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In pursuit of a GABA(A) alpha5-subtype-selective inverse agonist to enhance cognition, a series of 6,7-dihydro-2-benzothiophen-4(5H)-ones has been identified as a novel class of GABA(A) receptor ligands. These thiophenes have higher binding affinity for the GABA(A) alpha5 receptor subtype compared to the GABA(A) alpha1, alpha2, and alpha3 subtypes, and several analogues exhibit high GABA(A) alpha5 receptor inverse agonism. 6,6-Dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one (43) has been identified as a full inverse agonist at the GABA(A) alpha5 receptor and is functionally selective over the other major GABA(A) receptor subtypes. 43 readily penetrates into the CNS to give selective occupancy of GABA(A) alpha5 receptors. In addition, 43 enhances cognitive performance in rats in the delayed 'matching-to-place' Morris water maze test-a hippocampal-dependent memory task-without the convulsant or proconvulsant activity associated with nonselective, GABA(A) receptor inverse agonists.

The structures of ternary complexes of human inositol monophosphatase with inhibitory Gd3+ and either D- or L-myo-inositol 1-phosphate have been determined to 2.2-2.3 A resolution using X-ray crystallography. Substrate and metal are bound identically in each active site of the phosphatase dimer. The substrate is present at full occupancy, while the metal is present at only 35% occupancy, suggesting that Li+ from the crystallization solvent partially replaces Gd3+ upon substrate binding. The phosphate groups of both substrates interact with the phosphatase in the same manner with one phosphate oxygen bound to the octahedrally coordinated active site metal and another oxygen forming hydrogen bonds with the amide groups of residues 94 and 95. The active site orientations of the inositol rings of D- and L-myo-inositol 1-phosphate differ by rotation of nearly 60 degrees about the phosphate ester bond. Each substrate utilizes the same key residues (Asp 93, Ala 196, Glu 213, and Asp 220) to form the same number of hydrogen bonds with the enzyme. Mutagenesis experiments confirm the interaction of Glu 213 with the inositol ring and suggest that interactions with Ser 165 may develop during the transition state. The structural data suggest that the active site nucleophile is a metal-bound water that is activated by interaction with Glu 70 and Thr 95. Expulsion of the ester oxygen appears to be promoted by three aspartate residues acting together (90, 93, and 220), either to donate a proton to the leaving group or to form another metal binding site from which a second Mg2+ coordinates the leaving group during the transition state.

myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolase, EC 3.1.3.25) is an attractive target for mechanistic Investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of Its Ihibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human inositol monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence ofMn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabiing the leaving inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.In the phosphatidylinositol second-messenger pathway, receptor-activated phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate, to form two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca2+ and protein kinase C-mediated signal transduction, respectively (1-3). Inositol 1,4,5-trisphosphate is subsequently metabolized in a series of pathways, regenerating inositol for reincorporation into inositol phospholipids, thereby maintaining this signaling cycle. myo-Inositol monophosphatase (IMPase; myo-l-phosphate phosphohydrolase, EC 3.1.3.25) catalyzes the last hydrolytic step in these pathways, regenerating myo-D-inositol from myo-D-inositol 1-phosphate [Ins(l)P], Ins(3)P, or Ins(4)P (4).IMPase, a homodimer of two 30-kDa subunits, requires Li+ parameters gave contacts in agreement with those seen in the Cambridge Crystallographic Database (13), but to achieve similarly satisfactory results the van der Waals radius parameter of Gd3+ was reduced to 2.1 A. Hydrogens were added to the protein by using the Sybyl BIOPOLY ADDH command. The standard Sybyl TIP3P water model was used for crystallographically observed water molecules. Charges were taken from the standard Kollman parameter set for water molecules and protein residues. Starting coordinates for dianionic Ins(l)P were obtained from the crystal structure of the IMPase/Gd/Ins(l)P complex, supplemented with hydrogens at idealized sites on the inositol moiet...

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.

Since lithium inhibits IMPase and modulates phosphatidylinositol (PtdIns) cell signalling at therapeutically relevant concentrations (0.5-1.0 mM), IMPase has attracted attention as a putative molecular target for lithium in the treatment of manic depression. IMPase is a homodimer, with each subunit organised in an a/]a/]ot arrangement of a-helices and /]-sheets, and this type of structure seems crucial to the two-metal catalysed mechanism in which an activated water molecule serves as a nucleophile. Lithium appears to inhibit the enzyme following substrate hydrolysis by occupying the second metal binding site before the phosphate group can dissociate from its interaction with the site 1 metal. The understanding of IMPase structure and the mechanism of substrate hydrolysis and lithium inhibition should be useful in the development of novel inhibitors which may prove clinically useful in the treatment of manic depression.Key words: Inositol monophosphatase; Lithium; Enzyme inhibitor; Enzyme structure; Enzyme mechanism ium on signal transduction mechanisms, the modulation of the phosphatidylinositol (PtdIns)-linked cell signalling pathway ( Fig. 1) has attracted most attention, primarily due to the fact that most of the effects of lithium on this pathway occur at therapeutically relevant concentrations of lithium (0.5-1.0 raM). More specifically, it is proposed that lithium inhibits inositol monophosphatase (IMPase; a crucial enzyme in the recycling of inositol from the inositol polyphosphate second messengers ( Fig. 1)), thereby causing a depletion ofinositol and consequently a reduced synthesis of PtdIns. This in turn ultimately results in an attenuation of the production of intracellular second messengers in response to extracellular stimuli [6]. Consequently, IMPase is an attractive therapeutic target and accordingly has been subjected to detailed structural and mechanistic analysis which will be reviewed in the present article. Sequence of inositol monophosphatase Inositol monophosphatase: Putative therapeutic target for lithiumAlthough lithium (generally administered as lithium carbonate) is a very effective treatment for manic depression, it nevertheless has appreciable side effects and a narrow range between therapeutic (0.5-1.0 raM) and toxic (>2 raM) plasma lithium concentrations [1]. Consequently, plasma lithium concentrations have to be monitored in patients receiving lithium therapy. Given these limitations, it is possible that compounds which mimic the mechanism of action of lithium might represent novel treatments for manic depression that are devoid of the side effect and toxicity profile which may be due to millimolar concentrations of lithium interfering non-specifically with a number of key physiological functions (e.g. electrolyte homeostasis, ion transport, etc.).In order to try and develop compounds which mimic the therapeutic actions of lithium, it is first necessary to establish what the mechanism of action of lithium actually is. Unfortunately, although the use of lithium in the tr...

Since inhibition of myo-inositol monophosphatase (EC 3.1 3.25) by lithium ions and the resulting attenuation of phosphatidylinositol cycle activity may be the mechanism by which lithium exerts its therapeutic effect in the treatment of manic depression, it is of great interest to understand the mechanism of the enzyme and how lithium and other metals interact with it. Divalent magnesium is essential for enzyme activity, whereas Li' and high concentrations of Mg" act as unconipetitive inhibitors with respect to substrate. From the recently solved crystal structure of the human enzyme, several amino acid residues in the active site were targeted for mutagenesis studies. Nine singleresidue substituted mutants were characterized with regard to catalytic parameters. Mg" dependence, and Li' inhibition. In addition, a terbium fluorescence assay was developed to determine the metal binding properties of the wild-type and mutant enzymcs. Although nonc of these mutations affected K, for substrate subs~antially, the mutations Glu70+Gln, Glu70+Asp, A s p 9 0 j A s n and Thr95+Ala, in which residues within coordinating distance of the active site metal were modified, all resulted in large reductions in catalytic activity. The position of Glu70 in the crystal structure further suggests that this residue may be involved in activating water for nucleophilic attack on the substrate. The mutations Lys36+Ile, Asp90+Asn, Thr95+Ala, Thr95+Ser, His217+Gln, and Cys2 18+Ala all resulted in parallel reductions in both lithium and magnesium affinity, suggesting that Li' and Mg' ' share a common binding site.Although lithium ion has been used as a treatment for manic depression for over 40 years (Cadc, 1949). the molecular basis for its thcrapeutic effects remains uncertain. Evidence in reccnt ycars (Hallcher and Sherman, 1980: Nahorski et al., 1992; Atack ct al., 1992) has shown that Li' interferes with thc dcphosphorylation of inositol monophosphatcs in the phosphatidylinositol (PtdIns) secondary inessenger pathway (Berridge and Irvine, 1989;Berridge et al., 1989). While a direct link between lithium's cfrccts on the PtdIns cyclc and on manic depression remains to be proved, this ubiquitous intracellular cycle is linked to multiple signal transduction pathways (Berridge and Trvine, 1989; Fisher el al., 1992) and it is no1 surprising that interference with it should have profound effects.In the PtdIns cycle, receptor-activated phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate, to form two second messengers, diacylglycerol and inositol 1,4,5-Currvspondrnce t o S . .I. Pollack. Merck Sharp

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