Design of potent inhibitors for human brain memapsin 2 (β-secretase) [1]

Full text of DOI:10.1021/ja000300g

3522
J. Am. Chem. Soc. 2000, 122, 3522-3523
Communications to the Editor
Design of Potent Inhibitors for Human Brain
Memapsin 2 (â-Secretase)
Arun K. Ghosh,† Dongwoo Shin,† Debbie Downs,‡
Gerald Koelsch,‡ Xinli Lin,‡ Jacques Ermolieff,‡ and
Jordan Tang*,‡,§
Department of Chemistry, UniVersity of Illinois
at Chicago, Chicago, Illinois
Protein Studies Program, Oklahoma Medical Research
Foundation and Department of Biochemistry and
Molecular Biology, UniVersity of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma 73104
ReceiVed January 27, 2000
Figure 1. Structures of OM99-1 (Val-Asn-Leu*Ala-Ala-Glu-Phe) and
OM99-2 (Glu-Val-Asn-Leu*Ala-Ala-Glu-Phe) inhibitors. The asterisk in
the sequence designates the hydroxyethylene transition-state isostere.
The generation of the 40/42-residue amyloid â (Aâ) peptide
in human brain by proteolysis of the membrane anchored
â-amyloid precursor protein (APP) is a key event in the
progression of Alzheimer’s disease.1 Proteases involved in the
production of Aâ peptide are known as γ- and â-secretases.
â-Secretase, which catalyzes the rate-limiting step in Aâ produc-
tion, hydrolyzes an easily accessible site in the luminal side of
APP, and is regarded as the major therapeutic target for the design
of inhibitor drugs. Our laboratory recently cloned a human brain
aspartic protease called memapsin 2, which we demonstrated to
be the long sought â-secretase.2 Several other laboratories also
independently discovered the same enzyme.3 The new knowledge
on kinetics and specificity of memapsin 22 has enabled us to
design and test two potent inhibitors for human memapsin 2.
The â-secretase site of APP (SEVKM/DAEFR) is hydrolyzed
poorly (kcat/Km ) 40 s-1 M-1) by recombinant memapsin 2.
However, the same site from the Swedish mutant APP (SEVNL/
DAEFR) is an excellent substrate (kcat/Km ) 2450 s-1 M-1).2 Thus,
for the initial design of memapsin 2 inhibitors, we utilized the
template of the â-secretase site of Swedish APP with a change
of P1′ Asp to Ala. Not only does the specificity at the P1′ site
indicate that Ala is a highly preferred residue,2 such a change
also reduces polarity and increases lipophilicity of the inhibitor,
factors important for blood-brain barrier penetration.4 The peptide
bond between P1 and P1′ sites in the inhibitors is replaced by a
hydroxyethylene transition-state isostere, which is a highly
effective transition-state analogue for the inhibition of aspartic
proteases.5 The structures of two of the initially designed
inhibitors, OM99-1 (1) and OM99-2 (2), are shown in Figure 1.
The strategy for the synthesis of these inhibitors is to first
synthesize Leu*Ala dipeptide isostere (the asterisk represents
hydroxyethylene isostere) which was then used in solid-state
Scheme 1a
a Reagents and conditions: (a) LiAlH4, Et2O, -40 °C, 30 min (86%);
(b) LDA, HCtC-CO2Et, THF, -78 °C, 30 min, then 4, -78 °C, 1 h
(42%); (c) H2, Pd-BaSO4, EtOAc; (d) AcOH, PhMe, reflux, 6 h (74%);
(e) LiHMDS, MeI, THF, -78 °C, 20 min (76%); (f) aqueous LiOH,
THF-H2O, 23 °C, 10 h; (g) TBDMSCl, imidazole, DMF, 24 h (90%);
(h) CF3CO2H, CH2Cl2, 0 °C, 1.5 h; (i) Fmoc-OSu, aqueous NaHCO3,
dioxane, 23 °C, 8 h (61%).
† University of Illinois at Chicago.
‡ Protein Studies Program.
§ Department of Biochemistry and Molecular Biology.
peptide synthesis6 of the inhibitors. The synthesis of Leu*Ala is
outlined in Scheme 1. Commercial Boc-leucine was converted
to Weinreb amide 3 by treatment with isobutyl chloroformate and
N-methylpiperidine followed by treatment of the resulting mixed
anhydride with N,O-dimethylhydroxylamine.7 Reduction of 3 with
lithium aluminum hydride in diethyl ether provided the aldehyde
4 which was reacted with lithium propiolate derived from the
treatment of ethyl propiolate and lithium diisopropylamide to
(1) (a) Selkoe, D. Trends Cell Biol. 1998, 8, 447-453. (b) Selkoe, D. Nature
1999, 399A, 23-31. (c) Sinha, S.; Lieberburg, I. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 11049-11053.
(2) Lin, X.; Koelsch, G.; Wu, S.; Downs, D.; Dashti, A.; Tang, J. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 1456-1460.
(3) (a) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E.
A.; et al. Science 1999, 286, 735-741. (b) Yan, R.; Bienkowski, M. J.; Shuck,
M. E.; Miao, H.; Tory, M. C.; et al. Nature 1999, 402, 533-537. (c) Sinha,
S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.; et al. Nature
1999, 402, 537-540. (d) Hussain, I.; Powell, D.; Howlett, D. R.; Tew, D. G.;
Meek, T. D.; et al. Mol. Cell. Neurosc. 1999, 14, 419-427.
(4) Kearney, B. P.; Aweeka, F. T. Neurol. Clin. 1999, 17, 883-900.
(5) (a) Marciniszyn, J., Jr.; Hartsuck, J. A.; Tang, J. J. Biol. Chem. 1976,
251, 7088-7094. (b) Vacca, J. P. Methods Enzymol. 1994, 241, 311-334.
(c) Greenlee, W. J. Med. Res. ReV. 1990, 10, 173-236.
(6) The solid-state peptide synthesis of OM99-1 and OM99-2 was carried
out at the Molecular Biology Resource Center on the campus of the University
of Oklahoma Health Science Center.
(7) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 32, 3815-3819.
10.1021/ja000300g CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/23/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 14, 2000 3523
furnish the acetylenic alcohol 5 as an inseparable mixture of
diastereomers (5.8:1) in 42% isolated yield.8 Catalytic hydrogena-
tion of 5 over Pd/BaSO4 followed by acid-catalyzed lactonization
of the resulting γ-hydroxy ester with a catalytic amount of acetic
acid in toluene at reflux provided the γ-lactones 6 and 7 in 74%
yield. The isomers were separated by silica gel chromatography
by using 40% ethyl acetate in hexane as the eluent. For
introduction of the methyl group at C-2, the lactone 7 was
alkylated with methyl iodide stereoselectively. Thus, generation
of the dianion of lactone 7 with lithium hexamethyldisilazide (2.2
equiv) in tetrahydrofuran at -78 °C (30 min) and alkylation with
methyl iodide (1.1 equiv) for 30 min at -78 °C, followed by
quenching with propionic acid (5 equiv), provided the desired
alkylated lactone 8 (76% yield) along with a small amount (<5%)
of the corresponding epimer.9 The stereochemical assignment of
alkylated lactone 8 was made based on extensive 1H NMR NOE
experiments.
Figure 2. Inhibition of memapsin 2 activity toward fluorogenic substrate
10. Ki values are described in the text.
disease, its inhibition is of significance pharmacologically.
Pepstatin A has been shown to be a weak inhibitor of memapsin
2.2 One of the two synthetic decatetrapeptides with statine at the
C-terminus was reported to inhibit â-secretase with an IC50 near
30 nM.3c The pharmaceutical potential of having the transition-
state isostere (statine) at the C-terminus remains to be seen.
Therefore, the inhibitors reported here represent the first designed
potent inhibitors for memapsin 2. To evolve the current compound
into a new generation of memapsin 2 inhibitors with drug
potentials will require a reduction of molecular size and optimiza-
tion of lipophilicity to penetrate the blood-brain barrier. We have
also in preliminary studies considered the selectivity question.
The most serious selectivity demand for memapsin 2 inhibitors
is to be distinguished from human intracellular aspartic proteases
such as cathepsin D. The Ki of cathepsin D against OM99-2 is
48 nM, about 5-fold higher than that for memapsin 2. It is
expected that as the molecular sizes are reduced in the new
generation of inhibitors, the selectivity will be enhenced since
the subsites P1, P2, P1′, and P2′ are the most important specificity
determinants in aspartic proteases. It is also noteworthy that
memapsin 2 inhibitor drugs may not need to reach the potency
of the HIV protease inhibitors (<1 nM), which is required to
completely abolish HIV replication. Since memapsin 2 may have
important physiological functions unrelated to APP hydrolysis,2
potency at which some of these functions are preserved may be
a desirable feature of inhibitor design. Further investigations aimed
at improvement of potency, reduction of molecular size, and
structure-based design of specific ligands are underway in our
laboratories.
Aqueous lithium hydroxide promoted hydrolysis of the lactone
8 followed by protection of the γ-hydroxyl group with tert-
butyldimethylsilyl chloride in the presence of imidazole and
(dimethylamino)pyridine in dimethylformamide afforded the acid
9 in 90% yield. Selective removal of the BOC-group was affected
by treatment with trifluoroacetic acid in dichloromethane at 0 °C
for 1 h. Treatment of the resulting amine salt with commercial
Fmoc-succinimide derivative in dioxane in the presence of
aqueous NaHCO3 provided the Fmoc-protected L*A isostere 10
in 65% yield after chromatography. Protected isostere 10 was
utilized in the solid-state peptide synthesis of inhibitors 1 and 2.
In this synthesis, 10 was inserted in a coupling step as for other
amino acid residues. After the last residue coupling step, the
peptide was cleaved from the solid-state resin using 95%
trifluoroacetic acid, which also removed all the side-chain
protecting groups including the silyl group of 10. Inhibitors
OM99-1 and OM99-2 were separately purified in reverse-phase
HPLC and the mass was verified by mass spectrometry.
The purified OM99-1 and OM99-2 were tested for inhibition
of recombinant human memapsin 2 prepared from E. coli
expression.2 Figure 2 shows the inhibition profile of OM99-1 and
OM99-2 against memapsin 2.10 Both of these inhibition curves
are characteristic of tight-binding inhibitors. The Ki values
calculated11 were 6.84 × 10-8 M ( 2.72 × 10-9 M for OM99-1
and 9.58 × 10-9 M ( 2.86 × 10-10 M for OM99-2.
The above results demonstrated that highly potent inhibitors
of human memapsin 2 have been designed and synthesized from
the current available specificity information. Since memapsin 2
cut APP to produce the Aâ peptide involved in Alzheimer’s
Acknowledgment. Financial support for this work was provided by
the National Institute of Health (GM 53386 to A.K.G.) and from
Oklahoma Medical Research Foundation (OMRF). J.T. is the holder of
the J.G. Puterbaugh Chair in Biomedical Research at the OMRF. The
authors thank Dr. Ken Jackson of the Warren Medical Research
Foundation, University of Oklahoma Health Sciences Center, for as-
sistance in peptide synthesis.
(8) Fray, A. H.; Kaye, R. L.; Kleinman, E. F. J. Org. Chem. 1986, 51,
4828-4833.
(9) For similar alkylation of γ-lacones, see: Ghosh, A. K.; Fidanze, S. J.
Org. Chem. 1998, 63, 6146-6154 and references therein.
(10) Memapsin 2 inhibition was measured using recombinant enzyme
produced from E. coli expression as described in ref 2. A fluorogenic substrate
Arg-Glu(EDANS)-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(Dabcyl)-Arg was
used with 0.47 µM of the enzyme in 0.1 M Na-acetate + 5% dimethyl
sulfoxide, pH 4.5 at 37 °C. The excitation wavelength was 350 nm and the
emission wavelength was 490 nm. Details of this assay will be described
elsewhere.
Supporting Information Available: Experimental procedures for the
synthesis of Leu-Ala dipeptide isostere and solid-phase synthesis of
OM99-1 and OM99-2 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
(11) Bieth, J. Bayer-Symposium V: Proteinase Inhibitors; Springer-
Verlag: Berlin, 1994; pp 463-469.
JA000300G