Note: Descriptions are shown in the official language in which they were submitted.
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WO 02/16574 PCT/EP01/09102
Methods for identifying specifically cleavable peptides and use of such
peptide sequences
Description
The invention relates to methods for finding and identifying specifically
cleavable peptides having a defined amino acid sequence by using
proteolytically active solutions and to the use of said peptides for specific
release of chemical active substances and for diagnostics.
US 5981200 describes a method for detecting specifically cleaved peptides
by using a specific protein construct which fluoresces when cleaved
proteolytically. The method is based on fluorescence resonance energy
technique (FRET). This method has the advantage that it can also be used
for in vivo detection of protein cleavage events. A disadvantage, on the
other hand, is the fact that only specific peptides can be labeled or that, in
the case of studying different labeled proteins, assigning the cleavage
event precisely to the relevant sequence would be very complicated. Said
method has another disadvantage in that relatively large amounts of
labeled substrate have to be used in order to obtain a detectable signal.
Another method which may be used for selective identification of enryme
substrates is phage display (documented, for example, in WO 97/47314).
This method has the disadvantage that the phage display peptide libraries,
in contrast to other surface-represented peptide libraries, have a smaller
number of possible different sequences and the subsequent work-up of the
phage particles and determination of the identified substrates is relatively
complicated.
It is the object of the present invention to develop a method for finding and
identifying specifically proteolytically cleavable peptides and to provide
substances which allow a targeted release of chemical active substances.
The object is achieved by a method for identifying specifically
proteolytically cleavable peptides, which comprises the following method
steps:
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a) incubating a library of fusion molecules comprising a peptide and a
nucleic acid encoding said peptide with a proteolytically active
sample,
b) isolating the proteolytically removed parts of said fusion molecules,
c) determining the sequence of the nucleic acid part of the isolated
fusion molecules.
The method of the invention is based on using fusion molecules which
contain a phenotypic peptide part and a genotypic nucleic acid part which
has a sequence encoding said peptide part. The peptide part is linked to
the nucleic acid part via a suitable linker. The linker used is preferably a
protein acceptor, for example puromycin, which is covalently bound to the
nucleic acid part. The linker may comprise further components such as, for
example, a noncoding nucleic acid sequence, preferably a polyA
sequence. In a preferred embodiment, the nucleic acid part contains further
regions which are constant in its sequence and are located on one or both
sides of the coding region. These constant nucleic acid regions may serve,
for example, as primer binding sites for carrying out a PCR or as restriction
enzyme cleavage sites. The coding region of the nucleic acid part of the
fusion molecules contains a variable part which codes for at least two
amino acids, preferably for at least four, particularly preferably for seven
to
twelve, amino acids. To this, further constant coding nucleotide sequences
may be added. The constant nucleic acid sequences may encode peptide
sections which allow the peptidic part of the fusion molecule to be readily
attached to a solid surface or which generate interesting structural
elements. Examples of such structural elements are epitopes for
antibodies, tags for purifying or for detecting the constructs, or elements
which determine particular tertiary structures.
Such fusion molecules may be prepared, for example, according to
W098/31700 which describes a system in which a protein acceptor, for
example a puromycin, is bound to the nucleic acid, preferably RNA, via a
suitable linker. This makes it possible, shortly before in vitro translation
of
the RNA into the corresponding protein has finished, to bind the
synthesized protein covalently to its encoding RNA and thus to
characterize it in more detail. Examples of comparable systems which can
be used for the present invention are described in DE 1964637201,
W098/16636, US 5843701 and W094/13623.
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In a preferred embodiment of the method of the invention, the fusion
molecules are attached to a surface (support) via their peptidic part. The
term "support" means in accordance with the present invention material
which is present in solid or else gel-like form. Examples of suitable support
materials are ceramic, metal, in particular semiconductors, noble metal,
glasses, plastics, crystalline materials or thin layers of the support, in
particular of said materials, and (bio)molecular filaments such as cellulose
and structural proteins. However, suitable supports are not only flat
materials but also particles such as, for example, materials for column
chromatography, which can be loaded with proteins, or beads made of
organic polymers. The support is generally loaded covalently, quasi-
covalently, supramolecularly or physically.
The peptide part of the fusion molecules may be attached to the support,
for example, via biotin-streptavidin binding but specific domains of the
peptide part of the fusion molecules, such as, for example, metal-binding
domains (e.g. His tag), terminal cysteine residues or domains containing
epitopes which can be recognized by specific antibodies, can mediate such
an attachment.
In the method of the invention, preference is given to libraries whose fusion
molecules contain all possible permutations with respect to the variable
part of the peptide sequence. Practically still manageable libraries of such
fusion proteins are approx. 10'3 different sequences in size and thus are
103-104 times (e.g. phage display) or 106-10' times (classical, chemically
synthesized libraries) larger than other known peptide libraries. In order to
cover all permutations, preference is given to a variable peptide part of less
than 11 amino acids, and particular preference is given to variable peptide
parts composes of seven or eight amino acids. It is also possible, of
course, to construct libraries whose fusion molecules contain longer
variable peptide parts; in this case, however, the wide variety of sequences
can no longer be covered completely. Since the proteases known to date,
which cleave specific peptide bonds, require a recognition sequence of at
least four amino acids, preference is given to libraries containing a variable
peptide part of at least four amino acids.
The library of fusion molecules is exposed to a proteolytically active
sample, either in solution or, preferably, attached to a support. A suitable
proteolytically active sample, presample or comparison sample is
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especially tissue-specific extracts of animal, human or plant origin, extracts
of particular cell compartments, such as, for example, cytosolic extracts,
subcellular extracts, extracts of membrane components, extracellular
extracts, or mixtures of such extracts. Of particular interest are also
extracts of diseased tissue, for example of carcinomas. It is, however, also
possible to use extracts which represent the entire proteolytic activity or
parts thereof of an organism, in particular of viruses, microorganisms such
as bacteria or protists. Proteolytically active samples, presamples or
comparison samples which may be used are also solutions containing
known proteases.
The library is incubated with the proteolytically active sample preferably
under physiological conditions, preferably at temperatures between 0°C
and 45°C. For this purpose, the extracts to be tested for their
proteolytic
activity may be used directly or the extracts are taken up in a suitable
solvent such as, for example, physiological saline and used for incubation.
During incubation the proteases contained in the sample cleave the
variable peptide parts of the fusion molecules. This results in a specific
proteolytic cleavage pattern which is characteristic for the sample extract
used. The fusion molecule parts removed by cleavage are encoded by the
nucleic acid part.
After incubating the library with the proteolytically active sample to be
studied, the fusion molecule parts removed by proteolytic cleavage are
isolated and the sequence of the coding nucleic acid part is determined.
This identifies the peptide sequences cleavable by said sample.
After incubation of library and proteolytically active extract in solution,
the
cut fusion proteins can be isolated as follows. The nucleic acid regions of
the cut fusion proteins can be isolated from those of the uncut fusion
proteins, for example, by making use of a constant structural feature, for
example a known epitope for an antibody, in the particular peptide part
which, due to proteolytic cleavage, is no longer linked to the rest of the
fusion molecule including the nucleic acid part. For this purpose, the
mixture is subjected to an affinity chromatography by utilizing said
antibody, for example. The nucleic acid part of the cut fusion proteins
cannot bind, is separated from the nucleic acids of the uncut fusion
proteins and is thus present in the eluate. It is also possible to use other
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structural features such as, for example, a His tag or Strep tag for the
removal. The fusion molecules retained on the affinity matrix may be eluted
and used further in solution or, advantageously, directly in the attached
state.
After incubation of the attached library with extract, different methods may
be used to isolate the cut fusion proteins.
In a preferred embodiment of the method of the invention, after incubation
of the attached library with the extract, the nucleic acid part of the cut
fusion molecules are obtained directly in the eluate.
In the simplest case, the sequences of the cleaved peptides can be
identified directly by PCR amplification of the nucleic acid parts of the
cleaved fusion proteins, present in the eluate, followed by cloning and
sequencing. It is, however, also possible first to purify the cleaved fusion
proteins from other components present in the eluate by first another
affinity chromatography and making use of specific structures in the
peptide part or nucleic acid part of the cleaved fusion proteins. After
eluting
the cleaved fusion proteins from the chromatographic material, for example
by incubation with a KOH solution, the cleaved peptides may be
sequenced again by means of PCR, cloning anb sequencing.
In addition to this, a number of further chromatographic or electrophoretic
methods for isolating the fusion molecule parts removed by proteolytic
cleavage are possible. For this purpose, it is advantageous if the fusion
molecules are labeled or modified for easier identification. Thus, for
example, the nucleic acid part of the fusion molecules may be, for example,
fluorescently labeled or radiolabeled. Magnetic labeling of the nucleic acid
part of the fusion molecules is particularly preferred and may also be used
in connection with fluoresence labeling or radiolabeling. The fusion
molecule parts removed by cleavage may be magnetically isolated very
easily and selectively. The proteolytic pattern is evaluated, for example, via
hybridization of the isolated nucleic acid sequences on a biochip as is
available, for example, from Affymetrix or Nanogen. Thus it is possible to
analyze directly the isolated mixture of fusion molecule parts removed by
cleavage and containing different nucleic acid sequences.
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When preparing the extracts to be studied, attention must be paid to
isolating the proteases in their active form. At the same time, however,
DNA-degrading nucleases should be inactivated as far as possible.
For this purpose, the proteolytically active sample may be worked up
mechanically as follows: the complete tissue is purified, where appropriate,
by washing the outside in cold buffer (50 mM Tris-HCI, pH 7.5, 2 mM
EDTA, 150 mM NaCI, 0.5 mM DTT; Dignam, J.D., Preparation of Extracts
from Higher Eukaryotes. From: Deutscher, M.P. (ed.) Guide to Protein
Purification. Methods in Enzymology, Vol 182, Academic Press, (1990).
Page 194-202). The tissue is cut up in cold buffer and, where appropriate,
is removed from components such as, for example, connective tissue, skin
and blood vessels. The tissue pieces may be admixed with homogenization
buffer according to Kobayashi et al. (Kobayashi, H., Fujishiro, S., Terao, T.,
Cancer Res. (1994) 54, 6539-6548): 0.01 M HEPES buffer, pH 7.2, 0.25 M
sucrose, 0.5% Triton X-100 in a volume ratio from 1:2 to 1:5 (w/w) and
processed to a homogenate in an appropriate tissue homogenizes (see
below) with ice cooling. The method must be adapted to the desired tissue
type. Examples of suitable tissue homogenizers are:
- Mixer: liver, for example, can easily be mashed in a mixer and is
then further strained through a close-meshed nylon net. A fine
tissue homogenate is formed.
- "Potted' homogenizes: rotating glass rod which is inserted tightly
in a fitting vessel so that the tissue between the vessel wall and
the glass rod is crushed. Constant cooling must be ensured.
- Dispersers: homogenizers with rotating blades in a shaft.
The homogenized tissue is admixed with further homogenization buffer and
incubated with shaking at 4 - 8°C for up to 45 min. The tissue
homogenate
is then centrifuged at 4°C and 16,000 g and the supernatant is used for
analysis according to the method of the invention either directly or after
further purification, for example via column chromatography or dialysis.
If frozen material is used instead of fresh tissue, the method should be
modified. Frozen tissue should not just be left to thaw, since in this case
proteases released from lysosomes inactivate the enzymes. Preferably, the
tissue is pulverized with constant cooling in a mortar using liquid nitrogen
and the powder is freeze-dried in a lyophilizes overnight. The dry powder
can then be used further.
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An example of a suitable processing of brain tissue and separation thereof
into nuclear fraction, cytosolic fraction, membrane fraction and cytoskeletal
fraction is stated below. The extracts obtained may be used as
proteolytically active samples, presamples or comparison samples either
directly or with addition of further auxiliary substances.
Brains, for example from mice, of several preparations (approx. 50) are
introduced into approx. 25 ml of solution buffer 1 (SB1 ) (20 mM Tris-HCI
pH 7.5; 150 mM NaCI, 1 mM EDTA, 1 mM EGTA) and homogenized for 1
min using an Ultraturrax at maximum speed. Alternatively, the Potter glass
homogenizer is used in the case of less material.
A nuclear fraction may be isolated by centrifuging the homogenate at
1000 x g at 4°C for 10 min. The supernatant is collected for further
preparations. The centrifugate (nuclear pellet) is resuspended in 12 ml of
SB 1 and centrifuged as before. The nuclear pellet is resuspended in SB2
(20 mM Tris-HCI pH 7.5; 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1 % NP-
40).
The collected supernatants may be used for isolating and purifying a
cytosolic fraction. For this purpose, they are centrifuged at 100,000 x g at
4°C for 1 h. The supernatant represents the extract containing
cytosolic
proteins.
The membrane fraction is isolated by washing the pellet from removing the
cytosolic fraction three times in SB1 and centrifuging at 100,000 x g at
4°C
for 1 h. After the third washing step, the pellet is resuspended in 15 ml of
SB2 and solubilized overnight, followed by centrifugation at 100,000 x g at
4°C for 1 h. The supernatant forms the membrane fraction.
A cytoskeletal fraction is obtained by washing the pellet from removing the
membrane fraction three times in SB2 and centrifuging at 100,000 x g at
4°C for 1 h. After the third washing step, the pellet is resuspended in
SB3
(12.5 mM Tris-HCI pH 7.5; 0.4% SDS) and solubilized at 95°C. This is
followed by a final centrifugation at 20,800 x g at room temperature for 15
min. The supernatant contains the cytoskeletal fraction.
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Proteolytically active extracts containing secreted proteases are obtained
from cells and tissues by culturing, for example, human cells or cell lines in
cell culture as monolayers. The cells are washed three times with cell
culture medium. The medium is then replaced with fresh medium, with the
secretory proteases being secreted into the medium at 37°C. A suitable
secretion time is 1 to 3 h. Tissue pieces or tissue sections may be
processed in the same way.
Tissue fluid can be obtained from tissue assemblages by carefully cutting
up, for example, organs or tissue with scissors and scalpel. The tissue
pieces are washed several times with tissue buffer. The tissue pieces or
else small organs are incubated in physiological buffer or medium at
37°C
for 1-3 hours. After incubation, the tissue pieces are allowed to sediment in
a 15 ml conical vessel, and the medium is removed and can be studied
further.
Alternatively, the tissue may be transferred into centrifugation vessels and
centrifuged gently. The supernatant contains the proteases of the interstitial
fluid from the interstitial spaces.
Alternatively, it is also possible to obtain protein extracts according to the
following method: cutting up the tissue in ice-cold RIPA buffer (50 mM Tris-
HCI, pH 7.4; 1 % NP-40, 0.25% sodium deoxycholate, 150 mM NaCI, 1 mM
EGTA, 1 mM PMSF, 1 mM Na3V04, 1 mM NaF and in each case 1 ~glml
aprotonin, leupeptin and pepstatin), freezing the cut-up tissue and
subsequent crushing in a mortar. The tissue is homogenized by adding
RIPA buffer in a volume ratio of 1 to 2 and the mixture is then thawed.
After 15 min, the lysate is centrifuged at 13,000 x g and 4°C for
10 min.
The lysate may be divided into aliquots and may be shock-frozen and
stored in liquid nitrogen.
In a further modified method, the proteolytic activity of an unknown sample
may be determined in comparison with a comparison sample. The method
for differential determination of the proteolytic activity comprises the
following method steps:
a) incubating a library of fusion molecules comprising a peptide and a
nucleic acid encoding said peptide with a proteolytically active
sample,
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b) incubating the same library of fusion molecules in at least one
parallel mixture with at least one proteolytically active comparison
sample,
c) isolating the proteolyticaily removed parts of said fusion molecules,
d) constructing a differential nucleic acid bank,
e) determining the sequence of the nucleic acids determined.
Differential determination of the proteolytically removed nucleic acid parts
of the fusion molecules may be carried out, for example, by applying an
excess of single-stranded nucleic acid parts which have been isolated from
the comparison sample to a suitable support, saturating the free binding
sites of the support followed by hybridization with single-stranded nucleic
acid parts which have been isolated from the sample. The removable not
hybridized nucleic acid parts specifically represent only peptide sequences
cut in the sample.
In another method of the invention, the proteolytic activity of a sample is
determined in comparison with at least one presample. Successive
incubations of the fusion molecule library with presample and sample make
it possible to determine the differential proteolytic activity of said
presample
and sample. The method comprises the following method steps:
a) incubating a library of fusion molecules comprising a peptide and a
nucleic acid encoding said peptide with at least one proteolytically
active presample,
b) removing the fusion molecule parts removed by cleavage,
c) incubating the thus prepared library with a proteolytically active
sample,
d) isolating the fusion molecule parts removed by proteolytic cleavage,
e) determining the sequence of the nucleic acid part of the removed
fusion molecules.
In preferred embodiments of all three method variants of the invention, the
nucleic acid parts of the proteolytically cleaved and isolated fusion
molecule parts are multiplied by means of PCR. For this purpose, fusion
molecules are used whose nucleic acid part have on both sides of the
coding nucleic acid sequence constant nucleic acid sequences as primer
binding sites.
In order to increase greatly both the amount of fusion molecule cut in the
extract studied and the specificity of the selection step (cleavage in the
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proteolytically active extract), preference is given in the method of the
invention to amplifying the isolated nucleic acid of the cleaved fusion
proteins by means of PCR and transcribing it in vitro. Starting from the
RNA prepared in this way, a new library of fusion proteins is prepared
again by methods already described and is incubated with the same extract
containing proteolytic activities. This cycle can be repeated several times.
In a further variant of the method of the invention, it is possible to carry
out
PCR, in vitro transcription and/or in vitro translation in one or more of the
cycles described with a higher than normal error rate. This increases the
available peptide sequences to be tested (in vitro protein evolution).
Likewise, the variant of directed in vitro protein evolution makes it possible
to alter in a rapid and simple manner known cleavage sequences such that
different kinetic properties with respect to the cutting proteases are
produced.
Further additives such as, for example, specifically acting protease
inhibitors or nuclease inhibitors or unspecific RNA and/or DNA for
saturating possible nucleases may be added to the proteolytically active
sample. The addition of an inhibitor specifically acting against said
proteases, such as, for example, the addition of pepstatin A, is
recommended in particular, if the sample extracts used contain
unspecifically cutting lysosomal or endosomal proteases (acidic peptidases
such as aspartate peptidases). Specific inhibition of exoproteases may also
be advantageous in order to prevent degradation of the proteins contained
in the sample, comparison sample or presample, in particular of the other
proteases and peptidic cofactors thereof. The attached fusion molecules
themselves need not be protected against exoproteases, since no free N-
or C-terminal peptide ends are present. Degradation of the peptide moiety
of the proteolytically cleaved fusion molecules is unimportant for the further
course of the method.
The removal of nuclease activities from tissue extracts to protect the
nucleic acid parts of the fusion molecules may be achieved by treating
freshly prepared tissue homogenate with RNAse inhibitors or DNAse
inhibitors already during work-up of said tissue and subsequent steps. In
another embodiment, the nuclease inhibitors may be coupled covalently to
particles or suitable column materials. Nucleases binding to surface-
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coupled nuclease inhibitors are thus removed from the remaining tissue
homogenate. An alternative to nuclease inhibition is protection of the
nucleic acid moiety of fusion molecules by auxiliary substances. The
auxiliary substances enclose the DNA and RNA, form a complex and thus
protect said DNA and RNA against nucleases (Katayose, S. (1998) J.
Pharm. Sci., 87: 160-163). Said auxiliary substances are in particular
polyethyleneimine or PEG-PLL but may also be proteins or chaparones.
Protection against nucleases is likewise possible by using artificial nucleic
acids such as, for example, PNA in the fusion molecule. In order to
increase stability, the nucleic acids may also be chemically modified, for
example by methylation.
The incubation times of library and extract depend on the proteolytic
activity of the sample used. In an extended embodiment, the methods of
the invention are carried out repeatedly with varying incubation times. The
advantage of this is that it is possible to make kinetic statements regarding
the sample and the peptidic substrates thereof which include the library of
fusion molecules. Peptidic substrates which are proteolytically cleaved with
short incubation times have a high rate constant with respect to
proteolytically active sample components.
Another simple possibility to make kinetic statements about the proteolytic
activity of the sample is either to vary the concentration of the library
fusion
molecules which constitute the peptidic substrates or, preferably, to vary
the concentration of the sample intended for incubation.
A great advantage of the method of the invention is the fact that the
proteolytic activity of the sample studied can be described
phenomenotogically, i.e. without knowing all of the proteolyticaNy active
components of the sample. When comparing various samples, it does not
matter whether a different proteolytic activity is based on the presence of
various proteases or on direct mutation of a protease, which is rather rare,
or whether it is caused here by faulty regulation of a protease gene or a
protease inhibitor or protease activator. The present method can also
record alterations in proteolytic activity which are induced via extracellular
signals. It is known that, for example, binding of ligands such as hormones
to transmembrane receptors can alter cytosolic kinase and/or phosphatase
activities in a cell. The degree of phosphorylation plays a decisive part in
the regulation of enzyme activities in cells and thus has a decisive
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influence on protease activity. A deregulated intracellular phosphatase
activity and, coupled thereto, protease activity play a decisive part, for
example, in the formation of cancer.
Especially advantageous is the differential determination of specifically
cleavable peptides with respect to extracts of healthy and diseased tissue
and also of healthy tissue and tissue infected with pathogens. Finding
specifically cleavable peptides when comparing individual organisms is
likewise of particular interest.
Furthermore, the methods of the invention may be used to determine
rapidly and easily peptide sequences which are cut, in particular
specifically cut, by the sample studied. On the other hand, sequences
which are not subgect to proteolytic cleavage by the sample studied can
likewise be found.
Apart from identifying specifically proteolytically cleavable peptide
sequences, it is also possible to use the above-described methods for
finding specifically acting inhibitors or activators, without needing to know
for the individual case the proteases to be inhibited. For this purpose, a
potential inhibitor or activator is simply added to the sample solution and
the differential proteolytic activity between sample and samples containing
potential inhibitors or activators is determined. A reduction in the number of
cleaved sequences indicates an inhibitor and an increase in the number of
cleaved sequences indicates an activator.
In a preferred embodiment, inhibitors are identified by using a preselected
library of fusion molecules which have been obtained after several
selection cycles using the proteolytically active sample. Alternatively, it is
also possible to use the specifically cleavable peptides identified by the
methods of the invention directly for screening of inhibitors, for example in
a FRET assay.
Thus a method for screening of protease inhibitors is available, which can
be carried out rapidly and easily.
The rapid and simple screening of protease inhibitors makes it also
possible to combine many effective inhibitors in a mixture of active
substances, in order to prevent the appearance of resistances.
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The present invention further relates to proteolytically cleavable substances
which contain a peptide sequence obtainable using one of the methods
described.
The specifically proteolytically cleavable peptide sequences may be used
for synthesizing specifically acting inhibitors. For this purpose, for
example,
the peptide sequence is chemically modified or one or more a-amino acids
of the peptide are replaced with ~i-amino acids or L-amino acids are
replaced with D-amino acids in order to prevent proteolysis (see, for
example, Werder M. & Hauser H. (1999) Helvetica Chimica Acta, 82, 1774-
1783). The specifically proteolytically cleavable peptides which can be
identified using the present method may be used for constructing drug
delivery systems in order to achieve selective release of active substance
at the target site. The target site may be, for example, a particular organ,
tissue, a particular cell type, a subcellular compartment or may be
diseased, compared with healthy, tissue or microbially infected, compared
with uninfected, tissues or cells.
In the case of an active substance which can be activated by protease
cleavage, the specifically proteolytically cleavable substances may be used
as covalently bound inhibitors. The specifically proteolytically cleavable
peptides may also be used as linkers in order to link an active substance to
a targeting ligand such as, for example, a specific antibody or a specific
receptor ligand. However, it is also possible to construct active substance
loaded systems which are closed with specifically proteolytically cleavable
substances (Minko T. et al., (2000) Int. J. Cancer, 86: 108-117).
The variant of directed in vitro protein evolution makes it possible to alter
known peptidic cleavage sequences such that different cleavage kinetics
are produced. This makes it possible to control the time profile of active
substance release.
Consequently, it is possible, with the aid of the specifically proteolytically
cleavable peptides and via a target-controlled release of active substance,
to control more efficiently diseases in which, compared with the healthy
state, altered proteolytic activities are present at the desired site of
action
or which are caused by a faulty regulation or mutation of specific proteases
or activators or inhibitors thereof. Examples of diseases with disturbed
protease activity are asthma, osteoporosis, cancers such as leukemia,
breast cancer, bowel cancer, stroke, neuronal disorders such as
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Alzheimer's disease, arthritis, pancreatitis, hypertension, thromboses, colds
and schistosomiasis.
The targeted release of active substances can minimize the side effects of
said active substances and the amount of active substance to be used can
be reduced, owing to the altered distribution profile in the organism.
Especially interesting are pharmaceutical active ingredients, for example
against bacteria, fungi, viruses or against diseased tissue cells, but also
herbicides, fungicides or insecticides may be used.
Another application of the method of the invention is the use as diagnostic
assay. The assay may be based, for example, on a comparison of the
profile of the proteolytic activity of extracts toward a fusion molecule
library
with defined composition between diseased tissue, for example from
cancer cells, and a sample to be studied. However, preference is given to
assays which, compared to a comparison sample or presample are based
on peptides proteolytically cleavable exclusively by diseased tissue. In
such a selective assay, the sequencing step which is intended to follow
removal of the fusion molecule parts removed by cleavage can be
dispensed with. In this case, the presence of a particular disease-specific
protease activity may be identified using a hybridization probe. Such a
method may be highly integrated by using in such an assay many
cleavable peptides identified as disease-specific markers.
Alternatively, it is also possible to use the specifically cleavable peptides
identified by the methods of the invention directly for the screening of
inhibitors, for example in a FRET assay. The specifically cleavable
sequences may also be used in vivo as substrate for FRET assays.
The peptides specifically cleavable by a proteolytically active sample may
also be used for isolating the protease cleaving said peptide sequence
(see, for example, Li Y.M. (2000) Nature, 405: 689-694).
US 5843701, for example, describes a method applicable for isolating
serine proteases. Serine proteases are protein enzymes which catalyze
hydrolysis of peptide bonds within proteins. Frequently, owing to the
specific peptide linkage within the substrate, the target protein is
selectively
cut. The serine proteases include, inter alia, tissue plasminogen activator,
trypsin, elastase, chymotrypsin, thrombin and plasmin. Many disease
stages can be treated with serine protease inhibitors, such as, for example,
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blood clotting disorders. Elastase inhibitors can reduce the clinical
progression of emphysemas.
Proteases may be isolated by coupling the specifically proteolytically
cleavable peptides, determined according to the methods described above,
to column material, for example, by using standard methods and carrying
out an affinity chromatography of the proteolytically active samples on said
column material. The buffer used during the binding cycles must not be
denatured so as not to inactivate the protease. The proteases interacting
with the peptides can additionally be bound to the attached peptides via
common crosslinking methods, if required. For this purpose, it is also
possible to use ~i-amino acid-containing cleavage sequences which can be
specifically recognized but cannot be cleaved by proteases.
The invention is clarified by some exemplary embodiments stated below:
Example 1:
Preparation of fusion molecules comprising a peptide with a known
protease cleavage site and a nucleic acid encoding said peptide.
General remarks:
2 types of fusion molecules were prepared, firstly fusion molecules
containing a peptide sequence which is specifically cleaved by the
protease thrombin ("thrombin fusion molecules"), and secondly fusion
molecules containing a peptide sequence which is specifically cleaved by
matrix metalloprotease-3 (MMP-3) ("MMP-3 fusion molecules"). The
preparation of fusion molecules of this type is described, for example, in
W 098/31700.
The fusion molecules prepared in this way have protease cleavage sites for
MMP-3 or for thrombin.
The MMP-3 protease cuts between Glu and Leu, and thrombin protease
cuts between Arg and Ser. The peptide sequences used and the
corresponding nucleotide sequence are listed below.
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Protease cleavage sites:
MMP-3 cleavage site (Nagase, H., 1995, Methods Enzymol. 248,449-470)
Protein (AS): Arg-Pro-Lys-Pro-Val-Glu- ; Leu-Trp-Arg-Lys (Seq.
IDNo.1)
DNA (bp): AGA CCA AAA CCC GTT GAG CTC 'IGG AGA AAG (Seq.
ID No. 2)
Thrombin cleavage site (Le Bonniec, B.F., 1996, Biochemistry 35, 7114-
7122)
Protein (AS): Val-Pro-Arg- ; Ser-Phe-Arg (Seq. ID No. 3)
DNA (bp): GTT CCA AGA AGC TTC AGG (Seq. ID NO. 4)
1.1 Preparation of MMP-3 template DNA and thrombin template DNA
via PCR
250 ng of DNA template, 25 pmol of 5' and 3' primers, 10 mM dNTPs, 5 p.1
of 10 x PCR buffer and 5 U/wl Taq DNA polymerase were combined in a 50
lul reaction mixture (buffer and enzyme from Promega, Madison, USA). The
PCR was carried out in a Biometra T gradient cycler: 1 min 94°C, 21
x (0.5
min 94°C, 0.5 min 55°C, 0.5 min 72°C).
Primer and template:
5'Strep Tag primer (90 bp):
5'-taa tac gac tca cta tag gga caa tta cta ttt aca att aca atg tgg tcc cac ccc
cag ttc gag aag agt ggc tca agc tca gga tca-3' (Seq. ID No. 5)
3'MMP-3 primer (44 bp):
5'-ttt taa ata gcg gat get act agg cta gac cca gag cta ccc ga-3' (Seq. ID No.
6)
3'-thrombin primer (44 bp):
5'-ttt taa ata gcg gat get act agg cta gac cca gag gat cct ga-3' (Seq. ID No.
7)
MMP-3 template:
5'-agt ggc tca agc tca gga tca gga tct ggt aga cca aaa ccc gtt gag ctc tgg
aga aag cac cat cac cat cac cat gga agt ggc tcg ggt agc tct ggg tct agc-3'
(Seq. ID No. 8)
Thrombin template:
5'-agt ggc tca agc tca gga tca gga tct ggt gtt cca aga agc ttc agg cac cat
cac cat cac cat gga agt ggc tca gga tcc tct ggg tct agc-3' (Seq. ID No. 9)
CA 02420065 2003-02-19
. 17 .
Fig. 1 shows the PCR products on a 2% agarose ethidium bromide gel.
Lane 1: 50 by DNA marker, Lane 2: MMP-3 DNA, Lane 3: thrombin DNA,
Lane 4: 100 by DNA marker.
As Fig. 1 shows, MMP-3 DNA and thrombin DNA were successfully
amplified. The PCR products have the expected molecular weight of 199
by for MMP-3 DNA and 189 by for thrombin DNA.
1.2 In vitro transcription of MMP-3 and thrombin DNA
100 pmol of DNA were used in the transcription reaction (Ribomax
T7/Promega, Madison, USA). The DNA was incubated with 5 x T7 buffer,
rNTPs and T7 RNA pofymerase in a 500 w1 mixture at 37°C for 4 hours.
RNA was purified by means of phenol/chloroform extraction and analyzed
on a 6% urea gel (Novex GeU Invetrogen, Groningen, Netherlands).
Fig. 2 shows the RNA obtained after transcription on a 6% urea gel.
Lane 1: MMP-3 RNA, Lane 2: thrombin RNA
As Fig. 2 shows, RNA can be detected, as expected, after the transcription
reaction. The RNA is not degraded.
1.3 Ligation of RNA with puromycin linker via UV crossiinking
3 nmol of RNA, 4.5 nmol of puromycin (Pu) linker (PEG = polyethylene
glycol, Pso = psoralen) having the sequence
5'-(Pso)2'OMe-U AGC GGA UGC AAA AAA AAA AAA AAA AAA PEG
PEG CC-(Pu)-3' (Seq. ID No. 10 represents nucleotides 1 to 28)
(lntreractiva/Ulm), 12 w( of 10x ligase buffer (1 M NaCI, 250 mM Tris pH
7.5) were combined in a 120 ~I mixture and incubated at 85°C for 5
minutes and then at room temperature for 10 minutes. UV crosslinking was
carried out at 366 nm for 15 minutes (handheld UV tamp by LTF-
Labortechnik, 12 W).
Ligation efficiency was checked on a 5% urea-agarose gel.
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Fig. 3 shows analysis of the ligation reaction on a 5% urea-TBE gel.
Lane 1: MMP-3 RNA, Lane 2: MMP-3 RNA with puromycin linker, Lane 3:
thrombin RNA, Lane 4: thrombin RNA with puromycin linker. (The signal L
is caused by the color marker).
After ligation of the puromycin linker to the RNA, a distinct increase in the
molecular weight is observed, indicating that thrombin RNA and MMP-3
RNA has been linked successfully to puromycin.
1.4 In vitro translation
8 ~I of linker-RNA were mixed with 200 ~I of reticulocyte lysate (Promega,
Madison, USA L416X), 5 ~I of ~S-methionin (Hartmann, 10.8 p.M, specific
activity: the specific activity is 72181 dpm/pmol)), 6 p1 of amino acid mix
(without methionin, 1 mM, Promega) and 80 ~I of H20 were mixed and then
incubated at 30°C for 30 minutes. Addition of 130 ~I of 2 M KCI and 75
~,I of
MgCl2 was followed by another incubation at 30°C for 30 minutes.
The fusion molecules obtained were purified using oligo dT cellulose
(Amersham Pharmacia, Freiburg, Germany) (removal of all those
components of the in vitro translation mixture which contain no polyA RNA
sequences).
1.5 Reverse transcriptase reaction
The purified 200 ~.I fusion molecules were mixed with 2.5 p1 of 100 ~,M
reverse primer and incubated at 80°C for 5 minutes. After cooling the
reaction mixture on ice, 50 p,1 of 5x strand buffer, 20 p1 of dNTPs (in each
case 10 mM), 2.5 p.1 of RT-Superscript II (all from Promega, Madison, USA)
were added and the mixture was incubated at 42°C for 40 minutes.
Example 2:
Proteolytic cleavage of the fusion molecules by thrombin and MMP-3 in
solution
The fusion molecules prepared were incubated with the corresponding
proteases and the reaction products were analyzed (Fig. 4). 10 nM MMP-3
(Sigma, Deisenhofen, Germany) were added to 5 pmol of fusion molecule
containing the MMP-3 cleavage site and the mixture was incubated in
MMP-3 buffer (50 mM Tris pH 7, 150 mM NaCI, 10 mM CaCl2) in a total
CA 02420065 2003-02-19
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volume of 15 ~I at 37°C for 45 minutes. 1 U of thrombin (1 NIH unit =
0.324 pg, Sigma, Deisenhofen, Germany) was added to 5 pmol of fusion
molecule containing the thrombin cleavage site and incubated in thrombin
buffer (50 mM Tris pH 8, 150 mM NaCI) in a total volume of 15 p1 at
37°C
for 45 minutes. The starting fusion molecules and the fusion molecules
after proteolytic cleavage were analy2ed using a phosphoimager, after
SDS PAGE (Fig. 4)
Fig. 4 shows the fusion molecule digest with thrombin and MMP-3 in
solution:
Phosphoimager image after 4-20°l° Tris-glycine SDS PAGE.
Lane 1: "MMP-3 fusion molecules" prior to digest;
Lane 2: "thrombin fusion molecules" prior to digest;
Lane 3: "thrombin fusion molecules" + thrombin
Lane 4: "thrombin fusion molecules" prior to digest
Lane 5: "MMP-3 fusion molecules" + MMP-3
Lane 6: "MMP-3 fusion molecules" prior to digest.
(The bands i show fusion molecules, the bands ii show peptidic by
products and the bands iii are caused by N-terminal fusion molecule
fragments.)
The "MMP-3 and thrombin fusion moiecutes» were proteolytically cleaved
by MMP-3 protease and thrombin protease, respectively.
Example 3
Isolation and detection of the proteolytically cleaved "thrombin fusion
molecules" and amplification of the nucleic acid moiety
Specifically proteolytically cleaved fusion molecules were isolated and
identified by carrying out three successive method steps. Fig. 5 shows
diagrammatically the method steps according to this example for identifying
specifically cleavable peptides:
Step 1. Coupling of the fusion molecules to Streptactin-Sepharose
(support material) via an N-terminal Strep tag
Step 2. Proteolytic cleavage of the "thrombin fusion molecules" on the
support material by thrombin.
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Step 3. Isolation of the cleaved C-terminal fusion molecule fragments
and detection of said fragments after PCR amplification of the
nucleic acid moiety.
Optionally, the isolated fragments may be purified via their His tag by
means of affinity chromatography to nickel particles prior to analysis.
3.1 Coupling of "thrombin fusion molecules" to Streptactin Sepharose
50 ~I of "thrombin fusion molecules" (corresponding to approx. 5 pmol)
were [lacuna] with 50 ~.I of Streptactin Sepharose, washed 5x in Streptactin
buffer (150 mM NaCI, 100 mM Tris pH 8) and then incubated at 4°C for 2
hours. Unbound fusion molecules were removed by washing five times with
Streptactin buffer.
3.2 Proteolytic digest of the "thrombin fusion molecules" by thrombin
The fusion molecules bound to Streptactin Sepharose were resuspended in
80 ~I of water and incubated with 10 p1 of 10x thrombin buffer (1.5 M NaCI,
500 mM Tris pH 8) and 10 ~.I of 100 U/~,I thrombin at 37°C for 45
minutes.
As a control, the same reaction was carried out without thrombin.
3.2.1 Isolation and detection of the bound "thrombin fusion moleculesn
After the incubation period, the reaction mixture was centrifuged (1 minute
at 3000 rpm). The pelleted Streptactin Sepharose containing the N-terminal
fragment of the cleaved fusion molecules was washed three times with
Streptactin buffer and then resuspended in 80 ~I of water.
The "thrombin fusion molecules" bound to Streptactin Sepharose (control
mixture) or the N-terminal fragments bound after incubation with thrombin
were fractionated by SDS PAGE and visualized by means of
phosphoimaging (Fig. 6).
Fig. 6 shows "thrombin fusion molecules" and fusion molecule fragments
bound to Streptactin Sepharose. 15 ~I of resuspended Streptactin
Sepharose were mixed in each case with 5 ~.I of Lammli loading buffer,
heated at 82°C for 5 min and fractionated by means of 4-20% Tris
glycine
SDS PAGE. The gel was then dried at 80°C for 2 hours and
visualized by
means of phosphoimaging.
CA 02420065 2003-02-19
-21 -
Lane 1: "thrombin fusion molecules" after incubation with thrombin. Lane 2:
"thrombin fusion molecules" without incubation with thrombin (control).
(Band i shows the infact fusion molecule, bands ii are from peptidic by-
products and band iii is caused by the N-terminal fusion molecule
fragment.).
After incubating the "thrombin fusion molecules" with thrombin, only the N-
terminal fragments (labeled with 35S-methionin) remain bound to
Streptactin Sepharose.
3.2.2 Detection of the nucleic acid moieties of the "thrombin fusion
moleculesn by means of PCR analysis
After removing Streptactin Sepharose, 15 ~I of the supernatant or 15 ~,I of
resuspended Streptactin Sepharose were used for the PCR. Apart from the
15 ~I samples, the 50 ~,l PCR mixtures also contained 5 ~I of 5' and 3'
thrombin primers (5 pmoU~.l), 10 mM dNTPs, 5 ~I of 10 x PCR buffer and
5 U/~I Taq DNA polymerase (buffer and enzyme from Promega, Madison,
USA). The amplification was carried out under the following conditions:
1 min 94°C, 21 x (0.5 min 94°C, 0.5 min 55°C, 0.5 min
72°C). The PCR
products obtained are depicted in Fig. 7.
Fig. 7 shows the PCR products of the "thrombin fusion molecules" after 2%
agarose gel electrophoresis. Lanes 1 and 2 show the products as black
signals on a light background and lanes 3 and 4, owing to a different
imaging technique, show the products as white signals on a dark
background.
Lane 1: °thrombin fusion molecules" + thrombin; supernatant.
Lane 2: "thrombin fusion molecules" without thrombin (control);
supernatant.
Lane 3: "thrombin fusion molecules" + thrombin; bound to Streptactin
Sepharose.
Lane 4: "thrombin fusion molecules" without thrombin; bound to Streptactin
Sepharose.
Consequently, it is shown that "thrombin fusion molecules" are cut by
incubation with thrombin. After removing the N-terminal fragment which
contains no nucleic acid moiety, the C-terminal moiety remains in the
supernatant. Consequently, a strong PCR amplicon is obtained in the
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supernatant (see, for example, lane 1, Fig. 7) but in the pellet only a very
weak PCR amplicon (e.g. lane 3, Fig. 7) is obtained. In the control reaction,
the "thrombin fusion molecules" were not incubated with thrombin.
Consequently, the fusion molecules remain bound to Streptactin
Sepharose and a strong PCR amplicon is obtained in the pellet (e.g. lane
4, Fig. 7), while the PCR amplicon of the supernatant (e.g. lane 2, Fig. 7) is
only very weak.
r CA 02420065 2003-02-19
SEQUENCE LISTING
<110> Xzillion GmbH & Co KG
<120> Methods for identifying specifically cleavable peptides and
use of such peptide sequences
<130> 200at19.wo
<140>
<141>
<150> 10041238.6
<151> 2000-08-22
<160> 10
<170> Patentln Ver. 2.1
<210> 1
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Description of artificial sequence: MMP-3 cleavage site
<400> 1
Arg Pro Lys Pro Val Glu Leu Trp Arg Lys
1 S 10
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: MMP-3 cleavage site-
encoding DNA
<400> 2
agaccaaaac ccgttgaqct ctggagaaag 30
<210> 3
<211> 6
<212> PRT
<213> Artificial sequence
CA 02420065 2003-02-19
2
<220>
<223> Description of artificial sequence: thrombin cleavage site
<400> 3
Val Pro Arg Ser Phe Arg
1 5
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence
<220> '
<223> Description of artificial sequence: thrombin cleavage site-
encoding DNA
<400> 4
gttccaagaa gcttcagg
1R
<210> 5
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 5' Strep tag primer
<400> 5
taatacgact cactataggg acaattacta tttacaatta caatgtggtc ccacccccag 60
ttcgagaaga gtggctcaag ctcaggatca g0
<210> 6
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 3' 1~IP-3 primer
<400> 6
ttttaaatag cggatgctac taggctagac ccagagctac ccga qq
<210> 7
<211> 44
<212> DNA
<213> Artificial sequence
CA 02420065 2003-02-19
3
<220>
<223> Description of artificial sequence: 3' thrombin primer
<400> 7
ttttaaatag cggatgctac taggctagac ccagaggatc ctga 44
<210> 8
<211> 108
<212> DNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: 1~IP-3 template
<400> 8
agtggctcaa gctcaggatc aggatctggt agaccaaaac ccgttgagct ctggagaaag 60
caccatcacc atcaccatgg aagtggctcq qgtagctctg ggtctagc 108
<210> 9
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: thrombin template
<400> 9
agtggctcaa gctcaggatc aggatctggt gttccaagaa gcttcaggca ccatcaccat 60
caccatggaa gtggctcagg atcctctggg tctagc 96
<210> 10
<211> 28
<212> RNA
<213> Artificial sequence
<220>
<223> Description of artificial sequence: puromycin-linker-RNA
part
<400> 10
uagcggaugc aaaaaaaaaa aaaaaaaa 2g
