Design, synthesis and X-ray structure of protein-ligand complexes: Important insight into selectivity of memapsin 2 (β-secretase) inhibitors

Article abstract of DOI:10.1021/ja058636j

Structure-based design, synthesis, and X-ray structure of protein-ligand complexes of memapsin 2 are described. The inhibitors are designed specifically to interact with S₂- and S₃-active site residues to provide selectivity over mem

Full text of DOI:10.1021/ja058636j

Published on Web 04/01/2006
Design, Synthesis and X-ray Structure of Protein-Ligand Complexes:
Important Insight into Selectivity of Memapsin 2 (â-Secretase) Inhibitors
Arun K. Ghosh,*,† Nagaswamy Kumaragurubaran,† Lin Hong,‡,§ Hui Lei,‡ Khaja Azhar Hussain,§
Chun-Feng Liu,‡ Thippeswamy Devasamudram,‡ Vajira Weerasena,‡ Robert Turner,‡
Gerald Koelsch,‡,§ Geoffrey Bilcer,‡ and Jordan Tang§,
Departments of Chemistry and Medicinal Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907, Zapaq Inc.,
Oklahoma City, Oklahoma 73104, Protein Studies Program, Oklahoma Medical Research Foundation and
Department of Biochemistry and Molecular Biology, UniVersity of Oklahoma Health Science Center,
Oklahoma City, Oklahoma 73104
Received December 20, 2005; E-mail: akghosh@purdue.edu
The proteolytic enzyme memapsin 2 (â-secretase, BACE-1) has
emerged as a leading target for therapeutic intervention of Alzhei-
mer’s disease (AD).1 It is one of two proteases that cleave the
â-amyloid precursor protein (APP) to generate the 40/42 residue
amyloid-â peptide (Aâ). The excess level of Aâ leads to formation
of amyloid plaques and neurofibrillary tangles in the brain.2,3 The
neurotoxicity of Aâ eventually leads to the death of neurons,
inflammation of the brain, dementia, and AD.4 On the basis of initial
kinetics and substrate specificity data,5 we designed a number of
potent inhibitors incorporating a nonhydrolyzable Leu-Ala hy-
droxyethylene dipeptide isostere.6 One such inhibitor is OM99-2
(1, Figure 1) which has a Ki value of 1.6 nM for human memapsin
2.6a An X-ray crystal structure of 1-bound memapsin 2 was
determined at 1.9 Å resolution.7 The structure provided molecular
Figure 1. Structure of Inhibitors 1 and 2.
insight into the ligand-binding site interactions of the memapsin
2 active site.
Subsequently, our preliminary structure-activity relationship
studies led to the design of potent peptidomimetic inhibitor 2 with
a Ki value of 2.5 nM against memapsin 2.6b However, it displayed
no selectivity over memapsin 1 (BACE-2) or cathepsin D. From a
therapeutic point of view, the selectivity of memapsin 2 inhibitors
over other human aspartic proteases is expected to be important,
especially memapsin 1 and cathepsin D.
Memapsin 1, which has specificity similarity with memapsin 2,8
has independent physiological functions. Cathepsin D, which is
abundant in all cells, plays an important role in cellular protein
catabolism.9 Its inhibition would likely consume inhibitor drugs as
well as lead to probable toxicity.
Figure 2. Structure of Inhibitors 3-6.
The synthesis of inhibitors 3-6 is outlined in Scheme 1.
Coupling of previously described6 Leu-Ala dipeptide isostere 7 with
valine derivatives 8 and 9 using EDC and HOBt in the presence of
i-Pr2NEt provided derivatives 10 and 11 (71-95%). Urethanes 12
and 13 were prepared by treatment of 2,5-dimethylpyrazolylmetha-
nol with triphosgene in CH2Cl2 followed by addition of methionine
and methylcysteine methyl esters to provide the corresponding
urethanes.10 Saponification of the resulting methyl esters with
aqueous lithium hydroxide provided 12 and 13 (36-44%). Removal
of Boc and TBS groups by exposure of 10 and 11 to trifluoroacetic
acid and coupling of the resulting amines with the corresponding
acids using EDC and HOBt afforded inhibitors 3 and 4 (40-64%).
Oxidation of sulfide 4 with mCPBA in a mixture (6:1) of CH2Cl2
and MeOH furnished sulfone 5 (86%). Acid 14 was prepared by
alkoxycarbonylation10 of 2,5-dimethyl-4-oxazolemethanol11 and
methylcysteine methyl ester followed by saponification of the
resulting ester (see Supporting Information for details). It was
converted to inhibitor 6 by analogous procedures described above.
The X-ray structure of 1-bound memapsin 2 revealed a number
of critical ligand-binding site interactions in the S2- and S3-subsites.7
Based upon examination of this X-ray structure and a model of
memapsin 1, it appears that the residues in the S2- and S3-subsites
may be suitable for building selectivity over memapsin 1 and
cathepsin D. Herein we report our structure-based design of novel
memapsin 2 inhibitors that incorporate methylsulfonyl alanine as
the P2-ligand and pyrazole and oxazole-derived heterocycles as the
P3-ligands (see Figure 2). The corresponding inhibitors have
exhibited enhanced potency against memapsin 2 and excellent
selectivity over memapsin 1 and cathepsin D. Furthermore, the
protein-ligand X-ray structure of the pyrazole-bearing inhibitor
provided important molecular insight into the specific cooperative
ligand-binding site interactions for selectivity design.
† Purdue University.
‡ Zapaq Inc.
§ Oklahoma Medical Research Foundation and Department of Biochemistry
and Molecular Biology, University of Oklahoma Health Science Center.
Department of Biochemistry and Molecular Biology, University of Oklahoma
Health Science Center.
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J. AM. CHEM. SOC. 2006, 128, 5310-5311
10.1021/ja058636j CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Synthesis of Inhibitors 3-6
Figure 3. Inhibitor-5-bound X-ray structure of memapsin 2.
On the basis of this molecular insight into the selectivity, we
designed the oxazolylmethyl P3-ligand for inhibitor 6 (MW 659).
This provided by far the most potent (M2 Ki 0.12 nM) and selective
inhibitor (>3800-fold over M1 and >2500-fold over CD). We have
also determined the cellular inhibition of memapsin 2 by inhibitors
5 and 6 in Chinese hamster ovary cells. They have shown an
average cellular IC50 value of 1.4 and 1.7 µM respectively,
compared to an IC50 value of 45 µM for 1.13
Table 1. Ki Values and Selectivities for Inhibitors 2-6a
selectivity ratios
In conclusion, our structure-based design led to the development
of very potent and highly selective memapsin 2 inhibitors.
Furthermore, our X-ray structural analysis of protein-inhibitor
complexes has uncovered potentially important molecular interac-
tions useful in the design of selectivity against other aspartyl
proteases. Additional studies to further elucidate the role of these
and other interactions important for selectivity are in progress.
compd
M2 (nM)
M1 (nM)
CD (nM)
M1/M2
CD/M2
IC50 (µM)
2
3
4
5
6
2.5
14
4.4
0.3
0.12
1.2
811
161
356
458
nd
25
15
131
304
-
58
36
-
1.8
3
436
>2500
6.5
nd
nd
1.4
1.7
1186
>3800
a Data represent the mean value of 3-5 determinations; memapsin 2
(M2), memapsin 1 (M1), cathepsin D (CD); nd, not determined.
Acknowledgment. Financial support by the National Institutes
of Health (AG 18933) is gratefully acknowledged.
Potencies of various inhibitors were determined against recom-
binant memapsin 2, memapsin 1, and human cathepsin D. The
results are shown in Table 1. As shown, inhibitor 2 with P3-Boc-
Val is more potent for memapsin 1 than memapsin 2. Incorporation
of pyrazolylmethyl urethane in place of P3-Boc-Val provided
inhibitor 3 having a >5-fold reduction in potency for memapsin 2
compared to inhibitor 2. Inhibitor 3 also showed significantly
reduced activity against M1 with a Ki value of 811 nM (58-fold
selectivity over M1) but showed little selectivity for M2 over CD.
Inhibitor 4 with a P3′-isobutylamide and P2-methylcysteine has
shown a 3-fold enhancement of M2 potency. It has also shown
>36-fold selectivity over M1 and a modest 3-fold selectivity over
CD. Based upon inhibitor models, the P2-methionine in 3 or P2-
methylcysteine side chain in 4 does not appear to be forming a
hydrogen bond with Arg-235 of memapsin 2. However, the
corresponding P2-sulfone of 4 appears to be in close proximity to
Arg-235 of memapsin 2. Indeed, oxidation of the P2-methyl cysteine
provided inhibitor 5 (MW 658) with very impressive potency (M2
Ki 0.3 nM) and selectivity for memapsin 2. It displayed 1186-fold
selectivity over M1 and 436-fold selectivity over CD.
To gain further molecular insight, the 5-bound memapsin 2 X-ray
structure was determined at 1.8 Å resolution (Figure 3).12 As it
appears from the structure, one of the P3-pyrazole nitrogens is within
hydrogen-bonding distance to Thr-232 with one of the dimethyl
groups effectively filling in the shallow hydrophobic pocket in the
S3-subsite and the other occupying the hydrophobic S3-site. In
addition, the P2-sulfone functionality is within hydrogen-bonding
distance to Arg-235 as well as with a tightly bound water molecule
in the S2-site. These interactions of the P2 and P3 ligands are
presumably responsible for the observed enhanced selectivity for
inhibitor 5 compared to that for inhibitor 4.
Supporting Information Available: Experimental procedures and
spectral data for 3-6, 10-14, and X-ray information and complete ref
3a. This material is available free of charge via the Internet at http://
pubs.acs.org.
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