Development of clickable active site-directed photoaffinity probes for γ-secretase

Article abstract of DOI:10.1016/j.bmcl.2012.02.027

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visuali

Full text of DOI:10.1016/j.bmcl.2012.02.027

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2997–3000
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of clickable active site-directed photoaffinity
probes for c-secretase
Christina J. Crump a,b, , Christopher W. am Ende c, , T. Eric Ballard c,d, Nikolay Pozdnyakov e,
c,d,
Martin Pettersson c, De-Ming Chau a,b, Kelly R. Bales e, Yue-Ming Li a,b, , Douglas S. Johnson
⇑
⇑
a Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
b Department of Pharmacology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA
c Neuroscience Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340, USA
d Neuroscience Chemical Biology, Pfizer Worldwide Research and Development, Groton, CT 06340, USA
e Neuroscience Research Unit, Pfizer Worldwide Research and Development, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed clickable active site-directed photoaffinity probes for c-secretase which incorporate a
Received 19 January 2012
Revised 8 February 2012
Accepted 10 February 2012
Available online 20 February 2012
photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated con-
jugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-
azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and val-
idated specific labeling to presenilin–1 N-terminal fragment (PS1-NTF), the active site aspartyl protease
component within the c-secretase complex. Additionally, we were able to identify signal peptide pepti-
dase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased
fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach
Keywords:
c
-Secretase
Photoaffinity labeling
Click chemistry
expands the utility of
c-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be
tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe
for target pull down and/or fluorescent imaging applications.
Clickable photoprobe
Alzheimer’s disease
Ó 2012 Elsevier Ltd. All rights reserved.
Photoaffinity labeling (PAL) is a powerful method to covalently
capture the protein targets of small molecules from a variety of
matrices.1,2 PAL has enabled critical studies elucidating the mech-
anism of action of compounds that either inhibit or modulate c-
secretase, an intramembrane aspartyl protease that contributes
complement of physiologically relevant substrates,8 is not com-
pletely understood.9
We designed and prepared clickable photoaffinity probes 4 and
5 based on the active site-directed GSIs L458 (1), L646 (2) and L505
(3).6 A benzophenone was incorporated into these probes to effect
covalent modification of the target upon irradiation. Rather than
incorporate a reporter group into the photoaffinity probe directly,
we choose to strategically incorporate a terminal alkyne to allow
for click chemistry-mediated conjugation with various azide-re-
porter tags (e.g., rhodamine-azide for fluorescence detection or
biotin-azide for avidin enrichment) after the probe is cross-linked
to the target.10 This approach expands the utility of GSI photo-
affinity probes in that labeled proteins can be tagged with any
number of azide-linked reporter groups using a single clickable
photoaffinity probe for target pull down and fluorescent imaging
applications. We recently utilized this approach to show that
to forming amyloid-b peptides and is a major target for Alzhei-
mer’s disease therapy.3
c-Secretase is a complex of four different
integral membrane proteins (presenilin, nicastrin, Aph-1 and
Pen-2)4 and heterogeneity within the subunit composition has
limited the ability of investigators to isolate purified enzyme for
subsequent structural biology studies.5 Active site-directed photo-
reactive hydroxyethylene
L646 (2) and L505 (3) were instrumental in elucidating that the
catalytic domain of
-secretase is contained within presenilin.6
Furthermore, these photoprobes have been used to visually cap-
ture active
-secretase at the plasma membrane.7 Yet, the underly-
c-secretase inhibitors (GSIs) such as
c
c
ing cell biology of the active
c-secretase complex, including its full
piperidine acetic acid
c-secretase modulators (GSMs) directly bind
to PS1-NTF.11
⇑
Corresponding authors.
E-mail addresses: liy2@mskcc.org (Y.-M. Li), doug.johnson@pfizer.com
(D.S. Johnson).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.027

2998
C. J. Crump et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2997–3000
With the clickable photoaffinity probes 4 and 5 in hand, we
determined their in vitro potencies in CHO-APP cells and in a
cell-free HeLa membrane assay using a recombinant amyloid pre-
cursor protein (APP) substrate (Table 1).15,16 Both 4 and 5 are po-
OH
O
H
N
BocHN
NH2
1
N
H
(L458)
O
O
tent
c-secretase inhibitors with IC50 values of 2.3 and 0.2 nM,
respectively, in HeLa membranes. However, they exhibit a signifi-
cant difference in a cell-based setting. Although probe 5 maintains
good cellular potency (4 nM) and is improved in comparison to the
biotinylated compound 3, the cellular potency of 4 is quite low,
which is likely due to poor cell permeability. This is consistent with
previous studies which indicate that 3 is able to penetrate the cell
O
H
N
2
R =
R =
OH
O
biotin
H
N
H
H
N
(L646)
N
N
R
H
O
O
O
4
plasma membrane and access intracellular
c-secretase to inhibit
APP processing, while 2 cannot.7
O
Next, we developed and optimized procedures for photoaffinity
labeling of 4 and 5 and tested the efficiency of the click reaction. To
H
N
3
R =
R =
biotin
OH
O
(L505)
H
N
H
N
this end, we incubated HeLa cell membranes (800
at 37 °C for 1 h with either 2 or 4 in the presence or absence of
competitor compound 1 (1 M). After incubation, membrane frac-
lg/1.2 mL PBS)
BocHN
N
H
R
5
O
O
l
tions were photoirradiated at ꢀ350 nm for 30 min at 4 °C followed
by centrifugation to remove excess reagents. Cell pellets were
resuspended in PBS (500 lL) and subjected to click chemistry con-
The synthesis of clickable photoprobes 4 and 5 began with
preparation of the right-hand side dipeptide incorporating the
clickable alkyne handle (Scheme 1). Treatment of commercially
available phthalimide 6 with hydrazine in methanol provided
aminohexyne 7 in excellent yield.12 Amide coupling of 7 to either
Leu-Phe (8) or Leu-Bpa (9) dipeptides followed by removal of the
Boc protecting group furnished dipeptides 10 and 11 that incorpo-
rated the clickable handle.
ditions employing TCEP (1.0 mM), CuSO4 (1.0 mM), TBTA (0.1 mM)
and biotin-azide (0.1 mM–Invitrogen) for 1 h at room temperature
with slight agitation. HeLa membranes were centrifuged to remove
excess reagents and the resulting cell pellets were resuspended
into 1Â RIPA buffer. The resulting solubilized labeled membranes
were affinity enriched with streptavidin beads, eluted with loading
buffer and separated using SDS–PAGE followed by Western blot
analysis with anti-PS1-NTF antibody (Fig. 1). Clickable L646 analog
4 displayed efficient PS1-NTF photolabeling and the click reaction
effectively appended biotin post-labeling for affinity enrichment.
Importantly, PS1-NTF labeling was blocked when the parent non-
photoreactive GSI L458 (1) compound was included in the reaction
demonstrating specific labeling. The labeling efficiency was
approximately 2 to 3-fold less for 4 which probably reflects the
efficiency of the click reaction with biotin-azide and/or loss of pro-
tein during the click chemistry/precipitation step. Assuming com-
parable crosslinking efficiency for 2 and 4, it would appear that
the click chemistry conjugation with biotin-azide proceeds in
approximately 30–50% yield.
We next compared the labeling of PS1-NTF, PS1-CTF and signal
peptide peptidase (SPP), a related aspartyl intramembrane prote-
ase17 that is known to have cross-reactivity with GSIs,18 with both
clickable photoprobes 4 and 5. As anticipated, probe 4 (based on
L646) strongly labeled PS1-NTF versus CTF and also displayed min-
imal labeling of SPP (Fig. 2).6 Probe 5 (based on L505) confirmed
the expected preference for PS1-CTF with minimal PS1-NTF label-
ing,6 but also revealed significant labeling of SPP.
Next, we focused on the hydroxyethylene segment of the mol-
ecule. Lactone 12 was synthesized from L-phenylalanine via a se-
quence similar to that previously reported.13 Subsequent Boc
deprotection followed by amidation with benzophenone-4-carbox-
ylic acid provided lactone 13 in 52% yield over 2 steps (Scheme 2).
While initial attempts to access 14 proved challenging due to rel-
actonization of the unprotected alcohol intermediate, careful con-
trol of pH and temperature during manipulations allowed access to
14 in good overall yield. Furthermore, we found that the reaction
worked better on larger scales (>1 g).14 Treatment of 14 with EDCI,
HOBt and the alkyne-containing dipeptide 10 provided the TBS
protected probe in 91% yield. Deprotection under standard TBAF
conditions afforded the desired clickable photoprobe 4.
Photoprobe 5 was constructed in a similar fashion to that
shown for 4; however, in this instance, lactone 12 was hydrolyzed
and silyl protected to supply 15 in good overall yield (Scheme 3).
Again, the unprotected alcohol was susceptible to relactonization
and care was taken to minimize exposure to acidic conditions.
An amide coupling with the Leu-Bpa dipeptide 11 joined the requi-
site clickable alkyne and photoprobe portions of the GSI. Removal
of the TBS group was accomplished in 95% yield to provide 5.
Furthermore, we analyzed the labeled proteins in an unbiased
fashion by click chemistry with TAMRA-azide followed by in-gel
R
O
BocHN
OH
O
N
H
8
: R = H
R
9: R = C(O)Ph
O
O
a
b, c
H
N
H2N
N
H2N
N
H
4
4
O
O
10
6
7
: R = H
11: R = C(O)Ph
Scheme 1. Reagents and conditions. (a) NH2NH2, MeOH, 96%; (b) 8 or 9, HATU, DIPEA, DMF, 77–92%; (c) TFA, DCM, 92–95%.

C. J. Crump et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2997–3000
2999
O
O
O
O
O
H
a, b
BocHN
N
O
12
13
O
OTBS
H
N
f, g
c, d, e
OH
O
O
14
O
OH
O
H
H
N
H
N
N
N
H
O
O
O
4
Scheme 2. Reagents and conditions. (a) TFA, DCM, rt; (b) benzophenone-4-carboxylic acid, EDCI, HOBt, DIPEA, DMF, 52% over 2 steps; (c) LiOH, H2O, DME; (d) TBSCl,
Imidazole, DMF; (e) MeOH, 60% over 3 steps; (f) 10, EDCI, HOBt, DIPEA, DMF, 91%; (g) TBAF, THF, 60%.
kDa
O
Multimer
Mono
OTBS
O
a, b, c
- 100
BocHN
BocHN
OH
SPP
O
12
15
- 40
O
OH
O
- 35
- 25
d, e
H
N
H
PS1-NTF
BocHN
N
N
H
O
O
5
PS1-CTF
Scheme 3. Reagents and conditions. (a) LiOH, H2O, DME; (b) TBSCl, Imidazole,
DMF; (c) MeOH, 75% over 3 steps; (d) 11, EDCI, HOBt, DIPEA, DMF, 88%; (e) TBAF,
THF, 95%.
- 15
Competition: 1 (1 µM)
+
-
+
-
4 (20 nM)
5 (20 nM)
Figure 2. HeLa membranes were photolabeled with 4 or 5 (20 nM) in the presence
or absence of 1 (1 M), followed by click chemistry with biotin-azide, streptavidin
pull down, and Western blot analysis with PS1-NTF, PS1-CTF and SPP antibodies.
l
Table 1
Potencies of 1–5 for inhibiting production of Ab42
Compound
Ab42 IC50 (nM)
Ab42 IC50 (nM)
(HeLa membranes)
(CHO-APP)
fluorescence detection (Fig. 3). After click chemistry, the fluores-
cently-tagged probe-labeled HeLa membranes were acetone pre-
cipitated to remove excess reagents, re-solubilized in loading
buffer, and separated on SDS–PAGE. Gels were then scanned for
fluorescent detection of labeled proteins followed by Coomassie
blue staining for confirmation of equal protein loading in each of
the lanes (Fig. 3). Our in-gel fluorescence labeling results support
the disparate labeling profiles of 4 and 5, confirming an increase
in labeling of PS1-NTF versus CTF for probe 4 and increase in
PS1-CTF versus NTF labeling for 5. For example, probe 4 shows a
predominate band at ꢀ30 kDa which is consistent with the label-
ing of PS1-NTF that was observed by Western blot analysis, while
probe 5 shows a predominate band at ꢀ18 kDa which is consistent
with the preferential labeling of PS1-CTF (Fig. 2). Bands that could
correspond to fluorescent labeling of SPP (monomer and oligomer
forms) were also observed in the gels for 4 and 5. In each case these
specific bands were competed by the addition of GSI L458 (1).
1
2
3
4
5
0.3
1.0
0.3
2.3
0.2
36
817
30
1000
4.0
PS1-NTF
Competition: 1 (1 µM)
+
-
-
+
4 (20 nM)
2 (20 nM)
Figure 1. HeLa membranes were photolabeled with 2 or 4 (20 nM) in the presence
or absence of 1 (1 M), followed by click chemistry with biotin-azide (in the case of
4), streptavidin pull down, and Western blot analysis with PS1-NTF antibody.
l

3000
C. J. Crump et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2997–3000
Fluorescence Coomassie
Fluorescence
Coomassie
75
75
50
50
37
37
PS1-NTF
PS1-NTF
25
25
20
PS1-CTF
20
PS1-CTF
15
15
1 (1 µM)
1 (1 µM)
+
-
+
-
+
-
+
-
4 (20 nM)
4 (20 nM)
5 (20 nM)
5 (20 nM)
Figure 3. HeLa membranes were photolabeled with 4 or 5 (20 nM) in the presence or absence of 1 (1
lM), followed by click chemistry with TAMRA-azide, in-gel fluorescence,
and Coomassie blue gel staining.
Robertson, D. W.; Cordell, B.; Stern, A. M.; Olson, R. E.; Zaczek, R. J. Biol. Chem.
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In summary, we have developed two potent clickable active
site-directed GSI photoaffinity probes (4 and 5) and demonstrated
efficient PS1 labeling within the c-secretase complex. Utilizing the
terminal acetylene, we were able to demonstrate conjugation with
both biotin- and TAMRA-azide for affinity enrichment/WB studies
and in-gel fluorescence detection. In addition, clickable L505 ana-
log 5 was shown to be cell penetrant and may be a useful tool
for imaging c-secretase. Efforts to this end are under investigation
and results will be disclosed in due course.
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Acknowledgments
8. Haapasalo, A.; Kovacs, D. M. J. Alzheimers Dis. 2011, 25, 3.
9. De Strooper, B.; Annaert, W. Annu. Rev. Cell Dev. Biol. 2010, 26, 235.
10. For examples of clickable photoaffinity probes see, (a) Ballell, L.; Alink, K. J.;
Slijper, M.; Versluis, C.; Liskamp, R. M.; Pieters, R. J. ChemBioChem 2005, 6, 291;
(b) Sieber, S. A.; Niessen, S.; Hoover, H. S.; Cravatt, B. F. Nat. Chem. Biol. 2006, 2,
274; (c) Salisbury, C. M.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
1171; (d) MacKinnon, A. L.; Garrison, J. L.; Hegde, R. S.; Taunton, J. J. Am. Chem.
Soc. 2007, 129, 14560; (e) Kalesh, K. A.; Sim, D. S.; Wang, J.; Liu, K.; Lin, Q.; Yao,
S. Q. Chem. Commun. 2010, 46, 1118; (f) Shi, H.; Cheng, X.; Sze, S. K.; Yao, S. Q.
Chem. Commun. 2011, 47, 11306; (g) Eirich, J.; Orth, R.; Sieber, S. A. J. Am. Chem.
Soc. 2011, 133, 12144.
We thank Leslie Pustilnik for measuring Ab42 in CHO-APP cells.
This work is supported by NIH grants 1R01NS076117 and
2R01AG026660 (YML), the American Health Assistance Foundation
(YML), Pfizer (YML), Mr. William H. Goodwin and Mrs. Alice Good-
win and the Commonwealth Foundation for Cancer Research, the
Experimental Therapeutics Center of MSKCC, and the William Ran-
dolph Hearst Fund in Experimental Therapeutics. CJC was sup-
ported by Institutional Training Grant T32 GM073546-01A1.
11. Crump, C. J.; Fish, B. A.; Castro, S. V.; Chau, D.-M.; Gertsik, N.; Ahn, K.; Stiff, C.;
Pozdnyakov, N.; Bales, K. R.; Johnson, D. S.; Li, Y.-M. ACS Chem. Neurosci. 2011,
2, 705.
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