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Br J Pharmacol
2018 Feb 01;1753:419-428. doi: 10.1111/bph.14087.
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Towards functional selectivity for α6β3γ2 GABAA receptors: a series of novel pyrazoloquinolinones.
Treven M
,
Siebert DCB
,
Holzinger R
,
Bampali K
,
Fabjan J
,
Varagic Z
,
Wimmer L
,
Steudle F
,
Scholze P
,
Schnürch M
,
Mihovilovic MD
,
Ernst M
.
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BACKGROUND AND PURPOSE: The GABAA receptors are ligand-gated ion channels, which play an important role in neurotransmission. Their variety of binding sites serves as an appealing target for many clinically relevant drugs. Here, we explored the functional selectivity of modulatory effects at specific extracellular α+/β- interfaces, using a systematically varied series of pyrazoloquinolinones.
EXPERIMENTAL APPROACH: Recombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABA-elicited currents by the newly synthesized and reference compounds were investigated by the two-electrode voltage clamp method.
KEY RESULTS: We identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at α6β3γ2 GABAA receptors with nearly no residual activity at the other αxβ3γ2 (x = 1-5) subtypes. This modulation was independent of affinity for α+/γ- interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular α+/β- interfaces.
CONCLUSION AND IMPLICATIONS: These results constitute a major step towards a potential selective positive modulation of certain α6-containing GABAA receptors, which might be useful to elicit their physiological role. Furthermore, these studies pave the way towards insights into molecular principles that drive positive versus negative allosteric modulation of specific GABAA receptor isoforms.
Figure 1. PQ structures. Top left: PQ scaffold with labels for rings A to D and residue numbering (R7 and R8 on ring A; R'2 (o), R'3 (m), R'4 (p) on ring D). Top row: 1 is a p‐methoxy compound with unsubstituted ring A. R8 = chloro compounds are derived from varying the position of the methoxy group at ring D. Bottom row: compound variants with different residues in R'3 m‐position.
Figure 2. (A–D) GABAA receptor subtype activity profile of compounds with systematically varying positions of the methoxy‐group on ring D (compound 1, compound 2: p‐methoxy; compound 3: m‐methoxy; compound 4: o‐methoxy) at αxβ3γ2 (x = 1–6) GABAA receptors. Y‐axis shows % modulation of currents elicited by a GABA concentration amounting to 3–5% of maximum GABA currents per cell. For the purpose of structure–activity comparison, some datasets are reproduced: compound 1 at αxβ3γ2 (x = 1,2,3 and 5) reproduced with permission (Ramerstorfer et al.,
2011). Compound 2 at αxβ3γ2 (x = 1–6) reproduced with permission (Varagic et al.,
2013a). (E) Sample recording of GABA currents and co‐application of increasing concentrations of compound 3, from an oocyte injected with α6β3γ2 subunits. (F) Co‐application of compound 3 (10 and 30 μM) can inhibit positive GABA current modulation by 1 μM of compound 2.
Figure 3. Compound 4 is unable to block modulatory effects of compound 3 at α6β3γ2. (A) Modulation by compound 3 (3 μM) is unaffected by co‐application of compound 4 (n.s. = not significant; n = 9; P > 0.05 all groups vs. control before, one‐way ANOVA with Dunnett's multiple comparison test). (B) Sample recording of one oocyte sequentially exposed to 3 μM of compound 3 + increasing concentrations of compound 4 (one experiment from data presented in A).
Figure 4. Conformational analysis of compounds 2 (A), 3 (B) and 4 (C). Position of the methoxy substituent on ring D influences the dihedral angle φ between the planes of rings A–C and ring D. Number of calculated conformations: P = 2, m = 3, o = 4 (see Supporting Information Table S1). Methoxy substitution in o‐position rotates ring D by up to ~20° (green arrow).
Figure 5. (A) Screening of a series of compounds 5–9 at α6β3γ2 GABAA receptors (see bottom row in Figure 1). At 1 μM compound concentration, compounds 3 (dashed blue line representing the fitted curve of Figure 2c), 5 and 9 show more than 150% modulation of GABA EC3–5 currents. At 10 μM compound concentrations, compounds 5, 8 and 9 show strongest modulation of GABA EC3–5 currents, comparable to compound 3. (B, C) Subunit profile of compounds 5 (B) and 9 (C) at αxβ3γ2 (x = 1–6) GABAA subunit combinations. Note that some receptors, particularly α1β3γ2, are positively modulated by compound 5 but negatively modulated by compound 9. (D) Effective selectivity of 10 μM compound at α6β3γ2 over α1β3γ2. Modulation at α1β3γ2 was calculated as fraction of the modulation at α6β3γ2 [baseline (100%) = 0, efficacy at α6β3γ2 = 1; all signs positive] and subtracted from 1. (E) Separate experiment comparing effects of compound 5 and compound 9 at α1β3 versus α1β3γ2 receptors, demonstrating independence from the γ subunit.
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