Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA).

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.

The positive allosteric modulator BPAM344 and L-glutamate introduce an active-like structure of the ligand-binding domain of GluK2.

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.

Recovery from desensitization in GluA2 AMPA receptors is affected by a single mutation in the N-terminal domain interface.

AMPA-type ionotropic glutamate receptors (AMPARs) are central to various neurological processes, including memory and learning. They assemble as homo- or heterotetramers of GluA1, GluA2, GluA3, and GluA4 subunits, each consisting of an N-terminal domain (NTD), a ligand-binding domain, a transmembrane domain, and a C-terminal domain. While AMPAR gating is primarily controlled by reconfiguration in the ligand-binding domain layer, our study focuses on the NTDs, which also influence gating, yet the underlying mechanism remains enigmatic.

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 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are 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 to test agonists, antagonists, and positive allosteric modulators of GluK1-3.

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 μM) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344).

Differences between the GluD1 and GluD2 receptors revealed by GluD1 X-ray crystallography, binding studies and molecular dynamics.

Ionotropic glutamate receptors are ligand-gated ion channels essential for fast excitatory neurotransmission in the brain. In contrast to most other members of the iGluR family, the subfamily of delta receptors, GluD1 and GluD2, does not bind glutamate but glycine/D-serine. GluD1 is widely expressed in the brain and the inner ear, where it is required for high-frequency hearing.

Development of Thiochroman Dioxide Analogues of Benzothiadiazine Dioxides as New Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors.

On the basis of the activity of 1,2,4-benzothiadiazine 1,1-dioxides as positive allosteric modulators of AMPA receptors, thiochroman 1,1-dioxides were designed applying the isosteric replacement concept. The new compounds expressed strong modulatory activity on AMPA receptors , although lower than their corresponding benzothiadiazine analogues. The pharmacokinetic profile of three thiochroman 1,1-dioxides (, , ) was examined after oral administration, showing that these compounds freely cross the blood-brain barrier.

Structural basis for positive allosteric modulation of AMPA and kainate receptors.

This paper summarizes the present knowledge on how positive allosteric modulators (PAMs) interact with the ligand-binding domain (LBD) of AMPA and kainate receptors, based on structure determinations. AMPA and kainate receptors belong to the family of ionotropic glutamate receptors that are responsible for mediating the majority of fast excitatory neurotransmission. These receptors have been related to brain disorders, e.

Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2.

Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e.

Ionotropic Glutamate Receptor GluA2 in Complex with Bicyclic Pyrimidinedione-Based Compounds: When Small Compound Modifications Have Distinct Effects on Binding Interactions.

()-2-Amino-3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic acid (AMPA) receptors comprise an important class of ionotropic glutamate receptors activated by glutamate in the central nervous system. These receptors have been shown to be involved in brain diseases, for example, Alzheimer's disease and epilepsy. To understand the functional role of AMPA receptors at the molecular level and their potential as targets for drugs, development of tool compounds is essential.

-(7-(1-Imidazol-1-yl)-2,3-dioxo-6-(trifluoromethyl)-3,4-dihydroquinoxalin-1(2)-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.

Nanoscale Mobility of the Apo State and TARP Stoichiometry Dictate the Gating Behavior of Alternatively Spliced AMPA Receptors.

Neurotransmitter-gated ion channels are allosteric proteins that switch on and off in response to agonist binding. Most studies have focused on the agonist-bound, activated channel while assigning a lesser role to the apo or resting state. Here, we show that nanoscale mobility of resting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPA receptors) predetermines responsiveness to neurotransmitter, allosteric anions and TARP auxiliary subunits.

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.

Crystal Structures of Potent Dimeric Positive Allosteric Modulators at the Ligand-Binding Domain of the GluA2 Receptor.

The ionotropic glutamate receptor GluA2 is considered to be an attractive target for positive allosteric modulation for the development of pharmacological tools or cognitive enhancers. Here, we report a detailed structural characterization of two recently reported dimeric positive allosteric modulators, TDPAM01 and TDPAM02, with nanomolar potency at GluA2. Using X-ray crystallography, TDPAM01 and TDPAM02 were crystallized in the ligand-binding domain of the GluA2 flop isoform as well as in the flip-like mutant N775S and the preformed dimer L504Y-N775S.

N1-Substituted Quinoxaline-2,3-diones as Kainate Receptor Antagonists: X-ray Crystallography, Structure-Affinity Relationships, and in Vitro Pharmacology.

Among the ionotropic glutamate receptors, the physiological role of kainate receptors is less well understood. Although ligands with selectivity toward the kainate receptor subtype GluK1 are available, tool compounds with selectivity at the remaining kainate receptor subtypes are sparse. Here, we have synthesized a series of quinoxaline-2,3-diones with substitutions in the N1-, 6-, and 7-position to investigate the structure-activity relationship (SAR) at GluK1-3 and GluK5.

Small-angle neutron scattering studies on the AMPA receptor GluA2 in the resting, AMPA-bound and GYKI-53655-bound states.

The AMPA receptor GluA2 belongs to the family of ionotropic glutamate receptors, which are responsible for most of the fast excitatory neuronal signalling in the central nervous system. These receptors are important for memory and learning, but have also been associated with brain diseases such as Alzheimer's disease and epilepsy. Today, one drug is on the market for the treatment of epilepsy targeting AMPA receptors, a negative allosteric modulator of these receptors.

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 GluA2( Q) (calcium flux experiment).

( S)-2-Amino-3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic Acid (AMPA) and Kainate Receptor Ligands: Further Exploration of Bioisosteric Replacements and Structural and Biological Investigation.

Starting from 1-4 and 7 structural templates, analogues based on bioisosteric replacements (5a-c vs 1, 2 and 6 vs 7) were synthesized for completing the SAR analysis. Interesting binding properties at GluA2, GluK1, and GluK3 receptors were discovered. The requirements for GluK3 interaction were elucidated by determining the X-ray structures of the GluK3-LBD with 2 and 5c and by computational studies.

7-Phenoxy-Substituted 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Nanomolar Potency.

We report here the synthesis of 7-phenoxy-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides and their evaluation as AMPA receptor positive allosteric modulators (AMPApams). The impact of substitution on the phenoxy ring and on the nitrogen atom at the 4-position was examined. At GluA2(Q) expressed in HEK293 cells (calcium flux experiment), the most potent compound was 11m (4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, EC = 2.

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D O) when investigated by neutrons.

Structure and Affinity of Two Bicyclic Glutamate Analogues at AMPA and Kainate Receptors.

Ionotropic glutamate receptors (iGluRs) are involved in most of the fast excitatory synaptic transmission in the central nervous system. These receptors are important for learning and memory formation, but are also involved in the development of diseases such as Alzheimer's disease, epilepsy and depression. To understand the function of different types of iGluRs, selective agonists are invaluable as pharmacological tool compounds.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region.

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g.

Identification and Structure-Function Study of Positive Allosteric Modulators of Kainate Receptors.

Kainate receptors (KARs) consist of a class of ionotropic glutamate receptors, which exert diverse pre- and postsynaptic functions through complex signaling regulating the activity of neural circuits. Whereas numerous small-molecule positive allosteric modulators of the ligand-binding domain of ()-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid (AMPA) receptors have been reported, no such ligands are available for KARs. In this study, we investigated the ability of three benzothiadiazine-based modulators to potentiate glutamate-evoked currents at recombinantly expressed KARs.