Cunde Wang

Neurosteroid Access to the GABAA Receptor

GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Synthesis of chiral ligands derived from the Betti base and their use in the enantioselective addition of diethylzinc to aromatic aldehydes

Abstract A novel procedure for selective direct N , N -alkylation of the chiral Betti base was developed, and a new family of chiral ligands, ( S )-1-(α-cycloaminobenzyl)-2-naphthols, were prepared. The ligands with five- and six-membered cyclic amines showed highly efficient asymmetric induction in the addition of diethylzinc to aromatic aldehydes in 93–96% yields and 91–99% ee.

The Influence of Neuroactive Steroid Lipophilicity on GABAA Receptor Modulation: Evidence for a Low-Affinity Interaction

Anesthetic steroids with actions at gamma-aminobutyric acid type A receptors (GABA(A)Rs) may access transmembrane domain binding site(s) directly from the plasma cell membrane. Accordingly, the effective concentration in lipid phase and the ability of the steroid to meet pharmacophore requirements for activity will both contribute to observed steady-state potency. Furthermore, onset and offset of receptor effects may be rate limited by lipid partitioning. Here we show that several GABA-active steroids, including naturally occurring neurosteroids, of different lipophilicity differ in kinetics and potency at GABA(A)Rs. The hydrophobicity ranking predicted relative potency of GABA(A)R potentiation and predicted current offset kinetics. Kinetic offset differences among steroids were largely eliminated by gamma-cyclodextrin, a scavenger of unbound steroid, suggesting that affinity differences among the analogues are dwarfed by the contributions of nonspecific accumulation. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-tagged fluorescent analogue of the low-lipophilicity alphaxalone (C17-NBD-alphaxalone) exhibited faster nonspecific accumulation and departitioning than those of a fluorescent analogue of the high-lipophilicity (3alpha,5alpha)-3-hydroxypregnan-20-one (C17-NBD-3alpha5alphaA). These differences were paralleled by differences in potentiation of GABA(A)R function. The enantiomer of C17-NBD-3alpha5alphaA, which does not satisfy pharmacophore requirements for steroid potentiation, exhibited identical fluorescence kinetics and distribution to C17-NBD-3alpha5alphaA, but was inactive at GABA(A)Rs. Simple simulations supported our major findings, which suggest that neurosteroid binding affinity is low. Therefore both specific (e.g., fulfilling pharmacophore requirements) and nonspecific (e.g., lipid solubility) properties contribute to the potency and longevity of anesthetic steroid action.

Novel preparation of non-racemic 1-[α-(1-azacycloalkyl)benzyl]-2-naphthols from Betti base and their application as chiral ligands in the asymmetric addition of diethylzinc to aryl aldehydes

A novel route for the preparation of non-racemic 1-[α-(1-azacycloalkyl)benzyl]-2-naphthols was developed, which involves regioselective N-cycloalkylation of the Betti base with dials in the presence of NaBH3CN to give 1-azacycloalka[2,1-b]oxazine followed by the selective cleavage of a C–O bond with LiAlH4. As a new family of chiral ligands, their application in the enantioselective addition of diethylzinc to aryl aldehydes was tested. The ligands incorporating pyrrolidine and piperidine led to highly efficient asymmetric induction to give products in up to 96% yield and 99% ee.

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions.

Neuroactive steroids are potent modulators of GABAA receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABAA receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABAA receptor, such as (3α,5α)‐3‐hydroxypregnan‐20‐one (3α5αP, allopregnanolone).

Neurosteroid migration to intracellular compartments reduces steroid concentration in the membrane and diminishes GABA-A receptor potentiation

Neurosteroids are potent modulators of GABA‐A receptors. We have examined the time course of development of potentiation of α1β2γ2L GABA‐A receptors during coapplication of GABA and an endogenous neurosteroid (3α,5α)‐3‐hydroxypregnan‐20‐one (3α5αP). The simultaneous application of 3α5αP with 5 μm GABA resulted in a biphasic rising phase of current with time constants of 50–60 ms for the rapid phase and 0.3–3 s for the slow phase. The properties of the rapid phase were similar at all steroid concentrations but the time constant of the slower phase became successively shorter as the steroid concentration was increased. Potentiation developed very rapidly (τ= 130 ms) when cells were preincubated with 300 nm 3α5αP before application of GABA + 3α5αP, and in outside‐out patch recordings, suggesting that steroid diffusion to intracellular compartments competes with receptor potentiation by depleting the cell membrane of steroid. Very low steroid concentrations (3–5 nm) potentiated GABA responses but the effects took minutes to develop. Intracellular accumulation of a fluorescent steroid analogue followed a similar time course, suggesting that slow potentiation results from slow accumulation within plasma membrane rather than indirect effects, such as activation of second messenger systems. In cell‐attached single‐channel recordings, where 3α5αP is normally applied through the pipette solution, addition of steroid to the bath solution dramatically shifted the steroid potentiation concentration–effect curve to lower steroid concentrations. We propose that bath‐supplied steroid compensates for the diffusion of pipette‐supplied steroid out of the patch to the rest of the cell membrane and/or intracellular compartments. The findings suggest that previous studies overestimate the minimum concentration of steroid capable of potentiating GABA actions at GABA‐A receptors. The results have implications for the physiological role of endogenous neurosteroids.

Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light

Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the β configuration at the C2 position of (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone, 3α5αP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.

Effects on Membrane Capacitance of Steroids with Antagonist Properties at GABAA Receptors

We investigated the electrophysiological signature of neuroactive steroid interactions with the plasma membrane. We found that charged, sulfated neuroactive steroids, those that exhibit noncompetitive antagonism of GABA(A) receptors, altered capacitive charge movement in response to voltage pulses in cells lacking GABA receptors. Uncharged steroids, some of which are potent enhancers of GABA(A) receptor activity, produced no alteration in membrane capacitance. We hypothesized that the charge movements might result from physical translocation of the charged steroid through the transmembrane voltage, as has been observed previously with several hydrophobic anions. However, the charge movements and relaxation time constants of capacitive currents did not exhibit the Boltzmann-type voltage dependence predicted by a single barrier model. Further, a fluorescently tagged analog of a sulfated neurosteroid altered membrane capacitance similar to the parent compound but produced no voltage-dependent fluorescence change, a result inconsistent with a strong change in the polar environment of the fluorophore during depolarization. These findings suggest that negatively charged sulfated steroids alter the plasma membrane capacitance without physical movement of the molecule through the electric field.

Preparation of (5α, 13α)-D-azasteroids as key precursors of a new family of potential GABAA receptor modulators

Abstract Three groups of (5α,13α)- d -azasteroids, (5α,13α)-3-hydroxy-17a-aza- d -homoandrostans ( 12 ), (5α,13α)-3-hydroxy-17-aza- d -homoandrostans ( 15 ), and (5α,13α)-3-hydroxy-17-azaandrostans ( 17 ), were designed and synthesized as key precursors for the further preparation of a new family of potential GABA A receptor modulators from commercially available natural steroids (5α)-3-hydroxyandrostane-17-ones ( 7 ).

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors

RationaleWhile neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified.ObjectivesThis study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors.ResultsTwo neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C–H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1β2γ2L subunits, modulated [35S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [3H]CW12 and [3H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and β2 subunits or β3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific.ConclusionsCW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.