Guanidino Acids Act as ρ1 GABAC Receptor Antagonists

Abstract

GABA(C) receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, beta-alanine and taurine were evaluated at human rho(1)GABA(C) receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-guanidinopropionic acid (IC(50) = 2.2 microM) and guanidinoacetic acid (IC(50) = 5.4 microM; K (B) = 7.75 microM [pK (B) = 5.11 +/- 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the beta-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure-activity-relationship profile required in developing novel therapeutic agents.

Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

“…The wild-type human GABA-A r1 receptor had a GABA EC 50 of 9.4 6 0.6 mM with a Hill coefficient of 1.1 6 0.2 (Table 1). The described wild-type GABA-A r1 receptor EC 50 is within the range of reported GABA-A r1 receptor GABA EC 50 values expressed in the HEK293T cell line (Kusama et al, 1993;Amin and Weiss, 1994;Greka et al, 2000;Harrison and Lummis, 2006;Chebib et al, 2009). …”

“…Borosilicate glass capillary tubes (Sutter Instrument Company, Novato, CA) were pulled to a resistance of 8-12 MV using a pipette puller (P-87 pipette puller, Sutter Instrument Co.). Recording patch electrodes were filled with internal solution consisting of (in mM): CsCl (120), tetraethylammonium chloride (20), CaCl 2 (1), MgCl 2 (2) EGTA (11), HEPES (10), and adjusted to pH 7.2 using N-methyl-D-glucamine (Chebib et al, 2009). Cells were perfused with external solution containing (in mM): NaCl (137), KCl (5.4) CaCl 2 (1.8) MgCl 2 (1) HEPES (5), adjusted to pH 7.4 (Chebib et al, 2009).…”

“…Recording patch electrodes were filled with internal solution consisting of (in mM): CsCl (120), tetraethylammonium chloride (20), CaCl 2 (1), MgCl 2 (2) EGTA (11), HEPES (10), and adjusted to pH 7.2 using N-methyl-D-glucamine (Chebib et al, 2009). Cells were perfused with external solution containing (in mM): NaCl (137), KCl (5.4) CaCl 2 (1.8) MgCl 2 (1) HEPES (5), adjusted to pH 7.4 (Chebib et al, 2009). Amiloride hydrochloride hydrate, GMQ, picrotoxin, and GABA were obtained from Sigma-Aldrich (St. Louis, MO).…”

“…The observed mixed pharmacology extends to endogenous guanidine compounds, which have exhibited different modulatory effects on the heteromeric GABA-A receptors than GABA-A r1 receptor (Neu et al, 2002;Chebib et al, 2009). In the inherited disorder guanidinoacetate methyltransferase deficiency, there is a buildup of guanidinoacetate, the immediate precursor to creatine that can directly activate the heteromeric GABA-A receptors (Neu et al, 2002).…”

“…In the inherited disorder guanidinoacetate methyltransferase deficiency, there is a buildup of guanidinoacetate, the immediate precursor to creatine that can directly activate the heteromeric GABA-A receptors (Neu et al, 2002). Where guanidinoacetic acid acts as an agonist in the heteromeric GABA-A receptors, this compound antagonizes the GABA-A r1 receptor (Chebib et al, 2009). Furthermore, guanidine compounds that modulate the acid-sensing ion channel influence Cys-loop receptor activity.…”