Image-guided neural activity manipulation with a paramagnetic drug.

Targeted manipulations of neural activity are essential approaches in neuroscience and neurology, but monitoring such procedures in the living brain remains a significant challenge. Here we introduce a paramagnetic analog of the drug muscimol that enables targeted neural inactivation to be performed with feedback from magnetic resonance imaging. We validate pharmacological properties of the compound in vitro, and show that its distribution in vivo reliably predicts perturbations to brain activity.

4,5-Substituted 3-Isoxazolols with Insecticidal Activity Act as Competitive Antagonists of Housefly GABA Receptors.

The insect GABA receptor (GABAR), which is composed of five RDL subunits, represents an important target for insecticides. A series of 4,5-disubstituted 3-isoxazolols, including muscimol analogues, were synthesized and examined for their activities against four splice variants (ac, ad, bc, and bd) of housefly GABARs expressed in Xenopus oocytes. Muscimol was a more potent agonist than GABA in all four splice variants, whereas synthesized analogues did not exhibit agonism but rather antagonism in housefly GABARs. The introduction of bicyclic aromatic groups at the 4-position of muscimol and the simultaneous replacement of the aminomethyl group with a carbamoyl group at the 5-position to afford six 4-aryl-5-carbamoyl-3-isoxazolols resulted in compounds that exhibited significantly enhanced antagonism with IC50 values in the low micromolar range in the ac variant. The inhibition of GABA-induced currents by 100 µM analogues was approximately 1.5-4-fold greater in the ac and bc variants than in the ad and bd variants. 4-(3-Biphenylyl)-5-carbamoyl-3-isoxazolol displayed competitive antagonism, with IC50 values of 30, 34, 107, and 96 µM in the ac, bc, ad, and bd variants, respectively, and exhibited moderate insecticidal activity against houseflies, with an LD50 value of 5.6 nmol/fly. These findings suggest that these 3-isoxazolol analogues are novel lead compounds for the design and development of insecticides that target the orthosteric site of housefly GABARs.

Synthesis and biological evaluation of 4-(aminomethyl)-1-hydroxypyrazole analogues of muscimol as γ-aminobutyric acid(a) receptor agonists.

A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA(A) receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. The unsubstituted 4-AHP analogue (2a) (EC(50) 19 µM, R(max) 69%) was a moderately potent agonist at human α(1)ß(2)γ(2) GABA(A) receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α(1)ß(2)γ(2) GABA(A) receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α(1)ß(2)γ(2) over ρ(1) GABA(A) receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.

Protein disulfide isomerase in ALS mouse glia links protein misfolding with NADPH oxidase-catalyzed superoxide production.

Protein disulfide isomerase (PDI) is an oxidoreductase assisting oxidative protein folding in the endoplasmic reticulum of all types of cells, including neurons and glia. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), up-regulation of PDI is an important part of unfolded protein response (UPR) that is thought to represent an adaption reaction and thereby protect the neurons. Importantly, studies on animal models of familial ALS with mutant Cu/Zn superoxide dismutase 1 (SOD1) have shown that the mutant SOD1 in astrocytes or microglia strongly regulates the progression of the disease. Here, we found an early up-regulation of PDI in microglia of transgenic (tg) mutant SOD1 mice, indicating that in addition to neurons, UPR takes place in glial cells in ALS. The observation was supported by the finding that also the expression of a UPR marker GADD34 (growth arrest and DNA damage-inducible protein) was induced in the spinal cord glia of tg mutant SOD1 mice. Because mutant SOD1 can cause sustained activation of NADPH oxidase (NOX), we investigated the role of PDI in UPR-induced NOX activation in microglia. In BV-2 microglia, UPR resulted in NOX activation with increased production of superoxide and increased release of tumor necrosis factor-α. The phenomenon was recapitulated in primary rat microglia, murine macrophages and human monocytes. Importantly, pharmacological inhibition of PDI or its down-regulation by short interfering RNAs prevented NOX activation in microglia and subsequent production of superoxide. Thus, results strongly demonstrate that UPR, caused by protein misfolding, may lead to PDI-dependent NOX activation and contribute to neurotoxicity in neurodegenerative diseases including ALS.

GABA(A) receptor ligands and their therapeutic potentials.

The GABA(A) receptor system is implicated in a number of neurological diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP or isoguvacine. Using recombinant GABA(A) receptors, functional selectivity has been shown for a number of compounds such as the GABA(A)agonists imidazole-4-acetic acid and THIP, showing highly subunit-dependent potency and maximal response. In the light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogues of 4-PIOL, a low-efficacy partial GABA(A) agonist, have been performed. In this connection, a series of GABA(A) ligands has been developed showing pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effect. Only little information about direct acting GABA(A) receptor agonists in clinical studies is available. Results from clinical studies on the effect of the GABA(A) agonist THIP on human sleep pattern shows that the functional consequences of a direct acting agonist are different from those seen after administration of GABA(A) receptor modulators.

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.

Thiomuscimol, a new photoaffinity label for the GABAA receptor.

Thiomuscimol inhibits [3H]muscimol binding to brain GABAA receptors. Exposure of Ag(+)-treated membrane preparations to UV radiation at 254 nm for 40 min in the presence of thiomuscimol (10(-5) M) produced a 20-30% irreversible decrease in high-affinity [3H]muscimol binding sites. The photoaffinity labeling of thiomuscimol was inhibited by GABA (10(-4) M) added prior to exposure to UV light. The data show that thiomuscimol can label the GABAA receptor site and that the ligand can be used as a photoaffinity label for purification and identification of GABA binding sites within the GABAA receptor complex.

Identification of a second glycine-like fragment on the strychnine molecule.

Strychnine is a complex molecule that inhibits the physiological actions of glycine, an important inhibitory neurotransmitter in the spinal cord, brain stem, and other areas of many vertebrates. Since 1987, we have employed atomistic molecular modeling tools to find an explanation at the molecular level for how this antagonism works. We have located a second glycine-like fragment in the strychnine molecule that, when compared to glycine in a three pair atom analysis, provides an excellent topological and electronic charge congruence. The topological congruence in the second glycine-like fragment is much better than with the first fragment reported in 1987 when using a truncated strychnine molecule in the quantum mechanical analysis. A fourth negative atom, a characteristic of antagonists which we reported earlier (Aprison and Lipkowitz: J Neurosci Res 30:442-446, 1991; Aprison and Lipkowitz: J Neurosci Res 31:166-174, 1992) was found in strychnine. This result follows the pattern reported recently for the three weak glycine antagonists N,N-dimethylmuscimol, N-methyl-THIP, and iso-THAO, a bicyclic 5-isoxazolol zwitterion.

Molecular pharmacology of gamma-aminobutyric acid type A receptor agonists and partial agonists in oocytes injected with different alpha, beta, and gamma receptor subunit combinations.

Using systematic combination of alpha 1, alpha 3, and alpha 5 with beta 1, beta 2, and beta 3, together with gamma 1, gamma 2, and gamma 3, we have investigated the contributions of the various alpha, beta, and gamma subunits to the pharmacology of gamma-aminobutyric acid (GABA)A agonists. We have characterized GABA, (RS)-dihydromuscimol, piperidine-4-sulfonic acid, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol with recombinant human GABAA receptors expressed in Xenopus oocytes. Our observations indicate that the alpha subunit is the major determinant of efficacy for partial GABAA agonists. When alpha 1 and alpha 3 or alpha 1 and alpha 5 are coexpressed, the alpha 1 subunit determines the maximum efficacy, whereas the affinity is determined by the entire combination of subunits. Thus, the results of the present study demonstrate that the pharmacology of GABAA agonists is dependent on the subunit composition of the GABAA receptor complex. Functional GABAA receptors containing two different alpha subunits show pharmacological profiles distinctly different from those of receptors containing a single alpha subtype, indicating that two different alpha subunits can be coexpressed in one functional GABAA receptor complex.

Condensation of muscimol or thiomuscimol with aminopyridazines yields GABA-A antagonists.

Ten analogs of muscimol and thiomuscimol in which the amino function was delocalized in an amidinic system were prepared by N2 alkylation of 6-aryl-3-aminopyridazines with (chloromethyl)isoxazole or (chloromethyl)isothiazole derivatives. These muscimol and thiomuscimol derivatives show potent binding properties for GABA-A receptors (they displace [3H]GABA and [3H]gabazine) and provoke convulsions after iv injections. They fit well with the model pharmacophore proposed by our group for the GABA-A antagonists and show similar structure-activity profiles to that of the pyridazinyl-GABAs.

Muscimol and N,N-dimethylmuscimol: from a GABA agonist to a glycine antagonist.

By using molecular modeling methods, a molecular mechanism was identified which can explain how the incorporation of two methyl groups in place of two hydrogen atoms on the terminal nitrogen atom of muscimol can not only convert this potent agonist at GABAnergic receptors to an inactive molecule at these receptors, but also can convert this new derivative to an antagonist of glycine at glycinergic receptors. This insight into the molecular mechanism operative in the conversion of physiological function provides a basis for understanding how a single molecule may be able to act at both the GABA- and glycine-inhibitory receptors.

Enantioselectivity at the physiologically active GABAA receptor.

A subfraction of cortical tissue from rat brain, containing membrane vesicles was prepared freshly with added protease inhibitors and antioxidant. The preparation was used to measure stimulation of transmembrane 36C1- flux and inhibition of bicuculline-sensitive [3H] muscimol binding by (+)-(S) and (-)-(R) enantiomers of dihydromuscimol at 30 degrees in physiological salt solution. Displacement of bound [3H]muscimol and stimulation of 36Cl- flux appeared in the 0.1-10 microM concentration range of the enantiomers, channel gating, however, required rather high concentrations. Degrees of enantioselectivity for channel gating, desensitization of and binding to GABAA receptors were estimated by the concentration ratios of dihydromuscimol enantiomers, [(-)-(R)]/[(+)-(S)], at the same level of response or displacement. Different enantioselectives were observed for channel gating (6 +/- 3), receptor binding (3 +/- 2) and desensitization (no selectivity). The low and concentration-dependent enantioselectives found for channel gating and receptor binding can be explained by desensitization and heterogeneity of GABAA receptors.

Inhibition of GABA uptake in the rat hippocampal slice.

Pharmacological manipulations known to inhibit GABA uptake prolonged GABA-evoked conductance increases in CA1 pyramidal cells in the rat hippocampal slice preparation. Treatments included reduction of extracellular sodium and exposure to cis-4-OH-nipecotic acid, nipecotic acid or L-2,4-diaminobutyric acid (all at 1 mM). These effects contrast with the results obtained with 4-OH-isonipecotic acid, an inactive structural analog of nipecotic acid, which had no effect on the time-course of GABA responses. 4,5,6,7-Tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THPO), an impotent but selective inhibitor of GABA uptake into glia, did not prolong GABA-evoked responses. The effect of sodium reduction depended on the distance between the source of GABA and its receptors, as predicted for an uptake-limited response. GABA-receptor agonists that are poor substrates for GABA uptake (muscimol, thiomuscimol, piperidine-4-sulphonic acid, isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP) evoked very long conductance changes that were not further prolonged by uptake inhibitors. These results demonstrate the presence of a functional GABA uptake system in the hippocampal slice. The accessibility of hippocampal GABAergic synapses and the known susceptibility of the hippocampus to epileptiform events suggest that the hippocampal slice could be a valuable CNS preparation to study the role of GABA uptake in synaptic physiology.

GABA agonists. Resolution, absolute stereochemistry, and enantioselectivity of (S)-(+)- and (R)-(-)-dihydromuscimol.

(RS)-5-(Aminomethyl)-2-isoxazolin-3-ol (dihydromuscimol, DHM) is a potent 4-aminobutyric acid (GABA) agonist, the inhibitory effects of which on neurons are sensitive to the antagonist bicuculline methochloride (BMC), and it also interacts with the GABA uptake system in vitro. (S)-(+)-DHM (4) and (R)-(-)-DHM (5) were obtained in optically pure forms via resolution of tert-butyloxycarbonyl-protected DHM (1) using cinchonidine as the only resolving agent. The optical purity and absolute stereochemistry of 4 and 5 were established by chemical correlation to the (S)-(+) enantiomer of 3-hydroxy-4-aminobutyric acid (GABOB). While 4 was a specific and potent BMC-sensitive GABA agonist in vivo and in vitro, possibly the most potent GABA agonist so far described, the inhibition of GABA uptake by DHM proved to reside exclusively in the (R)-(-) enantiomer (5). The affinity of 5 for BMC-sensitive GABA receptor sites in vitro was some 50 times lower than that of 4. Compounds 4 and 5 can be considered semirigid isosteres of the conformationally flexible GABA analogues (S)-(+)- and (R)-(-)-GABOB, respectively, which show a very low degree of enantioselectivity with respect to GABA synaptic mechanisms. This correlation between the degree of enantioselectivity and conformational mobility of chiral GABA analogues might be of importance for the design of new drugs with specific actions at synapses at which GABA is the transmitter.

Glycine antagonists structurally related to muscimol, THIP, or isoguvacine.

Microelectrophoretic methods were used to study the effects on cat spinal neurones of a number of compounds structurally related to the gamma-aminobutyric acid (GABA) agonists muscimol, THIP, and isoguvacine. While N-methylmuscimol was an agonist at bicuculline methochloride-sensitive GABA receptors, somewhat weaker than GABA and THIP, neither N,N-dimethylmuscimol nor N-methyl-THIP interfered significantly with GABA receptors in vivo or binding sites in vitro. Both N,N-dimethylmuscimol and N-methyl-THIP, however, reversibly antagonized the depressant action of glycine. The seven-membered ring analogues of THIP, namely THIA (5,6,7,8-tetrahydro-4H-isoxazolo[5,4-c]azepin-3-ol), THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol) and iso-THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol), also blocked neuronal inhibition by glycine, iso-THAZ being the most potent compound. The conformationally mobile isomer of THAZ and iso-THAZ, 3-PYOL (5-(3-pyrrolidinyl)-3-isoxazolol), was a much less selective glycine antagonist, being also an antagonist of GABA, 3,4-TAZA (2,5,6,7-tetrahydro-1H-azepine-4-carboxylic acid) and 4,5-TAZA (2,3,6,7-tetrahydro-1H-azepine-4-carboxylic acid), which are amino acid analogues of THIA and THAZ, respectively, and ring homologues of isoguvacine, were also shown to be glycine antagonists. The mechanism of action of the present class of zwitterionic glycine antagonists is unknown. The compounds are much less potent than strychnine.

GABA agonists. Development and interactions with the GABA receptor complex.

This review describes the development of GABA receptor agonists with no detectable affinity for other recognition sites in GABA-mediated synapses. The key compounds are THIP, isoguvacine, and piperidine-4-sulphonic acid (P4S), developed via extensive structural modifications of the potent but not strictly specific GABA agonist muscimol. The structural parameters, which have to be considered in the design of GABA agonists are discussed on the basis of the structures and biological activities of these GABA agonists and a number of related compounds. A model, which summarizes our present knowledge of the structure of the postsynaptic GABA receptors complex, is presented, and the interaction of GABA agonists with various sites in this complex is discussed. Of particular interest are the effects of GABA agonists on the binding of diazepam to the benzodiazepine binding site, assumed to be a structural unit of the GABA receptor complex. While rigid molecules like THIP are capable of activating the GABA receptors, a certain degree of conformational mobility of GABA agonists apparently is a prerequisite for stimulation of diazepam binding in vitro at 0 degree C. The findings suggest that GABA receptor functions involve conformational changes of certain elements, including the attempts to develop GABA agonists with desirable pharmacokinetic and toxicological characteristics. While muscimol is a toxic compound, THIP is well tolerated by animals, and in contrast to isoguvacine, THIP penetrates into the brain after systemic administration to animals, a difference which can be explained on the basis of their protolytic properties. The attempts to develop pro-drugs of isoguvacine capable of penetrating the blood-brain barrier with subsequent decomposition in the brain tissue to isoguvacine are described.

Inhibitors of the GABA uptake systems.

This review describes a novel class of heterocyclic GABA uptake inhibitor with no affinity for the GABA receptors. The parent compound nipecotic acid is a potent inhibitor of neuronal and glial GABA uptake, and nipecotic acid is a substrate for the transport carriers concerned. The structurally related cyclic amino acids guvacine and cis-4-hydroxynipecotic acid are also potent inhibitors of both GABA transport systems. Even minor structural alterations of these compounds result in considerable or complete loss of activity. Whereas homonipecotic acid is a weak but selective inhibitor of glial GABA uptake, homoguvacine is virtually inactive. Similarly the lower homologues of nipecotic acid and guvacine, beta-proline and 3-pyrroline-3-carboxylic acid, respectively, show some selectivity with respect to inhibition of glial GABA uptake, but these compounds are much weaker than the parent compounds. The bicyclic compounds THPO and THAO, in which the carboxyl groups of nipecotic acid and homonipecotic acid have been replaced by 3-isoxazolol units are moderately potent and practically specific inhibitors of glial GABA uptake. cis-4-Mercaptonipecotic acid is considerably weaker than the closely related analogue cis-4-hydroxynipecotic acid, but the former compound may interact irreversibly with the GABA transport carriers. The results demonstrate a pronounced substrate specificity of the glial and in particular the neuronal GABA transport system. It is evident that the GABA molecule is transported in a conformation different from that, in which it activates its receptors. These findings are of importance for the development of drugs for selective pharmacological regulation of the functions of central GABA-mediated synapses in certain neurological diseases.