During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimer's disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of γ-secretase (Aβ40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

The amyloid-␤ (A␤) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-␤ precursor protein (APP) through consecutive proteolytic cleavages by ␤-site APP-cleaving enzyme and ␥-secretase. Unexpectedly ␥-secretase inhibitors can increase the secretion of A␤ peptides under some circumstances. This "A␤ rise" phenomenon, the same inhibitor causing an increase in A␤ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the A␤ rise depends on the ␤-secretase-derived C-terminal fragment of APP (␤CTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of A␤, known as "p3," formed by ␣-secretase cleavage, did not exhibit a rise. In addition to the A␤ rise, low ␤CTF or C99 expression decreased ␥-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, ␥-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of A␤ under conditions of low substrate expression. The A␤ rise was also observed in rat brain after dosing with the ␥-secretase inhibitor BMS-299897. The A␤ rise and potency shift are therefore relevant factors in the development of ␥-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the A␤ rise, including the "incomplete processing" and endocytic models, are discussed.Evidence suggests that the amyloid-␤ (A␤) 9 peptide plays a key role in Alzheimer disease. A␤ is generated by proteolytic processing of the amyloid-␤ precursor protein (APP) through consecutive cleavages by the ␤-site APP-cleaving enzyme (BACE) and ␥-secretase. APP is cleaved by BACE to form a ␤-secretase-derived C-terminal fragment of APP (␤CTF), which undergoes further cleavage by ␥-secretase to form A␤. In addition, APP is cleaved by ␣-secretase to form ␣CTF, which undergoes ␥-secretase cleavage to produce an N-terminally truncated form of A␤ known as "p3." Using the conventional amino acid numbering of A␤, BACE cleavage leads to A␤ peptides with N-terminal ends at positions 1 and 11, whereas ␣-secretase leads to p3 peptides with an N-terminal end at position 17. Cleavage of ␤CTF and ␣CTF by ␥-secretase produces a mixture of different C termini in the resulting A␤ and p3 peptides. For example, the predominant ␥-secretase cleavage of ␤CTFs at position 40 produces A␤-(1-40) and A␤-(11-40), whereas other ␥-secretase cleavage sites produce a range of less abundant A␤ peptides, such as the disease-associated A␤-(1-42) (1, 2).

Pantothenic acid and ␤-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be ratelimiting for ␤-alanine and pantothenic acid biosynthesis. Overexpression of FMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required ␤-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 and YIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in ␤-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for ␤-alanine biosynthesis; spe1⌬ , spe2⌬ , spe3⌬ , and spe4⌬ are all ␤-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the ␤-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms.Pantothenic acid (vitamin B 5 ) is a metabolic precursor to coenzyme A (CoA) and acyl carrier protein, which are cofactors required by a large number of metabolic enzymes. Biosynthesis of pantothenic acid occurs in microbes and plants only, whereas animals obtain it in their diet. In bacteria, it is synthesized by the condensation of pantoate, derived from 2-oxoisovalerate, an intermediate in valine biosynthesis, and ␤-alanine, produced by the decarboxylation of L-aspartate (1, 2). In Escherichia coli, four genes, panB, panC, panD, and panE, encode the four enzymes required for pantothenic acid biosynthesis, as illustrated in Fig.

Thermosensitive mutations in the DBF2 gene arrest the cell cycle during nuclear division. Although the chromatin has divided in arrested cells, an elongated mitotic spindle is present and Cdc28 protein kinase activity remains high, indicating that nuclear division is incomplete. By execution point analysis we show that Dbf2 carries out an essential cell cycle function after the metaphase to anaphase transition and is therefore required during anaphase and/or telophase. This cell cycle stage‐specific requirement for the function of Dbf2 coincides with the cell cycle regulation of Dbf2/Dbf20 protein kinase activity, which can be detected in immunoprecipitates containing Dbf2 or Dbf20. The kinase activity is specific for serine/threonine residues and Dbf2 accounts for the bulk of the activity, with Dbf20 playing a minor role. Furthermore, Dbf2 is a phosphoprotein and, significantly, the dephosphorylated form appears with the same cell cycle timing as the kinase activity, suggesting a role for dephosphorylation in the activation mechanism. In addition, we show that the DBF2 transcript, which is under cell cycle control, is expressed in advance of the activation of the kinase, but that cell cycle‐regulated expression of the mRNA is not required for activation of the Dbf2 kinase during M phase. Thus, Dbf2/Dbf20 kinase activity is precisely regulated in the cell cycle by a post‐translational mechanism and phosphorylates its target substrates for an event that occurs during anaphase and/or telophase.