Tritium and deuterium labelling of a kainate receptor antagonist and evaluation as a radioligand.

Kainate receptors play a crucial role in mediating synaptic transmission within the central nervous system. However, the lack of selective pharmacological tool compounds for the GluK3 subunit represents a significant challenge in studying these receptors. Recently presented compound 1 stands out as a potent antagonist of GluK3 receptors, exhibiting nanomolar affinity at GluK3 receptors and strongly inhibiting glutamate-induced currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with Kb values of 65 and 39 nM, respectively. This study presents the synthesis of two potent GluK3-preferring iodine derivatives of compound 1, serving as precursors for radiolabelling. Furthermore, we demonstrate the optimisation of dehalogenation conditions using hydrogen and deuterium, resulting in [2H]-1, and demonstrate the efficient synthesis of the radioligand [3H]-1 with a specific activity of 1.48 TBq/mmol (40.1 Ci/mmol). Radioligand binding studies conducted with [3H]-1 as a radiotracer at GluK1, GluK2, and GluK3 receptors expressed in Sf9 and rat P2 membranes demonstrated its potential applicability for selectively studying native GluK3 receptors in the presence of GluK1 and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-blocking ligands.

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

Stereoselective Synthesis of New (2S,3R)-3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid Analogues Utilizing a C(sp3)-H Activation Strategy and Structure-Activity Relationship Studies at the Ionotropic Glutamate Receptors.

Competitive antagonists for ionotropic glutamate receptors (iGluRs) are highly valuable tool compounds for studying health and disease states in the central nervous system. However, only few subtype selective tool compounds are available and the discovery of antagonists with novel iGluR subtype selectivity profiles remains a profound challenge. In this paper, we report an elaborate structure-activity relationship (SAR) study of the parental scaffold 2,3-trans-3-carboxy-3-phenyl-proline by the synthesis of 40 new analogues. Three synthetic strategies were employed with two new strategies of which one being a highly efficient and fully enantioselective strategy based on C(sp3)-H activation methodology. The SAR study led to the conclusion that selectivity for the NMDA receptors was a general trend when adding substituents in the 5'-position. Selective NMDA receptor antagonists were obtained with high potency (IC50 values as low as 200 nM) and 3-34-fold preference for GluN1/GluN2A over GluN1/GluN2B-D NMDA receptors.

A Diversity Oriented Synthesis Approach to New 2,3-trans-Substituted l-Proline Analogs as Potential Ligands for the Ionotropic Glutamate Receptors.

Discovery of chemical tools for the ionotropic glutamate receptors continues to be a challenging task. Herein we report a diversity-oriented approach to new 2,3-trans-l-proline analogs whereby we study how the spatial orientation of the distal carboxylate group influences the binding affinity and receptor class and subtype selectivity. In total, 10 new analogs were synthesized and 14 stereoisomers characterized in binding assays at native rat ionotropic glutamate receptors, and at cloned human homomeric kainic acid (KA) receptor subtypes GluK1-3. The study identified isoxazole analogs 3d,e, which displayed selectivity in binding at native N-methyl-d-aspartate (NMDA) receptors over native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and KA receptors, in the high nanomolar to low micromolar range. Furthermore, analogs 3i-A/B showed a preference in binding affinity for GluK3 over GluK1,2. Finally, analog 3j displayed high nanomolar affinity for native NMDA receptors as well as for homomeric GluK3 receptors.

N-(7-(1H-Imidazol-1-yl)-2,3-dioxo-6-(trifluoromethyl)-3,4-dihydroquinoxalin-1(2H)-yl)benzamide, a New Kainate Receptor Selective Antagonist and Analgesic: Synthesis, X-ray Crystallography, Structure-Affinity Relationships, and in Vitro and in Vivo Pharmacology.

Selective pharmacological tool compounds are invaluable for understanding the functions of the various ionotropic glutamate receptor subtypes. For the kainate receptors, these compounds are few. Here we have synthesized nine novel quinoxaline-2,3-diones with substitutions in the 7-position to investigate the structure-activity relationship at kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Compound 11 exhibited the highest binding affinity across GluK1-3 while having selectivity toward kainate vs AMPA receptors. Compound 11 potently inhibited glutamate evoked currents at homomeric GluK1 and GluK3 receptors in HEK293 cells with Kb values of 65 and 39 nM, respectively. The binding mode of 11 in the ligand binding domain of GluK1 was investigated by X-ray crystallography, revealing that 11 stabilizes the receptor in an open conformation, consistent with its demonstrated antagonism. Furthermore, 11 was tested for analgesic effects in the mouse tail flick test where it significantly increased tail flick latency at doses where 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]-quinoxaline-7-sulfonamide (NBQX) was ineffective.

Use of the 4-Hydroxytriazole Moiety as a Bioisosteric Tool in the Development of Ionotropic Glutamate Receptor Ligands.

We report a series of glutamate and aspartate analogues designed using the hydroxy-1,2,3-triazole moiety as a bioisostere for the distal carboxylic acid. Compound 6b showed unprecedented selectivity among ( S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtypes, confirmed also by an unusual binding mode observed for the crystal structures in complex with the AMPA receptor GluA2 agonist-binding domain. Here, a methionine (Met729) was highly disordered compared to previous agonist-bound structures. This observation provides a possible explanation for the pharmacological profile. In the structure with 7a, an unusual organization of water molecules around the bioisostere arises compared to previous structures of ligands with other bioisosteres. Aspartate analogue 8 with the hydroxy-1,2,3-triazole moiety directly attached to glycine was unexpectedly able to activate both the glutamate and glycine agonist-binding sites of the N-methyl-d-aspartic acid receptor. These observations demonstrate novel features that arise when employing a hydroxytriazole moiety as a bioisostere for the distal carboxylic acid in glutamate receptor agonists.