Enhancing Action of Positive Allosteric Modulators through the Design of Dimeric Compounds.

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 GluA2o( 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-2 H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC50 = 13 nM) was successfully cocrystallized with the GluA2o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

Discovery and assessment of water soluble coumarins as inhibitors of the coagulation contact pathway.

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.

Fully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach.

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.