Probing pockets S2-S4′ of the γ-secretase active site with (hydroxyethyl)urea peptidomimetics

Article abstract of DOI:10.1016/j.bmcl.2004.01.077

(Hydroxyethyl)urea peptidomimetics are potent inhibitors of γ-secretase that are accessible in a few synthetic steps. Systematic alteration of P2-P4′ revealed that the corresponding S2-S4′ active site pockets accommodate a variety of substituents, consistent with the fact that this protease cleaves a variety of single-pass membrane proteins; however, phenylalanine is not well tolerated at P2′. A compound spanning P2-P3′ was identified as a low nM inhibitor of γ-secretase activity both in cells and under cell-free conditions.

Full text of DOI:10.1016/j.bmcl.2004.01.077

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1935–1938
Probing pockets S2–S40 of the ꢀ-secretase active site with
(hydroxyethyl)urea peptidomimetics
William P. Esler, Chittaranjan Das and Michael S. Wolfe*
Center for Neurologic Diseases, Harvard Medical School and Brigham and Womens Hospital, Boston, MA 02115, USA
Received 4 December 2003; accepted 26 January 2004
Abstract—(Hydroxyethyl)urea peptidomimetics are potent inhibitors of g-secretase that are accessible in a few synthetic steps.
Systematic alteration of P2–P40 revealed that the corresponding S2–S40 active site pockets accommodate a variety of substituents,
consistent with the fact that this protease cleaves a variety of single-pass membrane proteins; however, phenylalanine is not well
tolerated at P20. A compound spanning P2–P30 was identified as a low nM inhibitor of g-secretase activity both in cells and under
cell-free conditions.
# 2004 Published by Elsevier Ltd.
The production and self-association of the 38–43 resi-
due amyloid-b peptide (Ab) are essential steps in the
pathogenesis of Alzheimer0s disease (AD) and are thus
considered appropriate points for therapeutic interven-
tion.1 In particular, the two proteases, b- and g-secretases,
responsible for cutting Ab out of its precursor protein
(APP) have emerged as principal targets.2 b-Secretase is
a membrane-anchored aspartyl protease in the pepsin
family and generates the N-terminus of Ab (Fig. 1,
top).3 Knockout of b-secretase eliminates Ab produc-
tion in mice with little or no apparent developmental
defects,4 suggesting the enzyme is a suitable therapeutic
target. Moreover, the co-crystallization of b-secretase
with a transition-state analogue inhibitor5 has made this
protease amenable to structure-based design.
served transmembrane aspartates in PS essential for
activity and the suggestion that PS is the catalytic com-
ponent of the protease.11 Affinity reagents based on
these inhibitors label PS, strongly supporting this
hypothesis.12,13 g-Secretase has recently been discovered
to be a complex of three other membrane proteins in
addition to PS.14 This enzyme is a founding member of
an emerging family of proteases with membrane-
embedded active sites.15
Although g-secretase is also considered a key target, its
biology has proven to be much more complicated. This
protease somehow accomplishes a hydrolysis in the
middle of the APP transmembrane domain to generate
the C-terminus of Ab (Fig. 1, top). AD-causing muta-
tions in presenilin-1 and-2 (PS1 and PS2) affect the site
of g-secretase cleavage in APP to overproduce the more
deleterious 42-residue form of Ab.6 Knockout of the PS
genes eliminates g-secretase activity7 but also causes
embryonic lethality in mice.8 Transition-state analogue
inhibitors first suggested that g-secretase is an aspartyl
protease9,10 and led to the identification of two con-
Figure 1. Top: Proteolysis of APP by b- and g-secretases produce Ab.
Bottom: Structure of hydroxyethylene (1) and (hydroxyethyl)urea (2)
peptidomimetic inhibitors of g-secretase.
* Corresponding author. Tel.: +1-617-525-5511; fax: +1-617-525-
5252; e-mail: mwolfe@rics.bwh.harvard.edu
0960-894X/$ - see front matter # 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.01.077

1936
W. P. Esler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1935–1938
Small molecule inhibitors have been indispensable tools
for identifying g-secretase, elucidating its mechanism of
action, understanding its biological roles, and deter-
mining its potential as a therapeutic target. Inhibitors
used as molecular probes include difluoro ketones and
alcohols,9,16 hydroxyethylenes,10 benzodiazepines,17
and (more recently) helical peptides that mimic the
conformation of the transmembrane substrate.18
The hydroxyethylene peptidomimetics (e.g., 1, Fig. 1,
bottom) have been particularly useful for probing the
enzyme active site.12,19 However, the synthesis of the
dipeptide isostere building blocks is cumbersome.19 To
simplify the synthesis and access a variety of related
analogues, we replaced the chiral carbon atom in P10
with an achiral nitrogen. The resulting series of (hydro-
xyethyl)urea peptidomimetics (e.g., 2, Fig. 1) has pro-
vided potent inhibitors as well as feedback about the
nature of the S2–S40 pockets of g-secretase. In parti-
cular, we systematically altered positions P2, P10, P20,
P30, and P40 with small, medium, and large hydrophobic
residues (alanine, valine, leucine, and phenylalanine) to
explore the steric limits of the corresponding pockets
(Scheme 1).
Table 1. Structures of (hydroxyethyl)ureas varied at P10–P30 and
their abilities to lower Ab production from APP-transfected CHO cells
Compd
R10
R20
P30
IC50 (mM)a
6
7
8
9
10
11
12
13
14
15
16
2
Me
i-Pr
i-Bu
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
i-Bu
i-Bu
i-Bu
i-Bu
Me
i-Pr
i-Bu
Bn
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Ala
Val
Leu
Phe
—
3.1
22
0.40
0.40
0.14
0.40
0.39
18
2.0
0.22
0.65
0.39
8.0
17
a Values are means of three experiments.
coupling of a-amino esters with HATU in the presence
of diisopropylethylamine (DIPEA) in DMF. N-terminal
extension to P2 (e.g, 23, Table 2) was accomplished by
removal of the N-butoxycarbonyl (Boc) group with
TFA followed by coupling with Boc-protected amino
acids, again using HATU and DIPEA in DMF.
We previously reported a series of difluoro ketone pep-
tidomimetics varied in the P1 position, which provided
evidence for a large S1 pocket in the active site of g-
secretase.20 This, in combination with the report of 1 by
Shearman and colleagues,10 led us to begin the synthesis
with the commercially available epoxide 3, which would
provide analogues with the benzyl substituent (i.e.,
phenylalanine side chain) in P1. Following the method
of Getman et al., who developed (hydroxyethyl)urea
peptidomimetics as inhibitors of HIV protease,21 this
epoxide was opened with several different alkylamines
in high yield (80–99%) by refluxing in isopropanol for
16 h. The amino alcohols 4 were then treated with iso-
cyanates (in turn obtained from a-amino methyl esters
and phosgene22) to yield the (hydroxyethyl)urea P1–P20
isosteres 5 in near quantitative yields. C-terminal exten-
sion to incorporate P30 and P40 (e.g., 2 and 18, Tables 1
and 2, respectively) was accomplished by hydrolysis of
the methyl ester with lithium hydroxide and subsequent
These compounds were tested for their ability to block
Ab production from Chinese hamster ovary (CHO) cells
stably transfected with human APP.9,20,23 Reduction of
Ab from cells by these particular compounds reflects
inhibition of g-secretase, not b-secretase, for several
reasons. First, the length and amino acid identity of
these peptidomimetics is already known to strongly dis-
favor inhibition of b-secretase.24 Second, the identifica-
tion of b-secretase inhibitors that work in cell culture
has been quite challenging.25 Third, selected compounds
in this series elevate APP g-secretase substrates in cells
(data not shown) and block g-secretase activity in cell-
free assays (see below). (Hydroxyethyl)ureas varied in
the P10 position showed better potency with larger sub-
stituents (leucine and phenylalanine side chains; that is,
8 and 9), with IC50 values of 400 nM, suggesting a large
complementary S10 pocket. In contrast, phenylalanine
was clearly disfavored in the P20 position (13), by nearly
two orders of magnitude compared with alanine, valine,
and leucine (10–12). Thus, the S20 active site pocket
apparently cannot accommodate the larger benzyl sub-
stituent. Alterations of the P30 position revealed a slight
preference for the valine residue (15, IC50=220 nM),
and removal of P30 altogether resulted in substantial
loss of potency (17, IC50 ꢀ8000 nM). The phenylala-
nine and leucine residues were well tolerated (2 and 16),
suggesting that the S30 pocket is also relatively large and
can accommodate a variety of residues.
Surprisingly, extension to the P40 position did not lead
to any gain of potency compared to no residue at all
(Table 2, 15 versus 18–22). These results suggest either
that S40 is a very large pocket or that the P40 residues
are not interacting with the protease. In contrast,
0
Scheme 1. Reagents and conditions: (a) R1 -NH2, i-PrOH, reflux;
0
0
(b) OCN-CH(R2 )CO2Me; (c) aq LiOH, THF; H2N-CH(R3 )CO2Me,
HATU, DIPEA, DMF.

W. P. Esler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1935–1938
1937
Table 2. Structures of (hydroxyethyl)ureas varied at P2 and P40 and
their abilities to lower Ab production from APP-transfected CHO cells
xyethyl)ureas displayed an approximately 15- to 20-fold
loss of potency. These results are largely consistent with
the findings of Shearman and colleagues, who observed
that the same stereochemical preference for hydroxy-
ethylene peptidomimetics,10 although the loss of
potency on inversion of the alcohol stereocenter was
much more dramatic (several orders of magnitude) for
these compounds.
Compd
P2
P40
IC50 (mM)a
15
18
19
20
21
22
23
24
25
26
—
—
—
—
—
—
Ala
Val
Leu
Phe
—
Ala
Val
Leu
Ile
Phe
—
—
—
0.22
0.67
0.53
0.54
0.27
0.25
0.15
0.07
0.23
0.50
In conclusion, (hydroxyethyl)urea peptidomimetics are
readily accessible and convenient tools for studying g-
secretase. Indeed, we have already utilized such com-
pounds for the affinity isolation and characterization of
the protease complex,27 and for testing the ability of
other inhibitors to affect the g-secretase active site.29
These inhibitors reveal a remarkable lack of clear spe-
cificity in the S2 through S40 pockets. Our findings are
consistent with the discovery that this protease can
cleave a variety of integral membrane proteins (e.g., the
APP and Notch families, Notch ligands, the CD44 and
Erb-B4 receptors, E-cadherins).30 However, the phenyl-
alanine residue appears to be strongly disfavored in the
P20 position, consistent with a previous report that arti-
ficial V715F mutagenesis of APP, two residues from the
cleavage site generating the 42-residue Ab, lowers pro-
duction of this Ab variant.31 Similarly, mass spectral
analysis of the Notch counterpart of Ab identified sev-
eral products resulting from transmembrane proteolysis,
but not the fragment that would have been generated
from phenylalanine in the P20 position.32 These obser-
vations validate the use of (hydroxyethyl)urea peptido-
mimetics as probes for the topography of the g-secretase
active site.
—
a Values are means of three experiments.
extension of the N-terminus into the P2 position resul-
ted in a substantial increase in potency, with installation
of the valine substituent (24) leading to a compound
with a 70 nM IC50 value in this cell-based assay. This
compound was further tested in a cell-free g-secretase
assay, which involves detergent-solubilized membrane
preparations from HeLa cells and a recombinant APP-
based substrate called C100-Flag.26,27 Compound 24
(Boc-VFÉFLV-OMe) could block the generation of the
proteolytic products AICD-Flag (Fig. 2) and Ab (not
shown) with an IC50 of about 1 nM.
We also prepared compounds with inverted alcohol
stereochemistry for selected inhibitors (2, 15, and 16).
The 2R,3S diastereomeric epoxide (27) was not com-
mercially available but could be prepared in several
steps from l-phenylalanine (Scheme 2) according to the
method of Luly et al.28 The diastereomeric (hydro-
Acknowledgements
We thank Ms. Bing Zheng for carrying out Ab ELISAs
and Ms. Thekla Diehl for assistance with cell cultures.
This work was supported by grant NS41355 from the
N.I.H. to M.S.W. and a New Investigator Award from
the Alzheimer0s Association to W.P.E.
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Figure 2. Ability of 24 (Boc-VFÉFLV-OMe) to inhibit g-secretase
activity in a cell-free assay. Conversion of recombinant substrate
C100-Flag to AICD-Flag was visualize by western blot with M2 anti-
Flag antibodies.
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