JP2003508080A - Compositions for detecting proteases in biological samples and methods of using the same - Google Patents

Abstract

  (57) [Summary] The present invention provides novel reagents whose fluorescence is increased in the presence of a particular protease. The reagent comprises a characteristically folded peptide backbone, wherein each end of said backbone is conjugated to a fluorophore. When the folded peptide is cleaved by digestion with a protease, the fluorophore provides a high intensity fluorescent signal at visible wavelengths. These protease indicators are particularly suitable for the detection of protease activity in biological samples, especially frozen tissue fragments. Because of their high fluorescent signal at visible wavelengths. The present invention also provides a method for detecting protease activity in a field frozen fragment.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a new class of fluorescence generating compositions that enhance fluorescence levels in the presence of active proteases. Those fluorogenic protease indicators typically fluoresce in the visible wavelengths and, therefore, are very useful for the detection and localization of protease activity in biological samples.

BACKGROUND OF THE INVENTION Proteases refer to many types of proteolytic enzymes that catalytically hydrolyze peptide bonds. The main groups of proteases are metalloproteases,
Includes serine proteases, cysteine proteases and aspartic proteases. Proteases, especially serine proteases, are responsible for many physiological processes,
For example, it is associated with blood coagulation, fertilization, inflammation, hormone production, immune response and fibrinolysis.

Many pathologies are caused by and can be characterized by alterations in the activity of certain proteases and their inhibitors. For example, emphysema,
Arthritis, thrombosis, cancer metastasis and some forms of hemophilia result from defective regulation of serine protease activity (eg Textbook of Biochemistry with Clinical
Correlations, John Wiley and Sons, Inc. NY (1993)). In the case of viral infection, the presence of viral protease in the infected cells has been identified. Such viral proteases include, for example, the HIV protease associated with AIDS and the NS3 protease associated with hepatitis C. These viral proteases play an important role in the viral life cycle.

Proteases have been thought to be associated with cancer metastasis. Enhanced synthesis of the protease urokinase has been associated with increased metastatic potential in many cancers.
Urokinase activates plasmin from plasminogen, which is ubiquitous in the extracellular space, and its activation can cause the degradation of proteins in the extracellular matrix invading metastatic tumor cells. Plasmin can also activate collagenase, thus promoting the degradation of collagen in the basement membrane surrounding the capillaries and lymphatic system, thereby allowing the entry of tumor cells into the target tissue (Dano, Adv. Cancer. Res., 44: 139 (1985)).

The unambiguous measurement of altered activity of a particular protease is of clinical significance in the treatment and management of the underlying medical condition. However, proteases cannot be easily assayed. Typical approaches include ELISA with antibodies that bind to proteases, or RIA with various labeled substrates. Assays with their natural substrates are difficult and expensive to perform.
Currently available synthetic substrate assays are expensive, insensitive, and non-selective. In addition, many "indicator" substrates require large amounts of protease, which leads in part to the self-destruction of the protease.

Recent approaches to protease detection have involved cleavage-induced spectroscopic changes in a distant chromogen or fluorophore located at the P1 'position (the amino acid position on the carboxyl side of the cleavable peptide bond). Depends (eg, US Pat. No. 4,557,8
62 and 4,648,893). However, many proteases require 2 or 3 amino acid residues on either side of the scissile bond for recognition of the protease (because a particular protease requires no more than 6 amino acid residues). , And therefore their approach lacks protease specificity.

However, recently, a fluorogenic indicator composition has been developed in which the “donor” fluorophore is a peptide () which is the binding site for the HIV protease.
(7 amino acids) and its peptide are linked to the "receptor" chromophore by a short bridge containing a linker that connects the fluorophore and the chromophore (Wang et al., Tetra. Len.
S. 45: 6493 to 6496 (1990)). The donor fluorophore signal is quenched by the receptor fluorophore through a process that appears to involve resonance energy transfer (RET). Cleavage of the peptide resulted in separation of the fluorophore and chromophore, ie elimination of the quench, and subsequent signal was measured from the donor fluorophore.

The design of the bridge between the donor and the receptor has led to relatively ineffective quenching, which limits the sensitivity of the assay. Furthermore, the chromophore and / or fluorophore absorbed light in the UV range, which reduces sensitivity for detection in biological samples that typically contain molecules that strongly absorb light in the UV range. It shows high signal levels when cleaved, and very close signal levels when not compromised, exhibits a high degree of protease specificity, and operates exclusively in the visible range, thereby rendering them biological samples. A fluorogenic protease indicator suitable for use in is desired. The composition of the present invention is
Provides those and other benefits.

SUMMARY OF THE INVENTION The present invention provides novel reagents that enhance the fluorescence of certain proteases in the presence thereof. Their fluorescent protease indicators provide a high intensity fluorescent signal at visible wavelengths when they are digested by proteases. Due to their high fluorescent signal at visible wavelengths, their protease indicators are particularly suitable for the detection of protease activity in biological samples, especially frozen tissue fragments and cultured or newly isolated cells. Appropriate.
Measurements can be performed using a fluorescence microscope for tissue samples, cells, etc. and a flow cytometer or microscope for cell suspension samples.

The fluorogenic protease indicator of the present invention is a composition suitable for detecting the activity of a protease. Those compositions have the following general formula:

[Chemical 2]

[Wherein P is 2 to about 15, preferably 2 to about 12, preferably 2 to about 10, preferably 2 to about 8, 2 to about 6, or A peptide comprising a protease binding site for a protease, consisting of 2 to about 4 amino acids;
F ¹ and F ² are fluorophores; S ¹ and S ² are peptide spacers ranging in length from 1 to about 50 amino acids; i and r are independently 0 or 1; and C ¹
And C ² is a conformational determining region comprising a peptide ranging in length from 1 to about 8, more preferably 1 to about 6 amino acids].

[0012] Each conformation-determining region introduces bends into the composition, or limits the degree of freedom of the peptide backbone, thereby creating a fluorophore with a separation of less than about 100 Å. Juxtapose. If any of the spacers (S ¹ and S ² ) are present, they are linked to the protease binding site by a peptide attached to the α carbon atom of the terminal amino acid. Therefore, when n is 1, S ^1, through terminal α-amino group of C ^1, is connected to the C ¹ by a peptide bond, and when k is 1, S ² peptide through terminal α carboxyl group of C ² It is linked to C ² by a bond.

Amino acid residues comprising the protease binding site are conventionally numbered relative to the peptide bond that is hydrolyzed by the particular protease. Thus, the first amino acid residue on the amino side of the cleaved peptide bond is designated P ₁ and the first amino acid residue on the carboxyl side of the cleaved peptide bond is designated P ₁ '. . The numbering of residues increases as the distance from the hydrolyzed peptide bond increases. Thus, the amino acid protease binding region 4 comprises an amino acid which was named _{P 2 -P 1 -P 1 '-P} 2', and the protease cleaves the bond region between P ₁ and P ₁ '.

In a particularly preferred embodiment, the fluorescent light source composition of the present invention is a composition of formula (II) and formula (V) as described herein. Preferred fluorophore indicators of the present invention have a determinant region, and optionally a spacer as described herein. In the most preferred embodiment, the composition has a single type of fluorophore. Preferred fluorophores for these "homolabeled" compositions include H
Includes a fluorophore that forms a type dimer. Particularly preferred fluorophores are about 300 nm and 800
nm, more preferably between about 310 nm and 750 nm, most preferably about 315 nm and about
It has an excitation wavelength between 700 nm.

In another aspect, the invention provides a method of detecting the activity of a protease. The method comprises contacting a protease with one or more protease indicators described herein. In a particularly preferred embodiment, "
"Contact" in tissue sections, or in tissue, blood, urine, saliva or other biological fluids,
In a material, culture, or cell suspension selected from the group consisting of biological samples including, but not limited to, lymph, biopsy. Detection methods include methods selected from the group consisting of fluorescence microscopy, fluorescence microplate reader, flow cytometry, fluorometry, absorption spectroscopy.

In a preferred composition, F ¹ can be 5- and / or 6-carboxytetramethylrhodamine and F ² can be rhodamine X acetamide. These compositions may be attached to a solid support or liquid such as membranes or liposomes.

In another aspect, any of the above compositions can be used in a method for detecting protease activity in a sample. The sample can be, for example, a sample of "stock" protease used in research or industry, or it can be a biological sample. Accordingly, the present invention provides for the protease in a sample to be contacted with a sample and any of the above compositions, and then detecting the change in fluorescence of the fluorogenic composition in which an increase in fluorescence is indicative of protease activity. Methods for detecting activity are provided. Said sample is preferably a biological sample including biological fluids such as saliva or blood, tissue samples such as biopsies or fragments, and cell samples as biopsies or in culture.
Particularly preferred are tissue fragments, cultured cells, cultured tissue and the like.

In yet another aspect, the invention provides a method for binding a molecule to a cell. The method comprises providing a molecule having at least two fluorophore molecules and a hydrophobic group attached thereto; and contacting the cell with the molecule, thereby introducing the molecule into the cell. In one embodiment, the method comprises providing a molecule having at least two large flat hydrophobic fluorophores and a hydrophobic group attached thereto. Preferred molecules include polypeptides, nucleic acids, lipids, oligosaccharides. Suitable fluorophores and hydrophobic groups are described herein. Preferred cells include mammalian cells.

In yet another aspect of the present invention, the present invention provides a protease (or nuclease,
Methods for screening test substances for their ability to modulate lipases) are provided. This method involves contacting a protease or cells containing the protease with a test substance, contacting the protease with a fluorescent source indicator as described herein, and detecting the signal or non-existence of the signal produced by the fluorescent source composition. Detecting the presence, wherein the signal produced by the protease or cell contacted with the test substance as compared to a control (eg, a negative control) in which the protease or cell is contacted with a low concentration of said test substance. Indicates that the test substance regulates the activity of protease. In a preferred embodiment, the control is the absence of test substance.

[0020] Typically, an increase in the signal generated by the protease or cells contacted with the test substance as compared to the control indicates an increase in the activity of said protease by the test compound, and A decrease in signal (eg, fluorescence) produced by the contacted protease or cells indicates a decrease in protease activity by the test substance. In certain embodiments, the protease is contacted with the fluorogenic composition after removal of the test substance. This method makes it possible to put a test substance which regulates the activity of the protease into a database which comprises a list of test substances which regulate the protease. In various embodiments, detecting comprises detecting intracellular signals (eg, by microscopy, flow cytometry, etc.). In certain particularly preferred embodiments, the detection involves high throughput of whole cells (
high throughput).

Definitions The term "protease binding site" means an amino acid sequence which is specifically recognized and cleaved by a protease. The protease binding site comprises a peptide bond that is hydrolyzed by a protease, and the amino acid residue linked by the peptide bond is said to form its cleavage site. The amino acids represent P ₁ and P ₁ 'for residues on the amino and carboxyl sides of the hydrolyzed bond, respectively.

Fluorophores are molecules that absorb light at a characteristic wavelength and then most typically, re-emit light at a different characteristic wavelength. Fluorophores are well known to those of skill in the art and include, but are not limited to, chelating agents with rhodamine and rhodamine derivatives, fluorescein and fluorescein derivatives, coumarin, and lanthanide ion series. Fluorophores are distinct from chromophores that absorb light but do not re-emit light.

“Peptides” and “polypeptides” have the α carbon atoms linked through a peptide bond formed by the condensation reaction between the α carbon carbonyl group of one amino acid and the amino group of another amino acid. A chain of amino acids. The terminal amino acid at one end of the chain (the amino terminus) has a free amino group, and the terminal amino acid at the other end of the chain (amino
The carboxyl end) has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated as N-terminus) refers to a free α-amino group on an amino acid at the end of a peptide, or at any other position within the peptide. The α-amino group of an amino acid (imino group when involved in peptide bond) is meant. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other position within a peptide. Peptides can also be essentially any polyamino acid, such as peptidomimetics,
For example, but not limited to, amino acids linked by an ether bond to an amide bond.

The polypeptides described herein are written with an amino terminus on the left and a carboxyl terminus on the right. Amino acids comprising the peptide component of the invention are numbered relative to the protease cleavage site, and the number increases sequentially with distance in the carboxyl and amino directions from the cleavage site. Residues on the carboxyl site are designated by a "^'" as in P ₁ ' or by a letter and subscript indicating the region where they are located. The "^'" indicates that the residue is located on the carboxyl side of the cleavage site.

The term “residue” or “amino acid” as used herein means an amino acid incorporated into a peptide. Amino acids are naturally occurring amino acids,
And, unless otherwise stated, it can include known analogs of naturally occurring amino acids that can function in a manner similar to the naturally occurring amino acids. The term "domain" or "region" means a characteristic region of a polypeptide. Domains are defined by specific structural features, such as β-turns, α-helices, or β-pleated sheets, by characteristic constituent amino acids (eg, preferential hydrophobic or hydrophilic amino acids, or repetitive amino acid sequences), or folded conformations. It can be characterized by its localization in a particular region of a polypeptide. A region or domain, as used herein, consists of a series of contiguous amino acids.

The term “protease activity” or “protease activity” means the cleavage of peptides by proteases. Protease activity involves the "digestion" of one or more peptides into many small peptide fragments. The protease activity of a particular protease can result in hydrolysis at a particular peptide binding site that is specifically recognized by the particular protease. The particular protease may be characterized by the production of peptide fragments bearing particular terminal amino acid residues.

The term “test substance” means a substance to be screened in one or more of the assays described herein. The substance can be essentially any chemical compound. They can exist as a single isolated compound,
Alternatively, it can be a member of a chemical (eg, combinatorial) library.
In certain preferred embodiments, the test substance will be a small organic molecule. The term "small organic molecule" means a molecule of comparable size to the organic molecules commonly used in medicine. The term does not include biological macromolecules (eg, proteins, nucleic acids, etc.). Preferred small organic molecules are up to about 5000 Da, more preferably 20
It ranges up to 00 Da, and most preferably up to about 1000 Da.

The term “database” means a means for recording or reproducing information.
The database can include any convenient medium, including, but not limited to, paper systems, card systems, mechanical systems, electrical systems, optical systems, magnetic systems. Preferred databases include electronic (eg, computer-based). Computer systems for use in storing and manipulating databases are well known to those skilled in the art, but include "personal computer systems", mainframe systems, one or more Internet or intranets stored on special hardware. Including but not limited to distributed glands.

The term “biological sample”, as used herein, means a sample obtained from a component of an organism (eg, a cell or tissue) and / or from an in vitro cell or tissue culture. The sample is a sample of any biological tissue or fluid (eg blood,
Plasma, lymph, spinal fluid, urine, saliva, etc.) samples. Biological samples also include sections of organs or tissues, such as frozen sections taken for histological purposes. Some amino acids referred to herein are described by the following abbreviations:

[0030]

[Table 1]

Other abbreviations used herein include “Fm” for Fmoc (9-fluorenylmethoxycarbonyl) group, “Ac” for N (α) -acetyl group, “daa” (“
d "represents the d isomer of aa), and" Z "representing a benzoxycarbonyl group.

SPECIFIC DESCRIPTION Fluorescent Indicator of Protease Activity The present invention provides novel fluorescent molecules useful for detecting protease activity in a sample. The fluorogenic protease indicators of the present invention generally include
It includes a fluorophore (donor) linked to a "receptor" molecule by a peptide having an amino acid sequence that is recognized and cleaved by a particular protease. Donor fluorophores are typically excited by incident radiation at a particular wavelength that re-emits at a different (longer) wavelength. When the donor fluorophore is maintained in close proximity to the receptor molecule, the receptor absorbs light re-emitted by the fluorophore, thereby
Quench the fluorescent signal of the donor molecule, or the hypothetical donor and acceptor form a complex that absorbs the incident light and does not emit irradiation energy until the complex is destroyed.

In this latter embodiment, quenching occurs even if the two fluorophores are different or the same. Thus, in addition to the double-labeled peptide with two different fluorophores as shown in Example 1, a double-labeled peptide with the same fluorophore can also be used as a protease indicator ( See, eg, Example 6). Cleavage of the backbone (eg peptide) linking the two fluorophores or chromophores results in separation of the two molecules, release of the quenching effect and increased fluorescence or altered spectral properties.

In one basic application, the fluorogenic molecules of the invention are used to assay the activity of purified proteases produced as reagents (eg, in buffer solution) for experimental or industrial use. Can be used. Proteases, like many other enzymes, lose activity over time, especially when they are stored in their active form. In addition, many proteases naturally exist in inactive precursor forms (eg zymogens) which, prior to use, must themselves be activated by hydrolysis of a particular peptide bond to produce the active form of the enzyme. It is recognized that proteases are active, as the degree of activation is variable, and the proteases lose activity over time, and often in certain applications:
Before using a particular protease, it is often desirable to quantify its activity.

Previous approaches to recognizing and quantifying protease activity have involved mixing an aliquot of protease with its substrate and digesting for a period of time.
And then involves measuring the amount of the digested protein, at most typically by HPLC. This approach is time consuming, uses expensive reagents, requires many steps, and is labor intensive. In contrast, the fluorogenic reagents of the present invention allow the rapid determination of protease activity in minutes in a single step process. An aliquot of the protease to be tested is simply added to, or contacted with, the fluorogenic reagent of the invention, and subsequent changes in fluorescence are monitored (e.g., a fluorometer or a fluoromicrometer). Using a plate reader).

In addition to determining protease activity in “reagent” solutions, the fluorescent composition of the present invention can also be used to detect protease activity in biological samples. The term “biological sample”, as used herein, means a sample obtained from an organism or from a component (eg, cell) of an organism. The sample can be of any biological tissue or fluid. Sometimes the sample may be a "clinical sample" which is a sample derived from a patient. Such samples include, but are not limited to, saliva, blood, blood cells (eg, white blood cells), tissue or fine needle biopsy samples, urine, ascites, and pleural effusion, or cells therefrom. Not done. Biological samples also include pieces of tissue, such as frozen pieces taken for histological purposes.

The fluorescent protease indicators that have been described so far are typically
Absorbs light in the ultraviolet range (eg Wang, et al., Supra). Therefore, they are unsuitable for the sensitive detection of protease activity in biological samples that typically contain components that absorb in the UV range (eg proteins). In contrast, in a preferred embodiment, the fluorescent indicator of the present invention comprises
It absorbs and emits in the visible range (400 nm to about 750 nm). Therefore, their signals are not readily quenched by the activation of the fluorophore, ie absorption of light, or interfered with by background molecules; thus, they are easily detected in biological samples.

Furthermore, in contrast to previous fluorogenic protease indicators, which often use a fluorophore and a quenching chromophore, the indicator of the present invention comprises two fluorophores (
That is, the same two fluorophores that effectively form a ground-state dimer when linked by one of the fluorophores as donors and receptors), fluorophores and chromophores, or the peptide backbone of the invention. Can be used. Fluorophore pairs may be selected that exhibit a higher degree of quenching than the chromophore / fluorophore combinations described thus far.

In fact, previous compositions have been limited to relatively low potency fluorophores (Wang et al., Supra) due to the low degree of quenching provided by paired chromophores. In contrast, the fluorogenic protease indicators of the present invention use a high potency fluorophore and achieve a high degree of quenching while providing a strong signal when quenching is released by cleavage of the peptide substrate. be able to. The high signal allows the detection of very low levels of protease activity. Therefore, the fluorogenic protease indicators of the present invention are particularly suitable for in situ detection of protease activity.

[0040]   Preferred fluorescent protease indicators of the present invention have the following general formula:

[Chemical 3]

Where P is a peptide containing a protease binding site, F ¹ and F ² are fluorophores, C ¹ and C ² are conformational determining regions, and S ¹ and S ² Is any peptide spacer, F ¹ is a donor fluorophore or chromophore, and F ² is an acceptor fluorophore or chromophore, or conversely, F ² is a donor fluorophore or chromophore. Yes, and F ¹ is an acceptor fluorophore or chromophore,
Alternatively, F ¹ and F ² can be the same (fluorophore or chromophore). The protease binding site provides the amino acid sequence (peptide) that is recognized and cleaved by the protease whose activity the indicator is designed to show. The protease binding site is typically a peptide ranging in length from 2 amino acids to about 12 amino acids, 2 to about 10, 2 to about 8, 2 to about 6, or 2 to about 4 amino acids.

[0042]   A preferred conformational determining region is to introduce a bend into the molecule, or
Alternatively, it is an amino acid sequence that limits the degree of freedom of the peptide main chain. Two conformations
The combined effect of each decision area is C¹ And C² The amino terminus and cal
The juxtaposition of the fluorophore attached to the boxy end. Therefore, the fluorophore is preferred
In other words, they are located adjacent to each other at a distance of about 100Å or less. Fluorescent group (F¹ And F² 
) Are typically conformations, although they can be attached to a linker.
It is directly joined to the decision area. If present, any spacer (S¹ And S ²  ), The composition on a solid support, or a component of a biological sample (eg, cell membrane).
) To the composition. Spacers can also be additional or other
Functionality can be provided. For example, the spacer can be
The amino acid GY can be included to provide an optical signal for easy detection.
It

The substantially conformational determining region enhances the protease specificity of the composition. Amino acid sequences comprising conformational determinants are typically inaccessible to enzymes due to steric hindrance with each other and with the associated fluorophore. In contrast, the protease binding site is relatively unobstructed by either the fluorophore or the conformation determining region, and thus the protease is easily accessible.

II. Protease Binding Site In a preferred embodiment, the protease binding site and the conformation determining region form a contiguous amino acid sequence (peptide). The protease binding site is
An amino acid sequence that is recognized and cleaved by a particular protease. It is well known that various proteases cleave peptide bonds flanking specific amino acids. Thus, for example, trypsin cleaves a peptide bond following a basic amino acid, such as arginine and lysine, and chymotrypsin
It cleaves peptide bonds following large hydrophobic amino acid residues such as tryptophan, phenylalanine, tyrosine and leucine. Serine proteases cleave peptide bonds following small hydrophobic residues such as alanine.

However, certain proteases will not cleave any bonds in proteins that have the correct contiguous amino acids. Rather, the protease is
It is specific for a particular amino acid sequence that acts as a recognition domain for each particular protease. Without being bound by a particular theory, the choice of a particular protease for a particular cleavage site over many other possible sites in a folded globular protein is the amino acid sequence of that potential cleavage site and also It seems that their conformation and conformational flexibility can be largely determined.

Thus, for example, a single-stranded folded protein ribonuclease-A to ribonuclease-S (a non-covalent complex of two polypeptide chains) with a restricted proteolytic product, for example a protease called subtilisin. Body). Similarly, a single-stranded staphylococcus (Stap
A double-stranded non-covalent complex, Staphylococcus nuclease-T, is obtained from hylococcus nuclease. Another example of the selection of a particular protease for one substrate over another is human fibroblast-type collagenase. This protease favors type I over type III soluble collagen even if both type I and type III soluble collagen substrates contain the same collagenase-sensitive Gly-Ile or Gly-Leu bond (eg, Brikedal- Hansen etc.,
(1993) Crit. Rev. in Oral Biology and Medicine 4: 197-250).
.

Any amino acid sequence which contains a recognition domain and is therefore capable of being recognized and cleaved by a protease is suitable for the “protease binding site” of the fluorescent protease indicator composition of the present invention. Known protease substrate sequences and peptide inhibitors of proteases have an amino acid sequence that is cleaved or recognized by the particular protease that they inhibit. Thus, the known substrate and inhibitor sequences provide suitable basic sequences for use in the protease recognition region. A number of protease substrate and inhibitor sequences suitable for use as the protease binding domain in the compositions of the invention are shown in Table 2. One of ordinary skill in the art will recognize that this is not a complete listing, and that other protease substrate or inhibitor sequences may be used.

Amino acid residues containing protease binding sites are conventionally numbered relative to the peptide bond that is hydrolyzed by the particular protease. Accordingly, the first amino acid residue on the amino side of the cleaved peptide bond is designated as P _1, and the first amino acid residue on the carboxyl side of the cleaved peptide bond is designated as P ₁ ' It The numbering of the residues increases with the distance away from the hydrolyzed peptide bond. Thus, four amino acid protease binding region comprises an amino acid called _{P 2 -P 1 -P 1 '-P} 2', and protease cleaves the bond region between P ₁ and P ₁ '.

In a preferred embodiment, the protease binding region of the fluorogenic protease indicator of the present invention is selected to be symmetrical with respect to the cleavage site. Therefore,
For example, if the binding region is Ile-Pro-Met-Ser-Ile (eg α-1 anti-trypsin) and the cleavage occurs between Met and Ser, then a four amino acid residue binding region based on this sequence Is

[0050]

[Chemical 4]

It is Other examples of binding domains selected from longer sequences are provided in Table 2. The remaining amino or carboxyl residues that are not present within the protease binding domain can remain as part of the conformational determining region subject to certain restrictions, as explained below. Thus, in the present example, the amino terminal Ile can be incorporated into the B05449 (2), C ¹ conformation determining region. Various amino acid substitutions can be made to amino acids comprising the protease binding domain to enhance binding specificity, eliminate reactive side chains, or reduce the conformational entropy of the molecule (less freedom). . Thus, for example, it is sometimes desirable to replace a methionine (Met) residue bearing an oxidizable sulfur with norleucine. Thus, in the example given, the preferred protease binding region has the following sequence:

[0052]

[Chemical 5] Will have.

Conformation Determining Regions The conformation determining regions (C ¹ and C ² ) are the protease cleavages that anchor the peptide backbone of the fluorogenic protease indicator molecule of the present invention and introduce a bend therein. Peptide regions at either end of the region. The combination of the two conformation determining regions and the relatively linear protease cleavage region results in a "U
It produces a roughly U-shaped molecule with a cleavage site at the "shaped base (center). The term U-shaped is, of course, a rough approximation, ie a juxtaposition of the fluorophores in close proximity (eg about 100Å or less). Means to be held relatively fixed below.

In one embodiment, amino acids such as proline (Pro) and α-aminobutyric acid
Both (Aib) are chosen to introduce bending into the peptide molecule and enhance the anchoring of the peptide backbone. The C ¹ and C ² domains are chosen such that the U-shaped “arm” is fixed and the bound fluorescers are separated and located adjacent to each other for a distance of about 100 Å or less. In order to maintain the required rigidity of the peptide backbone and the arrangement of the fluorophores, the conformation determining region is preferably 4 or less amino acids in length, or, on the other hand, about It is 18 or more amino acids in length and forms a stable α-helix conformation or β-pleated sheet.

A) Tetrapeptide Binding Site Compositions In a preferred embodiment, the peptide backbone of the fluorogenic protease indicator of the present invention comprises a tripeptide C ¹ region, a tetrapeptide P region and a single amino acid or dipeptide C ² region. Will.

[0056]   Those compounds have the formula:

[Chemical 6]

[0057]   [In the formula, Y is

[Chemical 7] Which is one of them?)

Can be represented by In these expressions, the peptide binding region is -P ₂ -P ₁
-P ₁ '-P _2' - indicated as, and conformation determining regions C ¹ and each amino acid residue is -C of ^{_{C 2 1 5 -C 1 4 -C}} 1 3 - and -C ² ₃ -C Shown as ² ₄ −. The C ² region can be either an amino acid or a dipeptide. Whether the C ² region is a dipeptide or an amino acid, the F ² fluorophore and S ² spacer, when present, are always attached to the carboxyl-terminal residue of C ² . When the spacer is present in the C ² region, it is attached to the α-carboxyl group by a peptide bond to C ²
The carboxyl-terminal residue of is attached.

As indicated above, the conformation determining region typically comprises an amino acid residue, such as proline (Pro), which introduces a bend into the molecule and enhances its rigidity. However, if the terminal residues of the protease binding region (P) are themselves bend-creating residues, for example proline, residues that create bend at positions close to P in the C-region bound to their ends. Those skilled in the art will understand that it is not necessary to place Therefore, the conformation-determining region first determines the protease binding region, as described above, the "remaining" residues present in the conformation-determining region, and, if necessary, those residues according to the following guidelines. Designed by modifying the group:

1. When the P ₂ 'site is not Pro, C ² is a dipeptide (formula III) Pro-Cys, Aib-C
ys, Pro-Lys, or Aib-Lys, and conversely, when the P ₂ 'site is Pro,
C ² is a single amino acid residue (Formula IV) Cys or Lys. 2. If P ₂ site is not Pro, C ¹ is _{^{Asp-C 1 4 -Pro, Asp}} -C 1 4 -Aib, Asp-Aib-
It is a tripeptide consisting of Pro, Asp-Pro-C ¹ ₃ , Asp-Aib-C ¹ ₃ , Asp-Pro-Aib or Asp-Aib-Aib, and when the P ₂ site is a Pro residue, the group C ¹ is a tripeptide consisting of Asp-C ¹ ₄ -C ¹ _3, or Asp-C ¹ ₄ -Aib. 3. When P ₃ (C ¹ ₃₎ residue is a Pro, C ¹ is Asp-C ¹ ₄ -Pro or Asp-Aib-Pro
Is a tripeptide consisting of

4. P ₄ (C ¹ ₄₎ if residue is Pro, C ¹ is Asp-Pro-C ¹ ₃ or Asp-Pro-Aib
Is a tripeptide consisting of 5. If P ₂ and C ¹ ₃ is not proline both, C ¹ is _{^{Asp-Pro-C 1 3,}} Asp-A
_{^{ib-C 1 3, Asp-}} C 1 4 -Pro, Asp-C 1 4 -Aib, a tripeptide consisting of Asp-Pro-Aib or Asp-Aib-Pro. As mentioned above, either methionine (Met) can be replaced by norleucine (Nle). Many suitable peptide backbones consisting of C ¹ and C ² are provided in Table 2.

[0062]

[Table 2]

[0063]

[Table 3]

[0064]

[Table 4]

B) Indicators with Other Binding Sites In another preferred embodiment, the binding sites (P) range from 2 to about 12 amino acids in length. A rather large conformational determinant region can sufficiently limit the degree of freedom of the indicator molecule, ie its fluorophore is bound [
Recognition] It was a finding of the present invention that it is appropriately quenched regardless of the amino acid sequence of the domain (P). In one preferred embodiment, the compositions are

[0066]   Formula V:

[Chemical 8]

[0067]   Including compounds represented by In this formula, P is a protease binding site.
A peptide comprising and comprising 2 to about 12 amino acids, F¹ And F ²  Is the fluorophore, where F¹ Is the amino terminal amino acid of the composition (excluding the spacer).
Bound to no acid, and F² Is attached to the carboxyl terminal amino acid. S¹ Over
And S² Is a peptide space ranging from 1 to about 50 amino acids in length, if present.
Sir and S¹ Is attached to the amino-terminal amino acid, if present, and
Then S² Is attached to the carboxyl-terminal amino acid, if present. Subscript
The letters i, j, k, m, n, o, p, q and r are independently 0 or 1.

In a particularly preferred embodiment, aa ¹ and aa ¹⁰ are independently selected from the group consisting of lysine, ornithine and cysteine; aa ² , aa ³ , aa ⁸ and aa ⁹ are Asp, Glu.
, Lys, ornithine, Arg, citrulline, homocitrulline, Ser, homoserine, Thr and Tyr independently selected from the group consisting of amino acids or dipeptides; aa ⁵ , aa ⁴ , aa ⁶ and aa ⁷ are proline, 3 Independently selected from the group consisting of, 4-dehydroproline, hydroxyproline, α-aminoisobutyric acid and N-methylalanine;

X is Gly, βAla, γAbu, Gly-Gly, Ahx, βAla-Gly, βAla-βAla, γAbu-Gly, βAl
a-γAbu, Gly-Gly-Gly, γAbu-γAbu, Ahx-Gly, βAla-Gly-Gly, Ahx-βAla, βAl
a-βAla-Gly, Gly-Gly-Gly-Gly, Ahx-γAbu, βAla-βAla-βAla, γAbu-βAla-Gl
y, γAbu-γAbu-Gly, Ahx-Ahx, γAbu-γAbu-βAla, and Ahx-Ahx-Gly; Y is Gly, βAla, γAbu, Gly-Gly, Ahx, Gly-βAla, βAla -βAla,
Gly-γAbu, γAbu-βAla, Gly-Gly-Gly, γAbu-γAbu, Gly-Ahx, Gly-Gly-βAla,
βAla-Ahx, Gly-βAla-βAla, Gly-Gly-Gly-Gly, γAbu-Ahx, βAla-βAla-βAla,
Gly-βAla-γAbu, Gly-γAbu-γAbu, Ahx-Ahx, βAla-γAbu-γAbu, and Gly-Ah
selected from the group consisting of x-Ahx.

[0070] If i is 1, S ¹ is coupled to aa ¹ by a peptide bond through terminal α-amino group of aa ^1; if and γ is 1, S ^2, the terminal α carboxyl group of aa ¹⁰ Through a peptide bond to aa ¹⁰ . When one or more of those amino acids is absent, the fluorophore is attached to the remaining terminal amino acid. The amino acid backbone of such particularly preferred compositions is listed in Tables 3 and 4.

[0071]

[Table 5]

[0072]

[Table 6]

[0073]

[Table 7]

[0074]

[Table 8]

[0075]

[Table 9]

[0076]

[Table 10]

[0077]

[Table 11]

[0078]

[Table 12]

[0079]

[Table 13]

[0080]

[Table 14]

[0081]

[Table 15]

[0082]

[Table 16]

IV. Fluorophores Fluorophores excited by incident light absorb the light and then re-emit the light at a different (long) wavelength. However, in the presence of a second class of molecules known as "receptors", the light emitted by the so-called donor fluorophore is absorbed by the receptor, thereby quenching the donor's fluorescent signal. Thus, as opposed to the fluorophore / chromophore, the use of two fluorophores allows a clear assessment of the overlap between the emission spectrum of the donor and the excitation spectrum of the receptor. This facilitates the design of the peptide backbone allowing optimization of quenching.
This results in a highly efficient donor / receptor pair that facilitates detection of low concentrations of protease activity. Thus, although a fluorophore / chromophore combination is suitable, in a preferred embodiment, the fluorogenic protease inhibitors of the present invention will include two fluorophores.

The “donor” and “receptor” molecules are typically chosen as a matched pair so that the absorption spectrum of the receptor molecule overlaps as broadly as possible with the emission spectrum of the donor molecule. In addition, the donor and receptor fluorophores preferably have absorption and emission spectra of the donor molecule in the visible range (400 nm to about 700 nm).
nm). The fluorophore thereby provides a detectable signal in the biological sample and thus facilitates detection of protease activity in biological fluids, tissue homogenates, tissue fragments and the like. The emission spectra, absorption spectra and chemical compositions of many fluorophores are well known to those skilled in the art (eg Handbook of Fluorescent Probes and Research Chemicals,
See RP Hauglond, ed., Which is incorporated herein by reference.)
.

Preferred fluorophore pairs include rhodamine derivatives. Thus, for example, 5-carboxytetramethylrhodamine or 5- and / or 6-carboxytetramethylrhodamine (9- (2,5-dicarboxyphenyl) -3,6-bis- (dimethylamino) xanthylium chloride (5 -TMR) and 9- (2,6-dicarboxyphenyl) -3,6-bis- (dimethylamino) xanthylium chloride (6-TMR)), succinimidyl esters of (Molecular Probes,

C211 and C1171) (Formula VI), available from Eugene, Oregon, USA, are particularly preferred donor molecules:

[Chemical 9]

[0087]   And Rhodamine X acetamide (R492 from Molecular Probes) (Formula VII)

[Chemical 10]

Or 5- and / or 6-carboxy-X-rhodamine (9- (2,5-dicarboxyphenyl) -2,7-dimethyl-3,6-bis- (ethylamino) xanthene (5-DER ) And 9- (2,6-dicarboxyphenyl) -2,7-dimethyl-3,6-bis- (ethylamino) xanthene (6-DER)), mixed isomers obtained as C1309. Body (C1309 from Molecular Probes
) Is a particularly preferred receptor molecule.

The fluorophores are such that the excitation and emission of both members of this donor / receptor pair are at visible wavelengths, the molecule has a high excitation coefficient and the molecule has a high fluorescence yield in solution. Therefore, it is particularly preferable. Excitation coefficient is a measure of light absorption by a luminophore at a particular wavelength, and is therefore related to its ability to quench a signal, where fluorescence yield is the ratio of absorbed light to re-emitted light. , And is a measure of the efficiency of the fluorophore, and thus affects the sensitivity of the protease indicator.

Other preferred fluorophores include 9- (2-carboxyphenyl) -2,7-dimethyl-3,6-bis (ethylamino) xanthylium, 9- (2-carboxyphenyl) -3,6. -Bis (dimethylamino) xanthylium, and 9- (2-carboxyphenyl) -xanthylium, but are not limited thereto. Of course, although not at all preferred, fluorophores that absorb and emit in the UV range can also be used in the protease indicators of the present invention. One particularly preferred UV absorbing pair of the fluorophore is the following 7-hydroxy-4-methylcoumarin-3-acetic acid (Formula VIII) as a donor molecule:

[0091]

[Chemical 11]

And 7-diethylamino-3-((4′-iodoacetyl) amino) phenyl) -4-methylcoumarin (formula IX) as a receptor molecule:

[0093]

[Chemical 12]

Is Those and other fluorophores are available from many manufacturers, such as Molecular Pr.
Commercially available from obes (Eugene, Oregon, USA). It is a surprising finding that a fluorophore with a matched absorption and emission spectrum is not required under the practice of the invention. In fact, a single species of fluorophore is capable of quenching itself when linked to the polypeptide backbone of the invention at positions dominated by F ¹ and F ² . Moreover, this quenching is fully opened when the peptide backbone is cleaved.

Although not based on any particular theory, it is believed that quenching is achieved by the formation of ground state dimers, where the electron orbitals of the two fluorophores interact, leading to reversible quenching. It is the limited conformational entropy of the peptide backbone of the present invention that brings the fluorophore close enough to effectively form the ground state dimer.

Particularly preferred molecules from H-type dimers. The formation of H-type dimers by fluorescent molecules is described by Packard et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 1164.
0-11645. This H-type dimer is characterized by an excitation band in the absorption spectrum and fluorescence quenching (eg, Valdes-Aguilera).
(1989) Acc. Chem. Res., 22: 171-177 and Packard et al. (1996) Proc.
Natl. Acad. Sci. USA, 93: 11640-11645). Therefore, in a preferred embodiment, the protease indicator of the present invention is
Only a single type of fluorophore is included, more preferably a fluorophore capable of forming an H-type dimer.

NorFes is an undecapeptide containing a recognition sequence and cleavage site for the serine protease elastase. When NorFes was doubly labeled with different fluorophores on opposite sites of the amino acid sequence, the fluorescence was quenched due to the formation of intramolecular ground state dimers. The spectral features of those dimers could be predicted by excitation theory.

The decrease in dimer / monomer ratio with increasing temperature indicated intermolecular attraction between the dye molecules. The free energy of activation of destruction of homodimers composed of tetramethylrhodamine was at least 1.7 Kcal / mol and, in the case of diethylrhodamine, 2.4 Kcal / mol. Due to the intermolecular attraction of the fluorophores that form the excited dimer, the binding amino acid sequences deviate from the optimal sequences described herein. Thus, if an excitation-forming fluorophore is used, amino acid substitutions can be made in the "backbone" described herein, and activity can still be maintained.

Particularly preferred excitation-forming fluorophores include, but are not limited to, carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin and carbocyanine dyes. In this embodiment, only a single fluorophore is used, so there is no need to match the emission or absorption spectra. Therefore, a wide variety of fluorophores can be effectively used. Moreover, the use of a single fluorophore greatly simplifies synthetic chemistry.

The homo-double labeled indicators of the invention (indicators labeled with a single species of fluorophore) also allow the detection of enzyme activity by absorption as well as fluorescence measurements. Since the blue-shifted excitation band (or blue-shifted absorption maximum) in the absorption spectrum indicates H-dimer formation, and the fluorescence digestion occurs simultaneously with the latter, the measurement of the absorption spectrum is a diagnostic tool in the correct setting. As may be sufficient. When the doubly labeled protease indicator is cleaved by a particular protease, the H-type dimer is destroyed.
The blue-shifted absorption maximum associated with the H-type dimer is then lost. Therefore, when measuring the absorption intensity in the blue-shifted excitation band, H
As the -type dimer is destroyed, the absorption intensity is expected to decrease, whereas the absorption intensity at the monomer maximum peak is expected to increase.

Preferred for use in certain high throughput screening systems are the indicators of the present invention formulated with rhodamine, or cyanine dyes such as cyanine and cyanine analogs. A particularly preferred embodiment uses carbocyanine dyes, more preferably dialkylated carbocyanine dyes such as those described in Example 6. Suitable cyanine dyes include N-ethyl-N '-[5- (N "-succinimidyloxycarbonyl) pentyl] indocarbocyanine chloride and N-ethyl-N'-[5
-(N "-succinimidyloxycarbonyl) pentyl] -3,3,3 ', 3-tetramethyl-2,
Includes, but is not limited to, 2-indodicarbocyanine chloride.

V. Preparation of Fluorogenic Protease Indicator The fluorescent luminescent protease indicator of the present invention first comprises a peptide backbone, namely a protease cleavage site (P), two conformational determining regions (C ¹ and C ² ), and the presence thereof. Then, it is preferably prepared by synthesizing spacers (S ¹ and S ² ). The fluorophore is then chemically conjugated to the peptide. The fluorophores are preferably directly conjugated to the peptide, however they are also attached to the peptide through a linker. Finally, when the fluorogenic protease indicator is attached to a solid support, it is then chemically conjugated, either directly or through a linker, via a spacer (S ¹ or S ² ) to the solid support.

A) Peptide Backbone Preparation Solid phase peptide synthesis in which the C-terminal amino acid of the sequence is bound to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence is the peptide backbone of a compound of the invention. Is a preferred method for preparing. Techniques for solid phase synthesis are described in: Barany and Merrifield, Solid-Phase Peptide Synthesis:
pp. 3 to 284, The Peptides: Analysis, Synthesis, Biology, Vol. 2: Specia
l Methods in Peptide Synthesis, Part A., Merrifield, et al, J. Am. Chem.
Soc. 85, 2149-2156 (1963), and Gross and Meienhofer, eds. Academic Pre
ss, NY, 1980 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed.
Pierce Chem. Co., Rockford, III. (1984); these are incorporated herein by reference. Solid phase synthesis is most easily accomplished on commercially available peptide synthesizers using FMOC or TBOC chemistry. The chemical synthesis of the peptide component of the fluorescent assistant protease indicator is described in detail in Examples 1 and 2.

In a particularly preferred embodiment, peptide synthesis is performed using Fmoc synthetic chemistry. The side chains of Asp, Ser, Thr and Tyr are preferably protected with t-butylthio and the side chains of Cys residues are protected with S-trityl and St-butylthio and Lys residues are preferably Protected with t-Boc, Fmoc and 4-methyltrityl for lysine residues. Appropriately protected amino acid reagents are commercially available. The use of multiple protecting groups allows for selective deblocking and attachment of the fluorophore to any particular desired side chain.

Thus, for example, t-Boc deprotection is achieved with TFA in dichloromethane, Fmoc deprotection is achieved with 20% (v / v) piperidine in DMF or N-methylpyrrolidone, And deprotection of 4-methyltrityl is 1-5% (v / v) TFA in water.
, Or with 1% TFA or 5% triisopropylsilane in DCM. St-Butyl thio deprotection was achieved with aqueous mercaptoethanol (10%), t-butyl and t-boc, and S-trityl deprotection were TFA: phenol:
Achieved using water: thioanisole: ethanedithiol (85: 5: 5: 2.5: 2.5),
And t-butyl and t-Boc deprotection is achieved with TFA: phenol: water (95: 5: 5). Detailed synthesis, deprotection, and fluorophore attachment methods are provided in Examples 1 and 2.

On the other hand, the peptide component of the fluorescent protease indicator of the present invention may also be synthesized using recombinant DNA technology. Briefly, a DNA molecule encoding the desired amino acid sequence is described in Beaucage and Carruthers, Tetra. Letts. 22: 1859.
~ 1862 (1981), the solid-state phosphoramidite method, Matleucci, et al.
, J. Am. Chem. Soc., 103: 3185 (1981), both incorporated herein by reference, or known to those skilled in the art. Are chemically synthesized by other methods. Preferably, DNA can be synthesized using standard β-cyanoethyl phosphoramidite based on commercially available DNA synthesizers using standard methods.

Oligonucleotides can be purified, if necessary, by techniques well known to those of skill in the art. Typical purification methods include gel electrophoresis, anion exchange chromatography (eg Mono-Q columns, Pharmacia-LKB, Piscataway, New Jersey, USA) or reverse phase high performance liquid chromatography (HPLC), provided that It is not limited to these. Methods of protein and peptide purification are well known to those of skill in the art. For a review of standard methods, see Methods in Enzymology, Volume 182:
See Guide to Protein Purification, M. Deutscher, ed. (1990), pages 619-626, which is incorporated herein by reference.

The oligonucleotide may be annealed by a complementary oligonucleotide, or
It can be converted to double-stranded DNA by polymerization with a DNA polymerase. The DNA is then inserted into a vector under the control of a promoter and used to transform host cells so that the cells express the encoded peptide sequence. Methods of peptide cloning and expression are well known to those of skill in the art. For example, Samb
rook, et al., Molecular Cloning: A Laboratory Manual (2nd Ed., Vols. 1
-3, Cold Spring Harbor Laboratory (1989)), Methods in Enzymology, Vol.
152: Guids to Molecular Cloning Technigues (Berger and Kimmel (eds.),
San Diego: Academic Press, Inc. (1987)), or Current Protocols in Molec
ular Biology, (Ausubel, et al. (eds.), Greens Publishing and Wiley-Inte
See rscience, New York (1987), which are incorporated herein by reference.

B) Linkage of Fluorophores to the Peptide Backbone The fluorophore is linked to the peptide backbone by any of a number of means well known to those of skill in the art. In a preferred embodiment, the fluorophore is linked from a reactive site on the fluorophore to a reactive group on the peptide, such as a terminal amino or carboxyl group, or a reactive group on the amino acid side chain, such as an amino, hydroxyl, or carboxyl moiety. Is directly connected to. Many fluorophores usually contain suitable reactive sites. On the other hand, the fluorophore can be derivatized to provide reactive sites for linkage to other molecules. Fluorophores derivatized with functional groups for attachment to a second molecule are available from various manufacturers. Derivatization can be by simple substitution of groups on the fluorophore itself, or by conjugation to a linker. Various linkers are well known to those of skill in the art and are discussed below.

As stated above, in a preferred embodiment, the fluorophore is directly linked to the peptide backbone of the protease indicator. Thus, for example, the 5'-carboxytetramethylrhodamine (C2211) fluorophore is a
It can be linked to aspartic acid through an amino group. Rhodamine X acetamide (
The iodoacetamide group of R492) can be linked by reaction of cysteine with a sulfhydryl group, as shown in formula VI. Means for making such a connection are well known to those of skill in the art, and details of one such connection are provided in Example 1.

Those skilled in the art will appreciate that if a peptide spacer (S ¹ or S ² ) is present (as discussed below), the fluorophore is preferably the spacer itself, respectively C ¹ or C ^2.
As to form the terminal amino and carboxyl groups and the peptide chain, it will appreciate that it is connected to the conformation determining regions through a reactive group on the side chain terminal amino acids of C ¹ or C ^2.

C) Selection of Spacer Peptides and Linkage to Solid Supports The fluorescent protease indicators of the present invention can be obtained in solution or linked to a solid support. A "solid support" is one that does not dissolve or react with any of the components present in the solution used for assaying protease activity with the fluorogenic protease indicators of the present invention. , And any solid material that provides a functional group for attachment of the fluorogenic molecule.

Solid support materials are well known to those skilled in the art and include silica, controlled porosity glass (CPG), polystyrene, polystyrene / latex, carboxyl-modified Teflon®, dextran, derivatized multi-layers. Including but not limited to sugars such as agar bearing amino, carboxyl or sulfhydryl groups, various plastics such as polyethylene, acrylics, and the like. "Semi-solid" supports, such as lipid membranes as found in cells and liposomes, are also used. Those skilled in the art will appreciate that solid supports may be derivatized with functional groups (eg hydroxyl, amine, carboxyl, ester, and sulfhydryl) to provide reactive sites for attachment of linkers or direct attachment of peptides. You will understand that.

The fluorescent aid protease indicator can be linked directly to the solid support through a fluorophore or through a peptide bond containing the indicator. Most preferred is linkage through the peptide backbone. If it is desired to link to the solid support through a peptide backbone, the peptide backbone can include additional peptide spacers (designated S ¹ or S ² in Formula I). The spacer is present at the amino or carboxyl terminus of the peptide backbone and is from about 1 to about 50 amino acids, more preferably 1 to about 50 amino acids.
It can be about 20 and most preferably 1 to about 10 amino acids in length. Particularly preferred spacers are Asp-Gly-Ser-Gly-Gly-Gly-Glu-Asp-Glu-Lys, Lys-Glu.
-Asp-Gly-Gly-Asp-Lys, Asp-Gly-Ser-Gly-Glu-Asp-Glu-Lys, and Lys-Glu-Asp
-Glu-Gly-Ser-Gly-Asp-Lys.

The amino acid composition of the peptide spacer is not critical if the spacer acts to separate the active components of the molecule from the support, thereby preventing unwanted interactions. However, the amino acid composition of the spacer can be selected to provide an amino acid (eg, cysteine or lysine) having a side chain to which the linker or solid support itself is easily attached. On the other hand, the linker or the solid support itself, S ¹
Can be attached to the amino terminus of S ² or the carboxyl terminus of S ² .

In a preferred embodiment, the peptide spacer is actually linked to the solid support by a linker. The term “linker” as used herein means a molecule (eg, solid support, fluorophore, etc.) that can be used to link a peptide to other molecules. The linker is a homobifunctional molecule that provides a first reactive site capable of forming a covalent bond with the peptide and a second reactive site capable of forming a covalent bond with a reactive group on the solid support. Covalent attachment to the peptide (spacer) is through either the terminal carboxyl or amino groups, or by a reactive group on the amino acid side chain (eg, through a disulfide bond to cysteine).

Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers or peptide linkers. As mentioned above, linkers can be linked to carboxyl and amino terminal amino acids through their terminal carboxyl or amino groups or through their reactive side groups.

Particularly preferred linkers are capable of forming covalent bonds to amino groups, carboxyl groups, or sulfhydryls. Amino-linked linkers include reactive groups such as carboxyl groups, isocyanates, isothiocyanates, esters, haloalkyls, and the like. Carboxyl-linked linkers can form reactive groups such as various amines, hydroxyls and the like. Finally, sulfhydryl- / linker linkers include reactive groups such as sulfhydryl groups, acrylates, isothiacinates, isocyanates and the like.

Particularly preferred linkers are for linking amino groups (for example amino groups found on lysine residues in peptides) by sulfhydryl groups found on solid supports, or amino groups found on solid supports. Include a sulfo MBO (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester) to link sulfhydryl groups (eg, found on cysteine residues of peptides). Other particularly preferred linkers include EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and bis- (sulfosuccinimidyl suberate). Other suitable linkers are well known to those of skill in the art.

[0120]   The fluorogenic compound of the present invention is characterized in that the donor fluorophore is on a solid support after cleavage of the molecule.
Or the donor fluorophore enters the solution after cleavage, S. ¹  Or S² It can be attached to the solid support through any of the spacers. Former field
If so, the support is then assayed for fluorescence to detect protease activity.
, And in the latter case, the solution is fluorescently detected to detect protease activity.
It is essay.

VI. Detection of Protease Activity The present invention also provides methods for using a fluorogenic protease indicator to detect protease activity in various contexts. Accordingly, in one aspect, the invention provides a method of using a fluorogenic indicator to establish or quantify the protease activity of a stock solution of a protease used for experimental or industrial purposes. Prior to use, confirmation of the protease activity of a stock solution of proteases is generally that proteases often lose activity over time (eg, through autohydrolysis) or are of varying degrees when activated from zymogen precursors. Recommended because it shows activation of.

Assaying stock solutions for protease activity simply involves adding a fixed amount of stock solution to the fluorogenic protease indicator of the present invention and measuring the subsequent increase in fluorescence. Stock solutions and fluorogenic indicators can also be bound and assayed in a "digestion buffer" that optimizes the activity of proteases.
Suitable buffers for assaying protease activity are well known to those of skill in the art. In general, a buffer will be chosen whose pH corresponds to the optimum pH for the particular protease.

For example, a particularly suitable buffer for assaying elastase activity consists of 50 mM sodium phosphate, 1 mM EDTA, pH 8.9. The measurement is most easily performed by a fluorometer, and a device that provides an "excitation" light source for the fluorophore and then measures the light emitted at a particular wavelength. Comparison with a control indicator solution lacking protease provides a measure of protease activity. The activity level can be accurately quantified by generating a standard curve for the protease / indicator combination in which the rate of change of fluorescence produced by a protease solution of known activity is determined.

Although detection of the fluorogenic compound is preferably accomplished using a fluorometer, detection can be accomplished by various other methods well known to those of skill in the art. Thus, for example, the fluorophore of the present invention emits in the visible wavelengths so that detection can simply be performed by visual inspection of the fluorescence in response to excitation by the light source. Detection can also be performed by the image analysis system, using a video camera coupled to a digitizer or other image acquisition system. Detection can also be performed by visualization through a filter under a fluorescent microscope. The microscope provides the signal visualized by the operator. However, the signal can be recorded on photographic film or using a video analysis system. The signal can also be simply quantified in real time using either an image analysis system or simply a photometer.

Thus, for example, a basic assay for protease activity of a sample is to suspend or dissolve the sample in a buffer (at the optimum pH for the particular protease to be assayed) and use the fluorogenic protease indicators of the invention. Would be added to the buffer and monitoring the resulting change in fluorescence using a spectrophotometer. The spectrophotometer will be set to excite the donor fluorophore at the excitation wavelength of the donor fluorophore and detect the resulting fluorescence at the emission wavelength of the donor fluorophore.

In another aspect, the protease activity indicators of the invention can be used for the detection of protease activity in a biological sample. Therefore,
In a preferred embodiment, the invention provides an isolated biological sample, such as saliva, blood, blood cells, tumor biopsies and the like, or cells or tissues in culture,
Or a fragment thereof is not embedded and provides a method for detecting protease activity in a non-fixed fragment. The signal can be quantified using a fluorescence microscope, fluorescence microplate reader, fluorometer, or flow cytometer.

A) Ex Vivo Assay of Isolated Biological Samples In an aspect of 1, the present invention provides a method of detecting protease activity in an isolated biological sample. This can be determined by simply contacting the sample with a fluorescent aid protease indicator of the present invention and monitoring the change in indicator fluorescence over time. The sample may be suspended in "digestion buffer" as described above. The sample may also be cleared of cell debris prior to analysis, eg by centrifugation.

When the fluorescent aid protease indicator is bound to a solid support, the assay involves contacting the sample solution with a solid support bearing the indicator. If the indicator is linked to the solid support by the side of the molecule bearing the donor fluorophore, the fluorescence of the support due to digestion of the indicator will be monitored over time by any of the above means. Conversely, if the receptor molecule fluorophore is bound to a solid support, the test solution is passed over the solid support and then the resulting luminescence of the test solution (due to the cleaved fluorophore )
Is measured. Donor and acceptor pairs are present on both solid supports and liquids,
It can be replaced by the same fluorophore. This latter approach is particularly suitable for high throughput automated assays.

B) In situ Assay of Histological Fragments In another aspect, the invention provides a method for detecting in situ protease activity in histological fragments. This method of detecting protease activity in tissue offers substantial advantages over prior art methods (eg, specific stains, antibody labels, etc.). This is because, unlike the simple labeling approach, in situ assays with protease indicators show actual activity rather than the simple presence or absence of protease. The protease is
Often, those inactive precursors ((
Zymogen) present in tissues. Therefore, conventional labeling approaches do not provide information about physiological conditions as well as tissue protease activity.

In situ assays generally provide a tissue fragment (preferably a frozen fragment), contacting the fragment with one of the fluorescent protease indicators of the present invention and visualizing the resulting fluorescence. Comprising: Visualization is preferably
It is performed using a fluorescence microscope. The fluorescence microscope supplies an "excitation" light source to induce the fluorescence of the "donor" fluorophore. Microscopes are typically equipped with filters to optimize the detection of the resulting fluorescence. Thus, for example, for the fluorescence aid protease indicator described in Example 1, a typical filter cube for a Nikon microscope has an excitation filter (λ = 550 ± 12 nm), a dichroic mirror (λ = 580 nm) and interference. -Will include an emission filter (λ = 580 ± 10 nm). As mentioned above, the microscope may be equipped with a camera, photometer or image acquisition system.

The fragment is preferably cleaved as a frozen fragment because immobilization or embedding destroys protease activity in the sample. The fluorescent indicator can be introduced into the fragment in a number of ways. For example, the fluorescent aid protease indicator can be provided in a buffer solution, as described above, which is applied to the tissue section. On the other hand, the fluorogenic protease indicator may be supplied as a semi-solid medium such as a gel or agar spread on a tissue sample.
The gel helps retain moisture in the sample while providing a signal in response to protease activity.

Fluorogenic protease indicators can also be supplied conjugated to polymers, such as plastic films, that can be used in methods similar to Western blot development. The plastic film is placed on the tissue sample on the slide and the fluorescence due to the cleaved indicator molecules is seen in the sample tissue under the microscope. Typically, the tissue sample should be incubated for the time that the endogenous protease cleaves the fluorescent protease indicator. Incubation times will range from about 10-60 minutes at temperatures up to and including 37 ° C.

[0133] C) in onsite assay further embodiment of the cells in cultures and cell suspensions derived from tissue and biopsy samples, the present invention is, tissue, biopsy samples, or biological fluids (e.g. Methods of detecting in situ protease activity of cells in cultures, cell suspensions or adherent cell layers derived from saliva, blood, urine, lymph, plasma, etc.). In a preferred embodiment, cultured cells are grown on chamber slides or in suspension and then transferred to histology slides by cytocentrifugation. Similarly, cell suspensions are prepared according to standard methods and transferred to histology slides.

In one preferred embodiment, slides are washed with phosphate buffered solution and coated with a semi-solid polymer or solution containing a fluoresced protease indicator. The slides are incubated at 37 ° C for the time required for the endogenous protease to cleave the protease indicator. The slides are then examined under a fluorescence microscope with the appropriate filters as described above. In another preferred embodiment, cells are incubated with a protease indicator at 37 ° C, then washed with buffer and transferred to glass capillaries and examined under a fluorescent microscope. When a flow cytometer is used to quantify intracellular enzyme activity, cells with a fluorogenic indicator are simply diluted with buffer after incubation at 37 ° C and analyzed.

VII. Screening for Modulation of Protease Activity In certain preferred embodiments, the present invention provides methods for screening for modulation of protease activity. A modulator of protease activity is
Factors that increase, decrease, or eliminate the activity of a protease, or increase, decrease, or eliminate the availability of a protease at a particular site (eg, in particular a cell or location in a cell) (eg, , Compound). A modulator of protease activity can act directly on the protease, or indirectly, for example, by altering the availability or activity of the enzyme that activates the subject protease. .

In a preferred embodiment, these methods basically involve contacting the “subject” protease or cells containing the subject protease with one or more test agents. Alternatively, the protease or cell is contacted with one or more indicator compounds of the invention. A difference in the signal produced by the indicator compound in the presence of the test factor as compared to a control using the test factor at lower concentrations or no test factor indicates that the test factor modulates protease activity. .

[0137] Typically, there is a difference between the activity seen when the test factor is present or when the test factor is previously applied and the activity of the (usually negative) control. If, preferably, the difference is statistically significant (eg, greater than 80%, preferably greater than about 90%, more preferably greater than about 98%, most preferably greater than about 99%), as positive Scored. Most preferred "
A "positive" assay shows a difference from the negative control of at least 1.2 fold, preferably at least 1.5 fold, more preferably at least 2 fold, most preferably 4 fold or even 10 fold.

These assays can be performed in vitro using one or more proteases of interest and one or more compounds of the present invention in a suitable buffer system. The test factor can be added to the buffer system to detect changes in the signal of the indicator. Additionally or alternatively, the "test" assay can simply be compared to an assay of the same buffer system lacking the test agent (negative control). These assays can be performed in cells cultured in vivo, tissues cultured, or cells / tissue in an organism. One or more cell-permeability indicators of the present invention are introduced into test cells. A cell, tissue, or organism is contacted with one or more test agents and the change in the indicator signal generated by the one or more test agents is detected as described herein.

A) Test Factor Virtually any test factor can be screened according to the methods of the invention. Such factors include, but are not limited to, nucleic acids, proteins, sugars, polysaccharides, glycoproteins, lipids, and small organic molecules. The term small organic molecule means a molecule of comparable size to typical, commonly used pharmaceutically organic molecules. The term excludes biological macromolecules (eg, proteins, nucleic acids, and others). Preferred small organic molecules are up to about 5000 Da, more preferably
It ranges in size up to 2000 Da, most preferably up to about 1000 Da.

In general, new chemical entities with efficacious properties identify compounds with some desired property or activity (referred to as “lead compounds”) and create variants of the lead compounds, Generated by assessing the properties and activities of the mutant compounds of. However, the current trend is to reduce the time scale for all aspects of drug discovery. High Throughput Screening (HTS) methods have been used in place of conventional lead compound identification methods because large numbers can be tested rapidly and efficiently.

In one preferred embodiment, the high throughput screening method comprises providing a library containing a large number of potential therapeutic compounds (candidate compounds).
Such "combinatorial chemical libraries" are then identified in one or more assays as described herein to display members of the library displaying the desired characteristic activity (especially Chemical species or subclass). The compounds thus identified can act as common “lead compounds” or can themselves be used as potential or actual therapeutic agents.

Combinatorial chemical libraries include a large number of chemical (building block), eg
It is an assembly of various compounds generated by chemical synthesis or biological synthesis by combining reagents. For example, combining a linear combinatorial chemical library, eg, a set of chemical building blocks (ie, multiple amino acids in a polypeptide), called amino acids in all possible ways for a given compound length. A polypeptide (eg, mutein) library is formed by.
Millions of compounds can be synthesized through the combinatorial mixing of such chemical building blocks. For example, one systematic combinatoric mix of 100 compatible chemical building blocks could theoretically synthesize 1 × 10 ⁸ tetramer compounds or 1 × 10 ¹⁰ pentamer compounds, according to one commentator. (Gallop et al. (1994) 3
7 (9): 1233-1250).

Preparation of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, eg, the following references: US Pat. No. 5,010,175, F).
urka (1991) Int. J. Pept. Prot. Res. 37: 487-493, Houghton et al. (1991)
) Nature 354: 84-88). Peptide synthesis is by no means the only approach contemplated and contemplated for use with the present invention. Also, other chemical methods that generate chemically diverse libraries can be used. Such chemical methods include, but are not limited to:

Peptides (PCT publication No. WO 91/19735, December 26, 1991), encoded peptides (PCT publication No. WO 93/20242, October 14, 1993), random bio-oligomers (PCT publication) No.WO 92/00091, January 9, 1992), Benzodiazepine (US Pat. No. 5)
, 288,514), diversomers such as hydantoin, benzodiazepines and dipeptides (Hbbs et al. (1993) Proc. Natl. Acad. Sci. U.
SA 90: 6909-6913), vinylogous polypeptide (Hagihara et al.
1992) J. Amer. Chem. Soc. 114: 6568), non-peptidic peptidomimetics with beta-D-glucose scaffolding (H.
irschmann et al. (1992) J. Amer. Chem. Soc. 114: 9217-9218), a similar organic synthesis method for small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 11).
6: 2661), oligocarbamates (Cho et al. (1993) Science 261: 1303), and / or peptidyl phosphonates (Campbell et al. (1994) J. Org. Chem. 59: 6.
58).

See generally the following references: Gordon et al. (1994) J. Med. Chem. 37: 1.
385, nucleic acid libraries (see, eg, Stratagene, Corp.), peptide nucleic acid libraries (see, eg, US Pat. No. 5,539,083), antibody libraries (eg, Vaughn et al. (1996) Nature Biotechnology, 14 (3): 309. -314), and PC
T / US 96/10287), carbohydrate libraries (see, eg, Liang et al. (1996) Science, 274: 1520-1522, and US Pat. No. 5,593,853).
, And libraries of small organic molecules (see, eg, benzodiazepines, Baum (1993) C & EN, Jan 18, p. 33, isopenoids, US Pat. No. 5,569,588, thiazolidinones and metathiazanones, US patents). Number 5,549,974
, Pyrrolidine, U.S. Patent No. 5,525,735 and U.S. Patent No. 5,519,134, Morpholino Compounds, U.S. Patent No. 5,506,337, Benzodiazepines, U.S. Patent No. 5,288,514.
And other).

Equipment for preparing combinatorial libraries is commercially available (see, eg, 357 MPS, 390 MPS, Advanced Chem. Tech., Symphony, Rainin, Mass., Louisville, KY). Woburn, 433A, Appl
ied Biosystems, Foster City, CA, 9090 Plus, Milli
pores, Bedford, Mass.).

Also, a number of well known robotic systems have been developed for solution phase chemistry. These systems include, but are not limited to, automated workstations such as automated synthesizers (Takeda Chemical).
Industries, LTD., Osaka, Japan) and numerous robotic systems using robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass .; Orca, Hewlett-Packerd, Palo Alto, CA). These are manual synthesis operations performed by chemists. Mimics the VentureTM platform, an ultra-high throughput synthesizer capable of running 576-9,600 simultaneous reactions from start to finish (see Advanced Chem. Tech., Inc., Louisville, Kentucky).

Any of the above-described devices are suitable for use with the present invention. The nature of these devices (if used) and the implementation of changes to them will be apparent to those skilled in the art so that these devices can operate as described herein. In addition, a number of combinatorial libraries are themselves commercially available (see, eg: ComGenex, Princeton, NJ, As).
inex, Moscow, Ru, Tripos, Inc., St. Louis, Missouri, ChemStar, Ltd
, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek
Biosciences, Columbia, Maryland, and others).

B) High Throughput Screens Assays for any protease activity described herein and / or modulators of protease activity or other cleavage activity or other cleavage activity (eg,
Modulators of glycosylase activity, nuclease activity, lipase activity, and others) allow for high throughput screening. The preferred assay is
The alteration of the signal produced by the indicator of the invention in response to the presence of the test compound is detected.

These assays do not necessarily have to screen a single test agent at a time. On the contrary, at least to facilitate high throughput screening,
A single assay can be performed using two, preferably at least 5, more preferably at least 10, most preferably at least 20 test compounds. If the assay is positive, subsequent assays can be performed with a subset of test agents until an active test agent is identified.

High throughput assays for optical signals (eg fluorescence, altered spectra, and others), high throughput assays for various reporter gene products, are well known to those of skill in the art. For example, flow cytometers and multi-well fluorometers are commercially available. In Example 17, PE Biosystems FN in a fluorescent, homogeneous, multiplex, live cell and bead based screening assay for the assay of the present invention.
7 illustrates the use of the ATTM System 8100, an automated, macro-confocal high throughput screening (HTS) system.

VIII. Other Indicator Compositions As described above, fluorescent molecules that are covalently attached on opposite sides of the backbone (eg, peptide cleavage sites) are self-interacting (eg, through the formation of dimers).
It was the discovery of the present invention that it can be quenched by. Thus, in one embodiment, the indicator molecule can be prepared using a single fluorophore rather than a matched donor-acceptor pair. Also, as explained above, particularly preferred fluorophores are those that form H-type dimers (eg, carboxyrhodamine 11
0, carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, and carbocyanine dyes).

In a preferred embodiment, using combinatorial determining regions (CDRs) according to the invention,
While making dual-labeled peptide indicators with a single species of fluorophore, the use of such dual-labeled fluorophore is not limited to peptide substrates comprising a combinatorial determinant region. In contrast, the homo-dual labeled indicator system described herein can be used with virtually any peptide backbone that provides a backbone that allows the formation of "dimers" of fluorophores (mutual quenching). Is. Thus, according to the methods described herein, the fluorescence resonance energy transfer system (FR
Previously known peptide backbone indicators using the ET) (acceptor / donor) system can be designed with a single fluorophore instead.

However, the use of single species labeled indicators is not limited to peptide-based compositions. Conversely, "homo-double labeled" indicator molecules can utilize a variety of backbones including, but not limited to: nucleic acid backbones, oligosaccharide backbones, lipid backbones, and others. For example, conjugation methods for attaching fluorophores to amino acids, peptides, proteins, nucleic acids, oligonucleotides, sugars, polysaccharides, proteoglycans, lipids, glycolipids and lipopolysaccharides are described in Hermanson, (1995) Biocon.
jugate Techniques, Academic Press New York, NY, Kay M. et al., (1995)
Biochemistry, 34: 293-300, and Stubbs, et al., (1996) Biochemistry 35:
937-947.

A) Nucleic Acid Indicator The homo-double labeled nucleic acid backbone provides an effective indicator for nucleic acid hybridization and / or endonuclease activity. In this aspect, the nucleic acid backbone is self-quenching (eg, H-) at the 3'and 5'ends (either through a direct bond or indirectly through a linker (eg, peptide)).
Type dimer-forming) Labeled by the fluorophore. The nucleic acid backbone comprises a self-complementary region and is thereby selected to form a hairpin or other self-hybridized conformation that brings the fluorophore closer so that self-quenching occurs.

When the indicator (probe) thus formed is hybridized to a complementary target nucleic acid, self-hybridization is eliminated, the fluorophore is separated and the fluorescent signal produced by the molecule. Rises. On the other hand, fluorescently labeled nucleic acid backbones can be used to assay for nuclease activity (eg restriction endonuclease or ribozyme activity). When the nucleic acid backbone is cleaved by nucleases (eg, by restriction endonuclease recognition of target sites on the backbone), the fluorophore is dissociated again and the fluorescent signal is enhanced. Tyagi and Kramer et al. (1996) Natur for methods of selecting appropriate nucleic acid backbones.
e Biotechnology, 14: 303-308.

Homo-double labeled fluorescent DNA probes can be used for the detection, localization or quantification of target DNA sequences under various circumstances. Thus, for example, the nucleic acid indicators of the invention can be used for rapid detection of amplification products in nucleic acid amplification (eg PCR) reactions. Here, an indicator is selected that has a backbone complementary to the region of the amplification product. As the amplification product is produced, the indicator hybridizes to the product and the fluorescent signal activity of the PCR solution increases. Nucleic acid indicators can be used as hybridization or nuclease activity indicators under various other circumstances.

For example, in in situ hybridization (eg FISH), genomic DN
Mapping of A can be accomplished using fluorescent probes to target specific regions within the chromosome (see, eg, Meyne (1993) Chromosome mapping by fluoresce.
nt in situ hybridization, pp 263-268 In: Methods in Nonradioactive Dete
ction GC Howard, ed., Appleton & Lange, Norwalk, Connecticut; Morriso
n (1992) Deiection of energy transfer and fluorescence quenching, pp. 31
1-352 In: Nonisotopic DNA Probes Techniques LJ Kricka, ed. Academic P
ress, New York; and Varani (1995) Annu. Rev. Biophys. Biomol. Struct. 2
4: 379-404).

In another embodiment, the self-quenching fluorophore can be used to assay the interaction of two molecules (eg, protein-protein, protein-nucleic acid, ligand-receptor, etc.). . In this embodiment, one fluorophore is attached to one molecule (eg, a protein) and a second fluorophore is attached to a second molecule (eg, a second nucleic acid or nucleic acid binding protein). When the two molecules bind, the fluorophores are juxtaposed and digest each other (eg, through the formation of H-type dimers). The use of a donor-acceptor resonance energy transfer system to measure the interaction of two molecules is described by Bannwarth et al., Helve.
tica Chimica Acta. (1991) 74: 1991-1999, Bannwarth et al. (1991), Helvet
ica ChimicaActa. 74: 2000-2007, and Bannwarth et al., European patent application D439036A2.

B) Oligosaccharide Indicators Homo-double labeled oligosaccharide backbone indicators allow detection of glycosidase activity and identification of lecithin binding proteins. The fluorophore can be directly conjugated to the oligosaccharide or glycopeptide backbone or attached through a linker (eg, peptide). Oligosaccharides and / or glycopeptides can be chemically synthesized, recombinantly expressed, or isolated from natural sources, such as fetuin and other glycoproteins, by proteolytic fragmentation of the parent glycoprotein.

As in the case with oligonucleotides, oligosaccharide-specific structures can be chosen for the detection of specific glycosidases, enzymes that hydrolyze the bond between two sugar molecules. When a particular oligosaccharide or lecithin is selected to find its lecithin-binding protein, the enhanced fluorescence can distort the two dyes or distort the relative orientation of the two dyes. Shows the complexation phenomenon that destroys H-type dimers. Their effect results in enhanced fluorescence from homo-double labeled probes.

C) Lipid Indicator Lipids, glycolipids or lipopolysaccharides are labeled with a self-quenching (eg H-type dimer forming) fluorophore and attached to liposomes or other lipid (eg biological) membranes. When added, a decrease in fluorescence is indicative of H-type dimer formation, and the extent of such fluorescence intensity would be an indication of the amount of H-type dimer formation. Due to the relative fluidity of lipid membranes, self-quenching fluorophores are stable H
Capable of interacting with a type of dimer (eg approaching a space of about 6 to about 10Å).

When a membrane activator is added, for example, an agent that affects either the hydrodynamics of the membrane or the permeability to the test compound, the observed change in fluorescence intensity is the change in membrane fluidity or permeability. The ability of the test compound to denature is shown. Accordingly, such labeled lipids are useful in drug screening and liquid-drug delivery vehicle development. Similarly, the lipid-based probes of the invention can be used to probe the extent of lipid / protein interactions as well.

IX. It was also a discovery of the present invention that the attachment of a hydrophobic protecting group to a cell uptake polypeptide of a polypeptide enhances uptake of that polypeptide by cells. The effect is most pronounced when the polypeptide also carries a fluorophore, more preferably two fluorophores (see Example 9). However, in certain preferred embodiments, the fluorophore can be doubled as a hydrophobic group. Preferred hydrophobic groups include:
But not limited to:

Fmoc, 9-fluoreneacetyl group (Fa), 1-fluorenecarboxyl group, 9-fluorenecarboxyl group, and 9-fluorene-1-carboxyl group, benzyloxycarbonyl, xanthyl (Xan), trityl (Trt), 4-methyltrityl (Mtt)
, 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), mesitylene-2-sulfonyl (Mts), 4,4'-dimethoxybenzhydryl (Mbh), tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), benzyloxy (BzlO), benzyl (Bzl) ), Benzoyl (
Bz),

3-nitro-2-pyridinesulfenyl (Npys), 1- (4,4-dimethyl-
2,6-Dioxocyclohexylidene) ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z)
, 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl
(Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-
Butoxymethyl (Bum), t-butoxy (tBUO), t-butyl (tBu), acetyl (A
c), and trifluoroacetyl (TFA). The hydrophobic group can be coupled to the molecule of interest (eg, indicator or inhibitor) at essentially any position.

In a preferred embodiment, the hydrophobic group is coupled in a position that does not interfere with the recognition / binding of the molecule of interest by its cognate binding partner (eg protease). In a particularly preferred embodiment where the molecule of interest is a polypeptide, the hydrophobic groups are terminally attached. The hydrophobic group can be directly coupled to the molecule of interest or can be coupled through a linker. Suitable linkers for coupling hydrophobic groups are well known in the art.

Accordingly, the invention provides methods for delivering molecules (eg, polypeptides, oligonucleotides, oligosaccharides, lipids, etc.) into cells. The method provides a molecule (eg, a polypeptide) to be provided having attached at least two fluorophore molecules and a hydrophobic group, more preferably an Fmoc group, and then the molecule and the cell. Contacting with.

If the peptide, oligonucleotide, oligosaccharide or lipid is to be delivered in vivo for diagnostic endpoints or for therapeutic purposes, it has reduced toxicity or no toxicity at all. It will be appreciated that photogroups and hydrophobic groups are preferred. Thus, in a preferred embodiment, the fluorophore can be replaced by a non-toxic molecule with little or no biological activity. A preferred molecule is a fused ring compound that acts as a linker connecting the two ends of the molecule to be supplied. Particularly preferred fused ring compounds bring the spaces of excited dimers closer together.

Most preferred fused ring compounds include, but are not limited to, steroids. The relatively flat and hydrophobic fluorophores known for the formation of H-type dimers include the similarly hydrophobic and structurally rigid and / or flat fusions found in, for example, steroid molecules. Ring compounds, ie, steroid derivatives smaller than the complete steroid molecule, where 2
The ~ 3 fused 6-membered ring molecules can be cross-linked through conventional cross-linking agents to provide a size and overall hydrophobicity comparable to Fmoc and other hydrophobic groups described herein. The toxicity of such hybrid molecules is expected to be low because a safe metabolic pathway exists for large molecules composed of their small building blocks. In a preferred embodiment, the hydrophobic molecule is about 17 × 12Å in size.

If the peptide is to be delivered in vivo, a reduced toxic or non-toxic fluorophore is preferred. The toxicity of many fluorophores is well known to those of skill in the art (eg, Haugland, Handbook of Fluorescent Probes and Research Chemicals, 6
th Ed., Molecular Probes, Eugene, OR. (1996)). Furthermore, toxicity (eg LD ₅₀ ) can be readily determined according to standard methods well known to those skilled in the art. In the most preferred embodiment, the fused ring compound is a fused steroid, for example Latt et al., Structures XI and XII shown in (1965) J. Am. Chom. Soc. , Where -OR and -OR ₂ can act as activated points of attachment for the ends of peptides, nucleic acids or other molecules that it is desired to transport into the cell.

As indicated above, cellular uptake of most any molecule will be enhanced by the attachment of a hydrophobic group and a fluorophore or steroid crosslinker. Thus, suitable molecules include substantially any molecule which it is desired to introduce into a cell. Particularly preferred molecules are polypeptides (eg, protease inhibitors of the invention), and nucleic acids (eg, oligonucleotide HIV inhibitors (see, eg, Jing (1997) Biochem., 36: 12498-12505), ribozymes, peptides). Nucleic acid, and the like), but not limited thereto.

X. Activity Detection Kit The present invention also provides a kit for the detection of protease activity in a sample. The kit comprises one or more containers containing the fluorogenic protease indicators of the present invention. The indicator can be supplied in solution or attached to a solid support. Thus, the kit may include an indicator solution or indicator "dipstick", blotter paper, culture medium and the like. The kit can also include an indicator cartridge for use in an automated protease activity detector, where the fluorogenic indicator is attached to the solid support by the "receptor" fluorophore side.

The kit can further include instructions that teach the method and describe the use of the components of the kit. In addition, the kit may also include other reagents, buffers, various concentrations of protease inhibitors, stock proteases (for production of standard curves, etc.), culture media, disposable cuvettes and the like, according to the present invention. Included to aid in detection of protease activity using the emerging protease indicator. Additionally or alternatively, it will be appreciated that the kit comprises any of the other indicators described herein (eg, nucleic acid based indicators, oligosaccharide indicators, lipid indicators, etc.). . In this case, the kit will facilitate the detection of a particular activity / compound / interaction in which the particular indicator backbone is the substrate or binder.

XI. Protease Inhibitors It was also a discovery of the present invention that protease indicators could also act as protease inhibitors. Protease inhibitors and protease substrates share several basic properties, such as the ability to bind to the catalytic substrate binding site of a protease and the ability to form a relatively stable complex with the protease. Therefore, many common substrates or fragments thereof exhibit competitive substrate inhibition at higher concentrations. The inhibition is competitive because the inhibitor binds to the same substrate binding site of the protease, which competes with the native substrate for binding to the catalytic domain of the protease.

The present invention provides three novel approaches for the design of protease inhibitors. In the first approach, the normal substrate is redesigned so that it binds the protease well, but has a reduced (slow or absent) hydrolysis rate. Slow hydrolysis rates are achieved by introducing altered (different) conformations and / or conformational flexibility in the protease recognition domain. After the substrate (eg, native) binds to the substrate-binding site of the protease, the conformation of the peptide bond between P ₁ and P ₁ 'will become the transfer conformation of the peptide bond hydrolysis reaction of a given protease. Is distorted.

If this peptide bond and adjacent peptide bonds are altered so that they are not distorted, the rate of hydrolysis is relative to a substrate whose cleavage site peptide bond is easily distorted to the desired transfer conformation. Will be lowered. This approach is shown in Example 16, where it shows how protease recognition, the rate of hydrolysis of a substrate can be altered without changing the amino acid sequence.

In the second approach, inhibitors are generated by replacing critical P ₁ or P ₁ 'residues that make it difficult to distort the cleavage site peptide bond. Usually, the amino acid side chains of P ₁ and P ₁ 'specifically interact with the side chains of the protease catalytic domain. Their specific interactions facilitate the coordination of peptide bond distortions into the transfer conformation of hydrolysis reactions. So, for example, CP
When the critical P ₁ residue of the aspartic acid residue in the P32 protease substrate is replaced by uncharged asparagine, the normal interaction between the substrate and the protease is that the denatured substrate is the substrate binding site of the protease. It doesn't happen even when bound to.

Again, this leads to slower or zero hydrolysis rates. An example of the P ₁ residue substitution effect in the design of inhibitors is shown by the properties of the DEVN peptide (
See, for example, Figure 5 and Example 12). The biological conformation in which the substrate DEVN is an inhibitor is shown in Example 13. Additional evidence is given in Example 15 that the peptide DEVN binds to proteases. The P ₁ 'residue is a charged amino acid side chain, or a structurally rigid residue as shown in Table 3 (eg proline), ie DVV CC SMS (normal substrate) and DVVC C PdMS (inhibitor). The substrate sequence for the hepatitis C virus protease substrate NS3 NS5A / 5B of can be modified to introduce. The underlined residue is the P ₁ residue.

In a third approach, the amide bond between the P ₁ and P ₁ 'residues of the substrate results in a non-hydrolyzable chemical bond, such as the same amino acid side chain for the P ₁ and P ₁ ' residues. It may be changed to an ether, a thioether, a methylene bond, or an alkylene (C = C) or ether bond (COC (= O)) (but not limited to). Moreover, an amide bond, Retroinverso (retroinverso) binding, or other pseudo-amino acid binding, for example to replace the carbonyl group with a CH ₂ group CH ₂ -NH or C (= O) -
Can be replaced by S.

Examples The present invention is illustrated by the following examples. The examples are for illustrative purposes and are not a limitation of the present invention. Example 1 Synthetic peptide synthesis and derivatization of a fluorogenic molecule for detection of protease activity was performed by the method described in PCT publication pct / us98 / 03000 (wo98 / 37226). Incorporate in the specification)
.

Example 2. Fluorogenic protease indicators show strong signal when digested.
The extent of cleavage was determined by assaying for the appearance of indicator cleavage products in the presence of proteases, to show that the fluorogenic protease indicators of the present invention that supply the enzyme are readily digested by proteases.

Formula F ¹ -Asp-Ala-Ile-Pro-Nle-Ser-Ile-Pro-Cys-F ² (wherein F ¹ is the donor fluorophore (5 ′ linked to the aspartic acid through the α-amino group) -Carboxytetramethylrhodamine (C2211)), and F ² is a receptor fluorophore (Rhodamine X acetamide (R492)) linked through the sulfhydryl group of cysteine), about 1 μg of a protease indicator, 50 mM phosphorus. It was dissolved in a buffer consisting of sodium acidate, 1 mM EDTA (pH 8.9). To this solution was added 1 unit of elastase. The solution was analyzed by HPLC before and about 30 minutes after the addition of elastase. Digestion was performed at 37 ° C. The components separated by HPLC were collected at a wavelength of 550 nm which allowed detection of both the 5-TMR and R492 fluorophores.
Monitored at a wavelength of 580 nm which allows detection of the R492 fluorophore.

The results are shown in FIG. 1, which shows the HPLC profile of the fluorogenic protease indicator solution before and after addition of protease elastase. Figure 1 (a
) Shows HPLC before addition of elastase showing a single peak representing intact fluorogenic protease inhibitors. After addition of elastase (Fig. 1 (
b) and 1 (c)), there was no trace of a single peak in the late elution (Fig. 1 (a)) indicating complete digestion of the fluorogenic protease indicator. In addition, Figures 1 (b) and 1 (c)
The two predominant peaks in s suggest that digestion occurred predominantly at a single site. There are some small peaks indicating lower digestion at other sites within the peptide sequence, however, the significant preference of only two digestion peaks makes their second site not readily accessible to elastase. Indicates that

The change in the emission spectrum of the fluorogenic protease indicator after addition of elastase protease was monitored using the SLM Spectrofluorometer Model 48000 with the slit width set at 4 nm on both the excitation and emission sides. did. All measurements were performed at 37 ° C. The spectra in FIG. 2 show the spectra before (a) and after (b) addition of elastase.
) Of the fluorogenic protease indicator and the time-dependent increase in emission intensity of the indicator donor fluorophore after addition of elastase is plotted in FIG.

Fluorogenic protease inhibitors show more than a 10-fold increase in fluorescence at 589 nm after treatment with elastase protease (FIG. 2 (a) compared to FIG. 2 (b)).
, And a more than 5-fold increase in fluorescence occurred within 1000 seconds of the beginning of exposure to the protease. The change in intensity between the treated and untreated indicators is, to some extent, a function of the slit width used, as they represent the signal integrated through a particular slit width. Thus, if a wider slit width (eg, 8 or 16 nm slit) is used, an even higher signal will be provided in response to digestion.

Example 3. Fluorescence signal was due to dequenching of intramolecular energy. To show that the increase in fluorescence observed after protease treatment is due to dequenching of intramolecular energy. The signal generated by elastase digestion of the fluorogenic protease indicator was compared to the signal generated by elastase treatment of the same peptide backbone bound to either F ¹ (5-TMR) or F ² (R492). The change in the fluorescence intensity of the donor fluorophore after addition of 1 unit of elastase to an equiconcentration of the double fluorophore molecule and two single fluorophore molecules was investigated.

The results are shown in FIG. The double fluorophore molecule initially shows almost complete quenching, followed by a dramatic increase in fluorescence after addition of elastase, which reaches a constant value approximately 30 minutes after addition of elastase. It is shown (Fig. 4 (a)). In contrast, the two single fluorophore molecules showed virtually no initial quenching and no significant change in fluorescence after addition of elastase. In fact, the fluorescence level corresponded to that of a fully digested double-fluorescent indicator molecule (Fig. 4 (b)). These results show that the increase in fluorescence intensity of the fluorescent protease indicator is
This is due to the interruption of the resonance energy that is transferred intramolecularly from the donor fluorophore to the receptor fluorophore, and not the interaction between the fluorophore and the peptide backbone. This is based on binding to large or hydrophobic peptides
This is significant because the fluorescence of many hydrophobic fluorophores is quenched.

Example 4. Protease specificity is a function of indicator conformation.
Although not based on any particular theory, the fluorogenic protease indicators of the present invention are high due to their folded structure, and more particularly due to their relatively rigid U-shaped conformation. It appears to achieve a degree of protease specificity. The degree of extinction obtained from the molecule affects the average separation distance of the two fluorophores. Thus, if the protease indicator is present in a relatively unfolded or pliable state, the conditions that tend to cause the unfolded state (denaturation) are the fluorescence of the molecule in the absence of protease. Was expected to have little or no effect on. Conversely, if the molecule is relatively rigid, denaturing conditions enhance the fluorescent signal because it is expected that the mean separation of the fluorophore will be increased, thereby reducing the quenching effect. That would be expected.

Therefore, the effect of denaturing conditions on the fluorescence of the fluorescent light-sourced protease indicator in the absence of protease was determined. First, the change in fluorescence of the indicator of Example 1 as a function of the added chaotropic agent concentration (2M or 8M urea) was measured. When the fluorescent light-sourced protease indicator was denatured by the chaotropic agent, the fluorescence intensity increased with time and became flat due to the denaturation (unfolding) of the molecule.

These data show that the fluorescent folded protease indicators are stable folded conformations created by conformational determinants, as predicted by models based on energy minimization problem solving. It is present in the formation. The plateau fluorescence level indicates the remaining quenching of the fluorophore still linked by the fully denatured peptide backbone. Digestion of extended (denatured) peptides results in more than a two-fold increase in fluorescence, as the fluorophores can move further away from each other.

Example 5. Quenching and Opening of a Peptide Double Labeled with One Fluorophore The peptide backbone of the present invention double labeled with one fluorophore still achieves fluorescence quenching, thus Exhibiting quenching through other mechanisms besides resonance energy transfer is a surprising finding of the present invention. To assess that ground state dimerization and collisional quenching contribute to the overall observed quenching, a series of double-labeled peptides listed in Table 11 were synthesized.

In addition to comparing the absorption spectra of the dyes with the NorFes peptide labeled alone with the individual dyes, the emission spectra taken before and after cleavage were quenched by means other than resonance energy transfer (RET). % And the presence of fluorescence signal quenching were compared. The fluorophore was linked to the amino terminus through the α-amino group of the aspartic acid residue (D) and to the ε-amino group of lysine (K). Labeling was performed by displacement of the succinimidyl group linked to 6-TMR or DER. The structure of the peptide designated NorFES-KGY is as follows:

[0194]

[Chemical 13]

All double-labeled peptides, except fluorescein-NorFES-fluorescein, showed the presence of so-called ground state dimers, as determined from absorption spectroscopy. This was indicated by the shift of the absorption maximum to shorter wavelengths and the change in shape of the absorption spectrum when compared to the spectrum for the enzymatically digested, double-labeled sample. Based on cleavage by elastase, the ground-state dimer was destroyed and the resulting spectrum was the same as a solution containing the same concentration of each singly labeled peptide.

Although not based on any particular theory, the ground-state dimer formation observed in the compounds designed and synthesized according to the present invention is due to the fact that the U-shaped conformation of the peptide backbone renders the fluorophore extremely unlikely. It will be shown to bring them into close proximity, thus allowing the electron orbitals of the two fluorophores to overlap, resulting in reverse quenching through ground-state dimerization. It was a surprising finding that the polypeptides of the present invention allowed the formation of ground state dimers at significantly lower dye concentrations than previously observed.

For example, the ground state dimerization of free fluoroscein dye in solution is 0.74
Only observed at high concentrations above M, ground state dimerization of free eosin dye in solution was observed only at high concentrations above 2.8 × 10 ^-2 M (Forster and Konig, Ze
itschrift fur Electrochemie, 61: 344 (1957)), and ground state dimerization of rhodamine B dye in solution was only observed at concentrations above 6 × 10 ^-4 M (Arbeloa and Ojeda, Chemical Physics Letters, 87: 556 (1982)
checking). In contrast, in the present invention, the effect was observed at 4.0 × 10 ⁻⁷ M or at a concentration about 100 times lower than the reported value.

Observations of the ground state dimer for compounds synthesized according to the present invention are described in
Predicted significant levels of fluorescence quenching for double-labeled peptides having the same fluorophore as those compounds listed in the table. In fact, this prediction was confirmed.
That is, 6-TMR-NorFES-KGY-6-TMR, that is, a homo-double labeled peptide
Comparison with 6-TMR-NorFES-KGY-6-TMR shows that the degree of quenching is slightly higher in hetero- vs. homo- (94% vs 90%). However, the fluorescein derivative showed only 55% quenching. The symbols I _o and I _c for% fluorescence quenching (% Q) refer to the fluorescence intensity for intact labeled peptide and enzyme digested labeled peptide solution.

[0199]

[Table 17]

The substrate sequence is extended by one amino acid residue, and the fluorophore is attached through the episilon amino group on the side chain of the lysine residue without major concern for the amount of quenching observed. Obtained. In particular, this addition (a peptide designated K-NorFES-KGY) resulted in a slight increase in the cleavage rate and a very slight increase in% quenching for both hetero- and homo-double labeled peptides (K In the -NorFES-KGY peptide, the N-terminal labeling was through the epsilon amino group of lysine rather than at the α-amino terminus).

The rate of cleavage of those substrates by elastase also shows that the signal is 1/2 the maximum.
Was measured by recording the time after addition of protease (see Table 11). 3 homo-double labeled peptides, 6-TMR; DE
A comparison of two molecules of R and NorFES-KGY labeled with fluorescein (F1) shows the order of cleavage rates as follows: F1-NorFES-KGY-F1> 6-TMR-NorFES-KGY-6
-TMR> DER-NorFES-KGY-DER.

Example 6. Dye-dye dimers are formed in long peptides. Furthermore, we have found that (homo-double labeled) PAI-2, CS-1 (31 residue long peptides) and dyes. -Two DEVD-like peptides were synthesized and derivatized that do not allow dye dimer formation. The CS-1 peptide shows that in significantly longer peptides a dye-dye dimer structure can be formed. Note that this peptide contains four proline residues amino terminal to the putative cleavage site Ile-Leu bond. There is also one proline in the carboxyl domain. CS-1
The results from the peptides maintain a potentially large sequence between the two dyes (fluorophores). The amino acid sequences of two DEVE-like peptides that do not allow the formation of productive H-type dimers are F ₁ -DEVDGIDPK [F ₁ ] GY (SEQ ID NO: 2) and F ₁ -PDEVDGIDPK [F ₁ ] G
Y (SEQ ID NO: 3).

Example 7. Cellular uptake of substrates tested by flow cytometry and fluorescence microscopy The compounds listed in Table 12 were synthesized and assayed for cellular uptake. Substrate internalization of the substrate was determined by Jurkat cells (human acute T-cell leukemia system), HL-60 cells (human promyelocytic leukemia system), human lymphocyte system, A1.1 cells (murine T-cell system) and murine primary thymus. Tested with cells. The methods used to determine substrate uptake by viable cells are provided in Example 6 (for HPLC method), Example 2 (for fluorescence microscopy analysis), and Example 3 (for flow cytometry analysis). A summary of those analyzes on cellular uptake of substrate is provided in this example.

[0204]

[Table 18]

The data listed in Table 12 indicate that (1) the presence of only two fluorophores was not optimal for cell uptake, as indicated by constructs 2, 5, 7 and 9. (2) Addition of a 9-fluorenylmethoxycarbonyl (Fmoc) group at the α-amino group, and binding of only one fluorophore does not result in negligible cellular uptake (eg, compound 3); and ( 3)
Fluorophores and at least one Fmoc group allow for efficient cellular uptake of substrates (
Structures 1, 4, 6, 8, 10, 11 and 12).

Other experiments with two identical fluorophores and a protease substrate of the invention labeled with at least one additional hydrophobic group, such as the Fmoc group, fit this paradigm. Substitution of the Fmoc group with a low hydrophobic group and a small benzyloxycarbonyl group resulted in low levels of cellular uptake, but with hydrophobic groups such as DEVD.
Significantly better than the compound without Peptide Compound Structure 5.

The data show that Fmoc can be substituted with benzyloxycarbonyl, Z, or other hydrophobic groups such as: xanthyl (Xan), trityl (Trt), 4-
Methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3
6-trimethylbenzenesulfonyl (Mtr), mesitylene-2-sulfonyl (Mts)
, 4,4'-Dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7
, 8-Pentamethylchroman-6-sulfonyl (Pmc), 4-methylbenzyl (MeB
zl), 4-methoxybenzyl (MeOBzl), benzyloxy (BzlO), benzyl (B
zl), benzoyl (Bz),

3-Nitro-2-pyridinesulfenyl (Npys), 1- (4,4-dimethyl-
2,6-Dioxocyclohexylidene) ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z)
, 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl
(Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-
Butoxymethyl (Bam), t-butoxy (tBtO), t-butyl (tBu), acetyl (
Ac) and trifluoroacetyl (TFA).

When the acid group DEVD peptide on compound 5 was esterified with ethanol, this modified peptide did not show any enhanced cellular uptake by viable cells. Thus, the importance of the Fmoc group, and the two fluorophore-forming H-type dimers, are illustrated by this negative example.

Example 8. Esterases or Apopurotoshisu - Fluorescence microscopy analysis elastase substrate incubated cells with associated protease substrate, Fm-K [F1] DAIPNIuSIPK [F1] GY ( where, F1 is the carboxytetramethylrhodamine, Fm is an Fmoc , K [F1] is F1 covalently bound through the epsilon amino group of lysine (K), and Fm-K is a Fmoc group covalently bound at the α-amino group of the amino-terminal lysine residue). Used with -60 cells. The cells were treated with elastase substrate at varying concentrations ranging from 10 nM to 10 μM
Incubated for 60 minutes.

Next, the cells were diluted 5-fold with RPMI 1640 medium containing 5% serum or a phosphate buffer solution. The sample was centrifuged and washed again with 1 ml of wash solution. After centrifugation and removal of wash solution, the cell pellet was softened with approximately 25 μl of medium and the cells were transferred to glass capillaries. The tubules were then placed on glass microscope slides and examined under a fluorescence microscope using standard Rhodamine filters. For determination of apoptosis-related protease activity, listed in Example 8 10
Compounds (compound structures 2-13) at μM concentrations were incubated with cells for 30 minutes to 3 hours. The cells were then washed twice in the same manner. Using a glass capillary
The washed cells were transferred and examined under a fluorescent microscope.

Example 9. Flow cytometric analysis of cells incubated with apoptosis-related protease substrate The concentration of substrate used for flow cytometric analysis contains 4-10% fetal bovine serum.
10 μM in RPMI 1640 medium. During incubation with the selected substrates, cell densities ranged from 50,000 cells / ml to 4,000,000 cells / ml. Incubation time was 30 minutes to 3 hours at 37 ° C. and incubation volume was 50 μl to 2 ml. After incubating with the substrate for 30-60 minutes, the cell suspension was diluted 10-fold with ice-cold Hanks buffer solution (HBSS),
And then filtered through a nylon cloth sheet. The filtered cell suspension was submitted for flow cytometry analysis using an excitation source at 488 nm. Becton Dickenson,
Inc. Flow cytometric analysis FacSort was used for this flow cytometric analysis. Typically 10,000-30,000 phenomena were collected per sample.

Instrumental detector parameters were set using control cells without substrate incubation and samples with maximum expected fluorescent signal. For example, substrate compound # 11 Fm-CGD2D: Fm-K [F1] DBJGDEVDGIDGJPK [F1] GY (SEQ ID NO: 4
) (Where F1 is carboxytetramethylrhodamine; Fm is Fmoc, K
[F1] is F1 covalently linked through the epsilon amino group of lysine (K), NIu is norleucine, B is aminoisobutyric acid, and J is epsilon-aminocaproic acid) with Jurkat cells. After a minute of incubation, an increase in approximately 10 channels indicative of cellular uptake of substrate was measured. Note that Substrate # 11 was not completely quenched.

Therefore, a small amount of background fluorescence is expected from the intact substrate. Signals from cells activated with 1 μg / ml anti-Fas antibody, CH11 clone for 1-6 hours showed an increase in peak channel number. An increase of about 10 times the fluorescence intensity was observed. The cells contain apoptosis-inducing agents such as anti-Fas antibody and 50
This observed increase in fluorescence intensity was abolished when co-incubated with the CPP32 protease inhibitor ZVAD-fluoromethylketone at μM. This indicated that the signal from compound 11 was due to CPP32 protease activity that could be inhibited by ZVAD-FMK. Therefore, the observed fluorescence intensity in individual cells as determined by flow cytometry analysis served as a direct measure of intracellular CPP32 protease activity.

Example 10. Competitive Substrate Inhibitors Shown by Their Effect on Cell Lysate Hydrolysis of Apoptosis-Related Protease Substrates The level of CPP32 protease activity in 6-hour anti-Fas-stimulated Jurkat cell lysates was determined to be 50 μM substrate concentration of protease. Substrate DEVD-AF
Tested at 37 ° C with C, where AFC is aminofluoromethylcoumarin. The buffer used is 50 mM HEPES (Ph7.5, 10% w / v sucrose, 0.1% w
/ V CHAPS). Changes in fluorescence density were monitored by SLM 48000 Spectral Fluorometer. The rate of hydrolysis of DEVD-AFC was found to depend on the concentration of DEVD, DEVN and ICE substrates (Compounds 5, 7, and 9 in Table 12) present in the reaction mixture.

As the concentration of DEVD, DEVN and ICE increased to 25 μM, the rate of DEVD-AFC hydrolysis slowed down. Thus, the DEVD, DEVN and ICE substrates bind to the substrate binding site of the target protease, eg CPP32, and act as competitive inhibitors because their rate of hydrolysis is slower than that of the DEVD-AFC substrate. The substrate control peptide having its P ₁ residue mutagenized by the uncharged conservative residue Asn still retains the ability to bind to the protease substrate binding site and exhibit enzyme inhibition. It is surprising to find out.

Example 11. Substrate Delays and Inhibits Apoptosis Stimulation in Whole Cells Jurkat cells in RPMI 160 containing 10% fetal calf serum at 37 ° C. in a 5% CO ₂ atmosphere.
Proliferate in medium. When the serum content dropped to 4%, not only the Jurkat cell growth rate slowed down, but also a significant number of cells died within 36 hours. The cell density used was approximately 400,000 cells / ml. After 36 hours, control wells contained approximately 50% dead cells (trypan blue positive cells), but where 0.1 or 1
Compound # 11 (Table 12) “Fm-CGD2D” or Fm-K [F1] DBJGDEVDGIDGJP at a concentration of 0.0 μl
Wells containing K [F1] GY (SEQ ID NO: 6) showed only 10% or 8% non-viable cells. Thus, compound # 11, which exhibits effective cell uptake, delayed apoptosis in their Jurkat cells, where it acted as a CPP32 protease inhibitor or as a CPP32 activating protease inhibitor.

Example 12. Isolated anti -Fas antibody (37 2 hours at ℃, 1μg / ml) of intact and cleaved substrate fragments from cells induced to by apoptosis Jur
Kat cells were incubated with 10 μM substrate compound # 10 Fm-G2D2D. After incubation with this substrate for 1 hour, cells were washed with RPMI 1640 medium containing 4% serum (1 ml wash solution for every 100 μl incubation medium). Cells were washed 3 times and then lysed with cell lysis buffer containing Triton X-100. The cell lysate was then loaded onto a C ₄ reverse phase chromatography column and 0.0.
Analysis was performed using a water / acetonitrile eluent system containing 75% trifluoroacetic acid. Analysis showed the presence of intact substrate with two major new peaks eluting faster than intact substrate. The two recovered major peaks show a rhodamine absorption spectrum; therefore, they correspond to the two major substrate fragments produced on the basis of protease degradation of the substrate.

Example 13. Fluorescent signal DEVN (10 μM) from the DEVN substrate when mixed with a solution containing the target enzyme , a substrate control peptide that is a compound of Table 12, was used for protease digestion by apoptosis-activated Jurkat cell lysates. It was found to be resistant. Intensive digestion time did not result in a further increase in fluorescence intensity. HPLC reverse phase analysis of this reaction mixture confirmed the presence of the substrate which was not completely cleaved. Substitution of the P ₁ residue Asp by amino acid Asn uncharged resulted in conversion of protease substrate to the protease non-substrate.

This control peptide showed competitive substrate inhibition in experiments as described in Example 12. Furthermore, after addition of cell lysates, the fluorescence intensity monitored as a function of time initially showed a significant increase in fluorescence intensity, but after 15 minutes, this initial intensity level stabilized. Considering the absence of substrate cleavage by the protease present in cell lysates, the best explanation for this initial fluorescence intensity is due to the DEVN substrate binding to the protease and the substrate undergoing a conformational change. This conformational change involving the backbone of the substrate also affects the conformation of the two covalently linked fluorophore molecules with respect to each other in terms of average distance and relative orientation.

It has been found that the extent of the fluorescence quenching of those two fluorophores in the substrate structure is sensitive to their distance with respect to their dipoles and the particular orientation. Thus, any conformational change that affects those two aspects of the fluorescent reporting molecule is expected to affect the fluorescence quenching. Therefore, the conformational changes induced by the substrate binding to the substrate binding site of the protease were affected in the observed change in the initial fluorescence intensity, i.e. in the increase in the fluorescence intensity. Since the substrate cannot be cleaved, the increase in initial fluorescence intensity reaches a steady state. Exploiting this observed increase in fluorescence intensity due to a novel type of reading, eg, conformational change of the substrate rather than substrate cleavage as a degree of association between the substrate and its target binding molecule. You can

Example 14. Physiologically protease cleavage of suitable substrates for the various conformations determined domain (CDR) hydrolysis rate fluctuations Izure of predetermined proteases induced by changing the flexibility of the protease recognition domain by the amino acid sequence Sites can be classified into two cases. One is a serine protease inhibitor, such as a neutrophil elastase inhibitor, or α-1-antitrypsin, where the elastase recognition sequence is conserved fairly accurately by the remaining inhibitor molecule.

Based on cleavage by elastase, this protease-reactive site and its newly formed terminal residue showed significant conformation as revealed by high resolution crystal structure analysis of the reactive and cleaved inhibitor structures. Receive a change of home.
In the second type of protease cleavage site, the cleavage sequence is as present in free linear peptides in domains where the conformation is poorly defined or there is a significant amount of flexibility. Some degree of defined conformation, or maximally available backbone flexibility difference between the two potential substrates, may dictate the choice of a given protease for one substrate over another. It is said to bring.

Compounds 4 (Fm-DEVD), 10 (Fm-G2D2D) and 11 (Fm-CGD2D) show the same amount of forced conformational space or flexibility in a given substrate. We show how it can be introduced by a protease recognition domain, but by a different conformation determining domain or region (CDR), while retaining the bent forming function of that CDR. This example shows how the relative stiffness or flexibility of the central protease recognition domain can be altered by altering the conformational flexibility or stiffness of the CDRs.

The parent compound of Fm-DEVD has the following composition: Fmoc-K [F1] DB DEVDGID PK [F1] GY (SEQ ID NO: 7). Bold underlined letters are protease recognition sequences consisting of 7 amino acid residues. Compound # 10 contains two glycine extensions at each end of this protease recognition sequence. The central protease recognition domain is the eight-length residue GDEVDGID (SEQ ID NO: 8) because the glycine residue at the amino terminus is part of the native sequence. Naturally more flexible than other amino acids, such as alanine 2
The two glycine residues are less conformationally constrained, or conversely, compound 4 (Table 12).
It provides greater flexibility and thereby allows more bending when combined with Aib or Pro residues.

In addition to the methylene group present in glycine, the additional insertion of aminocaproic acid at both ends, along with the five methylene groups, further relaxed the forced conformation, and thus more than for the protease recognition domain GDEVDGID. Provides high flexibility. This progression of flexibility resulted in an increased rate of hydrolysis by the CPP32 protease, as CPP32 recognizes a more flexible protease recognition domain than elastase. Support for this reference is the CPP32 protease cleavage site in the proform of its physiological substrate, namely poly (ADP-ribose) polymerase,
PARP is located between two well-folded domains.

It is therefore expected that such protease cleavage sites may not be reliably retained or their conformation is less well defined than the rest of the molecule. Therefore, the introduction of flexible residues such as glycine, epsilon aminocaproic acid, β-alanine, and aminobutyric acid, in order to provide those structural features to the substrate, regulates backbone flexibility of the central protease recognition domain of the substrate. It is expected to play an important role in doing so. Those additional preferred residues for the conformational determinant domain are also predicted to confer the required bend-induced effect.

The observed altered hydrolysis rates for those three substrates lend themselves to success in controlling the flexibility of the protease recognition domain. This is manifested in the observed differences in proteolytic rates while maintaining the proper orientation for the two fluorophores that interact with each other through space. The importance of this conformational determinant domain in providing a means to regulate flexibility and in allowing orientation of the amino and carboxyl termini in appropriately tight spaces is due to the importance of those compounds (4 , 10 and 11).

Examples thereof provide tetrapeptides and pentapeptides comprising Lys-Asp-Aib-Gly or Lys-Asp-Aib-Ahx-Gly, where Ahx is episilon aminocaproic acid (ie, NH 3 ₂ - (CH _{2) 5} -COOH). The fluorophore is attached to the epicylon amino group of the leucine residue. The carboxyl-terminal CDR domain is a tripeptide Gly-Pr.
It is defined as o-Lys and the tetrapeptide Gly-Ahx-Pro-Lys. The hydrolysis rates were as follows: Compound 4 (Fm-DEVD: Fm-K [F1] DBDEVDGIDPK [F1] GY]) (SEQ ID NO: 9) and 10 (Fm-
G2D2D: Fm-K [F1] DBGDEVDGIDGPK [F1] GY) (SEQ ID NO: 9) was accelerated by 3 times.

As shown in FIG. 5, the hydrolysis rate was further measured by the aminocaproic acid (Ahx) adduct, compound 11 (Fm-CGD2D: Fm-K [F1] DB Ahx GDEVDGIDG Ahx PK [F1] GY).
(SEQ ID NO: 10)) resulted in about 3 times faster than the above glycine residue insertion.
Thus, an overall at least 9-fold increase in substrate hydrolysis rate was achieved (Table 12).
4 and 11) in.

Example 15. Structural features present invention fluorophores which form dimers intramolecular H- type in protease substrate homo - fluorophores that various possibilities for use of the double labeled fluorescent original composition, The strongest interrelationships between H-dimer formation and structural elements are, in order:
Delocalized charge, symmetry, and transition dipole size. Hydrophobicity was not observed to be the major determinant in this type of dimerization. In the experiments described herein, a new class of profluorescent protease substrates was designed and synthesized. Their novel fluorogenic indicators have spectral properties that are compatible with the excitation model; more specifically, the spectra of their polypeptides doubly labeled with rhodamine have blue-shifted absorption peaks and fluorescence. Indicates quenching, where both indicators are of H-dimer formation.

[0232] For example, NoreFes, an undecapeptide that is cleaved by the serine protease elastase, has six species on the opposite side of its cleavage site to identify structural elements of the dye that exhibit intramolecular H-type dimer formation. Homo-double labeled by the fluorophore. The absorption and fluorescence spectra of those six substrates obtained before and after enzymatic cleavage show the delocalized charge, the presence of subsequent symmetry, and the size of the lowest energy electron transfer dipole. Is an important factor in dimer formation. Surprisingly, there was no evidence that hydrophobic interactions were important in the fluorophore used in this study. The six fluorophores used in this study were rhodamine-X, tetramethylrhodamine, fluorescein, diethylaminocoumarin, hydroxycoumarin and pyrene.

Although the xanthene components of those two rhodamines (rhodamine-X, tetramethylrhodamine) have the same charge and symmetric structure, the distinguishing feature between them is that the tetramethylrhodamine's high transition dipole Size and low hydrophobicity. The spectra of the intact tetramethylrhodamine-derivatized substrate show that when comparing the absorption spectra of the two double-labeled intact peptides with the respective cleaved peptide solution spectra. It is noted that it exhibits a more pronounced charge than the X charge.

As shown above, the three conjugated ring components of fluorescein were charged at pH 9 compared to the two rhodamine derivatives with a +1 charge localized across the xanthene structure individually. Didn't. After separation of the dye (fluorescein) by degradation of the peptide, the loss of any significant shape change in the absorption spectrum is
Show the role for the charge in dimer formation. The little noticeable, but nevertheless measurable, quenching observed with this derivative is
Compared to the interaction consistent with previous studies of xanthene in solution, where either of the two rhodamines has a dissociation constant for dimer formation for fluorescein that is as much as 4 smaller than the dissociation constant for rhodamine, Directs a low, but measurable, interaction between the two fluoresceins.

The effect of dye symmetry was then tested with two coumarins, diethylaminocoumarin and hydroxycoumarin. This type of molecule does not contain symmetrical elements. Diethylaminocoumarin bears a delocalized positive charge on its two joined rings, similar to rhodamine, and hydroxycoumarin, similar to fluorescein, is neutral at pH 9. The diethylaminocoumarin-labeled NorFes spectrum shows a blue shift of 11 nm, and the hydroxycoumarin-labeled NorFes spectrum shows a slightly blue layer. The respective extinctions of intact peptides relative to the cleaved peptide solution, 76% and 28
% Is consistent with the importance of delocalized charge. Comparison of the less prominent spectra of the diethylaminocoumarin-derivatized peptide with that of xanthene supports the role of symmetry as a key element in H-dimer formation.

Finally, the role of hydrophobicity was investigated using pyrene, a fluorophore with S ₂ symmetry containing only carbon and hydrogen. No spectral changes are observed in either the absorption or fluorescence models, and the size of the transition dipole is extremely small. The results provide evidence for a possible role for hydrophobicity in H-dimer formation.

In summary, the strongest interrelationships between H-dimer formation and structural elements are, in turn, delocalized charge, symmetry, and transition dipole size. Hydrophobicity is
No major determinant was observed in this type of dimerization. The above examples are illustrative and do not limit the invention. Other variations of the invention will be readily apparent to those of ordinary skill in the art. All publications, patents and patent applications cited herein are hereby incorporated by reference.

Example 16 High Throughput Screening When the indicators of the present invention use fluorescent molecules that emit in the wavelength range from about 650 nm to about 720 nm, they are based on fluorescence, homogeneity, multiplexing, live cells and beads. Perkin Elmer Applied Biosystems FM for targeted screening assays
ATTM System 8100 Automated, Macro-Confocal Well suited for use in high throughput screening (HTS) systems.

Cells were plated at a density of 3-10 × 10 ³ cells / well in 96-well flat bottom plates. One population of cells was incubated with an apoptosis-inducing factor, such as staurosporine, at a concentration of about 1 μM for about 3-4 hours, and a second population was incubated with a vehicle, such as DMSO, for an equal amount of time. After the incubation period, PhiPhiLux (OncoImmunin, Inc.) cell-permeable fluorogenic substrate comprising two IC5 fluorophores was added at a final concentration of approximately 2 μM. Incubation was carried out for 1 hour. The plate was placed in the FMATTM8100 instrument and the number of fluorescent cells in each well was counted.

In a preferred embodiment, the system is simply “as is” by downward tuning the increase on the photomultiplier tube (decreasing detector sensitivity) until the desired signal is obtained relative to background levels. "used. Other modifications of the FMATTM8100 were made to enhance the utility of this instrumentation, especially to detect intracellular protease activity using the indicators of the invention. In one modification, the machine was modified so that a neutral density filter could be introduced on the emission side to reduce the total emission signal provided by the fluorescent indicator. This allows the detector to operate without reducing sensitivity. A continuously variable filter stepped filter (eg, filter wheel) allows selection of moderate signal reduction.

Variable pinholes, rather than fixed pinholes, can be provided in the FMAT ™ to allow selection of optical section thickness. Current instrumentation is fixed 100 μm
The optical section of is used. The preferred thickness range is 0.1 μM to 100 μm, and optical section thicknesses of about 10-20 μm are most preferred for use with the indicators of the present invention. Introducing the option of reduced image capture of bright fields and / or phases allows for the switching of protease indicator signals and images of cells. This facilitates measurement of the total number of cells in the field.

Increasing the FMAT magnification setting allows capture of subcellular localized images rather than lower resolution whole cell images. This facilitates localization of protease activity to specific subcellular organelles or domains. Introducing an ultraviolet laser excitation option with an existing helium-neon laser allows existing nuclear chromatins, such as the Hearchst dye, to be used to count nuclei, which facilitates cell counting.

The image analysis software provided with the FMAT 8100 can be modified.
Modifying the software to allow groups to be formed into one or more subpopulations, for example by applying various measured parameters, such as specific characteristic shapes, intensities, sizes, presence of specific markers, and others. can do. This allows, for example, one to correlate enzyme activity with one or more physiological parameters or markers. A modification of the software for the analysis of real-time captured images allows the number of cells or the number of subcellular features to be counted, which allows the device to be normalized to its data acquisition protocol.

The above examples are illustrative of the invention, but should not be construed as limiting the invention. Other variations of the invention will be readily apparent to those skilled in the art and are covered by the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference.

[Brief description of drawings]

FIG. 1A shows a D-NorFES-A protease inhibitor (F ¹ -Asp-Ala-Ile-Pro-Nle-Ser-Ile-P).
ro-Cys-F ² ) where F ¹ is the donor (D) fluorophore (5′-carboxytetramethylrhodamine (C2211) and F ² is the acceptor (A) fluorophore (rhodamine X acetamide (R492)) before and after addition of esterase, Figure 1A: HPLC before addition of elastase, showing post-elution peaks showing intact indicator molecules.

FIG. 1B shows a D-NorFES-A protease inhibitor (F ¹ -Asp-Ala-Ile-Pro-Nle-Ser-Ile).
-Pro-Cys-F ² ), wherein F ¹ is a donor (D) fluorophore (5′-carboxytetramethylrhodamine (C2211) and F ² is an acceptor (A) fluorophore (rhodamine X Figure 1B shows HPLC analysis of acetamide (R492) before and after addition of esterase, Figure 1B shows HPLC after addition of elastase, where both fluorophores absorb.
Detection at 550 nm is shown.

FIG. 1C shows a D-NorFES-A protease inhibitor (F ¹ -Asp-Ala-Ile-Pro-Nle-Ser-Ile.
-Pro-Cys-F ² ), wherein F ¹ is a donor (D) fluorophore (5′-carboxytetramethylrhodamine (C2211) and F ² is an acceptor (A) fluorophore (rhodamine X Fig. 1C shows HPLC analysis of acetamide (which is R492) before and after addition of esterase, and Fig. 1C shows HPLC after addition of elastase, which shows the maximum absorption of F ^2.
Detection at 80 nm is shown.

Figure 2A shows the emission spectrum of the D-NorFES-A- Fluorescent Light Source Protease Indicator before addition of elastase.

Figure 2B shows the emission spectrum of the D-NorFES-A- Fluorescent Light Source Protease Indicator after elastase addition.

FIG. 3 shows the increase over time of the fluorescein protease indicator of FIG. 1 as a function of time after addition of 1 unit of elastase.

FIG. 4A shows the fluorescence intensity of the donor fluorophore as a function of time after addition of 1 unit of elastase. Fluorescent light source protease indicator of Figure 1. D-NorFES
-A is an F ¹ -Asp-Ala-Ile-Pro-Nle-Ser-Ile-Pro-Cys-F ² protease indicator (wherein F ¹ is a donor fluorophore (5′-carboxytetramethylrhodamine (
C2211), and F ² is an acceptor fluorophore (Rhodamine X acetamide (
R492)). Each of D-NorFES and A-NorFES represents a molecule having the same peptide backbone, but providing only one of the two fluorophores.

FIG. 4B shows the fluorescence intensity of the donor fluorophore as a function of time after addition of 1 unit of elastase. FIG. 4B: Peptide backbone of the fluorescent light source protease of FIG. 1 labeled with either one of two fluorophores. D-NorFES-A is F ¹ -Asp-Ala-Ile-P
ro-Nle-Ser-Ile-Pro-Cys-F ² protease indicator, where F ¹ is a donor fluorophore (5′-carboxytetramethylrhodamine (C2211) and F ² is an acceptor fluorophore. (Rhodamine X acetamide (R492)).
Each of D-NorFES and A-NorFES represents a molecule having the same peptide backbone, but providing only one of the two fluorophores.

FIG. 5 shows the fluorescence of DEVD, DEVN and ICE substrates. Measurement buffer 50 mM HEPES buffer, pH 7.5, 10% (w / v) containing 1 μM substrate DEVD (Compound 2 of Example 8), DEVN (Compound 3 of Example 8) and ICE (Compound 5 of Example 8) ) Sucrose and 0.1% (w
/ V) (HAPS) to 100 μl, add 10 μl of Jurkat cell lysate,
And it incubated at 37 degreeC for 16 hours. This Jurkat cell lysate is
Prepared from cells stimulated with anti-Fas antibody at a concentration of 1 μg / ml for 6 hours. The fluorescence intensity of the substrate solution alone is shown in FIG. 5 as the horizontal line labeled as t = 0, and the fluorescence intensity of the mixture of cell lysate and substrate solution after 16 hours is shown as the vertical line, and t = Label as a 16-hour digest. Add 10 μl of cell lysate to 50 μM ZVAD-FM
Preincubated with K (benzoxycarbonyl-valanyl-alanyl-aspartyl-fluoromethylketone) for 30 minutes at 37 ° C and then added to the substrate solution.
The fluorescence intensity of this mixture after 16 hours is indicated by the bar labeled as ZVAD-FMK (inhibitor). Finally, the pre-incubated cell lysate was added to the substrate solution with iodoacetamide (alkylating agent for sulfhydryl groups) and PMSF (to inhibit serine proteases). The fluorescence intensity after 16 hours at 37 ° C is indicated by the bar labeled as iodoacetamide / PMSF. The DEVN substrate is a negative control substrate (P ¹
, Asp residues are replaced by Asn). CPP32 protease is P ¹
It requires that the residue is an aspartic acid residue. The activated lysates do not contain any other proteases that digest the DEVD substrate, as shown by the four bars in the DEVN substrate graph (Figure 5). This is because the 16-hour digestion intensity is the same as that of the substrate alone. The bar graph of DEVD substrate shows that activated cell lysates contain CPP32 protease and this protease activity is inhibited by ZVAD-FMK, a known CPP32 protease inhibitor. The contribution of other proteases to the digestion of DEVD substrates is very small, as shown by the difference between the intensity of ZVAD-FMK and iodoacetamide / PMSF rods.

FIG. Preferred dialkylated carbocyanine dyes for use in the method of the invention. X and Y are independently selected from the group consisting of (CH ₃ ) ₂ C, NH, O, S and the like. Preferably, N is greater than 0 and less than 20, more preferably N is greater than 0 and less than 10, and most preferably N is greater than 0 and less than about 5. In some embodiments, N is 1 or 2. R ¹ and R ² are independently selected from alkyl groups. R ³ to R ¹⁰ are independently H, alkyl, O-alkyl, Aruharaido, alkylated amine is selected from the group consisting of such amines. Z is an arbitrary counterion. In IC5, R ¹ is ethyl and R ² is 5- (N-carbonylpentyl). R ^{3 to} R ¹⁰ are H. X and Y are 3,3,
3 ', 3'-Tetramethyl (see, for example, IC5-OSu from Dojindo Laboratories).

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Packard, Beverly S.             Maryland 20852, United States,             Rockville, Pine Haven Terrace             10605 F term (reference) 4B063 QA01 QQ02 QQ03 QQ08 QQ20                       QQ36 QR41 QR48 QR66 QS11                       QS24 QS36 QX01 QX02

Claims (38)

[Claims] 1. The following formula: [Where P is DEVDGIN (SEQ ID NO: 11), (dO) DEVDGIN (SEQ ID NO: 12), DEVDGID
(SEQ ID NO: 13), LVEIDNG (SEQ ID NO: 14), GIETESGV (SEQ ID NO: 15), TGRT (SEQ ID NO: 16), VMTGRT (SEQ ID NO: 17), SEVKLDAEF (SEQ ID NO: 18), S (dE) VK (d
-L) DAE (dF) (SEQ ID NO: 19), EDVVCCS (SEQ ID NO: 20), EEVEGIN (SEQ ID NO: 21)
), D (dF) VDGIN, (dD) EV (dD) GIN, LVEIENG (SEQ ID NO: 22), GIETDSG (
SEQ ID NO: 23), GIETESG (SEQ ID NO: 24), LEHDGIN (SEQ ID NO: 25), LETDGIN (SEQ ID NO: 26), WEHDGIN (SEQ ID NO: 27), YVHDG (SEQ ID NO: 28), YVHDGIN (SEQ ID NO: 29), YVHDA (sequence) No. 30), TGRTG (SEQ ID NO: 31), S (dE) VK (dL) DAE (
dF) (SEQ ID NO: 32), IEPDS (SEQ ID NO: 33), PLGIAGI (SEQ ID NO: 34), IETDSGV
(SEQ ID NO: 35), SEVNLDAEF (SEQ ID NO: 36), YVHDAPV (SEQ ID NO: 37) and SQNY
A peptide selected from the group consisting of PIVQ (SEQ ID NO: 38); F1 and F2 are fluorophores, F1 is attached to the amino-terminal amino acid, and F2 is attached to the carboxyl-terminal amino acid S1 and S2, when present, are peptide spacers ranging in length from 1 to about 50 amino acids, S1 is attached to the amino-terminal amino acid when present, and S2 is a carboxyl when present. I, j, k, l, m, n, o, p, q, and r are independently 0 or 1; aa1 and aa10 are independently lysine, ornithine and Independently selected from the group consisting of cysteine; aa2, aa3, aa8, and aa9 are amino acids or Asp, Glu, Lys, ornithine, Arg
, Acitrutin, homociturtin, Ser, homoserine, Thr, and Tyr independently selected from the group consisting of dipeptides; aa5, aa4, aa6, and aa7 are proline, 3,4-dehydroproline, hydroxyproline, alphaaminoiso Independently selected from the group consisting of butyric acid and N-methylalanine; X is Gly, βAla, γAbu, Gly-Gly, Ahx, C7, βAla-Gly, βAla-βAla, γA
bu-Gly, βAla-γAbu, Gly-Gly-Gly, γAbu-γAbu, Ahx-Gly, βAla-Gly
-Gly, Ahx-βAla, βAla-βAla-Gly, Gly-Gly-Gly-Gly, Ahx-γAbu, β
Ala-βAla-βAla, γAbu-βAla-Gly, γAbu-γAbu-Gly, Ahx-Ahx, γAbu
-ΓAbu-βAla and Ahx-Ahx-Gly; Y is Gly, βAla, γAbu, Gly-Gly, Ahx, C7, Gly-βAla, βAla-βAla, Gly.
-ΓAbu, γAbu-βAla, Gly-Gly-Gly, γAbu-γAbu, Gly-Ahx, Gly-Gly-
βAla, βAla-Ahx, Gly-βAla-βAla, Gly-Gly-Gly-Gly, γAbu-Ahx, β
Ala-βAla-βAla, Gly-βAla-γAbu, Ahx-Ahx, βAla-γAbu-γAbu, and Gly-Ahx-Ahx; and when i is 1, S1 is the terminal alpha amino of aa1. A through a peptide bond through the group
S2 is bound to aa10 by a peptide bond passing through the terminal alpha carboxyl group of aa10 when bound to a1 and r is 1.]. 2. The composition of claim 1, wherein the carboxyl terminal amino and carboxylic acid groups are substituted with amides. 3. The composition of claim 1, wherein r is 0 and aa10 is a C-terminal amide group or free carboxylic acid group. 4. The amino acid sequences of Fa-KDPJGDEVDGINGJPKGY and Fm-KDPJGDEVDGIN.
GJPk amide, Fm-KDPJG (dO) DEVDGINGJPKGY, Fm-KDPJGDEVDGINGPKGY, Fm-KD
PGDEVDGINGJPKGY, Fm-KDPJGDEVDGIDGJPk amide, Fm-KDPJGLVEINDNGJPKGY, Fm
-KDPJGIETESGVGJPKGY, Fm-KDPJTGRTGPKGY, Fm-DPTGRTGPKGY, Fm-KDPVMTGRTG
JPKGY, Fm-KDPTGRTGJPKGY, Fm-KDPJGTGRTGJPKGY, Fm-KDPJGTGRTGPKGY, Fm-K
DPGTGRTGPKGY, Fm-KDPJGSEVKLGAEFGJPKGY, Fm-KDPJGS (dE) VK (dL) DAE (d-
F) GC5PKDDY, Fa-KDPJGEDVVCCSGJPKGY, KDPJGEEVEGINGJPKGY, KDPJGD (dF) VD
GINGJPKGY, KDPJG (dD) EV (dD) GINGJPKGY, KDPJGLVEIENGJPKGY, KDPJGIETDS
GJPKGY, KDPJGIETESGJPKGY, KDPJGLEHDGINGJPKGY, KDPJGLETDGNINGJPKGY, KDPJG
WEHDGINGJPKGY, KDPJGYVHDGJPKGY, KDPJGYVHDGINGJPKGY, KDPJGYVGHDAPKGY, KDP
JTGRTGJPKGY, KDPC3TGRTGPKGY, KDPC7TGRTGPKGY, KDPC5GS (dE) VK (dL) DAE
(DF) GJPKGY, KDPJGIEPDSGJPKGY, KDPJGPLGIAGIGJPKGY, and KDPJGSQNYPIVQ
The composition of claim 1 selected from the group consisting of GJPKGY. 5. The composition of claim 1, wherein F1 and F2 are the same fluorophore. 6. F1 and F2 have an excitation wavelength of about 315 to about 700 nm.
The composition according to. 7. The composition of claim 1, wherein the F1 molecule is attached through the α-amino group of the aa1 amino acid, or through the side chain amino group of the aa1 amino acid, or through the side chain sulfhydryl group of the aa1 amino acid. 8. The F2 molecule passes through the side chain amino group of aa10 amino acid, or aa10
2. The composition according to claim 1, which is linked through a carboxyl group of an amino acid or through a side chain sulfhydryl group of aa10 amino acid. 9. The phosphor is rhodamine X, 9- (2,5- (or 2,6) -dicarboxyphenyl) -3,6-bis (dimethylamino) xanthylium halide or other anion (TMR). ), 9- (2,5) -Dicarboxyphenyl) -2,7-dimethyl-3,6-bis (ethylamino) xanthylium halide or other anion (Rh6G), 9- (2,6)- Dicarboxyphenyl) -2,7-dimethyl-3,6-
Bis (ethylamino) xanthylium halide or other anion, 9- (2,5
-(Or 2,6) -dicarboxyphenyl) -3,6-bisamino-xanthylium halide or other anion (Rh110), 9- (2,5 (or 2,6) -dicarboxyphenyl) -3 -Amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, 9- (2,5-dicarboxyphenyl) -3
, 6-bis- (dimethylamino) xanthylium chloride (5-TMR), 9- (2
, 6-Dicarboxyphenyl) -3,6-bis- (dimethylamino) xanthylium chloride (6-TMR), 9- (2-carboxyphenyl) -2,7-dimethyl-3
, 6-bis (ethylamino) xanthylium, 9- (2-carboxyphenyl) -3
The composition of claim 1 selected from the group consisting of :, 6-bis (ethylamino) xanthylium, and 9- (2-carboxyphenyl) -xanthylium. 10. The composition of claim 1, wherein the composition has a hydrophobic group. 11. The composition according to claim 4, wherein the composition has a hydrophobic group. 12. The hydrophobic group is Fmoc, 9-fluoreneacetyl, 1-fluorenecarboxylic acid group, 9-fluorenecarboxylic acid group, and 9-fluorene-1-carboxylic acid group, benzyloxycarbonyl, xanthyl (Xan). , Trityl (Trt)
, 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,
3,6-Trimethyl-benzenesulfonyl (Mtr), mesitylene-2-sulfonyl (
Mts), 4,4-dimethoxybenzhydryl (Mbh), tosyl (Tos), 2, 2, 5, 7
, 8-Pentamethylchroman-6-sulfonyl (Pmc), 4-methylbenzyl (MeBz
l), 4-methoxybenzyl (MeOBzl), benzyloxy (MzlO), benzyl (Bz
l), 3-nitro-2-pyridinesulfonyl (Npys), 1- (4,4-dimethyl-2,6
-Diaxocyclohexylidene) ethyl (Dde), 2,6-dichlorobenzyl (2,
6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), 12. The composition of claim 11, selected from the group consisting of cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), acetyl (Ac), and trifluoroacetyl (TFA). 13. The composition of claim 12, wherein the hydrophobic group is Fmoc. 14. The composition according to claim 12, wherein the hydrophobic group is Fa. 15. The composition of claim 12, wherein the hydrophobic group is attached to the amino terminus of the molecule. 16. A method of detecting protease activity, which comprises contacting a protease with the composition of claim 1. 17. The method of claim 16, wherein the contacting is a histological section. 18. The method of claim 16, wherein said contacting is performed in cell culture. 19. The method of claim 16, wherein the contacting is seeded or cultured adherent cells. 20. The method of claim 16, wherein the contacting is performed in a cell suspension derived from a biological sample selected from the group consisting of tissue, blood, urine, saliva, lymph, biopsy. 21. The method of claim 16, wherein the detection is performed by a method selected from the group consisting of fluorescence microscopy, fluorescence microplate reader, flow cytometry, fluorometry, absorption spectroscopy. 22. Providing a molecule according to claim 1 attached to a hydrophobic group or at least one fused ring structure, and contacting the cell with the molecule, whereby the molecule enters the cell. A method of delivering a molecule into a cell, which comprises: 23. The hydrophobic group is Fmoc, 9-fluoreneacetyl, 1-fluorenecarboxylic acid group, 9-fluorenecarboxylic acid group, and 9-fluorene-1-carboxylic acid group, benzyloxycarbonyl, xanthyl (Xan). , Trityl (Trt)
, 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,
3,6-Trimethyl-benzenesulfonyl (Mtr), mesitylene-2-sulfonyl (
Mts), 4,4-dimethoxybenzhydryl (Mbh), tosyl (Tos), 2, 2, 5, 7
, 8-Pentamethylchroman-6-sulfonyl (Pmc), 4-methylbenzyl (MeBz
l), 4-methoxybenzyl (MeOBzl), benzyloxy (MzlO), benzyl (Bz
l), 3-nitro-2-pyridinesulfonyl (Npys), 1- (4,4-dimethyl-2,6
-Diaxocyclohexylidene) ethyl (Dde), 2,6-dichlorobenzyl (2,
6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), 23. The method of claim 22, selected from the group consisting of cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), acetyl (Ac), and trifluoroacetyl (TFA). 24. The phosphor is rhodamine X, 9- (2,5- (or 2,6)-
Dicarboxyphenyl) -3,6-bis (dimethylamino) xanthylium halide or other anion (TMR), 9- (2,5) -dicarboxyphenyl) -2,7-
Dimethyl-3,6-bis (ethylamino) xanthylium halide or other anion (Rh6G), 9- (2,6) -dicarboxyphenyl) -2,7-dimethyl-3,6
-Bis (ethylamino) xanthylium halide or other anion, 9- (2,
5- (or 2,6) -dicarboxyphenyl) -3,6-bisamino-xanthylium halide or other anion (Rh110), 9- (2,5 (or 2,6) -dicarboxyphenyl)- 3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, 9- (2,5-dicarboxyphenyl)-
3,6-bis- (dimethylamino) xanthylium chloride (5-TMR), 9- (2
, 6-Dicarboxyphenyl) -3,6-bis- (dimethylamino) xanthylium chloride (6-TMR), 9- (2-carboxyphenyl) -2,7-dimethyl-3
, 6-bis (ethylamino) xanthylium, 9- (2-carboxyphenyl) -3
23. The method of claim 22, selected from the group consisting of :, 6-bis (ethylamino) xanthylium, and 9- (2-carboxyphenyl) -xanthylium. 25. The phosphor is selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X and diethylaminocoumarin.
23. The method of claim 22. 26. The method of claim 22, wherein the cells are mammalian cells. 27. A protease or cells comprising a protease is contacted with a test agent, the protease is contacted with the fluorogenic composition of any one of claims 1-15, and the fluorogenic composition. The difference in the signal produced by the protease or cells contacted with the test factor, as compared to a control in which the signal or lack of signal produced by is detected, wherein the protease or cells are contacted with a lower concentration of the test factor. Demonstrating that said test factor modulates said protease activity, comprising screening a test factor for the ability to modulate protease activity. 28. The method of claim 27, wherein the lower concentration of the test factor is absent of the test factor. 29. The method of claim 27, wherein an increase in the signal produced by the protease or cell contacted with the test factor as compared to a control indicates that the test factor increases the protease activity. 30. The method of claim 27, wherein a decrease in the signal produced by the protease or cells contacted with the test agent as compared to a control indicates that the test agent reduces the protease activity. 31. The method of claim 27, wherein an increase in the signal produced by the protease or cell contacted with the test factor as compared to a control indicates that the test factor increases the protease activity. 32. The method of claim 27, wherein the protease is contacted with the fluorogenic composition in the presence of the test factor. 33. The method of claim 27, wherein the protease is contacted with the fluorogenic composition after removal of the test agent. 34. The method of claim 27, further comprising placing a test agent that modulates the protease activity in a database that includes a list of test agents that modulate the protease activity. 35. The detection according to claim 27, wherein the detection comprises detection of an intracellular signal.
The method described in. 36. The method of claim 27, wherein said detecting comprises microscopy. 37. The detection of claim 27, wherein the detection comprises flow cytometry.
The method described in. 38. The method of claim 27, wherein said detecting comprises high throughput screening of whole cells.