- Email: [email protected]
Synergistic inhibition of Aβ production by combinations of γ-secretase modulators
European Journal of Pharmacology xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Synergistic inhibition of Aβ production by combinations of γ-secretase modulators Alan S. Robertsona, Lawrence G. Ibena, Cong Weib, Jere E. Meredith Jr.a, Dieter M. Drexlerb, Martyn Banksc, Gregory D. Vitec, Richard E. Olsond, Lorin A. Thompsond, Charles F. Albrighta, ⁎ Michael K. Ahlijaniana, Jeremy H. Toyna, a
Neuroscience Biology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA Discovery Analytical Sciences, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA c Lead Discovery and Lead Profiling, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA d Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA b
A R T I C L E I N F O
A BS T RAC T
Keywords: Alzheimer Amyloid Combination index Lasalocid Presenilin Synergism
Alzheimer's disease is associated with the accumulation of amyloid-β (Aβ) in the brain. In particular, the 42amino acid form, Aβ1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aβ1-42 production without inhibiting the production of other forms of Aβ. Here we describe the novel discovery of synergistic inhibition of Aβ by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aβ peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aβ occurred only when one GSM was of the “acid GSM” structural class and the other was of the “non-acid GSM” class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aβ production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aβ. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target.
1. Introduction The 42-amino acid form of the amyloid-β peptide, Aβ1-42, is associated with Alzheimer's disease (Karran et al., 2011). It is therefore of interest that certain small molecules, known as γ-secretase modulators (GSM), can selectively decrease Aβ1-42 (for reviews see Bursavich et al., 2016; Crump et al., 2013; Hall and Patel, 2014; Tate et al., 2012). GSMs are structurally and functionally diverse: one type, the “acid GSMs,” includes non-steroidal anti-inflammatory drugs (NSAID), such as sulindac sulfide (Weggen et al., 2001) and related structures containing carboxylic acid moieties, such as “GSM-2” (Mitani et al., 2012). Typically, the acid GSMs decrease Aβ1-42 and increase Aβ1-38, but have little effect on production of Aβ1-37 or Aβ140. A second group of GSMs decreases both Aβ1-42 and Aβ1-40, while at the same time increasing both Aβ1-37 and Aβ1-38. We shall refer to
⁎
this structurally diverse group as “non-acid GSMs.” An example is BMS-869780 (Toyn et al., 2014). Additional GSMs include natural product-derived triterpenoids (Findeis et al., 2012), and endogenous steroids found in mammals, such as 3β-hydroxy-5-cholestenoic acid (Jung et al., 2013). In some cases, close GSM analogs increase Aβ1-42 and are known as “inverse GSMs” (Jung et al., 2013; Ohki et al., 2011). In general, however, there is a predictable correspondence between the GSM structural type and the effects on production of Aβ peptides. Photolabelling studies show that GSMs target presenilin, the catalytic subunit of γ-secretase (Crump et al., 2011; Ebke et al., 2011; Jumpertz et al., 2012; Ohki et al., 2011). Furthermore, photolabelling competition studies (Pozdnyakov et al., 2013) and enzyme cross competition analysis (Borgegard et al., 2012) suggest that GSMs may target two distinct binding sites. Consistent with an allosteric mode of action, GSMs cause conformational changes within the
Correspondence to: Yale University, Office of Development, 157 Church Street, New Haven, CT 06510, USA. E-mail address: [email protected] (J.H. Toyn).
http://dx.doi.org/10.1016/j.ejphar.2017.07.019 Received 21 February 2017; Received in revised form 3 July 2017; Accepted 5 July 2017 0014-2999/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Robertson, A.S., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.07.019
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
N Cl
O N
N
N N
N
NH
O
N
N
N
N H
N H
N H
OH
O
O
N N
O
NH N
O
O
O
F
N
F
F H N
N
N
F
O
HN
N
N
N N
N
N
N
N N N
F
CF
F COOH O
S
Me Me
O Me
N
H
HO
O OH
O
HO
H
OH O
H
F
Cl
HO
O H O
F
OH
β O O
HO
OH
S
F C
Cl O
N
N
CF
CF Cl
F
F C
COOH
F C
HO
O
Fig. 1. Chemical structures of compounds used in this study.
2. Materials and methods
presenilin protein, and within the complex between presenilin and its substrate, the β-C-terminal fragment (β-CTF) of the amyloid precursor protein (Ohki et al., 2011; Uemura et al., 2009, 2010, 2011). Furthermore, allosteric interactions can be inferred from photolabelling interactions between GSMs and γ-secretase inhibitors (GSI): a GSM can enhance photolabelling by a GSI-derived photoligand (Crump et al., 2011), and a GSI can enhance photolabelling by a GSM-derived photoligand (Pozdnyakov et al., 2013). However, there are no reports describing enhanced inhibition of Aβ production resulting from combinations of different modulators or inhibitors of γ-secretase. Here we describe the novel discovery of synergistic inhibition of Aβ production by pairwise combinations of GSMs. Synergistic inhibition is unexpected because GSMs acting individually do not inhibit Aβ production. In addition, we found the natural product lasalocid (Westley et al., 1970) to be a potent GSM, but lasalocid did not synergize with other GSMs. Our findings further elucidate the functional complexity of γ-secretase small molecule pharmacology, and suggest new approaches to advance the development of future GSMs.
2.1. Materials Many of the GSMs and GSIs used in this study were prepared at Bristol-Myers Squibb, including (S)-N-(4-(4-chloro-1H-imidazol-1-yl)3-methoxyphenyl)-9-(4-fluorophenyl)-6,7,8,9-tetrahydro-5H-[1,2,4] triazolo[1,5-a]azepin-2-amine (BMS-869780, Toyn et al., 2014); (S)-2((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-(((S,Z,Z)-3methyl-4-oxo-4,5-dihydro-3Hbenzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796, Prasad et al., 2007); (S)-7-(4-fluorophenyl)-N2(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481, Soares et al., 2016; Toyn et al., 2016); 6-benzyl-4-(3-methoxy-4-(4methyl-1H-imidazol-1-yl)benzyl)-N-methyl-1,6-dihydro-1,3,5-triazin2-amine (BMS-802299, Marcin et al., 2012); 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid (BMS-299897, Barten et al., 2005); (E)-1-[(1S)-1-(4fluorophenyl)ethyl]-3-[3-methoxy-4-(4-methyl-1H-imidazol-1-yl)benzylidene]piperidin-2-one (E2012, Nakano-Ito et al., 2013); (S,E,E)-2-
2
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 2. Cell cultures were treated with combinations of BMS-869780 and GSM-2 to determine effects on Aβ levels. (A) Aβ1-x levels shown as dose response of GSM-2 at constant concentrations of BMS-869780. (B) Aβ1-x levels shown as dose response of BMS-869780 at constant concentrations of GSM-2. (C) A contour plot summarizes the average values of Aβ1-x observed in 96-well format. (D) Aβ1-40 levels shown as dose response of GSM-2 at constant concentrations of BMS-869780. (E) Lactate dehydrogenase (LDH) levels in culture supernatants. (F) Aβ1-42 levels shown as dose response of GSM-2 at constant concentrations of BMS-869780. Error bars represent standard error between three replicate cultures.
(Toyn et al., 2014). Cell cultures were grown in 96-well format for the experiments shown in Figs. 2, 3, 4, and 6D-F, and grown in T-75 culture flasks for the experiments shown in Figs. 5, 6B and C. The final concentration of dimethyl sulfoxide in all cell cultures was 0.1%. MALDI-TOF mass spectroscopy of Aβ peptides immunoprecipitated from cell cultures, and immunoassays for Aβ1-37, Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-x, were carried out as previously described (Toyn et al., 2014). For western blotting of α-CTF and β-CTF, cell cultures were harvested, cell proteins were solubilized in sodium dodecyl sulfate electrophoresis sample buffer, total protein concentrations were quantified using a kit (EZQ Invitrogen catalog number R33200), and 10 μg total protein per sample was loaded per lane for western blotting, as previously described (Toyn et al., 2014). Lactate dehydrogenase (LDH) in cell cultures was quantified using a kit (BioVision, Milpitas, CA).
(2-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)vinyl)-8-(2(trifluoromethyl)phenyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine (GSM-A, Lu et al., 2012); 2-((2S,4R)-1-(((R)-1-(4-chlorophenyl)4-methylpentyl)-2-(4-(trifluoromethyl)phenyl)piperidin-4-yl)acetic acid (GSM-1, Page et al., 2007); {(2S,4R)-1-[(4R)-1,1,1-trifluoro-7methyloctan-4-yl]-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetic acid (GSM-2, Mitani et al., 2012); and (R)-2-(5-chloro-6-(2,2,2-trifluoroethoxy)-4'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)-3-cyclobutylpropanoic acid (EVP-0015962, Rogers et al., 2012). {(1Z)-5-fluoro-2methyl-1-[4-(methylsulfinyl)benzylidene]-1H-indene-3-yl}acetic acid (sulindac sulfide) was obtained from Sigma-Aldrich (cat# S3131). 3βhydroxy-5-cholestenoic acid, and 6-{(3R,4S,5S,7R)-7-[(2S,3S,5S)-5ethyl-5-((2R,5R,6S)-5-ethyl-5-hydroxy-6-methyltetrahydropyran-2yl)-3-methyltetrahydrofuran-2-yl]-4-hydroxy-3,5-dimethyl-6-oxononyl}-2-hydroxy-3-methylbenzoic acid (lasalocid) were obtained from Santa Cruz Biotechnology (cat.# sc-209761, lot# J2912, and cat.# sc362029, lot# L1012, respectively). Stock solutions were prepared at a concentration of 20 mM in dimethyl sulfoxide. Chemical structures of the compounds are illustrated in Fig. 1.
2.3. Handling of animals and in vivo experiments All experimental procedures with mice followed National Institutes of Health guidelines, and were authorized by and in compliance with policies of the Bristol-Myers Squibb Animal Care and Use Committee. Mice were housed with a 6:00 a.m. to 6:00 p.m. light/dark cycle and allowed free access to food and water. Mice were randomly assigned to drug treatment and vehicle control groups, and were randomized with respect to the order of dosing within each time group. Oral dosing and preparation of brain samples for Aβ immunoassays were carried out as previously described (Toyn et al., 2014). Briefly, 12-week old C57Bl/6
2.2. Cell cultures, Aβ immunoassays, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectroscopy, and western blotting Maintenance of human neuroglioma H4-APPsw cell cultures and treatments with compounds were carried out as previously described 3
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 3. Analysis of synergism between BMS-869780 and GSM-2 in cell cultures. (A) Aβ1-x isobolograms at 50% ( ), 75% ( ), and 90% ( Aβ1-x as a function of BMS-869780/GSM-2 ratio at 50% ( ), 75% ( ), and 90% (
) effect levels. (B) Combination index for
) effect levels. (C) Aβ1-40 isobolograms. (D) Combination index for Aβ1-40. (E) Aβ1-42
isobolograms. (F) Combination index for Aβ1-42.
D1 D + 2 =1 Dx1 Dx 2
wild type mice were given oral doses of BMS-869780 and GSM-2 at 25 mg/kg, or BMS-433796 at 10 mg/kg. Results of the Aβ assays were evaluated by ANOVA and post hoc Dunnett's test.
where D1 and D2 are the respective doses of drug 1 and drug 2 that in combination have x effect, and Dx1 and Dx2 are the respective doses of drug 1 acting alone and drug 2 acting alone that have x effect. A related formula can be used to give a quantitative measure of synergistic effect size known as the combination index (CI):
2.4. Evaluation of synergism using isobolograms and combination index Synergism is defined as an effect of a drug combination that is greater than expected from the effects of the individual drugs. Dose response curves for GSM-2 at fixed concentrations of BMS-869780, and dose response curves for BMS-869780 at fixed concentrations of GSM-2, were fitted using spline curves in Graphpad Prizm, and dose combinations resulting in 50%, 75%, or 90% decreased Aβ were determined by interpolation. The concentrations of each GSM in dose combinations having equal effects on Aβ were plotted on Cartesian coordinates to make isobolograms (Tallarida, 2006). In principle, summation of the effects of two inhibitory drugs, in the absence of synergism or antagonism, would result in an isobologram having a straight downward sloping line, known as the “line of additivity,” enclosing a triangular space in the upper right quadrant. The equation representing the line of additivity is:
CI =
D1 D αD1 D2 + 2 + Dx1 Dx 2 Dx1 Dx 2
where α is a factor ranging in value between 0 and 1 representing the extent to which two drugs act independently of one another (Chou and Talalay, 1984). For the calculations of CI reported here, a value of α = 1, representing the case in which two compounds act independently of one another, was used because it makes the most conservative assumptions with respect to detection of synergism, yielding the most conservative estimates for CI. In principle, a value of CI = 1 indicates the potency predicted from addition of the effects of each individual compound acting alone. Values of CI < 1 indicate possible synergism, whereas values of CI > 1 indicate possible antagonism, that is, a lessening of the effect of one compound in the presence of another 4
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
than Aβ1-23 in length. Cell cultures were treated with combinations of BMS-869780 and GSM-2 to explore the entire dose-response range. The dose responses of BMS-869780 at fixed concentrations of GSM-2 showed potent inhibition of Aβ1-x by the combination (Fig. 2A). Likewise, for the same experiment, the dose response curve of GSM2 plotted at fixed concentrations of BMS-869780 showed potent inhibition of Aβ1-x (Fig. 2B). A contour plot gives another view of how combinations of the two GSMs decreased or increased Aβ1-x (Fig. 2C). It can be seen that BMS-869780 on its own did not inhibit Aβ1-x at any concentration tested, and even shows an increase in Aβ1x at concentrations in the micromolar range. The increase is consistent with many previous observations of the effect of BMS-869780 on Aβ1x, and correlates with increases in Aβ1-37 and Aβ1-38 (Toyn et al., 2014). Aβ1-40 and Aβ1-42 both showed decreases in the presence of the GSMs (Fig. 2D and F). Cell health and viability, as evaluated by lactate dehydrogenase levels in the culture media, were not affected, except at the highest concentration, 10,000 nM, of GSM-2 (Fig. 2E). Thus, combinations of BMS-869780 and GSM-2 cause inhibition of Aβ1-x production. Synergistic effects can be evaluated using isobolograms, which are graphical plots of dose combinations having an equal level of effect (Tallarida, 2006). In principle, additive effects of two compounds result in a straight line of downward slope, whereas synergism would cause a leftward deviation, and antagonism a rightward deviation. Using data from the same experiment illustrated in Fig. 2, Aβ1-x isobolograms constructed for BMS-869780 and GSM-2 showed a dramatic deviation to the left, confirming strong synergism (Fig. 3A). Isobolograms for Aβ1-40 also show a strong leftward deviation, again indicating synergism (Fig. 3C). In contrast, the Aβ1-42 isobolograms showed no clear evidence of a leftward deviation, suggesting an additive effect for Aβ142 (Fig. 3E). While isobolograms are qualitatively compelling, a quantitative way to evaluate synergism uses the “combination index” (CI; Chou and Talalay, 1984). The Aβ1-x data illustrated in Fig. 2 were used to calculate CI values (see materials and methods). CI values were plotted against the BMS-869780/GSM-2 concentration ratio for combinations that yielded 50%, 75% and 90% Aβ1-x lowering (Fig. 3B). The minimum values observed, CI = 0.05-0.06, indicate a very high degree of synergism. The minimum CI value occurred at a molar ratio of GSM-2:BMS-869780 in the range 1.5-2.3. Furthermore, the CI values, and the molar ratio at which the maximum synergism occurred, were of similar magnitude across 50%, 75% and 90% Aβ1-x lowering. Aβ1-40 showed low CI values that depended on the extent of lowering, with minima ranging from CI = 0.7 for 50% lowering to CI = 0.2 for 90% lowering (Fig. 3D). Aβ1-42 showed less pronounced lowering of the CI values, suggesting no significant effect, and that lowering of Aβ142 is therefore not synergistic (Fig. 3F). CI values greater than CI = 1 were also observed, which may represent interference between the GSMs at certain concentrations. The possible significance of increased CI values was not evaluated further.
Fig. 4. Cell cultures were treated with representative GSMs. (A) Stack chart showing Aβ1-37 (purple), Aβ1-38 (blue), Aβ1-40 (green), and Aβ1-42 (red) from cell cultures treated individually with BMS-869780 at 2 μM, BMS-932481 at 2 μM, GSM-A at 2 μM, GSM-2 at 2 μM, EVP-0015962 at 2 μM, 3β-hydroxy-5-cholestenoic acid at 10 μM, sulindac sulfide at 25 μM, or BMS-299897 (GSI) at 1 μM. Combination treatments included 1 μM concentration of each compound, except for 3β-hydroxy-5-cholestenoic acid and sulindac sulfide, which were 10 μM and 25 μM, respectively. (B) Bar chart showing results of Aβ1-x ELISA from the same experiment illustrated in panel A, and additional results from an experiment using the GSMs, BMS-802299, E2012, and GSM1, at a concentration of 2 μM individually or 1 μM each in combinations, respectively. Error bars indicate standard deviation for mostly 3 and up to 6 replicate wells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
compound. As a guide to interpretation, a value of CI < 0.85 may indicate synergism, and a value of CI < 0.3 may indicate strong synergism (Bijnsdorp et al., 2011). To implement CI calculations for the GSM BMS-869780, which does not inhibit Aβ1-x when acting alone, the highest concentration tested (10,000 nM) was used in CI calculations for effects on Aβ1-x, which therefore yields an overestimate of CI, i.e. a conservative evaluation that gives an underestimate of synergism, but nonetheless facilitates inspection of patterns in the data.
3.2. Synergism requires the combination of one acid GSM with one non-acid GSM To find out if Aβ inhibition is a general property of GSM combinations, cell cultures were treated with a selection of different GSMs either individually or in pairwise combinations to evaluate the effects on Aβ production. Cell culture treatments included all pairwise combinations of BMS-869780, BMS-932481, GSM-A, GSM-2, EVP15962, 3β-hydroxy-5-cholestenoic acid, and sulindac sulfide, representing 3 non-acid GSMs and 4 acid GSMs (Fig. 4A). Treatments with the individual GSMs caused changes in the relative levels of Aβ1-37, Aβ1-38, Aβ1-40, and Aβ1-42, as expected. However, all 12 of the cultures treated with a combination of one acid GSM and one non-acid GSM resulted in lowering of Aβ levels. Combinations containing acid with acid, or non-acid with non-acid, as well as the individual GSMs, showed effects on the Aβ profile characteristic of the structural type of
3. Results 3.1. Synergistic inhibition of Aβ production by combinations of BMS869780 and GSM-2 To evaluate inhibition by combinations of GSMs, the Aβ1-x ELISA was used because it can detect all major soluble Aβ peptides greater 5
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 5. Cell cultures were treated with BMS-869780 (a), GSM-1 (b), GSM-2 (c), or BMS-299897 (d), either as individual compounds at a concentration of 1 μM or 2 μM, as indicated, or pairwise combinations at concentrations of 1 μM each. (A) Bar chart showing levels of Aβ1-42 levels (left axis, red bars) or Aβ1-x levels (right axis, black bars). (B) Stack chart showing Aβ1-37 (purple), Aβ1-38 (blue), Aβ1-40 (green), and Aβ1-42 (red). (C) Western blot showing α-CTF (arrow indicating lower band), and β-CTF (arrow indicating upper band). Error bars represent standard error for three replicate Aβ assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cause a switch to Aβ peptides shorter than Aβ1-24, which would be undetectable in this assay. Although truncated Aβ1-14, Aβ1-15, Aβ116, and Aβ1-17 can be readily detected in H4-APPsw cultures using MALDI-TOF, they are of low abundance, and treatment with combinations of GSMs did not show consistent increases in truncated Aβ peptides (not shown).
GSM, but did not decrease overall levels of Aβ. For comparison, the GSI BMS-299897 inhibited all four Aβ peptides (Fig. 4A). The Aβ1-x assay was evaluated in the same cultures, as well as additional cell cultures treated with combinations of the GSMs, BMS-802299, E2012, and GSM-1. Again, only the acid/non-acid GSM combinations showed Aβ1x lowering (Fig. 4B). In all, the combinations included 6 different pairs of non-acid GSMs, 4 different pairs of acid GSMs, and 15 different pairs containing one acid GSM and one non-acid GSM. Thus, all 10 of these GSMs exhibited synergism only when paired in combinations of one acid GSM and one non-acid GSM.
3.4. Lasalocid is a GSM that does not synergise with other GSMs To further address the question whether or not all GSMs exhibit synergism, if combined appropriately, we evaluated lasalocid (Westley et al., 1970), an Aβ1-42 lowering compound we identified in a BristolMyers Squibb high throughput screen of a natural product library. Lasalocid has an extended chemical structure containing 10 chiral centers and a single carboxylic acid group (Fig. 1). It has the functional characteristics of a GSM. First, in the automated Aβ1-42 and Aβ1-40 assays it exhibited potency in the nanomolar range, with greater potency for Aβ1-42, IC50 ca. 10 nM, than Aβ1-40, IC50 ca. 36 nM. Second, MALDI-TOF analysis of lasalocid-treated cell cultures showed a pronounced increase in Aβ1-37, a decrease in Aβ1-40, but no significant effect on Aβ1-38 (Fig. 6A-C). In ELISA assays, cell cultures given lasalocid alone exhibited strongly decreased Aβ1-42, intermediate decreases in Aβ1-x, and pronounced increases of up to 4-fold in Aβ1-37 (Fig. 6D-F). Combinations of lasalocid with six representative GSMs (3 acid GSMs and 3 non-acid GSMs) showed no additional lowering of Aβ1-x compared to lasalocid alone (Fig. 6E). For Aβ1-37, lasalocid caused an increase even in the presence of acid GSMs such as GSM-2 and 3β-hydroxy-5-cholestenoic acid, which by themselves cause less increase in Aβ1-37, suggesting a lack of interference between compounds. Thus, lasalocid is a unique GSM that showed no evidence of synergism or antagonism in combinations with other GSMs.
3.3. The mechanism of synergism involves inhibition of γ-secretase As further confirmation that Aβ1-x lowering represented inhibition of γ-secretase activity, we evaluated the effects of combinations of GSMs on α-/β-CTF substrate accumulation. Cell cultures were treated with pairwise combinations of BMS-869780, GSM-1 and GSM-2, or the same compounds individually. As a control for γ-secretase inhibition, cells were treated with the GSI BMS-299897. After treatment, Aβ levels were quantified, and cells were harvested for analysis of α-/β-CTF levels by western blotting. Aβ1-42 was decreased in all treated cultures relative to the dimethyl sulfoxide vehicle-treated culture, whereas Aβ1x was decreased only in cultures treated with the BMS-869780/GSM-1 and BMS-869780/GSM-2 combinations, or the GSI (Fig. 5A). All the GSMs caused changes in the relative amounts of Aβ1-37, Aβ1-38, Aβ140, and Aβ1-42, but the total quantity of these four peptides was decreased only for the BMS-869780/GSM-1 and BMS-869780/GSM-2 combinations, and for the GSI (Fig. 5B). Accumulation of α-CTF was most pronounced in cultures treated with the BMS-869780/GSM-1 and BMS-869780/GSM-2 combinations, and for the GSI (Fig. 5C). It is a quirk of the H4-APPsw cell line that β-CTF turnover is not affected even under conditions when Aβ production is fully blocked by γsecretase inhibition (Toyn et al., 2014), most likely due to the predominance of proteasomal and lysosomal degradation pathways in these cells (Bustamante et al., 2013). Thus, the observed α-CTF accumulation is consistent with γ-secretase inhibition by combinations of GSMs. Nevertheless, we did consider an alternative interpretation: in principle, the Aβ1-x ELISA can detect all Aβ peptides longer than Aβ1-24, so a hypothetical possibility is that GSM combinations might
3.5. Synergism did not translate in vivo To explore if GSM synergism can occur in vivo, we took advantage of the favorable oral pharmacokinetics and brain penetrance of BMS869780 and GSM-2 in mice. Groups of mice were dosed with BMS869780 and GSM-2 either as individual compounds or in combination. Additional groups of mice were given the GSI BMS-433796 for 6
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 6. Lasalocid is a GSM that does not exhibit synergistic inhibition when combined with other GSMs. (A) MALDI-TOF analysis of an equimolar mixture of synthetic Aβ peptides. (B) Aβ peptides in a cell culture treated with dimethyl sulfoxide vehicle. (C) Aβ peptides in a cell culture treated with lasalocid at a concentration of 300 nM. (D) To determine the effect on Aβ1-42, cell cultures were treated with lasalocid at 1 μM or 2 μM, as indicated, and six other GSMs as individual compounds at 2 μM, or pairwise combinations of lasalocid and the six GSMs at a concentration of 1 μM each. (E) Aβ1-42 levels as in panel D. (F) Aβ1-37 levels as in panel D. Error bars represent standard error.
tion, thus any lowering of Aβ1-x is by definition synergistic. Second, isobolograms, for 50%, 75%, and 90% Aβ1-x or Aβ1-40 lowering, showed curves with strong leftward curves, indicative of synergism. Third, calculation of the combination index (CI) over a range of combined GSM concentrations showed low values, indicating strong synergism. The CI analysis does not assume knowledge of mechanism, and merely answers the question whether or not the combined effect of two drugs deviates from the amount of inhibition predicted from the individual inhibitory effects of the two compounds. Fourth, 15 pairwise combinations of one acid GSM with one non-acid GSM consistently showed inhibition of Aβ production, whereas 10 combinations containing GSMs of the same structural class did not inhibit Aβ production, i.e. pairs consisting only of acid GSMs or pairs consisting only of non-acid GSMs were not synergistic. This consistency suggests that essentially any combination of one acid GSM and one non-acid GSM would be expected to be synergistic. Fifth, synergistic GSM combinations caused accumulation of Aβ precursors, APP-CTF, indicative of inhibition of
comparison. Brain Aβ1-42 and Aβ1-x levels were determined 3 h and 6 h after dosing (Fig. 7). All dose groups showed significant decreases in Aβ1-42 relative to the vehicle-only group at both time points, but only the GSI showed significant Aβ1-x lowering (Table 1). A small but significant decrease in Aβ1-42 was observed for the combination treatments relative to BMS-698780 treatment alone, however, for Aβ1-x there were no significant differences between any of the GSM groups (Table 1). Thus, in mouse brain under these conditions, synergism was not observed for the BMS-869780/GSM-2 combination. Additional combinations of GSMs were not tested in mice.
4. Discussion Our main finding was that certain combinations of GSMs can inhibit Aβ production. First, the combined effect of two GSMs was greater than expected given the effects of individual GSMs. Individual GSMs generally have no effect on the total amount of Aβ1-x produc7
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
neously acting like both an acid GSM and a non-acid GSM, based on the idea that its extended structure would allow it to interact with both types of GSM binding sites on γ-secretase. Consistent with this idea, we showed that lower concentrations of lasalocid decreased Aβ1-42 and increased Aβ1-37 in the manner of a GSM, but higher concentrations of lasalocid lowered all forms of Aβ in the manner of an acid/non-acid GSM combination. An intriguing prediction, based on the lasalocid results, is that the acid and non-acid GSM binding sites might be located in close proximity. However, we did not pursue mapping studies of GSM binding sites that would directly address the possible juxtaposition of GSM binding sites. Many representative GSMs have been shown to bind directly to presenilin, the catalytic subunit of γ-secretase (Crump et al., 2011; Ebke et al., 2011; Jumpertz et al., 2012; Ohki et al., 2011; Pozdnyakov et al., 2013). A priori, therefore, it seems likely that the synergistic effects of combinations of GSMs would also act directly through binding to the γ-secretase enzyme. This hypothesis is supported by the pharmacology of the multiple combinations that we evaluated: 15 acid/non-acid combinations that were synergistic, in contrast to six acid/acid GSM combinations and four non-acid/non-acid GSM combinations that were not synergistic. Given the broad structural diversity of so many compounds whose common features are their binding to presenilin and their modulation of γ-secretase, this pharmacological consistency strongly implicates a direct effect on the γ-secretase enzyme. It seems reasonable to suggest that the synergistic binding sites correspond to the sites detected by photoligation and GSM competition binding studies. Previous studies of GSM combinations considered only the effects on Aβ1-42, and no synergism was detected (Borgegard et al., 2012). We likewise found no evidence for a synergistic effect on Aβ1-42 production. Nevertheless, the studies by Borgegard et al. established independent GSM binding sites based on their analysis using a well-established binding model (Yonetani and Theorell, 1964). A similar analysis of our synergistic data for Aβ1-x and Aβ1-40 did not fit significantly to the same model (analysis not shown). Given that GSM combinations were synergistic for Aβ1-x and Aβ1-40, but not synergistic for Aβ1-42, an explanation based on synergistic changes in the binding affinities of GSMs seems unlikely, because, in that case, increased GSM binding should have affected the production of all forms of Aβ equally. A second possibility would be that synergistic pairs of GSMs might displace binding of substrate, i.e. substrate APPCTF would not be able to bind to the enzyme at the same time as an acid/non-acid GSM pair. However, GSMs individually do not compete with substrate binding, and they do not even inhibit enzyme activity, so it would be surprising if GSM combinations were found to inhibit substrate binding. Remarkably, evidence even suggests that GSMs have the opposite effect—causing increased substrate binding—because active site-directed ligands and GSMs exhibit mutual enhancements of binding to presenilin (Crump et al., 2011; Pozdnyakov et al., 2013). This gives some basis to speculate that synergistic GSM combinations would inhibit catalysis by causing an irreversible binding of substrate, product, or an intermediate. The mechanism of GSM synergism therefore remains unsolved, but it seems most likely that GSM combinations lead to a blockage in a critical aspect of the enzyme reaction cycle, rather than causing increased GSM affinity or decreased substrate affinity. GSM synergism suggests potential applications in drug discovery. A new screening method would now have a theoretic underpinning, allowing the sensitivity and convenience of low-volume assays for the abundantly secreted Aβ1-x or Aβ1-40, at the same time as an unambiguous test for the specific GSM mechanism of action. For example, a high-throughput screen could be performed in the presence of a GSM, and inhibitory hits could be re-screened in the absence of the GSM. Alternatively, screening of compounds in the presence or absence of a given GSM could be implemented at a secondary stage for the decisive identification of new GSMs. Such an approach would facilitate high data quality, cost-effectiveness, and accurate identification of GSM
Fig. 7. The BMS-869780/GSM-2 combination shows no evidence of synergism in vivo. Groups (n = 5) of mice were given BMS-869780 or GSM-2, either as individual compounds or the combination. Additional groups were given the GSI BMS-433796. (A) Bar chart showing brain levels of Aβ1-42 (red) and Aβ1-x (blue) in groups of mice 3 h after dosing. (B) Bar chart showing brain levels of Aβ1-42 (red) and Aβ1-x (blue) in groups of mice 6 h after dosing. Error bars represent standard error. The results of significance testing are summarized in Table 1. The label “both” indicates the combination dose of BMS-869780 and GSM-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 1 Summary of multiple comparisons between groups of mice using ANOVA and post hoc Dunnett's test. Aβ1-x
Vehicle vs. BMS-869780 Vehicle vs. GSM-2 Vehicle vs. both Vehicle vs. GSI d Both vs. BMS-869780 d Both vs. GSM-2 d Both vs. GSI
Aβ1-42
3h
6h
3h
6h
ns ns ns c ns ns c
ns ns ns c ns ns c
c c c c ns ns ns
c c c c b ns ns
ns, P > 0.05. P < 0.01. c P < 0.001. d “Both” indicates the combination dosing of GSM-2 and BMS-869780. b
APP processing by γ-secretase. In addition, we evaluated a natural product called lasalocid, not previously classified as a GSM. Lasalocid showed no evidence of synergism in combinations with acid or non-acid GSMs, possibly representing a distinct, third mechanism of action among the GSMs. An alternative, perhaps more parsimonious explanation, would be that lasalocid acts through the same mechanisms as other GSMs, simulta-
8
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Zhang, L., Leung, L., Becker, S.L., Tseng, E., Barricklow, J., Miller, E.H., Osgood, S., O’Neill, B.T., Brodney, M.A., Johnson, D.S., Pettersson, M., 2012. Cerebrospinal fluid amyloid-β (Aβ) as an effect biomarker for brain Aβ lowering verified by quantitative preclinical analyses. J. Pharmacol. Exp. Ther. 342, 366–375. Marcin, L.R., Higgins, M.A., Lentz, K.A., Grace, J.E., Toyn, J.H., Olson, R.E., Albright, C. F., Macor, J.E., Thompson, L.A., 2012. Bicyclic triazoles as modulators of γ-secretase for the treatment of Alzheimer’s disease, MEDI-9, In: 243th ACS National Meeting, San Diego, California. Mitani, Y., Yarimizu, J., Saita, K.K., Uchino, H., Akashiba, H., Shitaka, Y., Ni, K., Matsuoka, N., 2012. Differential effects between γ-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice. J. Neurosci. 32, 2037–2050. Nakano-Ito, K., Fujikawa, Y., Hihara, T., Shinjo, H., Kotani, S., Suganuma, A., Aoki, T., Tsukidate, K., 2013. E2012-induced cataract and its predictive biomarkers. Toxicol. Sci. 137, 249–258. Ohki, Y., Higo, T., Uemura, K., Shimada, N., Osawa, S., Berezovska, O., Yokoshima, S., Fukuyama, T., Tomita, T., Iwatsubo, T., 2011. Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1. EMBO J. 30, 4815–4824. Page, R.M., Baumann, K., Tomioka, M., Peréz-Revuelta, B.I., Fukumori, A., Jacobsen, H., Flohr, A., Luebbers, T., Ozmen, L., Steiner, H., Haass, C., 2007. Generation of Aβ38 and Aβ42 is independently and differentially affected by familial Alzheimer diseaseassociated presenilin mutations and γ-secretase modulation. J. Biol. Chem. 283, 677–683. Pozdnyakov, N., Murrey, H.E., Crump, C.J., Pettersson, M., Ballard, T.E., Am Ende, C.W., Ahn, K., Li, Y.-M., Bales, K.R., Johnson, D.S., 2013. . γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin. J. Biol. Chem. 288, 9710–9720. Prasad, C.V.C., Zheng, M., Vig, S., Bergstrom, C., Smith, W.D., Gao, Q., Yeola, S., Polson, C.T., Corsa, J.A., Guss, V.L., Loo, A., Wang, J., Sleczka, B.G., Dangler, C., Robertson, B.J., Hendrick, J.P., Roberts, S.B., Barten, D.M., 2007. Discovery of (S)-2-((S)-2(3,5-difluorophenyl)-2-hydroxyacetamido)-N-(((S,Z,Z)-3-methyl-4-oxo-4,5-dihydro3Hbenzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796): a γ-secretase inhibitor with Aβ lowering activity in a transgenic mouse model of Alzheimer's disease. Bioorg. Med. Chem. Lett. 17, 4006–4011. Rogers, K., Felsenstein, K.M., Hrdlicka, R., Tu, Z., Albayya, F., Lee, W., Hopp, S., Miller, M.-J., Spaulding, D., Yang, Z., Hodgdon, H., Nolan, S., Wen, M., Costa, D., Blain, J.F., Freeman, E., De Strooper, B., Vulsteke, V., Scrocchi, L., Zetterberg, H., Portelius, E., Hutter-Paier, B., Havas, D., Ahlijanian, A., Flood, D., Leventhal, L., Shapiro, G., Patzke, H., Chesworth, R., Koenig, G., 2012. Modulation of γ-secretase by EVP0015962 reduces amyloid deposition and cognitive deficits in Tg2576 mice. Mol. Neurodegen. 7, 61. Soares, H.D., Gasior, M., Toyn, J.H., Wang, J.S., Hong, Q., Berisha, F., Furlong, M.T., Raybon, J., Lentz, K.A., Sweeney, F., Zheng, N., Akinsanya, B., Berman, R.M., Thompson, L.A., Olson, R.E., Morrison, J., Drexler, D.M., Macor, J.E., Albright, C.F., Ahlijanian, M.K., AbuTarif, M., 2016. The γ-secretase modulator, BMS-932481, modulates Aβ peptides in the plasma and cerebrospinal fluid of healthy volunteers. J. Pharmacol. Exp. Ther. 358, 138–150. Tallarida, R.J., 2006. An overview of drug combination analysis with isobolograms. J. Pharmacol. Exp. Ther. 319, 1–7. Tate, B., McKee, T.D., Loureiro, R.M.B., Dumin, J.A., Xia, W., Pojasek, K., Austin, W.F., Fuller, N.O., Hubbs, J.L., Shen, R., Jonker, J., Ives, J., Bronk, B.S., 2012. Modulation of gamma-secretase for the treatment of Alzheimer's disease. Int. J. Alzheimer Dis. 2012, 210756. Toyn, J.H., Boy, K.M., Raybon, J., Meredith, J.E., Jr, Robertson, A.S., Guss, V., Hoque, N., Sweeney, F., Zhuo, X., Clarke, W., Snow, K., Denton, R.R., Zuev, D., Thompson, L.A., Morrison, J., Grace, J., Berisha, F., Furlong, M., Wang, J.-S., Lentz, K.A., Padmanabha, R., Cook, L., Wei, C., Drexler, D.M., Macor, J.E., Albright, C.F., Gasior, M., Olson, R.E., Hong, Q., Soares, H.D., AbuTarif, M., Ahlijanian, M.K., 2016. Robust translation of γ-secretase modulator pharmacology across preclinical species and human subjects. J. Pharmacol. Exp. Ther. 358, 125–137. Toyn, J.H., Thompson, L.A., Lentz, K.A., Meredith, J.E., Jr, Burton, C.R., Sankaranarayanan, S., Guss, V., Hall, T., Iben, L.G., Krause, C.M., Krause, R., Lin, X.-A., Pierdomenico, M., Polson, C., Robertson, A.S., Denton, R.R., Grace, J.E., Morrison, J., Raybon, J., Zhuo, X., Snow, K., Padmanabha, R., Agler, M., Esposito, K., Harden, D., Prack, M., Varma, S., Wong, V., Zhu, Y., Zvyaga, T., Gerritz, S., Marcin, L.R., Higgins, M.A., Shi, J., Wei, C., Cantone, J.L., Drexler, D.M., Macor, J.E., Olson, R.E., Ahlijanian, M.K., Albright, C.F., 2014. Identification and preclinical pharmacology of the γ-secretase modulator BMS-869780. Int. J. Alzheimer Dis. 2014, 431858. Uemura, K., Farner, K.C., Hashimoto, T., Nasser-Ghodsi, N., Wolfe, M.S., Koo, E.H., Hyman, B.T., Berezovska, O., 2010. Substrate docking to γ-secretase allows access of γ-secretase modulators to an allosteric site. Nat. Commun. 1, 130. Uemura, K., Farner, K.C., Nasser-Ghodsi, N., Jones, P., Berezovska, O., 2011. Reciprocal relationship between APP positioning relative to the membrane and PS1 conformation. Mol. Neurodegen. 6, 15. Uemura, K., Lill, C.M., Li, X., Peters, J.A., Ivanov, A., Fan, Z., De Strooper, B., Bacskai, B.J., Hyman, B.T., Berezovska, O., 2009. Allosteric modulation of PS1/γ-secretase conformation correlates with amyloid β42/40 ratio. PLoS One 4, e7893. Westley, J.W., Evans, R.H., Jr, Williams, T., Stempel, A., 1970. Structure of antibiotic X537A. Chem. Commun. 1970, 71–72. Weggen, S., Eriksen, J.L., Das, P., Sagi, S.A., Wang, R., Pietrzik, C.U., Findlay, K.A., Smith, T.E., Murphy, M.P., Bulter, T., Kang, D.E., Marquez-Sterling, M., Golde, T.E., Koo, E.H., 2001. A subset of NSAIDs lowers amyloidogenic Aβ42 independently of cyclooxygenase activity. Nature 414, 212–216. Yonetani, T., Theorell, H., 1964. Studies on liver alcohol dehydrogenase complexes. Arch. Biochem. Biophys. 106, 243–251.
mechanism of action. Another potential application would be in the evaluation of endogenous GSM activities that affect γ-secretase. It would be possible to discover or explore in vivo conditions that might enhance production of endogenous steroids at tissue levels that influence γ-secretase activity. Such conditions would be revealed by an inhibitory effect on Aβ1-x or Aβ1-40 after dosing a non-acid GSM. Again, the abundance of Aβ1-x and Aβ1-40 would lower assay volumes, decrease animal group sizes, and facilitate data quality relative to conventional Aβ1-42 measurements. In conclusion, this study provides clear evidence that essentially any combination of acid and non-acid GSM can cause synergistic inhibition of the main forms of Aβ, Aβ1-x and Aβ1-40, and suggests applications that may be of value in the discovery and development of new GSMs. Disclosure statement All authors are employees or former employees of Bristol-Myers Squibb. The experiments were carried out at Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06437, USA Acknowledgements The authors thank members of the Discovery Chemistry Department of Bristol-Myers Squibb for providing the GSMs and GSIs used in these studies. References Barten, D.M., Guss, V.L., Corsa, J.A., Loo, A., Hansel, S.B., Zheng, M., Munoz, B., Srinivasan, K., Wang, B., Robertson, B.J., Polson, C.T., Wang, J., Roberts, S.B., Hendrick, J.P., Anderson, J.J., Loy, J.K., Denton, R., Verdoorn, T.A., Smith, D.W., Felsenstein, K.M., 2005. Dynamics of β-amyloid reductions in brain, cerebrospinal fluid, and plasma of β-amyloid precursor protein transgenic mice treated with a γsecretase inhibitor. J. Pharmacol. Exp. Ther. 312, 635–643. Bijnsdorp, I.V., Giovannetti, E., Peters, G.J., 2011. Analysis of drug interactions. Methods Mol. Biol. 731, 421–434. Borgegard, T., Juréus, A., Olsson, F., Rosqvist, S., Sabirsh, A., Rotticci, D., Paulsen, K., Klintenberg, R., Yan, H., Waldman, M., Stromberg, K., Nord, J., Johansson, J., Regner, A., Parpal, S., Malinowsky, D., Radesater, A.-C., Li, T., Singh, R., Eriksson, H., Lundkvist, J., 2012. First and second generation γ-secretase modulators (GSMs) modulate amyloid-β (Aβ) peptide production through different mechanisms. J. Biol. Chem. 287, 11810–11819. Bursavich, M.G., Harrison, B.A., Blain, J.-F., 2016. Gamma secretase modulators: New Alzheimer's drugs on the horizon? J. Med. Chem. 59, 7389–7409. Bustamante, H.A., Rivera-Diccter, A., Cavieres, V.A., Muñoz, V.C., González, A., Lin, Y., Mardones, G.A., Burgos, P.V., 2013. Turnover of C99 is controlled by a crosstalk between ERAD and ubiquitin-independent lysosomal degradation in human neuroglioma cells. PLoS ONE 8, e83096. Chou, T.-C., Talalay, P., 1984. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. 22, 27–55. Crump, C.J., Fish, B.A., Castro, S.V., Chao, D.-M., Gertsik, N., Ahn, K., Stiff, C., Pozdnyakov, N., Bales, K.R., Johnson, D.S., Li, Y.-M., 2011. Piperidine acetic acid based γ-secretase modulators directly bind to presenilin-1. ACS Chem. Neurosci. 2, 705–710. Crump, C.J., Johnson, D.S., Li, Y.-M., 2013. Development and mechanism of γ-secretase modulators of Alzheimer's disease. Biochemistry 52, 3197–3216. Ebke, A., Luebbers, T., Fukumori, A., Shirotani, K., Haass, C., Baumann, K., Steiner, H., 2011. Novel γ-secretase enzyme modulators directly target presenilin protein. J. Biol. Chem. 286, 37181–37186. Findeis, M.A., Schroeder, F., McKee, T.D., Yager, D., Fraering, P.C., Creaser, S.P., Austin, W.F., Clardy, J., Wang, R., Selkoe, D., Eckman, C.B., 2012. Discovery of a novel pharmacological and structural class of gamma secretase modulators derived from the extract of Acteae racemosa. ACS Chem. Neurosci. 3, 941–951. Hall, A., Patel, T.R., 2014. . γ-Secretase modulators: current status and future directions. Prog. Med. Chem. 53, 101–145. Jumpertz, T., Rennhack, A., Ness, J., Baches, S., Pietrzik, C.U., Bulic, B., Weggen, S., 2012. Presenilin is the molecular target of acidic γ-secretase modulators in living cells. PLoS ONE 7, e30484. Jung, J.I., Ladd, T.B., Kukar, T., Price, A.R., Moore, B.D., Koo, E.H., Golde, T.E., Felsenstein, K.M., 2013. Steroids as γ-secretase modulators. FASEB J. 27, 3775–3785. Karran, E., Mercken, M., De Strooper, B., 2011. The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Disc 10, 698–712. Lu, Y., Riddell, D., Hajos-Korcsok, E., Bales, K., Wood, K.M., Nolan, C.E., Robshaw, A.E.,
9
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Synergistic inhibition of Aβ production by combinations of γ-secretase modulators Alan S. Robertsona, Lawrence G. Ibena, Cong Weib, Jere E. Meredith Jr.a, Dieter M. Drexlerb, Martyn Banksc, Gregory D. Vitec, Richard E. Olsond, Lorin A. Thompsond, Charles F. Albrighta, ⁎ Michael K. Ahlijaniana, Jeremy H. Toyna, a
Neuroscience Biology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA Discovery Analytical Sciences, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA c Lead Discovery and Lead Profiling, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA d Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06437, USA b
A R T I C L E I N F O
A BS T RAC T
Keywords: Alzheimer Amyloid Combination index Lasalocid Presenilin Synergism
Alzheimer's disease is associated with the accumulation of amyloid-β (Aβ) in the brain. In particular, the 42amino acid form, Aβ1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aβ1-42 production without inhibiting the production of other forms of Aβ. Here we describe the novel discovery of synergistic inhibition of Aβ by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aβ peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aβ occurred only when one GSM was of the “acid GSM” structural class and the other was of the “non-acid GSM” class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aβ production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aβ. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target.
1. Introduction The 42-amino acid form of the amyloid-β peptide, Aβ1-42, is associated with Alzheimer's disease (Karran et al., 2011). It is therefore of interest that certain small molecules, known as γ-secretase modulators (GSM), can selectively decrease Aβ1-42 (for reviews see Bursavich et al., 2016; Crump et al., 2013; Hall and Patel, 2014; Tate et al., 2012). GSMs are structurally and functionally diverse: one type, the “acid GSMs,” includes non-steroidal anti-inflammatory drugs (NSAID), such as sulindac sulfide (Weggen et al., 2001) and related structures containing carboxylic acid moieties, such as “GSM-2” (Mitani et al., 2012). Typically, the acid GSMs decrease Aβ1-42 and increase Aβ1-38, but have little effect on production of Aβ1-37 or Aβ140. A second group of GSMs decreases both Aβ1-42 and Aβ1-40, while at the same time increasing both Aβ1-37 and Aβ1-38. We shall refer to
⁎
this structurally diverse group as “non-acid GSMs.” An example is BMS-869780 (Toyn et al., 2014). Additional GSMs include natural product-derived triterpenoids (Findeis et al., 2012), and endogenous steroids found in mammals, such as 3β-hydroxy-5-cholestenoic acid (Jung et al., 2013). In some cases, close GSM analogs increase Aβ1-42 and are known as “inverse GSMs” (Jung et al., 2013; Ohki et al., 2011). In general, however, there is a predictable correspondence between the GSM structural type and the effects on production of Aβ peptides. Photolabelling studies show that GSMs target presenilin, the catalytic subunit of γ-secretase (Crump et al., 2011; Ebke et al., 2011; Jumpertz et al., 2012; Ohki et al., 2011). Furthermore, photolabelling competition studies (Pozdnyakov et al., 2013) and enzyme cross competition analysis (Borgegard et al., 2012) suggest that GSMs may target two distinct binding sites. Consistent with an allosteric mode of action, GSMs cause conformational changes within the
Correspondence to: Yale University, Office of Development, 157 Church Street, New Haven, CT 06510, USA. E-mail address: [email protected] (J.H. Toyn).
http://dx.doi.org/10.1016/j.ejphar.2017.07.019 Received 21 February 2017; Received in revised form 3 July 2017; Accepted 5 July 2017 0014-2999/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Robertson, A.S., European Journal of Pharmacology (2017), http://dx.doi.org/10.1016/j.ejphar.2017.07.019
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
N Cl
O N
N
N N
N
NH
O
N
N
N
N H
N H
N H
OH
O
O
N N
O
NH N
O
O
O
F
N
F
F H N
N
N
F
O
HN
N
N
N N
N
N
N
N N N
F
CF
F COOH O
S
Me Me
O Me
N
H
HO
O OH
O
HO
H
OH O
H
F
Cl
HO
O H O
F
OH
β O O
HO
OH
S
F C
Cl O
N
N
CF
CF Cl
F
F C
COOH
F C
HO
O
Fig. 1. Chemical structures of compounds used in this study.
2. Materials and methods
presenilin protein, and within the complex between presenilin and its substrate, the β-C-terminal fragment (β-CTF) of the amyloid precursor protein (Ohki et al., 2011; Uemura et al., 2009, 2010, 2011). Furthermore, allosteric interactions can be inferred from photolabelling interactions between GSMs and γ-secretase inhibitors (GSI): a GSM can enhance photolabelling by a GSI-derived photoligand (Crump et al., 2011), and a GSI can enhance photolabelling by a GSM-derived photoligand (Pozdnyakov et al., 2013). However, there are no reports describing enhanced inhibition of Aβ production resulting from combinations of different modulators or inhibitors of γ-secretase. Here we describe the novel discovery of synergistic inhibition of Aβ production by pairwise combinations of GSMs. Synergistic inhibition is unexpected because GSMs acting individually do not inhibit Aβ production. In addition, we found the natural product lasalocid (Westley et al., 1970) to be a potent GSM, but lasalocid did not synergize with other GSMs. Our findings further elucidate the functional complexity of γ-secretase small molecule pharmacology, and suggest new approaches to advance the development of future GSMs.
2.1. Materials Many of the GSMs and GSIs used in this study were prepared at Bristol-Myers Squibb, including (S)-N-(4-(4-chloro-1H-imidazol-1-yl)3-methoxyphenyl)-9-(4-fluorophenyl)-6,7,8,9-tetrahydro-5H-[1,2,4] triazolo[1,5-a]azepin-2-amine (BMS-869780, Toyn et al., 2014); (S)-2((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-(((S,Z,Z)-3methyl-4-oxo-4,5-dihydro-3Hbenzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796, Prasad et al., 2007); (S)-7-(4-fluorophenyl)-N2(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481, Soares et al., 2016; Toyn et al., 2016); 6-benzyl-4-(3-methoxy-4-(4methyl-1H-imidazol-1-yl)benzyl)-N-methyl-1,6-dihydro-1,3,5-triazin2-amine (BMS-802299, Marcin et al., 2012); 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid (BMS-299897, Barten et al., 2005); (E)-1-[(1S)-1-(4fluorophenyl)ethyl]-3-[3-methoxy-4-(4-methyl-1H-imidazol-1-yl)benzylidene]piperidin-2-one (E2012, Nakano-Ito et al., 2013); (S,E,E)-2-
2
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 2. Cell cultures were treated with combinations of BMS-869780 and GSM-2 to determine effects on Aβ levels. (A) Aβ1-x levels shown as dose response of GSM-2 at constant concentrations of BMS-869780. (B) Aβ1-x levels shown as dose response of BMS-869780 at constant concentrations of GSM-2. (C) A contour plot summarizes the average values of Aβ1-x observed in 96-well format. (D) Aβ1-40 levels shown as dose response of GSM-2 at constant concentrations of BMS-869780. (E) Lactate dehydrogenase (LDH) levels in culture supernatants. (F) Aβ1-42 levels shown as dose response of GSM-2 at constant concentrations of BMS-869780. Error bars represent standard error between three replicate cultures.
(Toyn et al., 2014). Cell cultures were grown in 96-well format for the experiments shown in Figs. 2, 3, 4, and 6D-F, and grown in T-75 culture flasks for the experiments shown in Figs. 5, 6B and C. The final concentration of dimethyl sulfoxide in all cell cultures was 0.1%. MALDI-TOF mass spectroscopy of Aβ peptides immunoprecipitated from cell cultures, and immunoassays for Aβ1-37, Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-x, were carried out as previously described (Toyn et al., 2014). For western blotting of α-CTF and β-CTF, cell cultures were harvested, cell proteins were solubilized in sodium dodecyl sulfate electrophoresis sample buffer, total protein concentrations were quantified using a kit (EZQ Invitrogen catalog number R33200), and 10 μg total protein per sample was loaded per lane for western blotting, as previously described (Toyn et al., 2014). Lactate dehydrogenase (LDH) in cell cultures was quantified using a kit (BioVision, Milpitas, CA).
(2-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)vinyl)-8-(2(trifluoromethyl)phenyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridine (GSM-A, Lu et al., 2012); 2-((2S,4R)-1-(((R)-1-(4-chlorophenyl)4-methylpentyl)-2-(4-(trifluoromethyl)phenyl)piperidin-4-yl)acetic acid (GSM-1, Page et al., 2007); {(2S,4R)-1-[(4R)-1,1,1-trifluoro-7methyloctan-4-yl]-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetic acid (GSM-2, Mitani et al., 2012); and (R)-2-(5-chloro-6-(2,2,2-trifluoroethoxy)-4'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)-3-cyclobutylpropanoic acid (EVP-0015962, Rogers et al., 2012). {(1Z)-5-fluoro-2methyl-1-[4-(methylsulfinyl)benzylidene]-1H-indene-3-yl}acetic acid (sulindac sulfide) was obtained from Sigma-Aldrich (cat# S3131). 3βhydroxy-5-cholestenoic acid, and 6-{(3R,4S,5S,7R)-7-[(2S,3S,5S)-5ethyl-5-((2R,5R,6S)-5-ethyl-5-hydroxy-6-methyltetrahydropyran-2yl)-3-methyltetrahydrofuran-2-yl]-4-hydroxy-3,5-dimethyl-6-oxononyl}-2-hydroxy-3-methylbenzoic acid (lasalocid) were obtained from Santa Cruz Biotechnology (cat.# sc-209761, lot# J2912, and cat.# sc362029, lot# L1012, respectively). Stock solutions were prepared at a concentration of 20 mM in dimethyl sulfoxide. Chemical structures of the compounds are illustrated in Fig. 1.
2.3. Handling of animals and in vivo experiments All experimental procedures with mice followed National Institutes of Health guidelines, and were authorized by and in compliance with policies of the Bristol-Myers Squibb Animal Care and Use Committee. Mice were housed with a 6:00 a.m. to 6:00 p.m. light/dark cycle and allowed free access to food and water. Mice were randomly assigned to drug treatment and vehicle control groups, and were randomized with respect to the order of dosing within each time group. Oral dosing and preparation of brain samples for Aβ immunoassays were carried out as previously described (Toyn et al., 2014). Briefly, 12-week old C57Bl/6
2.2. Cell cultures, Aβ immunoassays, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectroscopy, and western blotting Maintenance of human neuroglioma H4-APPsw cell cultures and treatments with compounds were carried out as previously described 3
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 3. Analysis of synergism between BMS-869780 and GSM-2 in cell cultures. (A) Aβ1-x isobolograms at 50% ( ), 75% ( ), and 90% ( Aβ1-x as a function of BMS-869780/GSM-2 ratio at 50% ( ), 75% ( ), and 90% (
) effect levels. (B) Combination index for
) effect levels. (C) Aβ1-40 isobolograms. (D) Combination index for Aβ1-40. (E) Aβ1-42
isobolograms. (F) Combination index for Aβ1-42.
D1 D + 2 =1 Dx1 Dx 2
wild type mice were given oral doses of BMS-869780 and GSM-2 at 25 mg/kg, or BMS-433796 at 10 mg/kg. Results of the Aβ assays were evaluated by ANOVA and post hoc Dunnett's test.
where D1 and D2 are the respective doses of drug 1 and drug 2 that in combination have x effect, and Dx1 and Dx2 are the respective doses of drug 1 acting alone and drug 2 acting alone that have x effect. A related formula can be used to give a quantitative measure of synergistic effect size known as the combination index (CI):
2.4. Evaluation of synergism using isobolograms and combination index Synergism is defined as an effect of a drug combination that is greater than expected from the effects of the individual drugs. Dose response curves for GSM-2 at fixed concentrations of BMS-869780, and dose response curves for BMS-869780 at fixed concentrations of GSM-2, were fitted using spline curves in Graphpad Prizm, and dose combinations resulting in 50%, 75%, or 90% decreased Aβ were determined by interpolation. The concentrations of each GSM in dose combinations having equal effects on Aβ were plotted on Cartesian coordinates to make isobolograms (Tallarida, 2006). In principle, summation of the effects of two inhibitory drugs, in the absence of synergism or antagonism, would result in an isobologram having a straight downward sloping line, known as the “line of additivity,” enclosing a triangular space in the upper right quadrant. The equation representing the line of additivity is:
CI =
D1 D αD1 D2 + 2 + Dx1 Dx 2 Dx1 Dx 2
where α is a factor ranging in value between 0 and 1 representing the extent to which two drugs act independently of one another (Chou and Talalay, 1984). For the calculations of CI reported here, a value of α = 1, representing the case in which two compounds act independently of one another, was used because it makes the most conservative assumptions with respect to detection of synergism, yielding the most conservative estimates for CI. In principle, a value of CI = 1 indicates the potency predicted from addition of the effects of each individual compound acting alone. Values of CI < 1 indicate possible synergism, whereas values of CI > 1 indicate possible antagonism, that is, a lessening of the effect of one compound in the presence of another 4
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
than Aβ1-23 in length. Cell cultures were treated with combinations of BMS-869780 and GSM-2 to explore the entire dose-response range. The dose responses of BMS-869780 at fixed concentrations of GSM-2 showed potent inhibition of Aβ1-x by the combination (Fig. 2A). Likewise, for the same experiment, the dose response curve of GSM2 plotted at fixed concentrations of BMS-869780 showed potent inhibition of Aβ1-x (Fig. 2B). A contour plot gives another view of how combinations of the two GSMs decreased or increased Aβ1-x (Fig. 2C). It can be seen that BMS-869780 on its own did not inhibit Aβ1-x at any concentration tested, and even shows an increase in Aβ1x at concentrations in the micromolar range. The increase is consistent with many previous observations of the effect of BMS-869780 on Aβ1x, and correlates with increases in Aβ1-37 and Aβ1-38 (Toyn et al., 2014). Aβ1-40 and Aβ1-42 both showed decreases in the presence of the GSMs (Fig. 2D and F). Cell health and viability, as evaluated by lactate dehydrogenase levels in the culture media, were not affected, except at the highest concentration, 10,000 nM, of GSM-2 (Fig. 2E). Thus, combinations of BMS-869780 and GSM-2 cause inhibition of Aβ1-x production. Synergistic effects can be evaluated using isobolograms, which are graphical plots of dose combinations having an equal level of effect (Tallarida, 2006). In principle, additive effects of two compounds result in a straight line of downward slope, whereas synergism would cause a leftward deviation, and antagonism a rightward deviation. Using data from the same experiment illustrated in Fig. 2, Aβ1-x isobolograms constructed for BMS-869780 and GSM-2 showed a dramatic deviation to the left, confirming strong synergism (Fig. 3A). Isobolograms for Aβ1-40 also show a strong leftward deviation, again indicating synergism (Fig. 3C). In contrast, the Aβ1-42 isobolograms showed no clear evidence of a leftward deviation, suggesting an additive effect for Aβ142 (Fig. 3E). While isobolograms are qualitatively compelling, a quantitative way to evaluate synergism uses the “combination index” (CI; Chou and Talalay, 1984). The Aβ1-x data illustrated in Fig. 2 were used to calculate CI values (see materials and methods). CI values were plotted against the BMS-869780/GSM-2 concentration ratio for combinations that yielded 50%, 75% and 90% Aβ1-x lowering (Fig. 3B). The minimum values observed, CI = 0.05-0.06, indicate a very high degree of synergism. The minimum CI value occurred at a molar ratio of GSM-2:BMS-869780 in the range 1.5-2.3. Furthermore, the CI values, and the molar ratio at which the maximum synergism occurred, were of similar magnitude across 50%, 75% and 90% Aβ1-x lowering. Aβ1-40 showed low CI values that depended on the extent of lowering, with minima ranging from CI = 0.7 for 50% lowering to CI = 0.2 for 90% lowering (Fig. 3D). Aβ1-42 showed less pronounced lowering of the CI values, suggesting no significant effect, and that lowering of Aβ142 is therefore not synergistic (Fig. 3F). CI values greater than CI = 1 were also observed, which may represent interference between the GSMs at certain concentrations. The possible significance of increased CI values was not evaluated further.
Fig. 4. Cell cultures were treated with representative GSMs. (A) Stack chart showing Aβ1-37 (purple), Aβ1-38 (blue), Aβ1-40 (green), and Aβ1-42 (red) from cell cultures treated individually with BMS-869780 at 2 μM, BMS-932481 at 2 μM, GSM-A at 2 μM, GSM-2 at 2 μM, EVP-0015962 at 2 μM, 3β-hydroxy-5-cholestenoic acid at 10 μM, sulindac sulfide at 25 μM, or BMS-299897 (GSI) at 1 μM. Combination treatments included 1 μM concentration of each compound, except for 3β-hydroxy-5-cholestenoic acid and sulindac sulfide, which were 10 μM and 25 μM, respectively. (B) Bar chart showing results of Aβ1-x ELISA from the same experiment illustrated in panel A, and additional results from an experiment using the GSMs, BMS-802299, E2012, and GSM1, at a concentration of 2 μM individually or 1 μM each in combinations, respectively. Error bars indicate standard deviation for mostly 3 and up to 6 replicate wells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
compound. As a guide to interpretation, a value of CI < 0.85 may indicate synergism, and a value of CI < 0.3 may indicate strong synergism (Bijnsdorp et al., 2011). To implement CI calculations for the GSM BMS-869780, which does not inhibit Aβ1-x when acting alone, the highest concentration tested (10,000 nM) was used in CI calculations for effects on Aβ1-x, which therefore yields an overestimate of CI, i.e. a conservative evaluation that gives an underestimate of synergism, but nonetheless facilitates inspection of patterns in the data.
3.2. Synergism requires the combination of one acid GSM with one non-acid GSM To find out if Aβ inhibition is a general property of GSM combinations, cell cultures were treated with a selection of different GSMs either individually or in pairwise combinations to evaluate the effects on Aβ production. Cell culture treatments included all pairwise combinations of BMS-869780, BMS-932481, GSM-A, GSM-2, EVP15962, 3β-hydroxy-5-cholestenoic acid, and sulindac sulfide, representing 3 non-acid GSMs and 4 acid GSMs (Fig. 4A). Treatments with the individual GSMs caused changes in the relative levels of Aβ1-37, Aβ1-38, Aβ1-40, and Aβ1-42, as expected. However, all 12 of the cultures treated with a combination of one acid GSM and one non-acid GSM resulted in lowering of Aβ levels. Combinations containing acid with acid, or non-acid with non-acid, as well as the individual GSMs, showed effects on the Aβ profile characteristic of the structural type of
3. Results 3.1. Synergistic inhibition of Aβ production by combinations of BMS869780 and GSM-2 To evaluate inhibition by combinations of GSMs, the Aβ1-x ELISA was used because it can detect all major soluble Aβ peptides greater 5
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 5. Cell cultures were treated with BMS-869780 (a), GSM-1 (b), GSM-2 (c), or BMS-299897 (d), either as individual compounds at a concentration of 1 μM or 2 μM, as indicated, or pairwise combinations at concentrations of 1 μM each. (A) Bar chart showing levels of Aβ1-42 levels (left axis, red bars) or Aβ1-x levels (right axis, black bars). (B) Stack chart showing Aβ1-37 (purple), Aβ1-38 (blue), Aβ1-40 (green), and Aβ1-42 (red). (C) Western blot showing α-CTF (arrow indicating lower band), and β-CTF (arrow indicating upper band). Error bars represent standard error for three replicate Aβ assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cause a switch to Aβ peptides shorter than Aβ1-24, which would be undetectable in this assay. Although truncated Aβ1-14, Aβ1-15, Aβ116, and Aβ1-17 can be readily detected in H4-APPsw cultures using MALDI-TOF, they are of low abundance, and treatment with combinations of GSMs did not show consistent increases in truncated Aβ peptides (not shown).
GSM, but did not decrease overall levels of Aβ. For comparison, the GSI BMS-299897 inhibited all four Aβ peptides (Fig. 4A). The Aβ1-x assay was evaluated in the same cultures, as well as additional cell cultures treated with combinations of the GSMs, BMS-802299, E2012, and GSM-1. Again, only the acid/non-acid GSM combinations showed Aβ1x lowering (Fig. 4B). In all, the combinations included 6 different pairs of non-acid GSMs, 4 different pairs of acid GSMs, and 15 different pairs containing one acid GSM and one non-acid GSM. Thus, all 10 of these GSMs exhibited synergism only when paired in combinations of one acid GSM and one non-acid GSM.
3.4. Lasalocid is a GSM that does not synergise with other GSMs To further address the question whether or not all GSMs exhibit synergism, if combined appropriately, we evaluated lasalocid (Westley et al., 1970), an Aβ1-42 lowering compound we identified in a BristolMyers Squibb high throughput screen of a natural product library. Lasalocid has an extended chemical structure containing 10 chiral centers and a single carboxylic acid group (Fig. 1). It has the functional characteristics of a GSM. First, in the automated Aβ1-42 and Aβ1-40 assays it exhibited potency in the nanomolar range, with greater potency for Aβ1-42, IC50 ca. 10 nM, than Aβ1-40, IC50 ca. 36 nM. Second, MALDI-TOF analysis of lasalocid-treated cell cultures showed a pronounced increase in Aβ1-37, a decrease in Aβ1-40, but no significant effect on Aβ1-38 (Fig. 6A-C). In ELISA assays, cell cultures given lasalocid alone exhibited strongly decreased Aβ1-42, intermediate decreases in Aβ1-x, and pronounced increases of up to 4-fold in Aβ1-37 (Fig. 6D-F). Combinations of lasalocid with six representative GSMs (3 acid GSMs and 3 non-acid GSMs) showed no additional lowering of Aβ1-x compared to lasalocid alone (Fig. 6E). For Aβ1-37, lasalocid caused an increase even in the presence of acid GSMs such as GSM-2 and 3β-hydroxy-5-cholestenoic acid, which by themselves cause less increase in Aβ1-37, suggesting a lack of interference between compounds. Thus, lasalocid is a unique GSM that showed no evidence of synergism or antagonism in combinations with other GSMs.
3.3. The mechanism of synergism involves inhibition of γ-secretase As further confirmation that Aβ1-x lowering represented inhibition of γ-secretase activity, we evaluated the effects of combinations of GSMs on α-/β-CTF substrate accumulation. Cell cultures were treated with pairwise combinations of BMS-869780, GSM-1 and GSM-2, or the same compounds individually. As a control for γ-secretase inhibition, cells were treated with the GSI BMS-299897. After treatment, Aβ levels were quantified, and cells were harvested for analysis of α-/β-CTF levels by western blotting. Aβ1-42 was decreased in all treated cultures relative to the dimethyl sulfoxide vehicle-treated culture, whereas Aβ1x was decreased only in cultures treated with the BMS-869780/GSM-1 and BMS-869780/GSM-2 combinations, or the GSI (Fig. 5A). All the GSMs caused changes in the relative amounts of Aβ1-37, Aβ1-38, Aβ140, and Aβ1-42, but the total quantity of these four peptides was decreased only for the BMS-869780/GSM-1 and BMS-869780/GSM-2 combinations, and for the GSI (Fig. 5B). Accumulation of α-CTF was most pronounced in cultures treated with the BMS-869780/GSM-1 and BMS-869780/GSM-2 combinations, and for the GSI (Fig. 5C). It is a quirk of the H4-APPsw cell line that β-CTF turnover is not affected even under conditions when Aβ production is fully blocked by γsecretase inhibition (Toyn et al., 2014), most likely due to the predominance of proteasomal and lysosomal degradation pathways in these cells (Bustamante et al., 2013). Thus, the observed α-CTF accumulation is consistent with γ-secretase inhibition by combinations of GSMs. Nevertheless, we did consider an alternative interpretation: in principle, the Aβ1-x ELISA can detect all Aβ peptides longer than Aβ1-24, so a hypothetical possibility is that GSM combinations might
3.5. Synergism did not translate in vivo To explore if GSM synergism can occur in vivo, we took advantage of the favorable oral pharmacokinetics and brain penetrance of BMS869780 and GSM-2 in mice. Groups of mice were dosed with BMS869780 and GSM-2 either as individual compounds or in combination. Additional groups of mice were given the GSI BMS-433796 for 6
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Fig. 6. Lasalocid is a GSM that does not exhibit synergistic inhibition when combined with other GSMs. (A) MALDI-TOF analysis of an equimolar mixture of synthetic Aβ peptides. (B) Aβ peptides in a cell culture treated with dimethyl sulfoxide vehicle. (C) Aβ peptides in a cell culture treated with lasalocid at a concentration of 300 nM. (D) To determine the effect on Aβ1-42, cell cultures were treated with lasalocid at 1 μM or 2 μM, as indicated, and six other GSMs as individual compounds at 2 μM, or pairwise combinations of lasalocid and the six GSMs at a concentration of 1 μM each. (E) Aβ1-42 levels as in panel D. (F) Aβ1-37 levels as in panel D. Error bars represent standard error.
tion, thus any lowering of Aβ1-x is by definition synergistic. Second, isobolograms, for 50%, 75%, and 90% Aβ1-x or Aβ1-40 lowering, showed curves with strong leftward curves, indicative of synergism. Third, calculation of the combination index (CI) over a range of combined GSM concentrations showed low values, indicating strong synergism. The CI analysis does not assume knowledge of mechanism, and merely answers the question whether or not the combined effect of two drugs deviates from the amount of inhibition predicted from the individual inhibitory effects of the two compounds. Fourth, 15 pairwise combinations of one acid GSM with one non-acid GSM consistently showed inhibition of Aβ production, whereas 10 combinations containing GSMs of the same structural class did not inhibit Aβ production, i.e. pairs consisting only of acid GSMs or pairs consisting only of non-acid GSMs were not synergistic. This consistency suggests that essentially any combination of one acid GSM and one non-acid GSM would be expected to be synergistic. Fifth, synergistic GSM combinations caused accumulation of Aβ precursors, APP-CTF, indicative of inhibition of
comparison. Brain Aβ1-42 and Aβ1-x levels were determined 3 h and 6 h after dosing (Fig. 7). All dose groups showed significant decreases in Aβ1-42 relative to the vehicle-only group at both time points, but only the GSI showed significant Aβ1-x lowering (Table 1). A small but significant decrease in Aβ1-42 was observed for the combination treatments relative to BMS-698780 treatment alone, however, for Aβ1-x there were no significant differences between any of the GSM groups (Table 1). Thus, in mouse brain under these conditions, synergism was not observed for the BMS-869780/GSM-2 combination. Additional combinations of GSMs were not tested in mice.
4. Discussion Our main finding was that certain combinations of GSMs can inhibit Aβ production. First, the combined effect of two GSMs was greater than expected given the effects of individual GSMs. Individual GSMs generally have no effect on the total amount of Aβ1-x produc7
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
neously acting like both an acid GSM and a non-acid GSM, based on the idea that its extended structure would allow it to interact with both types of GSM binding sites on γ-secretase. Consistent with this idea, we showed that lower concentrations of lasalocid decreased Aβ1-42 and increased Aβ1-37 in the manner of a GSM, but higher concentrations of lasalocid lowered all forms of Aβ in the manner of an acid/non-acid GSM combination. An intriguing prediction, based on the lasalocid results, is that the acid and non-acid GSM binding sites might be located in close proximity. However, we did not pursue mapping studies of GSM binding sites that would directly address the possible juxtaposition of GSM binding sites. Many representative GSMs have been shown to bind directly to presenilin, the catalytic subunit of γ-secretase (Crump et al., 2011; Ebke et al., 2011; Jumpertz et al., 2012; Ohki et al., 2011; Pozdnyakov et al., 2013). A priori, therefore, it seems likely that the synergistic effects of combinations of GSMs would also act directly through binding to the γ-secretase enzyme. This hypothesis is supported by the pharmacology of the multiple combinations that we evaluated: 15 acid/non-acid combinations that were synergistic, in contrast to six acid/acid GSM combinations and four non-acid/non-acid GSM combinations that were not synergistic. Given the broad structural diversity of so many compounds whose common features are their binding to presenilin and their modulation of γ-secretase, this pharmacological consistency strongly implicates a direct effect on the γ-secretase enzyme. It seems reasonable to suggest that the synergistic binding sites correspond to the sites detected by photoligation and GSM competition binding studies. Previous studies of GSM combinations considered only the effects on Aβ1-42, and no synergism was detected (Borgegard et al., 2012). We likewise found no evidence for a synergistic effect on Aβ1-42 production. Nevertheless, the studies by Borgegard et al. established independent GSM binding sites based on their analysis using a well-established binding model (Yonetani and Theorell, 1964). A similar analysis of our synergistic data for Aβ1-x and Aβ1-40 did not fit significantly to the same model (analysis not shown). Given that GSM combinations were synergistic for Aβ1-x and Aβ1-40, but not synergistic for Aβ1-42, an explanation based on synergistic changes in the binding affinities of GSMs seems unlikely, because, in that case, increased GSM binding should have affected the production of all forms of Aβ equally. A second possibility would be that synergistic pairs of GSMs might displace binding of substrate, i.e. substrate APPCTF would not be able to bind to the enzyme at the same time as an acid/non-acid GSM pair. However, GSMs individually do not compete with substrate binding, and they do not even inhibit enzyme activity, so it would be surprising if GSM combinations were found to inhibit substrate binding. Remarkably, evidence even suggests that GSMs have the opposite effect—causing increased substrate binding—because active site-directed ligands and GSMs exhibit mutual enhancements of binding to presenilin (Crump et al., 2011; Pozdnyakov et al., 2013). This gives some basis to speculate that synergistic GSM combinations would inhibit catalysis by causing an irreversible binding of substrate, product, or an intermediate. The mechanism of GSM synergism therefore remains unsolved, but it seems most likely that GSM combinations lead to a blockage in a critical aspect of the enzyme reaction cycle, rather than causing increased GSM affinity or decreased substrate affinity. GSM synergism suggests potential applications in drug discovery. A new screening method would now have a theoretic underpinning, allowing the sensitivity and convenience of low-volume assays for the abundantly secreted Aβ1-x or Aβ1-40, at the same time as an unambiguous test for the specific GSM mechanism of action. For example, a high-throughput screen could be performed in the presence of a GSM, and inhibitory hits could be re-screened in the absence of the GSM. Alternatively, screening of compounds in the presence or absence of a given GSM could be implemented at a secondary stage for the decisive identification of new GSMs. Such an approach would facilitate high data quality, cost-effectiveness, and accurate identification of GSM
Fig. 7. The BMS-869780/GSM-2 combination shows no evidence of synergism in vivo. Groups (n = 5) of mice were given BMS-869780 or GSM-2, either as individual compounds or the combination. Additional groups were given the GSI BMS-433796. (A) Bar chart showing brain levels of Aβ1-42 (red) and Aβ1-x (blue) in groups of mice 3 h after dosing. (B) Bar chart showing brain levels of Aβ1-42 (red) and Aβ1-x (blue) in groups of mice 6 h after dosing. Error bars represent standard error. The results of significance testing are summarized in Table 1. The label “both” indicates the combination dose of BMS-869780 and GSM-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 1 Summary of multiple comparisons between groups of mice using ANOVA and post hoc Dunnett's test. Aβ1-x
Vehicle vs. BMS-869780 Vehicle vs. GSM-2 Vehicle vs. both Vehicle vs. GSI d Both vs. BMS-869780 d Both vs. GSM-2 d Both vs. GSI
Aβ1-42
3h
6h
3h
6h
ns ns ns c ns ns c
ns ns ns c ns ns c
c c c c ns ns ns
c c c c b ns ns
ns, P > 0.05. P < 0.01. c P < 0.001. d “Both” indicates the combination dosing of GSM-2 and BMS-869780. b
APP processing by γ-secretase. In addition, we evaluated a natural product called lasalocid, not previously classified as a GSM. Lasalocid showed no evidence of synergism in combinations with acid or non-acid GSMs, possibly representing a distinct, third mechanism of action among the GSMs. An alternative, perhaps more parsimonious explanation, would be that lasalocid acts through the same mechanisms as other GSMs, simulta-
8
European Journal of Pharmacology xxx (xxxx) xxx–xxx
A.S. Robertson et al.
Zhang, L., Leung, L., Becker, S.L., Tseng, E., Barricklow, J., Miller, E.H., Osgood, S., O’Neill, B.T., Brodney, M.A., Johnson, D.S., Pettersson, M., 2012. Cerebrospinal fluid amyloid-β (Aβ) as an effect biomarker for brain Aβ lowering verified by quantitative preclinical analyses. J. Pharmacol. Exp. Ther. 342, 366–375. Marcin, L.R., Higgins, M.A., Lentz, K.A., Grace, J.E., Toyn, J.H., Olson, R.E., Albright, C. F., Macor, J.E., Thompson, L.A., 2012. Bicyclic triazoles as modulators of γ-secretase for the treatment of Alzheimer’s disease, MEDI-9, In: 243th ACS National Meeting, San Diego, California. Mitani, Y., Yarimizu, J., Saita, K.K., Uchino, H., Akashiba, H., Shitaka, Y., Ni, K., Matsuoka, N., 2012. Differential effects between γ-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice. J. Neurosci. 32, 2037–2050. Nakano-Ito, K., Fujikawa, Y., Hihara, T., Shinjo, H., Kotani, S., Suganuma, A., Aoki, T., Tsukidate, K., 2013. E2012-induced cataract and its predictive biomarkers. Toxicol. Sci. 137, 249–258. Ohki, Y., Higo, T., Uemura, K., Shimada, N., Osawa, S., Berezovska, O., Yokoshima, S., Fukuyama, T., Tomita, T., Iwatsubo, T., 2011. Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1. EMBO J. 30, 4815–4824. Page, R.M., Baumann, K., Tomioka, M., Peréz-Revuelta, B.I., Fukumori, A., Jacobsen, H., Flohr, A., Luebbers, T., Ozmen, L., Steiner, H., Haass, C., 2007. Generation of Aβ38 and Aβ42 is independently and differentially affected by familial Alzheimer diseaseassociated presenilin mutations and γ-secretase modulation. J. Biol. Chem. 283, 677–683. Pozdnyakov, N., Murrey, H.E., Crump, C.J., Pettersson, M., Ballard, T.E., Am Ende, C.W., Ahn, K., Li, Y.-M., Bales, K.R., Johnson, D.S., 2013. . γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin. J. Biol. Chem. 288, 9710–9720. Prasad, C.V.C., Zheng, M., Vig, S., Bergstrom, C., Smith, W.D., Gao, Q., Yeola, S., Polson, C.T., Corsa, J.A., Guss, V.L., Loo, A., Wang, J., Sleczka, B.G., Dangler, C., Robertson, B.J., Hendrick, J.P., Roberts, S.B., Barten, D.M., 2007. Discovery of (S)-2-((S)-2(3,5-difluorophenyl)-2-hydroxyacetamido)-N-(((S,Z,Z)-3-methyl-4-oxo-4,5-dihydro3Hbenzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796): a γ-secretase inhibitor with Aβ lowering activity in a transgenic mouse model of Alzheimer's disease. Bioorg. Med. Chem. Lett. 17, 4006–4011. Rogers, K., Felsenstein, K.M., Hrdlicka, R., Tu, Z., Albayya, F., Lee, W., Hopp, S., Miller, M.-J., Spaulding, D., Yang, Z., Hodgdon, H., Nolan, S., Wen, M., Costa, D., Blain, J.F., Freeman, E., De Strooper, B., Vulsteke, V., Scrocchi, L., Zetterberg, H., Portelius, E., Hutter-Paier, B., Havas, D., Ahlijanian, A., Flood, D., Leventhal, L., Shapiro, G., Patzke, H., Chesworth, R., Koenig, G., 2012. Modulation of γ-secretase by EVP0015962 reduces amyloid deposition and cognitive deficits in Tg2576 mice. Mol. Neurodegen. 7, 61. Soares, H.D., Gasior, M., Toyn, J.H., Wang, J.S., Hong, Q., Berisha, F., Furlong, M.T., Raybon, J., Lentz, K.A., Sweeney, F., Zheng, N., Akinsanya, B., Berman, R.M., Thompson, L.A., Olson, R.E., Morrison, J., Drexler, D.M., Macor, J.E., Albright, C.F., Ahlijanian, M.K., AbuTarif, M., 2016. The γ-secretase modulator, BMS-932481, modulates Aβ peptides in the plasma and cerebrospinal fluid of healthy volunteers. J. Pharmacol. Exp. Ther. 358, 138–150. Tallarida, R.J., 2006. An overview of drug combination analysis with isobolograms. J. Pharmacol. Exp. Ther. 319, 1–7. Tate, B., McKee, T.D., Loureiro, R.M.B., Dumin, J.A., Xia, W., Pojasek, K., Austin, W.F., Fuller, N.O., Hubbs, J.L., Shen, R., Jonker, J., Ives, J., Bronk, B.S., 2012. Modulation of gamma-secretase for the treatment of Alzheimer's disease. Int. J. Alzheimer Dis. 2012, 210756. Toyn, J.H., Boy, K.M., Raybon, J., Meredith, J.E., Jr, Robertson, A.S., Guss, V., Hoque, N., Sweeney, F., Zhuo, X., Clarke, W., Snow, K., Denton, R.R., Zuev, D., Thompson, L.A., Morrison, J., Grace, J., Berisha, F., Furlong, M., Wang, J.-S., Lentz, K.A., Padmanabha, R., Cook, L., Wei, C., Drexler, D.M., Macor, J.E., Albright, C.F., Gasior, M., Olson, R.E., Hong, Q., Soares, H.D., AbuTarif, M., Ahlijanian, M.K., 2016. Robust translation of γ-secretase modulator pharmacology across preclinical species and human subjects. J. Pharmacol. Exp. Ther. 358, 125–137. Toyn, J.H., Thompson, L.A., Lentz, K.A., Meredith, J.E., Jr, Burton, C.R., Sankaranarayanan, S., Guss, V., Hall, T., Iben, L.G., Krause, C.M., Krause, R., Lin, X.-A., Pierdomenico, M., Polson, C., Robertson, A.S., Denton, R.R., Grace, J.E., Morrison, J., Raybon, J., Zhuo, X., Snow, K., Padmanabha, R., Agler, M., Esposito, K., Harden, D., Prack, M., Varma, S., Wong, V., Zhu, Y., Zvyaga, T., Gerritz, S., Marcin, L.R., Higgins, M.A., Shi, J., Wei, C., Cantone, J.L., Drexler, D.M., Macor, J.E., Olson, R.E., Ahlijanian, M.K., Albright, C.F., 2014. Identification and preclinical pharmacology of the γ-secretase modulator BMS-869780. Int. J. Alzheimer Dis. 2014, 431858. Uemura, K., Farner, K.C., Hashimoto, T., Nasser-Ghodsi, N., Wolfe, M.S., Koo, E.H., Hyman, B.T., Berezovska, O., 2010. Substrate docking to γ-secretase allows access of γ-secretase modulators to an allosteric site. Nat. Commun. 1, 130. Uemura, K., Farner, K.C., Nasser-Ghodsi, N., Jones, P., Berezovska, O., 2011. Reciprocal relationship between APP positioning relative to the membrane and PS1 conformation. Mol. Neurodegen. 6, 15. Uemura, K., Lill, C.M., Li, X., Peters, J.A., Ivanov, A., Fan, Z., De Strooper, B., Bacskai, B.J., Hyman, B.T., Berezovska, O., 2009. Allosteric modulation of PS1/γ-secretase conformation correlates with amyloid β42/40 ratio. PLoS One 4, e7893. Westley, J.W., Evans, R.H., Jr, Williams, T., Stempel, A., 1970. Structure of antibiotic X537A. Chem. Commun. 1970, 71–72. Weggen, S., Eriksen, J.L., Das, P., Sagi, S.A., Wang, R., Pietrzik, C.U., Findlay, K.A., Smith, T.E., Murphy, M.P., Bulter, T., Kang, D.E., Marquez-Sterling, M., Golde, T.E., Koo, E.H., 2001. A subset of NSAIDs lowers amyloidogenic Aβ42 independently of cyclooxygenase activity. Nature 414, 212–216. Yonetani, T., Theorell, H., 1964. Studies on liver alcohol dehydrogenase complexes. Arch. Biochem. Biophys. 106, 243–251.
mechanism of action. Another potential application would be in the evaluation of endogenous GSM activities that affect γ-secretase. It would be possible to discover or explore in vivo conditions that might enhance production of endogenous steroids at tissue levels that influence γ-secretase activity. Such conditions would be revealed by an inhibitory effect on Aβ1-x or Aβ1-40 after dosing a non-acid GSM. Again, the abundance of Aβ1-x and Aβ1-40 would lower assay volumes, decrease animal group sizes, and facilitate data quality relative to conventional Aβ1-42 measurements. In conclusion, this study provides clear evidence that essentially any combination of acid and non-acid GSM can cause synergistic inhibition of the main forms of Aβ, Aβ1-x and Aβ1-40, and suggests applications that may be of value in the discovery and development of new GSMs. Disclosure statement All authors are employees or former employees of Bristol-Myers Squibb. The experiments were carried out at Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06437, USA Acknowledgements The authors thank members of the Discovery Chemistry Department of Bristol-Myers Squibb for providing the GSMs and GSIs used in these studies. References Barten, D.M., Guss, V.L., Corsa, J.A., Loo, A., Hansel, S.B., Zheng, M., Munoz, B., Srinivasan, K., Wang, B., Robertson, B.J., Polson, C.T., Wang, J., Roberts, S.B., Hendrick, J.P., Anderson, J.J., Loy, J.K., Denton, R., Verdoorn, T.A., Smith, D.W., Felsenstein, K.M., 2005. Dynamics of β-amyloid reductions in brain, cerebrospinal fluid, and plasma of β-amyloid precursor protein transgenic mice treated with a γsecretase inhibitor. J. Pharmacol. Exp. Ther. 312, 635–643. Bijnsdorp, I.V., Giovannetti, E., Peters, G.J., 2011. Analysis of drug interactions. Methods Mol. Biol. 731, 421–434. Borgegard, T., Juréus, A., Olsson, F., Rosqvist, S., Sabirsh, A., Rotticci, D., Paulsen, K., Klintenberg, R., Yan, H., Waldman, M., Stromberg, K., Nord, J., Johansson, J., Regner, A., Parpal, S., Malinowsky, D., Radesater, A.-C., Li, T., Singh, R., Eriksson, H., Lundkvist, J., 2012. First and second generation γ-secretase modulators (GSMs) modulate amyloid-β (Aβ) peptide production through different mechanisms. J. Biol. Chem. 287, 11810–11819. Bursavich, M.G., Harrison, B.A., Blain, J.-F., 2016. Gamma secretase modulators: New Alzheimer's drugs on the horizon? J. Med. Chem. 59, 7389–7409. Bustamante, H.A., Rivera-Diccter, A., Cavieres, V.A., Muñoz, V.C., González, A., Lin, Y., Mardones, G.A., Burgos, P.V., 2013. Turnover of C99 is controlled by a crosstalk between ERAD and ubiquitin-independent lysosomal degradation in human neuroglioma cells. PLoS ONE 8, e83096. Chou, T.-C., Talalay, P., 1984. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul. 22, 27–55. Crump, C.J., Fish, B.A., Castro, S.V., Chao, D.-M., Gertsik, N., Ahn, K., Stiff, C., Pozdnyakov, N., Bales, K.R., Johnson, D.S., Li, Y.-M., 2011. Piperidine acetic acid based γ-secretase modulators directly bind to presenilin-1. ACS Chem. Neurosci. 2, 705–710. Crump, C.J., Johnson, D.S., Li, Y.-M., 2013. Development and mechanism of γ-secretase modulators of Alzheimer's disease. Biochemistry 52, 3197–3216. Ebke, A., Luebbers, T., Fukumori, A., Shirotani, K., Haass, C., Baumann, K., Steiner, H., 2011. Novel γ-secretase enzyme modulators directly target presenilin protein. J. Biol. Chem. 286, 37181–37186. Findeis, M.A., Schroeder, F., McKee, T.D., Yager, D., Fraering, P.C., Creaser, S.P., Austin, W.F., Clardy, J., Wang, R., Selkoe, D., Eckman, C.B., 2012. Discovery of a novel pharmacological and structural class of gamma secretase modulators derived from the extract of Acteae racemosa. ACS Chem. Neurosci. 3, 941–951. Hall, A., Patel, T.R., 2014. . γ-Secretase modulators: current status and future directions. Prog. Med. Chem. 53, 101–145. Jumpertz, T., Rennhack, A., Ness, J., Baches, S., Pietrzik, C.U., Bulic, B., Weggen, S., 2012. Presenilin is the molecular target of acidic γ-secretase modulators in living cells. PLoS ONE 7, e30484. Jung, J.I., Ladd, T.B., Kukar, T., Price, A.R., Moore, B.D., Koo, E.H., Golde, T.E., Felsenstein, K.M., 2013. Steroids as γ-secretase modulators. FASEB J. 27, 3775–3785. Karran, E., Mercken, M., De Strooper, B., 2011. The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Disc 10, 698–712. Lu, Y., Riddell, D., Hajos-Korcsok, E., Bales, K., Wood, K.M., Nolan, C.E., Robshaw, A.E.,
9