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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.g. Alzheimer's disease and attention deficit hyperactivity disorder. PAMs are small molecules that potentiate AMPA and kainate receptor currents by interfering with receptor desensitization. Therefore, PAMs are considered to be of interest for the development of pharmacological tools. Whereas PAMs for AMPA receptors have been known for several years, only recently have PAMs for kainate receptors been reported. Today, >80 structures are available for AMPA receptors with PAMs. These PAMs bind at the interface between two LBD subunits in the vicinity of residue 775, which is important for functional differences between flip and flop isoforms of AMPA receptors. PAMs can be divided into five classes based on their binding mode. The most potent PAM reported to date belongs to class 3, which comprises dimerized PAMs. Three structures of the kainate receptor GluK1 were determined with PAMs belonging to class 2. One PAM enhances kainate receptor currents 5- to 59-fold but shows 100-fold lower potency compared to AMPA receptors. Selective PAMs for kainate receptors will be of great use as pharmacological tools for functional investigations in vivo and might potentially prove useful as drugs in controlling the activity of neuronal networks.

(S)-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. Th...

Development of pharmacological tools for the ionotropic glutamate receptors (iGluRs) is imperative for the study and understanding of the role and function of these receptors in the central nervous system. We report the synthesis of 18 analogues of (2S,3R)-2-carboxy-3-pyrrolidine acetic acid (3a), which explores the effect of introducing a substituent on the ε-carbon (3c−q). A new synthetic method was developed for the efficient synthesis of racemic 3a and applied to give expedited access to 13 racemic analogues of 3a. Pharmacological characterization was carried out at native iGluRs, cloned homomeric kainate receptors (GluK1−3), NMDA receptors (GluN1/GluN2A-D), and excitatory amino acid transporters (EAAT1−3). From the structure−activity relationship studies, several new ligands emerged, exemplified by triazole 3p-d1, GluK3-preferring (GluK1/GluK3 K(i) ratio of 15), and the structurally closely related tetrazole 3q-s3−4 that displayed 4.4−100-fold preference as an antagonist for the GluN1/GluN2A receptor (K(i) = 0.61 μM) over GluN1/GluN2B-D (K(i) = 2.7−62 μM).

We report the synthesis of a series of bis-functionalized b-peptoid oligomers of the hexamer length. This was achieved by synthesizing and incorporating protected amino- or azido-functionalized chiral building blocks into precursor oligomers by a trimer segment coupling strategy. The resulting hexamers were readily elaborated to provide target compounds displaying amino groups, carboxy groups, hydroxy groups, or triazolo-pyridines, which should enable metal ion binding. Analysis of the novel hexamers by CD spectroscopy and HSQC NMR spectroscopy revealed robust helical folding propensity in acetonitrile. CD analysis showed solvent-dependent degree of helical content in the structural ensembles when adding different ratios of protic solvents including aqueous buffer. These studies were enabled by the substantial increase in solubility compared to previously analyzed b-peptoid oligomers. This also allowed for investigation of the effect of pH on folding propensity of the amino- and carboxy-functionalized oligomers, respectively. Interestingly, we could demonstrate a reversible effect of sequentially adding acid and base, resulting in a switching between compositions of folded ensembles with varying helical content. We envision that the present discoveries can form the basis for the development of functional peptidomimetic materials responsive to external stimuli.

[Image: see text] 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. In all structures, one modulator molecule binds at the dimer interface with two characteristic hydrogen bonds being formed from the modulator to Pro515. Whereas the GluA2 dimers and modulator binding mode are similar when crystallized in the presence of l-glutamate, the shape of the binding site differs when no l-glutamate is present. TDPAM02 has no effect on domain closure in both apo and l-glutamate bound GluA2 dimers compared to structures without modulator.

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. Here, we report binding affinities of two bicyclic, conformationally restricted analogues of glutamate (CIP-AS and LM-12b) at AMPA (GluA2 and GluA3) and kainate receptor subunits (GluK1–3 and GluK5). Both CIP-AS and LM-12b were found to be GluK3-preferring agonists, with Ki of 6 and 22 nM, respectively, at recombinant GluK3 receptors. The detailed binding mode of CIP-AS and LM-12b in the ligand-binding domains of the AMPA receptor subunit GluA2 (GluA2-LBD) and the kainate receptor subunits GluK1 (GluK1-LBD) and GluK3 (GluK3-LBD) was investigated by X-ray crystallography. CIP-AS ...

Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full-length structure has been determined of GluK2 by cryo electron microscopy to 7.6 Å resolution as well as 84 high-resolution crystal structures of N-terminal domains and ligand-binding domains, including agonist and antagonist bound structures, modulatory ions and mutations. However, there are still many unanswered questions and challenges in front of us. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (Ki = 4 μM). In a functional assay, 1b displayed full antagonist activity with IC50 = 6 ± 2 μM. A crystal structure was obtained of 1b when bound in the ligand binding domain of GluK1. A domain opening of 13–14° was seen compared to the structure with glutamate, consistent with 1b being an antagonist. A structure–activity relationship study showed that the chemical nature of the tethering atom (C, O, or S) linking the pyrrolidine ring and the phenyl ring plays a key role in the receptor selectivity profile and that substituents on the phenyl ring are well accommodated by the GluK1 receptor.

Peptidomimetic foldamers adopting well-defined three-dimensional structures while being stable toward proteolysis are of interest in biomedical research, chemical biology, and biomimetic materials science. Despite their backbone flexibility, β-peptoids containing N-( S)-1-(1-naphthyl)ethyl ( Ns1npe) side chains can fold into unique triangular prism-shaped helices. We report herein the successful introduction of amino groups onto robustly folded β-peptoid helices by construction and incorporation of novel chiral building blocks. This is the first example of an X-ray crystal structure of a linear β-peptoid containing more than one type of side chain. We thus present a unique foldamer design comprising a robustly folded core with functionalized side chains protruding perpendicular to the helical axis to provide a highly predictable display of functional groups. This work paves the way for development of β-peptoid foldamers with a desired function, such as catalytic properties or as scaffolds enabling polyvalent display.

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. Pharmacological characterization at native and recombinant kainate and AMPA receptors revealed that compound 37 had a GluK3-binding affinity ( K

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., This research was generously supported by the Lundbeck Foundation (S.M., D.S.P., K.F., and J.S.K.), GluTarget (J.B., T.N.J.), Danscatt (S.M., K.F., J.S.K.), and European Union Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Grant Agreement No. 642720 (M.D.C).

Given the heterogeneity within the γ-aminobutyric acid (GABA) receptor and transporter families, a detailed insight into the pharmacology is still relatively sparse. To enable studies of the physiological roles governed by specific receptor and transporter subtypes, a series of GABA analogues comprising five-membered nitrogen- and sulfur-containing heterocycles as amine bioisosteres were synthesized and pharmacologically characterized at native and selected recombinant GABAA receptors and GABA transporters. The dihydrothiazole and imidazoline analogues, 5-7, displayed moderate GAT activities and GABAA receptor binding affinities in the mid-nanomolar range ( Ki, 90-450 nM). Moreover, they exhibited full and equipotent agonist activity compared to GABA at GABAA-αβγ receptors but somewhat lower potency as partial agonists at the GABAA-ρ1 receptor. Stereoselectivity was observed for compounds 4 and 7 for the GABAA-αβγ receptors but not the GABAA-ρ1 receptor. This study illustrates how subtle differences in these novel amino GABA bioisosteres result in diverse pharmacological profiles in terms of selectivity and efficacy.

Summary 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. Mobility at rest is regulated by alternative splicing of the flip/flop cassette of the ligand-binding domain, which controls motions in the distant AMPA receptor N-terminal domain (NTD). Flip variants promote moderate NTD movement, which establishes slower channel desensitization and robust regulation by anions and auxiliary subunits. In contrast, greater NTD mobility imparted by the flop cassette acts as a master switch to override allosteric regulation. In AMPA receptor heteromers, TARP stoichiometry further modifies these actions of the flip/flop cassette generating two functionally distinct classes of partially and fully TARPed receptors typical of cerebellar stellate and Purkinje cells.

The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 ( 2 ) and BPAM344 ( 3 ) into a hydroxyl group (BPAM557 ( 4 ) and BPAM521 ( 5 ), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from −4.9 ( 2 ) and −7.5 ( 3 ) to −6.2 ( 4 ) and −14.5 ( 5 ), but also a less favorable binding entropy (−TΔS, kcal/mol) from −2.3 ( 2 ) and −1.3 ( 3 ) to −0.5 ( 4 ) and 4.8 ( 5 ). Thus, the dissociation constants (K d , μ M) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 μ M L-glutamate at homomeric rat GluA2( Q ) i receptors with EC 50 values of 67.3 and 2.45 μ M, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5 . The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5 , whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5 . In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.

Positive allosteric modulators of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)-type ionotropic glutamate receptors are promising compounds for treatment of neurological disorders, for example, Alzheimer's disease. Here, we report synthesis and pharmacological evaluation of a series of mono-, di-, or trialkyl-substituted 7-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides, comprising in total 16 new modulators. The trisubstituted compounds 7b, 7d, and 7e revealed potent activity (EC2× = 2.7-4.3 μM; concentration of compound responsible for a 2-fold increase of the AMPA mediated response) as AMPA receptor potentiators in an in vitro cellular fluorescence assay (FLIPR). The 4-cyclopropyl compound 7f was found to be considerably less potent (EC2× = 60 μM), in contrast to previously described 4-monoalkyl-substituted benzothiadiazine dioxides for which the cyclopropyl group constitutes the best choice of substituent. 7b was subjected to X-ray structural analysis in complex with the GluA2 ligand-binding domain. We propose an explanation of the unexpected structure-activity relationship of this new series of mono-, di-, and trialkyl-substituted 1,2,4-benzothiadiazine 1,1-dioxide compounds. The methyl substituent in the 3-position directs the binding mode of the 1,2,4-benzothiadiazine 1,1-dioxide (BTD) scaffold. When a methyl substituent is present in the 3-position of the BTD, additional methyl substituents in both the 2- and 4-positions increase potency, whereas introduction of a 4-cyclopropyl group does not enhance potency of 2,3,4-alkyl-substituted BTDs. A hydrogen bond donor in the 2-position of the BTD is not necessary for modulator potency.

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. Antinociceptive potential was demonstrated for GluK1 partial agonist 3 and antagonist 7 (2 mg/kg ip).

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, EC50 = 2.0 nM). The Hill coefficient in the screening and the shape of the dimerization curve in small-angle X-ray scattering (SAXS) experiments using isolated GluA2 ligand-binding domain (GluA2-LBD) are consistent with binding of one molecule of 11m per dimer interface, contrary to most benzothiadiazine dioxides developed to date. This observation was confirmed by the X-ray structure of 11m bound to GluA2-LBD and by NMR. This is the first benzothiadiazine dioxide AMPApam to reach the nanomolar range.

Schizines A (1) and B (2), the first naturally occurring iminolactones (3,6-dihydro-2H-1,4-oxazin-2-one derivatives) to be reported, have been isolated from the fruiting bodies of Schizophyllym commune. In principle the 2-oxazinone moiety might have been formed by a reaction between the amino acid phenylalanine or tryptophan and an 2α-hydroxy-1-ketomarasmone. The alkaloids are unusual in that the carboxyl group of the amino acid precursor is preserved during the biosynthesis. The compounds showed some inhibition of the growth of cancer cells.

The physiological function of kainate receptors (GluK1-GluK5) in the central nervous system is not fully understood yet. With the aim of developing potent and selective GluK1 ligands, we have synthesized a series of new thiophene-based GluK1 agonists (6a-c) and antagonists (7a-d). Pharmacological evaluation revealed that they are selective for the GluK1 subunit, with 7b being the most subtype-selective ligand reported to date (GluK1 vs GluK3). The antagonist 7a was cocrystallized with the GluK1 ligand binding domain, and an X-ray crystallographic analysis revealed the largest flexibility in GluK1 ligand binding domain opening upon binding of a ligand seen to date. The results provide new insights into the molecular mechanism of GluK1 receptor ligand binding and pave the way to the development of new tool compounds for studying kainate receptor function.

A phytochemical investigation of Seidlitzia rosmarinus collected along the shoreline of the Gulf of Aqaba in the remote southern desert region of the Sinai peninsula has revealed the presence of the registered drug metformin (4). However, analysis of the C-14 content revealed the drug to be an anthropogenic contaminant. Consequently, natural product researchers should be aware that compounds isolated from plants might originate from environmental contamination rather than biosynthesis. The new natural product N-(4-hydroxyphenylethyl)-alpha-chloroferuloylamide was isolated as a mixture of the E and Z isomers along with a number of other well-established secondary metabolites.

Two 4-ethyl-substituted pyridothiadiazine dioxides belonging to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor positive allosteric modulators were cocrystallized with the GluA2 ligand binding domain in order to decipher the impact of the position of the nitrogen atom on their binding mode at the AMPA receptors. The latter was found to be very similar to that of previously described benzothiadiazine-type AMPA receptor modulators. The affinity of the two compounds for the receptor was determined by isothermal titration calorimetry. Accordingly, the synthesis and biological evaluation of novel 4-cyclopropyl-substituted pyridothiadiazine dioxides was performed and completed with the synthesis of the corresponding chloro-substituted 4-cyclopropyl-3,4-dihydro-2H-benzothiadiazine 1,1-dioxides. The "8-aza" compound 32 was found to be the most potent pyridothiadiazine-type AMPA receptor potentiator in vitro, whereas the 7-chloro-substituted compound 36c emerged as the most promising benzothiadiazine dioxide. Due to proper drug-likeness and low in vivo acute toxicity in mice, 36c was chosen for a more complete preclinical evaluation. The compound was able to easily cross the blood-brain barrier. In an in vivo object recognition test with CD1 mice, oral administration of 36c was found to significantly improve cognition performance at doses as low as 1 mg/kg.

We have efficiently synthesized 36 arylopeptoid dimers with ortho-, meta-, and para-substituted aromatic backbones and tert-butyl or phenyl side chains. The dimers were synthesized by using a “submonomer method” on solid phase, by applying a simplified common set of reaction conditions. X-ray crystallographic analysis of two of these dimers disclosed that the tert-butyl side chain invokes a cis amide conformation with a comparatively more closely packed structure of the surrounding aromatic backbone while the phenyl side chain results in a trans amide conformation with a more open, extended structure of the surrounding aromatic backbone.

Ionotropic glutamate receptor antagonists are valuable tool compounds for studies of neurological pathways in the central nervous system. On the basis of rational ligand design, a new class of selective antagonists, represented by (2S,4R)-4-(2-carboxyphenoxy)pyrrolidine-2-carboxylic acid (1b), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (K

Positive allosteric modulators of ionotropic glutamate receptors are potential compounds for treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind within the dimer interface of the ligand-binding domain (LBD) and stabilize the agonist-bound conformation, thereby slowing receptor desensitization and/or deactivation. Here we describe the synthesis and pharmacological testing at GluA2 of a new generation of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. The most potent modulator 3 in complex with GluA2-LBD-L483Y-N754S was subjected to structural analysis by X-ray crystallography, and the thermodynamics of binding was studied by isothermal titration calorimetry. Compound 3 binds to GluA2-LBD-L483Y-N754S with a Kd of 0.35 μM (ΔH = -7.5 kcal/mol and -TΔS = -1.3 kcal/mol). This is the first time that submicromolar binding affinity has been achieved for this type of positive allosteric modulator. The major structural factor increasing the binding affinity of 3 seems to be interactions between the cyclopropyl group of 3 and the backbone of Phe495 and Met496.

In the mammalian central nervous system, (S)-glutamate (Glu) is released from the presynaptic neuron where it activates a plethora of pre- and postsynaptic Glu receptors. The fast acting ionotropic Glu receptors (iGluRs) are ligand gated ion channels and are believed to be involved in a vast number of neurological functions such as memory and learning, synaptic plasticity, and motor function. The synthesis of 14 enantiopure 2,4-syn-Glu analogues 2b-p is accessed by a short and efficient chemoenzymatic approach starting from readily available cyclohexanone 3. Pharmacological characterization at the iGluRs and EAAT1-3 subtypes revealed analogue 2i as a selective GluK1 ligand with low nanomolar affinity. Two X-ray crystal structures of the key analogue 2i in the ligand-binding domain (LBD) of GluA2 and GluK3 were determined. Partial domain closure was seen in the GluA2-LBD complex with 2i comparable to that induced by kainate. In contrast, full domain closure was observed in the GluK3-LBD complex with 2i, similar to that of GluK3-LBD with glutamate bound.

A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA(A) receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. The unsubstituted 4-AHP analogue (2a) (EC(50) 19 μM, R(max) 69%) was a moderately potent agonist at human α(1)β(2)γ(2) GABA(A) receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α(1)β(2)γ(2) GABA(A) receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α(1)β(2)γ(2) over ρ(1) GABA(A) receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.

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 (S)-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. 4-cyclopropyl-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (BPAM344) potentiated glutamate-evoked currents of GluK2a 21-fold at the highest concentration tested (200 μM), with an EC50 of 79 μM. BPAM344 markedly decreased desensitization kinetics (from 5.5 to 775 ms), whereas it only had a minor effect on deactivation kinetics. 4-cyclopropyl-7-hydroxy-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (BPAM521) potentiated the recorded peak current amplitude of GluK2a 12-fold at a concentration of 300 μM with an EC50 value of 159 μM, whereas no potentiation of the glutamate-evoked response was observed for 7-chloro-4-(2-fluoroethyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (BPAM121) at the highest concentration of modulator tested (300 μM). BPAM344 (100 μM) also potentiated the peak current amplitude of KAR subunits GluK3a (59-fold), GluK2a (15-fold), GluK1b (5-fold), as well as the AMPA receptor subunit GluA1i (5-fold). X-ray structures of the three modulators in the GluK1 ligand-binding domain were determined, locating two modulator-binding sites at the GluK1 dimer interface. In conclusion, this study may enable the design of new positive allosteric modulators selective for KARs, which will be of great interest for further investigation of the function of KARs in vivo and may prove useful for pharmacologically controlling the activity of neuronal networks.

Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 A resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

A series of analogues of the (S)-2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) receptor agonist BnTetAMPA (5b) were synthesized and characterized pharmacologically in radioligand binding assays at native and cloned AMPA receptors and functionally by two-electrode voltage clamp electrophysiology at the four homomeric AMPA receptors expressed in Xenopus laevis oocytes. The analogues 6 and 7 exhibit very different pharmacological profiles with binding affinity preference for the subtypes GluA1 and GluA3, respectively. X-ray crystal structures of three ligands (6, 7, and 8) in complex with the agonist binding domain (ABD) of GluA2 show that they induce full domain closure despite their low agonist efficacies. Trp767 in GluA2 ABD could be an important determinant for partial agonism of this compound series at AMPA receptors, since agonist efficacy also correlated with the location of the Trp767 side chain.

A library of iminolactones was prepared by esterification of several 2-hydroxyketones with a number of N-protected d- and l-α-amino acids. Some of the hydroxyketones were of terpenoid origin while others were obtained via synthesis. After N-deprotection of the intermediate esters, the free amines spontaneously underwent condensation with the ketone to form iminolactones. Esters of (1S,2S,5S)-2-hydroxypinan-3-one with both d- and l-α-amino acids were partially epimerized at the α-carbon atom to give a diastereomeric ester mixture. Only iminolactones of l-amino acids were formed after cyclization of (1S,2S,5S)-2-hydroxypinan-3-one, and correspondingly only d-amino acid iminolactones were formed after reaction with (1R,2R,5R)-2-hydroxypinan-3-one. The protocol thus enables inversion of the absolute configuration of amino acids. Some members of the prepared library of iminolactones displayed significant anti-proliferative effects toward three cancer cell lines (EL4, MCF7, PC3) with insignificant effect on non-malign cell lines (McCoy, MCF10A, NIH3T3). Thus, iminolactones appear to be potential lead structures for preparation of drugs selectively affecting proliferation of malign cell lines.

International audience; Conformationally restricted glutamate analogues have been pharmacologically characterized at AMPA and kainate receptors and the crystal structures have been solved of the ligand (2S,10R,20S)-2-(20-carboxycyclobutyl) glycine (CBG-IV) in complex with the ligand binding domains of the AMPA receptor GluA2 and the kainate receptor GluK3. These structures show that CBG-IV interacts with the binding pocket in the same way as (S)-glutamate. The binding affinities reveal that CBG-IV has high affinity at the AMPA and kainate receptor subtypes. Appreciable binding affinity of CBG-IV was not observed at NMDA receptors, where the introduction of the carbocyclic ring is expected to lead to a steric clash with binding site residues. CBG-IV was demonstrated to be an agonist at both GluA2 and the kainate receptor GluK1. CBG-IV showed high affinity binding to GluK1 compared to GluA2, GluK2 and GluK3, which exhibited lower affinity for CBG-IV. The structure of GluA2 LBD and GluK3 LBD in complex with CBG-IV revealed similar binding site interactions to those of (S)-glutamate. No major conformational rearrangements compared to the (S)-glutamate bound conformation were found in GluK3 in order to accommodate CBG-IV, in contrast with GluA2 where a shift in lobe D2 binding site residues occurs, leading to an increased binding cavity volume compared to the (S)-glutamate bound structure.

Ionotropic glutamate receptors are key players in fast excitatory synaptic transmission within the central nervous system. These receptors have been divided into three subfamilies: the N-methyl-d-aspartic acid (NMDA), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) and kainate receptors. Kainate has previously been crystallized with the ligand binding domain (LBD) of AMPA receptors (GluA2 and GluA4) and kainate receptors (GluK1 and GluK2). Here, we report the structures of the kainate receptor GluK3 LBD in complex with kainate and GluK1 LBD in complex with kainate in the absence of glycerol. Kainate introduces a conformational change in GluK3 LBD comparable to that of GluK2, but different from the conformational changes induced in GluA2 and GluK1. Compared to their domain closures in a glutamate bound state, GluA2 and GluK1 become more open and kainate induces a domain closure of 60% and 62%, respectively, relative to glutamate (100%). In GluK2 and GluK3 with kainate, the domain closure is 88% and 83%, respectively. In previously determined structures of GluK1 LBD in complex with kainate, glycerol is present in the binding site where it bridges interlobe residues and thus, might contribute to the large domain opening. However, the structure of GluK1 LBD with kainate in the absence of glycerol confirms that the observed domain closure is not an artifact of crystallization conditions. Comparison of the LBD structures with glutamate and kainate reveals that contacts are lost upon binding of kainate in the three kainate receptors, which is in contrast to the AMPA receptors where similar contacts are seen. It was revealed by patch clamp electrophysiology studies that kainate is a partial agonist at GluK1 with 36% efficacy compared to glutamate, which is in between the published efficacies of kainate at GluK2 and AMPA receptors. The ranking of efficacies seems to correlate with LBD domain closures.

A series of racemic aryl-substituted phenylalanines was synthesized and evaluated in vitro at recombinant rat GluA1-3, at GluK1-3, and at native AMPA receptors. The individual enantiomers of two target compounds, (RS)-2-amino-3-(3,4-dichloro-5-(5-hydroxypyridin-3-yl)phenyl)propanoic acid 37 and (RS)-2-amino-3-(3'-hydroxybiphenyl-3-yl)propanoic acid 38, were characterized. (S)-37 and (R)-38 were identified as the only biologically active isomers, both being antagonists at GluA2 receptors with Kb of 1.80 and 3.90 μM, respectively. To address this difference in enantiopharmacology, not previously seen for amino acid-based AMPA receptor antagonists, X-ray crystal structures of both eutomers in complex with the GluA2 ligand binding domain were solved. The cocrystal structures of (S)-37 and (R)-38 showed similar interactions of the amino acid parts but unexpected and different orientations and interactions of the biaromatic parts of the ligands inside the binding site, with (R)-38 having a binding mode not previously identified for amino acid-based antagonists.

In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and GluA2(R)(o) receptors with an approximately 4-fold preference for GluA1/2 vs GluA3/4. In TEVC electrophysiological experiments (RS)-3h competitively antagonized GluA2(Q)(i) receptors. The X-ray structure of the active enantiomer (S)-3h in complex with GluA2-S1S2J showed a domain closure around 8°. Even though the nitro and the carboxyethyl groups of (S)-3h were both anchored to Tyr702 through a water H-bond network, these interactions only induced weak subtype selectivity. In spite of the fact that (S)-3h induced a domain closure close to that observed for partial agonists, it did not produce agonist responses at GluA2 receptors under nondesensitizing conditions. 2-Carboxyethylphenylalanine derivatives provide a new synthetic scaffold for the introduction of substituents that could lead to AMPA receptor subtype-selective ligands.

Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in the central nervous system. These receptors play an important role for the development and function of the nervous system, and are essential in learning and memory. However, iGluRs are also implicated in or have causal roles for several brain disorders, e.g. epilepsy, Alzheimer's disease, Parkinson's disease and schizophrenia. Their involvement in neurological diseases has stimulated widespread interest in their structure and function. Since the first publication in 1998 of the structure of a recombinant soluble protein comprising the ligand-binding domain of GluA2 extensive studies have afforded numerous crystal structures of wildtype and mutant proteins including different ligands. The structural information obtained combined with functional data have led to models for receptor activation and desensitization by agonists, inhibition by antagonists and block of desensitization by positive allosteric modulators. Furthermore, the structural and functional studies have formed a powerful platform for the design of new selective compounds.

A novel direct, flexible, and robust approach to the iso-ergoline tetracyclic system in which a five or six-membered ring is established by intramolecular carbene C–H insertion is reported. The protocol involves a two step conversion of an aromatic aldehyde to the corresponding hydrazone and without purification further conversion to the diazo-compound followed by thermal carbene formation and C–H insertion α to a nitrogen.

The excitatory amino acid transporters (EAATs) are expressed throughout the central nervous system, where they are responsible for the reuptake of the excitatory neurotransmitter (S)-glutamate (Glu). (1) Recently, we have reported the discovery of the first subtype selective EAAT1 inhibitor 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101) (1b) and presented an introductory structure-activity relationship (SAR) study. (2) Here, we present a detailed SAR by the design, synthesis, and pharmacological evaluation of analogues 1g-1t. By comparison of potencies of 1b, 1h, and 1i versus 1j, it is evident that potency is largely influenced by the chemical nature of the R(1) substituent. The study also demonstrates that any chemical change of the functional groups or a change to the parental scaffold results in the complete loss of inhibitory activity of the compounds at EAAT1. Finally, a bioavailability study of UCPH-101 determined the half-life to be 30 min in serum (rats) but also that it was not able to penetrate the blood-brain barrier to any significant degree.

Optimization of the anticancer activity for a class of compounds built on a 1,3-dihydroindole-2-one scaffold was performed. In comparison with recently published derivatives of oxyphenisatin the new analogues exhibited an equally potent antiproliferative activity in vitro and improved tolerability and activity in vivo. The best compounds from this series showed low nanomolar antiproliferative activity toward a series of cancer cell lines (compound (S)-38: IC(50) of 0.48 and 2 nM in MCF-7 (breast) and PC3 (prostate), respectively) and potent antitumor effects in well tolerated doses in xenograft models. The racemic compound (RS)-38 showed complete tumor regression at a dose of 20 mg/kg administered iv on days 1 and 7 in a PC3 rat xenograft.

The prevailing structural model for ligand activation of ionotropic glutamate receptors posits that agonist efficacy arises from the stability and magnitude of induced domain closure in the ligand-binding core structure. Here we describe an exception to the correlation between ligand efficacy and domain closure. A weakly efficacious partial agonist of very low potency for homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19), induced a fully closed iGluR5 ligand-binding core. The degree of relative domain closure, approximately 30 degrees , was similar to that we resolved with the structurally related high affinity agonist dysiherbaine and to that of l-glutamate. The pharmacological activity of MSVIII-19 was confirmed in patch clamp recordings from transfected HEK293 cells, where MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at a high concentration (1 mm) of MSVIII-19 (1% of mean glutamate-evoked currents). To determine the efficacy of the ligand quantitatively, we constructed concentration-response relationships for MSVIII-19 following potentiation of steady-state currents with concanavalin A (EC(50) = 3.6 microm) and on the nondesensitizing receptor mutant iGluR5-2b(Y506C/L768C) (EC(50) = 8.1 microm). MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in parallel recordings with glutamate. Molecular dynamics simulations and electrophysiological recordings confirm that the specificity of MSVIII-19 for iGluR5 is partly attributable to interdomain hydrogen bond residues Glu(441) and Ser(721) in the iGluR5-S1S2 structure. The weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine, together with altered stability of the interdomain interaction, may be responsible for the apparent uncoupling of domain closure and channel opening in this kainate receptor subunit.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) class of ionotropic glutamate receptors comprises four different subunits: iGluR1/iGluR2 and iGluR3/iGluR4 forming two subgroups. Three-dimensional structures have been reported only of the ligand-binding core of iGluR2. Here, we present two X-ray structures of a soluble construct of the R/G unedited flip splice variant of the ligand-binding core of iGluR4 (iGluR4i(R)-S1S2) in complex with glutamate or AMPA. Subtle, but important differences are found in the ligand-binding cavity between the two AMPA receptor subgroups at position 724 (Tyr in iGluR1/iGluR2 and Phe in iGluR3/iGluR4), which in iGluR4 may lead to displacement of a water molecule and hence points to the possibility to make subgroup specific ligands.

Replacement of the methyl group of the AMPA receptor agonist 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA) with a benzyl group provided the first AMPA receptor agonist, compound 7, capable of discriminating GluR2-4 from GluR1 by its more than 10-fold preference for the former receptor subtypes. An X-ray crystallographic analysis of this new analogue in complex with the GluR2-S1S2J construct shows that accommodation of the benzyl group creates a previously unobserved pocket in the receptor, which may explain the remarkable pharmacological profile of compound 7.

Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.

Herein we describe the first structure–activity relationship study of the broad-range iGluR antagonist (2S,3R)-3-(3-carboxyphenyl)pyrrolidine-2-carboxylic acid (1) by exploring the pharmacological effect of substituents in the 4, 4′, or 5′ positions and the bioisosteric substitution of the distal carboxylic acid for a phosphonic acid moiety. Of particular interest is a hydroxyl group in the 4′ position 2a which induced a preference in binding affinity for homomeric GluK3 over GluK1 (Ki = 0.87 and 4.8 μM, respectively). Two X-ray structures of ligand binding domains were obtained: 2e in GluA2-LBD and 2f in GluK1-LBD, both at 1.9 A resolution. Compound 2e induces a D1–D2 domain opening in GluA2-LBD of 17.3–18.8° and 2f a domain opening in GluK1-LBD of 17.0–17.5° relative to the structures with glutamate. The pyrrolidine-2-carboxylate moiety of 2e and 2f shows a similar binding mode as kainate. The 3-carboxyphenyl ring of 2e and 2f forms contacts comparable to those of the distal carboxylate in kainate.

Nine new eudesmanolides (1-9), two new guaianolides (12 and 13), and a new germacrane (10), along with a previously reported guaianolide (11), have been isolated from the roots of Thapsia nitida var. meridionalis. Thapsia nitida var. nitida also afforded compound 13 along with a new guaianolide (14). The structure of 13 was confirmed by X-ray crystallographic analysis. Compounds 1, 2, and 11-14 have been tested as potential inhibitors of the sarco- and endoplasmic Ca2+-dependent ATPases (SERCA) pump. None of them showed significant activities.

Overy, D. P., Phipps, R. K., Frydenvang, K., Larsen, T. O. (2006). Epi-Neozaline, a chemotaxonomic marker for Penicillium tulipae. Biochemical Systematics and Ecology, 34, (4), 345-348. Sponsorship: Danish Research Council (grant No. 9901295)