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J. Biol. Chem., Vol. 283, Issue 5, 2927-2938, February 1, 2008

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Identification of -Secretase Inhibitor Potency Determinants on Presenilin^*

From the Elan Pharmaceuticals Inc., South San Francisco, California 94080

Received for publication, October 29, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Production of amyloid β peptides (Aβ), followed by their deposition in the brain as amyloid plaques, contributes to the hallmark pathology of Alzheimer disease. The enzymes responsible for production of Aβ, BACE1 and -secretase, are therapeutic targets for treatment of Alzheimer disease. Two presenilin (PS) homologues, referred to as PS1 and PS2, comprise the catalytic core of -secretase. In comparing presenilin selectivity of several classes of -secretase inhibitors, we observed that sulfonamides in general tend to be more selective for inhibition of PS1-comprising -secretase, as exemplified by ELN318463 and BMS299897. We employed a combination of chimeric constructs and point mutants to identify structural determinants for PS1-selective inhibition by ELN318463. Our studies identified amino acid residues Leu¹⁷², Thr²⁸¹, and Leu²⁸² in PS1 as necessary for PS1-selective inhibition by ELN318463. These residues also contributed in part to the PS1-selective inhibition by BMS299897. Alanine scanning mutagenesis of areas flanking Leu¹⁷², Thr²⁸¹, and Leu²⁸² identified additional amino acids that affect inhibitor potency of not only these sulfonamides but also nonsulfonamide inhibitors, without affecting Aβ production and presenilin endoproteolysis. Interestingly, many of these same residues have been identified previously to be important for -secretase function. These findings implicate TM3 and a second region near the carboxyl terminus of PS1 aminoterminal fragment in mediating the activity of -secretase inhibitors. Our observations demonstrate that PS-selective inhibitors of -secretase are feasible, and such inhibitors may allow differential inhibition of Aβ peptide production and Notch signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The most common cause of dementia in the elderly is Alzheimer disease (AD),³ which bears pathological hallmarks of amyloid plaques and neurofibrillary tangles. Extensive genetic, biochemical, molecular, and cellular studies over the last 2 decades have implicated the production and deposition of Aβ peptides as a critical initiating event leading to the pathogenesis of AD (reviewed in Ref. 1). Aβ peptides are produced by the action of two enzymes, β-secretase and -secretase. The -secretase enzyme is a large molecular complex composed of four principal subunits, the presenilins (PS1 or PS2), nicastrin, Aph-1, and Pen-2 (2-6). -Secretase functions as an intramembrane-cleaving aspartyl protease (reviewed in Refs. 7-9).

In addition to processing of β-amyloid precursor protein (APP) and Aβ peptide production, -secretase is also involved in the processing of many additional Type I transmembrane proteins, most notably Notch (reviewed in Refs. 9 and 10). -Secretase cleavage of Notch leads to nuclear translocation of Notch intracellular domain and expression of downstream genes, a process referred to as Notch signaling. Notch signaling is critical for cellular differentiation during development, as well as tissue homeostasis in adults. Pharmacological inhibition of Notch signaling by nonselective -secretase inhibitors in adult mice has been demonstrated to affect tissue differentiation and cell homeostasis in multiple systems (e.g. gastrointestinal tract, pancreas, skin, and lymphocytes) (11-14), consistent with predictions from KO mouse models (15-22). Inhibition of Notch signaling by nonselective -secretase inhibitors is a potentially limiting issue for the clinical development of this class of therapeutics. Hence, a major objective of inhibitors targeting -secretase for treatment of AD is the development of selective inhibitors that can reduce Aβ peptide production, without significantly interfering with the processing of other substrates of -secretase, principally Notch.

Two presenilin homologues, referred to as PS1 and PS2, comprise the catalytic core of -secretase. Observations from KO mice show that Presenilin 1 containing -secretase contributes to 80% of total Aβ production in brain, whereas PS2 containing -secretase contributes to 20% of total Aβ (16, 17, 23, 24). Furthermore, the observation that PS1 KO mice exhibit perinatal lethality, whereas PS2 KO mice are viable suggests that PS2-selective inhibitors may spare Notch signaling while lowering Aβ appreciably to be viable therapeutics (16, 24-27).

We employed transformed fibroblasts from presenilin double-KO mice transiently transfected with Swedish APP751 (APPsw) + PS1 or APPsw + PS2 to identify PS-selective inhibitors. Among the different classes of inhibitors we tested, we noted that although most were equipotent for inhibition of Aβ production from PS1 or PS2 -secretase, sulfonamides exemplified by ELN318463 (this report) and BMS299897 (28-30) displayed selectivity for inhibition of PS1 -secretase. We exploited this observation to map determinants for PS1 selectivity of these sulfonamides using chimeric constructs and point mutants. The results of our studies identify 3 residues as necessary and sufficient for the observed PS-1 selectivity of sulfonamide ELN318463: Leu¹⁷², Thr²⁸¹, and Leu²⁸² in PS1. These residues also contribute in part to the observed selectivity of BMS299897. We performed alanine scanning mutagenesis in regions flanking these residues to identify additional residues in PS1 that affect the inhibitor potency of the two sulfonamides as well as that of DAPT and L-685,458. Our findings provide insights into the possible binding sites of these inhibitors and, furthermore, demonstrate that selective targeting of presenilins is a feasible strategy for inhibiting -secretase in an isoform-specific manner for treatment of AD.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemical Synthesis—Synthesis of (R)-3-(4-bromobenzylamino)azepan-2-one. D--Amino--caprolactam (63) (0.04 M) in methanol (100 ml) was treated with 4-bromobenzaldehyde (0.046 m), and the resulting solution was stirred at room temperature for 24 h. Polymer-supported borohydride (40 g; Aldrich on Amberlite IRA-400) was added in one portion, and a slight exotherm and some bubbling was observed. The mixture was stirred at room temperature for 20 h, and the resin was then filtered off and thoroughly washed with methanol. The combined filtrates were evaporated to dryness. The residue was dissolved in ethyl acetate, and the organic phase was washed with water, saturated aqueous sodium bicarbonate, water, and brine. The organic phase was then dried with sodium sulfate. Solids were filtered off, filtrate was evaporated, and residue was recrystallized from hot ethyl acetate (20 ml) and hexane (150 ml). The crystalline product was filtered off, washed briefly with cold ethyl acetate/hexane (1:1) mixture, and air-dried to yield 7.8 g of (R)-3-(4-bromobenzylamino)azepan-2-one having m/z = 299.0.

Synthesis of (R)-N-(4-bromobenzyl)-4-chloro-N-(2-oxoazepan-3-yl) benzenesulfonamide (ELN318463) and BMS299897—4-Chlorobenzenesulfonyl chloride (11.24 mmol, 2.37 g) was added in one portion to a 0 °C methylene chloride (40 ml) solution of (R)-3-(4-bromobenzylamino)-azepan-2-one (7.49 mmol, 2.23 g) and N,N-diisopropylethylamine (60 mmol, 8 ml). The reaction was warmed to room temperature, stirring overnight. Solvent was removed, and residue was chromatographed with silica gel eluting with 1:1 hexane in ethyl acetate to obtain 2.61 g of (R)-N-(4-bromobenzyl)-4-chloro-N-(2-oxoazepan-3-yl)benzenesulfonamide having m/z = 473.0. BMS299897 was synthesized as described by Smith et al. (30).

Molecular Cloning and Construction of Chimeras—Human PS1, PS2, and APPsw cDNA inserts were subcloned into pCF vector, which was modified from pcDNA3 (Invitrogen) by inserting the adenoviral tripartite leader sequence (31) 38 bp upstream of the starting ATG codon, between the cytomegalovirus promoter and the EcoRI site. Presenilin chimeras were constructed by blunt end ligation of PCR-amplified fragments (Pfu Turbo DNA polymerase kit; Stratagene), and sequence was verified prior to transfection into transformed fibroblasts derived from PS1/PS2 double knock-out mice. Alanine scanning mutagenesis of the C2 and C5 subregions was carried out with mutagenic primers and the QuikChange site-directed mutagenesis kit (Stratagene).

Cell Culture and Transfection—Mouse fibroblasts derived from the PS1^-/-/PS2^-/- double knock-out cells (dKO cells; gift from Dr. Bart De Strooper) (23) were grown at 37 °C under 10% CO₂ in Dulbecco's modified Eagle's medium containing 2-10% fetal bovine serum and 100 µg/ml penicillin/streptomycin (Invitrogen), and 2 µML-glutamine (Invitrogen). Transfections were performed using Nucleofector II (Amaxa GmbH, Germany) with about 1-10 million of PS1^-/-PS2^-/- cells, using transfection reagents supplied by the manufacturer. An RPMI-washed cell pellet was resuspended in 100 µl of Solution R. To this cell suspension, 1-2 µg of a DNA mixture composed of APPsw, (32), and PS1 (2) or PS2 (33) was added, and the cell-DNA mixture was electroporated immediately (program T-20). Electroporated cells were first suspended into 1 ml of warm RPMI and then transferred (within 2-5 min after the addition of RPMI) into 5-10 ml of Dulbecco's modified Eagle's medium with 10% fetal bovine serum, for plating into 96-well plates. For experiments using PS2, cells were plated at a density of 50,000/well in 96-well plates; for experiments involving PS1, cells were plated at a density of 15,000/well in 96-well plates. The cells were treated overnight (14-20 h) with different concentrations of -secretase inhibitors 1-3 h following plating. During the initial phases of this work, transfection efficiencies were monitored using green fluorescent protein reporter plasmid (90%) in conjunction with Aβ production from parallel transfections. The levels of Aβ produced were normalized for APP expression levels as well as PS expression levels, using Western blot analysis (see Figs. S1 and 9). Since the level of Aβ produced was highly consistent and reproducible across experiments with Nucleofector-mediated transfection under the experimental conditions described above (as illustrated in Fig. 8), in subsequent experiments with point mutant PS constructs shown in Figs. 5 and 6 and Tables 1 and 2, Aβ production from transfected cultures was used as a surrogate for transfection efficiency.

ELISAs were performed at room temperature using 50 µl of diluted or original conditioned medium from overnight culture of transfected cells, treated with -secretase inhibitors or Me₂SO control. Following a 1-h incubation to capture Aβ peptide, the plates were incubated sequentially (45 min/incubation) with biotinylated detecting antibody (0.5 mg/ml), followed by horseradish peroxidase-strepavidin and horseradish peroxidase substrate (one-step slow TMB-Elisa; Pierce; catalogue number 34024). Plates were washed between incubations with Tris-buffered saline plus 0.05% Tween 20. Substrate reactions were terminated with 2 NH₂SO₄, for absorbance readings with a SpectraMax Plus (Molecular Devices, Sunnyvale, CA).

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Sulfonamide inhibitors of -secretase show differential inhibition of PS1- and PS2-comprised -secretase. Inhibitor potencies were assessed in PS1-/-PS2-/- cells transfected with human PS1 + APPsw or PS2 + APPsw. Cells were treated with a range of inhibitor concentrations, and dose response of inhibition was determined by ELISA of Aβ in conditioned media.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We examined the inhibition potency of -secretase inhibitors against PS1- or PS2-comprised -secretase using transformed primary embryonic fibroblasts derived from PS1^-/- PS2^-/- knock-out mice (23) transfected with PS1 or PS2 expression constructs to reconstitute homogenous enzyme complexes in these cells, without the complication of endogenous presenilins. This experimental system allowed us to unambiguously compare functional properties of reconstituted wild type or mutant PS1 or PS2 -secretases. In each experiment, expression constructs for either human PS1 or PS2, together with APPsw, were transiently transfected into PS1^-/- PS2^-/- dKO cells. The transfected cells were then incubated with different concentrations of inhibitors overnight. Total Aβ levels in conditioned medium from transfected cells were then measured by ELISA.

Fig. 1 illustrates that among the classes of -secretase inhibitors we tested, sulfonamides show preferential inhibition of PS1--secretase, whereas nonsulfonamide inhibitors only have modest selectivity for PS1-versus PS2--secretase. The dose-response curves and EC₅₀ values from a representative experiment are shown in Fig. 1. The mean values from two independent experiments on PS1/PS2 selectivity of the inhibitors are shown in Fig. 3. ELN318463 is 51-fold more selective for PS1, and BMS299897 is 35-fold more selective for PS1, whereas L-685,458 is only 3-fold more selective for PS1, and DAPT is actually 2-fold more selective for PS2. Additional sulfonamide inhibitors of the type represented by ELN318463 also displayed preferential PS1 selectivity (data not shown).

The observation of the differential inhibition of PS1 versus PS2, mainly by the sulfonamide series of inhibitors, prompted us to examine the structural basis for this differential inhibition. We employed chimeric PS1/PS2 molecules (illustrated in Fig. 2) to map the domain(s) in PS1 responsible for differences in inhibitor potencies. Evaluation of an initial set of chimeric presenilin molecules revealed that the middle third of PS1 (residues 128-298) is both necessary and sufficient for its high potency inhibition by ELN318463 and BMS299897 (Fig. 3). For both ELN318463 and BMS299897, the EC₅₀ values of PS1/2B are similar to that of PS1, whereas EC₅₀ values of PS1/2A and PS1/2C are similar to those of PS2. More telling, inhibitor potencies against PS2/1C behaved just like PS1, in terms of its inhibition by ELN318463 and BMS299897, despite the fact that the majority of this construct is composed of PS2 sequence. As before (Fig. 1), nonsulfonamide inhibitors, such as DAPT and L-685,458, did not display >3-fold selectivity for PS1 nor PS2, and the chimeras did not reveal any consistent basis for this low level of selectivity.

We examined the region bounded by PS1 residues 128-298 in more detail by studying inhibitor sensitivity of additional "small fragment chimeras" (PS1/2C1, PS1/2C2, PS1/2C3, PS1/2C4, and PS1/2C5) (Fig. 2) for the two sulfonamide inhibitors. Expression, Aβ production, and endoproteolytic processing of the chimeric PS molecules are indistinguishable from the wild type counterpart (supplemental Fig. S1, B and C). The nonsulfonamide inhibitors were not examined further due to their lack of appreciable PS selectivity (Fig. 3). As shown in Fig. 4, it is clear that for ELN318463 and BMS-299897, only the C2 (containing PS2 residues 171-200 substituted for PS1 residues 165-194) and C5 subregion (containing PS2 residues 275-304 substituted for PS1 residues 269-298) chimeras reported inhibitor EC₅₀ values appreciably different from that of parental PS1. Thus, the C2 and C5 subregions largely account for the inhibitor selectivity between PS1 and PS2. The PS2 C2 region in the context of PS1 reduces the potency of Elan G 3-fold relative to wild type PS1, whereas the PS2 C5 region in the context of PS1 reduces the potency of Elan G 18-fold relative to PS1. It is noteworthy that the product of the -fold selectivity for ELN318463 reported by the C2 and C5 subregions (54-fold) is in close agreement with the average PS1 selectivity of ELN318463 from the experiments reported in Figs. 1, 3, and 4 (39-, 51-, and 65-fold, respectively).

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Schematic illustration of PS1/PS2 chimeras and sequence junctions of constructs used in these studies. PS1 contains 467 amino acid residues, and PS2 contains 448. Numbers in the table indicate beginning and end positions of each chimeric fragment in source human PS1 or PS2 sequences.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Mapping selectivity determinants for differential inhibition potency on presenilin using chimeric constructs. EC50 values (nM, ±S.E., n = 2 per compound) for inhibition potencies of different compounds are shown in the table. The results map selectivity determinants to the middle third of the PS1 molecule between residues 128 and 298.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. PS1 C2 and C5 subregions in the middle third of presenilin-1 are dominant contributors of selective inhibition of -secretase by sulfonamides. Potencies of compounds are from n = 2 independent experiments, all constructs were assayed in triplicate in each experiment, and values are reported as mean EC50 ± S.E.).

Since the C5 subregion (encompassing the C-terminal segment of presenilin NTF) makes a greater contribution for PS1/PS2 selectivity of ELN318463 (Fig. 4), we tested the five nonconserved residues within the C5 subregion (Fig. 5A) first for their contribution to the observed selectivity of the sulfonamides. We mutated and assayed five conversion mutant constructs in the context of PS1/2C2 (Fig. 5, B and C). For convenience, PS1/2C2 was used as the reference construct in place of PS1. The results obtained using PS1/2C2 as a reference are validated by the triple mutation constructs described below as well as by the alanine scanning of the C2 region (see "Discussion").

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Fine mapping selectivity determinants in the C5 subregion. A, amino acid sequence alignment in the C5 subregion between PS1 and PS2. The C5 subregion contains half of the loop connecting TM6 and the conserved segment following TM6 in PS1 or PS2. The amino acid residues differing between PS1 and PS2 are boxed and highlighted in red boldface type. B, diagram of parental construct PS1/2C2. The five arrows above the C5 subregion illustrate the 5 amino acid residues different between PS1 and PS2. Each of the five positions was mutated in the chimera PS1/2C2 backbone to convert a PS1 amino acid residue into a PS2 amino acid residue. C, EC50 values determined from cells individually transfected with the control construct PS1/2C2 or one of the five point mutants and treated with ELN318463 or BMS299897. The EC50 values (nM) are shown as mean ± S.E. (n = 2).

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Fine mapping selectivity determinants in the C2 subregion. A, amino acid sequence alignment in the C2 subregion between PS1 and PS2. The C2 region contains the majority of the transmembrane 3 (TM3) segment, indicated by the line above the sequences, and the loop between TM3 and TM4. The amino acid residues different between PS1 and PS2 are boxed and highlighted in red boldface type. B, diagram of parental construct, PS1(L282I), in which Leu282 is replaced by Ile from PS2. The arrows above the C2 region indicate the 7 amino acid residues different between PS1 and PS2. Each of the seven positions was mutated in the PS1(L282I) backbone to convert a PS1 amino acid residue into a PS2 amino acid residue. C, PS1/2C2(L282I) and PS1(L282I) are the same, except that, in the C2 region, PS1/2C2(L282I) harbors PS2 sequence, whereas PS1(L282I) harbors PS1 sequence. D, EC50 values determined from cells individually transfected with the control construct PS1/2C2(L282I) or one of the seven point mutants and treated with either ELN318463 or BMS299897. The EC50 values (nM) are shown as mean ± S.E. (n = 2).

We next queried the C2 subregion for dominant residues conferring PS1 selectivity. The C2 subregion in PS1 and PS2 encodes the majority of transmembrane domain 3 (TM3) as well as part of the loop connecting TM3 and TM4. Sequence alignment of PS1 and PS2 reveals seven nonconserved amino acid residues in the C2 subregion (Fig. 6A). Conversion mutants at each of these positions were generated in the context of PS1(L282I) (Fig. 6B) and assayed for the impact on inhibitor potency. We selected PS1(L282I), instead of wild type PS1, as the backbone for this analysis in order to amplify the relative contribution of any conversion mutant in the PS1 C2 subregion, which alone contributes 3-fold potency difference between PS1 and PS2. Inhibition potency of the sulfonamides against a particular conversion mutant in the C2 subregion of PS1 was referenced against the EC₅₀ value against PS1/2C2(L282I), a PS1/2C2 chimera molecule with the L282I mutation, as illustrated in Fig. 6C.

We employed PS1/2C2(L282I) as the reference construct for assessment of PS1 C2 region conversion mutants, because it established the bottom of the range of inhibitor potency for the conversion mutants. Thus, we reasoned that if any one of the seven amino acid residues in the PS1 C2 subregion contributes to the higher inhibition potency of ELN318463 against PS1, a conversion mutant of that PS1 residue into a PS2 residue, in the context of PS1(L282I), should lead to a decrease in potency (i.e. an increase in EC₅₀ value) comparable with that observed against PS1/2C2(L282I). Indeed, this is what we observed for PS1(L282I,L172M). Inhibitor potencies of both ELN318463 and BMS299897 were lowered 3-fold against this particular mutant (Fig. 6D), and the EC₅₀ values of both compounds are very similar to their potencies against PS1/2C2(L282I). In contrast, potencies of ELN318463 and BMS299897 are not affected by any of the other six conversion mutants in the C2 subregion.

The data strongly suggest that L172 in PS1 largely accounts for the contribution made by the C2 subregion to the high potency inhibition of PS1 for both ELN318463 and BMS299897. Alanine scanning mutagenesis of Leu¹⁷² in the PS1 context lowered the potency of ELN318463 2.6-fold relative to wild type PS1 (see Table 1 and the alanine scanning results below), in close agreement with the 3-fold effect observed in the context of PS1/2C2(L281), validating use of this construct as a reference. Finally, in our experiments analyzing the C2 subregion, the potencies of ELN318463 and BMS299897 against PS1/2C2(L282I) are within 2-fold of the potencies determined during analysis of the C5 region (Fig. 5), further illustrating the limited range of interexperimental variability we observed during the course of this work.

Having identified Leu¹⁷², Thr²⁸¹, and Leu²⁸² in PS1 as being primarily responsible for the high potency inhibition of PS1 by ELN318463 when tested individually and partly responsible for BMS299897, we next tested whether the 3 amino acid residues, when in combination, are both necessary and sufficient in determining PS1/PS2 selectivity for the two sulfonamides. We also included the two nonsulfonamide inhibitors, DAPT and L-685,458 as controls in this experiment to confirm that the effect is specific to sulfonamides. We prepared two composite mutants; triple-PS1 refers to PS1(L172M,T281P,L282I), and triple-PS2 refers to PS2 (M178L,P287T,I288L). The mutated amino acid residues in triple-PS2 correspond to the three identified amino acid residues in triple-PS1.

As revealed by the data shown in Fig. 7, the EC₅₀ for inhibition of triple-PS1 by ELN318463 is indistinguishable from wild type PS2. Thus, in terms of inhibitor sensitivity, triple-PS1 behaved like PS2, despite the fact that it is largely a PS1 molecule, with only 3 amino acid residues converted into the PS2 sequence (L172M,T281P,L282I). Similarly, the EC₅₀ for inhibition of triple-PS2 by ELN318463 is indistinguishable from wild type PS1, despite the fact that it is largely a PS2 molecule, with only 3 amino acid residues converted into PS1 sequence (M178L, P287T, and I288L). Furthermore, the -secretase activity of each triple-PS construct is indistinguishable from that of its wild type counterpart, as measured by Aβ production (supplemental Fig. S1D). The data strongly suggest that the 3 identified amino acid residues are both necessary and sufficient in determining PS1 > PS2 selectivity for ELN318463. For BMS299897, the triple mutations had a slightly less dramatic effect than that seen for ELN318463. Still, the effect was stronger than what one would have predicted based on the additive effects of single mutations in both the C2 and C5 regions (compare EC₅₀ values for BMS299897 in Fig. 6D and Fig. 5C with those in Fig. 7B for mutations L172M, T281P, and L282I). As expected, the triple mutations did not have a detectable effect on inhibition potencies of the nonsulfonamide inhibitors, DAPT and L-685,458, confirming that the effects of the three identified amino acid residues were specific to sulfonamide inhibitors.

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Triple-PS1 (PS1(L172M,T281P,L282I), which is PS1-based) and triple-PS2 (PS2(M178L, P287T,I288L), which is PS2-based). The table shows EC50 values ± S.E. determined for PS1 and PS2, as controls, and for the two triple mutants for two sulfonamide inhibitors (ELN318463 and BMS299897) as well as two nonsulfonamide inhibitors (DAPT and L-685,458).

View larger version (19K): [in this window] [in a new window]   FIGURE 8. Activity of alanine scanning mutants as assessed by Aβ production. The graphs above show the relative Aβ levels of the mutants, expressed as a ratio of Aβ produced from the mutant relative to wild type PS1. The activity of alanine scanning mutants in the C2 and C5 regions of PS was assessed by measuring total Aβ produced (1-x) from the mutant compared with total Aβ from wild type PS1 within the same experiment. The arrows denote the select mutations identified in Tables 1 and 2, which affected the potency of multiple compounds. The red arrows indicate mutants that increased potency >2-fold; black arrows indicate mutants that decreased potency >2-fold. The Y288A mutation is inactive. A, Aβ production from alanine scanning mutants in the C2 subregion relative to wild type PS1. B, Aβ production from alanine scanning mutants in the C5 subregion relative to wild type PS1. In A and B, the values for Aβ production are derived from n = 3 independent experiments, and the error bars show S.E. C, assessment of Aβ42/Aβ 1-x ratio from select alanine mutants of FAD residues (n = 2).

View larger version (30K): [in this window] [in a new window]   FIGURE 9. A, APP expression levels in cells transfected with different chimeric PS constructs were determined by semiquantitative Western blots using an APP C-terminal antibody (Sigma). The relative APP level from different PS chimeras is expressed relative to the signal intensity of APP from WT PS1. B, Western blot of cell lysates (10 µg/lane) from cells transfected with PS1 alanine scanning constructs, indicated above each lane, probed with PS1 NTF, PS1 C-terminal fragment, and APP C-terminal antibodies, respectively.

In addition to L166A discussed above, I162A and W165A had a modest effect (3-4-fold reduction of potency) on both sulfonamide and nonsulfonamide inhibitors, whereas S170A primarily affected sulfonamide inhibitors (lowered potency 2.6-4.5-fold). These mutants were indistinguishable from PS1 with respect to activity, as assessed by Aβ production (Fig. 8A). Although L172A mutation lowered potency of ELN318463 2.6-fold. consistent with the 3-fold effect of the L172M conversion mutant (Fig. 6), the result did not reach statistical significance in this experiment (p = 0.14).

Results from a similar alanine scanning mutagenesis of Leu²⁸²-flanking residues in the C5 subregion of PS1 are summarized in Table 2. Inhibitor potencies against 21 alanine mutants were determined as described above. Five residues in the C5 subregion affected potency of all four inhibitors tested: Leu²⁷¹, Arg²⁷⁸, Leu²⁸², Leu²⁸⁶, and Ile²⁸⁷ (denoted by black arrows in Table 2). Of particular note, R278A had the most dramatic effect on all of the compounds tested. This mutation reduced the inhibition potency for L-685,458 by 120-fold and about 10-fold for ELN318463, BMS299897, and DAPT. Arg²⁷⁸ is situated near the amino terminus of cytosol-localized hydrophobic region 7 (36-38). One study reported that R278T was associated with early onset FAD (39), but no further molecular or biochemical studies were reported for this PS1 FAD mutant. Two other FAD mutations, R278K and R278I, have been described at this site. Aβ production from this mutant was 50% of wild type PS1 (Fig. 8B). The mutant exhibited a 5-fold elevation in Aβ 42/total ratio over wild type PS1 (Fig. 8C), and a concomitant decrease in PS1 endoproteolysis (Fig. 9B). The FAD-associated L271A also affected both sulfonamide and nonsulfonamide inhibitor potencies.

The Y288A mutation is also noteworthy, since it led to complete inactivation of PS1, despite normal endoproteolysis (Fig. 9B). Our findings corroborate the functional significance of Tyr²⁸⁸ described previously (40). Interestingly, our mutational scan of two upstream residues, L286A and I287A, revealed an affect on the potency of all classes of inhibitors, without significantly affecting Aβ production (Fig. 8B). PS endoproteolysis was not affected by the L286A mutation (Fig. 9B). Consistent with FAD mutations at codon 286, L286A displayed elevation in Aβ42/total ratio (Fig. 8C).

In contrast with the T281P conversion mutation identified above (Figs. 5 and 7), the T281A mutation did not affect potency of ELN318463 in the context of PS1. However, consistent with the 6.5-fold loss of potency effected by the conversion mutation L282I (Fig. 5), L282A lowered the potency of ELN318463 10-fold, with lesser effects on the other three inhibitors. Interestingly, V272A also reveals a strong effect on potency of ELN318463 as L282A, with no effect on BMS299897 inhibition, suggesting that Val²⁷² may also be involved in ELN318463 activity. Aβ production was not affected by V272A, T281A, and L282A mutants relative to wild type PS1 (Fig. 8B). Taken together, these observations support our studies with chimeric constructs, which mapped residues in the C5 subregion as contributing to the PS1 selectivity of ELN318463.

Although our alanine scanning analysis of the C2 and C5 subregions was driven by our observation of PS1-selective inhibition by sulfonamides, we identified certain residues that affected potencies of DAPT as well as L-685,458. As noted, many of these residues coincided with sites of FAD mutations in the C2 and C5 subregions (see "Discussion"). Interestingly, many of the non-FAD residues that affected inhibitor potency also elevated the Aβ42/Aβ 1-x ratio (see Fig. S2 and "Discussion"). Furthermore, our study also identified certain residues that affect potencies of the nonsulfonamides exclusively. In this last category, P284A is noteworthy for affecting potency of DAPT and L-685,458, whereas alanine mutation of Arg²⁷⁸-flanking residues E277A and N279A, affected the potency of L-685,458 exclusively. Curiously, alanine mutation of Phe²⁸³, the residue upstream of Pro²⁸⁴, increased potency of BMS299897. Mutation of the FAD residue Glu²⁸⁰ lowered potency of ELN318463, DAPT, and L-685,458, with a concomitant reduction in Aβ production (relative to WT PS1) (Fig. 8B).

In summary, alanine scanning mutagenesis of the C2 and C5 subregions in PS1 largely corroborates our studies mapping selectivity determinants for sulfonamides to these regions. In addition, the experiments reveal determinants in common with sulfonamides that affect the potency of additional classes of -secretase inhibitors, namely DAPT and L-685,458.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We report the first observation of PS1-selective inhibition of -secretase by sulfonamides. Using a series of chimeric constructs and point mutations, we identify the structural determinants for PS1-selective inhibition by ELN318463 and, to a lesser extent, BMS299897. Chimeric constructs offer a powerful and convenient solution for identifying domains in a protein that contribute to a particular phenotype when there is significant divergence in primary sequence between homologues. Our chimera studies identified two subregions in PS1 (designated C2 and C5, encompassing TM3 and hydrophobic region 7 following TM6, respectively), as comprising the principal determinants for PS1 selectivity of sulfonamide inhibitors. The divergence between PS1 and PS2 in the C2 and C5 subregions is among the lowest between the two presenilins. This implies that if other regions share a similar degree of divergence with the C2 or C5 subregions between PS1 and PS2 and make similar contributions to inhibition potency of sulfonamides, they would have been revealed in our chimera studies.

We identified 3 amino acid residues in PS1, Leu¹⁷², Thr²⁸¹, and Leu²⁸², that are largely responsible for the observed PS1 selectivity of sulfonamide inhibitor ELN318463 and, to a lesser extent, BMS299897. Since we did not exhaustively test all double mutant combinations of these residues, we cannot state with certainty that all 3 residues are necessary for completely affecting the potency of the sulfonamides we tested. However, the double mutant we did test, PS1(L282I,L172M) in Fig. 6D, had an intermediate effect on the potency of both sulfonamides tested when compared with the triple PS1 mutant (Fig. 7). This observation suggests that the other combinations of double mutant PS constructs would likewise have had an intermediate effect on inhibitor potency.

The difference in inhibition potency between PS1 and PS2 by sulfonamides could reflect either a difference in compound binding affinity or a difference in allosteric inhibition subsequent to compound binding. Our experimental approach does not distinguish between the two possibilities. However, the highly conservative L282I mutation in PS1 led to a 6.4-fold reduction in inhibition potency for ELN318463, whereas a non-conservative T281P mutation of the adjacent residue, a potentially much more disruptive change, only led to a 2-fold decrease in potency for ELN318463 (Fig. 7C). This observation suggests that L282 in PS1 may make physical contact with ELN318463, whereas other amino acid residues (Leu¹⁷² and Thr²⁸¹ in PS1) influence potency of ELN318463 indirectly. Verification of this assumption must await direct binding studies with cross-linkable compounds. Alternatively, the impact of the triple mutants on inhibitor potency could reflect a difference in the underlying biology of the presenilins as described by Mastrangelo et al. (41) (e.g. a difference in subcellular localization or in affinity for interacting proteins that indirectly affect sensitivity to inhibitors).

Our conclusions regarding selectivity determinants from chimeric constructs and conversion point mutants in the C2 and C5 subregions were confirmed by the subsequent results with the triple mutants (triple-PS1 and triple-PS2). Likewise, the data from conversion mutants in the C2 and C5 subregions was corroborated by the results of the alanine scanning experiments in these regions. Specifically, alanine scanning of every residue throughout the C2 and C5 regions did not reveal additional nonconserved residues that affected inhibitor potency. The additional residues revealed by alanine scanning to affect inhibitor potencies in the C2 and C5 subregions were all conserved between PS1 and PS2. Overall, the alanine mutations did not grossly affect function, as measured by total Aβ levels normalized relative to that of wild type PS1 (Fig. 8, A and B). Two noteworthy exceptions in the C5 subregion that affected Aβ production, Arg²⁷⁸ and Tyr²⁸⁸, are discussed below.

Alanine scanning mutagenesis of conserved residues in the C2 and C5 subregions revealed a preponderance of FAD-associated amino acids among the residues that affected potencies of both sulfonamide and nonsulfonamide inhibitors. A concordance between FAD residues and inhibitor sensitivity has been noted previously using peptidomimetic (42), active site (43), and most recently nonsteroidal anti-inflammatory drug (44) classes of -secretase inhibitors. We therefore examined whether the FAD as well as non-FAD residues in the C2 and C5 subregions that affected inhibitor potencies in our alanine scanning studies impacted Aβ42/Aβtotal ratio. Interestingly, many (but not all) alanine mutants of FAD as well as non-FAD residues elevated the Aβ42/Aβ 1-x ratio (Figs. 8C and S2). The results from this analysis revealed that in addition to the known FAD residues noted in Tables 1 and 2, alanine mutation of non-FAD residues Trp¹⁶⁵, Phe²⁸³, and Ile²⁸⁷ also elevated Aβ42/Aβtotal ratios. In contrast, I162A and F283A did not result in elevation of the Aβ42/Aβtotal ratio relative to WT PS1. Thus, there is a strong (but not strict), concordance between inhibitor sensitivity and elevation of Aβ42/Aβtotal. We also observe that under our experimental conditions, alanine mutation of FAD residue Thr²⁷⁴ caused neither an elevated Aβ42/Aβtotal ratio nor a difference in inhibitor sensitivity relative to WT PS1 (Table 2 and Figs. S2 and S3).

Taken together, the observed concordance between inhibitor sensitivity and elevation of the Aβ42/Aβ 1-x ratio suggests that the different classes of inhibitors not only share common structural determinants affecting potency but also that these common determinants coincide with residues in PS1 previously known to be functionally significant. The mutants L166A, R278A, and L286A had very large (>5-fold) effects on the inhibition potency of all classes of inhibitors we tested. The FAD residue Leu¹⁶⁶ has been demonstrated to differentially influence (S3-like) and (S4-like) cleavage of substrates (35). The effects we observe upon alanine mutation of residue Arg²⁷⁸ (Aβ production and reduced presenilin endoproteolysis) are similar to the observations of Nakaya et al. (45) from the FAD R278I mutation of PS1, whereas mutagenesis of Leu²⁸⁶ has been documented to differentially affect Aβ42 production and Notch cleavage (46). Alanine mutation of Leu²⁷¹ also affected sulfonamide and active site inhibitor potencies and, to a lesser extent, DAPT, and the FAD L271V has been associated with increased expression of exon 8-deleted transcripts, producing nonfunctional protein with regard to GSK 3β and -binding activities of PS1 (47).

The second noteworthy exception of alanine mutation that affected Aβ production in our studies is Y288A, which resulted in undetectable levels of Aβ (despite normal PS1 expression and processing) (Fig. 9B). Our findings on -secretase activity and presenilinase processing of PS1 from this mutant are consistent with those of Laundon et al. (40), who reported that this mutation significantly lowered Aβ production from a APP-C99 substrate. Residues 286-288 lie proximal to the presenilinase cleavage site (48, 49), and in addition to the effects of Leu²⁸⁶ mutation on inhibitor potency noted above, alanine mutation of Ile²⁸⁷ also significantly affected inhibitor potencies. These residues fall within the PALIY motif identified by Laudon et al. (40) as important for assembly of PS1 NTF and C-terminal fragment into functional -secretase high molecular weight complex. The preponderance of residues in the conserved cytosolic loop/hydrophobic region 7 of PS1 that affect the potency of all classes of inhibitors tested reveals a new role for this domain and suggests that this region is an important site for effecting inhibition of -secretase by multiple classes of inhibitors.

The relationship among different types of inhibitors (sulfonamide, BMS299897; peptidic, DAPT; transition state isostere, L-685,458) revealed in our studies was not anticipated by earlier investigations into compound binding sites nor mechanism of action. For example, it was reported that BMS299897 did not compete with L-685,458, suggesting that sulfonamide inhibitors are allosteric inhibitors, in contrast to transition state isosteres, which directly bind to the enzyme's active site (50). Separately, it was suggested that DAPT mainly inhibits -site cleavage, whereas L-685,458 mainly inhibits - and -site cleavage (51).

On the other hand, our alanine scanning experiments revealed several amino acid residues in TM3 as well as in the C terminus of PS NTF that affected the inhibition potency of all of the inhibitors tested (discussed above). This finding suggests that TM3 (the C2 subregion) and the C terminus of PS NTF (the C5 subregion) may be in close proximity, analogous to recent reports based on competition studies (52) and cysteine scanning mutagenesis (53), which revealed that (a) the binding sites for DAPT and transition state analogues partially overlap and (b) the proximity of inhibitor-interacting residues in TMD6 and TMD7. Hence, our data are not inconsistent with the recent finding on the binding site for DAPT (53, 54). In fact, the two results may complement each other. Morohashi et al. (54) employed biochemical labeling to identify protein fragments that are in the vicinity of the DAPT-binding site and found that (a) DAPT mainly labels TMD7 of PS; (b) sulfonamide inhibitor can compete effectively with DAPT; and (c) L-685,458 attenuated DAPT labeling only at higher concentrations. In a follow-up study, Sato et al. (53) identified Leu²⁵⁰ in TMD6 and Leu³⁸³ in TMD7 as the primary residues for DAPT binding.

Hence, based on the information generated by this and the aforementioned studies, we can speculate that the binding pocket for -secretase inhibitors may be composed of residues from several regions of presenilin. It is conceivable that the TMD6 and TMD7 binding sites (identified by Morohashi et al. (54) and Sato et al. (53)) and the TMD3 and the C terminus of PS NTF found by us are close to each other in three-dimensional space and form an extended binding site for different classes of inhibitors. This extended binding site may be spatially close to the active site or even overlap with the active site (composed of the catalytic aspartates in TMD6 and TMD7), because mutations in the C2 and C5 subregions led to changes in potencies for transition state analogue inhibitor. Biochemical labeling is a more direct approach for identifying binding site residues than the inhibition potency studies we performed. Since inhibition potency difference can be due to either a difference in compound binding or a difference in subsequent allosteric changes, we cannot definitively conclude that those identified residues are directly involved in compound binding. Hence, our findings provide an initial mechanistic understanding of how inhibitors interact with the -secretase. More extensive structure-function studies (e.g. direct chemical labeling studies with different compounds) are needed to advance our understanding of inhibitor/-secretase interaction.

In summary, the novel observation of PS1-selective inhibition of -secretase by sulfonamides reported here illustrates the possibility of discovering isoform-selective -secretase inhibitors. Constitutive as well as conditional knock-out mouse studies have demonstrated that mice with selective ablation of different subunits exhibit different severity of Notch-deficient phenotypes, depending upon the age and subunit targeted (10, 41, 55-62). Hence, these studies suggest that isoform-selective inhibitors offer another avenue for circumventing Notch-related toxicity observed with first generation -secretase inhibitors (11-14).

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ Present address: 819 Epperson Way, Sugar Land, TX 77479.

² To whom correspondence should be addressed: Elan Pharmaceuticals Inc., 700 Gateway Blvd., S. San Francisco, CA 94080. Tel.: 650-877-0900; Fax: 650-877-7615; E-mail: guriq.basi{at}elan.com.

³ The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β peptide; APP, amyloid precursor protein; APPsw, Swedish mutant version of APP751 isoform; FAD, familial Alzheimer disease; dKO cells, double knock-out cells; NTF, N-terminal fragment; PS, presenilin; TMD, transmembrane domain; WT, wild type; ELISA, enzyme-linked immunosorbent assay.

	ACKNOWLEDGMENTS

 
We thank Ruth Motter, Pearl Tang, and Dora Kholodenlo for providing ELISA reagents, Chip Frigon for fluorescence-activated cell sorter counting of cells during optimization of transfection conditions, Gergeley Toth for initial discussion of mutation data, and Susanna Hemphill and Jowell Go for helping with biochemical characterization of the -secretase complex. We also thank Dr. Bart DeStrooper for the gift of the PS1/PS2 double knock-out cells.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Hardy, J., and Selkoe, D. J. (2002) Science 297, 353-356[Abstract/Free Full Text]
2. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T., Mar, L., Foncin, J. F., Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L., Nee, L., Chumakov, I., Pollen, D., Brookes, A., Sanseau, P., Polinsky, R. J., Wasco, W., Da Silva, H. A. R., Haines, J. L., Pericak-Vance, M. A., Tanzi, R. E., Roses, A. D., Fraser, P. E., Rommens, J. M., and St George-Hyslop, P. H. (1995) Nature 375, 754-760[CrossRef][Medline] [Order article via Infotrieve]
3. Rogaev, E. I., Sherrington, R., Rogaeva, E. A., Levesque, G., Ikeda, M., Liang, Y., Chi, H., Lin, C., Holman, K., Tsuda, T., Mar, L., Sorbi, S., Nacmias, B., Piacentini, S., Amaducci, L., Chumakov, I., Cohen, D., Lannfelt, L., Fraser, P. E., Rommens, J. M., and St George-Hyslop, P. H. (1995) Nature 376, 775-778[CrossRef][Medline] [Order article via Infotrieve]
4. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell, W. H., Yu, C. E., Jondro, P. D., Schmidt, S. D., Wang, K., Crowley, A. C., Fu, Y.-H., Guenette, S. Y., Galas, D., Nemens, E., Wijsman, E. M., Bird, T. D., Schellenerg, G. D., and Tanzi, R. E. (1995) Science 269, 973-977[Abstract/Free Full Text]
5. Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M., Apfeld, J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M. C., Parks, A. L., Xu, W., Li, J., Gurney, M., Myers, R. L., Himes, C. S., Hiebsch, R., Ruble, C., Nye, J. S., and Curtis, D. (2002) Dev. Cell 3, 85-97[CrossRef][Medline] [Order article via Infotrieve]
6. Goutte, C., Tsunozaki, M., Hale, V. A., and Priess, J. R. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 775-779[Abstract/Free Full Text]
7. Wolfe, M. S. (2006) Biochemistry 45, 7931-7939[CrossRef][Medline] [Order article via Infotrieve]
8. Steiner, H., and Haass, C. (2000) Nat. Rev. Mol. Cell Biol. 1, 217-224[CrossRef][Medline] [Order article via Infotrieve]
9. Fortini, M. E. (2002) Nat. Rev. Mol. Cell Biol. 3, 673-684[CrossRef][Medline] [Order article via Infotrieve]
10. Marjaux, E., Hartmann, D., and De Strooper, B. (2004) Neuron 42, 189-192[CrossRef][Medline] [Order article via Infotrieve]
11. Hyde, L. A., McHugh, N. A., Chen, J., Zhang, Q., Manfra, D., Nomeir, A. A., Josien, H., Bara, T., Clader, J. W., Zhang, L., Parker, E. M., and Higgins, G. A. (2006) J. Pharmacol. Exp. Ther. 319, 1133-1143[Abstract/Free Full Text]
12. Milano, J., McKay, J., Dagenais, C., Foster-Brown, L., Pognan, F., Gadient, R., Jacobs, R. T., Zacco, A., Greenberg, B., and Ciaccio, P. J. (2004) Toxicol. Sci. 82, 341-358[Abstract/Free Full Text]
13. Searfoss, G. H., Jordan, W. H., Calligaro, D. O., Galbreath, E. J., Schirtzinger, L. M., Berridge, B. R., Gao, H., Higgins, M. A., May, P. C., and Ryan, T. P. (2003) J. Biol. Chem. 278, 46107-46116[Abstract/Free Full Text]
14. Wong, G. T., Manfra, D., Poulet, F. M., Zhang, Q., Josien, H., Bara, T., Engstrom, L., Pinzon-Ortiz, M., Fine, J. S., Lee, H. J., Zhang, L., Higgins, G. A., and Parker, E. M. (2004) J. Biol. Chem. 279, 12876-12882[Abstract/Free Full Text]
15. Apelqvist, A., Li, H., Sommer, L., Beatus, P., Anderson, D. J., Honjo, T., Hrabe de Angelis, M., Lendahl, U., and Edlund, H. (1999) Nature 400, 877-881[CrossRef][Medline] [Order article via Infotrieve]
16. De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., Von Figura, K., and Van Leuven, F. (1998) Nature 391, 387-390[CrossRef][Medline] [Order article via Infotrieve]
17. Herreman, A., Hartmann, D., Annaert, W., Saftig, P., Craessaerts, K., Serneels, L., Umans, L., Schrijvers, V., Checler, F., Vanderstichele, H., Baekelandt, V., Dressel, R., Cupers, P., Huylebroeck, D., Zwijsen, A., Van Leuven, F., and De Strooper, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11872-11877[Abstract/Free Full Text]
18. Jensen, J., Pedersen, E. E., Galante, P., Hald, J., Heller, R. S., Ishibashi, M., Kageyama, R., Guillemot, F., Serup, P., and Madsen, O. D. (2000) Nat. Genet. 24, 36-44[CrossRef][Medline] [Order article via Infotrieve]
19. Murtaugh, L. C., Stanger, B. Z., Kwan, K. M., and Melton, D. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14920-14925[Abstract/Free Full Text]
20. Stanger, B. Z., Datar, R., Murtaugh, L. C., and Melton, D. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 12443-12448[Abstract/Free Full Text]
21. Swiatek, P. J., Lindsell, C. E., del Amo, F. F., Weinmaster, G., and Gridley, T. (1994) Genes Dev. 8, 707-719[Abstract/Free Full Text]
22. van Es, J. H., van Gijn, M. E., Riccio, O., van den Born, M., Vooijs, M., Begthel, H., Cozijnsen, M., Robine, S., Winton, D. J., Radtke, F., and Clevers, H. (2005) Nature 435, 959-963[CrossRef][Medline] [Order article via Infotrieve]
23. Herreman, A., Serneels, L., Annaert, W., Collen, D., Schoonjans, L., and De Strooper, B. (2000) Nat. Cell Biol. 2, 461-462[CrossRef][Medline] [Order article via Infotrieve]
24. Steiner, H., Duff, K., Capell, A., Romig, H., Grim, M. G., Lincoln, S., Hardy, J., Yu, X., Picciano, M., Fechteler, K., Citron, M., Kopan, R., Pesold, B., Keck, S., Baader, M., Tomita, T., Iwatsubo, T., Baumeister, R., and Haass, C. (1999) J. Biol. Chem. 274, 28669-28673[Abstract/Free Full Text]
25. Shen, J., Bronson, R. T., Chen, D. F., Xia, W., Selkoe, D. J., and Tonegawa, S. (1997) Cell 89, 629-639[CrossRef][Medline] [Order article via Infotrieve]
26. De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J., Goate, A., and Kopan, R. (1999) Nature 398, 518-522[CrossRef][Medline] [Order article via Infotrieve]
27. Donoviel, D. B., Hadjantonakis, A. K., Ikeda, M., Zheng, H., Hyslop, P. S., and Bernstein, A. (1999) Genes Dev. 13, 2801-2810[Abstract/Free Full Text]
28. Anderson, J. J., Holtz, G., Baskin, P. P., Turner, M., Rowe, B., Wang, B., Kounnas, M. Z., Lamb, B. T., Barten, D., Felsenstein, K., McDonald, I., Srinivasan, K., Munoz, B., and Wagner, S. L. (2005) Biochem. Pharmacol. 69, 689-698[CrossRef][Medline] [Order article via Infotrieve]
29. Barten, D. M., Guss, V. L., Corsa, J. A., Loo, A., Hansel, S. B., Zheng, M., Munoz, B., Srinivasan, K., Wang, B., Robertson, B. J., Polson, C. T., Wang, J., Roberts, S. B., Hendrick, J. P., Anderson, J. J., Loy, J. K., Denton, R., Verdoorn, T. A., Smith, D. W., and Felsenstein, K. M. (2005) J. Pharmacol. Exp. Ther. 312, 635-643[Abstract/Free Full Text]
30. Smith, D. W., Munoz, B., Srinivasan, K., Bergstrom, C. P., Prasad, V., Chaturvedula, P. V., Milind, S., Deshpande, M. S., Keavy, D. J., Lau, W. Y., Parker, M. F., Solan, C. P., Wallace, O. B., and Wang, H. H. (February 22, 2000) U. S. Patent 6967196
31. Berkner, K. L., Schaffhausen, B. S., Roberts, T. M., and Sharp, P. A. (1987) J. Virol. 61, 1213-1220[Abstract/Free Full Text]
32. Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve]
33. Levy-Lahad, E., Wijsman, E. M., Nemens, E., Anderson, L., Goddard, K. A., Weber, J. L., Bird, T. D., and Schellenberg, G. D. (1995) Science 269, 970-973[Abstract/Free Full Text]
34. Johnson-Wood, K., Lee, M., Motter, R., Hu, K., Gordon, G., Barbour, R., Khan, K., Gordon, M., Tan, H., Games, D., Lieberburg, I., Schenk, D., Seubert, P., and McConlogue, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1550-1555[Abstract/Free Full Text]
35. Moehlmann, T., Winkler, E., Xia, X., Edbauer, D., Murrell, J., Capell, A., Kaether, C., Zheng, H., Ghetti, B., Haass, C., and Steiner, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8025-8030[Abstract/Free Full Text]
36. Henricson, A., Kall, L., and Sonnhammer, E. L. (2005) FEBS J. 272, 2727-2733[CrossRef][Medline] [Order article via Infotrieve]
37. Laudon, H., Hansson, E. M., Melen, K., Bergman, A., Farmery, M. R., Winblad, B., Lendahl, U., von Heijne, G., and Naslund, J. (2005) J. Biol. Chem. 280, 35352-35360[Abstract/Free Full Text]
38. Oh, Y. S., and Turner, R. J. (2005) Am. J. Physiol. 289, C576-C581[CrossRef]
39. Kwok, J. B., Taddei, K., Hallupp, M., Fisher, C., Brooks, W. S., Broe, G. A., Hardy, J., Fulham, M. J., Nicholson, G. A., Stell, R., St George Hyslop, P. H., Fraser, P. E., Kakulas, B., Clarnette, R., Relkin, N., Gandy, S. E., Schofield, P. R., and Martins, R. N. (1997) Neuroreport 8, 1537-1542[Medline] [Order article via Infotrieve]
40. Laudon, H., Karlstrom, H., Mathews, P. M., Farmery, M. R., Gandy, S. E., Lundkvist, J., Lendahl, U., and Naslund, J. (2004) J. Biol. Chem. 279, 23925-23932[Abstract/Free Full Text]
41. Mastrangelo, P., Mathews, P. M., Chishti, M. A., Schmidt, S. D., Gu, Y., Yang, J., Mazzella, M. J., Coomaraswamy, J., Horne, P., Strome, B., Pelly, H., Levesque, G., Ebeling, C., Jiang, Y., Nixon, R. A., Rozmahel, R., Fraser, P. E., St George-Hyslop, P., Carlson, G. A., and Westaway, D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8972-8977[Abstract/Free Full Text]
42. Xia, W., Ostaszewski, B. L., Kimberly, W. T., Rahmati, T., Moore, C. L., Wolfe, M. S., and Selkoe, D. J. (2000) Neurobiol. Dis. 7, 673-681[CrossRef][Medline] [Order article via Infotrieve]
43. Ikeuchi, T., Dolios, G., Kim, S. H., Wang, R., and Sisodia, S. S. (2003) J. Biol. Chem. 278, 7010-7018[Abstract/Free Full Text]
44. Czirr, E., Leuchtenberger, S., Dorner-Ciossek, C., Schneider, A., Jucker, M., Koo, E. H., Pietrzik, C. U., Baumann, K., and Weggen, S. (2007) J. Biol. Chem. 282, 24504-24513[Abstract/Free Full Text]
45. Nakaya, Y., Yamane, T., Shiraishi, H., Wang, H. Q., Matsubara, E., Sato, T., Dolios, G., Wang, R., De Strooper, B., Shoji, M., Komano, H., Yanagisawa, K., Ihara, Y., Fraser, P., St George-Hyslop, P., and Nishimura, M. (2005) J. Biol. Chem. 280, 19070-19077[Abstract/Free Full Text]
46. Kulic, L., Walter, J., Multhaup, G., Teplow, D. B., Baumeister, R., Romig, H., Capell, A., Steiner, H., and Haass, C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5913-5918[Abstract/Free Full Text]
47. Kwok, J. B., Halliday, G. M., Brooks, W. S., Dolios, G., Laudon, H., Murayama, O., Hallupp, M., Badenhop, R. F., Vickers, J., Wang, R., Naslund, J., Takashima, A., Gandy, S. E., and Schofield, P. R. (2003) J. Biol. Chem. 278, 6748-6754[Abstract/Free Full Text]
48. Podlisny, M. B., Citron, M., Amarante, P., Sherrington, R., Xia, W., Zhang, J., Diehl, T., Levesque, G., Fraser, P., Haass, C., Koo, E. H., Seubert, P., St George-Hyslop, P., Teplow, D. B., and Selkoe, D. J. (1997) Neurobiol. Dis. 3, 325-337[CrossRef][Medline] [Order article via Infotrieve]
49. Steiner, H., Romig, H., Pesold, B., Philipp, U., Baader, M., Citron, M., Loetscher, H., Jacobsen, H., and Haass, C. (1999) Biochemistry 38, 14600-14605[CrossRef][Medline] [Order article via Infotrieve]
50. Tian, G., Ghanekar, S. V., Aharony, D., Shenvi, A. B., Jacobs, R. T., Liu, X., and Greenberg, B. D. (2003) J. Biol. Chem. 278, 28968-28975[Abstract/Free Full Text]
51. Zhao, G., Cui, M. Z., Mao, G., Dong, Y., Tan, J., Sun, L., and Xu, X. (2005) J. Biol. Chem. 280, 37689-37697[Abstract/Free Full Text]
52. Kornilova, A. Y., Das, C., and Wolfe, M. S. (2003) J. Biol. Chem. 278, 16470-16473[Abstract/Free Full Text]
53. Sato, C., Morohashi, Y., Tomita, T., and Iwatsubo, T. (2006) J. Neurosci. 26, 12081-12088[Abstract/Free Full Text]
54. Morohashi, Y., Kan, T., Tominari, Y., Fuwa, H., Okamura, Y., Watanabe, N., Sato, C., Natsugari, H., Fukuyama, T., Iwatsubo, T., and Tomita, T. (2006) J. Biol. Chem. 281, 14670-14676[Abstract/Free Full Text]
55. Feng, R., Rampon, C., Tang, Y. P., Shrom, D., Jin, J., Kyin, M., Sopher, B., Miller, M. W., Ware, C. B., Martin, G. M., Kim, S. H., Langdon, R. B., Sisodia, S. S., and Tsien, J. Z. (2001) Neuron 32, 911-926[CrossRef][Medline] [Order article via Infotrieve]
56. Yu, H., Saura, C. A., Choi, S. Y., Sun, L. D., Yang, X., Handler, M., Kawarabayashi, T., Younkin, L., Fedeles, B., Wilson, M. A., Younkin, S., Kandel, E. R., Kirkwood, A., and Shen, J. (2001) Neuron 31, 713-726[CrossRef][Medline] [Order article via Infotrieve]
57. Feng, R., Wang, H., Wang, J., Shrom, D., Zeng, X., and Tsien, J. Z. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 8162-8167[Abstract/Free Full Text]
58. Saura, C. A., Choi, S. Y., Beglopoulos, V., Malkani, S., Zhang, D., Shankaranarayana Rao, B. S., Chattarji, S., Kelleher, R. J., III, Kandel, E. R., Duff, K., Kirkwood, A., and Shen, J. (2004) Neuron 42, 23-36[CrossRef][Medline] [Order article via Infotrieve]
59. Qyang, Y., Chambers, S. M., Wang, P., Xia, X., Chen, X., Goodell, M. A., and Zheng, H. (2004) Biochemistry 43, 5352-5359[CrossRef][Medline] [Order article via Infotrieve]
60. Saura, C. A., Chen, G., Malkani, S., Choi, S. Y., Takahashi, R. H., Zhang, D., Gouras, G. K., Kirkwood, A., Morris, R. G., and Shen, J. (2005) J. Neurosci. 25, 6755-6764[Abstract/Free Full Text]
61. Tournoy, J., Bossuyt, X., Snellinx, A., Regent, M., Garmyn, M., Serneels, L., Saftig, P., Craessaerts, K., De Strooper, B., and Hartmann, D. (2004) Hum. Mol. Genet. 13, 1321-1331[Abstract/Free Full Text]
62. Li, T., Wen, H., Brayton, C., Laird, F. M., Ma, G., Peng, S., Placanica, L., Wu, T. C., Crain, B. J., Price, D. L., Eberhart, C. G., and Wong, P. C. (2007) J. Neurosci. 27, 10849-10859[Abstract/Free Full Text]
63. Neitzel, M., Dappen, M. S., and Marugg, J. (May 12, 2005) PCT International Application WO 2005042489

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