Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and gamma-aminobutyric acidA receptor function. 1994

J E Hawkinson, and C L Kimbrough, and D Belelli, and J J Lambert, and R H Purdy, and N C Lan
CoCensys, Inc., Irvine, California 92718.

Neuroactive steroids, including endogenously occurring metabolites of progesterone and deoxycorticosterone as well as their synthetic derivatives, are positive allosteric modulators of the gamma-aminobutyric acid (GABA)A receptor complex. They inhibit the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), enhance the binding of [3H]flunitrazepam, and potentiate GABA-evoked chloride currents and agonist-stimulated 36Cl- uptake. The structure-activity relationship for 31 neuroactive steroids and related compounds was explored by examining their relative ability to inhibit [35S]TBPS binding in rat brain cortical membranes. A free 3 alpha-hydroxy group is necessary for high potency inhibition. Whereas hydroxylation in the 21-position and subsequent esterification maintain activity, 11 alpha- or 12 alpha-hydroxylation greatly reduces activity. The rank order of potency for 17-position substitutions in the 5 alpha-reduced series is 17 beta-acetyl > 17 beta-cyano > 17 beta-methoxycarbonyl > 17 alpha-acetyl > 17-one > or = 17-oxime > or = 17 alpha-cyano. Introduction of a double bond between the 9- and 11-positions reduces potency, whereas a double bond in the 4-position reduces the maximal extent of inhibition. Comparing the activities of these neuroactive steroids and related compounds in the [35S]TBPS and [3H]flunitrazepam assays, there is a strong correlation between potency (r = 0.90, n = 17) and magnitude of modulation (r = 0.95, n = 31), indicating that the neuroactive steroid binding site is similarly coupled to the TBPS and benzodiazepine sites in rat cortex. However, there is a weaker correlation (r = 0.74-0.78, n = 31) between the degree of modulation in either binding assay and potentiation of muscimol-stimulated 36Cl- uptake in rat cortical synaptoneurosomes. Using an electrophysiological approach, stronger correlations (r = 0.89-0.94, n = 15) were observed between the magnitude of modulation in the binding assays and potentiation of GABA-evoked chloride currents in Xenopus oocytes expressing human alpha 1 beta 1 gamma 2L receptor complexes. Thus, neuroactive steroid modulation of [35S]TBPS and [3H]flunitrazepam binding is predictive of functional activity at the GABAA receptor complex.

UI MeSH Term Description Entries
D008564 Membrane Potentials The voltage differences across a membrane. For cellular membranes they are computed by subtracting the voltage measured outside the membrane from the voltage measured inside the membrane. They result from differences of inside versus outside concentration of potassium, sodium, chloride, and other ions across cells' or ORGANELLES membranes. For excitable cells, the resting membrane potentials range between -30 and -100 millivolts. Physical, chemical, or electrical stimuli can make a membrane potential more negative (hyperpolarization), or less negative (depolarization). Resting Potentials,Transmembrane Potentials,Delta Psi,Resting Membrane Potential,Transmembrane Electrical Potential Difference,Transmembrane Potential Difference,Difference, Transmembrane Potential,Differences, Transmembrane Potential,Membrane Potential,Membrane Potential, Resting,Membrane Potentials, Resting,Potential Difference, Transmembrane,Potential Differences, Transmembrane,Potential, Membrane,Potential, Resting,Potential, Transmembrane,Potentials, Membrane,Potentials, Resting,Potentials, Transmembrane,Resting Membrane Potentials,Resting Potential,Transmembrane Potential,Transmembrane Potential Differences
D011278 Pregnanes Saturated derivatives of the steroid pregnane. The 5-beta series includes PROGESTERONE and related hormones; the 5-alpha series includes forms generally excreted in the urine.
D011869 Radioligand Assay Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders). Protein-Binding Radioassay,Radioreceptor Assay,Assay, Radioligand,Assay, Radioreceptor,Assays, Radioligand,Assays, Radioreceptor,Protein Binding Radioassay,Protein-Binding Radioassays,Radioassay, Protein-Binding,Radioassays, Protein-Binding,Radioligand Assays,Radioreceptor Assays
D011963 Receptors, GABA-A Cell surface proteins which bind GAMMA-AMINOBUTYRIC ACID and contain an integral membrane chloride channel. Each receptor is assembled as a pentamer from a pool of at least 19 different possible subunits. The receptors belong to a superfamily that share a common CYSTEINE loop. Benzodiazepine-Gaba Receptors,GABA-A Receptors,Receptors, Benzodiazepine,Receptors, Benzodiazepine-GABA,Receptors, Diazepam,Receptors, GABA-Benzodiazepine,Receptors, Muscimol,Benzodiazepine Receptor,Benzodiazepine Receptors,Benzodiazepine-GABA Receptor,Diazepam Receptor,Diazepam Receptors,GABA(A) Receptor,GABA-A Receptor,GABA-A Receptor alpha Subunit,GABA-A Receptor beta Subunit,GABA-A Receptor delta Subunit,GABA-A Receptor epsilon Subunit,GABA-A Receptor gamma Subunit,GABA-A Receptor rho Subunit,GABA-Benzodiazepine Receptor,GABA-Benzodiazepine Receptors,Muscimol Receptor,Muscimol Receptors,delta Subunit, GABA-A Receptor,epsilon Subunit, GABA-A Receptor,gamma-Aminobutyric Acid Subtype A Receptors,Benzodiazepine GABA Receptor,Benzodiazepine Gaba Receptors,GABA A Receptor,GABA A Receptor alpha Subunit,GABA A Receptor beta Subunit,GABA A Receptor delta Subunit,GABA A Receptor epsilon Subunit,GABA A Receptor gamma Subunit,GABA A Receptor rho Subunit,GABA A Receptors,GABA Benzodiazepine Receptor,GABA Benzodiazepine Receptors,Receptor, Benzodiazepine,Receptor, Benzodiazepine-GABA,Receptor, Diazepam,Receptor, GABA-A,Receptor, GABA-Benzodiazepine,Receptor, Muscimol,Receptors, Benzodiazepine GABA,Receptors, GABA A,Receptors, GABA Benzodiazepine,delta Subunit, GABA A Receptor,epsilon Subunit, GABA A Receptor,gamma Aminobutyric Acid Subtype A Receptors
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D001921 Brain The part of CENTRAL NERVOUS SYSTEM that is contained within the skull (CRANIUM). Arising from the NEURAL TUBE, the embryonic brain is comprised of three major parts including PROSENCEPHALON (the forebrain); MESENCEPHALON (the midbrain); and RHOMBENCEPHALON (the hindbrain). The developed brain consists of CEREBRUM; CEREBELLUM; and other structures in the BRAIN STEM. Encephalon
D005445 Flunitrazepam A benzodiazepine with pharmacologic actions similar to those of DIAZEPAM that can cause ANTEROGRADE AMNESIA. Some reports indicate that it is used as a date rape drug and suggest that it may precipitate violent behavior. The United States Government has banned the importation of this drug. Fluridrazepam,Rohypnol,Fluni 1A Pharma,Flunibeta,Flunimerck,Fluninoc,Flunitrazepam-Neuraxpharm,Flunitrazepam-Ratiopharm,Flunitrazepam-Teva,Flunizep Von Ct,Narcozep,RO-5-4200,Rohipnol,Flunitrazepam Neuraxpharm,Flunitrazepam Ratiopharm,Flunitrazepam Teva,RO54200,Von Ct, Flunizep
D000494 Allosteric Regulation The modification of the reactivity of ENZYMES by the binding of effectors to sites (ALLOSTERIC SITES) on the enzymes other than the substrate BINDING SITES. Regulation, Allosteric,Allosteric Regulations,Regulations, Allosteric
D000731 Androstanes The family of steroids from which the androgens are derived.
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia

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