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J. Biol. Chem., Vol. 277, Issue 35, 31499-31505, August 30, 2002

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Linear Non-competitive Inhibition of Solubilized Human -Secretase by Pepstatin A Methylester, L685458, Sulfonamides, and Benzodiazepines^*

Gaochao Tian^§, Cynthia D. Sobotka-Briner^§, John Zysk^¶, Xiaodong Liu^¶, Cynthia Birr, Mark A. Sylvester^**, Philip D. Edwards^**, Clay D. Scott^§, and Barry D. Greenberg^¶

From the Departments of ^§ Lead Discovery, ^¶ Molecular Sciences, and ^** Chemistry, AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850 and the  Department of Enabling Science and Technology Biology, AstraZeneca Pharmaceuticals, Waltham, Massachusetts 02451

Received for publication, December 21, 2001, and in revised form, June 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

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 difluoro alcohol group, another aspartyl protease transition state isostere, are also potent inhibitors of -secretase (21). Given these results, it has been hypothesized that, like -secretase, -secretase is an aspartyl protease (17-22). Recent pepstatin-derived affinity chromatography of PS1 (23), photo affinity labeling of PS1 with transition state analog L685458 (12), and chemical affinity labeling of PS1 with difluoro peptidomimetics (24) further support -secretase as an aspartyl protease and have led to the tentative identification of presenilins as the catalytic components of -secretase.

Although these transition state analogs have been extensively used as tools to probe the structure and mechanism, as well as the biochemical and cellular functions, of -secretase, the mechanisms of -secretase inhibition by these compounds have not been elucidated. Among potent and specific -secretase inhibitors are also a number of small molecules of different structural classes, such as sulfonamides (25) and benzodiazepines (26). It is an intriguing question whether these non-aspartyl protease isosteres would inhibit -secretase by the same mechanism as the transition state isosteres. Elucidating the inhibitory mechanisms of these different classes of inhibitors may provide important insights into the catalytic mechanism of -secretase and help in the design of novel drugs.

Here we report the results from inhibition of solubilized cell-free human -secretase by pepstatin A methylester (PME), L685458, sulfonamides 1 and 2, and benzodiazepines 3 and 4 (Fig. 1). Unexpectedly, the transition state analogs PME and L685458 inhibited -secretase by a linear non-competitive inhibition mechanism, suggesting that substrate can bind to the enzyme while the active site is occupied by transition state analogs. Similar non-competitive inhibition was also observed for the non-aspartyl protease isosteres 1-4. To our knowledge, this is the first example of aspartyl protease transition state analogs displaying non-competitive inhibition kinetics. A number of theoretical models are discussed in an attempt to rationalize this phenomenon.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Chemical structures of PME, L685458, sulfonamides 1 and 2, and benzodiazepines 3 and 4.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

Materials-- CHAPS and CHAPSO were purchased from Pierce. Phosphatidylcholine and phosphatidylethanolamide were purchased from Avanti Polar-Lipids. A40 and rabbit anti-A40 (RAA40) were purchased from BIOSOURCE. Biotin-4G8 was from Senetek PLC. Dynabeads (M-280) were purchased from IGEN.

Compounds-- The inhibitors used in this work were all prepared in the Central Nervous System Chemistry Department, AstraZeneca Pharmaceuticals, Wilmington DE. PME was synthesized by methylation of pepstatin A. The synthesis of L685,458 was based on a compilation of previously published procedures (27-29). Compounds 1 and 2 were synthesized using a procedure similar to the one reported in the international patent publication by Smith et al. (25). Compounds 3 and 4 were prepared according to the protocols reported in the international patent publication by Wu et al. (26).

Preparation of Ruthenium-labeled Goat Anti-rabbit (GAR)-- Affinity-purified goat anti-rabbit IgG (Jackson ImmunoResearch) was ruthenylated using Origin TAG-NHS ester (IGEN) at a challenge ratio of 10, according to the manufacturer's procedure.

Preparation of C100-- C100 (a recombinant protein with an amino acid sequence identical to CTF but containing an extra methionine residue at the N terminus) was purified from Escherichia coli inclusion bodies and stored at 80 °C before use. A 300-bp C100 fragment was amplified by polymerase chain reaction from an APP695 cDNA template. This fragment was cloned into the vector pET-21a(+). The insert contained a stop codon to prevent the C terminus of C100 from fusing with the His tag of the vector. Transformation of BL21 (DE3)pLysS cells with pET-21-C100 was conducted following the manufacturer's instructions. Cells were incubated at 37 °C in LB broth with 100 µg/ml ampicillin and harvested 4 h after isopropyl-1--D-galactopyranoside (1 mM) induction by centrifugation at 6000 × g for 15 min. The resulting cell pellet (4 g) was resuspended by vortexing in 40 ml of TE buffer (20 mM Tris-HCl, pH 7.5, and 1 mM EDTA). This suspension was homogenized on ice with a 55-ml Wheaton glass-teflon homogenizer and sonicated with a Branson Sonifier 450 (10 times at a setting of 5 for 30 s each with a 30-s break in between). The solution was centrifuged at 23,000 × g for 10 min at 4 °C, and the pellet was resuspended in 40 ml of TE buffer, sonicated, and centrifuged again as above. The pellet was washed twice, resuspended with TE buffer (10 ml/g), layered over a 1-M sucrose cushion prepared in TE buffer (12.5 ml/g), and centrifuged at 23,000 × g for 10 min at 4 °C. After centrifugation, the top layer was recovered, and the centrifugation step was repeated. The pellets from the two sucrose centrifugations were combined and dissolved in 2 ml of 2% diethylamine containing 0.1% CHAPS and centrifuged at 16,000 × g for 10 min at 4 °C. The supernatant was dialyzed overnight at 4 °C in 10 mM Tris-HCl buffer, pH 7.5, containing 20% glycerol, 5% ethylene glycol, and 0.02% azide in a dialyzer (size cutoff: 3.5 kDa). The dialyzed solution was centrifuged at 289,000 × g for 30 min at 4 °C. SDS was added to the supernatant to a final concentration of 2%. Aliquots of 1.8 mM C100 solutions were stored at 80 °C until use. The substrate was diluted to a final concentration from 0 to 2 µM in reaction. At this highest concentration of substrate, the SDS content was only 0.002%, and there was no indication of inhibition of the activity of -secretase at this level of SDS.

Preparation of Detergent-solubilized Human -Secretase-- Hela cells (8A8) (previously harvested and stored as cell pellets at 80 °C) were weighed and suspended in phosphate-buffered saline mixed in 1:1 ratio with a wash buffer (100 mM Tris-HCl, pH 8.4, 2 M KCl, and 50 mM EDTA) containing a protease inhibitor mixture (final concentrations: 0.1 µM pepstatin A, 10 µM leupeptin, 0.1 µM MG 132, 10 µM E64, and 1 mM benzamidine). The suspended cells were disrupted with a Polytron homogenizer, and the membranes were washed and collected by high speed centrifugation. After the total protein concentration was determined, the membranes were suspended at 4 mg/ml protein in 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, the protease inhibitor mixture, and 2% CHAPSO. The extraction was allowed to proceed at 4 °C for >2 h but <24 h. The extraction solution was centrifuged at 38,000 × g at 4 °C for 30 min. The supernatant was aliquoted and stored at 80 °C before use.

Enzyme and Inhibition Kinetics-- All the reactions were run in 96-well microplates. Reactions of defined final volume were run in 25 mM MES, pH 6.5, containing C100 at a defined concentration, solubilized enzyme at 20-fold dilution from stock, inhibitor at a defined concentration diluted from a stock in ME₂SO (final concentration of ME₂SO was maintained at 5%), 1 mM EDTA, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.25% CHAPSO, 0.01% phosphatidylethanolamide, and 0.01% phosphatidylcholine. The -secretase activity was not affected by 5% ME₂SO, and control reactions were performed in the presence of 5% ME₂SO. The reactions were initiated by addition of enzyme, and 40-µl aliquots were quenched at different times by addition of 50 µl of 200 µM PME. A 100-µl detection solution containing 0.2 µg/ml RAA40, 0.25 µg/ml biotin-4G8, 0.018 µg/ml Ru-GAR, and 62.5 µg/ml Dynabeads was added per quenched reaction mixture. The 96-well plates were incubated at 4 °C and vibrated with a plate shaker for >8 h. The plates were measured for ECL counts in an IGEN ECL M8 Analyzer.

Data Analysis-- Time courses of -secretase reaction monitored by ECL assay were analyzed by linear regression to obtain the slopes in counts/min. This value was then converted to pM/min by an ECL standard curve of A40, the main product of the reaction. Enzyme initial rate () data were fit by non-linear least squares analysis to Michaelis-Menten Equation 1,

(Eq. 1)

where V_m and K_m are maximum velocity and the Michaelis-Menten constant, respectively, and S is substrate. Inhibition data were fit to Equations 2, 3, and 4, respectively, for competitive, non-competitive, or uncompetitive inhibition,

(Eq. 2)

(Eq. 3)

and

(Eq. 4)

where K_is is the inhibition constant for the inhibitor binding to the free enzyme and K_ii is the inhibition constant for the inhibitor binding to the ES complex. To analyze whether inhibition is linear, the double reciprocal plots obtained at different inhibitor concentrations were fit to a linear equation, and the slope or intercept was then re-analyzed either by linear Equation 5,

(Eq. 5)

where a and b are constants, or by a second order polynomial in Equation 6,

(Eq. 6)

where c is also a constant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

Kinetics of Solubilized -Secretase-- The primary goal of this study was to elucidate the inhibition mechanisms of known -secretase transition state analogs, PME and L685458, and small molecule inhibitors, sulfonamides and benzodiazepines. Because -secretase is quite complex structurally and has never been purified to homogeneity, it is crucial to characterize carefully the substrate kinetics in order to provide a framework for inhibition studies. The substrate used in this study was C100, a recombinant protein constructed according to the sequence of the -secretase-generated C-terminal fragment of APP (CTF), a natural substrate of -secretase. All the reactions were run at pH 6.5, the pH optimum under the reaction conditions employed in this study (data not shown). The reaction of -secretase with C100 was monitored by following the production of A40, the major product of the -secretase reaction, using the IGEN ECL technology. The initial rate was calculated as the slope of the reaction progress curve for A40 production. The total turnover was <1% even at the lowest substrate concentration (0.05 µM), so there was essentially no substrate depletion. The total product formed was <1 nM, and this product concentration was in the linear range of A40 detection as judged from the A40 standard curves (data not shown). The progress curves at pH 6.5 were linear for at least 2 h (Fig. 2A), suggesting the enzyme was stable during the experiments. This method for determination of initial rates was much more accurate than end point measurements and was used to perform all the kinetic experiments described in this study.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   A typical progress curve of -secretase reaction at pH 6.5, 22 °C (A) and the initial rate of solubilized human -secretase as a function of C100 concentration obtained at pH 6.5, 22 °C (B) or 37 °C (C). The open circles are experimental values, and the lines are theoretical values.

As shown in Fig. 2, B and C, the initial rate of the solubilized -secretase as a function of C100 concentration followed a typical hyperbola. This, as well as the apparent linearity in the progress curves for initial rate measurements as stated above, suggested that the reactions followed a typical steady state kinetic mechanism. The kinetic constants were calculated by fitting the initial rates to the Michaelis-Menten equation (see Equation 1), and the values are tabulated in Table I. The K_m values (0.12-0.39 µM) are comparable with the K_m value of 1 µM reported previously using a FLAG-tagged C100 substrate (14).

View this table: [in this window] [in a new window]   Table I Summary of kinetic properties Summary of kinetic properties of solubilized human -secretase and the kinetic properties for inhibition of solubilized human -secretase by PME, L685458, 1, 2, 3, and 4 at pH 6.5.

Linear Non-competitive Inhibition of -Secretase by PME-- Having established the nature of steady state kinetics for solubilized -secretase in reactions with C100 as substrate, we proceeded to determine the inhibition mechanism of PME, a transition state isostere of aspartyl proteases. Transition state analogs by definition are enzyme inhibitors that bind to the catalytic site of their target; therefore, such inhibitors generally display competitive inhibition. Unexpectedly, initial inhibition data obtained at four different inhibitor concentrations indicated non-competitive inhibition of -secretase by PME (data not shown). As a result of these data, we examined in greater detail the effect of inhibitor concentration on the slope and intercept of the double reciprocal plot by employing a total of eight inhibitor concentrations in a separate kinetic experiment, reasoning that the slope replot as a function of inhibitor concentration would be curved. A parabolic slope replot but a linear intercept replot would indicate that the inhibitor, while binding at the catalytic site as a transition state analog is expected to do, may also bind at an allosteric site, thereby causing the observed non-competitive inhibition (Appendix). On the other hand, linear slope and intercept replots would indicate that the inhibitor binds at a single, non-competitive binding site (31, 32). As shown in Fig. 3, the double reciprocal plot of inhibition by PME (Fig. 3A) again clearly indicated non-competitive inhibition. Importantly, both the slope (Fig. 3B) and intercept (Fig. 3C) replots were linear, consistent with a pure non-competitive model where just one inhibitor binds to enzyme, as illustrated by Equation 7.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   A, double reciprocal plots for inhibition of solubilized human -secretase by PME at pH 6.5 and 22 °C at [I] = 0 (dotted diamond), 0.0294 (dotted triangle), 0.0529 (dotted square), 0.0953 (dotted circle), 0.172 (diamond), 0.309 (triangle), 0.556 (square), and 1.00 (circle) µM. B, slope replot. C, intercept replot.

(Eq. 7)

The value of K_is, which is the inhibition constant for inhibitor binding to free enzyme (E), and the value of K_ii, which is the inhibition constant for inhibitor binding to the ES complex, were similar (Table I), suggesting that there was little interaction between the substrate C100 and PME in the ternary complex and minimal conformational change that would affect the inhibitor binding upon substrate binding or vice versa. To rule out the possibility that the apparent non-competitive inhibition might arise from artifacts due to substrate aggregation at room temperature, the kinetic experiments were repeated at 37 °C. As shown in Fig. 4A, the kinetic pattern for PME obtained at 37 °C was not different from that which was obtained at 22 °C (Fig. 3A), although the inhibition constants were slightly higher at high temperature (Table I). The linear progress curves in the presence of PME (data not shown) and the near total recovery of enzyme activity from dilution of the enzyme-PME complex (data not shown) ruled out the possibility of time dependence or irreversible inhibition of -secretase by PME as the cause of the observed non-competitiveness. Taken together, these data indicate that PME is a bona fide linear non-competitive inhibitor of -secretase.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Double reciprocal plots for inhibition of solubilized human -secretase at pH 6.5 and 37 °C by PME. A, at [I] = 0 (diamond), 0.5 (triangle), 1.0 (square), and 2.0 (circle) µM and by L685458. B, at [I] = 0 (diamond), 0.00375 (triangle), 0.0075 (square), and 0.015 (circle) µM.

Non-competitive Inhibition of -Secretase by L685458, Sulfonamides, and Benzodiazepines-- To determine whether this type of non-competitive inhibition is PME-specific, we investigated the inhibition kinetics for three other types of -secretase inhibitors. L685458, another aspartyl protease transition state analog, also displayed a non-competitive inhibition pattern, as shown in Fig. 4B. The data obtained for four non-transition state inhibitors, two sulfonamides (1 and 2) and two benzodiazepines (3 and 4), also indicated non-competitive inhibition (Fig. 5). As observed for PME, the values of K_is were also quite similar to the values of K_ii for each of these compounds (Table I), suggesting minimal interaction between substrate and inhibitor. These data demonstrate that the non-competitive inhibition kinetics observed with -secretase is not restricted to a certain class of compounds but is quite common among -secretase inhibitors.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Double reciprocal plots for inhibition of solubilized human -secretase at pH 6.5 and 22 °C. A, compound 1 at [I] = 0 (diamond), 0.0075 (triangle), 0.015 (square), and 0.030 (circle) µM. B, compound 2 at [I] = 0 (diamond), 0.125 (triangle), 0.250 (square), and 0.500 (circle) µM. C, compound 3 at [I] = 0 (diamond), 6.7 (triangle), 13.3 (square), and 40 (circle) µM. D, compound 4 at [I] = 0 (diamond), 0.0017 (triangle), 0.0033 (square), and 0.010 (circle) µM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

Several different structural classes of -secretase inhibitors have been extensively used as tools for biochemical and functional studies of -secretase. In this study, the mechanisms of -secretase inhibition by transition state analogs, PME and L685458, and non-transition state inhibitors, sulfonamides and benzodiazepines, were investigated. Surprisingly, both the transition state isosteres and the non-transition state inhibitors displayed linear non-competitive inhibition.

The simplest interpretation of the linear non-competitive inhibition by PME is that PME binds to a non-catalytic, allosteric site of -secretase, which can be adequately described by a simple kinetic scheme as shown by Equation 7. It is known that -secretase is a high molecular weight, macromolecular complex (13, 14). This complex contains at least two functional units, presenilin (1 or 2) and nicastrin (13). Furthermore, mature presenilin in the active -secretase complex is a heterodimer of two subunits, NTF and CTF, formed by endoproteolysis of the full-length presenilin (15, 16). Given this structural complexity, it would not be surprising if an inhibitor binds to a non-catalytic site to interrupt subunit interactions, thereby displaying non-competitive inhibition kinetics.

Yet to be proven, evidence exists to suggest strongly that -secretase is an aspartyl protease (12, 17-24). Therefore, the issue with this model of PME or L685458 binding to an allosteric site is the issue of a transition state analog binding exclusively at a non-catalytic site. Consistent with the inhibitor binding to a site that is different from the substrate-binding site, Wolfe and co-workers (33) recently reported that CTF, a natural substrate of -secretase, could be co-purified with PS1 from an aspartyl protease inhibitor affinity column. To interpret this result, they proposed that substrate first binds to a docking site on the enzyme and then moves to the catalytic site where it is subsequently cleaved (Fig. 6A). The inhibitor, however, can bind directly at the catalytic site, thus explaining the co-purification of PS1 and CTF by the transition state analog-based affinity chromatography. This model is consistent with the non-competitive kinetic inhibition results presented in this study. Alternatively, the initial substrate binding is not docking but a process that anchors a tail (for example, the N or C terminus) of the substrate. A conformational change of substrate bound at the anchor site may then occur, perhaps swinging the scissile bond into the catalytic site for cleavage (Fig. 6B).

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Proposed models for kinetic mechanism and inhibition of -secretase by transition state analogs. A, substrate (S) docking and shifting. Substrate first binds at the docking site on the enzyme and then shifts into the catalytic site. After or in concert with the docking or shifting, the docking site or the catalytic site changes its shape, which prevents a second substrate from binding to -secretase. However, the catalytic site is open to small molecules before the substrate shifting and is accessible to the inhibitor. Therefore, binding of the inhibitor at the catalytic site of the ES complex, with S bound at the docking site, causes non-competitive inhibition. B, substrate anchoring and swinging. Substrate first anchors itself at the anchoring site on the enzyme and then swings the rest of the substrate into the catalytic site. The enzyme does not need to change its conformation to prevent a second substrate from binding. The catalytic site is open before the substrate swinging step and is accessible to inhibitor. Binding of inhibitor at the catalytic site of ES complex with S anchored at the docking site causes non-competitive inhibition.

Several other possibilities exist that may also explain the non-competitive inhibition kinetics. For example, the inhibitor may bind to an intermediate on the catalytic pathway of the substrate, which would result in non-competitive inhibition. However, a substrate-derived intermediate, such as an acyl-enzyme complex, is not expected to be present along the catalytic pathway if -secretase is indeed an aspartyl protease. Another possibility would be that the active site is shaped as a channel with the docking site located at the entrance and the catalytic site at the end. The catalytic site would be accessible to the inhibitor while a substrate molecule is bound at the opening of the channel. The substrate binding process may also involve docking proteins that deliver specific substrates in much the same way E2 conjugases introduce specific substrates to E3 ligases (30). The docking proteins would have specific binding sites for the substrate and would share a common binding domain for -secretase.

These putative mechanisms are attractive in light of the data presented here, explaining the non-competitive inhibition kinetics of known aspartyl protease inhibitor isosteres, PME and L685458, and providing additional sites of interactions for non-transition state inhibitors such as the sulfonamides and benzodiazepines that also inhibit -secretase activity. Whether these other inhibitors block -secretase by binding at the catalytic or some other site awaits further investigation.

Typically, enzymes involved in intermediate metabolism have an active site that is generally compact, and the residues involved in binding of substrate are located close to catalytic groups. For such enzymes, transition state analogs usually inhibit the enzyme competitively. However, in the case of proteases, which catalyze reactions with proteins as substrate, groups involved in binding and catalysis may have greater spatial separation so that binding and catalysis may happen at different locations (Fig. 6). In such cases, transition state analogs may display non-competitive inhibition, whereas non-transition state analogs may show competitive inhibition if their binding interferes with substrate binding. For enzymes such as -secretase, which are capable of catalyzing reactions with different protein substrates, non-competitive transition state analogs will be unable to selectively inhibit reactions between different substrates; all the substrates will need to use the same catalytic machinery for reactions regardless of how they bind to the enzymatic machinery. On the other hand, non-transition state inhibitors could be selective for a particular substrate if different substrates bind or anchor at different sites. Such inhibitors will be competitive with the substrate that shares the binding pocket with the inhibitor and non-competitive with substrate that does not. In the case of -secretase, if APP and Notch are anchored either at different sites or on separate docking proteins associated with the enzymatic machinery, it may be possible to develop inhibitors that are selective for APP over Notch or vice versa by targeting the anchor site rather than the catalytic site.

	ACKNOWLEDGEMENTS

We thank Dr. Deborah Hartman for sponsoring the work described in this paper and for critical reading of the manuscript. We thank Michael T. Klimas, Thomas R. Simpson, James M. Woods, James Kang, Peter R. Bernstein, Robert D. Dedinas, Bruce T. Dembofsky, Michael Balestra, Jingbo Yan, and James D. Rosamond for contributions to the syntheses of compounds 1-4. We are indebted to Dr. Nathan Spear for preparing part of the ruthenium-labeled goat anti-rabbit antibody used in this study. Dr. David D. Aharony is acknowledged for useful comments and discussion. We thank Drs. Robert Bostwick and Ron P. Julien for technical expertise and useful discussions. Drs. Michael S. Wolfe and P. W. Esler are sincerely acknowledged for insightful discussions.

	FOOTNOTES

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

To whom correspondence should be addressed. Tel.: 302-886-8137; Fax: 302-886-4983; E-mail: gaochao.tian@astrazeneca.com.

Published, JBC Papers in Press, June 18, 2002, DOI 10.1074/jbc.M112328200

	ABBREVIATIONS

The abbreviations used are: A, -amyloid; APP, amyloid precursor protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPSO, 2-hydoxy-CHAPS; CTF, C-terminal fragment of presenilin; CTF, CTF of -secretase-cleaved APP (C83); CTF, CTF fragment of -secretase-cleaved APP (C99); NTF, N-terminal fragment of presenilin; PME, pepstatin A methylester; PS, presenilin; RAA40, rabbit anti-A40; MES, 4-morpholineethanesulfonic acid.

	APPENDIX

Assuming fast equilibria for the substrate and inhibitor bindings and assuming that the inhibitor binds to both the substrate-binding site and an allosteric site according to the following mechanism shown in Equation A1,

(Eq. A1)

the initial rate equation is then given by Equation A2.

(Eq. A2)

Thus the slope and intercept replots will be, respectively, a parabolic and linear function of [I] as shown in Equation A3,

(Eq. A3)

and Equation A4.

(Eq. A4)

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION APPENDIX REFERENCES

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				E. E. Clarke, I. Churcher, S. Ellis, J. D. J. Wrigley, H. D. Lewis, T. Harrison, M. S. Shearman, and D. Beher Intra- or Intercomplex Binding to the {gamma}-Secretase Enzyme: A MODEL TO DIFFERENTIATE INHIBITOR CLASSES J. Biol. Chem., October 20, 2006; 281(42): 31279 - 31289. [Abstract] [Full Text] [PDF]

				Y. Morohashi, T. Kan, Y. Tominari, H. Fuwa, Y. Okamura, N. Watanabe, C. Sato, H. Natsugari, T. Fukuyama, T. Iwatsubo, et al. C-terminal Fragment of Presenilin Is the Molecular Target of a Dipeptidic {gamma}-Secretase-specific Inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-Butyl Ester) J. Biol. Chem., May 26, 2006; 281(21): 14670 - 14676. [Abstract] [Full Text] [PDF]

				N. Kakuda, S. Funamoto, S. Yagishita, M. Takami, S. Osawa, N. Dohmae, and Y. Ihara Equimolar Production of Amyloid beta-Protein and Amyloid Precursor Protein Intracellular Domain from beta-Carboxyl-terminal Fragment by {gamma}-Secretase J. Biol. Chem., May 26, 2006; 281(21): 14776 - 14786. [Abstract] [Full Text] [PDF]

				A. Yamasaki, S. Eimer, M. Okochi, A. Smialowska, C. Kaether, R. Baumeister, C. Haass, and H. Steiner The GxGD motif of presenilin contributes to catalytic function and substrate identification of gamma-secretase. J. Neurosci., April 5, 2006; 26(14): 3821 - 3828. [Abstract] [Full Text] [PDF]

				S. Oddo, A. Caccamo, I. F. Smith, K. N. Green, and F. M. LaFerla A Dynamic Relationship between Intracellular and Extracellular Pools of A{beta} Am. J. Pathol., January 1, 2006; 168(1): 184 - 194. [Abstract] [Full Text] [PDF]

				Y. Nakaya, T. Yamane, H. Shiraishi, H.-Q. Wang, E. Matsubara, T. Sato, G. Dolios, R. Wang, B. De Strooper, M. Shoji, et al. Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid {beta}-Peptides J. Biol. Chem., May 13, 2005; 280(19): 19070 - 19077. [Abstract] [Full Text] [PDF]

				D. M. Barten, V. L. Guss, J. A. Corsa, A. Loo, S. B. Hansel, M. Zheng, B. Munoz, K. Srinivasan, B. Wang, B. J. Robertson, et al. Dynamics of {beta}-Amyloid Reductions in Brain, Cerebrospinal Fluid, and Plasma of {beta}-Amyloid Precursor Protein Transgenic Mice Treated with a {gamma}-Secretase Inhibitor J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 635 - 643. [Abstract] [Full Text] [PDF]

				D. Drygin, S. Barone, and C. F. Bennett Sequence-dependent cytotoxicity of second-generation oligonucleotides Nucleic Acids Res., December 16, 2004; 32(22): 6585 - 6594. [Abstract] [Full Text] [PDF]

				D. Beher, E. E. Clarke, J. D. J. Wrigley, A. C. L. Martin, A. Nadin, I. Churcher, and M. S. Shearman Selected Non-steroidal Anti-inflammatory Drugs and Their Derivatives Target {gamma}-Secretase at a Novel Site: EVIDENCE FOR AN ALLOSTERIC MECHANISM J. Biol. Chem., October 15, 2004; 279(42): 43419 - 43426. [Abstract] [Full Text] [PDF]

				B. O. Popescu, A. Cedazo-Minguez, E. Benedikz, T. Nishimura, B. Winblad, M. Ankarcrona, and R. F. Cowburn {gamma}-Secretase Activity of Presenilin 1 Regulates Acetylcholine Muscarinic Receptor-mediated Signal Transduction J. Biol. Chem., February 20, 2004; 279(8): 6455 - 6464. [Abstract] [Full Text] [PDF]

				M. Zayzafoon, S. A. Abdulkadir, and J. M. McDonald Notch Signaling and ERK Activation Are Important for the Osteomimetic Properties of Prostate Cancer Bone Metastatic Cell Lines J. Biol. Chem., January 30, 2004; 279(5): 3662 - 3670. [Abstract] [Full Text] [PDF]

				Y. Taniguchi, S.-H. Kim, and S. S. Sisodia Presenilin-dependent "{gamma}-Secretase" Processing of Deleted in Colorectal Cancer (DCC) J. Biol. Chem., August 15, 2003; 278(33): 30425 - 30428. [Abstract] [Full Text] [PDF]

				G. Tian, S. V. Ghanekar, D. Aharony, A. B. Shenvi, R. T. Jacobs, X. Liu, and B. D. Greenberg The Mechanism of {gamma}-Secretase: MULTIPLE INHIBITOR BINDING SITES FOR TRANSITION STATE ANALOGS AND SMALL MOLECULE INHIBITORS J. Biol. Chem., August 1, 2003; 278(31): 28968 - 28975. [Abstract] [Full Text] [PDF]

				O. Berezovska, P. Ramdya, J. Skoch, M. S. Wolfe, B. J. Bacskai, and B. T. Hyman Amyloid Precursor Protein Associates with a Nicastrin-Dependent Docking Site on the Presenilin 1-{gamma}-Secretase Complex in Cells Demonstrated by Fluorescence Lifetime Imaging J. Neurosci., June 1, 2003; 23(11): 4560 - 4566. [Abstract] [Full Text] [PDF]

				Y. Takahashi, I. Hayashi, Y. Tominari, K. Rikimaru, Y. Morohashi, T. Kan, H. Natsugari, T. Fukuyama, T. Tomita, and T. Iwatsubo Sulindac Sulfide Is a Noncompetitive gamma -Secretase Inhibitor That Preferentially Reduces Abeta 42 Generation J. Biol. Chem., May 9, 2003; 278(20): 18664 - 18670. [Abstract] [Full Text] [PDF]

				A. Y. Kornilova, C. Das, and M. S. Wolfe Differential Effects of Inhibitors on the gamma -Secretase Complex. MECHANISTIC IMPLICATIONS J. Biol. Chem., May 2, 2003; 278(19): 16470 - 16473. [Abstract] [Full Text] [PDF]

				T. Ikeuchi, G. Dolios, S.-H. Kim, R. Wang, and S. S. Sisodia Familial Alzheimer Disease-linked Presenilin 1 Variants Enhance Production of Both Abeta 1-40 and Abeta 1-42 Peptides That Are Only Partially Sensitive to a Potent Aspartyl Protease Transition State Inhibitor of "gamma -Secretase" J. Biol. Chem., February 21, 2003; 278(9): 7010 - 7018. [Abstract] [Full Text] [PDF]

				L. Toulokhonova, W. J. Metzler, M. R. Witmer, R. A. Copeland, and J. Marcinkeviciene Kinetic Studies on beta -Site Amyloid Precursor Protein-cleaving Enzyme (BACE). CONFIRMATION OF AN ISO-MECHANISM J. Biol. Chem., February 7, 2003; 278(7): 4582 - 4589. [Abstract] [Full Text] [PDF]

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