Synthesis of GABAA receptor agonists and evaluation of their alpha-subunit selectivity and orientation in the GABA binding site

Abstract

Drugs used to treat various disorders target GABA A receptors. To develop alpha subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at alpha 2-, alpha 3-, and alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in alpha 2-, alpha 4-, and alpha 6-containing receptors and potentiated alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants alpha 1F64C and alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing alpha subtype selective GABA mimetic drugs.

Figure 1

Structures of GABA, muscimol, THIP and 4-PIOL.

Figure 2

Dose-response curves as measured against [³H]muscimol binding to cortical (squares) and cerebellar (circles) membranes. Binding data were normalized to the binding in the absence of any inhibitor set to 100%. Error bars indicate the S.E.M for at least three independent tissue preparations.

Figure 3

Whole-cell recordings of HEK 293 cells expressing recombinant rat α_iβ₃γ₂ (i = 1–6) GABA_A receptors. Currents were normalized to the maximally GABA-induced current at the approximate EC₁₀₀. To test the intrinsic activity, different concentrations of the 5-(4-piperidyl)-1,3,4-oxadiazol-derivates tested were applied to the cells. Error bars indicate the S.E.M for at least four cells.

Figure 4

Whole-cell recordings of HEK 293 cells expressing recombinant rat α_iβ₃γ₂ (i = 1–6) GABA_A receptors. Currents were normalized to the GABA concentrations specific for the receptor subtype under in vitro conditions. Different concentrations of the 5-(4-piperidyl)-1,3,4-oxadiazol-derivates tested were co-applied with GABA concentrations around the EC₂₅. Error bars indicate the S.E.M for at least four cells.

Figure 5

Picrotoxinin blocks 6a induced currents in α₁β₂ wt receptors. The first trace shows a 6a induced current. The second trace was recorded during a 20 s application of picrotoxinin, immediately followed by a co-application of 6a and picrotoxinin.

Figure 6

MTSES⁻ reaction rate with the α₁F64Cβ₂ cysteine mutant. A, EC₅₀ GABA current traces were recorded initially and after each brief application of 10 μM MTSES⁻ (↓). Currents during MTSES⁻application (↓) are not shown. B, GABA test currents were normalized to the initial GABA current (I_max) and plotted versus cumulative MTSES⁻ exposure time. Data were fit to a monoexponential decay function.

Figure 7

Protection assay shows that GABA and 6a protect α₁F64Cβ₂ receptors from reaction with MTSES⁻. A, MTSES⁻ modification of α₁F64Cβ₂ in the absence of agonist. Two GABA test pulses were applied to demonstrate the stability of the GABA current. At the downward arrow marked MTSES_non-sat 10 μM MTSES⁻ was applied for 12 s. Following washout a GABA test pulse was recorded. The GABA test current (third trace) was reduced by 87%. At the downward arrow marked MTSES_sat 20 μM MTSES⁻ was applied for 50 s to bring the MTSES⁻ reaction to completion. Following washout a final GABA test pulse (fourth trace) was applied. Currents during MTSES⁻ applications (↓) are not shown. B, GABA protects α₁F64Cβ₂ from modification by MTSES⁻. The same series of reagents are applied as in panel A, except that the MTSES_non-sat was coapplied with 3.6 mM GABA. The GABA current elicited by the next GABA test pulse (middle trace) is significantly larger than the GABA current after the MTSES_non-sat application in panel A, indicating that the presence of GABA significantly reduced the extent of reaction with the non-saturating concentration of MTSES⁻. C, 6a protects α₁F64Cβ₂ from modification by MTSES⁻. The same series of reagents are applied as in panel A, except that the MTSES_non-sat was coapplied with 10 mM 6a. The GABA current elicited by the next GABA test pulse (middle trace) is significantly larger than the GABA current after the MTSES_non-sat application in panel A, indicating that the presence of 6a significantly reduced the extent of reaction with the non-saturating concentration of MTSES⁻. Currents during MTSES⁻ application (↓) with or without agonist are not shown. Duration of application of GABA EC₅₀ test pulses are indicated by black horizontal bars above the current traces.

Figure 8

Summary of the protection assay with α₁F64Cβ₂ (black bars), α₁R66Cβ₂ (clear and striped bars), and α₁S68Cβ₂ (grey bars). Bars indicate the average percent inhibition of GABA test currents following the application of a non-saturating concentration of MTSES⁻ either in the absence of agonist or in the presence of EC₉₀ GABA or 6a (10 or 30 mM). We infer that a reagent, GABA or 6a, protected a mutant from reaction with MTSES⁻ if the extent of inhibition by MTSES⁻ coapplied with either GABA or 6a is significantly less than the extent of inhibition by MTSES⁻ applied alone. Conditions where the co-application of GABA or 6a are significantly different than the effect of MTSES⁻ application alone are indicated by *; (*, P<0.014; ***, P<0.0001) by one way ANOVA and Fisher’s PLSD. For α₁R66Cβ₂ 30 μM MTSEA-biotin reduced the subsequent GABA test currents by 93%. Application of 30 μM MTSEA-biotin with 30 mM 6a reduced the subsequent GABA test currents by 83%. The limited supply of 6a precluded further experiments.

Figure 9

6a reacted with α₁R66Cβ₂. A, Currents recorded from an oocyte expressing α₁R66Cβ₂. Alternating 10-s applications of 30 mM 6a [indicated by (↓)] and 5.5 mM GABA test currents (bars above current traces) resulted in a progressive decrease in the GABA test currents. The decrease eventually plateaued at which time a 12-s application of 10 mM MTSES⁻ (↓) had no effect indicating that all accessible cysteine had reacted with 6a. Reduction by a 20-s application of 10 mM DTT (↓) led to complete recovery of the GABA test current magnitude. Currents during application of 6a, MTSES⁻ and DTT are not shown. B, Application of the oxygen analogue 5a (30 mM, 10 s) (↓) to an oocyte expressing α₁R66Cβ₂ did not decrease the subsequent GABA test currents. Currents during 5a application are not shown. C, Reaction rate of 6a with α₁R66Cβ₂. GABA test currents were normalized to the initial GABA test current, plotted as a function of cumulative duration of 6a application and fitted to a monoexponential decay function.

Figure 10

Homology model of the GABA_A receptor agonist binding site based on the AChBP structure (PDB 1UW6). A, View of the principle side of the β₂ subunit GABA binding site (light blue) and of the complementary side of the α₁ subunit GABA binding site (dark blue) showing backbone in ribbon form. Side chains of residues mentioned in the text are shown in wireframe format. B, GABA binding site showing backbone in ribbon form with 6a and α₁R66C shown in spacefilling format with CPK colors. The close proximity of the sulfurs (yellow) in 6a and α₁Cys66 is consistent with the observed reaction between 6a and α₁Cys66. α₁F64 is shown in green spacefilling format. The close proximity between α₁F64 and 6a is consistent with the steric protection of the cysteine substituted at this position. In contrast, α₁S68 (pink colored spacefilling format) is not in close proximity to 6a. C, View of nicotine bound in the AChBP binding site (PDB 1UW6) from the same perspective as in panel B. Backbone is shown in ribbon form and nicotine in white-color spacefilling format. Side chains of AChBP residues aligned with the GABA_A cysteine mutants discussed in the text and shown in panel C are in wireframe format. Nicotine interacts with the homologous β strand adjacent to the β strand containing the residue in this model aligned with GABA_A α₁R66.

Figure 11

A, Structures of 6a and 5a (left column) and of GABA and muscimol (right column). B, Structures of the compounds in panel A with atomic distances between the basic nitrogen atom and other polar atoms in the respective molecules. Distances were measured after energy minimization (Chemsketch 5.12, ACD Inc., Toronto, Ontario, Canada). CPK color scheme used, carbon, white; nitrogen, blue; oxygen, red; sulfur, yellow. C, Electrostatic potential mapped onto the van der Waals surface of GABA (top) and 6a (bottom) with stick representation of molecules. Red indicates negative electrostatic potential and blue is positive potential. Image generated using Spartan. Note the similarity of the overall electrostatic potential, especially the distance between the positively charged nitrogen in GABA or 6a to the negative carboxylate (GABA) or the oxadiazolthione moeity (6a).

Scheme 1^a

^a Reaction conditions: (a) Et₃N, Boc₂O in CH₂Cl₂ or NaHCO₃, Boc₂O in water; (b) NH₂NH₂; (c) CDI to obtain 3; CS₂ to obtain 4; (d) 2.3 N ethanolic HCl; (e) PhCH₂NCX; (f) 2% NaOH; (g) H₃CC(OC₂H₅)₃; (h) HBr/HAc.

Scheme 2^a

^a Reaction conditions: (b) NH₂NH₂; (i) CH₂O, HCOOH to obtain 7a; PhCH₂Cl to obtain 7b; (k) triphosgene to obtain 9a,b; CS₂ to obtain 10a,b; (l) HC(OC₂H₅)₃.

Scheme 3^a

^a Reaction conditions as given in Scheme 1 and Scheme 2.

Scheme 4^a

^a Reaction conditions as given in Scheme 1 and Scheme 2.