Rational Design of Positive Allosteric Modulators Selective for the Kainate Receptors

Kainate receptors (KARs) are important regulators of glutamatergic transmission, but they remain far
less explored than AMPA receptors, largely because selective small-molecule positive allosteric modulators
(PAMs) are still scarce. At the start of this work, BPAM344 was the only validated benzothiadiazine-based
KAR PAM, but it still showed substantial AMPA receptor activity and its structure–activity relationships
(SAR) were poorly understood. This thesis therefore aimed to develop the first coherent SAR for
benzothiadiazine derivatives selective for KARs and to provide new pharmacological tools for future drug
development.
An integrated strategy combining literature analysis, rational design, modular synthesis, and biological
evaluation on recombinant GluA2 and GluK1-3 receptors was used. A first series of fourth-substituted
benzothiadiazines bearing aliphatic or benzylic side chains was synthesized to probe the KAR allosteric site
and shift activity away from AMPA receptors. Fluorescence assays showed that simple aliphatic substituents
were largely inactive, whereas benzylic groups combining a para hydrogen-bond acceptor with a meta-fluoro
substituent could potentiate GluK1-3 while reducing GluA2 activity.
To clarify these SARs, X-ray crystal structures of GluK1-LBD and GluA2o-LBD with representative
ligands were obtained. These structures show that the benzothiadiazine core binds in a conserved
hydrophobic cleft at the LBD dimer interface and that the sulfonamide nitrogen forms a conserved hydrogen
bond. They also reveal a specific interaction between a carboxylic acid substituent and His792 in GluK1,
which is absent in GluA2 and helps explain the observed selectivity.
Although solubility limitations and the qualitative nature of the assay prevented reliable EC50
determination for many compounds, the high-micromolar potentiation observed supports these ligands as
proof-of-concept KAR PAMs. Overall, this work redirects the benzothiadiazine scaffold toward KAR
modulation and establishes structural and medicinal chemistry foundations for the design of more potent and
subtype-selective KAR PAMs.