Identification of a small molecule nonpeptide active site β-secretase inhibitor that displays a nontraditional binding mode for aspartyl proteases

Article abstract of DOI:10.1021/jm049388p

A small molecule nonpeptide inhibitor of β-secretase has been developed, and its binding has been defined through crystallographic determination of the enzyme-inhibitor complex. The molecule is shown to bind to the catalytic aspartate residues in an unprecedented manner in the field of aspartyl protease inhibition. Additionally, the complex reveals a heretofore unknown 83 subpocket that is created by the inhibitor. This structure has served an important role in the design of newer β-secretase inhibitors.

Full text of DOI:10.1021/jm049388p

J. Med. Chem. 2004, 47, 6117-6119
6117
Identification of a Small Molecule
Nonpeptide Active Site â-Secretase
Inhibitor That Displays a Nontraditional
Binding Mode for Aspartyl Proteases
Craig A. Coburn,*,‡ Shawn J. Stachel,‡ Yue-Ming Li,†
Diane M. Rush,‡ Thomas G. Steele,‡
Figure 1. Nonpeptide BACE-1 inhibitor 1.
Elizabeth Chen-Dodson,‡ M. Katharine Holloway,‡
Min Xu,‡ Qian Huang,‡ Ming-Tain Lai,‡
Jillian DiMuzio,‡ Ming-Chih Crouthamel,‡
Xiao-Ping Shi,‡ Vinod Sardana,‡ Zhongguo Chen,‡
Sanjeev Munshi,‡ Lawrence Kuo,‡
mimic the putative enzymatic reaction intermediate and
result in potent inhibition. To date, a number of â-secre-
tase inhibitors based on this design principle have been
published and several have been cocrystallized with a
truncated version of BACE-1.5 However, â-secretase
inhibitors based on this strategy must ultimately over-
come the historical problems associated with peptidelike
structures such as low oral bioavailability, poor blood-
brain barrier permeability, and susceptibility to P-
glycoprotein transport.6
Gergely M. Makara,§ D. Allen Annis,§
Praveen K. Tadikonda,§ Huw M. Nash,§
Joseph P. Vacca,‡ and Tong Wang§
Departments of Medicinal Chemistry,
Biological Chemistry, Structural Biology and Molecular
Systems, Merck Research Laboratories,
West Point, Pennsylvania 19486-0004, and
Weary of the difficulties associated with advancing a
substrate-based lead, we chose to focus on the identifi-
cation of a nonpeptide inhibitor from the outset of the
program. Our efforts to identify such inhibitors hinged
on the ability to rapidly assess potential inhibitors at
high enzyme concentrations. Toward this end, we ap-
plied the Automated Ligand Identification System
(ALIS) technology developed by scientists at NeoGenesis
as a tool for lead generation.7 The high throughput
screening campaign involved incubation of BACE-1 with
mass-encoded compound libraries followed by auto-
mated microscale size exclusion chromatography. Com-
pounds that bound to BACE-1 were dissociated from the
enzyme during subsequent reverse phase HPLC, ana-
lyzed by mass spectroscopy, and identified according to
the appropriate mass information embedded in each
library. Through application of this strategy, a single
1,3,5-trisubstituted aromatic structure emerged from the
multimillion compound library! This structure was
identified as the aminopentyl oxyacetamide (1), a com-
pound that reproducibly inhibited â-secretase in five
different enzymatic assays at IC50 values equal to 25
µM.8 Selective inhibition of BACE-1 compared to related
mammalian aspartyl proteases (BACE-2, renin, cathe-
psin D) was also observed. This small molecule (MW )
506), nonpeptide inhibitor was found to be a reversible
inhibitor of BACE-1 and was competitive with the
known inhibitor, Stat-Val (KTEEISEVN (statine)-
VAEF).9 All of these data taken together gave us
confidence that compound 1 was indeed a novel active
site inhibitor of â-secretase.
Department of Chemistry, NeoGenesis Pharmaceuticals,
840 Memorial Drive, Cambridge, Massachusetts 02139
Received July 29, 2004
Abstract: A small molecule nonpeptide inhibitor of â-secre-
tase has been developed, and its binding has been defined
through crystallographic determination of the enzyme-inhibi-
tor complex. The molecule is shown to bind to the catalytic
aspartate residues in an unprecedented manner in the field
of aspartyl protease inhibition. Additionally, the complex
reveals a heretofore unknown S3 subpocket that is created by
the inhibitor. This structure has served an important role in
the design of newer â-secretase inhibitors.
Over the past five years there has been considerable
progress in understanding the etiology of Alzheimer’s
disease (AD). A key advance was the identification of
the enzyme, â-secretase (Asp-2, BACE-1), that plays a
critical role in the amyloid cascade.1 BACE-1 is a unique
member of the pepsin family of aspartyl proteases that
initiates the production of the amyloidogenic Aâ peptide,
the principle component of the senile plaques found in
postmortem analysis of AD patients. Furthermore,
recent reports have demonstrated a direct correlation
between increased â-secretase activity and Aâ produc-
tion in AD brain tissue.2 Genetic deletion of â-secretase
in mice has also been shown to prevent Aâ production
and subsequent Aâ related events.3 Since these knock-
out mice are otherwise healthy, inhibition of BACE-1
has emerged as an attractive therapeutic target for the
treatment of AD.
Efforts to achieve pharmacological inhibition of as-
partyl proteases have a long history driven by interest
in HIV and renin programs.4 Most of the successful
inhibitors have evolved by truncation of a substrate-
based polypeptide and concomitant replacement of the
cleavable amide bond by a noncleavable transition state
isostere. Such replacements for the scissile amide bond
Efforts aimed at improving the potency of the lead
compound focused on each of the three subunits of the
benzenoid core. Subtle modifications to the length as
well as the atomic composition of the oxyacetamide side
chain all resulted in loss of activity. SAR at the tertiary
amide site revealed that several amide surrogates, such
as the 2-cyanophenyl group (2) or the sulfonate ester
(3), could be employed to increase the potency of the
inhibitor (Table 1). The addition of a fluorine atom at
the 4-position of the R-methylbenzamide served to
increase potency an additional 2-fold when compared
to 3. Also interesting was the requirement for the
* Address correspondence to Craig A. Coburn, Tel.: (215)-652-5511,
E-mail: craig_coburn@merck.com.
† Present address: Sloan-Kettering Cancer Center, Box 459, 1275
York Ave., New York, NY 10021.
‡ Merck & Co., Inc.
§ NeoGenesis.
10.1021/jm049388p CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/30/2004

6118 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Table 1. SAR of BACE-1 Inhibitors
Letters
compound stereochem R1
R2
BACE-1 IC50 (µM)
1
2
3
4
5
R
R
R
S
R
H
H
H
H
F
C(O)N(allyl)2
2-CN-Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
25 ( 2.2
21 ( 1.5
3.4 ( 0.42
>100
1.4 ( 0.28
Figure 3. Hydrogen bonding network for BACE-1/inhibitor
5 complex.
structure revealed that the P3 R-methyl group packs
firmly against Ile110 while orienting the p-fluorophenyl
ring toward S3 thus creating a novel S3 subpocket (S3sp).
This finding is unexpected for BACE-1, as small hydro-
phobic groups are normally preferred at S3 in the
natural substrate (valine) and previously reported
substrate-based inhibitors.5 This phenomena, however,
is not completely unprecedented and is observed in the
binding of aliskiren to renin.15 Other notable features
of this unique structure include: (1) coiling of the
terminal aminopentyl chain back into the S1 pocket,
anchored by hydrogen bonds to the backbone carbonyl
oxygens of Gln73, Lys107, and Phe108; (2) weak hydro-
gen bonding between one of the sulfonate oxygens and
Arg235, a residue unique to BACE-1 and BACE-2; and
(3) a cation-π interaction between Lys321 and the
aromatic face of the benzylsulfonate substituent.
Our high throughput screening effort has resulted in
the identification of a low micromolar nonpeptide in-
hibitor of BACE-1. The unprecedented binding mode of
this novel inhibitor coupled with the structural informa-
tion gleaned therein has guided our drug design effort
toward more potent BACE-1 inhibition.
Figure 2. X-ray crystal (1.8 Å) structure of oxyacetamide 5
in the BACE-1 active site.
R-stereochemistry at the chiral R-methyl group to
maintain good enzymatic activity. Compound 5 dis-
played an IC50 versus BACE-1 of 1.4 µM (n ) 3) in an
electrochemilluminescence (ECL) enzyme inhibition as-
say. Comparative inhibition against related aspartyl
proteases was negligible (Cat D, renin > 500 µM) and
the IC50 against BACE-2 was 137 µM.
Using a human BACE-1 variant expressed in E. coli,
we were able to obtain a single crystal of the BACE-1/
inhibitor 5 complex at a resolution of 1.8 Å. To enable
crystallization in a highly ordered lattice, a construct
that contained two mutations, K75A and E77A, was
employed. The kinetic and binding properties of this
mutant were unchanged from those of the wild type
similarly expressed and purified.10 Details of our X-ray
crystallographic studies on both the apo and bound
forms of human BACE-1 will be given separately.
Note Added after ASAP Posting. An additional co-
author, Tong Wang, was added to the author byline in
the version of the manuscript posted October 30, 2004.
The new version was posted November 5, 2004.
Supporting Information Available: Experimental pro-
cedure for the synthesis of compound 5. PDB file for BACE-
1/inhibitor 5 complex has been deposited with the Protein Data
Bank (PDB identifier 1TQF). This material is available free
of charge via the Internet at http://pubs.acs.org.
Examination of the enzyme/inhibitor complex re-
vealed that compound 5 occupies the S4 to S1 subsites
of BACE-1 and does not have any interaction with the
‘prime-side’ of the enzyme. Most notably, compound 5
does not show any direct contact with the catalytic
aspartic acids, Asp32 and Asp228 (Figure 2). Instead,
inhibitor 5 forms a hydrogen bond from the oxyaceta-
mide NH to a water molecule situated between the
aspartyl dyad (see Figure 3) that is analogous to the
catalytic water observed in the apo crystal structures
of other aspartyl proteases, e.g. renin,11 cathepsin D,12
and endothiapepsin.13 With the exception of a recently
reported Ser-Thr dipeptide ligand for endothiapepsin
during a 12-year crystallization period, this water-
mediated binding mode is unique among aspartyl pro-
tease inhibitors.14 Regarding the stereochemistry of the
R-methyl-benzamide ligand, analysis of the crystal
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