U.S. patent application number 09/845226 was filed with the patent office on 2002-08-22 for inhibitors of memapsin 2 and use thereof.
This patent application is currently assigned to Oklahoma Medical Research Foundation. Invention is credited to Ghosh, Arun K., Hong, Lin, Koelsch, Gerald, Tang, Jordan J.N..
Application Number | 20020115600 09/845226 |
Document ID | / |
Family ID | 27538142 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115600 |
Kind Code |
A1 |
Koelsch, Gerald ; et
al. |
August 22, 2002 |
Inhibitors of memapsin 2 and use thereof
Abstract
Methods for the production of purified, catalytically active,
recombinant memapsin 2 have been developed. The substrate and
subsite specificity of the catalytically active enzyme have been
determined. The substrate and subsite specificity information was
used to design substrate analogs of the natural memapsin 2
substrate that can inhibit the function of memapsin 2. The
substrate analogs are based on peptide sequences, shown to be
related to the natural peptide substrates for memapsin 2. The
substrate analogs contain at least one analog of an amide bond
which is not capable of being cleaved by memapsin 2. Processes for
the synthesis of two substrate analogues including isosteres at the
sites of the critical amino acid residues were developed and the
substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2
is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID
NO:28) with the Leu-Ala peptide bond substituted by a
transition-state isostere hydroxyethylene group (FIG. 1). The
inhibition constant of OM99-2 is 1.6.times.10.sup.-9 M against
recombinant pro-memapsin 2. Crystallography of memapsin 2 bound to
this inhibitor was used to determine the tliree dimensional
structure of the protein, as well as the importance of the various
residues in binding. This information can be used by those skilled
in the art to design new inhibitors, using commercially available
software programs and techniques familiar to those in organic
chemistry and enzymology, to design new inhibitors to memapsin 2,
useful in diagnostics and for the treatment and/or prevention of
Alzheimer's disease.
Inventors: |
Koelsch, Gerald; (Oklahoma
City, OK) ; Tang, Jordan J.N.; (Edmond, OK) ;
Hong, Lin; (Oklahoma City, OK) ; Ghosh, Arun K.;
(River Forest, IL) |
Correspondence
Address: |
Patrea L. Pabst
Arnall Golden & Gregory, LLP
2800 One Atlantic Center
1201 West Peachtree Street
Atlanta
GA
30309-3450
US
|
Assignee: |
Oklahoma Medical Research
Foundation
|
Family ID: |
27538142 |
Appl. No.: |
09/845226 |
Filed: |
April 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09845226 |
Apr 30, 2001 |
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09603713 |
Jun 27, 2000 |
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60141363 |
Jun 28, 1999 |
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60168060 |
Nov 30, 1999 |
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60177836 |
Jan 25, 2000 |
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60178368 |
Jan 27, 2000 |
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60210292 |
Jun 8, 2000 |
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Current U.S.
Class: |
514/17.8 ;
435/184; 514/20.1; 530/326 |
Current CPC
Class: |
C07K 1/1136 20130101;
C07K 5/1021 20130101; A61P 43/00 20180101; Y02A 90/26 20180101;
C12N 9/6478 20130101; Y02A 90/10 20180101; A61P 25/28 20180101;
C07K 2299/00 20130101; C07K 5/06026 20130101; Y10S 514/879
20130101; C07K 5/06043 20130101; C07K 5/0806 20130101; C12N 9/6421
20130101; A61K 38/00 20130101; A61K 39/00 20130101 |
Class at
Publication: |
514/12 ; 435/184;
530/326 |
International
Class: |
A61K 038/17; A61K
038/00 |
Claims
We claim:
1. An inhibitor of catalytically active memapsin 2 which binds to
the active site of the memapsin 2 defined by the presence of two
catalytic aspartic residues and substrate binding cleft.
2. The inhibitor of claim 1 comprising an isostere of the active
site of memapsin 2.
3. The inhibitor of claim 2 comprising a molecule having the
general form X-
L.sub.4-P.sub.4-L.sub.3-P.sub.3-L.sub.2-P.sub.2-L.sub.1-P.sub.1-L.sub.-
0-P.sub.1'-L.sub.1'-P.sub.2'-L.sub.2'-P.sub.3'-L.sub.3'-P.sub.4'L.sub.4'-Y-
, wherein Px represent the substrate specificity position relative
to the cleavage site which is represented by an -LO-, and Lx
represent the linking regions between each substrate specificity
position, Px, and wherein L.sub.0 is a non-hydrolyzable bond and
P1'is -R.sub.1CR.sub.3-, wherein R.sub.1 is a group smaller than
CH.sub.2OH (side chain of serine), and at least two other P
positions are a hydrophobic group.
4. The inhibitor of claim 3 which is OM99-1.
5. The inhibitor of claim 3 which is OM99-2.
6. The inhibitor of claim 3 having the structure of FIG. 11.
7. The inhibitor of claim 3 having the structure of FIG. 12.
8. The inhibitor of claim 3 having the structure of FIG. 13.
9. The inhibitor of claim 3 having the structure of FIG. 14.
10. The inhibitor of claim 1 having an K.sub.i of less than or
equal to 10.sup.-7 M.
11. The inhibitor of claim I which binds to crystallized enzyme
characterized by the parameters in Table 2 when bound to
OM-99-2.
12. The inhibitor of claim 13 having a K.sub.i of less than or
equal to 10.sup.-6 M.
13. The inhibitor of claim 11 having a K.sub.i of less than or
equal to 2 nM.
14. The inhibitor of claim 13 having a K.sub.i of less than or
equal to 2 nM.
15. The inhibitor of claim 11 having a root mean square difference
of less than or equal to 0.5 .ANG. for the side chain and backbone
atoms for amino acids 18-379 of memapsin 2.
16. The inhibitor of claim 1 which is permeable to the blood brain
barrier.
17. The inhibitor of claim 1 which blocks cleavage by memapsin 2
under physiological conditions.
18. The inhibitor of claim 1 which is a non-amino acid small
molecule.
19. The inhibitor of claim 18 having a molecular weight of less
than 800 Daltons.
20. A method of synthesis of a Leu*Ala dipeptide isostere.
21. A method for treating a patient to decrease the likelihood of
developing or the progression of Alzheimer's disease comprising
administering to the individual an effective amount of an inhibitor
of memapsin 2 having an K; of less than or equal to 10.sup.-7 M or
which binds to crystallized enzyme characterized by the parameters
in Table 2 when bound to OM-99-2.
22. The method of claim 21 wherein the inhibitor is administered
orally.
23. The method of claim 21 wherein the inhibitor blocks cleavage of
APP.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims priority to U.S.S.N. 60/141,363
filed Jun. 28, 1999 by Lin, et al., U.S.S.N. 60/168,060 filed Nov.
30, 1999 by Lin, et al., U.S.S.N. 60/177,836 filed Jan. 25, 2000 by
Lin, et al., U.S.S.N. 60/178,368 filed Jan. 27, 2000 by Lin, et a.,
and U.S.S.N. 60/210,292 filed Jun. 8, 2000 by Lin Hong, et al., the
teachings of which are incorporated by reference herein.
[0002] This invention is in the area of the design and synthesis of
specific inhibitors of the aspartic protease Memapsin 2
(beta-secretase) which are useful in the treatment and/or
prevention of Alzheimer's Disease.
[0003] Alzheimer's disease (AD) is a degenerative disorder of the
brain first described by Alios Alzheimer in 1907 after examining
one of his patients who suffered drastic reduction in cognitive
abilities and had generalized dementia (The early story of
Alzheimer's Disease, edited by Bick et al. (Raven Press, New York
1987)). It is the leading cause of dementia in elderly persons. AD
patients have increased problems with memory loss and intellectual
functions which progress to the point where they cannot function as
normal individuals. With the loss of intellectual skills the
patients exhibit personality changes, socially inappropriate
actions and schizophrenia (A Guide to the Understanding of
Alzheimer's Disease and Related Disorders, edited by Jorm (New York
University Press, New York 1987). AD is devastating for both
victims and their families, for there is no effective palliative or
preventive treatment for the inevitable neurodegeneration.
[0004] AD is associated with neuritic plaques measuring up to 200
.mu.m in diameter in the cortex, hippocampus, subiculum,
hippocampal gyrus, and amygdala. One of the principal constituents
of neuritic plaques is amyloid, which is stained by Congo Red
(Fisher (1983); Kelly Microbiol. Sci. 1(9):214-219 (1984)). Amyloid
plaques stained by Congo Red are extracellular, pink or
rust-colored in bright field, and birefringent in polarized light.
The plaques are composed of polypeptide fibrils and are often
present around blood vessels, reducing blood supply to various
neurons in the brain.
[0005] Various factors such as genetic predisposition, infectious
agents, toxins, metals, and head trauma have all been suggested as
possible mechanisms of AD neuropathy. Available evidence strongly
indicates that there are distinct types of genetic predispositions
for AD. First, molecular analysis has provided evidence for
mutations in the amyloid precursor protein (APP) gene in certain
AD-stricken families (Goate et al. Nature 349:704-706 (1991);
Murrell et al. Science 254:97-99 (1991); Chartier-Harlin et al.
Nature 353:844-846 (1991); Mullan et al., Nature Genet. 1:345-347
(1992)). Additional genes for dominant forms of early onset AD
reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature
376:775-778 (1995); Levy-Lahad et al., Science 269:973-977 (1995);
Sherrington et al., Nature 375:754-760 (1995)). Another loci
associated with AD resides on chromosome 19 and encodes a variant
form of apolipoprotein E (Corder, Science 261:921-923 (1993)).
[0006] Amyloid plaques are abundantly present in AD patients and in
Down's Syndrome individuals surviving to the age of 40. The
overexpression of APP in Down's Syndrome is recognized as a
possible cause of the development of AD in Down's patients over
thirty years of age (Rumble et al., New England J. Med.
320:1446-1452 (1989); Mann et al., NeurobioL Aging 10:397-399
(1989)). The plaques are also present in the normal aging brain,
although at a lower number. These plaques are made up primarily of
the amyloid .beta. peptide (A.beta.; sometimes also referred to in
the literature as .beta.-amyloid peptide or , peptide) (Glenner and
Wong, Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is
also the primary protein constituent in cerebrovascular amyloid
deposits. The amyloid is a filamentous material that is arranged in
beta-pleated sheets. A.beta. is a hydrophobic peptide comprising up
to 43 amino acids.
[0007] The determination of its amino acid sequence led to the
cloning of the APP cDNA (Kang et al., Nature 325:733-735 (1987);
Goldgaber et al., Science 235:877-880 (1987); Robakis et al., Proc.
Natl. Acad. Sci. 84:4190-4194 (1987); Tanzi et al., Nature
331:528-530 (1988)) and genomic APP DNA (Lemaire et al., Nucl.
Acids Res. 17:517-522 (1989); Yoshikai et al., Gene 87, 257-263
(1990)). A number of forms of APP cDNA have been identified,
including the three most abundant forms, APP695, APP751, and
APP770. These forms arise from a single precursor RNA by alternate
splicing. The gene spans more than 175 kb with 18 exons (Yoshikai
et al. (1990)). APP contains an extracellular domain, a
transmembrane region and a cytoplasmic domain. A.beta. consists of
up to 28 amino acids just outside the hydrophobic transmembrane
domain and up to 15 residues of this transmembrane domain. A.beta.
is normally found in brain and other tissues such as heart, kidney
and spleen. However, A.beta. deposits are usually found in
abundance only in the brain.
[0008] Van Broeckhaven et al., Science 248:1120-1122 (1990), have
demonstrated that the APP gene is tightly linked to hereditary
cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch
families. This was confirmed by the finding of a point mutation in
the APP coding region in two Dutch patients (Levy et al., Science
248:1124-1128 (1990)). The mutation substituted a glutamine for
glutamic acid at position 22 of the A.beta. (position 618 of
APP695, or position 693 of APP770). In addition, certain families
are genetically predisposed to Alzheimer's disease, a condition
referred to as familial Alzheimer's disease (FAD), through
mutations resulting in an amino acid replacement at position 717 of
the full length protein (Goate et al. (1991); Murrell et al.
(1991); Chartier-Harlin et al. (1991)). These mutations
co-segregate with the disease within the families and are absent in
families with late-onset AD. This mutation at amino acid 717
increases the production of the A.beta..sub.1-42 form of A.beta.
from APP (Suzuki et al., Science 264:1336-1340 (1994)). Another
mutant form contains a change in amino acids at positions 670 and
671 of the full length protein (Mullan et al. (1992)). This
mutation to amino acids 670 and 671 increases the production of
total A.beta. from APP (Citron et al., Nature 360:622-674
(1992)).
[0009] APP is processed in vivo at three sites. The evidence
suggests that cleavage at the .beta.-secretase site by a membrane
associated metalloprotease is a physiological event. This site is
located in APP 12 residues away from the lumenal surface of the
plasma membrane. Cleavage of the .beta.-secretase site (28 residues
from the plasma membrane's lumenal surface) and the
.beta.-secretase site (in the transmembrane region) results in the
40/42-residue .beta.-amyloid peptide (A .beta.), whose elevated
production and accumulation in the brain are the central events in
the pathogenesis of Alzheimer's disease (for review, see Selkoe, D.
J. Nature 399:23-31 (1999)). Presenilin 1, another membrane protein
found in human brain, controls the hydrolysis at the APP
(.beta.-secretase site and has been postulated to be itself the
responsible protease (Wolfe, M. S. et al., Nature 398:513-517
(1999)). Presenilin 1 is expressed as a single chain molecule and
its processing by a protease, presenilinase, is required to prevent
it from rapid degradation (Thinakaran, G. et al., Neuron 17:181-190
(1996) and Podlisny, M. B., et al., Neurobiol. Dis. 3:325-37
(1997)). The identity of presenilinase is unknown. The in vivo
processing of the .beta.-secretase site is thought to be the
rate-limiting step in A .beta. production (Sinha, S. &
Lieberburg, I., Proc. Natl. Acad. Sci, USA, 96:11049-11053 (1999)),
and is therefore a strong therapeutic target.
[0010] The design of inhibitors effective in decreasing amyeloid
plaque formation is dependent on the identification of the critical
enzyme(s) in the cleavage of APP to yield the 42 amino acid
peptide, the A.beta..sub.1-42 form of A.beta.. Although several
enzymes have been identified, it has not been possible to produce
active enzyme. Without active enzyme, one cannot confirm the
substrate specificity, determine the subsite specificity, nor
determine the kinetics or critical active site residues, all of
which are essential for the design of inhibitors.
[0011] Memapsin 2 has been shown to be beta-secretase, a key
protease involved in the production in human brain of beta-amyloid
peptide from beta-amyloid precursor protein (for review, see
Selkoe, D. J. Nature 399:23-31 (1999)). It is now generally
accepted that the accumulation of beta-amyloid peptide in human
brain is a major cause for the Alzheimer's disease. Inhibitors
specifically designed for human memapsin 2 should inhibit or
decrease the formation of beta-amyloid peptide and the progression
of the Alzheimer's disease.
[0012] Memapsin 2 belongs to the aspartic protease family. It is
homologous in amino acid sequence to other eukaryotic aspartic
proteases and contains motifs specific to that family. These
structural similarities predict that memapsin 2 and other
eukaryotic aspartic proteases share common catalytic mechanism
Davies, D. R., Annu. Rev. Biophys. Chem. 19, 189 (1990). The most
successful inhibitors for aspartic proteases are mimics of the
transition state of these enzymes. These inhibitors have
substrate-like structure with the cleaved planar peptide bond
between the carbonyl carbon and the amide nitrogen replaced by two
tetrahedral atoms, such as hydroxyethylene [-CH(OH)-CH.sub.2-],
which was originally discovered in the structure of pepstatin
(Marciniszyn et al., 1976).
[0013] However, for clinical use, it is preferable to have small
molecule inhibitors which will pass through the blood brain barrier
and which can be readily synthesized. It is also desirable that the
inhibitors are relatively inexpensive to manufacture and that they
can be administered orally. Screening of thousands of compounds for
these properties would require an enormous effort. To rationally
design memapsin 2 inhibitors for treating Alzheimer's disease, it
will be important to know the three-dimensional structure of
memapsin 2, especially the binding mode of an inhibitor in the
active site of this protease.
[0014] It is therefore an object of the present invention to
provide purified, recombinant, and active memapsin 2, as well as
its substrate and subsite specificity and critical active site
residues.
[0015] It is a further object of the present invention to provide
compositions and methods for synthesis of inhibitors of memapsin
2.
[0016] It is a still further object of the present invention to
provide compositions that interact with memapsin 2 or its substrate
to inhibit cleavage by the memapsin 2 which can cross the blood
brain barrier (BBB).
[0017] It is therefore an object of the present invention to
provide means for rational design and screening of compounds for
inhibition of mamapsin 2.
SUMMARY OF THE INVENTION
[0018] Methods for the production of purified, catalytically
active, recombinant memapsin 2 have been developed. The substrate
and subsite specificity of the catalytically active enzyme have
been determined. The active enzyme and assays for catalytic
activity are useful in screening libraries for inhibitors of the
enzyme.
[0019] The substrate and subsite specificity information was used
to design substrate analogs of the natural memapsin 2 substrate
that can inhibit the function of memapsin 2. The substrate analogs
are based on peptide sequences, shown to be related to the natural
peptide substrates for memapsin 2. The substrate analogs contain at
least one analog of an amide (peptide) bond which is not capable of
being cleaved by memapsin 2. Processes for the synthesis-of two
substrate analogues including isosteres at the sites of the
critical amino acid residues were developed and the substrate
analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on
an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with
the Leu-Ala peptide bond substituted by a transition-state isostere
hydroxyethylene group. The inhibition constant of OM99-2 is
1.6.times.10.sup.-9 M against recombinant pro-memapsin 2.
Crystallography of memapsin 2 bound to this inhibitor was used to
determine the three dimensional structure of the protein, as well
as the importance of the various residues in binding.
[0020] This information can be used by those skilled in the art to
design new inhibitors, using commercially available software
programs and techniques familiar to those in organic chemistry and
enzymology, to design new inhibitors. For example, the side chains
of the inhibitors may be modified to produce stronger interactions
(through hydrogen bonding, hydrophobic interaction, charge
interaction and/or van der Waal interaction) in order to increase
inhibition potency. Based on this type of information, the residues
with minor interactions may be eliminated from the new inhibitor
design to decrease the molecular weight of the inhibitor. The side
chains with no structural hindrance from the enzyme may be
cross-linked to lock in the effective inhibitor conformation. This
type of structure also enables the design of peptide surrogates
which may effectively fill the binding sites of memapsin 2 yet
produce better pharmaceutical properties.
[0021] The examples demonstrate the production of catalytically
active enzyme, design and synthesis of inhibitors, and how the
crystal structure was obtained. The examples thereby demonstrate
how the methods and materials described herein can be used to
screen libraries of compounds for other inhibitors, as well as for
design of inhibitors. These inhibitors are useful in the prevention
and/or treatment of Alzheimer's disease as mediated by the action
of the beta secretase memapsin 2, in cleaving APP.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts the plasmid construct of vector
pET-lla-memapsin 2-Tl and pET-lla-memapsin 2-T2. The T7 promotor,
amino acid sequence from the vector (T7 protein) (SEQ ID NO:3), and
the beginning and ending of the memapsin 2 T1 and T2 construct are
shown. Construct promemapsin 2-T1 was used in the preparation of
protein for crystallization and includes residues l1-15v which are
derived from vector pET-lla. Residues 1p-48p are putative
pro-peptide. Residues 1-393 correspond to the mature protease
domain and C-terminal extension. The residue numbering of memapsin
2 starts at the aligned N-terminal position of pepsin (FIG. 3).
[0023] FIG. 2A is a graph of the initial rate of hydrolysis of
synthetic peptide swAPP (see Table 1) by M2.sub.pd at different pH.
FIG. 2B is a graph of the relative k.sub.cat/K.sub.m values for
steady-state kinetic of hydrolysis of peptide substrates by
M2.sub.pd.
[0024] FIGS. 3A and 3B are the chemical structures of memapsin 2
inhibitors, OM99-2 and OM99-1.
[0025] FIG. 4A is a graph of the inhibition of recombinant memapsin
2 by OM99-1. FIG. 4B is a graph of the inhibition of recombinant
memapsin 2 by OM99-2.
[0026] FIGS. 5A-E are photographs of crystals of recombinant
memapsin 2-OM99-2 complex.
[0027] FIG. 6 is a stereo view of crystal structure of memapsin 2
protease domain with bound OM99-2. The polypeptide backbone of
memapsin 2 is shown as a ribbon diagram. The N-lobe and C-lobe are
blue and yellow, respectively, except the insertion loops
(designated A to G, see FIG. 2) in the C-lobe are magenta and the
C-terminal extension is green. The inhibitor bound between the
lobes is shown in red.
[0028] FIG. 7 is a stereo view of comparison of the
three-dimensional structures of memapsin 2 and pepsin. The
molecular surface of the former is significantly larger by the
insertion of surface loops and helix and the C-terminal extension.
Chain tracing of human memapsin 2 is dark blue and is grey for
human pepsin. The light blue balls represent identical residues
which are topologically equivalent. The disulfide bonds are shown
in red for memapsin 2 and orange for pepsin. The C-terminal
extension is in green.
[0029] FIG. 8 is a schematic presentation of interaction between
OM99-2 and memapsin 2 protease domain. The S.sub.3' and S.sub.4'
subsites are not defined.
[0030] FIG. 9 is a stereo presentation of interactions between
inhibitor OM99-2 (orange) and memapsin 2 (light blue). Nitrogen and
oxygen atoms are marked blue and red, respectively. Hydrogen bonds
are indicated in yellow dotted lines. Memapsin 2 residues which
comprise the binding subsites are included. Residues P.sub.4,
P.sub.3, P.sub.2, P.sub.1 and P.sub.1' (defined in FIG. 8) of
OM99-2 are in an extended conformation. Inhibitor chain turns at
residue P2'which makes a distinct kink at this position. The
backbone of P.sub.3' and P.sub.4'directs the inhibitor to exit the
active site.
[0031] FIG. 10 are schematics of the cross linking between P.sub.3
Val and P.sub.1 Leu side chains in the design of new inhibitors for
memapsin 2 based on the current crystal structure. R and R' at
positions P.sub.2 and P.sub.1' indicate amino acid side chains.
Other structural elements of inhibitor are omitted for clarity.
[0032] FIG. 11 are schematics of the cross linking between P.sub.4
Glu and P.sub.2 Asn side chains in the design of new inhibitors for
memapsin 2 based on the current crystal structure. R at position
P.sub.3 indicates amino acid side chain. Other structural elements
of inhibitor are omitted for clarity.
[0033] FIG. 12 is a schematic of the design for the side chain at
the P.sub.1' subsite for the new memapsin 2 inhibitors based on the
current crystal structure. Arrows indicate possible interactions
between memapsin 2 and inhibitor. Other structural elements of
inhibitor are omitted for clarity.
[0034] FIG. 13 is a schematic of the design of two six-membered
rings in the inhibitor structure by the addition of atoms A and B.
The ring formation involves the P.sub.1-Leu side chain the the
peptide backbone near P.sub.1, P.sub.2, and P.sub.3. The new bonds
are in dotted lines. A methyl group can be added to the beta-carbon
of P.sub.1-Leu. Other structural elements of inhibitor are omitted
for clarity.
DETAILED DESCRIPTION OF THE INVENTION
[0035] I. Preparation of Catalytically Active Recombinant Memapsin
2 Cloning and Expression of Memapsin 2
[0036] Memapsin 2 was cloned and the nucleotide (SEQ ID NO. 1) and
predicted amino acid (SEQ ID NO. 2) sequences were determined, as
described in Example 1. The cDNA was assembled from the fragments.
The nucleotide and the deduced protein sequence are shown in SEQ ID
NOs. 1 and 2, respectively. The protein is the same as the aspartic
proteinase 2 (ASP2) described in EP 0 855 444 A by SmithKline
Beecham Pharmaceuticals, (published Jul. 29, 1998), and later
reported by Sinha, et al., Nature 402, 537-540 (December 1999) and
Vassar, et al., Science 286, 735-741 (Oct. 22, 1999).
[0037] Pro-memapsin 2 is homologous to other human aspartic
proteases. Based on the alignments, Pro-memapsin 2 contains a pro
region, an aspartic protease region, and a trans-membrane region
near the C-terminus. The C-terminal domain is over 80 residues
long. The active enzyme is memapsin 2 and its proenzyme is
pro-memapsin 2.
[0038] Refolding Catalytically Active Enzyme
[0039] In order to determine the substrate specificity and to
design inhibitors, it is necessary to express catalytically active
recombinant enzyme. No other known proteases contain a
transmembrane domain. The presence of transmembrane domains makes
the recombinant expression of these proteins less predictable and
more difficult. The transmembrane region often needs to be removed
so that secretion of the protein can take place. However, the
removal of the transmembrane region can often alter the structure
and/or function of the protein.
[0040] The starting assumption was that the region of memapsin 2
that is homologous with other aspartic proteases would
independently fold in the absence of the transmembrane domain, and
would retain protease activity in the absence of the C-terminal
transmembrane region. The transmembrane region appears to serve as
a membrane anchor. Since the active site is not in the
transmembrane region and activity does not require membrane
anchoring, memapsin 2 was expressed in E. coli in two different
lengths, both without the transmembrane region, and purified, as
described in Example 3. The procedures for the culture of
transfected bacteria, induction of synthesis of recombinant
proteins and the recovery and washing of inclusion bodies
containing recombinant proteins are essentially as described by Lin
et al., (1994). Refolding was not a simple matter, however. Two
different refolding methods both produced satisfactory results. In
both methods, the protein was dissolved in a strong
denaturing/reducing solution such as 8 M urea/100 mM
beta-mercaptoethanol. The rate at which the protein was refolded,
and in what solution, was critical to activity. In one method, the
protein is dissolved into 8 M urea/100 mM beta-mercaptoethanol then
rapidly diluted into 20 volumes of 20 mM-Tris, pH 9.0, which is
then slowly adjusted to pH 8 with 1 M HCI. The refolding solution
was then kept at 4.degree. C. for 24 to 48 hours before proceeding
with purification. In the second method, an equal volume of 20 mM
Tris, 0.5 mM oxidized/1.25 mM reduced glutathione, pH 9.0 is added
to rapidly stirred pro-memapsin 2 in 8 M urea/10 mM
beta-mercaptoethanol. The process is repeated three more times with
1 hour intervals. The resulting solution is then dialyzed against
sufficient volume of 20 mM Tris base so that the final urea
concentration is 0.4 M. The pH of the solution is then slowly
adjusted to 8.0 with 1 M HCI.
[0041] The refolded protein is then further purified by column
chromatography, based on molecular weight exclusion, and/or elution
using a salt gradient, and analyzed by SDS-PAGE analysis under
reduced and non-reduced conditions.
[0042] II. Substrate Specificity and Enzyme Kinetics of Memapsin
2
[0043] Substrate Specificity
[0044] The tissue distribution of the memapsin 2 was determined, as
described in Example 2. The presence of memapsin 2 (M2) in the
brain indicated that it might hydrolyze the .beta.-amyloid
precursor protein (APP). As described below, detailed enzymatic and
cellular studies demonstrated that M2 fits all the criteria of the
.beta.-secretase.
[0045] The M2 three-dimensional structure modeled as a type I
integral membrane protein. The model suggested that its globular
protease unit can hydrolyze a membrane anchored polypeptide at a
distance range of 20-30 residues from the membrane surface. As a
transmembrane protein of the brain, APP is a potential substrate
and its beta-secretase site, located about 28 residues from the
plasma membrane surface, is within in the range for M2
proteolysis.
[0046] A synthetic peptide derived from this site (SEVKM/DAEFR)
(SEQ ID NO:4) was hydrolyzed by M2.sub.pd (modified M2 containing
amino acids from Ala .sup.8P to Ala.sup.326) at the beta-secretase
site (marked by /). A second peptide (SEVNL/DAEFR) (SEQ ID NO:5)
derived from the APP beta-secretase site and containing the
`Swedish mutation` (Mullan, M. et aL, Nature Genet. 2:340-342
(1992)), known to elevate the level of alpha-beta production in
cells (Citron, M. et al., Nature 260:672-674 (1992)), was
hydrolyzed by M2.sub.pd with much higher catalytic efficiency. Both
substrates were optimally cleaved at pH 4.0. A peptide derived from
the processing site of presenilin 1 (SVNM/AEGD) (SEQ ID NO:6) was
also cleaved by M2.sup.pd with less efficient kinetic parameters. A
peptide derived from the APP gamma-secretase site (KGGVVIATVIVK)
(SEQ ID NO:7) was not cleaved by M2.sub.pd. Pepstatin A inhibited
M2.sub.pd poorly (IC.sub.50 approximately approximately 0.3 mM).
The kinetic parameters indicate that both presenilin 1 (k.sub.cat,
0.67 s.sup.-1; K.sub.m, 15.2 mM; k.sub.cat/K.sub.m, 43.8
s.sup.-1MN.sup.-1) and native APP peptides (k.sub.cat/K.sub.m, 39.9
s .sup.-1M.sup.-) are not as good substrates as the Swedish APP
peptide (k.sub.cat, 2.45 s.sup.-1,K.sub.m, 1 mM; k.sub.cat/K.sub.m,
2450 s .sup.-1M.sup.-1).
[0047] To determine if M2 possesses an APP beta-secretase function
in mammalian cells, memapsin 2 was transiently expressed in HeLa
cells (Lin, X., et al., FASEB J 7:1070-1080 (1993)), metabolically
pulse-labeled with .sup.35S-Met, then immunoprecipitated with
anti-APP antibodies for visualization of APP-generated fragments
after SDS-polyacrylamide electrophoresis and imaging. SDS-PAGE
patterns of immuno-precipitated APP N.beta.-fragment (97 kD band)
from the conditioned media (2 h) of pulse-chase experiments showed
that APP was cleaved by M2. Controls transfected with APP alone and
co-transfected with APP and M2 with Bafilomycin A1 added were
performed. SDS-PAGE patterns of APP BC-fragment (12 kD) were
immunoprecipitated from the conditioned media of the same
experiment as discussed above. Controls transfected with APP alone;
co-transfected with APP and M2; co-transfected with APP and M2 with
Bafilomycin A1; transfections of Swedish APP; and co-transfections
of Swedish APP and M2 were performed. SDS-PAGE gels were also run
of immuno-precipitated M2 (70 kD), M2 transfected cells;
untransfected HeLa cells after long time film exposure; and
endogenous M2 from HEK 293 cells. SDS-PAGE patterns of APP
fragments (100 kD betaN-fragment and 95 kD betaN-fragment)
recovered from conditioned media after immuno-precipitation using
antibodies specific for different APP regions indicated that
memapsin 2 cleaved APP.
[0048] Cells expressing both APP and M2 produced the 97 kD APP beta
N-fragment (from the N-terminus to the beta-secretase site) in the
conditioned media and the 12 kD betaC-fragment (from the
beta-secretase site to the C-terminus) in the cell lystate.
Controls transfected with APP alone produced little detectable
betaN-fragment and no beta C-fragment. Bafilomycin A1, which is
known to raise the intra-vesicle pH of lysosomes/endosomes and has
been shown to inhibit APP cleavage by beta-secretase (Knops, J. et
al., J. Biol Chem. 270:2419-2422 (1995)), abolished the production
of both APP fragments beta N-and beta C-in co-transfected cells.
Cells transfected with Swedish APP alone did not produce the beta
C-fragment band in the cell lysate but the co-transfection of
Swedish APP and M2 did. This Swedish beta C-fragment band is more
intense than that of wild-type APP. A 97-kD beta N-band is also
seen in the conditioned media but is about equal intensity as the
wild-type APP transfection.
[0049] These results indicate that M2 processes the beta-secretase
site of APP in acidic compartments such as the endosomes. To
establish the expression of transfected M2 gene, the pulse-labeled
cells were lysed and immuno-precipitated by anti-M2 antibodies. A
70 kD M2 band was seen in cells transfected with M2 gene, which has
the same mobility as the major band from HEK 293 cells known to
express beta-secretase (Citron, M. et al., Nature 260:672-674
(1992)). A very faint band of M2 is also seen, after a long film
exposure, in untransfected HeLa cells, indicating a very low level
of endogenous M2, which is insufficient to produce betaN-or
betaC-fragments without M2 transfection. Antibody
alpha-beta.sub.1-17, which specifically recognizes residues 1-17 in
alpha-beta peptide, was used to confirm the correct beta-secretase
site cleavage. In cells transfected with APP and M2, both beta
N-and beta N-fragments are visible using an antibody recognizing
the N-terminal region of APP present in both fragments. Antibody
Abeta, .sub.1-7 recognize the beta N-fragment produced by
endogenous beta-secretase in the untransfected cells. This antibody
was, however, unable to recognize the betaN-fragment known to be
present in cells co-transfected with APP and M2. These observations
confirmed that betaN-fragment is the product of beta-secretase site
cut by M2, which abolished the recognition epitope of
alpha-beta.sub.1-7.
[0050] The processing of APP by M2 predicts the intracellular
colocalization of the two proteins. HeLa cells co-expressing APP
and M2 were stained with antibodies directed toward APP and M2 and
visualized simultaneously by CSLM using a 100x objective. Areas of
colocalization appeared in yellow.
[0051] Immunodetection observed by confocal microscopy of both APP
and M2 revealed their colocalization in the superimposed scans. The
distribution of both proteins is consistent with their residence in
lysosomal/endosomal compartments.
[0052] In specificity studies, it was found that M2.sub.pd cleaved
its pro peptide (2 sites) and the protease portion (2 sites) during
a 16 h incubation after activation (Table 1). Besides the three
peptides discussed above, M2.sub.pd also cleaved oxidized bovine
insulin B chain and a synthetic peptide Nch. Native proteins were
not cleaved by M2.sub.pd.
[0053] The data indicate that human M2 fulfills all the criteria of
a beta-secretase which cleaves the beta-amyloid precursor protein
(APP): (a) M2 and APP are both membrane proteins present in human
brain and co-localize in mammalian cells, (b) M2 specifically
cleaves the beta-secretase site of synthetic peptides and of APP in
cells, (c) M2 preferentially cleaves the beta-secretase site from
the Swedish over the wild-type APP, and (d) the acidic pH optimum
for M2 activity and bafilomycin A1 inhibition of APP processing by
M2 in the cells are consistent with the previous observations that
beta-secretase cleavage occurs in acidic vesicles (Knops, J., et
al., J. BioL Chem. 270:2419-2422 (1995)). The spontaneous
appearance of activity of recombinant pro-M2 in an acidic solution
suggests that, intracellularly, this zymogen can by itself generate
activity in an acidic vesicle like an endosome.
[0054] II. Design and Synthesis of Inhibitors
[0055] Design of Substrate Analogs for Memapsin 2.
[0056] The five human aspartic proteases have homologous amino acid
sequences and have similar three-dimensional structures. There are
two aspartic residues in the active site and each residue is found
within the signature aspartic protease sequence motif,
Asp-Thr/Ser-Gly- (SEQ ID NO:8). There are generally two homologous
domains within an aspartic protease and the substrate binding site
is positioned between these two domains, based on the
three-dimensional structures. The substrate binding sites of
aspartic proteases generally recognize eight amino acid residues.
There are generally four residues on each side of the amide bond
which is cleaved by the aspartic protease.
[0057] Typically the side chains of each amino acid are involved in
the specificity of the substrate/aspartic protease interaction. The
side chain of each substrate residue is recognized by regions of
the enzyme which are collectively called sub-sites. The generally
accepted nomenclature for the protease sub-sites and their
corresponding substrate residues are shown below, where the double
slash represents the position of bond cleavage.
1 Protease sub-sites S4 S3 S2 S1 S1' S2' S3' S4' Substrate residues
P4 P3 P2 P1//P1' P2' P3' P4'
[0058] While there is a general motif for aspartic protease
substrate recognition, each protease has a very different substrate
specificity and breadth of specificity. Once the specificity of an
aspartic protease is known, inhibitors can be designed based on
that specificity, which interact with the aspartic protease in a
way that prevents natural substrate from being efficiently cleaved.
Some aspartic proteases have specificities which can accommodate
many different residues in each of the sub-sites for successful
hydrolysis. Pepsin and cathepsin D have this type of specificity
and are said to have "broad" substrate specificity. When only a
very few residues can be recognized at a sub-site, such as in
renin, the aspartic protease is said to have a stringent or narrow
specificity.
[0059] The information on the specificity of an aspartic protease
can be used to design specific inhibitors in which the preferred
residues are placed at specific sub-sites and the cleaved peptide
bond is replaced by an analog of the transition-state. These
analogs are called transition state isosteres. Aspartic proteases
cleave amide bonds by a hydrolytic mechanism. This reaction
mechanism involves the attack by a hydroxide ion on the
.beta.-carbon of the amino acid. Protonation must occur at the
other atom attached to the .beta.-carbon through the bond that is
to be cleaved. If the .beta.-carbon is insufficiently electrophilic
or the atom attached to the bond to be cleaved is insufficiently
nucleophilic the. bond will not be cleaved by a hydrolytic
mechanism. Analogs exist which do not mimic the transition state
but which are non-hydrolyzable, but transition state isosteres
mimic the transition state specifically and are
non-hydrolyzable.
[0060] Transition state theory indicates that it is the transition
state intermediate of the reaction which the enzyme catalyzes for
which the enzyme has its highest affinity. It is the transition
state structure, not the ground state structure, of the substrate
which will have the highest affinity for its given enzyme. The
transition state for the hydrolysis of an amide bond is tetrahedral
while the ground state structure is planar. A typical
transition-state isostere of aspartic protease is
--CH(OH)--CH.sub.2--, as was first discovered in pepstatin by
Marciniszyn et al. (1976). The transition-state analogue principles
have been successfully applied to inhibitor drugs for human
immunodeficiency virus protease, an aspartic protease. Many of
these are currently in clinical use. Information on the structure,
specificity, and types of inhibitors can be found in Tang, Acid
Proteases, Structure, Function and Biology, Adv. in Exptl. Med.
Biol. vol. 95 (Plenum Press, NY 1977); Kostka, Aspartic Proteinases
and their Inhibitors (Walter de Gruyter, Berlin 1985); Dunn,
Structure and Functions of the Aspartic Proteinases, Adv. in Exptl.
Med. Biol. 306 (Plenum Press, NY 1991); Takahashi, Aspartic
Proteases, Structure, Function, Biology, Biomedical Implications,
Adv. in Exptl. Med. Biol. 362 (Plenum Press, NY 1995); and James,
Aspartic Proteinases, Retroviral and Cellular Enzymes, Adv. in
Exptl. Med. Biol. 436 (Plenum Press, NY 1998)).
[0061] Substrate analog compositions are generally of the general
formula
X-L.sub.4-P.sub.4-L.sub.3-P.sub.3-L.sub.2-P.sub.2-L.sub.1-P.sub.1-L.sub.0-
-P.sub.1',-L.sub.1',-P.sub.2'-L.sub.2'-P.sub.3'-L.sub.3'-P.sub.4'L.sub.4'--
Y. The substrate analog compositions are analogs of small peptide
molecules. Their basic structure is derived from peptide sequences
that were determined through structure/function studies. It is
understood that positions represented by P.sub.X represent the
substrate specificity position relative to the cleavage site which
is represented by an -L.sub.o-. The positions of the compositions
represented by L.sub.X represent the linking regions between each
substrate specificity position, P.sub.X.
[0062] In a natural substrate for memapsin 2, a P.sub.x-L.sub.x
pair would represent a single amino acid of the peptide which is to
be cleaved. In the present general formula, each P.sub.X part of
the formula refers to the .alpha.carbon and side chain functional
group of each would be amino acid Thus, the P.sub.X portion of an
P.sub.X-L.sub.X pair for alanine represents HC-CH.sub.3. The
general formula representing the P.sub.X portion of the general
composition is --R.sub.1CR.sub.3--.
[0063] In general R.sub.1 can be either CH.sub.3 (side chain of
alanine), CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
(CH.sub.3)CH(CH.sub.2 CH.sub.3) (side chain of isoleucine),
CH.sub.2(Indole) (side chain of tryptophan), CH.sub.2(Benzene)
(side chain of phenylalanine), CH.sub.2CH.sub.2SCH.sub.- 3 (side
chain of methionine), H (side chain of glycine), CH.sub.2OH (side
chain of serine), CHOHCH.sub.3 (side chain of threonine),
CH.sub.2(Phenol) (side chain of tyrosine), CH.sub.2SH (side chain
of cysteine), CH.sub.2CH.sub.2CONH.sub.2 (side chain of glutamine),
CH.sub.2CONH.sub.2 (side chain of asparagine),
CH.sub.2CH.sub.2CH.sub.2CH- .sub.2NH.sub.2 (side chain of lysine),
CH.sub.2CH.sub.2CH.sub.2NHC(NH)(NH.- sub.2) (side chain of
arginine), CH.sub.2 (Imidazole) (side chain of histidine),
CH.sub.2COOH (side chain of aspartic acid), CH.sub.2CH.sub.2COOH
(side chain of glutamic acid), and functional natural and
non-natural derivatives or synthetic substitutions of these.
[0064] It is most preferred that R.sub.3 is a single H. In general,
however, R.sub.3 can be alkenyl, alkynal, alkenyloxy, and
alkynyloxy groups that allow binding to memapsin 2. Preferably,
alkenyl, alkynyl, alkenyloxy and alkynyloxy groups have from 2 to
40 carbons, and more preferably from 2 to 20 carbons, from 2 to 10
carbons, or from 2 to 3 carbons., and functional natural and
non-natural derivatives or synthetic substitutions of these.
[0065] The L.sub.X portion of the P.sub.X-L.sub.X pair represents
the atoms linking the P.sub.X regions together. In a natural
substrate the L.sub.X represents the .beta.-carbon attached to the
amino portion of what would be the next amino acid in the chain.
Thus, L.sub.X would be represented by -CO-NH-. The general formula
for L.sub.X is represented by R.sub.2. In general R.sub.2 can be
CO-HN (amide), CH(OH)(CH.sub.2) (hydroxyethylene), CH(OH)CH(OH)
(dihydroxyethylene), CH(OH)CH.sub.2NH (hydroxyetlhylamine),
PO(OH)CH.sub.2 (phosphinate), CH.sub.2NH, (reduced amide). It is
understood that more than one L-maybe an isostere as long as the
substrate analog functions to inhibit aspartic protease
function
[0066] Ls which are not isosteres may either be an amide bond or
mimetic of an amide bond that is noni-hydrolyzable.
[0067] X and Y represent molecules which are not typically involved
in the ecognition by the aspartic protease recognition site, but
which do not interfere ith recognition. It is preferred that these
molecules confer resistance to the degradation of the substrate
analog. Preferred examples would be amino acids coupled to the
substrate analog through a non-hydrolyzable bond. Other preferred
compounds would be capping agents. Still other preferred compounds
would be compounds which could be used in the purification of the
substrate analogs such as biotin.
[0068] As used herein, alkyl refers to substituted or unsubstituted
straight, branched or cyclic alkyl groups; and alkoxyl refers to
substituted or unsubstituted straight, branched or cyclic alkoxy.
Preferably, alkyl and alkoxy groups have from 1 to 40 carbons, and
more preferably from 1 to 20 carbons, from 1 to 10 carbons, or from
1 to 3 carbons.
[0069] As used herein, alkenyl refers to substituted or
unsubstituted straight chain or branched alkenyl groups; alkynyl
refers to substituted or unsubstituted strai ht chain or branched
alkynyl groups; alkenyloxy refers to substituted or unsubstituted
straight chain or branched alkenylxy; and alkynyloxy refers to
substituted or unsubstituted straight chain or branched alkynyloxy.
Preferably, alkenyl, alkynyl, alkenyloxy and alkynyloxy groups have
from 2 to 40 carbons, and more preferably from 2 to 20 carbons,
from 2 to 10 carbons, or from 2 to 3 carbons.
[0070] As used herein, alkaryl refers to an alkyl group that has an
aryl substituent; aralkyl refers to an aryl group that has an alkyl
substituent; heterocyclic-alkyl refers to a heterocyclic group with
an alkyl substituent; alkyl-heterocyclic refers to an alkyl group
that has a heterocyclic substituent.
[0071] The substituents for alkyl, alkenyl, alkynyl, alkoxy,
alkenyloxy, and alkynyloxy groups can be halogen, cyano, amino,
thio, carboxy, ester, ether, thioether, carboxamide, hydroxy, or
mercapto. Further, the groups can optionally have one or more
methylene groups replaced with a heteroatom, such as O.NH or S.
[0072] A number of different substrates were tested and analyzed,
and the cleavage rules for Memapsin 2 were determined. The results
of the substrates which were analyzed are presented in Table 1 and
the rules determined from these results are summarized below. (1)
The primary specificity site for a memapsin 2 substrate is subsite
position, P.sub.1' This means that the most important determinant
for substrate specificity in memapsin 2 is the amino acid, S1'.
P.sub.1' must contain a small side chain for memapsin 2 to
recognize the substrate. Preferred embodiments are substrate
analogs where R.sub.1 of the P.sub.1' position is either H (side
chain of glycine), CH.sub.3 (side chain of alanine), CH.sub.2OH
(side chain of serine), or CH.sub.2OOH (side chain of aspartic
acid). Embodiments that have an R1 structurally smaller than
CH.sub.3 (side chain of alanine) or CH.sub.2OH (side chain of
serine) are also preferred. (2) There are no specific sequence
requirements at positions P.sub.4, P.sub.3, P.sub.2, P.sub.1,
P.sub.2', P.sub.3', and P.sub.4' Each site can accommodate any
other amino acid residue in singularity as long as rule number 3 is
met. (3) At least two of the remaining seven positions, P.sub.4,
P.sub.3, P.sub.2, P.sub.1, P.sub.2', P.sub.3', and P.sub.4', must
have an R.sub.1 which is made up of a hydrophobic residue. It is
preferred that there are at least three hydrophobic residues in the
remaining seven positions, P.sub.4, P.sub.3, P.sub.2, P.sub.1,
P.sub.2', P.sub.3', and P.sub.4'. Preferred RI groups for the
positions that contain a hydrophobic group are CH.sub.3 (side chain
of alanine), CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
(CH.sub.3)CH(CH.sub.2 CH.sub.3) (side chain of isoleucine),
CH.sub.2(INDOLE) (side chain of tryptophan), CH.sub.2(Benzene)
(side chain of phenylalanine), CH.sub.2CH.sub.2SCH.sub.- 3 (side
chain of methionine) CH.sub.2(Phenol) (side chain of tyrosine). It
is more preferred that the hydrophobic group be a large hydrophobic
group. Preferred R.sub.1s which contain large hydrophobic groups
are CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
(CH.sub.3)CH(CH.sub.2 CH.sub.3) (side chain of isoleucine),
CH.sub.2(Indole) (side chain of tryptophan), CH.sub.2(Benzene)
(side chain of phenylalanine), CH.sub.2CH.sub.2SCH.sub.- 3 (side
chain of methionine) CH.sub.2(Phenol) (side chain of tyrosine). It
is most preferred that positions with a hydrophobic R.sub.1 are
CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
CH.sub.2(Benzene) (side chain of phenylalanine),
CH.sub.2CH.sub.2SCH.sub.3 (side chain of methionine), or
CH.sub.2(Phenol) (side chain of tyrosine). (4) None of the eight
positions, P.sub.4, P.sub.3, P.sub.2, P.sub.1, P.sub.1,'P.sub.2',
P.sub.3', and P.sub.4' may have a proline side chain at its R1
position. (5) Not all subsites must have an P represented in the
analog. For example, a substrate analog could have
X-P.sub.2-L.sub.1-P1-L.sub.o-P.sub.1'-L.sub.1'-P.sub.2'-L.sub.-
2'-P.sub.3'-L.sub.3'-Y or it could have
X-L.sub.1-P.sub.1-L.sub.o-P.sub.1'-
-L.sub.1'-P.sub.2'-L.sub.2'-P.sub.3'-L.sub.3'-P.sub.4'L.sub.4'-Y.
[0073] Preferred substrate analogs are analogs having the sequences
disclosed in Table 1, with the non-hydrolyzable analog between P1
and P1'.
[0074] Combinatorial Chemistry to Make Inhibitors
[0075] Combinatorial chemistry includes but is not limited to all
methods for isolating molecules that are capable of binding either
a small molecule or another macromolecule. Proteins,
oligonucleotides, and polysaccharides are examples of
macromolecules. For example, oligonucleotide molecules with a given
function, catalytic or ligand-binding, can be isolated from a
complex mixture of random oligonucleotides in what has been
referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992). One
synthesizes a large pool of molecules bearing random and defined
sequences and subjects that complex mixture, for example,
approximately 10.sup.15 individual sequences in 100 .mu.g of a 100
nucleotide RNA, to some selection and enrichment process. Through
repeated cycles of affinity chromatography and PCR amplification of
the molecules bound to the ligand on the column, Ellington and
Szostak (1990) estimated that 1 in 10.sup.10 RNA molecules folded
in such a way as to bind a small molecule dyes. DNA molecules with
such ligand-binding behavior have been isolated as well (Ellington
and Szostak, 1992; Bock et al, 1992).
[0076] Techniques aimed at similar goals exist for small organic
molecules, proteins and peptides and other molecules known to those
of skill in the art. Screening sets of molecules for a desired
activity whether based on libraries of small synthetic molecules,
oligonucleotides, proteins or peptides is broadly referred to as
combinatorial chemistry.
[0077] There are a number of methods for isolating proteins either
have de novo activity or a modifed activity. For example, phage
display libraries have been used for a number of years. A preferred
method for isolating proteins that have a given fimction is
described by Roberts and Szostak (Roberts R. W. and Szostak J. W.
Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997). Another
preferred method for combinatorial methods designed to isolate
peptides is described in Cohen et al. (Cohen B. A., et al., Proc.
Natl. Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes a
modified two-hybrid technology. Yeast two-hybrid systems are
usefuil for the detection and analysis of protein:protein
interactions. The two-hybrid system, initially described in the
yeast Saccharomyces cerevisiae, is a powerful molecular genetic
technique for identifying new regulatory molecules, specific to the
protein of interest (Fields and Song, Nature 340:245-6 (1989)).
Cohen et al., modifed this technology so that novel interactions
between synthetic or engineered peptide sequences could be
identified which bind a molecule of choice. The benefit of this
type of technology is that the selection is done in an
intracellular environment. The method utilizes a library of peptide
molecules that attach to an acidic activation domain. A peptide of
choice, for example an extracellular portion of memapsin 2 is
attached to a DNA binding domain of a transcriptional activation
protein, such as Gal 4. By performing the Two-hybrid technique on
this type of system, molecules that bind the extracellular portion
of memapsin 2 can be identified.
[0078] Screening of Small Molecule Libraries
[0079] In addition to these more specialized techniques,
methodology well known to those of skill in the art, in combination
with various small molecule or combinatorial libraries, can be used
to isolate and characterize those molecules which bind to or
interact with the desired target, either memapsin 2 or its
substrate. The relative binding affinity of these compounds can be
compared and optimum inhibitors identified using competitive or
non-competitive binding studies which are well known to those of
skill in the art. Preferred competitive inhibitors are
non-hydrolyzable analogs of memapsin 2. Another will cause
allosteric rearrangements which prevent memapsin 2 from functioning
or folding correctly.
[0080] Computer assisted Rational Drug Design
[0081] Another way to isolate inhibitors is through rational
design. This is achieved through structural information and
computer modeling. Computer modeling technology allows
visualization of the three-dimensional atomic structure of a
selected molecule and the rational design of new compounds that
will interact with the molecule. The three-dimensional construct
typically depends on data from x-ray crystallographic analyses or
NMR imaging of the selected molecule. The molecular dynamics
require force field data. The computer graphics systems enable
prediction of how a new compound will link to the target molecule
and allow experimental manipulation of the structures of the
compound and target molecule to perfect binding specificity. For
example, using NMR spectroscopy, Inouye and coworkers were able to
obtain the structural information of N-terminal truncated TSHK
(transmembrane sensor histidine kinases) fragments which retain the
structure of the individual sub-domains of the catalytic site of a
TSHK. On the basis of the NMR study, they were able to identify
potential TSHK inhibitors (U.S. Pat. No. 6,077,682 to Inouye).
Another good example is based on the three-dimensional structure of
a calcineurin/FKBP 12/FK506 complex detennined using high
resolution X-ray crystallography to obtain the shape and structure
of both the calcineurin active site binding pocket and the
auxiliary FKBP12/FK506 binding pocket (U.S. Pat. No. 5,978,740 to
Armistead). With this information in hand, researchers can have a
good understanding of the association of natural ligands or
substrates with the binding pockets of their corresponding
receptors or enzymes and are thus able to design and make effective
inhibitors.
[0082] Prediction of molecule-compound interaction when small
changes are made in one or both requires molecular mechanics
software and computationally intensive computers, usually coupled
with user-friendly, menu-driven interfaces between the molecular
design program and the user. Examples of molecular modeling systems
are the CHARMm and QUANTA programs, Polygen Corporation, Waltham,
Mass. CHARMm performs the energy minimization and olecular dynamics
functions. QUANTA performs the construction, graphic odeling and
analysis of molecular structure. QUANTA allows interactive
construction, modification, visualization, and analysis of the
behavior of molecules with each other.
[0083] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen, et al., 1988
Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57
(Jun. 16, 1988); McKinaly and Rossmann, 1989 Annu. Rev.
Pharmacol.sub.--Toxiciol. 29, 111-122; Perry and Davies, OSAR:
Quantitative Structure-Activity Relationships in Drug Designpp.
189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R.
Soc. Lond. 236, 125-140 and 141-162; and, with respect to a model
enzyme for nucleic acid components, Askew, et al., 1989 J. Am.
Chem. Soc. 111, 1082-1090. Other computer programs that screen and
graphically depict chemicals are available from companies such as
BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga,
Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario.
[0084] Although described above with reference to design and
generation of compounds which could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds which alter substrate binding or enzymatic
activity.
[0085] Screening of Libraries
[0086] Design of substrate analogs and rational drug design are
based on knowledge of the active site and target, and utilize
computer software programs that create detailed structures of the
enzyme and its substrate, as well as ways they interact, alone or
in the presence of inhibitor. These techniques are significantly
enhanced with x-ray crystallographic data in hand. Inhibitors can
also be obtained by screening libraries of existing compounds for
those which inhibit the catalytically active enzyme. In contrast to
reports in the literature relating to memapsin 2, the enzyme
described herein has activity analogous to the naturally produced
enzyme, providing a means for identifying compounds which inhibit
the endogenous activity. These potential inhibitors are typically
identified using high throughput assays, in which enzyme, substrate
(preferably a chromogenic substrate) and potential inhibitor
(usually screened across a range of concentrations) are mixed and
the extent of cleavage of substrate determined. Potentially useful
inhibitors are those which decrease the amount of cleavage.
[0087] II. Methods of Diagnosis and Treatment
[0088] Inhibitors can be used in the diagnosis and treatment and/or
prevention of Alzheimer's disease and conditions associated
therewith, such as elevated levels of the forty-two amino acid
peptide cleavage product, and the accumulation of the peptide in
amyeloid plaques.
[0089] Diagnostic Uses
[0090] The substrate analogs can be used as reagents for
specifically binding to memapsin 2 or memapsin 2 analogs and for
aiding in memapsin 2 isolation and purification or
characterization, as described in the examples. The inhibitors and
purified recombinant enzyme can be used in screens for those
individuals more genetically prone to develop Alzheimer's
disease.
[0091] Therapeutic Uses
[0092] Recombinant human memapsin 2 cleaves a substrate with the
sequence LVNMIAEGD (SEQ ID NO:9). This sequence is the in vivo
processing site sequence of human presenilins. Both presenilin 1
and presenilin 2 are integral membrane proteins. They are processed
by protease cleavage, which removes the N terminal sequence from
the unprocessed form. Once processed, presenilin forms a two-chain
heterodimer (Capell et al., J. Biol. Chem. 273, 3205 (1998);
Thinakaran et al., Neurobiol. Dis. 4, 438 (1998); Yu et al.,
Neurosci Lett. 2;254(3):125-8 (1998)), which is stable relative to
the unprocessed presenilins. Unprocessed presenilines are quickly
degraded (Thinakaran et al., J. Biol. Chem. 272, 28415 (1997);
Steiner et al., J. Biol. Chem. 273, 32322 (1998)). It is known that
presenilin controls the in vivo activity of beta-secretase, which
in turn cleaves the amyloid precursor protein (APP) leading to the
formation of alpha-beta42. The accumulation of alpha-beta42 in the
brain cells is known to be a major cause of Alzheimer's disease
(for review, see Selkoe, 1998). The activity of presenilin
therefore enhances the progression of Alzheimer's disease. This is
supported by the observation that in the absence of presenilin
gene, the production of alpha-beta42 peptide is lowered (De
Strooper et al., Nature 391, 387 (1998)). Since unprocessed
presenilin is degraded quickly, the processed, heterodimeric
presenilin must be responsible for the accumulation of alpha-beta42
leading to Alzheimer's disease. The processing of presenilin by
memapsin 2 would enhance the production of alpha-beta42 and
therefore, further the progress of Alzheimer's disease. Therefore a
memapsin 2 inhibitor that crosses the blood brain barrier can be
used to decrease the likelihood of developing or slow the
progression of Alzheimer's disease which is mediated by deposition
of alpha-beta42. Since memapsin 2 cleaves APP at the beta cleavage
site, prevention of APP cleavage at the beta cleavage site will
prevent the build up of alpha-beta42.
[0093] Vaccines
[0094] The catalytically active memapsin 2 or fragments thereof
including the active site defined by the presence of two catalytic
aspartic residues and substrate binding cleft can be used to induce
an immune response to the memapsin 2. The memapsin 2 is
administered in an amount effective to elicit blocking antibodies,
i.e., antibodies which prevent cleavage of the naturally occurring
substrate of memapsin 2 in the brain. An unmodified vaccine may be
useful in the prevention and treatment of Alzheimer's disease. The
response to the vaccine may be influenced by its composition, such
as inclusion of an adjuvant, viral proteins from production of the
recombinant enzyme, and/or mode of administration (amount, site of
administration, frequency of administration, etc). Since it is
clear that the enzyme must be properly folded in order to be
active, antibody should be elicited that is active against the
endogenous memapsin 2. Antibodies that are effective against the
endogenous enzyme are less likely to be produced against the enzyme
that is not properly refolded.
[0095] Pharmaceutically Ace-ptable Carriers
[0096] The inhibitors will typically be administered orally or by
injection. Oral administration is preferred. Alternatively, other
formulations can be used for delivery by pulmonary, mucosal or
transdermal routes. The inhibitor will usually be administered in
combination with a pharmaceutically acceptable carrier.
Pharmaceutical carriers are known to those skilled in the art. The
appropriate carrier will typically be selected based on the mode of
administration. Pharmaceutical compositions may also include one or
more active ingredients such as antimicrobial agents,
antiinflammatory agents, and analgesics.
[0097] Preparations for parenteral administration or administration
by injection include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Preferred
parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
and electrolyte replenishers (such as those based on Ringer's
dextrose).
[0098] Formulations for topical (including application to a mucosal
surface, including the mouth, pulmonary, nasal, vaginal or rectal)
administration may include ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders. Formulations for these
applications are known. For example, a number of pulmonary
formulations have been developed, typically using spray drying to
formulate a powder having particles with an aerodynanmic diameter
of between one and three microns, consisting of drug or drug in
combination with polymer and/or surfactant.
[0099] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0100] Peptides as described herein can also be administered as a
pharmaceutically acceptable acid-or base-addition salt, formed by
reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuiric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0101] Dosages
[0102] Dosing is dependent on severity and responsiveness of the
condition to be treated, but will normally be one or more doses per
day, with course of treatment lasting from several days to several
months or until the attending physician determines no further
benefit will be obtained. Persons of ordinary skill can determine
optimum dosages, dosing methodologies and repetition rates.
[0103] The dosage ranges are those large enough to produce the
desired effect in which the symptoms of the memapsin 2 mediated
disorder are alleviated (typically characterized by a decrease in
size and/or number of amyloid plaque, or by a failure to increase
in size or quantity), or in which cleavage of the alpha-beta 42
peptide is decreased. The dosage can be adjusted by the individual
physician in the event of any counterindications.
[0104] The present invention will be further understood by
reference to the following non-limiting examples.
[0105] Example 1. Cloning of memapsin 2.
[0106] 1. Cloning and nucleotide sequence ofpro-memapsin 2.
[0107] New sequences homologous to human aspartic proteases were
found in the following entries in the EST IMAGE database: AA136368
pregnant uterus ATCC 947471, AA207232 neurepithelium ATCC 214526,
and R55398 human breast ATCC 392689. The corresponding bacterial
strains: #947471 #214526, and # 392689 containing the EST sequences
were obtained from the ATCC (Rockville, Md.). The sequencing of
these clones obtained from ATCC confirmed that they contained
sequences not identical to known human aspartic proteases. The
completed sequences of these clones assembled into about 80% of
prepro-M2 cDNA. Full length cDNAs of these clones were obtained
using the following methods.
[0108] The Human Pancreas Marathon-Ready cDNA (Clontech), which is
double-strand cDNA obtained by reverse-transcription, primer
addition, and second strand synthesize of mRNA from human tissues,
was used as template for PCR amplification. An adapter primer (AP1)
and a nested adapter primer (AP2) were used for 5'- and 3'-RACE
PCR. For PCR the 5'-region of the memapsin 2 cDNA, primers API and
NHASPRI were used. Primers for the 3'end of the cDNA are NHASPF2
and AP 1. The middle of the cDNA was amplified by primers NHASPF1
and NHASPR2. The sequence for the primers is as follows:
2 NHASPF1: GGTAAGCATCCCCCATGGCCCCAACGTC (SEQ ID NO:10), NHASPR1:
GACGTTGGGGCCATGGGGGATGCTTACC (SEQ ID NO: 11), NHASPF2:
ACGTTGTCTTTGATCGGGCCCGAAAACGAATTGG (SEQ ID NO:12), NHASPR2:
CCAATTCGTTTTCGGGCCCGATCAAAGACAACG (SEQ ID NO:13), AP1:
CCATCCTAATACGACTCACTATAGGGC (SEQ ID NO:14), and AP2:
ACTCACTATAGGGCTCGAGCGGC (SEQ ID NO:15)
[0109] Memapsin 2 was also cloned from a human pancreas library
(Quick-Screen Human cDNA Library Panel) contained in lambda-gt10
and lambda-gt11 vectors. The primers from the vectors, GT10FWD,
GT10REV, GT11FWD, and GT11REV, were used as outside primers. The
sequence of the primers used was:
3 GT10FWD: CTTTTGAGCAAGTTCAGCCTGGTTAA (SEQ ID NO:16), GT10REV:
GAGGTGGCTTATGAGTATTTCTTCCAGGGTA (SEQ ID NO:17), GT11FWD:
TGGCGACGACTCCTGGAGCCCG (SEQ ID NO:18), GT11REV:
TGACACCAGACCAACTGGTAATGG (SEQ ID NO:19).
[0110] In addition, memapsin 2 cDNA was amplified directly from the
human pancreatic lambda-gt10 and lambda-gt11 libraries. The
sequence of the primers was:
4 PASPN1: catatgGCGGGAGTGCTGCCTGCCCAC (SEQ ID NO:20) and NHASPC1:
ggatccTCACTTCAGCAGGGAGATGTCATCAGCAAAGT (SEQ ID NO:21).
[0111] The amplified memapsin 2 fragments were cloned into an
intermediate PCR vector (Invitrogen) and sequenced.
[0112] The assembled cDNA from the fragments, the nucleotide and
the deduced protein sequence are shown in SEQ ID NO 1 and SEQ ID NO
2.
[0113] Pro-memapsin 2 is homologous to other human
aspartic.proteases. Based on the alignments, Pro-memapsin 2
contains apro region, an aspartic protease region, and a
trans-membrane region near the C-terminus. The active enzyme is
memapsin 2 and its pro-enzyme is pro-memapsin 2.
[0114] Example 2. Distribution of memapsin 2 in human tissues.
[0115] Multiple tissue cDNA panels from Clontech were used as
templates for PCR amplification of a 0.82 kb fragment of memapsin 2
cDNA. The primers used for memapsin 2 were NHASPF1 and NHASPR2.
Tissues that contain memapsin 2 or fragments of memapsin 2 yielded
amplified PCR products. The amount of amplified product indicated
that memapsin 2 is present in the following organs from most
abundant to least abundant: pancreas, brain, lung, kidney, liver,
placenta, and heart. Memapsin 2 is also present in spleen,
prostate, testis, ovary, small intestine, and colon cells.
[0116] Example 3. Expression of pro-memapsin 2 CDNA in E. coli,
refolding and purification of pro-memapsin 2.
[0117] The pro-memapsin 2 was PCR amplified and cloned into the
BamHI site of a pET11a vector. The resulting vector expresses
pro-memapsin 2 having a sequence from Ala-8p to Ala 326. FIG. 1
shows the construction of two expression vectors, pET11-memapsin
2-T1 (hereafter T1) and pET11-memapsin 2-T2 (hereafter T2). In both
vectors, the N-terminal 15 residues of the expressed recombinant
proteins are derived from the expression vector. 29 Pro-memapsin 2
residues start at residue Ala-16. The two recombinant pro-memapsin
2s have different C-terminal lengths. Clone T1 ends at Thr-454 and
clone T2 ends at Ala-419. The T1 construct contains a C-terminal
extension from the T2 construct but does not express any of the
predicted transmembrane domain.
[0118] Expression of recombinant proteins and recovery of inclusion
bodies
[0119] The T1 and T2 expression vectors were separately transfected
into E coli strain BL21 (DE3). The procedures for the culture of
transfected bacteria, induction for synthesis of recombinant
proteins and the recovery and washing of inclusion bodies
containing recombinant proteins are essentially as previously
described (Lin et al., 1994).
[0120] Three different refolding methods have produced satisfactory
results.
[0121] (i) The rapid dilution method.
[0122] Pro-memapsin 2 in 8 M urea/100 mM beta-mercaptoethanol with
OD.sub.280nm=5 was rapidly diluted into 20 volumes of 20 mM-Tris,
pH 9.0. The solution was slowly adjusted into pH 8 with 1 M HCI.
The refolding solution was then kept at 4.degree. C. for 24 to 48
hours before proceeding with purification.
[0123] (ii) The reverse dialysis method
[0124] An equal volume of 20 mM Tris, 0.5 mM oxidized/1.25 mM
reduced glutathione, pH 9.0 is added to rapidly stirred
pro-memapsin 2 in 8 M urea/10 mM beta-mercaptoethanol with
OD.sub.280 nm=5. The process is repeated three more times with 1
hour intervals. The resulting solution is then dialyzed against
sufficient volume of 20 mM Tris base so that the final urea
concentration is 0.4 M. The pH of the solution is then slowly
adjusted to 8.0 with 1 M HCI.
[0125] iii The preferred method for refolding.
[0126] Inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM
Glycine, 1 mM EDTA, 100 mM beta-mercaptoethanol, pH 10.0. The
OD.sub.280 of the inclusion bodies are adjusted to 5.0 with the 8 M
urea solution without beta-mercaptoethanol. The final solution
contains the following reducing reagents: 10 mM
beta-mercaptoethanol, 10 mM DTT (Dithiothreitol), 1 mM reduced
glutathion, and 0.1 M oxidized glutathion. The final pH of the
solution is 10.0.
[0127] The above solution is rapidly diluted into 20 volumes of 20
mM Tris base, the pH is adjusted to 9.0, and the resulting solution
is kept at 4.degree. C. for 16 hr. The solution is equilibrated to
room temperature in 6 hr, and the pH is adjusted to 8.5. The
solution is returned to 4.degree. C. again for 18 hr.
[0128] The solution is again equilibrated to room temperature in 6
hr, and the pH is adjusted to 8.0. The solution is returned to
4.degree. C. again for 4 to 7 days.
[0129] The refolding procedures are critical to obtain an
enzymically active preparation which can be used for studies of
subsite specificity of M2, to analyze inhibition potency of M2
inhibitors, to screen for inhibitors using either random structural
libraries or existing collections of compound libraries, to produce
crystals for crystallography studies of M2 structures, and to
produce monoclonal or polyclonal antibodies of M2.
[0130] Purification of recombinant pro-memapsin 2-T2
[0131] The refolded material is concentrated by ultrafiltration,
and separated on a SEPHACRYL.TM. S-300 column equilibrated with 20
mM Tris.HCI, 0.4 M urea, pH 8.0. The refolded peak (second peak)
from the S-300 column can be further purified with a FPLC
RESOURCE-Q.TM. column, which is equilibrated with 20 mM Tris-HCI,
0.4 M urea, pH 8.0. The enzyme is eluted from the column with a
linear gradient of NaCl. The refolded peak from S-300 can also be
activated before further purification. For activation, the
fractions are mixed with equal volume 0.2 M Sodium Acetate, 70%
glycerol, pH 4.0. The mixture is incubated at 22.degree. C. for 18
hr, and then dialyzed twice against 20 volumes of 20 mM Bis-Tris,
0.4 M urea, pH 6.0. The dialyzed materials are then further
purified on a FPLC RESOURCE-Q.TM. column equilibrated with 20
Bis-Tris, 0.4 M urea, pH 6.0. The enzyme is eluted with a linear
gradient of NaCl.
[0132] SDS-PAGE analysis of the S-300 fractions under reduced and
non-reduced conditions indicated that Pro-memapsin 2 first elutes
as a very high molecular weight band (greater than about 42 kD)
under non-reduced conditions. This indicates that the protein is
not folded properly in these fractions, due to disulfide cross
linking of proteins. Subsequent fractions contain a protein of
predicted pro-memapsin 2-T2 size (about 42 kDa). The pro-enzyme
obtained in these fractions is also proteolytically active for
auto-catalyzed activation. These fractions were pooled and
subjected to chromatography on the FPLC RESOURCE.TM. column eluted
with a linear gradient of NaCl. Some fractions were analyzed using
SDS-PAGE under non-reducing conditions. The analysis showed that
fractions 6 and 7 contained most of the active proteins, which was
consistent with the first FPLC peak containing the active protein.
The main peak was coupled to a shoulder peak, and was present with
repeated purification with the same RESOURCE.TM. Q column. The main
shoulder peaks were identified as active pro-memapsin 2 that exist
in different conformations under these conditions.
[0133] Example 4. Proteolytic activity and cleavage-site
preferences of recombinant memapsin 2.
[0134] The amino acid sequence around the proteolytic cleavage
sites was determined in order to establish the specificity of
memapsin 2. Recombinant pro-memapsin 2-T1 was incubated in 0.1 M
sodium acetate, pH 4.0, for 16 hours at room temperature in order
to create autocatalyzed cleavages. The products were analyzed using
SDS-polyacrylamide gel electrophoresis. Several bands which
corresponded to molecular weights smaller than that of pro-memapsin
2 were observed. The electrophoretic bands were trans-blotted onto
a PVDF membrane. Four bands were chosen and subjected to N-terminal
sequence determination in a Protein Sequencer. The N-terminal
sequence of these bands established the positions of proteolytic
cleavage sites on pro-memapsin 2.
[0135] In addition, the oxidized 13-chain of bovine insulin and two
different synthetic peptides were used as substrates for memapsin 2
to determine the extent of other hydrolysis sites. These reactions
were carried out by auto-activated pro-memapsin 2 in 0.1 M sodium
acetate, pH 4.0, which was then incubated with the peptides. The
hydrolytic products were subjected to HPLC on a reversed phase C-18
column and the eluent peaks were subjected to electrospray mass
spectrometry for the determination of the molecular weight of the
fragments. Two hydrolytic sites were identified on oxidized insulin
B-chain (Table 1). Three hydrolytic sites were identified from
peptide NCH-gamma. A cleavage site was observed in synthetic
peptide PS1-gamma, whose sequence (LVNMAEGD) (SEQ ID NO:9) is
derived from the beta-processing human presenilin 1 (Table 1).
5TABLE 1 Substrate Specificity of Memapsin 2 Site # Substrate P4 P3
P2 P1 P1' P2' P3' P4' 1 Pro- R G S M A G V L aa 12-18 of SEQ ID No.
3 2 memapsin 2 G T Q H G I R L aa 23-30 of SEQ ID No. 3 3 S S N F A
V G A aa 98-105 of SEQ ID No. 3 4 G L A Y A E I A aa 183-190 of SEQ
ID No. 3 5 Oxidized H L C{circumflex over ( )} G S H L V
C{circumflex over ( )} is cysteic acid; 6 insulin B- C{circumflex
over ( )} G E R G F F Y SEQ ID No. 22 chain' SEQ ID No. 23 7
Synthetic V G S G V Three sites cleaved in a peptide: 8 peptide* V
G S G V L L VGSGVLLSRK (SEQ ID 9 G V L L S R K NO:30) SEQ ID No. 24
SEQ ID No. 25 SEQ ID No. 26 10 Peptide** L V N M A E G D SEQ ID No.
9
[0136] Example 5. Activation of pro-menaspsin 2 and enzyme
kinetics.
[0137] Incubation in 0.1 M sodium acetate, pH 4.0, for 16 h at
22.degree. C. auto-catalytically convertered pro-M2.sub.pd to
M2.sub.pd. For initial hydrolysis test, two synthetic peptides were
separately incubated with pro-M2.sub.pd in 0.1 M Na acctate, pH 4.0
for different periods ranging from 2 to 18 h. The incubated samples
were subjected to LCIMS for the identification of the hydrolytic
products. For kinetic studies, the identified HPLC (Beckman System
Gold) product peaks were integrated for quantitation. The K.sub.m
and k.sub.cat values for presenilin 1 and Swedish APP peptides
(Table 1) were measured by steady-state kinetics. The individual
K.sub.m and k.sub.cat values for APP peptide could not be measured
accurately by standard methods, so its k.sub.cat/K.sub.m value was
measured by competitive hydrolysis of mixed substrates against
presenilin 1 peptide (Fersht, A. "Enzyme Structure and Mechanism",
2.sup.nd Ed., W. H. Freeman and Company, New York. (1985)).
[0138] The results are shown in FIGS. 2A and 2B. The conversion of
pro-M2.sub.pd at pH 4.0 to smaller fragments was shown by
SDS-polyacrylamide electrophoresis. The difference in migration
between pro-M2.sub.pd and converted enzyme is evident in a mixture
of the two. FIG. 2A is a graph of the initial rate of hydrolysis of
synthetic peptide swAPP (see Table 1) by M2.sub.pd at different pH.
FIG. 2B is a graph of the relative k.sub.cat/K.sub.m values for
steady-state kinetic of hydrolysis of peptide substrates by
M2.sub.pd.
[0139] Example 6. Expression in Mammalian cells.
[0140] Methods
[0141] PM2 cDNA was cloned into the EcoRV site of vector pSecTag A
(Invitrogen). Human APP cDNA was PCR amplified from human placenta
8-gt11 library (Clontech) and cloned into the Nhel and XbaI sites
of pSecTag A. The procedure for transfection into HeLa cells and
vaccinia virus infection for T7-based expression are essentially
the same as described by Lin, X., FASEB J. 7:1070-1080 (1993).
[0142] Transfected cells were metabolically labeled with 200
microCi .sup.35S methionine and cysteine (TransLabel; ICN) in 0.5
ml of serum-free/methionine-free media for 30 min, rinsed with 1 ml
media, and replaced with 2 ml DMEM/10% FCS. In order to block
vesicle acidification, Bafilomycin A1 was included in the media
(Perez, R. G., et al., J. Biol. Chem 271:9100-9107 (1996)). At
different time points (chase), media was removed and the cells were
harvested and Iysed in 50 mM Tris, 0.3 M NaCl, 5 mM EDTA, 1% Triton
X-100, pH 7.4, containing 10 mM iodoacetamide, 10 :M TPCK, 10 :M
TLCK, and 2 microg/ml leupeptin. The supernatant (14,000 x g) of
cell lysates and media were immunoadsorbed onto antibody bound to
protein G sepharose (Sigma). Anti-APP N-terminal domain antibody
(Chemicon) was used to recover the betaN-fragment of APP and
anti-alpha-beta.sub.1-17 antibody (Chemicon, recognizing the
N-termninal 17 residues of alpha-beta) was used to recover the 12
kDa .beta. C-fragment. The former antibody recognized only
denatured protein, so media was first incubated in 2 mM
dithiothrietol 0.1% SDS at 55.degree. C. for 30 min before
immunoabsorption. Samples were cooled and diluted with an equal
volume of cell lysis buffer before addition of anti-APP N-terminal
domain (Chemicon). Beads were washed, eluted with loading buffer,
subjected to SDS-PAGE (NOVEX.TM.) and visualized by autoradiogram
or phosphorimaging (Molecular Dynamics) on gels enhanced with
Amplify (Amersham). Immunodetection of the betaN-fragment was
accomplished by transblotting onto a PVDF membrane and detecting
with anti-alpha-betal-.sub.1-17 and chemiluminescent substrate
(Amersham).
[0143] Results
[0144] HeLa cells transfected with APP or M2 in 4-well chamber
slides were fixed with acetone for 10 min and permeabilized in 0.2%
Triton X-100 in PBS for 6 min. For localizing M2, polyclonal goat
anti-pro-M2.sub.pd antibodies were purified on DEAE-sepharose 6B
and affinity purified against recombinant pro-M2.sub.pd immobilized
on Affigel (BioRad). Purified anti-pro-M2.sub.pd antibodies were
conjugated to Alexa568 (Molecular Probes) according to the
manufacturer's protocol. Fixed cells were incubated overnight with
a 1:100 dilution of antibody in PBS containing 0.1% BSA and washed
4 times with PBS. For APP, two antibodies were used. Antibody A
.delta. .sub.1-17 (described above) and antibody A.delta.
.sub.17-42, which recognizes the first 26 residues following the
beta-secretase cleavage site (Chemicon). After 4 PBS washes, the
cells were incubated overnight with an anti-mouse FITC conjugate at
a dilution of 1:200. Cells were mounted in Prolong anti-fade
reagent (Molecular Probes) and visualized on a Leica TCS confocal
laser scanning microscope.
[0145] Example 7: Design and Synthesis of OM99-1 and OM99-2.
[0146] Based on the results of specificity studies of memapsin 2,
it was predicted that good residues for positions P1 and P1'would
be Leu and Ala. It was subsequently determined from the specificity
data that P1'preferred small residues, such as Ala and Ser.
However, the crystal structure (determined below in Example 9)
indicates that this site can accommodate a lot of larger residues.
It was demonstrated that P1'of memapsin 2 is the position with the
most stringent specificity requirement where residues of small side
chains, such as Ala, Ser, and Asp, are preferred. Ala was selected
for P1'mainly because its hydophobicity over Ser and Asp is favored
for the penetration of the blood-brain barrier, a requirement for
the design of a memapsin 2 inhibitor drug for treating Alzheimer's
disease. Therefore, inhibitors were designed to place a
transition-state analogue isostere between Leu and Ala (shown as
Leu*Ala, where * represents the transition-state isostere
-CH(OH)-CH.sub.2-) and the subsite P4, P3, P2, P2', P3'and P4'are
filled with the beta-secretase site sequence of the Swedish mutant
from the beta-amyloid protein. The structures of inhibitors OM99-1
and OM99-2 are shown below and in FIGS. 3A and 3B,
respectively:
6 OM99-1: Val-Asn-Leu*Ala-Ala-Glu-Phe (SEQ.ID NO.27) OM99-2:
Glu-Val-Asn-Leu*Ala-Ala-Glu-Phe (SEQ.ID NO.28)
[0147] The Leu* Ala dipeptide isostere was synthesized as
follows:
[0148] The Leu-Ala dipeptide isostere for the M.sub.2-inhibitor was
prepared from L-leucine. As shown in Scheme 1, L-leucine was
protected as its BOC-derivative 2 by treatment with BOC.sub.2O in
the presence of 10% NaOH in diethyl ether for 12 h. Boc-leucine 2
was then converted to Weinreb amide 3 by treatment with isobutyl
chcloroformate and N-methylpiperidine followed by treatment of the
resulting mixed anhydride with N,O-dimethylhydroxylamine 1
[0149] (Nahm and Weinreb, Tetrahedron Letters 1981, 32, 3815).
Reduction of 3 with lithium aluminum hydride in diethyl ether
provided the aldehyde 4. Reaction of the aldehyde 4 with lithium
propiolate derived from the treatment of ethyl propiolate and
lithium diisopropylamide afforded the acetylenic alcohol 5 as an
inseparable mixture of diastereomers (5.8:1) in 42% isolated yield
(Fray, Kaye and Kleinman, J. Org. Chem. 1986, 51, 4828-33).
Catalytic hydrogenation of 5 over Pd/BaSO.sub.4 followed by
acid-catalyzed lactonization of the resulting gamma-hydroxy ester
with a catalytic amount of acetic acid in toluene at reflux,
furnished the gamma-lactone 6 and 7 in 73% yield. The isomers were
separated by silica gel chromatography by using 40% ethyl acetate
in hexane as the eluent. 2
[0150] Introduction of the methyl group at C-2 was accomplished by
stereoselective alkylation of 7 with methyl iodide (Scheme 2).
Thus, generation of the dianion 10 of lactone 7 with lithium
hexamethyldisilazide (2.2 equivalents) in tetrahydrofuran at
-78.degree. C. (30 min) and alkylation with methyl iodide (1.1
equivalents) for 30 min at -78.degree. C., followed by quenching
with propionic acid (5 equivalents), provided the desired alkylated
lactone 8 (76% yield) along with a small amount (less than 5%) of
the corresponding epimer (Ghosh and Fidanze, 1998 J. Org. Chem.
1998, 63, 6146-54). The epimeric cis-lactone was removed by column
chromatography over silica gel using a mixture (3:1) of ethyl
acetate and hexane as the solvent system. The stereochemical
assignment of alkylated lactone 8 was made based on extensive
.sup.1H-NMR NOE experiments Aqueous lithium hydroxide promoted
hydrolysis of the lactone 8 followed by protection of the
gamma-hydroxyl group with tert-butyldimetlhylsilyl chloride in the
presence of imidazole and dimethylaminopyridine in
dimethylformamide afforded the acid 9 in 90% yield after standard
work-up and chromatography. Selective removal of the BOC-group was
effected by treatment with trifluoroacetic acid in dichloromethane
at 0.degree. C. for 1 h. The resulting amine salt was then reacted
with commercial (Aldrich, Milwaukee) Fmoc-succinimide derivative in
dioxane in the presence of aqueous NaHCO.sub.3 to provide the
Fmoc-protected L*A isostere 10 in 65% yield after chromatography.
Protected isostere 10 was utilized in the preparation of a random
sequence inhibitor library.
[0151] Experimental procedure
[0152] N-(tert-Butoxycarbonyl)-L-Leucine (2).
[0153] To the suspension of 10 g (76.2 mmol ) of L-leucine in 140
mL of diethyl ether was added 80 mL of 10% NaOH. After all solid
dissolves, 20 mL (87.1 mmol) of BOC.sub.2O was added to the
reaction mixture. The resulting reaction mixture was stirred at
23.degree. C. for 12 h. After this period, the layers were
separated and the aqueous layer was acidified to pH 1 by careful
addition of 1 N aqueous HCl at 0.degree. C. The resulting mixture
was extracted with ethyl acetate (3.times.100 mL). The organic
layers were combined and washed with brine and dried over anhydrous
Na.sub.2SO.sub.4. The solvent was removed under reduced pressure to
provide title product which was used directly for next reaction
without further purification (yield, 97%). .sup.1H NMR (400 MHz,
CDCl.sub.3).delta.4.89 (broad d, 1H, J =8.3 Hz), 4.31 (m, 1H),
1.74-1-49 (m, 3H), 1.44 (s, 9H), 0.95 (d, 6H, J =6.5 Hz).
[0154] N-(tert-Butoxycarbonyl)-L-leucine-N'-methoxy-N'-methyla-mide
(3).
[0155] To a stirred solution of N,O-dimethylhydroxyamine
hydrochloride (5.52 g, 56.6 mmol) in dry dichloromethane (25 mL)
under N.sub.2 atmosphere at 0.degree. C., -methylpiperidine (6.9
mL, 56.6 mmol) was added dropwise. The resulting mixture was
stirred at 0.degree. C. for 30 min. In a separate flask,
N-(tert-butyloxycarbonyl)-L-leucine (I) (11.9 g, 51.4 mmol) was
dissolved in a mixture of THF (45 mL) and dichloromethane (180 mL)
under N.sub.2 atmosphere. The resulting solution was cooled to
-20.degree. C. To this solution was added 1-methylpiperidine (6.9
mL, 56.6 mmol) followed by isobutyl chloroformate (7.3 mL, 56.6
mmol). The resulting mixture was stirred for 5 minutes at
-20.degree. C. and the above solution of N,O-dimethylhydroxyamine
was added to it. The reaction mixure was kept -20.degree. C. for 30
minutes and then warmed to 23.degree. C. The reaction was quenched
with water and the layers were seperated. The aqueous layer was
extracted with dichloromethane (3.times.100 mL). The combined
organic layers were washed with 10% citric acid, saturated sodium
bicarbonate, and brine. The organic layer was dried over anhydrous
Na.sub.2SO.sub.4 and concentrated under the reduced pressure. The
residue was purified by flash silica gel chromatography (25% ethyl
acetate/hexane) to yield the title compound 3 (13.8 g, 97%) as a
pale yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.5.06
(broad d, 1H, J. =9.1 Hz), 4.70 (m, 1H), 3.82 (s, 3H), 3.13 (s,
3H), 1.70 (m, 1H), 1.46-1.36 (m, 2H) 1.41 (s, 9H), 0.93 (dd, 6H,
J=6.5, 14.2 Hz).
[0156] N-(tert-Butoxycarbonyl)-L-leucinal (4).
[0157] To a stirred suspension of lithium aluminum hydride (770 mg,
20.3 mmol) in dry diethyl ether (60 mL) at -40.degree. C. under
N.sub.2 atmosphere, was added
N-tert-butyloxycarbonyl-L-leucine-N'-methoxy-N'-met- hylamide (5.05
g, 18.4 mmol) in diethyl ether (20 mL). The resulting reaction
mixture was stirred for 30 min. After this period, the reaction was
quenched with 10% NaHSO.sub.4 solution (30 mL). The resulting
reaction mixture was then warmed to 23.degree. C. and stirred at
that temperature for 30 min. The resulting solution was filtered
and the filter cake was washed by two portions of diethyl ether.
The combined organic layers were washed with saturated sodium
bicarbonate, brine and dried over anhydrous MgSO.sub.4. Evaporation
of the solvent under reduced pressure afforded the title aldehyde 4
(3.41 g) as a pale yellow oil. The resulting aldehyde was used
immediately without further purification. .sup.1H NMR (400 MHz,
CDCl.sub.3).delta.9.5 (s, 1H), 4.9 (s, 1H), 4.2 (broad mn, 1H),
1.8-1.6 (m, 2H), 1.44 (s, 9H), 1.49-1.39 (m, 1H), 0.96 (dd, 6H,
J=2.7, 6.5 Hz).
[0158] Ethyl (4S,5S)-and
(4R,5S)-5-[(tert-Butoxycarbonyl)amino]-4-hydroxy--
7-melhyloct-2-ynoale (5).
[0159] To a stirred solution of diisopropylamine (1.1 mL, 7.9 mmol)
in dry THF (60 mL) at 0.degree. C. under N.sub.2 atmosphere, was
added n-BuLi (1.6 M in hexane, 4.95 mL, 7.9 mmol) dropwise. The
resulting solution was stirred at 0.degree. C. for 5 min and then
warmed to 23.degree. C. and stirred for 15 min. The mixture was
cooled to -78.degree. C. and ethyl propiolate (801 .mu.L) in THF (2
mL) was added dropwise over a period of 5 min. The mixture was
stirred for 30 min, after which N-Boc-L-leucinal 4 (1.55 g, 7.2
mmol) in 8 mL of dry THF was added. The resulting mixture was
stirred at -78.degree. C. for 1 h. After this period, the reaction
was quenched with acetic acid (5 mL) in THF (20 mL). The reaction
mixure was warmed up to 23.degree. C. and brine solution was added.
The layers were separated and the organic layer was washed with
saturated sodium bicarbonate and dried over Na.sub.2SO.sub.4.
Evaporation of the solvent under reduced pressure provided a
residue which was purified by flash silica gel chromatography (15%
ethyl acetate/hexane) to afford a mixture (3:1) of acetylenic
alcohols 5 (0.96 g, 42%). .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta.4.64 (d, 1H, J=9.0 Hz), 4.44 (broad s, 1H), 4.18 (m, 2H ),
3.76 (m, 1H), 1.63 (m, 1H), 1.43-1.31 (m, 2H), 1.39 (s, 9H),
1.29-1.18 (m, 3H), 0.89 (m, 6H).
[0160] (5S, 1S') -5-[1
'-[(tert-Butoxycarbonyl)amino]-3'-methylbutyl]
-dihydrofuran-2(3H)-one (7).
[0161] To a stirred solution of the above mixture of acetylenic
alcohols (1.73 g, 5.5 mmol ) in ethyl acetate (20 mL) was added 5%
Pd/BaSO.sub.4 (1 g). The resulting mixture was hydrogenated at 50
psi for 1.5 h. After this period, the -catalyst was filtered off
through a plug of Celite and the filtrate was concentrated under
reduced pressure. The residue was dissolved in toluene (20 mL) and
acetic acid (100 .mu.L). The reaction mixure was refluxed for 6 h.
After this period, the reaction was cooled to 23.degree. C. and the
solvent was evaporated to give a residue which was purified by
flash silica gel chromatography (40% diethyl ether I hexane) to
yield the (5S, 1S')-gamma-lactone 7 (0.94 g, 62.8 and the (SR,
1S')-gamma-lactone 6 (0.16 g, 10.7%). Lactone 7: .sup.1H NMR (400
MHz, CDCl.sub.3) .delta.4.50-4.44 (m, 2H), 3.84-3.82 (m, 1H), 2.50
(t, 2H, J=7.8 Hz), 2.22-2.10 (m, 2H), 1.64-1.31 (m, 3H), 1.41 (s,
9H), 0.91 (dd, 6H, J =2.2, 6.7 Hz); .sup.13CNMR (75 MHz,
CDCl.sub.3) .delta.177.2, 156.0, 82.5, 79.8, 51.0,42.2, 28,6, 28.2,
24.7, 24.2, 23.0, 21.9.
[0162] (3R, 5S, [1'S)-5-]
-[(tert-Butoxycarbonyl)amino)]-3'-methylbut-vl]3- -methyl
dihydrofuran-2(3H)-one (8).
[0163] To a stirred solution of the lactone 7 (451.8 mg, 1.67 mmol)
in dry THF (8 mL) at -78.degree. C. under N.sub.2 atmosphere, was
added lithium hexamethyldisilazane (3.67 mL, 1.0 M in THF) over a
period of 3 min. The resulting mixture was stirred at -78.degree.
C. for 30 min to generate the lithium enolate. After this period,
MeI (228 .mu.L) was added dropwise and the resulting mixture was
stirred at -78.degree. C. for 20 min. The reaction was quenched
with saturated aqueous NH.sub.4Cl solution and was allowed to warm
to 23.degree. C. The reaction mixture was concentrated under
reduced pressure and the residue was extracted with ethyl acetate
(3.times.100 mL). The combined organic layers were washed with
brine and dried over anhydrous Na.sub.2SO.sub.4. Evaporation of the
solvent afforded a residue which was purified by silica gel
chromatography (15% ethyl acetate/hexane) to furnish the alkylated
lactone 8 (0.36 g, 76%) as an amorphous solid. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta.4.43 (broad t, 1H, J =6.3 Hz), 4.33 (d, 1H,
J=9.6 Hz), 3.78 (m, 1H), 2.62 (m, 1H), 2.35 (m, 1H), 1.86 (m, 1H),
1.63-1.24 (m, 3H), 1.37 (s, 9H), 1.21 (d, 3H, J=7.5 Hz), 0.87 (dd,
6H, J=2.6, 6.7 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.180.4,
156.0, 80.3, 79.8, 51.6, 41.9, 34.3, 32.5, 28.3, 24.7,23.0,21.8,
16.6.
[0164] (2R, 4S,
5S)-5-[(tert-Butoxycarbonyl)amino]-4-[(tert-butyldimeth
-ylsilyl)oxy ]-2, 7-dimethyloctanoic acid (9).
[0165] To a stirred solution of lactone 8 (0.33 g, 1.17 mmol ) in
THF (2 mL) was added 1 N aqueous LiOH solution (5.8 mL). The
resulting mixture was stirred at 23.degree. C. for 10 h. After this
period, the reaction mixture was concentrated under reduced
pressure and the remaining aqueous residue was cooled to 0.degree.
C. and acidified with 25% citric acid solution to pH 4. The
resulting acidic solution was extracted with ethyl acetate
(3.times.50 mL). The combined organic layers were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated to yield the
corresponding hydroxy acid (330 mg) as a white foam. This hydroxy
acid was used directly for the next reaction without fuirther
purification.
[0166] To the above hydroxy acid (330 mg, 1.1 mmol) in anhydrous
DMF was added imidazole (1.59 g, 23.34 mmol) and
tert-butyldimethylchlorosilane (1.76 g, 11.67 mmol). The resulting
mixture was stirred at 23.degree. C. for 24 h. After this period,
MeOH (4 mL) was added and the mixture was stirred for 1 h. The
mixure was diluted with 25% citric acid (20 mL) and was extracted
with ethyl acetate (3.times.20 mL). The combined extracts were
washed with water, brine and dried over anhydrous Na.sub.2SO.sub.4.
Evaporation of the solvent gave a viscous oil which was purified by
flash chromatography over silica gel (35% ethyl acetate/hexane) to
afford the silyl protected acid 9 (0.44 g, 90%). IR (neat)
3300-3000 (broad), 2955, 2932, 2859, 1711 cm.sup.-1; .sup.1H NMR
(400 MHz, DMSO-d.sup.6, 343 K) delta 6.20 (broad s, 1 H), 3.68 (m,
1H), 3.51 (broad s, 1H), 2.49-2.42 (m, 1H), 1.83 (t, 1H, J10.1 Hz),
1.56 (m, 1H), 1.37 (s, 9H), 1.28-1.12 (m, 3H), 1.08 (d, 3H, J=7.1
Hz), 0.87 (d, 3H, J=6.1 Hz) 0.86 (s, 9 H), 0.82 (d, 3H, J=6.5 Hz),
0.084 (s, 3H), 0.052 (s, 3H).
[0167] (2R, 4S,
5S)-5-[(luorenylmethyloxycarbonyl)amino]-4-[(tert-butyldi-- methyl
silyl)oxy]-2, 7-dimethyloctanoic acid (10).
[0168] To a stirred solution of the acid 9 (0.17 g, 0.41 mmol) in
dichloromethane (2 mL) at 0.degree. C. was added trifluoroacetic
acid (500 .mu.L). The resulting mixture was stirred at 0.degree. C.
for 1 h and an additional portion (500 .mu.L) of trifluoroacetic
acid was added to the reaction mixture. The mixture was stirred for
an additional 30 min and the progress of the reaction was monitored
by TLC. After this period, the solvents were carefully removed
under reduced pressure at a bath temperature not exceeding
5.degree. C. The residue was dissolved in dioxane (3 mL) and
NaHCO.sub.3 (300 mg) in 5 mL of H.sub.2O. To this solution was
added Fmoc-succinimide (166.5 mg, 0.49 mmol) in 5 mL of dioxane.
The resulting mixture was stirred at 23.degree. C. for 8 h. The
mixure was then diluted with H.sub.2O (5 mL) and acidified with 25%
aqueous citric acid to pH 4. The acidic solution was extracted with
ethyl acetate (3.times.50 mL). The combined extracts were washed
with brine, dried over Na.sub.2SO.sub.4 and concentrated under
reduced pressure to give a viscous oil residue. Purification of the
residue by flash chromatography over silica gel afforded the
Fmoc-protected acid 10 (137 mg, 61%) as a white foam. .sup.1H NMR
(400 MHz, DMSO-d.sup.6, 343 K) .delta.7.84 (d, 2H, J =7.4 Hz), 7.66
(d, 2H, J=8 Hz), 7.39 (t, 2H, J=7.4 Hz), 7.29 (m, 2H), 6.8 (s, 1H),
4.29-4.19 (m, 3H), 3.74-3.59 (m, 2H), 2.49 (m, 1H), 1.88 (m, 1H),
1.58 (m, 1H), 1.31-1.17 (m, 3H), 1.10 (d, 3H, J=7.1 Hz), 0.88 (s,
9H), 0.82 (d, 6H,J=6.2 Hz), 0.089 (s, 3H), 0.057 (s, 3H).
[0169] The synthesis of OM99-1 and 0M99-2 were accomplished using
solid state peptide synthesis procedure in which Leu*Ala was
incorporated in the fourth step. The synthesized inhibitors were
purified by reverse phase HPLC and their structure confirmed by
mass spectrometry.
[0170] Example 8. Inhibition of Memapsin 2 by OM99-1 and
OM99-2.
[0171] Enzyme activity was measured as described above, but with
the addition of either OM99-1 or OM99-2. OM99-1 inhibited
recombinant memapsin 2 as shown in FIG. 5A. The Ki calculated is
3.times.10.sup.-8 M. The substrate used was a synthetic fluorogenic
peptide substrate. The inhibition of OM99-2 on recombinant memapsin
2 was measured using the same fluorogenic substrate. The Ki value
was determined to be 9.58.times.10.sup.-9 M, as shown in FIG.
5B.
[0172] These results demonstrate that the predicted subsite
specificity is accurate and that inhibitors can be designed based
on the predicted specificity.
[0173] The residues in P1 and P1'are very important since the M2
inhibitor must penetrate the blood-brain barrier (BBB). The choice
of Ala in P1'facilitates the penetration of BBB. Analogues of Ala
side chains will also work. For example, in addition to the methyl
side chain of Ala, substituted methyl groups and groups about the
same size like methyl or ethyl groups can be substituted for the
Ala side chain. Leu at P1 can also be substituted by groups of
similar sizes or with substitutions on Leu side chain. For
penetrating the BBB, it is desirable to make the inhibitors
smaller. One can therefore use OM99-1 as a starting point and
discard the outside subsites P4, P3, P3'and P4'. The retained
structure Asn-Leu*Ala-Ala (SEQ ID NO:29) is then further evolved
with substitutions for a tight-binding M2 inhibitor which can also
penetrate the BBB. Example 9. Crystallization and X-ray diffraction
study of the protease domain of human memapsin 2 complexed to a
specifically designed inhibitor, OM99-2.
[0174] The crystallization condition and preliminary x-ray
diffraction data on recombinant human memapsin 2 complexed to
OM99-2 were determined.
[0175] Production of Recombinant Memapsin 2
[0176] About 50 mg of recombinant memapsin 2 was purified as
described in Example 3. For optimal crystal growth, memapsin 2 must
be highly purified. Memapsin 2 was over-expressed from vector
pET11a-M2pd. This memapsin 2 is the zymogen domain which includes
the pro and catalytic domains to the end of the C-terminal
extension but does not include the transmemhrane and the
intracellular domains. The vector was transfected into E. coli BL21
(DE3) and plated onto ZB agar containing 50 mg/liter ampicillin. A
single colony was picked to inoculate 100 ml of liquid ZB
containing 5 mg ampicillin and cultured at 30.degree. C., for 18
hours, with shaking at 220 RPM. Aliquots of approximately 15 ml of
the overnight culture were used to inoculate each 1 liter of LB
containing 50 mg of ampicillin. Cultures were grown at 37.degree.
C., with shaking at 180 RPM, until an optical density at 600 nm
near 0.8 was attained. At that time, expression was induced by
addition of 119 mg of IPTG to each liter of culture. Incubation was
continued for 3 additional hours post-induction.
[0177] Bacteria were harvested, suspended in 50 mM Tris, 150 mM
NaCl, pH 7.5 (TN buffer), and lysed by incubation with 6 mg
lysozyme for 30 minutes, followed by freezing for 18 hours at
-20.degree. C. Lysate was thawed and made to 1 mM MgCl.sub.2 then
1000 Kunitz units of DNAse were added with stirring, and incubated
for 30 min. Volume was expanded to 500 ml with TN containing 0.1%
Triton X-100 (TNT buffer) and lysate stirred for 30 minutes.
Insoluble inclusion bodies containing greater than 90% memapsin 2
protein were pelleted by centrifugation, and washed by resuspension
in TNT with stirring for 1-2 hours. Following three additional TNT
washes, the memapsin 2 inclusion bodies were dissolved in 40 ml of
8 M urea, 1 mM EDTA, 1 mM glycine, 100 mM Tris base, 100 mM
beta-mercaptoethanlol (8 M urea buffer). Optical density at 280 nm
was measured, and volume expanded with 8 M urea buffer to achieve
final O.D. near 0.5, with addition of sufficient quantity of
beta-mercaptoethanol to attain 10 mM total, and 10 mM DTT, 1 mM
reduced glutathione, 0.1 mM oxidized glutathione. The pH of the
solution was adjusted to 10.0 or greater, and divided into four
aliquots of 200 ml each. Each 200 ml was rapidly-diluted into 4
liters of 20 mM Tris base, with rapid stirring. The pH was adjusted
immediately to 9.0, with 1 M HCl, and stored at 4.degree. C.
overnight. The following morning the diluted memapsin 2 solution
was maintained at room temperature for 4-6 hours followed by
adjusting pH to 8.5 and replacing the flasks to the 4.degree. C.
room. The same procedure was followed the next day with adjustment
of pH to 8.0.
[0178] This memapsin 2 solution was allowed to stand at 4.degree.
C. for 2-3 weeks. The total volume of approximately 16 liters was
concentrated to 40 mls using ultra-filtration (Millipore) and
stir-cells (Amicon), and centrifuged at 140,000 xg at 30 minutes in
a rotor pre-equilbrated to 4.degree. C. The recovered supernatant
was applied to a 2.5.times.100 cm column of S-300 equilibrated in
0.4 M urea, 20 mM Tris-HCI, pH 8.0, and eluted with the same buffer
at 30 ml/hour. The active fraction of memapsin 2 was pooled and
further purified in a FPLC using a 1 ml Resource-Q (Pharmacia)
column. Sample was filtered, and applied to the Resource-Q column
equilibrated in 0.4 M urea, 50 mM Tris-HCI, pH 8.0. Sample was
eluted with a gradient of 0-1 M NaCl in the same buffer, over 30 ml
at 2 ml/min. The eluents containing memapsin 2 appeared near 0.4 M
NaCl which was pooled for crystallization procedure at a
concentration near 5 mg/ml.
[0179] The amino-terninal sequence of the protein before
crystallization showed two sequences starting respectively at
residues 28p and 30p. Apparently, the pro peptide of recombinant
pro-memapsin 2 had been cleaved during the preparation by a yet
unidentified proteolytic activity.
[0180] The activation of the folded pro-enzyme to mature enzyme,
memapsin 2, was carried out as described above, i.e., incubation in
0.1 M sodium acetate pH 4.0 for 16 hours at 22.degree. C. Activated
enzyme was further purified using anion-exchange column
chromatography on Resource-Q anion exchange column. The purity of
the enzyme was demonstrated by SDS-gel electrophoresis. At each
step of the purification, the specific activity of the enzyme was
assayed as described above to ensure the activity of the
enzyme.
[0181] Preliminary Crystallization with 0M99-2
[0182] Crystal trials were performed on purified memapsin 2 in
complex with a substrate based transition-state inhibitor OM99-2
with a Ki=10 nM. OM99-2 is equivalent to eight amino-acid residues
(including subsites S4, S3, S2, S1 S1', S2', S3'and S4'in a
sequence EVNLAAEF) with the substitution of the peptide bond
between the S1 and S1'(L-A) by a transition-state isostere
hydroxyethylene. Purified M2 was concentrated and mixed with 10
fold excessive molar amount of inhibitor. The mixture was incubated
at room temperature for 2-3 hours to optimize the inhibitor
binding. The crystallization trial was conducted at 20.degree. C.
using the hanging drop vapor diffusion procedure. A systemic search
with various crystallization conditions was conducted to find the
optimum crystallization conditions for memapsin 2/OM99-2 inhibitor
complex. For the first step, a coarse screen aimed at covering a
wide range of potential conditions were carried out using the
Sparse Matrix Crystallization Screen Kits purchased from Hampton
Research. Protein concentration and temperature were used as
additional variables. Conditions giving promising (micro) crystals
were subsequently used as starting points for optimization, using
fine grids of pH, precipitants concentration etc.
[0183] Crystals of memapsin-inhibitor complex were obtained at 30%
PEG 8000, 0.1 M NaCocadylate, pH 6.4. SDS gel electrophoresis of a
dissolved crystal verified that the content of the crystal to be
memapsin 2. Several single crystals (with the sizes about 0.3
mm.times.0.2 mm.times.0.1 mm) were carefully removed from the
cluster for data collection on a Raxis IV image plate. These
results showed that the crystals diffract to 2.6 .ANG.. A typical
protein diffraction pattern is shown in FIG. 6. An X-ray image
visualization and integration software-Denzo, was used to visualize
and index the diffraction data. Denzo identified that the primitive
orthorhombic lattice has the highest symmetry with a significantly
low distortion index. The unit cell parameters were determined
as:=a=89.1 .ANG., b=96.6 .ANG., c=134.1 .ANG.,
.alpha.=.beta.=.gamma.=90.degree. C. There are two memaspin
2/OM99-2 complex per crystallographic asymmetric unit, the V.sub.m
of the crystal is 2.9 .ANG..sup.3/Da. Diffraction extinction
suggested that the space group is P2.sub.12.sub.12.sub.1.
[0184] With diffraction of the current crystal to 2.6 .ANG., the
crystal structure obtained from these data has the potential to
reach atomic solution, i.e., the three-dimensional positions of
atoms and chemical bonds in the inhibitor and in memapsin 2 can be
deduced. Since memapsin 2 sequence is homologous with other
mammalian aspartic proteases, e.g., pepsin or cathepsin D, it is
predicted that the three dimensional structures of memapsin 2 will
be similar (but not identical) to their structures. Therefore, in
the determination of x-ray structure from the diffraction data
obtained from the current crystal, it is likely the solution of the
phase can be obtained from the molecular replacement method using
the known crystal structure of aspartic proteases as the search
model.
[0185] Further Crystallization Studies
[0186] Concentrated memapsin 2 was mixed with 10-fold molar
excessive of the inhibitor. The mixture was incubated at room
temperature for 2-3 hours to optimize inhibitor binding, and then
clarified with a 0.2 micron filter using centrifugation. Crystals
of memapsin 2-inhibitor complex were grown at 20.degree. C. by
hanging drop vapor diffusion method using equal volumes of
enzyme-inhibitor and well solution. Crystals of quality suitable
for diffraction studies were obtained in two weeks in 0.1 M sodium
cacodylate, pH 7.4, 0.2 M (NH4)2SO4, and 22.5% PEG8000. The typical
size of the crystals was about 0.4 .times.0.4.times.0.2
mm.sup.3.
[0187] Diffraction data were measured on a Raxis-IV image plate
with a Rigaku X-ray generator, processed with the HKL program
package [Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307 (1997)]
A single crystal of approximately 0.4.times.0.4.times.0.2 mm.sup.3
in size was treated with a cryo-protection solution of 25% PEG8000,
20% glycerol, 0.1 M sodium-cacodylate pH 6.6, and 0.2 M
(N.sub.4)2SO4 and then flash-cooled with liquid nitrogen to about
-180.degree. C. for data collection. Diffraction was observed to at
least 1.9 .ANG.. The crystal form belongs to space group P2.sub.1
with two memapsin 2/OM99-2 complexes per crystallographic
asymmetric unit and 56% solvent content.
[0188] Molecular replacement was performed with data in the range
of 15.0-3.5 .ANG. using program AmoRe, CCP4 package [Navaza, J.,
Acta Crystallog. Sect. A. 50, 157 (1994)]. Pepsin, a human aspartic
protease with 22% sequence identity, was used as the search
model(PDB id 1psn). Rotation and translation search, followed by
rigid body refinement, identified a top solution and positioned
both molecules in the asymmetric unit. The initial solution had a
correlation coefficient of 22% and an R-factor of 0.51. The
refinement was carried out using the program CNS [Brunger et al.,
Acta Crystallogr. Sect. D, 54, 905 (1998)]. 10% of reflections were
randomly selected prior to refinement for R.sub.free monitoring
[Bruger, A.T., X-PLOR Version 3.1: A system for X-ray
Crystallography and NMR, Yale University Press, New Haven, Conn.
(1992)]. Molecular graphics program [Jones, T.A., et al., Improved
methods for building protein models in electron denisty maps and
location of errors in these models. Acta Crystallogr. Sect. A 47,
110 (1991)] was used for map display and model building. From the
initial pepsin model, corresponding amino acid residues were
changed to that of memapsin 2 according to sequence alignment. The
side chain conformations were decided by the initial electron
density map and a rotomer library. This model was refined using
molecular dynamics and energy minimization function of CNS [Bruger,
A.T., et al., Acta Crystallogr. Sect. D, 54, 905 (1998)]. The first
cycle of refinement dropped the R.sub.working to 41% and the
R.sub.free to 45%. At this stage, electron densities in the omit
map clearly showed the inhibitor configuration in the active site
cleft. Structural features unique to memapsin 2 in chain tracing,
secondary structure, insertions, deletions and extensions (as
compared to the search model) are identified and constructed in
subsequent iterations of crystallographic refinement and map
fitting. The inhibitor was built into the corresponding electron
density.
[0189] About 440 solvent molecules were then gradually added to the
structure as identified in the
.vertline.Fo.vertline.-.vertline.Fe.vertli- ne. map contoured at
the 3 sigma level. Non-crystallographic symmetry restriction and
averaging were used in early stages of refinement and model
building. Bulk solvent and anisotropic over-all B factor
corrections were applied through the refinement. The final
structure was validated by the program PROCHECK Laskowski, R.A. et
al., J. Appl. Crystallog. 26, 283 (1993) which showed that 95% of
the residues are located in the most favored region of the
Ramachandran plot. All the main chain and side chain parameters are
within or better than the standard criteria. The final
R.sub.working and R.sub.free are 18% and 22% respectively.
Refinement statistics are listed in Table 2.
7TABLE 2 Data Collection and Refinement Statistics A. Data
Statistics Space group P2.sub.1 Unit Cell (a, b, and c in ) 53.7,
85.9, 109.2 (, , and in degrees) 90.0, 101.4, 90.0 Resolution ( )
25.0-1.9 Number of observed reflections 144, 164 Number of unique
reflections 69, 056 R.sub.merge.sup.a 0.061 (0.25) Data
completeness (%) (25.0-1.9 ) 90.0 (68.5) <I/(I)> 13.7 (3.0)
B. Refinement Statistics R.sub.working.sup.b 0.186 R.sub.free.sup.b
0.228 RMS deviation from ideal values Bond length ( ) 0.014 Bond
angle (Deg) 1.7 Number of water molecules 445 Average B-factor (
.sup.2) Protein 28.5 Solvent 32.2 .sup.aR.sub.merge = .sub.hkl
i.vertline.I.sub.hkl, i-<I.sub.hkl>.vertline./
.sub.hkl<I.sub.hkl>, where I.sub.hkl, i is the intensity of
the ith # measurement and <I.sub.hkl> is the weighted mean of
all measurements of I.sub.hkl. .sup.bR.sub.working (free) =
.vertline.F.sub.o.vertline.-.vertline.F.sub.c.vertline./
.vertline.F.sub.o.vertline., where F.sub.o and F.sub.c are the
observed and calculated # structure factors. Numbers in parentheses
are the corresponding numbers for the highest resolution shell #
(2.00-1.9 .ANG.). Reflections with F.sub.o/ (F.sub.o) >= 0.0 are
included in the refinement and R factor calculation.
[0190] Memasin 2 Crystal Structure.
[0191] The bilobal structure of memapsin 2 (FIG. 7) is
characteristic of aspartic proteases (Tang, J., et al., Nature 271,
618-621 (1978)) with the conserved folding of the globular core.
The substrate binding cleft, where the inhibitor is bound (FIG. 7),
is located between the two lobes. A pseudo two-fold symmetry
between the N-(residues 1-180) and C-(residues 181-385) lobes (FIG.
7), which share 61 superimposable atoms with an overall 2.3 .ANG.
rms deviation using a 4 .ANG. cutoff. The corresponding numbers for
pepsin are 67 atoms and 2.2 .ANG.. Active-site Asp.sup.32 and
Asp.sup.228 and the surrounding hydrogen-bond network are located
in the center of the cleft (FIG. 7) anid are conserved with the
typical active-site conformation (Davies, D. R., Annu. Rev.
Biophys. Chem. 19, 189 (1990)). The active site carboxyls are,
however, not co-planar and the degree of which (500) exceeds those
observed previously.
[0192] Compared to pepsin, the conformation of the N-lobe is
essentially conserved (Sielecki et al., 1990). The most significant
structural differences are the insertions and a C-terminal
extension in the C-lobe. Four insertions in helices and loops (FIG.
7) are located on the adjacent molecular surface. Insertion F,
which contains four acidic residues, is the most negatively charged
surface on the molecule. Together, these insertions enlarged
significantly the molecular boundary of memapsin 2 as compared to
pepsin (FIG. 8). These surface structural changes may have function
in the association of memapsin 2 with other cell surface
components. Insertions B and E are located on the other side of the
molecule (FIG. 7). The latter contains a beta-strand that paired
with part of the C-terminal extension G. A six-residue deletion
occurs at position 329 on a loop facing the flap on the opposite
side of the active-site cleft, resulting in an apparently more
accessible cleft. Most of the C-terminal extension (residues
359-393) is in highly ordered structure. Residues 369-376 form a
beta structure with 7 hydrogen bonds to strand 293-299, while
residues 378-383 form a helix (FIGS. 7 and 8). Two disulfide pairs
(residues 155/359 and 217/382) unique to memapsin 2 fasten both
ends of the extension region to the C-lobe. This C-terminal
extension is much longer than those observed previously and is
conformationally different [Cutfield, S. M., et al., Structure 3,
1261 (1995); Abad-Zapatero, C., et al., Protein Sci. 5, 640 (1996);
Symersky, J. et al., Biochemistry 36, 12700 (1997); Yang, J., et
al., Acta Crystallogr. D 55, 625 (1999)]. The last eight residues
(386-393) are not seen in the electron density map; they may form a
connecting stem between the globular catalytic domain and the
membrane anchoring domain.
[0193] Of the 21 putative pro residues only the last six, 43p-48p,
are visible in the electron density map. The remainders are likely
mobile. Pro-memapsin expressed in mammalian cell culture has an
N-terminus position at Glu.sup.33p. However, an Arg-Arg sequence
present at residues 43p-44p is a frequent signal for pro-protein
processing, e.g., in prorenin (Corvol, P. et al., Hypertension 5,
13-9 (1983)). Recombinant memapsin 2 derived from this cleavage is
fully active. The mobility of residues 28p-42p suggests that they
are not part of the structure of mature memapsin 2.
[0194] Memapsin 2-OM99-2 Interaction.
[0195] The binding of the eight-residue inhibitor OM99-2 in the
active-site cleft shares some structural features with other
aspartic protease-inhibitor complexes [Davies, D. R., Annu. Rev.
Biophys. Chem. 19 189 (1990); Bailey and Cooper, (1994); Dealwis et
al., (1994)]. These include four hydrogen bonds between the two
active-site aspartics to the hydroxyl of the transition-state
isostere, the covering of the flap (residues 69-75) over the
central part of the inhibitor and ten hydrogen bonds to inhibitor
backbone (FIG. 9). Most of the latter are highly conserved among
aspartic proteases [Davies, D. R. Annu. Rev. Biophys. Chem. 19, 189
(1990); Bailey and Cooper, (1994); Dealwis et al., (1994)] except
that hydrogen bonds to Gly.sup.11 and Tyr.sup.198 are unique to
memapsin 2. These observations illustrate that the manner by which
memapsin 2 transition-state template for substrate peptide backbone
and mechanism of catalysis are similar to other aspartic proteases.
These common features are, however, not the decisive factors in the
design of specific memapsin 2 inhibitors with high selectivity.
[0196] The observation important for the design of inhibitor drugs
is that the memapsin 2 residues in contact with individual
inhibitor side chains (FIG. 9) are quite different from those for
other aspartic proteases. These side chain contacts are important
for the design of tight binding inhibitor with high selectivity.
Five N-terminal residues of OM99-2 are in extended conformation
and, with the exception of P.sub.1' Ala, all have clearly defined
contacts (within 4 .ANG. of an inhibitor side chain) with enzyme
residues in the active-site cleft (FIG. 9).
[0197] The protease S4 subsite is mostly hydrophilic and open to
solvent. The position of inhibitor P.sub.4 Glu side chain is
defined by hydrogen bonds to Gly.sup.11 and to P.sub.2 Asn (FIG. 9)
and the nearby sidechains of Arg.sup.235 and Arg.sup.307, which
explains why the absence of this residue from OM99-2 cause a
10-fold increase in K.sub.i. Likewise, the protease S2 subsite is
relatively hydrophilic and open to solvent. Inhibitor P.sub.2 Asn
side chain has hydrogen bonds to P.sub.4 Glu and Arg.sup.235. The
relatively small S2 residues Ser.sup.325 and Ser.sup.327 (Gln and
Met respectively in pepsin) may fit a side chain larger than Asn.
Memapsin 2 S.sub.1 and S3 subsites, which consist mostly of
hydrophobic residues, have conformations very different from pepsin
due to the deletion of pepsin helix h.sub.H2 (Dealwis, et al.,
(1994)). The inhibitor side chains of P.sub.3 Val and P.sub.1 Leu
are closely packed against each other and have substantial
hydrophobic contacts with the enzyme (FIG. 9), especially P.sub.3
interacts with Tyr.sup.71 and Phe.sup.108. In the beta-secretase
site of native APP, the P.sub.2 and P.sub.1 residues are Lys and
Met respectively. Swedish mutant APP has Asn and Leu in these
positions respectively, resulting in a 60-fold increase of
k.sub.cat /K.sub.m over that for native APP and an early onset of
AD described by Mullan, M., et al. [Nat. Genet. 2, 340 (1992)]. The
current structure suggests that inhibitor P.sub.2 Lys would place
its positively charge in an unfavorable interaction with
Arg.sup.235 with a loss of hydrogen bond to Arg.sup.235, while
P.sub.1 Met would have less favorable contact with mnemapsin 2 than
does leucine in this site (FIG. 10). No close contact with memapsin
2 was seen for P.sub.1 ' Ala and an aspartic at this position, as
in API), may be accommodated by interacting with Arg.sup.228.
[0198] The direction of inhibitor chain turns at P.sub.2' and leads
P.sub.3' and P.sub.4' toward the protein surface (FIG. 10). As a
result, the side-chain position of P.sub.2' Ala deviates from the
regular extended conformation. The side chains of P.sub.3' Glu and
P.sub.4' Phe are both pointed toward molecular surface with little
significant interaction with the protease (FIG. 10). The relatively
high B-factors (58.2 .ANG..sup.2 for Glu and 75.6 .ANG..sup.2 for
Phe) and less well-defined electron density suggests that these two
residues are relatively mobile, in contrast to the defined
structure of the S.sub.3' and S.sub.4' subsites in renin-inhibitor
(CH-66) complex (Dealwis et al., 1994). The topologically
equivalent region of these renin subsites (residues 292-297 in
pepsin numbering) is deleted in memapsin 2. These observations
suggest that the conformation of three C-terminal residues of
OM99-2 may be a functional feature of memapsin 2, possibly a way to
lead a long protein substrate out of the active-site cleft.
[0199] Example 10: Using The Crystal Structure to Design
Inhibitors.
[0200] Pharmaceutically acceptable inhibitor drugs normally post a
size limit under 800 daltons. In the case of memapsin 2 inhibitors,
this requirement may even be more stringent due to the need for the
drugs to penetrate the blood-brain barrier [Kearney and Aweeka,
(1999)]. In the current model, well defined subsite structures
spending P.sub.4 to P.sub.2' provide sufficient template areas for
rational design of such drugs. The spacial relationships of
individual inhibitor side chain with the corresponding subsite of
the enzyme as revealed in this crystal structure permits the design
of new inhibitor structures in each of these positions. It is also
possible to incorporate the unique conformation of subsites
P.sub.2', P.sub.3' and P.sub.4' into the selectivity of memapsin 2
inhibitors. The examples of inhibitor design based on the current
crystal structure are given below. Example A: Since the side chains
of P.sub.3 Val and P.sub.1 Leu are packed against each other and
there is no enzyme structure between them, cross-linking these side
chains would increase the binding strength of inhibitor to memaspin
2. This is because when binding to the enzyme, the cross-linked
inhibitors would have less entropy difference between the free and
bound forms than their non-cross-linked counterparts [Khan, A. R.,
et al., Biochemistry, 37, 16839 (1998)]. Possible structures of the
cross-linked side chains include those shown in FIG. 11. Example B:
The same situation exits between the P4 Glu and P2 Asn. The current
crystal structure shows that these side chains are already hydrogen
bonded to each other so the cross linking between them would also
derive binding benefit as described in the Example A. The
cross-linked structures include those shown in FIG. 12. Example C:
Based on the current crystal structure, the P1'Ala side chain may
be extended to add new hydrophobic, Van der Waals and H-bond
interactions. An example of such a design is diagramed in FIG. 13.
Example D: Based on the current crystal structure, the polypeptide
backbone in the region of P1, P2, and P3, and the side chain of
P1-Leu can be bridged into rings by the addition of two atoms (A
and B in FIG. 14). Also, a methyl group can be added to the
beta-carbon of the P1-Leu (FIG. 14).
[0201] Modifications and variations of the methods and materials
described herein will be obvious to those skilled in the art and
are intended to come within the scope of the appended claims.
Sequence CWU 1
1
31 1 3252 DNA Homo sapiens 1 gcgggagtgc tgcctgccca cggcacccag
cacggcatcc ggctgcccct gcgcagcggc 60 ctggggggcg cccccctggg
gctgcggctg ccccgggaga ccgacgaaga gcccgaggag 120 cccggccgga
ggggcagctt tgtggagatg gtggacaacc tgaggggcaa gtcggggcag 180
ggctactacg tggagatgac cgtgggcagc cccccgcaga cgctcaacat cctggtggat
240 acaggcagca gtaactttgc agtgggtgct gccccccacc ccttcctgca
tcgctactac 300 cagaggcagc tgtccagcac ataccgggac ctccggaagg
gtgtgtatgt gccctacacc 360 cagggcaagt gggaagggga gctgggcacc
gacctggtaa gcatccccca tggccccaac 420 gtcactgtgc gtgccaacat
tgctgccatc actgaatcag acaagttctt catcaacggc 480 tccaactggg
aaggcatcct ggggctggcc tatgctgaga ttgccaggcc tgacgactcc 540
ctggagcctt tctttgactc tctggtaaag cagacccacg ttcccaacct cttctccctg
600 cagctttgtg gtgctggctt ccccctcaac cagtctgaag tgctggcctc
tgtcggaggg 660 agcatgatca ttggaggtat cgaccactcg ctgtacacag
gcagtctctg gtatacaccc 720 atccggcggg agtggtatta tgaggtgatc
attgtgcggg tggagatcaa tggacaggat 780 ctgaaaatgg actgcaagga
gtacaactat gacaagagca ttgtggacag tggcaccacc 840 aaccttcgtt
tgcccaagaa agtgtttgaa gctgcagtca aatccatcaa ggcagcctcc 900
tccacggaga agttccctga tggtttctgg ctaggagagc agctggtgtg ctggcaagca
960 ggcaccaccc cttggaacat tttcccagtc atctcactct acctaatggg
tgaggttacc 1020 aaccagtcct tccgcatcac catccttccg cagcaatacc
tgcggccagt ggaagatgtg 1080 gccacgtccc aagacgactg ttacaagttt
gccatctcac agtcatccac gggcactgtt 1140 atgggagctg ttatcatgga
gggcttctac gttgtctttg atcgggcccg aaaacgaatt 1200 ggctttgctg
tcagcgcttg ccatgtgcac gatgagttca ggacggcagc ggtggaaggc 1260
ccttttgtca ccttggacat ggaagactgt ggctacaaca ttccacagac agatgagtca
1320 accctcatga ccatagccta tgtcatggct gccatctgcg ccctcttcat
gctgccactc 1380 tgcctcatgg tgtgtcagtg gcgctgcctc cgctgcctgc
gccagcagca tgatgacttt 1440 gctgatgaca tctccctgct gaagtgagga
ggcccatggg cagaagatag agattcccct 1500 ggaccacacc tccgtggttc
actttggtca caagtaggag acacagatgg cacctgtggc 1560 cagagcacct
caggaccctc cccacccacc aaatgcctct gccttgatgg agaaggaaaa 1620
ggctggcaag gtgggttcca gggactgtac ctgtaggaaa cagaaaagag aagaaagaag
1680 cactctgctg gcgggaatac tcttggtcac ctcaaattta agtcgggaaa
ttctgctgct 1740 tgaaacttca gccctgaacc tttgtccacc attcctttaa
attctccaac ccaaagtatt 1800 cttcttttct tagtttcaga agtactggca
tcacacgcag gttaccttgg cgtgtgtccc 1860 tgtggtaccc tggcagagaa
gagaccaagc ttgtttccct gctggccaaa gtcagtagga 1920 gaggatgcac
agtttgctat ttgctttaga gacagggact gtataaacaa gcctaacatt 1980
ggtgcaaaga ttgcctcttg aattaaaaaa aaactagatt gactatttat acaaatgggg
2040 gcggctggaa agaggagaag gagagggagt acaaagacag ggaatagtgg
gatcaaagct 2100 aggaaaggca gaaacacaac cactcaccag tcctagtttt
agacctcatc tccaagatag 2160 catcccatct cagaagatgg gtgttgtttt
caatgttttc ttttctgtgg ttgcagcctg 2220 accaaaagtg agatgggaag
ggcttatcta gccaaagagc tcttttttag ctctcttaaa 2280 tgaagtgccc
actaagaagt tccacttaac acatgaattt ctgccatatt aatttcattg 2340
tctctatctg aaccaccctt tattctacat atgataggca gcactgaaat atcctaaccc
2400 cctaagctcc aggtgccctg tgggagagca actggactat agcagggctg
ggctctgtct 2460 tcctggtcat aggctcactc tttcccccaa atcttcctct
ggagctttgc agccaaggtg 2520 ctaaaaggaa taggtaggag acctcttcta
tctaatcctt aaaagcataa tgttgaacat 2580 tcattcaaca gctgatgccc
tataacccct gcctggattt cttcctatta ggctataaga 2640 agtagcaaga
tctttacata attcagagtg gtttcattgc cttcctaccc tctctaatgg 2700
cccctccatt tatttgacta aagcatcrca cagtggcact agcattatac caagagtatg
2760 agaaatacag tgctttatgg ctctaacatt actgccttca gtatcaaggc
tgcctggaga 2820 aaggatggca gcctcagggc ttccttatgt cctccaccac
aagagctcct tgatgaaggt 2880 catctttttc ccctatcctg ttcttcccct
ccccgctcct aatggtacgt gggtacccag 2940 gctggttctt gggctaggta
gtggggacca agttcattac ctccctatca gttctagcat 3000 agtaaactac
ggtaccagtg ttagtgggaa gagctgggtt ttcctagtat acccactgca 3060
tcctactcct acctggtcaa cccgctgctt ccaggtatgg gacctgctaa gtgtggaatt
3120 acctgataag ggagagggaa atacaaggag ggcctctggt gttcctggcc
tcagccagct 3180 gcccmcaagc cataaaccaa taaamcaaga atactgagtc
taaaaaaaaa aaaaaaaaaa 3240 aaaaaaaaaa aa 3252 2 488 PRT Homo
sapiens Purified Memapsin 2 2 Ala Gly Val Leu Pro Ala His Gly Thr
Gln His Gly Ile Arg Leu Pro 1 5 10 15 Leu Arg Ser Gly Leu Gly Gly
Ala Pro Leu Gly Leu Arg Leu Pro Arg 20 25 30 Glu Thr Asp Glu Glu
Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val 35 40 45 Glu Met Val
Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val 50 55 60 Glu
Met Thr Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp 65 70
75 80 Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe
Leu 85 90 95 His Arg Tyr Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg
Asp Leu Arg 100 105 110 Lys Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys
Trp Glu Gly Glu Leu 115 120 125 Gly Thr Asp Leu Val Ser Ile Pro His
Gly Pro Asn Val Thr Val Arg 130 135 140 Ala Asn Ile Ala Ala Ile Thr
Glu Ser Asp Lys Phe Phe Ile Asn Gly 145 150 155 160 Ser Asn Trp Glu
Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg 165 170 175 Pro Asp
Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr 180 185 190
His Val Pro Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro 195
200 205 Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile
Ile 210 215 220 Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp
Tyr Thr Pro 225 230 235 240 Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile
Ile Val Arg Val Glu Ile 245 250 255 Asn Gly Gln Asp Leu Lys Met Asp
Cys Lys Glu Tyr Asn Tyr Asp Lys 260 265 270 Ser Ile Val Asp Ser Gly
Thr Thr Asn Leu Arg Leu Pro Lys Lys Val 275 280 285 Phe Glu Ala Ala
Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys 290 295 300 Phe Pro
Asp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala 305 310 315
320 Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met
325 330 335 Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro
Gln Gln 340 345 350 Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln
Asp Asp Cys Tyr 355 360 365 Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly
Thr Val Met Gly Ala Val 370 375 380 Ile Met Glu Gly Phe Tyr Val Val
Phe Asp Arg Ala Arg Lys Arg Ile 385 390 395 400 Gly Phe Ala Val Ser
Ala Cys His Val His Asp Glu Phe Arg Thr Ala 405 410 415 Ala Val Glu
Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr 420 425 430 Asn
Ile Pro Gln Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val 435 440
445 Met Ala Ala Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val
450 455 460 Cys Gln Trp Arg Cys Leu Arg Cys Leu Arg Gln Gln His Asp
Asp Phe 465 470 475 480 Ala Asp Asp Ile Ser Leu Leu Lys 485 3 503
PRT Homo sapiens Pro-memapsin 2 3 Met Ala Ser Met Thr Gly Gly Gln
Gln Met Gly Arg Gly Ser Met Ala 1 5 10 15 Gly Val Leu Pro Ala His
Gly Thr Gln His Gly Ile Arg Leu Pro Leu 20 25 30 Arg Ser Gly Leu
Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu 35 40 45 Thr Asp
Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu 50 55 60
Met Val Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu 65
70 75 80 Met Thr Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val
Asp Thr 85 90 95 Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His
Pro Phe Leu His 100 105 110 Arg Tyr Tyr Gln Arg Gln Leu Ser Ser Thr
Tyr Arg Asp Leu Arg Lys 115 120 125 Gly Val Tyr Val Pro Tyr Thr Gln
Gly Lys Trp Glu Gly Glu Leu Gly 130 135 140 Thr Asp Leu Val Ser Ile
Pro His Gly Pro Asn Val Thr Val Arg Ala 145 150 155 160 Asn Ile Ala
Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser 165 170 175 Asn
Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro 180 185
190 Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His
195 200 205 Val Pro Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe
Pro Leu 210 215 220 Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly Ser
Met Ile Ile Gly 225 230 235 240 Gly Ile Asp His Ser Leu Tyr Thr Gly
Ser Leu Trp Tyr Thr Pro Ile 245 250 255 Arg Arg Glu Trp Tyr Tyr Glu
Val Ile Ile Val Arg Val Glu Ile Asn 260 265 270 Gly Gln Asp Leu Lys
Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser 275 280 285 Ile Val Asp
Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe 290 295 300 Glu
Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe 305 310
315 320 Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala
Gly 325 330 335 Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr
Leu Met Gly 340 345 350 Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile
Leu Pro Gln Gln Tyr 355 360 365 Leu Arg Pro Val Glu Asp Val Ala Thr
Ser Gln Asp Asp Cys Tyr Lys 370 375 380 Phe Ala Ile Ser Gln Ser Ser
Thr Gly Thr Val Met Gly Ala Val Ile 385 390 395 400 Met Glu Gly Phe
Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly 405 410 415 Phe Ala
Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala 420 425 430
Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn 435
440 445 Ile Pro Gln Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val
Met 450 455 460 Ala Ala Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu
Met Val Cys 465 470 475 480 Gln Trp Arg Cys Leu Arg Cys Leu Arg Gln
Gln His Asp Asp Phe Ala 485 490 495 Asp Asp Ile Ser Leu Leu Lys 500
4 10 PRT Artificial Sequence Description of Artificial Sequence
Primer 4 Ser Glu Val Lys Met Asp Ala Glu Phe Arg 1 5 10 5 10 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 5 Ser Glu Val Asn Leu Asp Ala Glu Phe Arg 1 5 10 6 8 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Peptide 6 Ser Val Asn Met Ala Glu Gly Asp 1 5 7 12 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 7 Lys
Gly Gly Val Val Ile Ala Thr Val Ile Val Lys 1 5 10 8 4 PRT Homo
sapiens 8 Asp Thr Ser Gly 1 9 8 PRT Homo sapiens 9 Leu Val Asn Met
Ala Glu Gly Asp 1 5 10 28 DNA Artificial Sequence Description of
Artificial Sequence Primer 10 ggtaagcatc ccccatggcc ccaacgtc 28 11
28 DNA Artificial Sequence Description of Artificial Sequence
Primer 11 gacgttgggg ccatggggga tgcttacc 28 12 34 DNA Artificial
Sequence Description of Artificial Sequence Primer 12 acgttgtctt
tgatcgggcc cgaaaacgaa ttgg 34 13 33 DNA Artificial Sequence
Description of Artificial Sequence Primer 13 ccaattcgtt ttcgggcccg
atcaaagaca acg 33 14 27 DNA Artificial Sequence Description of
Artificial Sequence Primer 14 ccatcctaat acgactcact atagggc 27 15
23 DNA Artificial Sequence Description of Artificial Sequence
Primer 15 actcactata gggctcgagc ggc 23 16 26 DNA Artificial
Sequence Description of Artificial Sequence Primer 16 cttttgagca
agttcagcct ggttaa 26 17 31 DNA Artificial Sequence Description of
Artificial Sequence Primer 17 gaggtggctt atgagtattt cttccagggt a 31
18 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 18 tggcgacgac tcctggagcc cg 22 19 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 19 tgacaccaga ccaactggta
atgg 24 20 27 DNA Artificial Sequence Description of Artificial
Sequence Primer 20 catatggcgg gagtgctgcc tgcccac 27 21 38 DNA
Artificial Sequence Description of Artificial Sequence Primer 21
ggatcctcac ttcagcaggg agatgtcatc agcaaagt 38 22 8 PRT Artificial
Sequence Description of Artificial Sequence Oxidized Insulin
B-chain 22 His Leu Xaa Gly Ser His Leu Val 1 5 23 8 PRT Artificial
Sequence Description of Artificial Sequence Oxidized Insulin
B-chain 23 Xaa Gly Glu Arg Gly Phe Phe Tyr 1 5 24 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Peptide 24
Val Gly Ser Gly Val 1 5 25 7 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Peptide 25 Val Gly Ser Gly Val Leu
Leu 1 5 26 7 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 26 Gly Val Leu Leu Ser Arg Lys 1 5 27 7
PRT Artificial Sequence Description of Artificial Sequence
Inhibitors 27 Val Asn Leu Ala Ala Glu Phe 1 5 28 8 PRT Artificial
Sequence Description of Artificial Sequence Inhibitors 28 Glu Val
Asn Leu Ala Ala Glu Phe 1 5 29 4 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Peptide 29 Asn Leu Ala
Ala 1 30 10 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Peptide 30 Val Gly Ser Gly Val Leu Leu Ser Arg
Lys 1 5 10 31 326 PRT Homo sapiens Amino acids 2-5, 6-9, 13-20,
25-32, 65-67, 69-74, 79-87, 89-91, 99-106, 119-122, 150-154,
164-167, 180-183, 191-194, 196-199, 201-204, 210-214, 221-223,
258-262, 265-269, and 275-278 are Beta Strands 31 Val Asp Glu Gln
Pro Leu Glu Asn Tyr Leu Asp Met Glu Tyr Phe Gly 1 5 10 15 Thr Ile
Gly Ile Gly Thr Pro Ala Gln Asp Phe Thr Val Val Phe Asp 20 25 30
Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Val Tyr Cys Ser Ser Leu 35
40 45 Ala Cys Thr Asn His Asn Arg Phe Asn Pro Glu Asp Ser Ser Thr
Tyr 50 55 60 Gln Ser Thr Ser Glu Thr Val Ser Ile Thr Tyr Gly Thr
Gly Ser Met 65 70 75 80 Thr Gly Ile Leu Gly Tyr Asp Thr Val Gln Val
Gly Gly Ile Ser Asp 85 90 95 Thr Asn Gln Ile Phe Gly Leu Ser Glu
Thr Glu Pro Gly Ser Phe Leu 100 105 110 Tyr Tyr Ala Pro Phe Asp Gly
Ile Leu Gly Leu Ala Tyr Pro Ser Ile 115 120 125 Ser Ser Ser Gly Ala
Thr Pro Val Phe Asp Asn Ile Trp Asn Gln Gly 130 135 140 Leu Val Ser
Gln Asp Leu Phe Ser Val Tyr Leu Ser Ala Asp Asp Gln 145 150 155 160
Ser Gly Ser Val Val Ile Phe Gly Gly Ile Asp Ser Ser Tyr Tyr Thr 165
170 175 Gly Ser Leu Asn Trp Val Pro Val Thr Val Glu Gly Tyr Trp Gln
Ile 180 185 190 Thr Val Asp Ser Ile Thr Met Asn Gly Glu Ala Ile Ala
Cys Ala Glu 195 200 205 Gly Cys Gln Ala Ile Val Asp Thr Gly Thr Ser
Leu Leu Thr Gly Pro 210 215 220 Thr Ser Pro Ile Ala Asn Ile Gln Ser
Asp Ile Gly Ala Ser Glu Asn 225 230 235 240 Ser Asp Gly Asp Met Val
Val Ser Cys Ser Ala Ile Ser Ser Leu Pro 245 250 255 Asp Ile Val Phe
Thr Ile Asn Gly Val Gln Tyr Pro Val Pro Pro Ser 260 265 270 Ala Tyr
Ile Leu Gln Ser Glu Gly Ser Cys Ile Ser Gly Phe Gln Gly 275 280 285
Met Asn Leu Pro Thr Glu Ser Gly Glu Leu Trp Ile Leu Gly Asp Val 290
295 300 Phe Ile Arg Gln Tyr Phe Thr Val Phe Asp Arg Ala Asn Asn Gln
Val 305 310 315 320 Gly Leu Ala Pro Val Ala 325
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