An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates

Journal of Neuroscience Methods 213 (2013) 14–21

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Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth

Basic Neuroscience

An improved cell-based method for determining the ␥-secretase enzyme activity against both Notch and APP substrates Timothy D. McKee ∗ , Robyn M.B. Loureiro, Jo Ann Dumin, Vladislav Zarayskiy, Barbara Tate Satori Pharmaceuticals, 281 Albany Street, Cambridge, MA 02139, United States

h i g h l i g h t s    

SUP-T1 assay provides simultaneous measurement of NICD production and A␤ inhibition. Higher levels of APP result in more potent IC50 ’s for certain classes of ␥-secretase inhibitors. Several reported Notch-sparing GSIs inhibit NICD production at IC50 ’s similar to A␤ lowering in the SUP-T1 dual substrate assay. GSMs do not affect Notch, but do alter APP processing.

a r t i c l e

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Article history: Received 7 August 2012 Received in revised form 28 November 2012 Accepted 29 November 2012 Keywords: ␥-Secretase Notch A␤ Alzheimer’s Disease Modulator NICD

a b s t r a c t ␥-Secretase modulators (GSM), which reduce amyloidogenic A␤42 production while maintaining total A␤ levels, and Notch-sparing ␥-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer’s Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic A␤ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC50 ’s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a ␥-secretase substrate. When combined with a sensitive method of measuring A␤ production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC50 ’s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and A␤42 lowering for therapeutically promising GSIs and GSMs. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Alzheimer’s Disease (AD) is a severe neurodegenerative disease defined by the key pathological features of neurofibrillary tangles and amyloid plaques. The amyloid hypothesis suggests that these neuropathologies are the downstream effects of a cascade initiated by ␤-amyloid (A␤) peptides (Hardy and Selkoe, 2002), which results in the formation of amyloid plaques, neurofibrillary tangles, synaptic loss, neurodegeneration and cognitive decline (Hardy and

Abbreviations: GSI, ␥-secretase inhibitor; GSM, ␥-secretase modulator; NICD, Notch intracellular domain; ␤-Amyloid, A␤; 2B, 7CHO-2B7. ∗ Corresponding author. Tel.: +1 617 547 0022; fax: +1 617 547 0661. E-mail address: [email protected] (T.D. McKee). 0165-0270/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneumeth.2012.11.011

Higgins, 1992; Selkoe, 1991). In an attempt to slow or prevent AD, multiple approaches are being investigated to disrupt the amyloid cascade. These include immunotherapy approaches directed at the clearance of A␤, as well as the inhibition of ␤-secretase (BACE) and ␥-secretase to reduce the production of A␤. Early therapeutic approaches focused on the amyloid hypothesis included inhibition of ␥-secretase. However, when small molecule GSI were administered in vivo, severe side effects were observed, including an increase in intestinal goblet cell number, a decrease in intrathymic differentiation and lymphocyte development disruption (Milano et al., 2004; Searfoss et al., 2003; Wong et al., 2004). The inhibition of ␥-secretase prevents the processing of other ␥-secretase substrates, such as Notch which has been implicated in the adverse events described above (Fre et al., 2005; Jensen et al., 2000; van Es et al., 2005). The processing of Notch to NICD

Table 1 Comparison of Notch selectivity for multiple small molecules directed toward gamma-secretase in SUP-T1 and CHO-2B7 cells. Compound class

Company

Name/ID

Peptide-based GSI

Elan

DAPT

Structure

IC50 for A␤42 in Sup-T1 cells (nM)

O

H N

F

CH3

O

O

N H

O

CH3 CH3 CH3

300

IC50 for A␤42 in CHO-2B7 cells (nM)

20

Selectivity over Notch in dual assay

Reported selectivity over Notch

1.2

0.5 (Martone et al., 2009)

2.6

0.9 (Martone et al., 2009)

1

0.6 (Chavez-Gutierrez et al., 2012) 1.2 (Martone et al., 2009)

0.8

16.8 (Martone et al., 2009) 2.5 (Chavez-Gutierrez et al., 2012)

0.6

15 (Henley et al., 2009) 0.8 (Martone et al., 2009) 0.14 (Chavez-Gutierrez et al., 2012)

0.6

193 (Gillman et al., 2010) 4 (Chavez-Gutierrez et al., 2012)

F

F O

Lilly

LY411575

CH3

Peptide-based GSI

Merck

L-685,458

H3C H3C

CH3 N H

N

Me

0.2

0.07

O

OH

H N

O

O

H N

H N

CH 3 O

O

O NH 2 N H CH 3 O

135

34

CH 3

CF3 CF3

HO HN 2nd gen. GSI

Wyeth

GSI-953 begacestat

SO2

289

2.5

S Cl

OH 2nd gen. GSI

Lilly

LY450139 semagacestat

H3 C CH3 O

O

H N

CH3

CF3 H 2N

2nd gen. GSI

BMS

BMS-708163 avagacestat

O

N H

700

N O

17

CH3

T.D. McKee et al. / Journal of Neuroscience Methods 213 (2013) 14–21

Peptide-based GSI

F

Cl O S N O

36 N

F N

O

0.2

15

NA

>33 (Hashimoto et al., 2010)

SPI-1865

N H3C

E-2012

JNJ-40418677

CF3

Cl

GSM1

Undisclosed

OMe CF3

N

O H3C

Me

CH3

Me N

CO 2 H

Structure Name/ID

OH

CF3

O

N

CH3

F

50

75

100

>180

>400 100

>20 250 59

57

IC50 for A␤42 in Sup-T1 cells (nM)

100

IC50 for A␤42 in CHO-2B7 cells (nM)

>350

Selectivity over Notch in dual assay

>30 (Ohki et al., 2011)

>50 (Van Broeck et al., 2011)

T.D. McKee et al. / Journal of Neuroscience Methods 213 (2013) 14–21

Reported selectivity over Notch

16

(Notch intracellular domain) is critical for cell development and differentiation (Miele and Osborne, 1999). Understanding that the toxicities observed in vivo were due to a lack of Notch processing led to the identification of “Notch-sparing” ␥-secretase inhibitors (Gillman et al., 2010; Kreft et al., 2008; Lanz et al., 2010; Mayer et al., 2008). These inhibitors were reported to potently inhibit A␤ peptide formation, while only inhibiting Notch processing at significantly higher drug levels. To determine the potencies to inhibit ␥-secretase processing of each substrate, multiple cell lines expressing each substrate were used (Wolfe, 2012). In addition, the potency to inhibit A␤ production is often tested in a cell line that over-expresses APP, while the inhibition of Notch processing is often assessed in a cell line with native levels of expression (Kreft et al., 2008; Mayer et al., 2008). To determine if there is a selectivity window, the potency differences for inhibiting the processing of these two substrates are compared. The use of two different cell lines to establish selectivity and project safety margins is especially concerning since an IC50 shift has been observed for some GSIs depending upon the level of expression of APP in the cell line used (Burton et al., 2008). The reason for this shift is not clear. It is possible that APP over-expression could stabilize the complex in such a way that it increases the binding affinity of the inhibitor. Regardless of the cause, the IC50 ’s for A␤ lowering are more potent in the APP over-expressing cells, which would likely result in an apparently larger in vitro selectivity window verses Notch processing. With these factors in mind, we have developed a novel dual substrate assay system in which both A␤ and Notch activities can be assessed in the same cell line, SUP-T1. This cell line does not overexpress APP. It also produces a truncated Notch receptor fragment that is a viable ␥-secretase substrate, not requiring an initial sheddase cleavage (Ellisen et al., 1991; Weng et al., 2003). Expression of the truncated receptor allows the stable and consistent production of NICD in cultures. This is opposed to other cell lines where Notch production needs to be stimulated by cytokines or LPS, which can vary among experiments (Liao et al., 2004; Tsao et al., 2011). Reporter based cellular systems have also been utilized for Notch activity, but these systems do not allow for the simultaneous measurement of A␤ (Martone et al., 2009). In these studies, we compare the effects of multiple compounds, including a novel Satori GSM (Table 1) for their effect on A␤38 , A␤40 , A␤42 and Notch in the dual substrate activity assay. 2. Materials and methods 2.1. Compounds DAPT, L-684,548 and LY450139 were purchased from Calbiochem, Tocris and Atomaxchem, respectively. LY411575, GSI953, BMS-708163, MK-GSM1, JNJ-40418677 and E-2012 were prepared according to published methods. SPI-1865 was prepared at Satori Pharmaceuticals.

Company

Merck

J&J

Eisai

Satori

Compound class

GSM

GSM

GSM

GSM

Table 1 (Continued)

2.2. Cell culture SUP-T1 cells (ATCC) were cultured in T75 flasks in RPMI media (Mediatech 10-041-CV) supplemented with 10% FBS and penicillin/streptomycin at 37 ◦ C in a 5% CO2 atmosphere. One hour prior to drug treatment six well plates were seeded with 1.5 mL of media containing 2% FBS and cells at a density of 1.0 × 106 cells/mL. Test compounds in DMSO were diluted 100-fold directly into the media with the cells and incubated for 18 h at 37 ◦ C. After treatment, 100 ␮L aliquots of treated cells were assayed for viability with the Promega Cell Titer Glo assay system. The conditioned media and cells were further processed to measure A␤ levels and NICD levels.

T.D. McKee et al. / Journal of Neuroscience Methods 213 (2013) 14–21

CHO-2B7 cells (Mayo Clinic) are Chinese hamster ovary cells stably transfected with human ␤APP 695 wt (Haugabook et al., 2001; Murphy et al., 2000). The cells were cultured in Ham’s F12 media (Thermo Fisher SH30026.01) supplemented with 10% FBS, 0.25 mg/mL Zeocin and penicillin/streptomycin at 37 ◦ C in a 5% CO2 atmosphere. For compound treatment, cells were plated in 96-well plates at a density of 1.0 × 105 cells/mL and allowed grow to 100% confluence over 2 days. Test compounds in DMSO were diluted 100-fold directly into the media before adding to the cells. Immediately prior to adding compound-containing media to the cells, they were washed once with 1XPBS. Conditioned media from CHO2B7 cells were collected after 5 h of treatment and the levels of A␤ peptides were assessed as described below. Cell viability was determined using a luminescent assay based on quantitation of the ATP (CellTiter-Glo, Promega). 2.3. Solid phase extraction Wells of 30 mg Oasis HLB 96-well extraction plates (Waters Corporation) were activated by addition of 1 mL of methanol followed by rinsing with 1 mL of water utilizing a vacuum plate manifold. A total of 1 mL of SUP-T1 conditioned media was then added. Wells were then washed with 2 mL of 10% methanol and then with 2 mL of 30% methanol. Samples were eluted into sample collection tubes by adding 250 ␮L of 90% methanol with 2% ammonium hydroxide to each well. Eluted samples were concentrated to dryness under vacuum without heating. 2.4. Measurement of Aˇ Conditioned media from 2B7 cells was collected and diluted with 1 volume of MSD blocking buffer (1% BSA in MSD wash buffer). Dried films of conditioned media from SUP-T1 cells after solid phase extraction were re-suspended with 75 ␮L of MSD blocking buffer (1% BSA in MSD wash buffer) and transferred to blocked MSD Human (6E10) A␤ 3-Plex plates. Plates were incubated for 2 h at room temperature with orbital shaking followed by washing and reading according to the manufacturer’s instructions (SECTOR® Imager 2400 Meso Scale Discovery, Gaithersburg, MD). A␤ concentrations were converted to percent vehicle values and used to construct dose response curves which were fitted to 3 parameter curves using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA. 2.5. NICD assay The remaining SUP-T1 cells were washed twice in PBS and then lysed with Promega reporter lysis buffer (E397A) containing a complete protease inhibitor cocktail (Roche 04 693 116 011) for 1 h at 4 ◦ C. Lysates were spun at 5000 RPM for 5 min and supernatants were collected and total protein levels were measured and adjusted to 1–2 ␮g/mL total protein using the BCA total protein assay (Thermo Scientific 23225). NICD levels were then measured with a cleavage specific Notch1 sandwich ELISA (Cell Signaling 7194) according to manufacturer’s instructions. ELISA signals were converted to percent vehicle values and used to construct dose response curves which were fitted to 3 parameter curves using GraphPad. 3. Results 3.1. APP expression levels alter Aˇ potencies We first wanted to demonstrate which compounds were susceptible to an IC50 shift with APP over-expression. We examined the potency of the compounds for A␤42 in two different cell lines. The

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CHO-2B7 cells (2B7) are a stably transfected cell line which overexpresses APP, leading to high levels of A␤ peptides (Haugabook et al., 2001). In our hands these cells express 400 pg/mL of A␤42 when plated at 1.0 × 105 on day 1 and media is harvested following a 5 h incubation on day 3. The second cell line used is the SUP-T1 cells, which express relatively low levels of APP and therefore make significantly less A␤ peptides than the 2B7 cells. When incubated over-night at a density of 1.0 × 106 cells/mL, the cells make 3 pg/mL of A␤42 . As shown in Table 1, all of the GSIs we tested have an increased potency in the over-expressing 2B7 cells. Both peptidomimetics (DAPT, LY411575) show a minor shift of 15- and 7-fold. Similarly, the transition state analog L-685,458 only shifted 4-fold. Conversely, the newer “second generation” GSIs represented by begacestat, semagacestat and avagacestat exhibited a significant shift with APP over-expression. In the 2B7 cells, semagacestat was 41 times more potent, while begacestat and avagacestat were 115 and 180 times more potent. These data suggest that the use of APP over-expressing cell lines may result in an over estimation of the potencies for many compounds which would result in an aberrant in vitro selectivity window between APP and Notch processing. In contrast to the GSIs, the GSMs do not show a potency shift in over-expressing cell lines. 3.2. SUP-T1 dual substrate assay A variety of small molecules designed either to inhibit or modulate ␥-secretase was selected and examined in the SUP-T1 cell line. These compounds have varying effects on the ␥-secretase complex and provide significant structural and physiochemical diversity. The compounds utilized are described in Table 1 including their previously reported window for Notch selectivity and the selectivity margin calculated using our assay. Compounds were tested in either in the SUP-T1 cell line, which has a physiologic level of expression of APP, or the CHO-2B7 cells, an APP over-expressing line, to examine which compounds show a potency shift. An increased potency for A␤ lowering was observed with GSIs in the over-expressing cells, but not the GSMs. When the IC50 from these over-expressing lines is compared to the Notch potency, a safety window appears to exist. When the IC50 derived from the SUP-T1 cells is used, there is no safety window even for those GSIs which are thought to be Notch-sparing. Reported values from the literature are also shown. 3.3. Peptide based -secretase inhibitors in the SUPT-1 dual substrate assay As positive controls for the SUPT-1 dual substrate assay, several GSIs which have been reported to impact Notch processing were tested. In this group of compounds, we tested a transition state mimic (L-685,458) and two non-transition state peptidomimetics (DAPT and LY411575). Classical transition state compounds interfere with the initial ␧-cleavage of APP by ␥-secretase, while non-transition state peptidomimetic analogs such as DAPT and LY411575 show a preference for inhibiting the ␦- and ␥-cleavages (Qi-Takahara et al., 2005; Zhao et al., 2004). As shown in Fig. 1, all three compounds completely inhibit A␤40 and A␤42 production as effectively as they inhibit NICD production in the SUPT-1 cells. Due to the low levels of A␤38 expression upon treatment with inhibitors, A␤38 could only be tracked in the DAPT treatment with an IC50 of 196 nM. The selectivity window between NICD and A␤42 production for DAPT was calculated to be 1.2-fold, 2.6-fold for LY411575 and 1-fold for L-685,458. This demonstrates that the dual substrate assay is capable of simultaneously measuring the inhibition of both APP and Notch processing by ␥-secretase inhibitors.

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Fig. 1. Dual substrate assay detects inhibition of A␤ and NICD production. DAPT (A), LY411575 (B) and L-685,458 (C) were assessed in the dual substrate assay using the SUP-T1 cells following an 18 h incubation. IC50 ’s were determined for NICD, A␤38 , A␤40 and A␤42 along with cell viability.

3.4. Second generation inhibitors in the SUPT-1 dual assay To further characterize the assay, we tested begacestat (GSI953), semagacestat (LY450139), and avagacestat (BMS-708163) in the dual substrate assay. Begacestat was reported to have a 16.8fold safety margin, which had been determined using a stable CHO cell line expressing APP for A␤ production and a second CHO line transfected with a construct containing a constitutively active mouse Notch and a reporter vector (Martone et al., 2009). In our hands, the compound lowered A␤38 , A␤40 and A␤42 with the IC50 for A␤42 of 289 nM, approximately 23-fold less potent that previously reported (Martone et al., 2009). As shown in Fig. 2A, in our assay the compound also decreased A␤40 and A␤38 with similar potencies. When the IC50 for NICD production was examined, the potency was once again similar to that for A␤42 . When a selectivity window is calculated there is less than 1-fold difference (0.8) which is within the margin of error. However if we used the A␤42

IC50 (25 nM) from our 2B7 cells to calculate the selectivity window, the compound appears to have a margin of 9, closer to the previously report margin of 16.8 (Martone et al., 2009). Semagacestat was reported to have a small margin between the IC50 for A␤42 and NICD inhibition. Depending on the assays utilized to derive the margin, a range of 0.14- to 15-fold has been reported (Chavez-Gutierrez et al., 2012; Henley et al., 2009). It is unclear why this compound generates such a wide range of results across laboratories. In our assay, semagacestat inhibited A␤38 , A␤40 and A␤42 production (Fig. 2B) with an IC50 for A␤42 lowering of 700 nM which is comparable to that for NICD production with an IC50 of 450 nM. These data indicate there is no selectivity window between the substrates. Yet, if the 2B7 cell A␤42 IC50 value (17 nM) is used for the selectivity calculation, there is a 26-fold window. Avagacestat was reported to have the largest window between the two activities with a 193-fold margin of A␤ lowering over

T.D. McKee et al. / Journal of Neuroscience Methods 213 (2013) 14–21

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Fig. 2. Testing 2nd generation inhibitors. Begacestat/GSI-953 (A), semagacestat/LY450139 (B) and avagacestat/BMS-708163 (C) were assessed in the dual substrate assay using the SUP-T1 cells following an 18 h incubation. IC50 ’s were determined for NICD, A␤38 , A␤40 and A␤42 along with cell viability.

inhibition of Notch processing (Gillman et al., 2010). The compound was initially tested for APP processing using the H4-8Sw cells, which is a H4 neuroglioma cell line stably expressing the Swedish mutant of APP751 resulting in over-expression of APP. To assess the effect on Notch processing, a cellular assay using a luciferase reporter and a Notch1-E construct was employed (Gillman et al., 2010). The determination of IC50 ’s in these two cellular assays suggests a selectivity ratio of 193 existed between to the two activities. In the SUPT-1 dual assay, the IC50 for A␤42 was found to be 36 nM, while the IC50 for Notch was 23 nM (Fig. 2C) suggesting that the compound is more potent or equally potent for both activities. However, in the overexpressing 2B7 cells the A␤42 IC50 was found to be 0.2 nM, a 180-fold shift in potency (Table 1) and if used to calculate a selectivity margin suggests that a 115-fold margin exists. Together these data indicate that the level of APP expression can drive the selectivity margin between Notch and APP processing. With the use of low A␤42 production, there is no selectivity window for this compound, whereas

when APP over-expression results in a significant margin being calculated. 3.5. Testing of -modulators in the SUPT-1 dual substrate assay To further investigate the usefulness of the dual assay, several ␥-modulators were tested. ␥-Modulators, unlike the inhibitors, do not affect the production of all A␤ peptides and reportedly do not inhibit Notch (Pissarnitski, 2007). In the dual substrate assay, the Merck compound, GSM1 showed a lowering of A␤42 , A␤40 was unaffected and A␤38 is significantly increased. As expected with a modulator, when we examine NICD inhibition, no inhibition was seen until concentrations were reached that also showed cell toxicity similar to previously published data (Ohki et al., 2011). A correlation with cell viability indicts that the reduction in NICD is not inhibition (Fig. 3A), but rather non-specific effects. The next modulator, JNJ-40418677 (Van Broeck et al., 2011), increased A␤38 and decreased A␤42 , but had little effect on A␤40

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Fig. 3. Modulators do not affect NICD production, but lower A␤42 . The effects of 4 GSMs were tested in the SUP-T1 cells. Following an 18 h incubation, IC50 ’s were determined for NICD, A␤38 , A␤40 and A␤42 along with cell viability. Important to note that in some cases GSMs will increase levels of A␤38 and where appropriate the IC50 is calculated for that increase.

and Notch until ␮M levels were attained and once again cell viability decreased (Fig. 3B). The IC50 for A␤42 production was found to be 58 nM. A selectivity margin was not calculated since the alterations in Notch appear to be due to toxicity, not inhibition. E-2012, another modulator was also tested (Hashimoto et al., 2010). In the dual substrate assay, E-2012 demonstrated an increase in A␤38 , lowering of A␤40 and a decrease in A␤42 (Fig. 3C). The IC50 for A␤42 lowering was 50 nM. A Notch IC50 was not determined, since any decrease in NICD production correlated with a decrease in viability, indicating that E-2012 does not affect Notch processing. The last ␥-modulator tested is from the Satori series. This series has a novel chemical scaffold, which originated from a natural product, black cohosh root. The scaffold has been modified to build in more drug-like properties and oral bioavailability. Unlike the previous ␥-modulators, this series has a unique A␤ profile where both A␤38 and A␤42 are decreased, A␤40 is unaffected and both A␤37 and A␤39 are increased (Xia et al., 2011). When tested in the dual assay, SPI-1865 had an IC50 of 629 nM for A␤40 , 75 nM for A␤42 (Fig. 3D). Due to the compounds ability to decrease A␤38 production, the levels of A␤38 were too low to measure an IC50 . Any effect on NICD production correlated with a loss in cell viability. These data demonstrate that SPI-1865, even with its unique A␤ profile is a modulator and does not inhibit Notch processing. 4. Discussion Identifying a safe therapeutic to lower amyloid levels, specifically A␤42 , has been a challenging problem for the pharmaceutical industry. While ␥-secretase has been targeted by many companies, a safe and efficacious compound has not yet been identified. Part of the challenge resides in understanding how to safely inhibit the processing of APP while not effecting the processing of the other ␥secretase substrates, especially Notch, inhibition of which results in many of the side effects observed with GSIs. Although companies have tried to determine if compounds spare NICD production while inhibiting A␤ production, the assays are typically performed in two separate cell lines, which can result in an over-estimation of the selectivity margin. This problem is further compounded by the use of cell lines with vastly different levels of APP expression. The

potency of some compounds is significantly impacted by substrate concentration, as shown in Table 1. To avoid this pitfall, we have modified and combined several protocols to develop a dual substrate assay. SUP-T1 cells express a native level of APP and a constitutively active Notch substrate. Unlike cell lines that need stimulation or are stably transfected with truncated Notch and a reporter vector, the SUP-T1 cells have a mutation that results in a ready-made gamma substrate without the need for a prior sheddase cleavage (Ellisen et al., 1991). Thus, both APP and Notch are expressed at consistent levels over multiple passages of SUP-T1 cells and ␥-secretase processing of both substrates can be measured simultaneously in the same cell. A more accurate ratio of APP to Notch processing is generated in this single cell system. Using a number of literature reference compounds, we have compared the reported margins of selectivity between APP and Notch processing. The peptidomimetic GSIs were not reported to be selective, but acted as a positive control in the validation of the assay. By using these compounds, we show that the dual substrate assay can detect the inhibition of A␤ and NICD production. Similarly, the GSM data demonstrate that the assay can also identify compounds that only affect the processivity of ␥-secretase, thus simultaneously allowing for the reduction of some products, typically the longer A␤ forms (A␤42 ), and not affecting others like NICD and A␤40 , while increasing the shorter A␤ forms to keep the total A␤ pool constant (Weggen et al., 2001). The most interesting data are from the recent GSI clinical candidates. The GSI, semagacestat (LY450139), did not have a large margin between A␤ and Notch inhibition in the dual substrate assay and showed Notch-related adverse events in the clinic, including gastrointestinal symptoms and skin cancer (Lilly, 2010; Selkoe, 2011). A second GSI, begacestat (GSI-953), was reported to have a 16-fold selectivity window (Martone et al., 2009). In the dual substrate assay begacestat was equally capable of inhibiting NICD and A␤42 production. The difference between the previously reported selectivity and the current report can be attributed to an approximately 10-fold shift in potency in the A␤42 IC50 in the dual substrate assay. Avagacestat (BMS-708163) was reported to be 193fold more selective for A␤42 than Notch based on two separate

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cell-based assays (Gillman et al., 2010) although in both preclinical and clinical studies Notch related toxicity at doses of 100 mg/day or greater have been reported (Hopkins, 2012). In our dual substrate assay, avagacestat was equally potent for both APP and Notch processing with IC50 ’s of 36 nM and 23 nM, respectively. Our data correspond with data from Chavez-Gutierrez et al., in which all three compounds were tested in a solubilized ␥-secretase assay. In their assay, the compounds also demonstrated little, if any selectivity over Notch processing. Our data shows that the dual substrate assay is capable of assessing a wide range of chemical matter. The data further suggests that measuring the potencies for both APP and Notch processing simultaneously in the same cell line provides a more accurate estimation of selectivity and will potentially identify compounds with safety concerns earlier in the drug discovery process. Acknowledgements We would like to thank Satori Medicinal Chemistry and Kelly Ames, Business Manager at Satori Pharmaceuticals, Inc. for their support. References Burton CR, Meredith JE, Barten DM, Goldstein ME, Krause CM, Kieras CJ, et al. The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression. J Biol Chem 2008;283:22992–3003. Chavez-Gutierrez L, Bammens L, Benilova I, Vandersteen A, Benurwar M, Borgers M, et al. The mechanism of gamma-secretase dysfunction in familial Alzheimer disease. EMBO J 2012;31:2261–74. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991;66:649–61. Fre S, Huyghe M, Mourikis P, Robine S, Louvard D, Artavanis-Tsakonas S. Notch signals control the fate of immature progenitor cells in the intestine. Nature 2005;435:964–8. Gillman KW, Starrett JE, Parker MF, Xie K, Bronson J, Marcin L, et al. Discovery and evaluation of BMS-708163, a potent, selective and orally bioavailable ␥secretase inhibitor. ACS Med Chem Lett 2010;1:120–4. Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade hypothesis. Science 1992;256:184–5. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353–6. Hashimoto T, Ishibashi A, Hagiwara H, Murata Y, Takenaka O, Miyagawa T. E2012: a novel gamma-secretase modulator-pharmacology part. Alzheimer’s Dement 2010;6:S242. Haugabook SJ, Yager DM, Eckman EA, Golde TE, Younkin SG, Eckman CB. High throughput screens for the identification of compounds that alter the accumulation of the Alzheimer’s amyloid beta peptide (Abeta). J Neurosci Methods 2001;108:171–9. Henley DB, May PC, Dean RA, Siemers ER. Development of semagacestat (LY450139), a functional gamma-secretase inhibitor, for the treatment of Alzheimer’s disease. Expert Opin Pharmacother 2009;10:1657–64. Hopkins CR. ACS chemical neuroscience molecule spotlight on begacestat (GSI-953). ACS Chem Neurosci 2012;3:3–4. Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, Ishibashi M, et al. Control of endodermal endocrine development by Hes-1. Nat Genet 2000;24:36–44. Kreft A, Harrison B, Aschmies S, Atchison K, Casebier D, Cole DC, et al. Discovery of a novel series of Notch-sparing gamma-secretase inhibitors. Bioorg Med Chem Lett 2008;18:4232–6. Lanz TA, Wood KM, Richter KE, Nolan CE, Becker SL, Pozdnyakov N, et al. Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014. J Pharmacol Exp Therap 2010;334:269–77.

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