The Mechanism of {gamma}-Secretase

MULTIPLE INHIBITOR BINDING SITES FOR TRANSITION STATE ANALOGS AND SMALL MOLECULE INHIBITORS*

Gaochao Tian {ddagger} §, Smita V. Ghanekar ¶, David Aharony ¶, Ashok B. Shenvi ||, Robert T. Jacobs ||, Xiaodong Liu ** and Barry D. Greenberg **

From the Departments of {ddagger}Lead Discovery, Neuroscience, **Molecular Sciences, and ||Chemistry, AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850

Received for publication, January 28, 2003 , and in revised form, April 24, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transition state analogs pepstatin methylester (PME) and L685458 have been shown to inhibit {gamma}-secretase non-competitively (Tian, G., Sobotka-Briner, C., Zysk, J., Liu, X., Birr, C., Sylvester, M. A., Edwards, P. D., Scott, C. W., and Greenberg, B. D. (2002) J. Biol. Chem. 277, 31499–31505). This unusual kinetics suggests physical separation of the sites for substrate binding and catalysis with binding of the transition state analogs to the catalytic site and not to the substrate binding site. Methods of inhibitor cross-competition kinetics and competition ligand binding were utilized to address whether non-transition state small molecule inhibitors, which also display non-competitive inhibition of {gamma}-secretase, inhibit the enzyme by binding to the catalytic site as well. Inhibitor cross-competition kinetics indicated competitive binding between the transition state analogs PME and L685458 and between small molecules arylsulfonamides and benzodiazepines, but non-competitive binding between the transition state analogs and the small molecule inhibitors. These results were indicative of two inhibitor binding sites, one for transition state analogs and the other for non-transition state small molecule inhibitors. The presence of two inhibitor binding sites for two different classes of inhibitors was corroborated by results from competition ligand binding using [3H]L685458 as the radioligand. Although L685458 and PME displaced the radioligand at the same concentrations as for enzyme inhibition, arylsulfonamides and benzodiazepines did not displace the radioligand at their Ki values, a result consistent with the presence of two inhibitor binding sites. These findings provide useful insights into the catalytic and regulatory mechanisms of {gamma}-secretase that may facilitate the design of novel {gamma}-secretase inhibitors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer's disease is characterized by formation of neurofibrillary tangles and amyloid plaques in the regions of the central nervous system that are involved in learning and memory (1). It is believed that accumulation of A{beta}1 in plaques or as soluble aggregates initiates a pathological cascade leading to synaptic dysfunction and neuronal toxicity, with neurodegeneration and dementia as the final outcome (1, 2). Therefore, strategies to reduce the level of brain A{beta} are being aggressively pursued as an approach likely to benefit Alzheimer's disease patients.

A{beta} is produced as the result of sequential proteolysis of a type I transmembrane protein APP by {beta}- and {gamma}-secretases. {beta}-Secretase cleaves APP in its extracellular domain at a site close to the membrane surface, a reaction that generates a membrane-bound APP C-terminal fragment of 99 amino acid residues (C99). A subsequent endoproteolysis within the transmembrane domain of C99 by {gamma}-secretase produces A{beta}. Whereas {beta}-secretase, an aspartyl protease, has been well characterized (36), the identity and structure of {gamma}-secretase, also thought to be an aspartyl protease (712), remains elusive, and its kinetic and catalytic mechanisms are poorly understood. To a large extent, this is due to the highly complicated structural organization of this unusual protease. In contrast to other known proteases, {gamma}-secretase is composed of a high molecular weight multicomponent complex of transmembrane proteins (13, 14). Primarily due to this structural complexity, the catalytic site and mechanism of action of {gamma}-secretase has not been unequivocally established. Early findings point to presenilin 1 or 2 as the catalytic subunit of {gamma}-secretase (15, 16). These multipass transmembrane proteins contain two essential aspartate residues in putatively adjacent transmembrane domains (15) and can be cross-linked by high affinity {gamma}-secretase inhibitors (1618). Recent advances have identified three additional proteins, nicastrin (19), aph-1 (20, 21), and pen-2 (20, 22), in the same multicomponent complex, whose co-expression with presenilin appears to be critical for {gamma}-secretase activity (1922). However, the precise roles of these additional protein subunits in the catalytic mechanism of {gamma}-secretase await further investigation.

Associated with the structural complexity of {gamma}-secretase is the versatility of this protease in cleaving several type I transmembrane proteins. In addition to APP processing, {gamma}-secretase is required for proteolytic activation of Notch receptor (2326), a signaling molecule essential for embryonic development of all metazoan species (27). Cleavage of Notch in the transmembrane domain by {gamma}-secretase generates Notch intracellular domain (NICD), which then translocates into the nucleus, where it regulates gene transcription (28, 29). The list of other potential protein substrates for {gamma}-secretase has recently been expanded to include ErbB4 (30, 31), E-cadherin (32), and CD44 (33). However, the mechanisms by which {gamma}-secretase reacts with these different substrates remains unknown. Given the importance of {gamma}-secretase in the Notch pathway, as well as its potential importance in pathways involving these other substrates, it is highly desirable to develop inhibitors selective for APP cleavage. Recent advances in understanding the kinetics and mechanisms of {gamma}-secretase activity suggest that this may be possible.

Transition state isosteres and small molecule inhibitors have been described for {gamma}-secretase. Recently, PME and L685458, which are transition state isosteres, have been shown to display non-competitive inhibition of {gamma}-secretase (34), suggesting an unprecedented enzyme kinetic mechanism that involves physical separation of substrate binding and catalysis (34). Inhibitors that bind to the catalytic site, as is normally expected for transition state isosteres, should impact similarly on the catalytic activities of the enzyme with different substrates. Inhibitors that target a substrate binding site or some other modulatory sites on {gamma}-secretase may, on the other hand, display substrate-selective effects (34). Interestingly, arylsulfonamides and benzodiazepines, which do not resemble transition state isosteres, also display non-competitive inhibition of {gamma}-secretase (34). This raised the question of whether these small molecule inhibitors also affect {gamma}-secretase activity by binding to the same site as the transition state isosteres or interact with {gamma}-secretase at some other as yet unknown binding sites.

To address this issue, we employed the methods of inhibitor cross-competition kinetics and competition ligand binding to characterize further the interactions of {gamma}-secretase with transition state analogs (PME and L685458) and small molecule inhibitors (arylsulfonamide 1 and benzodiazepine 2, also known as compound E) (see Fig. 1). Data obtained from inhibitor cross-competition kinetics suggested the presence on {gamma}-secretase of two inhibitor binding sites, one for binding of transition state isosteres and the other for non-transition state small molecule inhibitors. This observation was corroborated by results from competition ligand binding with the radioligand [3H]L685458. These findings provide useful insights into the catalytic and regulatory mechanisms of {gamma}-secretase that may facilitate the design of novel, and possibly substrate-selective, inhibitors.



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FIG. 1.
{gamma}-Secretase inhibitors used in the current study. Chemical structures of PME and L685458, which are transition state analogs of aspartyl proteases, and small molecule inhibitors arylsulfonamide 1 and benzodiazepine 2 (Compound E) are shown.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—CHAPS and CHAPSO were purchased from Pierce. Phosphotidylcholine and phosphotidylethanolamide were purchased from Avanti Polar Lipids. A{beta}40 and R{alpha}A{beta}40 were purchased from BIOSOURCE. Biotin-4G8 was from Senetek PLC. Dynabeads (M-280) were purchased from IGEN. Ruthenium-labeled G{alpha}R, C100, and detergent-solubilized human {gamma}-secretase in Tris-HCl at pH 8.4 were prepared as described previously (34). Preparation of the enzyme in a buffer of pH 6.5 was performed under the same conditions as described for the enzyme preparation at pH 8.4 (34) except that Tris-HCl was replaced with MES. PME, L685458, 1, and 2 were prepared as reported previously (34).

Synthesis of [3H]L685458 —[3H]L685458 was synthesized by tritiation, under catalytic conditions, of {1S-benzyl-4R-[1-(1-carbamoyl-2-phenyl-ethylcarbamoyl)-1S-3-methyl-but-3-enylcarbamoyl]-2R-hy droxy5-phenyl-pentyl}-carbamic acid tert-butyl ester (dehydroleucine derivative of L685458). The desired dehydroleucine derivative of L685458 was prepared by a method similar to that described for the synthesis of L685458 (35) except that 4,5-dehydro-Leu-Phe-NH2 was used instead of Leu-Phe-NH2.

Enzyme and Inhibition Kinetics—Enzyme and inhibition kinetics were performed using the methods as described previously (34) with minor modifications. All the reactions were run in 96-well microplates. Reactions with a final volume of 280 µl were run in 25 mM MES, pH 6.5, containing C100 at a defined concentration, solubilized enzyme at 20–40-fold dilution from stock (prepared at either pH 8.4 or pH 6.5), inhibitor or inhibitors at different combinations of final concentrations 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% phosphotidylethanolamide, and 0.01% phosphotidylcholine. The reactions were initiated by addition of enzyme, and 40-µl aliquots of the reaction mixture were quenched at six different times by addition of 50 µl of 200 µM PME. A 50-µl solution containing 0.2 µg/ml R{alpha}A{beta}40 and 0.25 µg/ml biotin-4G8 was added to each quenched reaction mixture. The solutions were shaken in 96-well plates at 4 °C for >7 h. Subsequently, 50 µl of a solution containing 0.018 µg/ml Ru-G{alpha}R and 62.5 µg/ml Dynabeads was added, and the plates were incubated at room temperature for 1 h, at which time they were measured for ECL counts in an IGEN ECL M8 analyzer.

Competition Ligand Binding—In a 96-well plate, 20 µl of [3H]L685458 (final concentration 1 nM) was mixed with 170 µl of solubilized {gamma}-secretase at a final 10-fold dilution of the stock (prepared at pH 6.5) and 10 µl of a test compound (with a final concentration in the range of 100 pM to 30 µM) in Me2SO or in Me2SO only. The mixture was incubated on a plate shaker at room temperature for 30 min, and then filtered through Beckman GF/B filters (soaked in 0.6% polyethyleneimmine) using a Packard Filtermate-196 and washed six times with 5 mM Tris-HCl, pH 7.4. After the filters dried, 30 µl of Microscint20 (Packard) was added to each well, and the plates were counted on a Packard TopCount. To assay binding of the radioligand itself, the same conditions were used as in the ligand competition studies, except that 12 concentrations (between 0.4 pM and 12 nM) of [3H]L685458 were used in the absence of test compound.

Data Analysis—Time courses of {gamma}-secretase reaction as monitored by ECL assay were analyzed by linear regression to obtain the slopes in counts/min. These values were then converted to pM/min by an ECL assay of an A{beta}40 standard containing C100 at the same concentration as used in the reaction.

IC50, the inhibitor concentration at which the enzyme activity is inhibited by 50%, was obtained by fitting inhibition data to,

(Eq. 1)
where %Act is calculated by dividing the ECL signal of a reaction mixture in the presence of inhibitor with the ECL signal obtained in its absence.

For inhibitor cross-competition analyses, the theoretical inhibition kinetics of two inhibitors, I1 and I2, is given by,

(Eq. 2)
where {nu}0 is the initial rate in the absence of inhibitor, Ki1 and Ki2 are inhibition constants for I1 and I2, respectively, and {alpha} is the constant defining the interaction between the two inhibitors. The value of {alpha} equals unity for binding of the two inhibitors to enzyme independently. An {alpha} value of <1 indicates that binding of one inhibitor facilitates the binding of the other. A value of >1 indicates that binding of one inhibitor makes the binding of the other more difficult. An infinitely large {alpha} indicates that bindings of the two inhibitors are mutually exclusive or competitive. In the latter case, Equation 2 reduces to,

(Eq. 3)
which describes competitive binding. The pattern of binding of two inhibitors may be analyzed graphically by using the reciprocal of Equation 2, which is a linear function of 1/{nu} versus [I1], as given by,

(Eq. 4)
Changes in [I2] will have a slope effect if {alpha} is close to unity but will have no slope effect if it is infinitely large. Therefore, if the two inhibitors bind simultaneously to the enzyme, the reciprocal plots will intercept on, or to the left of, the 1/{nu} axis. If binding of one inhibitor excludes the binding of the other, the reciprocal plots will be a set of parallel lines.

Data of radioligand [3H]L685458 binding to detergent extracts of HeLa cell membranes were analyzed by,

(Eq. 5)
where [L685458]b is the concentration of bound L685458, [L685458]bmax is the concentration of maximum bound ligand, and Kd is the dissociation constant for the binding. The ligand displacement data were analyzed by,

(Eq. 6)
where [L685458]t is the total concentration of L685458, and Ki is the dissociation constant for I.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibitor Cross-competition Kinetics—The cross-competition method can provide information on whether enzyme inhibition in the presence of two inhibitors may arise from simultaneous binding to independent sites or mutually exclusive binding to a single site or overlapping sites on the enzyme. In this study, all possible binary combinations of the four inhibitors (PME, L685458, arylsulfonamide 1, and benzodiazepine 2, shown in Fig. 1) were investigated using this approach. The data were analyzed according to Equation 2 to extract the kinetic constants, and the reciprocal 1/{nu} was plotted against [I1] to visualize the slope effect from varying [I2].

Variation of [PME] in both directions in a binary combination was first performed to validate the method of inhibitor cross-competition kinetics for {gamma}-secretase. As anticipated, variation of [PME] in one direction had no effect on the slope of the reciprocal plot obtained from variation of [PME] in the other direction, producing parallel plots as shown in Fig. 2A. The same parallel pattern was observed for the cross-competition between PME and L685458 (Fig. 2B), indicating competitive binding of PME and L685458. This result is consistent with the fact that both PME and L685458 are transition state isosteres, and therefore, are expected to bind to the same catalytic site of {gamma}-secretase. Cross-competition pairs formed between L685458 and either of the small molecule inhibitors produced an intercepting, or non-competitive, binding pattern (Fig. 3), indicating that the small molecule inhibitors bind to one or two sites different from the site for the transition state analogs. The pattern for the kinetics of cross-competition between 1 and 2 was, however, parallel (Fig. 4), suggesting that it is a single site to which both 1 and 2 bind. The relatively small size of the {alpha} constants (1.7–2.5) obtained for the competitions between L685458 and the small molecule inhibitors 1 or 2 (Table I) indicated that binding of L685458 had little effect on binding of the small molecule inhibitors at a different site. The cross-competition between PME and 1 or 2 also displayed an intercepting, non-competitive pattern (Fig. 5). However, the size of the {alpha} values was relatively large ({alpha} = 7–17, Table I), suggesting that binding of PME affected somewhat the binding of 1 or 2 to the other binding site, making it more difficult for 1 or 2 to bind.



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FIG. 2.
Reciprocal plots for inhibitor cross-competition between transition state isosteres PME and L685458. The graphs show cross-competition between PME and PME (A) at [PME] = 0 ({circ}), 0.17 ({square}), 0.5 ({triangleup}), and 1.5 ({diamond}) µM and between PME and L685458 (B) at [L685458] = 0 ({circ}), 0.67 ({square}), 2 ({triangleup}), and 6 ({diamond}) nM at pH 6.5, 22 °C. The lines are theoretical values calculated using Equation 4 with the parameters listed in Table I.

 


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FIG. 3.
Reciprocal plots for inhibitor cross-competition between L685458 and small molecule inhibitors. The graphs show cross-competition between L685458 and 1 (A) at [1] = 0 ({circ}), 1.1 ({square}), 3.3 ({triangleup}), and 10 ({diamond}) nM or 2 (B) at [2] = 0 ({circ}), 0.56 ({square}), 1.7 ({triangleup}), and 5 ({diamond}) nM at pH 6.5, 22 °C. The lines are theoretical values calculated using Equation 4 with the parameters listed in Table I.

 


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FIG. 4.
Reciprocal plots for inhibitor cross-competition between small molecule inhibitors. The graphs show cross-competition between 2 and 1 at [1] = 0 ({circ}), 1.1 ({square}), 3.3 ({triangleup}), and 10 ({diamond}) nM at pH 6.5, 22 °C. The lines are theoretical values calculated using Equation 4 with the parameters listed in Table I.

 

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TABLE I
Summary of kinetic inhibition constants obtained from inhibitor cross-competition kinetics

Cross-competition kinetics was performed at pH 6.5, 22 °C, with a combination of two inhibitors at various concentrations. The data were analyzed by non-linear least squares analysis using Equations 2 or 3 to obtain Ki1 and Ki2, the inhibition constants for inhibitors 1 or 2, respectively.

 


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FIG. 5.
Reciprocal plots for inhibitor cross-competition between PME and small molecule inhibitors. The graphs show cross-competition between PME and 1 (A) at [1] = 0 ({circ}), 1.1 ({square}), 3.3 ({triangleup}), and 10 ({diamond}) nM and between PME and 2 (B) at [2] = 0 ({circ}), 0.56 ({square}), 1.7 ({triangleup}), and 5 ({diamond}) nM at pH 6.5, 22 °C. The lines are theoretical values calculated using Equation 4 with the parameters listed in Table I.

 

Competition Ligand Binding—Competition ligand binding was conducted to characterize further the interaction of different {gamma}-secretase inhibitors with the enzyme. [3H]L685458 was synthesized and used as the radioligand. Binding to the enzyme was assayed by measuring the counts associated with the enzyme complex captured on filter-bottom 96-well microplates (see under "Experimental Procedures"). The time courses for binding of [3H]L685458 (2 nM) and its displacement in the presence of cold L685458 yielded a kon of 0.069 ± 0.01 nM1 min1 (or 0.14 ± 0.02 min1 at 2 nM [3H]L685458) (data not shown) and a koff of 0.017 ± 0.006 min1 (data not shown), which translate into a Kd of ~0.3 nM. This Kd value is in reasonable agreement with the Kd and Ki values from ligand binding and displacement experiments or IC50 values obtained from enzyme inhibition (Table II). An initial test indicated that maintaining a signal level sufficient for statistically meaningful analysis required an enzyme concentration that was no more than a 10-fold dilution of the stock, as compared with the 20–40-fold dilution used in inhibitor cross-competition kinetics. All subsequent ligand binding assays were then performed at this enzyme concentration. This initial test also indicated that for ligand displacement, 1 nM [[3H]L685458] was sufficient, and this concentration of radioligand was used for all the ligand displacement experiments.


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TABLE II
Summary of constants for ligand binding and displacement and inhibition of {gamma}-secretase at different enzyme concentrations

Ligand binding and displacement experiments were performed at [E] = 2.8 nM, and IC50 values were obtained with [E] = 0.7 or 2.8 nM at pH 6.5, 22 °C. The Kd was obtained by non-linear least squares analysis of data depicted in Fig. 6A according to Equation 5, and the Ki values were obtained by analyzing data depicted in Fig. 6B using Equation 6. The IC50 values were the results of analyzing inhibition data using Equation 1.

 



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FIG. 6.
Ligand binding and competition. A, [3H]L685458 binding to solubilized {gamma}-secretase. The circles represent experimental data, and the solid line represents the theoretical values calculated using Equation 5 with [[3H]L685458]bmax = 2.9 nM and Kd = 1 nM (Table II). B, displacement of [3H]L685458 by C100 ({triangledown}), PME ({triangleup}), L685458 ({diamond}), 1 ({circ}), and 2 ({square}) at pH 6.5, 22 °C. The solid lines are theoretical values calculated using Equation 6 with [[3H]L685458]t = 1nM, Kd = 1nM and the Ki values listed in Table II. C, [3H]L685458 binding to solubilized {gamma}-secretase in the absence ({triangleup}) and presence of 2 ({square}) or PME ({circ}). The solid lines represent the theoretical values calculated using Equation 5 with parameters listed in Table III.

 
The ligand binding curve obtained with a range of radioligand concentrations is depicted in Fig. 6A. Analysis of this data set using a hyperbolic equation (Equation 5) yielded a [[3H]L685458]bmax of 2.8 nM (data not shown), with an apparent Kd value of 1.0 ± 0.3 nM (Table II). This apparent Kd is comparable with the Ki value (1.9 ± 1.1 nM, Table II) obtained from displacement of the radioligand with the unlabeled ligand (Fig. 6B). Since the calculated maximum concentration of bound ligand is used to determine the concentration of enzyme active sites, the value for [[3H]L685458]bmax obtained in the ligand binding experiments indicated that the active enzyme concentration was about 2.8 nM. The fact that this value was close to the apparent Kd suggested that a partial depletion of free ligand might have happened at some ligand concentrations used. Thus, the Kd obtained in this manner (using Equation 5) might have somewhat underestimated the true affinities of the radioligand in these experiments, as well as the Ki values to be obtained later (using Equation 6) from subsequent competition ligand binding experiments. However, under partial ligand depletion conditions, such simple analyses would only have small perturbations in the true Ki values and would not have altered the conclusions regarding the sites of inhibitor binding. In support of this, the Ki values of 1.0–1.9 nM (Table II) obtained from the ligand binding or competition were statistically indistinguishable from the Ki values of 1.3–2.7 nM (Table I) obtained from kinetic inhibitor cross-competition, in which the enzyme concentration was 2–4-fold less than that used in ligand binding. Therefore, no data treatment with quadratic (for ligand binding) or cubic equations (for ligand competition) was applied to maintain the simplicity for data analysis.


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TABLE III
Summary of constants for ligand binding in the presence of 2 and PME

These experiments were performed at [E] = 2.8 nM in the absence or presence of 1 µM 2 or 30 µM PME. Kd was obtained by non-linear least squares analysis of data depicted in Fig. 6C according to Equation 5. Kd(calculated) was calculated using Kd(calculated) = Kd(L685458 alone)(1 + [I]/Ki), where I is either 2 or PME and Ki is the constant obtained from ligand displacement experiments (Table II).

 
There was no observed displacement of L685458 by C100, the substrate used for kinetic studies, at concentrations up to 5 µM, a value much greater than the Km for C100. This is consistent with the previous finding that transition state analogs inhibit {gamma}-secretase non-competitively (34).

Surprisingly, the Ki of 8.3 ± 2.3 µM (Table II) for PME in the displacement of L685458 was significantly greater than the values of 0.67–0.83 µM (Table I) obtained from the kinetic cross-competition. Radioligand binding performed in the presence of PME (30 µM) shifted the observed Kd for radioligand accordingly without perturbing Bmax (Fig. 6C and Table III). The size of the observed Kd is similar to the Kd calculated assuming binding of PME and L685458 at the same site. This result appeared to provide further support for the competitive binding between L685458 and PME, albeit suggesting that this competitive ligand binding occurred with a significantly higher Ki value than inhibitor cross-competition.

Recognizing that the only major difference in experimental conditions between kinetic cross-competition and ligand binding or displacement was the enzyme concentration (0.7–1.4 nM in kinetic inhibition and 2.8 nM in ligand binding), we assessed in more detail whether such differences in enzyme concentration have any effect on the enzymatic and inhibition kinetics, thereby causing the observed discrepancy in the Ki values obtained from inhibitor cross-competition and ligand displacement. As shown in Fig. 7, the enzyme activity was linear with [E] up to ~1.5 nM and then leveled off at higher enzyme concentrations. This perturbation in enzyme activity at higher enzyme concentrations resulted in a 15-fold increase in the IC50 value for PME (Table II), indicating that the inflated Ki value for PME obtained from ligand displacement was likely due to reduced affinity of the more concentrated enzyme for PME. The IC50 value for L685458, however, was essentially identical at both enzyme concentrations, as were the values for 1 or 2 (Table II). These data suggested that the increased [E] might have had a subtle structural effect in the binding site for the transition state analogs that was particular for PME. Taking this perturbation into account, the ligand competition results do appear to support the conclusion from inhibitor cross-competition kinetics of L685458 and PME binding at the same site. These results also demonstrate the importance of validating assay conditions thoroughly when developing series of inhibitors or studying kinetic mechanisms.



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FIG. 7.
Effect of enzyme concentration on the enzyme activity of {gamma}-secretase. A plot of initial rate ({nu}) as a function of the enzyme concentration at pH 6.5, 22 °C with [C100] = 0.6 µM is shown.

 

The competition ligand binding assay was subsequently applied to investigate whether bound [3H]L685458 could be displaced with the small molecule inhibitors 1 and 2. As shown in Fig. 6B, 1 did not reduce the bound radioligand at any of the inhibitor concentrations used (up to 30 µM), indicating that 1 did not bind at the site for L685458. On the other hand, 2 did show displacement of bound [3H]L685458 (Fig. 6B). This observed ligand competition by 2 was also supported by shifting of the observed Kd for radioligand in the presence of 1 µM 2 (Fig. 6C and Table III). However, the Ki value of 130 ± 50 nM (Table II) for displacement of L685458 by 2 was much greater than the Ki value of 1.2–2.5 nM (Table I) obtained from inhibitor cross-competition kinetics or the IC50 value of 0.6 nM from inhibition obtained at the same enzyme concentration (2.8 nM) (Table II). Therefore, inhibition of {gamma}-secretase by 2 could not have occurred via binding to the site for L685458 but rather via binding of 2 to a different site. These data are consistent with the results from cross-competition kinetics that 1 and 2 bind at a site different from the site for the transition state isosteres PME and L685458.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The non-competitive inhibition of {gamma}-secretase by the transition state isosteres PME and L685458, as reported previously (34), and the lack of observed displacement of L685458 by C100 as shown in the present study, are consistent with a mechanism in which substrate binding and catalysis occur at different locations on the enzyme and the catalysis requires a substrate movement after binding at the substrate binding site that swings the scissile bond into the catalytic site (34). This mechanism is supported by co-purification of C83, an alternative substrate of {gamma}-secretase of APP-derived fragment, using affinity columns prepared with derivatized transition state analogs (36). Delineation of the site on PS1 for binding of APP/C99 that is away from the proposed catalytic site on PS1 has also led to the proposal for spatial dissociation between substrate binding and catalysis that requires substrate movement for catalysis (37). By this mechanism, binding of the transition state analogs at the catalytic site would not necessarily interfere with the binding of substrate to the enzymatic complex (34).

The data presented in this study from inhibitor cross-competition kinetics and competition ligand binding indicate the presence on {gamma}-secretase of two inhibitor binding sites, one for the transition state isosteres PME and L685458 and the other for non-transition state small molecule arylsulfonamide and benzodiazepine inhibitors. It is conceivable that the site on {gamma}-secretase for binding transition state analogs is the site for catalysis. The question remaining is how the activity of {gamma}-secretase is inhibited by the small molecule inhibitors arylsulfonamides and benzodiazepines, which also display non-competitive inhibition of {gamma}-secretase (34) but bind at a site different from the site for transition state analogs (this study).

There are at least three ways to inhibit an enzyme where substrate binding and catalysis occur at different locations: (a) interfering with substrate binding, (b) affecting catalysis, and (c) blocking movement of substrate into the catalytic site. The first two are common enzyme inhibition mechanisms, whereas the third is only applicable for enzymes with separate substrate and catalytic sites. For such enzymes, catalysis requires movement, either by translocation or by swinging (34), of bound substrate into the catalytic site. Blocking substrate movement, which would display a non-competitive kinetic inhibition, may be accomplished either by inhibitor binding at a site located between the sites of substrate binding and catalysis to form a direct physical blockage or by a conformational change induced upon binding of inhibitor at a remote site.

The non-competitive inhibition of {gamma}-secretase demonstrated for arylsulfonamides and benzodiazepines (34) rules out the possibility that these inhibitors either bind at the substrate binding site or induce a conformational change in the substrate binding site when bound at a remote site. As a result, these small molecule inhibitors do not seem to inhibit {gamma}-secretase by interfering with substrate binding. Rather, the non-competitive inhibition displayed by these small molecule inhibitors (34) is consistent with inhibition by directly affecting catalysis. However, it is difficult to explain this in the context of their non-competitive cross-competition with the transition state isosteres (Figs. 3 and 5), which presumably (although not necessarily) bind at the catalytic site (34). In particular, the fact that the interaction factor {alpha} is near unity for cross-competition between L685458 and 1 or 2 (Table I) suggests that binding of the small molecule inhibitors causes little structural perturbation around the catalytic site where L685458 binds. As such, for the mechanism of affecting catalysis to be valid for the small molecule inhibitors, it is necessary either that the catalytic site be so spacious that it could accommodate both L685458 and a small molecule inhibitor simultaneously (the physical mechanism, Fig. 8A) or that the catalytic site must undergo such a delicate, partial structural distortion upon inhibitor binding at a remote site that the catalysis is prevented, whereas the integrity of the catalytic site for binding of L685458 is preserved (Fig. 8A, the conformational mechanism). Although these possibilities cannot be ruled out, in the absence of additional data, these scenarios do not seem very likely.



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FIG. 8.
Proposed mechanisms of inhibition of {gamma}-secretase by small molecule inhibitors. {gamma}-Secretase is a high molecular weight multicomponent complex containing presenilin, nicastrin, aph-1, and pen-2. For simplicity, presenilin is depicted as the catalytic subunit containing separate substrate binding and catalytic sites. The site for catalysis is also the binding site for transition state isosteres PME or L685458. The location for the second inhibitor site, a site for non-transition state small molecule inhibitors, is undefined. A, inhibition of {gamma}-secretase by affecting catalysis. This may be achieved either by a conformational mechanism where the catalytic site is distorted by inhibitor binding at a site away from the catalytic site or by a physical mechanism in which the inhibitor occupies a portion of the catalytic site, in both cases, without perturbing binding of the transition state analog L685458. B, inhibition of {gamma}-secretase by blocking substrate movement. The catalytic reaction proceeds with substrate binding at the substrate binding site followed by a motion of the part that contains the scissile bond into the catalytic site and subsequent bond cleavage (34). The reaction is inhibited either through a physical mechanism where the inhibitor binds at the region between the substrate binding and catalytic sites to form a physical blockage to the substrate movement or through a conformational mechanism by which the structure between the substrate and catalytic sites is distorted (stretched as it is depicted here as an example) such that the substrate movement into the catalytic site is no longer possible.

 

As {gamma}-secretase may catalyze its reaction through separated substrate binding and catalytic sites (34, 36, 37, and this study), it is possible that inhibition may be achieved by blocking the movement of substrate into the catalytic site. Analogous to the mechanism of affecting catalysis, blocking substrate movement may be achieved in at least two ways: 1) through a physical mechanism, where the inhibitor occupies a site located between the substrate binding site and the catalytic site to form a physical blockage to the substrate motion or 2) by a conformational mechanism, in which a structural distortion in the region between the substrate binding and catalytic sites results from binding of inhibitor remotely (Fig. 8B).

In addition to APP processing, {gamma}-secretase catalyzes endoproteolysis of Notch receptor (2326), a fundamentally important signal transduction molecule crucial for the development of all metazoans (27). Because of its biological importance, the cleavage activation of Notch by {gamma}-secretase is likely to be tightly regulated and substrate-specific given that in addition to APP, a number of other type I transmembrane proteins, such as ErbB4 (30, 31), E-cadherin (32), and CD44 (33), are also {gamma}-secretase substrates. Generally, substrate specificity of an enzyme cannot be modified by affecting catalysis alone since all substrates access the same catalytic machinery of the enzyme. For enzymes with substrate binding and catalysis at the same site, the only way of modulating substrate specificity is by modifying the affinity for different substrates. For enzymes with separated substrate binding and catalytic sites, substrate specificity may also be controlled by affecting substrate movement, if such a motion is substrate-specific. This is straightforward conceptually if multiple substrate binding sites exist, a concept that remains to be tested directly. It is a bit more difficult to conceptualize whether only a single substrate binding site exists, although still feasible in principle depending on the higher order structural features associated with the movement of different substrates into the catalytic site.

The feasibility of substrate selectivity for {gamma}-secretase is supported by these studies and two other recent reports (38, 39). Certain compounds containing the same 3,5-difluorophenyl substituent as 2 (38) and certain arylsulfonamides and benzodiazepines (39) have been reported to inhibit selectively APP processing over Notch cleavage by {gamma}-secretase. Since arylsulfonamides and benzodiazepines do not interfere with substrate binding (34), this observed substrate specificity for APP over Notch supports the hypothesis of blocking substrate movement as the mechanism of {gamma}-secretase inhibition. In contrast, the transition state isostere L685458 has been shown to inhibit APP and Notch processing with equal potency (39), consistent with the principle that substrate selectivity cannot result from affecting catalysis alone. Further investigation is needed to determine precisely how {gamma}-secretase is inhibited by the small molecule arylsulfonamide and benzodiazepine inhibitors and how these affect the processing of various substrates.

The separation of substrate binding and catalysis potentially offers a facile means for regulating and controlling the substrate specificity of {gamma}-secretase biologically. For drug discovery, separation of substrate binding and catalysis presents an opportunity for developing substrate-specific inhibitors. Such inhibitors for {gamma}-secretase, if discovered, would be more beneficial in principle to Alzheimer patients, sparing pathways that are otherwise unrelated to disease pathogenesis.


    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. Back

§ To whom correspondence should be addressed: Dept. of Lead Discovery, AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, DE 19850. Tel.: 302-886-8137; Fax: 302-886-4983; E-mail: gaochao.tian{at}astrazeneca.com.

1 The abbreviations used are: A{beta}, {beta}-amyloid; APP, amyloid precursor protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydoxy-1-propanesulfonate; MES, 4-morpholineethanesulfonic acid; C99, APP C-terminal fragment of 99 amino acid residues; C100, recombinantly produced C99 containing an additional methionine residue at its N terminus; ECL, electrochemiluminescence; G{alpha}R, goat anti-rabbit; PME, pepstatin A methylester; R{alpha}A{beta}40, rabbit anti-A{beta}40. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Deborah S. Hartman for sponsoring the work described in this study and Dr. Clay W. Scott for critical reading of the manuscript. We are grateful for Drs. Mark A. Sylvester, Philip D. Edwards, 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 contributing to the syntheses of compounds 1 and 2. We are indebted to Dr. John Zysk for providing HeLa cell membranes used in preparing detergent extracts of {gamma}-secretase.



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 DISCUSSION
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