Distinct Structural Features of Cyclothiazide Are Responsible for Effects on Peak Current Amplitude and Desensitization Kinetics at iGluR2 |
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Authors: | Helle Hald Philip K. Ahring Tommy Liljefors Jette S. Kastrup |
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Affiliation: | 1 Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark 2 NeuroSearch A/S, Drug Discovery, 93 Pederstrupvej, DK-2750 Ballerup, Denmark |
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Abstract: | Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. Upon glutamate application, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo rapid and almost complete desensitization that can be attenuated by positive allosteric modulators. The molecular mechanism of positive allosteric modulation has been elucidated previously by crystal structures of the ligand-binding core of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we investigate the structure and function of CTZ and three closely related analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and fast application patch-clamp electrophysiology. CTZ was the most efficacious and potent modulator of the four compounds on iGluR2(Q)i [Emax normalized to response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476% (NS5217) and EC50 in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48 (NS5217)]. The four modulators divide into three groups according to efficacy and desensitization kinetics: (1) CTZ increases the peak current efficacy twice as much as the three analogues and nearly completely blocks receptor desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate desensitization partially; (3) NS1493 has low efficacy but nearly completely blocks receptor desensitization. A hydrophobic substituent at the 3-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important for compound efficacy, with the following ranking: norbornenyl (bicyclo[2.2.1]hept-2-ene) > cyclopentyl > methyl. The replacement of the norbornenyl moiety with a significantly less hydrophobic cyclopentane ring increases the flexibility of the modulator as the cyclopentane ring adopts various conformations at the iGluR2 allosteric binding site. The main structural feature responsible for a nearly complete block of desensitization is the presence of an NH hydrogen bond donor in the 4-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ seems to be a major determinant of receptor desensitization kinetics. |
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Keywords: | AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid benzothiadiazine, 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine CHO, Chinese hamster ovary CTZ, cyclothiazide iGluRs, ionotropic glutamate receptors iGluR2i, the iGluR2 flip splice variant iGluR2o, the iGluR2 flop splice variant norbornenyl, bicyclo[2.2.1]hept-2-ene S1S2J, iGluR2o ligand-binding core S1S2J-N754S, iGluR2o ligand-binding core N754S mutant PDB, Protein Data Bank PEG, polyethylene glycol |
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