Discovery of a Novel Allosteric Modulator of 5-HT3 Receptors

Abstract

Background:The 5-HT 3 receptors belong to the Cys-loop receptor superfamily. Results: The novel 5-HT 3 antagonist PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is discovered, and its mechanism of action is delineated. Conclusion: PU02 is a potent and selective negative allosteric modulator of 5-HT 3 receptors acting through a transmembrane intersubunit site in the receptors. Significance: The study highlights the transmembrane subunit interface in the Cys-loop receptor as a hot spot for allosteric modulation.

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β− Interface

“…Inhibition was consistent with a two-site model as follows: a high affinity component (IC 50, H ϭ 1.7 Ϯ 0.5 M) that reduced the level of photolabeling to that observed in the presence of GABA, and a low affinity component with IC 50, L ϭ 38 Ϯ 8 M, similar to the affinity seen in the presence of GABA (Table 2). These results indicate that S-mTFD-MPPB binds in the presence of bicuculline to the ␥ ϩ -␤ Ϫ site with ϳ10-fold higher affinity than it binds to other intersubunit sites in the presence of bicuculline or GABA.…”

“…In addition to the ␥ ϩ -␤ Ϫ site, S-mTFD-MPPB also bound with ϳ10-fold lower affinity to the intersubunit TMD site in the 50 values of S-mTFD-MPPB in the presence of GABA or bicuculline provide evidence that agonist/antagonist binding at the orthosteric site in the purified ␣1␤3␥2 GABA A R shifts the receptor conformational equilibrium, presumably between desensitized and closed states in our photolabeling assays. Our results provide a simple explanation for why S-mTFD-MPPB inhibits ␣1␤3␥2 and potentiates ␣1␤3 GABA A Rs.…”

“…10C) was consistent with a single-site model (n H ϭ 1) with an IC 50 (7.5 Ϯ 1.6 M) close to that seen for inhibition of R- [ 3 H]mTFD-MPAB photolabeling (Table 2). However, in the presence of GABA, inhibition was characterized by n H ϭ 0.6 Ϯ 0.1 (IC 50 Fig. 5A) nor picrotoxinin (30 M) inhibited photolabeling in the hydrophilic fractions containing ␥M2.…”

“…Intersubunitbinding sites in the TMD for positive and negative allosteric modulators have been identified in nicotinic acetylcholine receptors and serotonin 5-HT 3 receptors containing cationselective channels (49,50). Also, general anesthetics of diverse chemical structure that act as GABA A R-positive allosteric modulators bind with varying selectivities to each of the intersubunit sites in the GABA A R TMD.…”

“…In contrast, at the ␤ ϩ -␣ Ϫ interface both S-mTFD-MPPB and R-mTFD-MPAB IC 50 values, the total anesthetic concentrations resulting in 50% inhibition of GABA A R photolabeling, were determined as described under "Experimental Procedures." Parameters were determined from fits of data from two independent experiments, each carried out in parallel in the presence of GABA or bicuculline.…”