Jana Sopkova-de Oliveira Santos

Toward safer thrombolytic agents in stroke: molecular requirements for NMDA receptor-mediated neurotoxicity

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-d-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (KD=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.

S100-annexin complexes: some insights from structural studies.

Several annexins have been shown to bind proteins that belong to the S100 calcium‐binding protein family. The two best‐characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N‐terminus and of S100C with annexin I N‐terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42–55), we propose a computer generated model for the entire annexin II/p11 complex.

Room‐Temperature ortho‐Alkoxylation and ‐Halogenation of N‐Tosylbenzamides by Using Palladium(II)‐Catalyzed CH Activation

The N-tosylcarboxamide group can direct the room-temperature palladium-catalyzed C-H alkoxylation and halogenation of substituted arenes in a simple and mild procedure. The room-temperature stoichiometric cyclopalladation of N-tosylbenzamide was first studied, and the ability of the palladacycle to react with oxidants to form C-X and C-O bonds under mild conditions was demonstrated. The reaction conditions were then adapted to promote room-temperature ortho-alkoxylations and ortho-halogenations of N-tosylbenzamides using palladium as catalyst. The scope and limitation of both alkoxylations and halogenations was studied and the subsequent functional transformation of the N-tosylcarboxamide group through nucleophilic additions was evaluated. This methodology offers a simple and mild route to diversely functionalized arenes.

S100 protein-annexin interactions: a model of the (Anx2-p11)(2) heterotetramer complex.

The (Anx2)(2)(p11)(2) heterotetramer has been implicated in endo- and exocytosis in vivo and in liposome aggregation in vitro. Here we report on the modelling of the heterotetramer complex using docking algorithms. Two types of models are generated-heterotetramer and heterooctamer. On the basis of the agreement between the calculated (X-ray) electron density and the observed projected density from cryo-electron micrographs on the one hand, and calculated energy criteria on the other hand, the heterotetramer models are proposed as the most probable, and one of them is selected as the best model. Analysis of this model at an atomic level suggests that the interaction between the Anx2 core and p11 has an electrostatic character, being stabilised primarily through charged residues.

Synthesis of new dipyrrolo- and furopyrrolopyrazinones related to tripentones and their biological evaluation as potential kinases (CDKs1-5, GSK-3) inhibitors.

We herein describe the synthesis of novel dipyrrolo- and furopyrrolopyrazinones related to highly cytotoxic tripentones and to their oximes. The synthetic pathway involved in particular a Curtius rearrangement and a subsequent cyclisation into the title pyrazinones. The biological evaluation towards various cyclin-dependent kinases (CDKs1-5, GSK-3) highlighted a weak inhibitory activity for the oximes whose SAR was studied by a molecular modeling study.

An efficient and straightforward access to sulfur substituted [2.2]paracyclophanes: application to stereoselective sulfenate salt alkylation.

A straightforward and high-yielding access to various [2.2]paracyclophanes possessing a sulfur-based functional group is reported, the key step being a SEAr reaction mediated by a sulfonium salt. The versatility of the methodology was exemplified by an original application in sulfenate salt chemistry, from which a remarkable chirality transfer was observed.

First evidence that oligopyridines, α-helix foldamers, inhibit Mcl-1 and sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies.

Apoptosis control defects such as the deregulation of Bcl-2 family member expression are frequently involved in chemoresistance. In ovarian carcinoma, we previously demonstrated that Bcl-xL and Mcl-1 cooperate to protect cancer cells against apoptosis and their concomitant inhibition leads to massive apoptosis even in the absence of chemotherapy. Whereas Bcl-xL inhibitors are now available, Mcl-1 inhibition, required to sensitize cells to Bcl-xL-targeting strategies, remains problematic. In this context, we designed and synthesized oligopyridines potentially targeting the Mcl-1 hydrophobic pocket, evaluated their capacity to inhibit Mcl-1 in live cells, and implemented a functional screening assay to evaluate their ability to sensitize ovarian carcinoma cells to Bcl-xL-targeting strategies. We established structure-activity relationships and focused our attention on MR29072, named Pyridoclax. Surface plasmon resonance assay demonstrated that pyridoclax directly binds to Mcl-1. Without cytotoxic activity when administered as a single agent, pyridoclax induced apoptosis in combination with Bcl-xL-targeting siRNA or with ABT-737 in ovarian, lung, and mesothelioma cancer cells.

Interpretation of honeybees contact toxicity associated to acetylcholinesterase inhibitors

The widespread use of different pesticides generates adverse effects on non target organisms like honeybees. Organophosphorous and carbamates kill honeybees through the inactivation of acetylcholinesterase (AChE), thereby interfering with nerve signaling and function. For this class of pesticides, it is fundamental to understand the relationship between their structures and the contact toxicity for honeybees. A Quantitative Structure-Activity Relationship (QSAR) study was carried out on 45 derivatives by a genetic algorithm approach starting from more than 2500 descriptors. In parallel, a new 3D model of AChE associated to honeybees was defined. Physicochemical properties of the receptor and docking studies of the derivatives allow understanding the meaningful of three descriptors and the implication of several amino acids in the overall toxicity of the pesticides.

Annexin A5 D226K structure and dynamics: identification of a molecular switch for the large-scale conformational change of domain III.

The domain III of annexin 5 undergoes a Ca2+‐ and a pH‐dependent conformational transition of large amplitude. Modeling of the transition pathway by computer simulations suggested that the interactions between D226 and T229 in the IIID–IIIE loop on the one hand and the H‐bond interactions between W187 and T224 on the other hand, are important in this process [Sopkova et al. (2000) Biochemistry 39, 14065–14074]. In agreement with the modeling, we demonstrate in this work that the D226K mutation behaves as a molecular switch of the pH‐ and Ca2+‐mediated conformational transition. In contrast, the hydrogen bonds between W187 and T224 seem marginal.

Synthesis and biological evaluation of new 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles as PI3Kα inhibitors.

A series of novel 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles was synthesized through a newly developed approach. All these compounds were evaluated against DYRK1A, CDK5 and PI3Kα and showed promising inhibitory activities against PI3Kα with most IC(50) values in the micromolar range. Among them, compound 18 was strongly considered as the most interesting compound with an IC(50) value of 0.091 μM. This series exhibited also significant anti-proliferative effects in various human cancer cell lines including those resulting in activation of the PI3K pathway.