Direct measurements of neurosteroid binding to specific sites on GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.

The Mechanism of Enantioselective Neurosteroid Actions on GABAA Receptors.

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that ent-ALLO potentiates GABA-elicited currents in α1ß3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-ALLO was prevented by the α1(Q242L) mutation in the intersubunit neurosteroid binding site between the ß3 and α1 subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that ent-ALLO binds weakly to the ß3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or ß3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the ß3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.

A Novel Mitragynine Analog with Low-Efficacy Mu Opioid Receptor Agonism Displays Antinociception with Attenuated Adverse Effects.

Mitragynine and 7-hydroxymitragynine (7OH) are the major alkaloids mediating the biological actions of the psychoactive plant kratom. To investigate the structure-activity relationships of mitragynine/7OH templates, we diversified the aromatic ring of the indole at the C9, C10, and C12 positions and investigated their G-protein and arrestin signaling mediated by mu opioid receptors (MOR). Three synthesized lead C9 analogs replacing the 9-OCH3 group with phenyl (4), methyl (5), or 3'-furanyl [6 (SC13)] substituents demonstrated partial agonism with a lower efficacy than DAMGO or morphine in heterologous G-protein assays and synaptic physiology. In assays limiting MOR reserve, the G-protein efficacy of all three was comparable to buprenorphine. 6 (SC13) showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning in mice. These results suggest the possibility of activating MOR minimally (G-protein Emax ≈ 10%) in cell lines while yet attaining maximal antinociception in vivo with reduced opioid liabilities.

Photo-oxidation and Thermal Oxidations of Triptycene Thiols by Aryl Chalcogen Oxides.

Oxidation of thiols yield sulfenic acids, which are very unstable intermediates. As sulfenic acids are reactive, they form disulfides in the presence of thiols. However, sulfenic acids also oxidize to sulfinic acids (-SO2H) and sulfonic acids (-SO3H) at higher concentrations of oxidants. Hydrogen peroxide is a commonly used oxidant for the oxidation of thiols to yield sulfenic acids. However, hydrogen peroxide also oxidizes other reactive functional groups present in a molecule. In this work, the reaction intermediates arising from the oxidation of sterically hindered thiols by aryl chalcogen oxides, dibenzothiophene S-oxide (DBTO), dibenzoselenophene Se-oxide (DBSeO), and dibenzotellurophene Te-oxide (DBTeO), were investigated. Photodeoxygenation of DBTO produces triplet atomic oxygen [O(3P)], which has previously shown to preferentially react with thiols over other functional groups. Similarly, aryl selenoxides have also shown that they can thermally react selectively with thiols at room temperature to yield disulfides. Conversely, aryl telluroxides have been reported to oxidize thiols to disulfides thermally with no selectivity toward thiols. The results from this study demonstrate that sulfenic acids are an intermediate in the oxidation of thiols by DBTeO and by photodeoxygenation of DBTO. The results also showed that the oxidation of thiols by DBSeO yields sulfonic acids. Triptycene-9-thiol and 9-fluorotriptycene-10-thiol were for the thiols used in this oxidation reaction. This work expands the list of oxidants that can be used to oxidize thiols to obtain sulfenic acids.

In vitro oxidations of low-density lipoprotein and RAW 264.7 cells with lipophilic O(3P)-precursors.

A beneficial property of photogenerated reactive oxygen species (ROS) is the capability of oxidant generation within a specific location or organelle inside a cell. Dibenzothiophene S-oxide (DBTO), which is known to undergo a photodeoxygenation reaction to generate ground state atomic oxygen [O(3P)] upon irradiation, was functionalized to afford localization within the plasma membrane of cells. The photochemistry, as it relates to oxidant generation, was studied and demonstrated that the functionalized DBTO derivatives generated O(3P). Irradiation of these lipophilic O(3P)-precursors in the presence of LDL and within RAW 264.7 cells afforded several oxidized lipid products (oxLP) in the form of aldehydes. The generation of a 2-hexadecenal (2-HDEA) was markedly increased in irradiations where O(3P) was putatively produced. The substantial generation of 2-HDEA is not known to accompany the production of other ROS. These cellular irradiation experiments demonstrate the potential of inducing oxidation with O(3P) in cells.