Cerebral deposition of amyloid ␤-protein (A␤) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A␤ is produced from the processing of amyloid ␤-protein precursor (APP) by ␤-and ␥-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike ␤-secretase, which is a monomeric aspartyl protease, ␥-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex. Pepstatin and L685458 are among several structural classes of ␥-secretase inhibitors identified so far. These compounds possess a hydroxyethylene dipeptide isostere of aspartyl protease transition state analogs, suggesting ␥-secretase may be an aspartyl protease. However, the mechanism of inhibition of ␥-secretase by pepstatin and L685458 has not been elucidated. In this study, we report that pepstatin A methylester and L685458 unexpectedly displayed linear non-competitive inhibition of ␥-secretase. Sulfonamides and benzodiazepines, which do not resemble transition state analogs of aspartyl proteases, also displayed potent, non-competitive inhibition of ␥-secretase. Models to rationalize how transition state analogs inhibit their targets by non-competitive inhibition are discussed. Accumulation and deposition of ␤-amyloid (A␤)1 peptides in the cerebral cortex is believed to be an early and central process in the pathogenesis of Alzheimer's disease. The A␤ peptides are generated from sequential proteolytic cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases, which are therefore considered important targets for therapeutic intervention. Molecular cloning (1-3) and crystallographic studies (4) have unequivocally established ␤-secretase as an aspartyl protease. However, the identity of ␥-secretase remains elusive.It is known that transmembrane proteins presenilin 1 (PS1) and presenilin 2 (PS2) are essential for intramembranous proteolytic ␥-cleavage of APP (5) and a few other ␥-secretase substrates such as Notch (6 -9) and ErbB4 (10). Evidence suggests that presenilins may have direct catalytic activity (11, 12), but recent reports indicate that this activity requires interactions between presenilins and other proteins such as nicastrin (13) and co-fractionates with a very high molecular weight complex (14). Mature presenilins themselves form subunit heterodimers between the N-and C-terminal fragments, which are generated from endoproteolytic cleavage of the full-length presenilin (15, 16). This complex membrane-bound molecular organization has hindered efforts to purify and reconstitute ␥-secretase activity.In the absence of purified enzyme and crystal structures, inhibition studies have played a prominent role in the understanding of the nature of ␥-secretase. ␥-secretase activity is sensitive to aspartyl protease transition state analogs such as the hydroxyl ethylene isosteres, pepstatin (17-19) and L685458 (20), typical aspartyl protease transition state inhibitors. Peptidomimetics containing a dif...

Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.

The preclinical pharmacology and pharmacokinetic properties of (2R)-6-methoxy-8-(4-methylpiperazin-1-yl)-N-(4-morpholin-4-ylphenyl)chromane-2-carboxamide (AZD3783), a potent 5-hydroxytryptamine 1B (5-HT 1B ) receptor antagonist, were characterized as part of translational pharmacokinetic/pharmacodynamic hypothesis testing in human clinical trials. The affinity of AZD3783 to the 5-HT 1B receptor was measured in vitro by using membrane preparations containing recombinant human or guinea pig 5-HT 1B receptors and in native guinea pig brain tissue. In vivo antagonist potency of AZD3783 for the 5HT 1B receptor was investigated by measuring the blockade of 5-HT 1B agonist-induced guinea pig hypothermia. The anxiolytic-like potency was assessed using the suppression of separation-induced vocalization in guinea pig pups. The affinity of AZD3783 for human and guinea pig 5-HT 1B receptor (K i , 12.5 and 11.1 nM, respectively) was similar to unbound plasma EC 50 values for guinea pig receptor occupancy (11 nM) and reduction of agonist-induced hypothermia (18 nM) in guinea pig. Active doses of AZD3783 in the hypothermia assay were similar to doses that reduced separation-induced vocalization in guinea pig pups. AZD3783 demonstrated favorable pharmacokinetic properties. The predicted pharmacokinetic parameters (total plasma clearance, 6.5 ml/min/kg; steady-state volume of distribution, 6.4 l/kg) were within 2-fold of the values observed in healthy male volunteers after a single 20-mg oral dose. This investigation presents a direct link between AZD3783 in vitro affinity and in vivo receptor occupancy to preclinical disease model efficacy. Together with predicted human pharmacokinetic properties, we have provided a model for the quantitative translational pharmacology of AZD3783 that increases confidence in the optimal human receptor occupancy required for antidepressant and anxiolytic effects in patients.

We describe herein the discovery of novel, de novo designed, 5-HT(1B) receptor antagonists that lack a basic moiety and that provide improved hERG and in vitro phospholipidosis profiles. We used a known 5-HT(1B) antagonist template as our starting point and focused on replacing the piperazine moiety. Pyrazole-based ideas were designed and synthesized among a small library of piperazine replacements. To our knowledge, these are the first potent, nonbasic, functionally active antagonists of the 5-HT(1B) receptor.

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ã-Secretase has been shown to be inhibited by the aspartyl protease transition state analogs pepstatin and L685458. Inhibition by these peptidic hydroxyethylenes has led to the suggestion that ã-secretase is an aspartyl protease. However, the mechanism of inhibition has not been elucidated. Here we report that pepstatin A methylester (PME) and L685458 unexpectedly display linear non-competitive inhibition of ã-secretase. Certain sulfonamides (see Fig. 1, 1 and 2) and benzodiazepines (3 and 4), which do not resemble transition state analogs of aspartyl proteases, also display potent non-competitive inhibition of ã-secretase. Kinetic results from cross-competition between various pairs of these compounds suggest that these inhibitors may bind to two different binding sites on ã-secretase, one site for transition state isosteres and the other for non-transition state inhibitors. Moreover, these results suggest that ã-secretase, if truly an aspartyl protease, demonstrates unique inhibition kinetics for this mechanistic class of proteases.

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