Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease.

The GABA A receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that ␣2-rather than ␣3-containing GABA A receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an ␣3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2Ј-difluoro-5Ј-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an ␣3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders ␣2-containing receptors BZ insensitive (␣2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of ␣3-containing GABA A receptors is sufficient to produce the anxiolytic effects of BZs and that ␣2 potentiation may not be necessary.

We present a comprehensive analysis of all ring systems (both heterocyclic and nonheterocyclic) in clinical trial compounds and FDA-approved drugs. We show 67% of small molecules in clinical trials comprise only ring systems found in marketed drugs, which mirrors previously published findings for newly approved drugs. We also show there are approximately 450 000 unique ring systems derived from 2.24 billion molecules currently available in synthesized chemical space, and molecules in clinical trials utilize only 0.1% of this available pool. Moreover, there are fewer ring systems in drugs compared with those in clinical trials, but this is balanced by the drug ring systems being reused more often. Furthermore, systematic changes of up to two atoms on existing drug and clinical trial ring systems give a set of 3902 future clinical trial ring systems, which are predicted to cover approximately 50% of the novel ring systems entering clinical trials.

7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo [4,3-b]pyridazine (TPA023) is a triazolopyridazine that binds with equivalent high (subnanomolar) affinity to the benzodiazepine binding site of recombinant human GABA A receptors containing an ␣1, ␣2, ␣3, or ␣5 subunit but has partial agonist efficacy at the ␣2 and ␣3 subtypes and essentially antagonist efficacy at the ␣1 and ␣5 subtypes. In rats, TPA023 gave time-and dose-dependent occupancy after oral dosing, with 50% occupancy corresponding to a dose of 0.42 mg/kg. It has anxiolytic-like activity in unconditioned (elevated plus maze) and conditioned (fear-potentiated startle and conditioned suppression of drinking) rat models of anxiety with minimum effective doses (MED; 1-3 mg/kg) corresponding to 70 to 88% occupancy. However, there was no appreciable sedation in a response sensitivity (chain-pulling) assay at a dose of 30 mg/kg, resulting in 99% occupancy. Similarly, TPA023 was robustly anxiolytic in the squirrel monkey conditioned emotional response assay, with a MED of 0.3 mg/kg, but did not produce any sedation in a lever-pressing test of sedation even at 10 mg/kg. TPA023 produced no impairment in performance in the mouse Rotarod assay, and there was only a mild interaction with ethanol. In addition to anxiolytic-like efficacy, TPA023 had anticonvulsant activity in a mouse pentylenetetrazole seizure model. Finally, TPA023 did not cause precipitated withdrawal in mice treated for 7 days with the nonselective agonist triazolam, nor did N-methyl-␤-carboline-3-carboxamide (FG 7142) precipitate withdrawal in mice treated for 7 days with TPA023. In summary, the novel ␣2/␣3-selective efficacy profile of TPA023 translates into a nonsedating anxiolytic profile that is distinct from nonselective agonists.GABA A receptors are ligand-gated chloride ionophores that mediate the fast synaptic as well as tonic extrasynaptic inhibitory effects of GABA (Mody and Pearce, 2004). They are pentameric assemblies of proteins (Cromer et al., 2002) derived from a family of 16 genes (␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , and ; Simon et al., 2004). Despite the theoretically large number of possible pentameric permutations that can arise from these 16 genes, only relatively few combinations are found within the central nervous system (McKernan and Whiting, 1996). Most of these native receptors contain ␣, ␤, and ␥ subunits in a 2:2:1 stoichiometry with an alternating ␣␤␣␤␥ arrangement as viewed from the synapse (Minier and Sigel, 2004).In addition to binding sites for their endogenous ligand, GABA A receptors also possess recognition sites for a number of pharmacologically active classes of compounds, including

t This approach had been previously used to prepare (E)-3-chloro-and (E)-3-bromo-2-methylprop-2-enoic acids. t9-BBN-OTf = 9-borabicycloC3.3. llnonyl trifluoromethanesulphonate, TBDMS-OTf = t-butyldimethylsilyl trifluoromethanesulphonate.

Intramembranous cleavage of the ␤-amyloid precursor protein by ␥-secretase is the final processing event generating amyloid-␤ peptides, which are thought to be causative agents for Alzheimer's disease. Missense mutations in the presenilin genes co-segregate with early-onset Alzheimer's disease, and, recently, a close biochemical linkage between presenilins and the identity of ␥-secretase has been established. Here we describe for the first time that certain potent ␥-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel ␥-secretase inhibitor, {1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl}-carbamic acid tertbutyl ester (CBAP), which not only physically interacts with PS1, but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments. This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. The subsequent use of CBAP as a biological tool to increase full-length PS1 levels in the absence of exogenous PS1 expression has provided evidence that wild-type PS1 endoproteolysis is not required either for PS1/␥-secretase complex assembly or trafficking. Furthermore, in cellbased systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the ␤-amyloid precursor protein cleavage and the S3 cleavage of the Notch receptor are inhibited by CBAP, an impairment of Trk receptor maturation was not observed.