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ABSTRACT: To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.
Bioorganic & medicinal chemistry letters 06/2008; 18(9):2765-70. · 2.65 Impact Factor
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ABSTRACT: The accuracy of the docking of a ligand in its modeled binding-site depends on the reliability of this model. To provide a model with experimental support, we have developed an engineered affinity labeling method combining cysteine-reactive probes with substitutions of putative site-lining residues into cysteines. This strategy amounts to building chemical sensors for the proximity of the substituted cysteines; it requires an activity or binding assay to monitor the irreversible occupancy of the site by the reactive ligand. Using affinity probes made reactive in different positions, the docking of the ligand can be inferred from the observed pattern of coupling reactions. The method involves three steps: ligand chemistry, mutagenesis and biological assays, which are detailed and scrutinized in the review: lead selection, ligand derivatization, and evaluation of the affinity probes (stability, reactivity and biological properties) for the ligands; positional selection and mutant properties for the cysteine substitutions; functional controls and assays for the analysis of the irreversible reactions. Examples illustrate the different criteria of concern; the data are interpreted in terms of binding-site structure and function. Potentially, the method can explore protein dynamics, since its targets are full-length membrane-inserted heteromeric proteins: it can detect subtype-dependent or activation-induced conformational changes.
Current Chemical Biology 08/2007; 1(3):271-277.
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ABSTRACT: Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABA(A) receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, alpha1H101Cbeta2gamma2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the alpha/gamma subunit interface, or if it occurs in both alpha1 subunits, exclusively reaction at the alpha/gamma subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15-1788 efficiently inhibited the covalent reaction in the presence of 100 microm GABA but only partially in its absence or in the presence of 10 microm GABA. It is concluded that Ro15-1788 efficiently protects activated and desensitized states, but not the resting state.
Journal of Neurochemistry 03/2005; 92(4):859-66. · 4.06 Impact Factor
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ABSTRACT: The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.
Journal of Biological Chemistry 07/2003; 278(26):24011-7. · 4.77 Impact Factor
