Biology subjects

Pirotte, B.

Publications and source records attributed to Pirotte, B..

4 recordsLinked to original sources

bioRxiv2023

Exploring thienothiadiazine dioxides as isosteric analogues of benzo- and pyridothiadiazine dioxides in the search of new AMPA and kainate receptor positive allosteric modulators

The synthesis and biological evaluation on AMPA and kainate receptors of new examples of 3,4-dihydro-2H-1,2,4-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxides is described. The introduction of a cyclopropyl chain instead of an ethyl chain at the 4-position of the thiadiazine ring was found to dramatically improve the potentiator activity on AMPA receptors, with compound 32 (BPAM395) expressing in vitro activity on AMPARs (EC2x = 0.24 {micro}M) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344). Interestingly, the 4-allyl-substituted thienothiadiazine dioxide 27 (BPAM307) emerged as the most promising compound on kainate receptors being a more effective potentiator than the 4-cyclopropyl-substituted thienothiadiazine dioxide 32 and supporting the view that the 4-allyl substitution of the thiadiazine ring could be more favorable than the 4-cyclopropyl substitution to induce marked activity on kainate receptors versus AMPA receptors. The thieno-analogue 36 (BPAM279) of the clinically tested S18986 (11) was selected for in vivo evaluation in mice as a cognitive enhancer due to a safer profile than 32 after massive per os drug administration. Compound 36 was found to increase the cognition performance in mice at low doses (1 mg/kg) per os suggesting that the compound was well absorbed after oral administration and able to reach the central nervous system. Finally, compound 32 was selected for co-crystallization with the GluA2-LBD (L504Y,N775S) and glutamate to examine the binding mode of thienothiadiazine dioxides within the allosteric binding site of the AMPA receptor. At the allosteric site, this compound established similar interactions as the previously reported BTD-type AMPA receptor modulators. HighlightsO_LIThe study explored AMPA/kainate receptor PAMs belonging to thienothiadiazine dioxides C_LIO_LIThe 4-cyclopropyl-substituted compound 32 was the most potent AMPA receptor modulator C_LIO_LI4-Allyl substitution improved activity and selectivity for kainate receptors C_LIO_LIThe tricyclic compound 36 expressed cognitive improvement in vivo in mice C_LIO_LICompound 32 was co-crystallized with GluA2-LBD to examine receptor binding mode C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/565294v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@fab46corg.highwire.dtl.DTLVardef@15b42dborg.highwire.dtl.DTLVardef@82740eorg.highwire.dtl.DTLVardef@916124_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Crystal structure of the GluK1 ligand-binding domain with kainate and the full-spanning positive allosteric modulator BPAM538

Kainate receptors play an important role in the central nervous system by mediating postsynaptic excitatory neurotransmission and modulating the release of the inhibitory neurotransmitter GABA through a presynaptic mechanism. To date, only three structures of the ligand-binding domain (LBD) of the kainate receptor subunit GluK1 in complex with positive allosteric modulators have been determined by X-ray crystallography, all belonging to class II modulators. Here, we report a high-resolution structure of GluK1-LBD in complex with kainate and BPAM538, which belongs to the full-spanning class III. One BPAM538 molecule binds at the GluK1 dimer interface, thereby occupying two allosteric binding sites simultaneously. BPAM538 stabilizes the active receptor conformation with only minor conformational changes being introduced to the receptor. Using a calcium-sensitive fluorescence-based assay, a 5-fold potentiation of the kainate response (100 M) was observed in the presence of 100 M BPAM538, whereas no potentiation was observed at GluK2. HighlightsO_LI1.9 [A] structure of the kainate receptor GluK1-LBD in complex with kainate and BPAM538 C_LIO_LIThe positive allosteric modulator BPAM538 occupies two binding sites C_LIO_LIThe binding mode is similar to class III modulators described for AMPA receptors C_LIO_LIBPAM538 prefers GluK1 over GluK2 C_LI

neuroscience↗

bioRxiv2023

Active-like structure of the ligand-binding domain of GluK2 with L-glutamate and the positive allosteric modulator BPAM344

Kainate receptors belong to the family of ionotropic glutamate receptors and contribute to the majority of fast excitatory neurotransmission. Consequently, they also play a role in brain diseases. Therefore, understanding how these receptors can be modulated is of importance. Our study provides a dimeric crystal structure of the ligand-binding domain of the kainate receptor GluK2 in complex with L-glutamate and the small molecule positive allosteric modulator, BPAM344, in an active-like conformation. The role of Thr535 and Gln786 for modulation of GluK2 by BPAM344 was investigated using a calcium-sensitive fluorescence-based assay on transiently transfected cells expressing GluK2 and mutants hereof. This study may aid design of tool compounds targeting kainate receptors, elucidating their potential as targets for treatment of brain diseases.

neuroscience↗

bioRxiv2023

Small molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: Development of screening assays and insight into GluK3 structure

The kainate receptors GluK1-3 belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain and associated with neurological and psychiatric diseases. How these receptors can be modulated by small molecule agents is not well-understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays at GluK1-3 for testing agonists, antagonists, and positive allosteric modulators. The EC50 of BPAM344 for potentiating the response of 100 {micro}M kainate was determined to 26.3 {micro}M at GluK1, 75.4 {micro}M at GluK2, and 639 {micro}M at GluK3. In the presence of 150 {micro}M BPAM344, domoate was found to be a potent agonist at GluK1 and GluK2 with EC50 of 0.77 {micro}M and 1.33 {micro}M, respectively. At GluK3, domoate acts as a very weak agonist or antagonist with IC50 of 14.5 {micro}M, in the presence of 500 {micro}M BPAM344 and 100 {micro}M kainate. Using H523A mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, zinc, sodium, and chloride ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in binding of zinc ions. Using electron microscopy, we show that in the presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement. This study may contribute to unravelling the potential of kainate receptors as targets for treatment of brain diseases.

neuroscience↗