A Combinatorial Approach to the Identification of Dipeptide Aldehyde Inhibitors of β-Amyloid Production

Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the ␥-secretase cleavage that generates the amyloid ␤ protein (A␤). It is not clear how PSs regulate ␥-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the ␥-secretase cleavage, ␥-secretase themselves, or regulators of intracellular trafficking that indirectly influence ␥-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit A␤ production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct ␥-secretases, we show that PS1 regulates multiple pharmacologically distinct ␥-secretase activities as well as inducible ␣-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be ␥-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.The 4-kDa amyloid ␤ protein (A␤) 1 deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid ␤ protein precursor (APP) (1-3). Generation of A␤ from APP requires two sequential proteolytic events: an initial cleavage at the amino terminus of the A␤ sequence referred to as ␤-secretase (4) and a subsequent cleavage at the carboxyl terminus known as ␥-secretase. Recently, a membrane-bound aspartic protease has been implicated as a ␤-secretase (5-8). However, the protease(s) responsible for ␥-secretase cleavage have not been identified. In addition, a third proteolytic activity referred to as ␣-secretase cleaves within the A␤ sequence to release a large secreted derivative (sAPP), thus precluding formation of full-length A␤. In mammalian cells, at least two members of the ADAM family (a disintegrin and metalloprotease) can contribute to the ␣-secretase activity (9, 10). Although full-length APP is not cleaved by ␥-secretase, APP carboxyl-terminal fragments (CTF) generated through cleavage of APP by either ␣-or ␤-secretase are both substrates for ␥-secretase, with cleavage of CTF␣ releasing a peptide referred to as p3 (A␤17-40 or A␤17-42) (1-3).␥-Secretase-catalyzed cleavages are of particular interest for a number of reasons. First, they are unusual in that the cleavage site of the substrate is predicted to lie within the transmembrane domain (TMD). Rather than primary amino acid sequence, position of the ␥-cleavage site with respect to the membrane appears to be the prime determinant of cleavage, with the length of the luminal TMD determining that position (11). Whether ␥-secretase actually cleaves residues within the membrane is a controversial topic. To date, there is no definitive evidence showing that any protease can cleave bonds when they are buried within a TMD. Second, altered ␥-secretase cleavage is implicated in the develo...

Section: ␥-40 and ␥-42 Secretase Activities Are Pharmacologically Andsupporting

confidence: 79%

Presenilin 1 Regulates Pharmacologically Distinct γ-Secretase Activities

Murphy

Uljon

Fraser

et al. 2000

“…This "A␤ rise" phenomenon, the same inhibitor causing an increase in A␤ at low concentrations but inhibition at higher concentrations, has been observed frequently (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). For peptide aldehyde inhibitors, some studies reported a rise that was specific for A␤42 (3)(4)(5)(6), whereas other studies reported a rise also for A␤40 in addition to A␤42 (7)(8)(9)(10). However, these studies also differed as to the pharmacological target proposed to mediate the effects on A␤; some authors considered only the protease calpain via an indirect effect on ␥-secretase (7,9,10), whereas others proposed a direct effect on ␥-secretase (3)(4)(5)(6)8).…”

mentioning

confidence: 72%

“…For peptide aldehyde inhibitors, some studies reported a rise that was specific for A␤42 (3)(4)(5)(6), whereas other studies reported a rise also for A␤40 in addition to A␤42 (7)(8)(9)(10). However, these studies also differed as to the pharmacological target proposed to mediate the effects on A␤; some authors considered only the protease calpain via an indirect effect on ␥-secretase (7,9,10), whereas others proposed a direct effect on ␥-secretase (3)(4)(5)(6)8). In one study, the A␤ rise was reported in isolated membrane preparations, suggesting a direct effect of peptide aldehydes on ␥-secretase (6).…”

mentioning

confidence: 99%

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

Burton

Meredith

Barten

et al. 2008

119

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).

“…Determination of A␤ Levels by Enzyme-linked Immunosorbent Assay-The ability of compounds to lower A␤ secretion was tested using Chinese hamster ovary cells overexpressing wild-type ␤ APP as described previously (20) and Roach et al 2 Briefly, confluent cells were incubated with a range of concentrations of compounds for 16 h in serum-free medium containing 0.2% bovine serum albumin. A␤ 1-40 in the resultant conditioned media was quantified by sandwich enzymelinked immunosorbent assay using a position 40 neoepitope-specific monoclonal antibody and a biotinylated monoclonal antibody directed to A␤ residues 10 -20.…”

Section: Methodsmentioning

confidence: 99%

Presenilin-1 and -2 Are Molecular Targets for γ-Secretase Inhibitors

Seiffert¹,

Bradley²,

Rominger³

et al. 2000

Self Cite

304

275

Presenilins are integral membrane protein involved in the production of amyloid ␤-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter ␥-secretase cleavage of the ␤-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A␤, the 4-kDa ␤-peptide. Here, we show that radiolabeled ␥-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A␤ formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N-and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of ␥-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective ␥-secretase inhibitors block A␤ formation by binding to presenilin-1 and -2.␤-Amyloid precursor protein (␤APP) 1 is a transmembrane protein that undergoes processing to A␤ by proteolytic activities known as ␤-and ␥-secretases (for review, see Refs. 1-3). The ␤-secretase cleavage occurs in the extracellular domain by a recently identified aspartyl protease variously termed BACE, memapsin, and Asp2 (4 -9), whereas the heterogeneous ␥-secretase cleavage occurs in the transmembrane domain (2, 10). Dominant mutations in either of the two human presenilin (PS-1 and PS-2) genes lead to familial Alzheimer's disease (AD). PS-1 and -2 are polytopic membrane proteins (for review, see Refs. 11-13). Presenilins are proteolytic processed. In vivo, only small amounts of the holoprotein can be detected, primarily in the nuclear envelope, whereas 30-kDa N-terminal and 20-kDa C-terminal fragments of presenilin are observed in all mammalian tissues and cell lines analyzed so far. Coimmunoprecipitation experiments revealed that presenilin fragments are assembled into a high molecular weight complex together with other proteins (for review see 11-13). The proposed mechanism through which the presenilin mutations cause AD is an alteration in the predominant ␥-secretase cleavage site which increases the amount of the longer, more amyloidogenic A␤ 1-42(43) fragments produced (11-13). A null mutation of the mouse PS-1 selectively reduces ␥-secretase activity (14), and site-directed mutagenesis of PS-1 and PS-2 at two conserved aspartyl residues, which resemble the catalytic center of aspartyl proteases, also reduces ␥-secretase activity (15, 16). These observations indicate that PS-1 and PS-2 either stimulate the activity of ␥-secretase by trafficking to appropriate cellular compartments, serve as cofactors of the ␥-secretase, or are ␥-secretase themselves.Here, we report that a series of potent and selective ␥-secretase inhibitors bind to mam...