersomes and Nicotinamide adenine dinucleotide phosphate reduced as cofactor, 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 offline 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 inline system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors.

Apolar trisubstituted derivatives of harmine show high antiproliferative activity on diverse cancer cell lines. However, these molecules present a poor solubility making these compounds poorly bioavailable. Here, new compounds were synthesized in order to improve solubility while retaining antiproliferative activity. First, polar substituents have shown a higher solubility but a loss of antiproliferative activity. Second, a Comparative Molecular Field Analysis (CoMFA) model was developed, guiding the design and synthesis of eight new compounds. Characterization has underlined the in vitro antiproliferative character of these compounds on five cancerous cell lines, combining with a high solubility at physiological pH, making these molecules druggable. Moreover, targeting glioma treatment, human intestinal absorption and blood brain penetration have been calculated, showing high absorption and penetration properties.

Highly functionalized 5-bromo-2-amino-1,3-thiazoles bearing various substituents could be easily prepared by a rapid and efficient one-pot method, using simple starting materials and mild conditions while avoiding the use of metal catalysts or inconvenient reagents such as elemental halogens. These useful products can serve as starting materials for other reactions or as pharmacologically interesting compounds. In our work we have shown that the resulting 5-bromothiazole compounds could lead to monoacylglycerol lipase (MAGL) inhibition in the μM range.

The bacterial cell wall and the enzymes involved in peptidoglycan synthesis are privileged targets for the development of novel antibacterial agents. In this work, a series of 1-(2-hydroxybenzoyl)-thiosemicarbazides inhibitors of D-Ala-D-Ala ligase (Ddl) were designed and synthesized in order to target resistant strains of bacteria. Among these, the 4-(3,4-dichlorophenyl)-1-(2-hydroxybenzoyl)-3-thiosemicarbazide 29 was identified as a potent Ddl inhibitor with activity in the micromolar range. This compound, possessing strong antimicrobial activity including against multidrug resistant strains, was proven to act through a bactericidal mechanism and demonstrated very low cytotoxicity on THP-1 human monocytic cell line. Inhibition of Ddl activity by 29 was confirmed in bacterio using UPLC-MS/MS by demonstrating an increase in D-Ala intracellular pools accompanied by a commensurate decrease in D-Ala-D-Ala. Further structure-activity relationships (SARs) studies provided evidence that the hydroxyl substituent in the 2-position (R 1) of the benzoylthiosemicarbazide scaffold is essential for the enzymatic inhibition. This work thus highlights the 1-(2-hydroxybenzoyl)-thiosemicarbazide motif as a very promising tool for the development of novel antibacterial compounds acting through an interesting mechanism of action and low cytotoxicity.

The “Journées Franco-Belges de Pharmacochimie” is a recognized two-day annual meeting on Medicinal Chemistry that is renowned for the advanced science presented, conviviality, and outstanding opportunities for senior and young scientists to exchange knowledge. Abstracts of plenary lectures, oral communications, and posters presented during the meeting are collected in this report., SCOPUS: ar.j, info:eu-repo/semantics/published

Background The dilute Russell's viper venom time (dRVVT) has been suggested for the assessment of the intensity of anticoagulation of all direct oral anticoagulants (DOACs). This study aimed to compare the performance of an optimized liquid-stable dRVVT-based DOAC assay (DRVV-DOAC) on clinical samples before and after mixing these with normal pooled plasma (NPP). Methods Forty-one apixaban, 25 dabigatran, 56 rivaroxaban and 49 vitamin K antagonists (VKAs) plasma samples were included for retrospective analysis. Plasma DOAC concentrations were determined by liquid chromatography coupled with tandem mass-spectrometry. INR was determined for all VKA samples. DRVV-DOAC was performed with an original ready-to-use reagent (Haematex Research ™) where plasma samples were tested neat and in a 1:1 mix with NPP. Results Plasma concentrations ranged from 1 to 406 ng/ml for apixaban, 0 to 386 ng/ml for dabigatran and 0 to 719 ng/ml for rivaroxaban. INR ranged from 2.2 to 6.1. DRVV-DOAC correlated well with plasma concentrations (r 2 = 0.70, 0.94, 0.63 (non-mixed procedure) and 0.77, 0.97, 0.86 (mixed procedure) for apixaban, dabigatran and rivaroxaban, respectively). DRVV-DOAC measurements in the normal range ruled out dabigatran and rivaroxaban concentrations above 30 and 50 ng/ml, but performance was lower for apixaban. DRVV-DOAC was sensitive to VKA samples but poorly reflected INR values. When VKA samples were mixed with NPP, DRVV-DOAC measurements decreased to values close to baseline clotting time. Conclusions DRVV-DOAC is a quick method which showed increased sensitivity compared with other phospholipid-rich dRVVT reagents already investigated. Mixing samples with NPP improved the specificity but reduced sensitivity, especially for apixaban.

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 μM 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.

With the aim to explore the interest of the thiosemicarbazide scaffold for the inhibition of the indoleamine 2,3-dioxygenase (IDO), a promising therapeutic target for anticancer immunotherapy, a series of 32 phenylthiosemicarbazide derivatives was prepared and their IDO inhibition evaluated. Our study demonstrated that among these derivatives, compound 14 characterized with a 4-cyanophenyl group on the thiosemicarbazide was the more potent IDO inhibitor in this series being endowed with an IC50 of 1.2 μM. The SAR depicted showed that substitution in the 3- and 4-position relative to the phenylthiosemicarbazide are very promising whereas substitution in the 2-position always leads to less potent or inactive derivatives. In fact the study highlighted a novel interesting scaffold for IDO inhibition for further development.

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.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance. IDO inhibition is thus an active area of research in drug development. Recently, our group has shown that tryptophan 2,3-dioxygenase (TDO), an unrelated hepatic enzyme also catalyzing the first step of tryptophan degradation, is also expressed in many tumors and that this expression prevents tumor rejection by locally depleting tryptophan. Herein, we report a structure-activity study on a series of 3-(2-(pyridyl)ethenyl)indoles. More than 70 novel derivatives were synthesized, and their TDO inhibitory potency was evaluated. The rationalization of the structure-activity relationships (SARs) revealed essential features to attain high TDO inhibition and notably a dense H-bond network mainly involving His(55) and Thr(254) residues. Our study led to the identification of a very promising compound (58) displaying good TDO inhibition (K(i) = 5.5 μM), high selectivity, and good oral bioavailability. Indeed, 58 was chosen for preclinical evaluation.

A magnetic beads based affinity-selection methodology towards the screening of acetylcholine binding protein (AChBP) binders in mixtures and pure compound libraries was developed. The methodology works as follows: after in solution incubation of His-tagged AChBP with potential ligands, and subsequent addition of cobalt (II)-coated paramagnetic beads, the formed bead-AChBP-ligand complexes are fetched out of solution by injection and trapping in LC tubing with an external adjustable magnet. Non binders are then washed to the waste followed by elution of ligands to a SPE cartridge by flushing with denaturing solution. Finally, SPE-LC–MS analysis is performed to identify the ligands. The advantage of the current methodology is the in solution incubation followed by immobilized AChBP ligand trapping and the capability of using the magnetic beads system as mobile/online transportable affinity SPE material. The system was optimized and then successfully demonstrated for the identification of AChBP ligands injected as pure compounds and for the fishing of ligands in mixtures. The results obtained with AChBP as target protein demonstrated reliable discrimination between binders with pKi values ranging from at least 6.26 to 8.46 and non-binders.

In this work, we aimed to build a 3D-model of NIK and to study the binding of pyrazolo[4,3-c]isoquinolines with a view to highlight the structural elements responsible for their inhibitory potency. However, in the course of this work, we unexpectedly found that the pyrazolo[4,3-c]isoquinolines initially reported as NIK inhibitors were neither inhibitors of this enzyme nor of the alternative NF-κB pathway, but were in fact inhibitors of another kinase, the TGF-β activated kinase 1 (TAK1) which is involved in the classical NF-κB pathway. © 2010 Elsevier Inc. All rights reserved.

The ex vivo testing emerges as an essential and critical step for the selection of the most promising prospective anticoagulant agents. The aim of the present study was to validate the thrombin generation assay as an ex vivo pharmacological screening test for measuring the anticoagulant behaviour and potency of molecules. The effects of six thrombin and/or factor Xa (FXa) inhibitors (argatroban, lepirudin, PPACK, enoxaparin, ZK-807834, fondaparinux) were investigated on the time course of thrombin catalytic activity triggered by the tissue factor pathway in platelet-poor plasma (PPP) of male healthy volunteers using the Calibrated Automated Thrombogram((R)) (CAT) method. In the presence of the anticoagulant drugs, the thrombin activity profiles were dose-dependently modified according to their specific enzyme inhibitory activity. ZK-807834 was the most potent drug for reducing the C(max) and the V(max) but also for prolonging the T(max). Lepirudin most efficiently delayed the lag time whereas enoxaparin was the most powerfully drug for diminishing the endogenous thrombin potential (ETP). In conclusion, the thrombin activity profile performed with the CAT method is a very rapid, suitable and reliable pharmacological tool for screening thrombin and/or FXa inhibitors whatever their inhibition mode. It consists of a powerful alternative for the classical PT clotting assay, especially regarding to the time course and the total amount of active thrombin generated. Last but not least, it provides insight into the mechanism of action of the compounds.

Novel 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives were synthesized and their potency in reducing the invasive behaviour of HT 1080 fibrosarcoma cells was evaluated. Structure-activity relationships were deduced from biological results and will be used in further design of new active compounds. In particular, the acetoxymethyl substituent found at the 6-position of previously described active compounds can be replaced by an acetamidomethyl substituent without loss of potency; while the presence of an aryl ester function at the 3-position was preferred to a thioester or an amide function to induce marked biological activity. This work confirms the interest of aryl esters of 6-substituted coumarin-3-carboxylic acids as potential new anti-cancer agents.

Beta-cyclodextrin (beta-CD) was monofunctionalized into its carboxylic derivative and then conjugated to the N-side of oxytocin (OT), a nonapeptide involved in human behavior and myometrium contraction. On isolated rat myometrium, this conjugate (beta-CD-OT) partly preserves the contracting activity of OT (EC(50) = 0.40 microM vs 1.7 nM). Moreover, the contraction induced frequency is also lowered by beta-CD-OT. This novel hydrophilic targeted carrier could form a host-guest complex with prostaglandins and their derivatives used as labor inducers or with anticancer drugs used in cervix and endometrial cancer. This strategy can improve the solubility, the stability, and/or the biological activity of these drugs as well as reducing their side-effects.

Introduction: Estimation of residual rivaroxaban plasma concentrations may be requested before invasive procedures and some patients at high thromboembolic risk will have a bridging therapy with heparins when rivaroxaban is interrupted. Objective: The objective of this study was to assess the performance of the STA-Liquid Anti-Xa assay (STA LAX) and the low and normal procedures of the Biophen Direct Factor Xa Inhibitors (DiXaI) assay, in patients with and without bridging with low-molecular-weight heparins (LMWHs). Materials and Methods: Seventy-nine blood samples were collected from 77 patients on rivaroxaban at CTROUGH or before an invasive procedure. Rivaroxaban plasma concentrations were estimated using Biophen DiXaI, Biophen DiXaI LOW, and STA LAX and compared to liquid chromatography coupled with mass spectrometry (LC-MS/MS) measurements. Stratifications were performed according to heparin bridging. Results: The Biophen DiXaI LOW and STA LAX showed better correlation with LC-MS/MS measurements than Biophen DiXaI in patients not bridged with LMWH (R: 0.97, 0.96, and 0.91, respectively). However, the performance of Biophen DiXaI LOW and STA LAX decreased when residual LMWH activity was present (R: 0.18 and 0.19 respectively) demonstrating that these tests are not specific to rivaroxaban. Conclusion: In patients not bridged with LMWH, we suggest to use the Biophen DiXaI LOW and STA LAX for the estimation of rivaroxaban concentrations

New 2-oxo-2H-1-benzopyran derivatives were prepared to optimize 2a,b, initially developed as mechanism-based α-chymotrypsin (α-CT) inhibitors, into potent and selective thrombin (THR) inhibitors. From this study, 22, characterized by a 2-(N-ethyl-2′-oxoacetamide)-5′- chlorophenyl ester side chain, was shown to be a good THR inhibitor (k i/K I = 3455 M -1·s -1), displaying an excellent selectivity profile against other serine proteases such as factor Xa, trypsin, and α-CT. Docking analysis of this compound into the different protein structures revealed the molecular basis responsible for its potency and selectivity.

The synthesis of novel coumarins bearing on the lateral side chain in the 3-position an amine or a guanidine group is described. In vitro evaluation highlighted 14d which possesses a meta aniline side chain as a very potent THR inhibitor. Surprisingly, the introduction of a guanidine moiety always led to a decrease in THR inhibiting properties. We, thus, used docking experiments to rationalize the SAR in the series. This study showed the crucial role of a conserved water molecule in the specificity pocket of THR during docking simulation in order to explain the inactivity of guanidine derivatives. © 2006 Elsevier Ltd. All rights reserved.

Recent developments in the field of haemostasis and thrombosis highlighted the crucial role of the tissue factor/factor VIIa complex (TF/FVIIa) in the initiation of coagulation processes. Nowadays, anticoagulant therapies involving heparin or coumarin derivatives, thrombin or factor Xa inhibitors are generally associated with side effects such as bleeding and thrombocytopenia. In this context, the inhibition of TF, FVIIa and their complex by efficient antithrombotic drugs represents a new strategy to reduce this bleeding and to prevent thrombosis events. Moreover, TF/FVIIa inhibition is shown to be useful in the treatment of biological processes independent of the clotting cascade such as angiogenesis and cancer. Among the natural and genetically engineered TF/FVIIa inhibitors, injections of the recombinant protein rNAPc2 show clinical improvements, such as reduced bleeding and thromboembolism, over classical drugs used in the therapy of coronary angioplasty and hip or knee replacement surgery. The knowledge of the 3D-structure of TF/FVIIa complex and examination of co-crystal data of some drugs bound to this complex led to the design and synthesis of numerous TF/FVIIa inhibitors. Among them, the p-amidinophenylurea 18 (Ki = 0.027 microM), the pyrimidinones PHA-927 (30, IC50 = 0.016 microM) and PHA-798 (31, IC50 = 0.014 microM) and the pyridinone 37 (IC50 = 0.052 microM) are highly potent inhibitors of the TF/FVIIa complex, deprived of activity towards thrombin (IC50 > 30-100 microM) and factor Xa (IC50 > 10-100 microM), other proteases involved in the coagulation cascade. Both pyrimidinones prevent arterial thrombosis in non-human primate models of thrombosis and represent a safe approach to anti-thrombotic therapy in patients with cardiovascular risk factors.

The Na(+)/H(+) exchanger (NHE) is involved in intracellular pH homeostasis of many mammalian cell types. To date seven NHE isoforms (NHE1-NHE7) have been identified. NHE1 is the most predominant isoform expressed in heart where it contributes to cardiomyocyte pH homeostasis. Although the NHE activation is essential for the restoration of physiological pH, hyperactivation of NHE1 during ischemia-reperfusion episodes disrupts the intracellular ion balance, leading to cardiac dysfunction and damage. Beside its ability to inhibit a conductive Na(+) channel and the Na(+)/Ca(++) exchanger, amiloride was the first drug described as NHE inhibitor. Double substitution of the nitrogen of the 5-amino group of amiloride gave DMA, EIPA, MIBA and HMA. Later, several acylguanidines were prepared to selectively inhibit NHE1. The replacement of the pyrazine ring of amiloride by a pyridine ring or by a phenyl increased the potency and the NHE selectivity. The simultaneous replacement of the pyrazine ring by a phenyl, of the 6-chloro by a sulfomethyl led to drugs such as HOE-694, cariporide, eniporide and BIIB-513 which also selectively inhibited NHE1. In the last decade several bicyclic guanidines were prepared: zoniporide, MS-31038, SM-20220, SM-20550, SMP-300, KB-R9032, BMS-284640, T-162559, TY-12533, S-3226 or SL-591227. Extensive pre-clinical studies indicated that NHE inhibitors afford substantial protection in different animal models of myocardial ischemia (MI) and reperfusion, but the results of clinical trials involving eniporide and cariporide were mixed.

In search for new anticancer agents, we have evaluated the antiinvasive and antimigrative properties of recently developed synthetic coumarin derivatives among which two compounds revealed important activity: 3-chlorophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate and 3-bromophenyl 6-acetoxymethyl-2-oxo-2H-1-benzopyran-3-carboxylate. Both drugs were able to inhibit cell invasion markedly in a Boyden chamber assay, the bromo derivative being more potent than the reference matrix metalloprotease (MMP) inhibitor GI 129471. In vivo, tumour growth was reduced when nude mice grafted with HT1080 or MDA-MB231 cells were treated i.p. 3 days week(-1) with the bromo coumarin derivative. These effects were not associated with the inhibition of urokinase, plasmin, MMP-2 or MMP-9. The mechanism of action of the drugs remains to be elucidated. However, these two coumarin derivatives may serve as new lead compounds of an original class of antitumour agents.

Novel coumarinic derivatives were synthesized and tested for their inhibitory potency toward α-CT and HLE. Cycloalkyl esters and amides were found to be essentially inactive on both enzymes. On the opposite, aromatic esters strongly inactivated α-CT whereas HLE was less efficiently inhibited with dichlorophenyl ester derivatives (k(inact)/K(I)=4000M-1 s-1 for 36). Representative examples of amide, ester, thioester and ketone derivatives were prepared in order to evaluate the influence of the link between the coumarinic ring and the phenyl side chain. The irreversible inactivation of α-CT by 6-chloromethyl derivatives should be due to alkylation of a histidine residue as suggested by the amino acid analysis of the modified chymotrypsin. Conversely the inhibition of HLE was transient. Intrinsic reactivity of coumarins has been calculated using a model of a nucleophilic reaction between the ligand and the couple methanol-water. From this calculation, it appears that differences in the inhibitory potency expressed by these molecules cannot only be explained by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack. Copyright (C) 2000 Elsevier Science Ltd.

Torasemide, a loop diuretic, has been reported to relax dog coronary artery precontracted by thromboxane A2 (TXA2), an endogenous prostanoid involved in cardiovascular and pulmonary diseases. N-cyano-N′-{[4-(3′-methylphenylamino)pyrid-3-yl]sulphonyl}homopiperidinoamidine (BM-144) and N-isopropyl-N′-[5-nitro-2-(3′-methylphenylamino)-benzenesulphonyl]urea (BM-500), chemically related to torasemide, have been examined for their TXA2 antagonism. The affinity (IC50, the concentration resulting in 50% inhibition) of BM-144 and BM-500 for the TXA2 receptor of washed platelets from man was 0.28 and 0.079 μM, respectively. This is better than for sulotroban (IC50 = 0.93 μM) but less than for SQ-29548 (IC50 = 0.021 μM), two TXA2 antagonists used as reference. The aggregation of platelets from man induced by arachidonic acid was prevented by BM-144 (IC50 = 9.0 μM) and by BM-500 (IC50 = 14.2 μM). Similar results were obtained by use of U-46619, a TXA agonist, as aggregating agent (BM-144, IC50 = 12.9 and BM-500, IC50 = 9.9 μM). The contracting effect of U-46619 on rat stomach strip was abolished by BM-144 (IC50 = 1.01 μM) and BM-500 (IC50 = 2–54 μM). Both drugs (BM-144: IC50 = 0.12 and BM-500: IC50 = 0.19 μM) also relaxed rat aorta precontracted by U-46619; both were more potent than sulotroban (IC50 = 1.62 μM). The two torasemide derivatives (100 μM) did not significantly affect the myo-stimulating effect of some prostaglandins (PGE2, PGI2, PGF2α) or aorta contraction elicited by KCl (30 mM). They did not modify rat diuresis after administration of a 30-mg kg−1 dose. In conclusion, BM-144 and BM-500 can be regarded as novel non-carboxylic TXA2 receptor antagonists and offer a novel template for the design of more potent molecules.

The lipophilic 1-cycloalkylamino-1-(pyrid-3-yl-sulfonamido)-2- nitroethylenes were synthesized as bioisosteres of BM-34, an anticonvulsant sulfonylthiourea. Compound 17 (ip) emerged from the maximal electroshock seizure (MES) test with a 50% effective dose (ED50) of 8.25 mg/kg. Its anticonvulsant profile was similar to that of phenytoin (ED50 = 9.51 mg/kg) and of BM-34 (ED50 = 1.19 mg/kg): active in theMES test and inactive in seizures induced by subcutaneous injection of pentetrazole, strychnine, bicuculline, picrotoxin, or N-methyl-D,L-aspartate. The neurotoxicity of 17 (TD50 = 113.8 mg/kg) was lower than that of phenytoin (TD50 = 65.5 mg/kg) but higher than that of BM-34 (TD50 = 147.2 mg/kg). Crystallographic study revealed that BM-401 (17) was a zwitterionic structure. Its sulfonamido nitroethylene side chain adopted a conformation which placed the two cycloalkyl rings face to face to form a single hydrophobic area.

Summary Sulfonylcyanoguanidines and sulfonamidonitroethylenes are bioisosteres of hypoglycemic sulfonylureas and were prepared and evaluated for their insulin release potency from rat pancreatic islets. At 25 μM, both bioisosteres of glibenclamide, sulfonylcyanoguanidine 22 and sulfonamidonitroethylene 23 , were as potent as their parent on insulin secretion.

Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), a hepatic enzyme catalyzing the first step of tryptophan degradation, is expressed in many tumors, thereby contributing to tumoral immune resistance.1 The complementary role of tryptophan catabolites has been demonstrated by others.2We set out to develop new, improved TDO inhibitors using as the starting point the only (unoptimized) series previously known in the literature.3 Herein, we describe the syntheses and structure-activity studies around a series of 3-alkenyl indoles and their derivatives.4 The TDO inhibitory potency was evaluated and rationalized by molecular modeling studies, while solubility, stability and oral bioavailability were determined for selected compounds. The most promising candidate was evaluated in a preclinical model in mice where, upon systemic treatment, it indeed reversed TDO-mediated tumoral immune resistance.5

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value.

The “Journées Franco-Belges de Pharmacochimie” is a recognized annual meeting in organic and medicinal chemistry known for the quality of scientific exchange and conviviality. Young researchers were encouraged to present their work and share ideas with senior scientists. Abstracts of plenary lectures, oral communications, and posters presented during the meeting are collected in this report.

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO) is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance.1 IDO inhibition has been an active area of research in drug development for a number of years.2 Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), an unrelated hepatic enzyme also catalyzing the first step of tryptophan degradation, is as well expressed in many tumors preventing their rejection by locally depleting tryptophan.3 The role of tryptophan catabolites was demonstrated by another group.4 Herein, we report the syntheses and structure-activity studies around a series of 3-(2-(pyridyl)ethenyl)indoles.5 Some 80 novel heterocyclic compounds were synthesized. Their TDO inhibitory potency was evaluated and rationalized by molecular modeling studies. The best candidate in terms of potency, selectivity, solubility and oral bioavailability was evaluated in a preclinical model in mice. Upon systemic treatment, the compound reversed TDO-mediated tumoral immune resistance.6 References This work was supported in part by FNRS-Télévie (7.4.543.07). 1) Uyttenhove, C., et al, Nat. Med. 2003, 9, 1269-1274. 2) Macchiarulo, A., et al, Amino Acids 2009, 37, 219-229; Röhrig, U. F., et al, J. Med. Chem. 2010, 53, 1172-1189. 3) Van den Eynde, B., et al, WO2010008427, 2010. 4) Opitz, C. A., et al, Nature 2011, 478, 197-203. 5) Dolusic, E., et al, J. Med. Chem. 2011, 54, 5320-5334. 6) Pilotte, L., et al, Proc. Natl. Acad. Sci. USA 2012, 109, 2497 - 2502.

To overcome the intrinsic resistance of cancer cells to apoptotic stimuli, we designed and synthesized approximately 50 novel β-carbolines structurally related to harmine. Harmine is known for its anticancer properties and is a DYRK1A inhibitor. Of the synthesized compounds, the most active in terms of growth inhibition of five cancer cell lines are cytostatic and approximately 100 times more potent than harmine but demonstrated no DYRK1A inhibitory activity. These novel β-carbolines display similar growth inhibitory activity in cancer cells that are sensitive and resistant to apoptotic stimuli. Using ChemGPS-NP, we found that the more active β-carbolines are all more lipophilic and larger than the less active compounds. Lastly, on the basis of the NCI human tumor cell line anticancer drug screen and the NCI COMPARE algorithm, it appears that some of these compounds, including 5a and 5k, seem to act as protein synthesis inhibitors. © 2012 American Chemical Society.