β‑Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC₅₀ values ∼1 μM at all three subtypes, this elaborate structure–activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue <b>4y</b> that displayed an IC₅₀ of 0.8 μM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the <i>m</i>-CF₃-phenyl analogue <b>4r</b> was a potent selective EAAT2-inhibitor (IC₅₀ = 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles

β‑Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC₅₀ values ∼1 μM at all three subtypes, this elaborate structure–activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue <b>4y</b> that displayed an IC₅₀ of 0.8 μM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the <i>m</i>-CF₃-phenyl analogue <b>4r</b> was a potent selective EAAT2-inhibitor (IC₅₀ = 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles

β‑Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC₅₀ values ∼1 μM at all three subtypes, this elaborate structure–activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue <b>4y</b> that displayed an IC₅₀ of 0.8 μM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the <i>m</i>-CF₃-phenyl analogue <b>4r</b> was a potent selective EAAT2-inhibitor (IC₅₀ = 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles

Identification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure–Activity Relationship Study

Screening of a small compound library at the three excitatory amino acid transporter subtypes 1–3 (EAAT1–3) resulted in the identification of compound (<i>Z</i>)-4-chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)­methyl)­furan-2-yl)­benzoic acid (<b>1a</b>) that exhibited a distinct preference as an inhibitor at EAAT1 (IC₅₀ 20 μM) compared to EAAT2 and EAAT3 (IC₅₀ > 300 μM). This prompted us to subject <b>1a</b> to an elaborate structure–activity relationship study through the purchase and synthesis and subsequent pharmacological characterization of a total of 36 analogues. Although this effort did not result in analogues with substantially improved inhibitory potencies at EAAT1 compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently available pharmacological tools in the EAAT field but also substantiates the notion that EAAT ligands not derived from α-amino acids hold considerable potential in terms of subtype-selective modulation of the transporters

Binding Mode of an α‑Amino Acid-Linked Quinoxaline-2,3-dione Analogue at Glutamate Receptor Subtype GluK1

Two α-amino acid-functionalized quinoxalines, <b>1a</b> (CNG-10301) and <b>1b</b> (CNG-10300), of a quinoxaline moiety coupled to an amino acid moiety were designed, synthesized, and characterized pharmacologically. While <b>1a</b> displayed low affinity at native AMPA, KA, and NMDA receptors, and at homomeric GluK1,3 receptors, the affinity for GluK2 was in the midmicromolar range (<i>K</i>_i = 136 μM), <b>1b</b> displayed low to midmicromolar range binding affinity at all the iGluRs (<i>K</i>_i = 9–126 μM). In functional experiments (outside-out patches excised from transfected HEK293T cells), 100 μM <b>1a</b> partially blocked GluK1 (33% peak response), while GluK2 was unaffected (96% peak response). Furthermore, <b>1a</b> was shown not to be an agonist at GluK1 and GluK2 at 100 μM. On the other hand, 100 μM <b>1b</b> fully antagonized GluK1 (8% peak response) but only partially blocked GluK2 (33% peak response). An X-ray structure at 2.3 Å resolution of <b>1b</b> in the GluK1-LBD (ligand-binding domain) disclosed an unexpected binding mode compared to the predictions made during the design phase; the quinoxaline moiety remains to act as an amino acid bioisostere, but the amino acid moiety is oriented into a new area within the GluK1 receptor. The structure of the GluK1-LBD with <b>1b</b> showed a large variation in domain openings of the three molecules from 25° to 49°, demonstrating that the GluK1-LBD is capable of undergoing major domain movements

Structure–Activity Relationship Study of Ionotropic Glutamate Receptor Antagonist (2<i>S</i>,3<i>R</i>)‑3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid

Herein we describe the first structure–activity relationship study of the broad-range iGluR antagonist (2<i>S</i>,3<i>R</i>)-3-(3-carboxyphenyl)­pyrrolidine-2-carboxylic acid (<b>1</b>) 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 <b>2a</b> which induced a preference in binding affinity for homomeric GluK3 over GluK1 (<i>K</i>_i = 0.87 and 4.8 μM, respectively). Two X-ray structures of ligand binding domains were obtained: <b>2e</b> in GluA2-LBD and <b>2f</b> in GluK1-LBD, both at 1.9 Å resolution. Compound <b>2e</b> induces a D1–D2 domain opening in GluA2-LBD of 17.3–18.8° and <b>2f</b> a domain opening in GluK1-LBD of 17.0–17.5° relative to the structures with glutamate. The pyrrolidine-2-carboxylate moiety of <b>2e</b> and <b>2f</b> shows a similar binding mode as kainate. The 3-carboxyphenyl ring of <b>2e</b> and <b>2f</b> forms contacts comparable to those of the distal carboxylate in kainate

Correction to Chemoenzymatic Synthesis of New 2,4-<i>syn</i>-Functionalized (<i>S</i>)‑Glutamate Analogues and Structure–Activity Relationship Studies at Ionotropic Glutamate Receptors and Excitatory Amino Acid Transporters

Correction to Chemoenzymatic Synthesis of New 2,4-<i>syn</i>-Functionalized (<i>S</i>)‑Glutamate Analogues and Structure–Activity Relationship Studies at Ionotropic Glutamate Receptors and Excitatory Amino Acid Transporter

Chemoenzymatic Synthesis of New 2,4-<i>syn</i>-Functionalized (<i>S</i>)‑Glutamate Analogues and Structure–Activity Relationship Studies at Ionotropic Glutamate Receptors and Excitatory Amino Acid Transporters

In the mammalian central nervous system, (<i>S</i>)-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-<i>syn</i>-Glu analogues <b>2b</b>–<b>p</b> is accessed by a short and efficient chemoenzymatic approach starting from readily available cyclohexanone <b>3</b>. Pharmacological characterization at the iGluRs and EAAT1–3 subtypes revealed analogue <b>2i</b> as a selective GluK1 ligand with low nanomolar affinity. Two X-ray crystal structures of the key analogue <b>2i</b> in the ligand-binding domain (LBD) of GluA2 and GluK3 were determined. Partial domain closure was seen in the GluA2-LBD complex with <b>2i</b> comparable to that induced by kainate. In contrast, full domain closure was observed in the GluK3-LBD complex with <b>2i</b>, similar to that of GluK3-LBD with glutamate bound

Design and Synthesis of a Series of l-<i>trans</i>-4-Substituted Prolines as Selective Antagonists for the Ionotropic Glutamate Receptors Including Functional and X‑ray Crystallographic Studies of New Subtype Selective Kainic Acid Receptor Subtype 1 (GluK1) Antagonist (2<i>S</i>,4<i>R</i>)‑4-(2-Carboxyphenoxy)pyrrolidine-2-carboxylic Acid

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 (2<i>S</i>,4<i>R</i>)-4-(2-carboxyphenoxy)­pyrrolidine-2-carboxylic acid (<b>1b</b>), for cloned homomeric kainic acid receptors subtype 1 (GluK1) was attained (<i>K</i>_i = 4 μM). In a functional assay, <b>1b</b> displayed full antagonist activity with IC₅₀ = 6 ± 2 μM. A crystal structure was obtained of <b>1b</b> 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 <b>1b</b> 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