WO2003099846A2

WO2003099846A2 - System and method for screening for protease inhibitors and proteases

Info

Publication number: WO2003099846A2
Application number: PCT/US2003/016417
Authority: WO
Inventors: Chen-Chen Kan; Ting-Jen Cheng
Original assignee: Keck Graduate Institute
Priority date: 2002-05-22
Filing date: 2003-05-22
Publication date: 2003-12-04
Also published as: AU2003241604A8; AU2003241604A1; WO2003099846A3

Abstract

The present invention provides simple and effective systems and methods for screening for protease inhibitors or proteases. In one aspect, it uses a reporter protein that comprises an apoptotic protein and a recognition sequence of a protease of interest within the apoptotic protein to evaluate the effects of candidate inhibitors on the activity of the protease of interest. In another aspect, it uses a reporter protein that comprises an apoptotic protein and a protease recognition sequence within the apoptotic protein to determine whether a candidate protease is capable of recognizing and cleaving within or near the protease recognition sequence.

Description

SYSTEM AND METHOD FOR SCREENING FOR PROTEASE INHIBITORS AND PROTEASES

BACKGROUND OF THE INVENTION

Field of the Invention This invention relates to a novel method and system for screening for protease inhibitors and cloning of proteases.

Description of the Related Art

To date, only 400-500 proteases have known functions and been characterized. It has been shown that proteases play important roles in normal cell functions and in diseases and are targets for certain drugs in the treatment of cancers, parasitic, fungal and viral infections, and inflammatory, immunological, respiratory, cardiovascular and neurodegenerative disorders (Leung et al., J. Med. Chem. 43: 305-41 , 2000). It is estimated that the human genome encoded between 1000 and 2000 proteases, among which many will be potential targets for drug discovery projects (Southan, Drug Discov. Today 6: 681-8, 2001 ; Southan, FEBS Lett. 498: 214-8, 2001). The first step leading to the design of the appropriate protease inhibitors for therapeutics is the characterization of novel protease genes in the human genome.

The traditional approach is the cloning and expression of the gene of interest. After preparation of cell-free extracts or purification of the expression proteins, the protein samples will be subjected to various in vitro assay systems to measure the activities of proteolytic enzymes. Most of these methods utilize a peptide or chemical substrate and are based on that the protease cleavage would release a covalently bound reporter group of a chromogenic, fluorogenic, or immunological activity, with each having advantages and disadvantages in terms of sensitivity, specificity, cost, assay time, equipment needs, and background level. Accordingly, there is a need for a quick, simple, and effective screening method for the detection of proteolytic activities of candidate proteins. In addition, there is a need in the art for effective screening for inhibitors of newly identified proteases as well as known proteases. For Example, human immunodeficiency virus (HIV) protease is one of the translated products from the HIV structural protein pol gene. This protease cleaves Gag and Gag-Pol precursor polyproteins at ten or more distinct cleavage sites; each site has a distinct amino acid sequence (Debouck et al., Drug Devel. Res. 21: 1-17, 1990). This post-translational processing of precursor polyproteins is necessary for HIV to be replication competent. As such, HIV protease is a target for chemotherapy of HIV patients. Ren and Lien, Prog. Drug Res. Spec: 1-34, 2001.

The present invention fulfills the need for effective cloning for proteases. In addition, the present invention fulfills the need for effective screening for protease inhibitors, such as HIV protease inhibitors.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1 depicts an exemplary gene cassette for testing HIV protease inhibitor.

. Figure 2 shows the amino acid sequence of the bait region of human α2-macroglobulin. Arrows indicate the cleavage sites by individual proteases. The abbreviations and corresponding proteases are as follows: CO: collagenase (Enghild et al., J. Biol. Chem. 264:8779-8785, 1989); CP: cathepsins (Virca et al., Hoppe-Seyler's Z. Physiol. Chem. 364:1297-1302, 1984); CS: chymosin (Mortensen et al., FEBS Lett. 135:295-300, 1981); CT: chymotrypsin (Mortensen et al., FEBS Lett. 735:295-300, 1981); E: elastase (Sottrup-Jensen et al., FEBS Lett. 727:167-173, 1981 ; Virca et al., Hoppe- Seyler's Z. Physiol. Chem. 364:1297-1302, 1984); HIVPR: HIV protease (Meier et al., Ann. N.Y. Acad. Sci. 737:431-433, 1994); MP: metalloproteases (Anai et al., Toxicon 36:1127-1139, 1998); PA: papain (Mortensen et al., FEBS Lett. 735:295-300, 1981); PL: human plasmin (Mortensen et al., FEBS Lett. 135:295- 300, 1981); PSA: prostate-specific antigen (Otto et al., J. Urol. 159:297-303, 1998); RP: arginyl protease (Rangarajan et al., Biol. Chem. 381:57-65, 2000); S: subtilisin (Mortensen et al., FEBS Lett. 135:295-300, 1981); SL: stromelysin (Enghild et al., J. Biol. Chem. 264:8779-8785, 1989); T: trypsin (Hall et al., Biochem. Biophys. Rec. Commun. 100:8-16, 1981); TH: thrombin (Mortensen et al., FEBS Lett. 135:295-300, 1981); TL: thermolysin (Mortensen et al., 1981); and V8: Staphylococcus aureus V8 protease (Hall et al., Biochem. Biophys. Rec. Commun. 100:8-16, 1981). DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a simple and effective system and method of screening for protease inhibitors and cloning proteases. In one aspect, it uses a reporter protein that comprises an apoptotic protein and a recognition sequence of a protease of interest within the apoptotic protein to evaluate the effects of candidate inhibitors on the activity of the protease of interest. The use of this reporter protein allows the screening for protease inhibitors by simply determining viability of host cells in which the reporter protein is present. In certain embodiments, the systems and methods of the present invention also employ another reporter protein to verify and/or refine the analysis of the effects of candidate inhibitors on the protease activity performed with a reporter protein that comprises an apoptotic protein.

In another aspect, the present invention uses a reporter protein that comprises an apoptotic protein and a protease recognition sequence within the apoptotic protein to evaluate the proteolytic activity of a candidate protease. The use of this reporter protein allows the screening for proteins with proteolytic activities by simply determining viability of host cells in which the chimeric protein is present. In certain embodiments, the systems and methods of the present invention also use another reporter protein to verify and/or refine the evaluation of the proteolytic activity of a candidate protease.

A. Definitions

Prior to providing a more detailed description of the present invention, it may be helpful to an understanding thereof to provide definitions as used herein, as follows. Additional definitions are also provided throughout the description of the present invention.

An "apoptotic protein" refers to a protein that either itself or a fragment (portion or domain) thereof, when present in a host cell, promotes cell death of the host cell. In certain embodiments, an apoptotic protein of the present invention is a protein that promotes apoptosis in an animal cell. "Apoptosis" refers to a form of cell death characterized by a condensation and subsequent fragmentation of the cell nucleus during which the plasma membrane remains intact.

"Cloning for proteases" refers to obtaining and/or identifying and/or isolating a nucleic acid molecule that codes for a protease. In the embodiments where the host cell is a bacterial cell, an exemplary apoptotic protein of the present invention is a Bax protein, which is an apoptotic protein in animal cells. When a Bax gene is expressed in a bacterial cell, it also promotes bacterial cell death. Another exemplary apoptotic protein of the present invention is FADD. An intact FADD protein is pro-apoptotic in animal cells, but does not promote cell death in bacteria. However, the N-terminal death effector domain (DED) of an FADD is capable of causing bacterial cell death.

A "reporter protein," as used herein, refers to a protein (e.g., an apoptotic protein and β-galactosidase) having a recognition sequence of a protease of interest within the protein to facilitate the screening of candidate protease inhibitors or candidate proteases as described in detail below. In some embodiments, the reporter protein is a protein that has already a recognition sequence of a protease of interest. In other embodiments, the reporter protein is produced by inserting a recognition sequence of a protease of interest into another protein that originally does not contain the protease recognition sequence.

A "protease recognition sequence" refers to a consecutive amino acid sequence that is recognized, and required for proteolytic cleavage, by a protease of interest. A protease recognition sequence may be coincident with the protease cleavage site (i.e., the site at which the cleavage by the protease occurs). In other words, the protease recognition sequence may include one or more amino acids on either side of the peptide bond to be hydrolyzed by the protease. Alternatively, the protease recognition sequence may be one, two or more amino acids distal, at the amino or carboxy terminus, to the cleavage site of the protease. In certain embodiments, a protease recognition sequence of the present invention is derived from the amino acid sequence of a naturally occurring substrate of the protease of interest.

An "expression vector" refers to a nucleic acid molecule capable of replication and expressing a gene of interest when transformed into a host cell.

A "host cell" refers to a cell into which a foreign nucleotide sequence is transformed or transfected. The host cell of the present invention includes a bacterial cell, a yeast cell, an insect cell, an mammalian cell, an animal cell other than the above animal cells, and a plant cell. An "α2-macroglobulin" refers to a wide-spectrum protease inhibitor that forms a complex with all of four major classes of protease (i.e., aspartic, serine, cysteine, and metallo-proteases) and in turn undergoes a drastic change in its structure. In mammals, α2-macroglobulin is a major plasma protease inhibitor. α2-macroglobulin contains a region (referred to as the "bait region") in which multiple protease recognition sequences are clustered. The cleavage at the bait region by a protease forms a thio-ester bond and triggers a conformational change in α2-macroglobulin. This conformational change, in turn, entraps the enzyme without blocking its active site Enghild et al., J. Biol. Chem. 264:8779-8785, 1989; Roberts, J. Med. 16: 129-224, 1985; Barret, Methods Enzymol. 80: 737-54, 1981 ; Chu et al., Ann. N. Y. Acad. Sci. 737: 291-307, 1994).

B. Methods and Systems for Screening for Protease Inhibitors

As noted above, in one aspect, the present invention provides a system and method of screening for protease inhibitors.

1. Overview

The method of the present invention uses a reporter protein that comprises an apoptotic protein and a recognition sequence of a protease of interest within the apoptotic protein for evaluating the effects of a candidate inhibitor on the protease of interest. More specifically, a nucleic acid that encodes the reporter protein is transformed or transfected into a host cell. The host cell also contains a nucleic acid that encodes the protease of interest. Under conditions that allow the expression of both the reporter protein and the protease, the protease cleaves the reporter protein within or near the protease recognition sequence and thus alters the apoptotic activity of the reporter protein. However, in the presence of an inhibitor of the protease, the reporter protein is protected from being cleaved by the protease, and its apoptotic activity of the reporter protein remains unaltered or less altered than that in the absence of the inhibitor. Thus, by monitoring the apoptotic activities of the reporter protein (e.g., by evaluating host cell vitality) in the presence and absence of a candidate inhibitor, one may determine whether the candidate inhibitor has any inhibitory effects on the protease of interest. 2. Reporter Proteins That Comprise Apoptotic Proteins

As described above, the present invention uses a reporter protein that comprises an apoptotic protein and a recognition sequence of a protease within the apoptotic protein for screening for or identifying inhibitors of the protease. A reporter protein must have an apoptotic activity in host cells different from its cleavage products. In addition, if the reporter protein contains an insertion of a protease recognition sequence (i.e., the apoptotic protein itself does not contain a recognition sequence of a protease of interest), the insertion should not change the apoptotic activity of the apoptotic protein. For instance, an exemplary reporter protein may be constructed by inserting an HIV recognition sequence into human Bax protein and used in a bacterial system for screening protease inhibitors. It was shown that when human Bax was expressed as low as 0.01% of total protein in E. coli, it was sufficient to cause cell death. Asoh et al., J. Biol. Chem. 273: 11384-91 , 1998. An HIV protease recognition sequence (e.g., at the PR/RT cleavage site) may be inserted into human Bax protein to replace amino acid residues 63 to 71 of the Bax protein because the deletion mutant BaxΔ63-71 was shown to retain apoptotic activity in bacteria. Asoh et al., supra. In addition, fragments of human Bax have been indicated to have no or reduced activities in inducing bacterial death. Ishibashi et al., Biochem. Biophys. Res. Comm. 243: 609-16, 1998.

When the above exemplary reporter protein is used, an expression vector that comprises a nucleic acid encoding of this reporter protein is used to transform bacterial cells. When HIV protease gene is also expressed in the bacterial cells, the reporter protein will be proteolytically attacked to produce two Bax fragments that contain partial HIV protease recognition sequences. The resulting Bax fragments have no or reduced activities in promoting or inducing bacterial death than the intact reporter protein. Accordingly, the bacterial cells remain alive or have a relatively low mortality rate. Conversely, when the HIV protease is inactive due to the inhibition of an HIV protease inhibitor, the reporter protein remains intact and induces bacterial cell death.

Another exemplary reporter protein useful in a bacterial system for screening protease inhibitors may be constructed by inserting an HIV protease recognition sequence into human FADD protein. It was shown that full-length FADD protein did not induce bacterial cell death, whereas the N-terminal DED (i.e., amino acids 1-88) of FADD did. Lee et al., Mol. Microbiol. 35: 1540-9, 2000. An HIV protease recognition sequence may be inserted into FADD at the junction between DED and the remaining portion of the FADD (i.e., the death domain (DD)) to provide a reporter protein. When a nucleic acid encoding the above reporter protein is expressed in bacterial cells, in the presence of an HIV protease, the reporter protein is cleaved into two fragments: one fragment having DED of FADD and a partial HIV protease recognition sequence, the other having DD of FADD and the remaining HIV protease recognition sequence. Because the fragment containing DED induces bacterial cell death, the presence of HIV protease causes no, or fewer and/or smaller bacterial colonies. Conversely, when HIV protease is inactive in the presence of an inhibitor, the reporter protein remains intact, and bacterial cells remain alive.

Besides Bax and FADD, any apoptotic proteins that meet the above requirements may be used to construct reporter proteins to screen for inhibitors of interest. Exemplary apoptotic proteins that may be used in the present invention include, but are not limited to, Apaf-1 , Bcl-xS, Bad, Bak, Bid, Bik, Blk, Bok, Egl-1 , Hrk, Nbk, Nip-3, and various caspases (e.g., caspases 1- 14).

For a given protease, any recognition sequence of the protease may be used to construct a reporter protein. The recognition sequence may be a portion of a naturally occurring protease substrate or may be an artificial polypeptide. For instance, the cleavage sites of HIV protease include the following polypeptides:

P17/P24: Ser Gin Asn Tyr Pro lie Val Gin P24/X: Ala Arg Val Leu Ala Glu Ala Met

X/p7: Ala Thr lie Met Met Gin Arg Gly

P7/p6: Pro Gly Asn Phe Leu Gin Ser Arg

P6/PR: Ser Phe Asn Phe Pro Gin He Thr

PR/RT: Thr Leu Asn Phe Pro lie Ser Pro RT5/RNase H: Ala Glu Thr Phe Tyr Val Asp Gly

RT/IN Arg Lys lie Leu Phe Leu Asp Gly

DEG1 Gin He Thr Leu Trp Gin Arg Pro

DEG2 Asp Thr Val Leu Glu Glu Met Ser

DEG3 Asp Gin He Leu He Glu lie Cys See, e.g., Debouk, AIDS Res. and Human Retroviruses 8: 153, 1992; U.S. Pat. No. 5,436,131. An exemplary protease recognition sequence of polio 3C protease is Met Glu Ala Leu Phe Gin Gly Pro Leu Gin Try Lys Asp. Pallai et al., J. Biol. Chem. 264: 9738-41 (1989). Additional protease recognition sequences may be found in Published U.S. Pat. Appl. 2002/0009715.

In certain embodiments, an amino acid sequence that comprises multiple protease recognition sequences may be present in a reporter protein. In such embodiments, the reporter protein may be used to screen for inhibitors of one or more proteases of which recognition sequence is present in the reporter protein. An exemplary amino acid sequence is the bait region of human α2-macroglobulin as shown in Figure 2. In certain embodiments, a protease recognition sequence may be a fragment of the bait region of an α2- macroglobulin, such as an amino acid fragment that contains most of the recognition sequences by various proteases in the bait region of human α2- macroglobulin. In other embodiments, a protease recognition sequence may comprise a portion of the bait region of an α2-macroglobulin having one, two or more, but not all the recognition sequences within the bait region. Such a portion may have 4-20, 5-15, or 6-8 amino acids.

The insertion of a cleavage site of a protease into an apoptotic reporter protein may be performed by any methods known in the art. For instance, the gene encoding an apoptotic reporter protein may be digested at a specific position, and an oligonucleotide encoding a cleavage site of a protease may be synthesized and ligated with the digested apoptotic reporter gene fragments at the specific position. In certain embodiments, when the cleavage sites are inserted into a reporter protein, one, two, or more additional amino acids flanking the cleavage sites may be introduced into the resulting reporter protein.

3. Proteases

The present invention may be used to screen for inhibitors of any proteases of interest. In addition to screening for inhibitors of naturally occurring proteases (including naturally occurring variants of wild type proteases), the present invention may also be used to screen for inhibitors of proteases that are artificially mutagenized but remain enzymatically active.

Exemplary proteases include those that have been implicated in human diseases. For instance, human renin is an aspartyle protease that has been implicated in hypertension. Imai et al., J. Biochem. 100: 425-32, 1986. The proteases trypsin and elastase are involved in the onset of emphysema. Cox and Levison, Am. Rev. Respir. Dis. 137: 371-5, 1988; Albin et al., Am. Rev. Respir. Dis. 135: 1281-5, 1987. Certain proteases are essential for the replication of microbial pathogens (e.g., poliovirus, HIV proteases), whereas other proteases are involved in the destructive effects of microbial pathogens in ways that do not involve replicative processes (e.g., collagenases from bacterium Clost dium histolylicum that participate in the invasiveness of the bacterium by destroying the connective tissue barriers of the host). Additional exemplary proteases include those of pathogenic organisms that are resistant to certain drugs. These proteases may be derived from wild type proteases during drug treatments of a host of the pathogenic organisms (e.g., HIV protease mutants).

4. Additional Reporters

In addition to using an apoptotic protein having a recognition sequence of a protease of interest as a reporter, in certain embodiments, the present invention also employs an additional reporter (or reporters) to verify or refine the inhibitor screening assays. The additional reporter may be, or derived from, any protein of which at least one of activities is detectable and that meets the following requirements. If the protein has already a recognition sequence of a protease of interest, the cleavage of the protein by the protease of interest at or near the recognition sequence must alter (i.e., increase, reduces or eliminates) the detectable activity of the protein. If the protein does not already have a recognition sequence of a protease of interest, this recognition sequence of the protease of interest needs to be inserted into the protein. Such an insertion must have no or little effects on the detectable activity of the protein, but the cleavage of the resulting reporter protein by the protease of interest must alter the detectable activity of the protein.

For instance, β-galactosidase with a protease recognition sequence inserted therein may be used as an additional reporter for the screening assays with bacterial cells as exemplary host cells. A protease recognition sequence (e.g., a p6/PR HIV protease cleavage site with a sequence of Val Ser Phe Asn Phe Pro Gin We Thr Leu) may be inserted to Sau I site at the codon number 80 of the β-glactosidase gene of E. coli. The resulting protein retains the β-galactosidase activity. The cleavage of the resulting protein by HIV protease that is co-expressed in E. coli causes the loss of β- galactosidase activity. See, U.S. Pat. No. 5,436,131; EP Pat. Appl. No. 0 421 109; and Baum et al., Proc. Natl. Acad. Sci. USA 87: 10023-7, 1990. Thus, in the presence of an HIV protease inhibitor, bacterial colonies that express HIV protease and contain the vector for expressing the above chimeric protein are blue on X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside)-containing medium plates. They are normally of white color due to the activity of the expressed HIV protease. The activity of β-galactosidase of the bacterial cells in the presence or absence of a protease inhibitor may also be quantified by various methods known in the art.

Additional exemplary proteins that may be utilized as, or in constructing, reporter proteins include, but are not limited to, alkaline phosphatase, β-glucuronidase, acetyltransferase, luciferase, green fluorescent protein, red fluorescent protein, aequorin, chloramphenicol acetyl transferase, horseradish peroxidase and variants thereof. "Variants" refers to proteins that contain amino acid deletion(s), insertion(s) or substitution(s) of an original protein but still retain the activities of the original protein.

5. Expression of Protease and Reporter Genes

Upon selection of an appropriate reporter protein (or reporter proteins), the expression of the nucleotide sequence(s) encoding the reporter protein(s) as well as the nucleotide sequence encoding a protease of interest in host cells may be accomplished by any methods known in the art. For general discussion of expressing foreign genes in a host cell, see, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NN., 2001.

In certain embodiments, the nucleotide sequence encoding an apoptotic protein with a recognition sequence of a protease of interest and the nucleotide sequence encoding the protease of interest are present in a single expression vector. In addition, if an additional reporter protein is used, the nucleic acid that encodes for the additional reporter may also be present in the same expression vector. Alternatively, the nucleic acid encoding reporter protein(s) and the nucleic acid encoding a protease of interest may be present in different vectors and subsequently co-transformed or co-transfected into a host cell.

In the embodiments where the nucleotide sequence encoding reporter protein(s) and the nucleotide sequence encoding the protease of interest are present in a single vector, these nucleic acids may be under the control of a single promoter, or they may be under the control of different promoters. The use of a single promoter allows coordinate regulation of the expression levels of reporter protein(s) and that of a protease of interest, whereas the use of different promoters enables differential regulation of the expression levels of reporter protein(s) and that of a protease of interest.

When the nucleotide sequences are under the control of a single promoter and the host cells are prokaryotic cells, the nucleotide sequences may be arranged to be transcribed to form a polycistronic mRNA, which is in turn translated to provide the reporter protein(s) and the protease of interest. Alternatively, the nucleotide sequences may be arranged into a gene cassette so that the cassette encodes a polyprotein that comprises the apoptotic protein with a protease recognition sequence, the protease of interest, and the additional reporter protein (if any) linked with each other via recognition sequences of the protease. The resulting polyprotein may be self-processed into individual protein components or fragments thereof by the action of its protease portion at or near the protease recognition sequence(s) within the junctions between the individual protein components and within the reporter protein(s). The above gene cassette is also applicable to screening systems that uses eukaryotic cells as host cells An exemplary gene cassette for testing HIV protease inhibitor in a bacterial system is illustrated in Figure 1. As indicated in this figure, the gene cassette encodes a polyprotein that comprises β-galactosidase, HIV and Bax proteins. There are four HIV protease recognition sequences present in the polyprotein: a p6/PR cleavage site within β-galactosidase, a p6/PR cleavage site between β-galactosidase and HIV protease, a PR/RT cleavage site between HIV protease and Bax protein, and a PR/RT cleavage site within Bax protein.

This gene cassette may be under the control of an inducible promoter (e.g., an IPTG (isopropylthiogalactopyranoside)-inducible Tac promoter) and transformed into an appropriate bacterial host strain (e.g., E. coli strain MC 1061 (araD139, Δ(ara, leu)7697, ΔlacX74, galU, galK, hsdR, strA). In the absence of an HIV protease inhibitor but in the presence of IPTG, the HIV protease portion of the polyprotein is active and cleaves the polyprotein at the above four cleavage sites to produce two β-galactosidase fragments, HIV protease, and two Bax protein fragments. 6. Protease Inhibitor Screening Assays

As described above, the effects of a candidate inhibitor on a protease of interest may be evaluated by monitoring the activities of reporter protein(s) in the presence or absence of the candidate inhibitor. For a reporter protein that comprises an apoptotic protein, cell viability (used exchangeably with "cell vitality") of host cells in which the reporter protein is present may be measured to determine the activities of the reporter protein. Methods of measuring host cell viability such as measuring bacterial cell viability are well known in the art, including counting the number of bacterial colonies, determining the sizes of bacterial colonies and the use of commercially available bacterial viability kit (e.g., BacLight bacterial viability kit, Molecular Probes). Baum et al., supra; Asoh et al., supra; Lee et al., supra; and Ishibashi et al., supra. The availability of simple and rapid methods for determining bacterial cell viability allows the present invention to be used effectively in screening for a large number of candidate inhibitors.

For additional reporter protein(s), any methods known in the art may be used in determining their activities. For instance, for using a β- galactosidase with a recognition sequence of a protease of interest as a reporter in a bacterial system, the activity of the reporter may be determined by plating the bacteria cells that express the gene encoding the reporter on a X- Gal-containing medium plate. If the β-galatosidase is active, the colonies of the bacterial cells are blue. Conversely, if the β-galactosidase is inactive, the bacterial colonies are white. The activity of this reporter protein may be further quantified by the use of O-nitrophenyl-β-D-galactoside (ONPG) or 4- methylumbelliferyl-β-D-galactoside (MUG) as a substrate. Rothstein et al., Proc. Natl. Acad. Sci. USA 77: 7372-6, 1980; Miller, Experiments in Molecular Genetics, Cold Spring Harbor, N Y., 1972; Roth, Methods Biochem. Anal. 17: 189-285, 1969; Youngman, in Plasmids: A practical approach (ed. K.G. Hardy), pp. 79-103, IRL Press, Oxford, United Kingdom, 1987. The use of the additional reporter protein(s) verifies and refines the inhibitor screening conducted by the use of a chimeric apoptotic protein as a reporter as described above.

A protease inhibitor screening system of the present invention may be initially tested using a known inhibitor of a protease of interest. For instance, examples of known HIV protease inhibitors include Amprenavir (AGENERASE®), Saquinavir (INVIRAS® and FORTOVASE®), Nelfinavir (VIRACEPT®), Ritonavir (NORVIR®) and Indinavir (CRIXIVAN®). For a general discussion of known HIV protease inhibitors, see Ren and Lien, supra. In addition, these known HIV protease inhibitors may be used as references for determining the relative inhibitory activity of a particular candidate protease inhibitor.

For a given candidate protease inhibitor, its effects on the activity of a reporter (or activities of reporters) may be determined by comparing the activity of the reporter in the presence of the candidate protease inhibitor with that in the absence of the candidate protease inhibitor. In addition, the effects of a candidate protease inhibitor at various concentrations on the activity of a reporter may be further obtained to determine the concentration (IC₅₀) at which the activity of the reporter is reduced to be 50% of the activity in the absence of the candidate protease inhibitor.

A protease inhibitor screening system according to the present invention may be further optimized. For instance, the expression level of the chimeric reporter protein(s) of the system as well as that of the protease of interest may be regulated to obtain the optimal sensitivity for inhibitor screening. Such regulation may be accomplished by placing the nucleic acid encoding the reporter protein(s) or the protease of interest under the control of promoters with different transcription-promoting strengths. Alternatively, if the promoter is inducible, the expression level of the gene(s) under its control may be regulated by changing the concentration of an inducing agent of the promoter.

In addition, one of ordinary skill in the art also appreciates that appropriate controls need to be performed when using the inhibitor screening system. For instance, host cells may be treated with a candidate inhibitor alone to determine its toxicity to these cells. Such toxicity should not be confused with the effects of the protease inhibitor when certain chimeric apoptotic proteins (e.g., FADD) are used as reporters.

C. Methods and Systems for Screening for Proteases In another aspect, the present invention provides a system and method of screening for proteases or evaluating proteolytic activity of candidate proteases. 1. Overview

The present invention uses a reporter protein that comprises an apoptotic protein and a protease recognition sequence within the apoptotic protein to determine whether a candidate protease can recognize and cleave within or near the protease recognition sequence. More specifically, a nucleic acid that encodes the reporter protein is transformed into a host cell. The host cell also contains a nucleic acid that encodes a candidate protease. Under conditions that allow the expression of both the reporter protein and the candidate protease, if the candidate protease is capable of recognizing the recognition sequence within the apoptotic protein, the reporter protein is thus cleaved and its apoptotic activity is altered. However, if the candidate protease is incapable of cleaving the recognition sequence within the apoptotic protein, the reporter protein remains intact and its apoptotic activity of the reporter protein remains unaltered. Thus, by monitoring the apoptotic activities of the reporter protein (e.g., by evaluating bacterial cell vitality) in host cells that express or do not express a nucleic acid that encodes the candidate protease, one may determine whether the candidate protease recognizes the protease recognition sequence and cleaves at or near the protease recognition sequence within the apoptotic protein.

2. Reporter Proteins Comprising Apoptotic Proteins

Reporter proteins that comprise apoptotic proteins useful for screening candidate protease inhibitors as described above can also be used for screening candidate proteases. The presence of apoptotic proteins in the reporter proteins enables a simple and convenient detection method for proteolytic activities by monitoring cell viability of bacterial cells co-expressing the reporter proteins and candidate proteases.

In certain embodiments, a reporter protein comprises multiple protease recognition sequences, such as the bait region of an α2-macroglobulin or fragments thereof. Such a reporter protein is especially useful because it can be used for screening for proteases with different substrate specificities.

3. Proteases

Any proteins that are suspected to have proteolytic activities may be tested using the method and system of the present invention. They include previously undiscovered protease or variants of previously known proteases having altered substrate specificity or proteolytic activities.

The nucleotide sequence that encode a candidate protease may be obtained from a cell known or suspected to express a protease. Cells suspected to express protease may be obtained, for example, from tissues known to produce substantial proteolysis, such as a tumor. Cells infected by a pathogen (e.g., an HIV virus) that express proteases, can also provide a nucleotide sequence encoding a candidate protease. Protease-expressing cells can be identified using known protease assays such as employing chromogenic or fluorescent substrates. Substrates and protease assay kits are commercially available from companies such as Promega, and Clontech.

Nucleotide sequences that encode candidate proteases can be present in plasmids of a cDNA expression library constructed from cells known to express a protease. Methods for preparing cDNA expression libraries are well known in the art, and any such methods can be used (see, Sambrook and Russell, supra).

As noted above, candidate proteases may be variants of previously known proteases having altered substrate specificity or proteolytic activities. The nucleotide sequence that encodes such a candidate protease may be purified from a source that is suspected to contain the candidate protease (e.g., HIV patients that undergo protease inhibitor treatments). Alternatively, the nucleotide sequence can be obtained by mutagenizing, either randomly or at specific sites, a nucleotide sequence encoding a known protease.

4. Additional Reporters

Additional reporters that comprise proteins other than apoptotic proteins useful for screening candidate protease inhibitors described above can also be used for screening candidate proteases. These additional reporters enable verification or refinement of the analysis performed by the use of reporter proteins that comprise apoptotic proteins.

In certain embodiments, a reporter protein having an amino acid sequence that comprises multiple protease recognition sequences, such as the bait region of an α2-macroglobulin or fragments thereof. Such a reporter protein is especially useful because it can be used for screening for proteases with different substrate specificities.

5. Expression of Protease and Reporter Genes

Upon selection of an appropriate reporter protein (or reporter proteins), the expression of the nucleotide sequence(s) encoding the reporter protein(s) as well as the nucleotide sequence encoding a protease of interest in host cells may be accomplished by any methods known in the art.

In certain embodiments, the nucleotide sequence encoding a candidate protease and the nucleotide sequence(s) encoding for reporter protein(s) may be present in different expression vectors. Such an arrangement allows convenient comparison of the activity of the reporter protein(s) in host cells that co-express the candidate protease and the reporter protein(s) with that in host cells that express only the reporter proteins but not the candidate protease. The difference between the activities of the reporter protein(s) in the above two types of bacterial cells indicates whether the candidate protease recognizes the protease recognition sequence in the reporter protein(s).

Alternatively, the nucleotide sequence encoding an apoptotic protein with a protease recognition sequence and the nucleotide sequence encoding the candidate protease are present in a single expression vector. In addition, if an additional reporter protein is used, the nucleotide sequence that encodes the additional reporter may also be present in the same expression vector. In certain embodiments, the nucleotide sequences encoding the reporter(s) and the candidate protease may be under the control of a single promoter, or they may be under the control of different promoters. The use of a single promoter allows coordinate regulation of the expression levels of reporter protein(s) and that of a candidate protease, whereas the use of different promoters enables differential regulation of the expression levels of reporter protein(s) and that of candidate protease. In embodiments where the nucleotide sequence encoding reporter(s) and the nucleotide sequence encoding a candidate protease are present in a single expression vector, an additional expression vector that comprises the nucleotide sequence(s) encoding the reporter(s), but not the nucleotide sequence encoding the candidate protease, may be needed. The activities of the reporter(s) in host cells transformed with this additional expression vector may be used as a reference for the activities of the reporter(s) in host cells transformed with an expression vector encoding both the candidate protease and the reporter(s). Ideally, the two vectors are identical in nucleotide sequences except in one aspect: One contains the nucleotide sequence encoding the candidate protease, and the other lacks the nucleotide sequence encoding the candidate protease.

It will be apparent that the choice of host cells is governed by the particular proteolytic activity to be assayed and by the particular protease recognition sequence present in the reporter protein(s). For a given protease activity (i.e., the activity of a protease that recognizes a particular protease recognition sequence and subsequently cleaves within or near the protease recognition sequence), the endogenous proteases (i.e., proteases normally expressed by the chosen host cells) must be unable to bind, and cleaves within or near, the recognition sequence in the reporter protein(s) to any significant extent. The proteolytic activity of an endogenous protease that recognizes the protease recognition sequence of the reporter protein(s) may be determined by transforming the selected host cell with only the nucleotide sequence(s) encoding the reporter protein(s) and then growing that host under conditions which cause expression of that nucleotide sequence(s) and which would cause expression of the candidate protease-encoding nucleotide sequence if that sequence were present.

Preferably, a host cell is deficient in various protease activities. Such a host cell is especially useful when reporter proteins comprises multiple protease recognition sequences, such as the bait region of an α2-macroglobulin or fragments thereof. An example of bacterial host strains deficient in protease activities is disclosed in U.S. Pat. No. 4,874,697.

An exemplary system for screening for proteases may comprise a protease-deficient bacterial strain, a first nucleotide sequence encoding a first reporter protein that comprises a BAX protein and a multiple protease recognition sequence from the bait region of an α2-macroglobulin or portions thereof, a second nucleotide sequence encoding a second reporter protein that comprises β-galactosidase and the same multiple protease recognition sequence as in the first reporter, a third nucleotide sequence encoding a candidate protease. The first, second and third nucleotide may be present in a single expression vector and under an inducible Tac promoter. The expression vector carries a bacterial origin of replication and a suitable selection marker (e.g., antibiotic resistant gene).

6. Protease Screening Assays

As described above, proteolytic activities of candidate proteases may be evaluated by comparing the activities of reporter protein(s) in host cells that express the candidate proteases with those in host cells that do not express the candidate proteases. The assays for determining reporter protein(s) are described above in the section related to protease inhibitor screening assays. A protease screening system may be validated by replacing the nucleotide sequence encoding a candidate protease with a known protease gene or a non-protease gene (e.g., actin) as a control. The expression of a known protease gene will result in the change in the activities of reporter protein(s), whereas the expression of a non-protease gene will not result in such a change.

All of the above U.S. patents, U.S. patent application publications, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference in their entirety. From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A nucleic acid comprising a nucleotide sequence that encodes a first reporter protein, said first reporter protein comprising an apoptotic protein and an amino acid sequence comprising a protease recognition sequence inserted within the apoptotic protein.

The nucleic acid of claim 1 wherein the apoptotic protein is

Bax.

3. The nucleic acid of claim 1 wherein the apoptotic protein is

FADD.

4. The nucleic acid of claim 1 wherein the protease recognition sequence is recognizable by a viral protease.

5. The nucleic acid of claim 4 wherein the protease recognition sequence is recognizable by an HIV protease.

6. The nucleic acid of claim 1 wherein the protease recognition sequence is the bait region of an α2-macroglobulin or a fragment thereof.

7. The nucleic acid of claim 6 wherein the α2-macroglobulin is human α2-macroglobulin.

8. The nucleic acid of claim 6 wherein the fragment of the bait region of the α2-macroglobulin is from 6 to 8 amino acids in length.

9. The nucleic acid of claim 1 further comprising a nucleotide sequence that encodes a protease that recognizes the protease recognition sequence within the apoptotic protein.

10. The nucleic acid of claim 1 or claim 9 further comprising a nucleotide sequence that encodes a second reporter protein that comprises

(i) a protein other than the apoptotic protein, and (ii) a protease recognition sequence within the other protein that is recognizable by a protease capable of cleaving the protease recognition sequence within the apoptotic protein.

11. The nucleic acid of claim 10 wherein the protease recognition sequence within the protein other than the apoptotic protein is the same as that within the apoptotic protein.

12. The nucleic acid of claim 10 wherein the protease recognition sequence within the protein other than the apoptotic protein is different from that within the apoptotic protein.

13. The nucleic acid of claim 10 wherein the protein other than the apoptotic protein is β-galactosidase.

14. An expression vector that comprises the nucleic acid of claim 1.

15. The expression vector of claim 14 wherein the apoptotic protein is Bax.

16. The expression vector of claim 14 wherein the apoptotic protein is FADD.

17. The expression vector of claim 14 wherein the protease recognition sequence is recognizable by a viral protease.

18. The expression vector of claim 17 wherein the protease recognition sequence is recognizable by an HIV protease.

19. The expression vector of claim 14 wherein the protease recognition sequence is the bait region of an α2-macroglobulin or a fragment thereof.

20. The nucleic acid of claim 19 wherein the α2-macroglobulin is human α2-macroglobulin.

21. The expression vector of claim 19 wherein the fragment of the bait region of the α2-macroglobulin is from 6 to 8 amino acids in length.

22. The expression vector of claim 14 further comprising a nucleotide sequence that encodes a protease that recognizes the protease recognition sequence.

23. The expression vector of claim 14 or claim 22 further comprising a nucleotide sequence that encodes a second reporter protein that comprises

(i) a protein other than the apoptotic protein, and (ii) a protease recognition sequence within the protein other than the apoptotic protein that is recognizable by a protease capable of cleaving the protease recognition sequence within the apoptotic protein.

24. The expression vector of claim 23 wherein the protein other than the apoptotic protein is β-galactosidase.

25. The expression vector of claim 14 wherein the nucleic acid is under the control of an inducible promoter.

26. The expression vector of claim 25 wherein the inducible promoter is a Tac promoter.

27. A host cell that comprises the expression vector of claim 14.

28. The host cell of claim 27 wherein the apoptotic protein is

Bax.

29. The host cell of claim 27 wherein the apoptotic protein is

FADD.

30. The host cell of claim 27 wherein the protease recognition sequence is recognizable by a viral protease.

31. The host cell of claim 30 wherein the protease recognition sequence is recognizable by an HIV protease.

32. The host cell of claim 27 wherein the protease recognition sequence is the bait region of an α2-macroglobulin or a fragment thereof.

33. The host cell of claim 32 wherein the α2-macroglobulin is human α2-macroglobulin.

34. The host cell of claim 32 wherein the fragment of the bait region of the α2-macroglobulin is from 6 to 8 amino acids in length.

35. The host cell of claim 27 wherein the expression vector further comprises a nucleotide sequence that encodes a protease that recognizes the protease recognition sequence within the apoptotic protein.

36. The host cell of claim 27 further comprising an expression vector that comprises a nucleotide sequence that encodes a protease that recognizes the protease recognition sequence within the apoptotic protein.

37. The host cell of claim 27 or claim 35 wherein the expression vector further comprises a nucleotide sequence that encodes a second reporter protein that comprises

38. The host cell of claim 27, claim 35 or claim 36 further comprising an expression vector that comprises a nucleotide sequence encoding a second reporter protein that comprises

39. The host cell of claim 27 wherein at least one of endogenous proteases in the cell is deficient.

40. The host cell of claim 27 wherein the host cell is selected from a bacterial cell, a yeast cell, an animal cell, and a plant cell.

41. The host cell of claim 40 wherein the host cell is a bacterial cell.

42. The host cell of claim 40 wherein the animal cell is a mammalian cell.

43. A protein encoded by the nucleic acid of any one of claims

1-13.

44. A method for identifying an inhibitor of a protease, comprising a. expressing in a host cell i. a first nucleotide sequence encoding a first reporter protein comprising an apoptotic protein that contains a recognition sequence of the protease, wherein the cleavage products of the reporter protein by the protease have an effect on the vitality of the host cell different from that of the intact reporter protein, and ii. a second nucleotide sequence encoding the protease; and b. measuring the vitality of the host cell in the presence or absence of a candidate compound, whereby the change of the vitality of the bacterial cell in the presence of the candidate compound with respect of that in the absence of the candidate compound indicates that the candidate compound is an inhibitor of the protease.

45. The method of claim 44 wherein the apoptotic protein is Bax.

46. The method of claim 44 wherein the apoptotic protein is FADD.

47. The method of claim 44 wherein the protease is a viral protease.

48. The method of claim 47 wherein the protease is an HIV protease.

49. The method of claim 44 wherein the protease recognition sequence is the bait region of an α2-macroglobulin or a fragment thereof.

50. The method of claim 49 wherein the α2-macroglobulin is human α2-macroglobulin.

51. The method of claim 49 wherein the fragment of the bait region of the α2-macroglobulin is from 6 to 8 amino acids in length.

52. The method of claim 44 wherein the first nucleotide sequence and the second nucleotide sequence are in a single expression vector.

53. The method of claim 52 wherein the first nucleotide sequence and the second nucleotide sequence are under the control of a single promoter.

54. The method of claim 44 further comprising the step of expressing in the bacterial cell a third nucleotide sequence that encodes a second reporter protein that comprises

55. The method of claim 54 wherein the protein other than the apoptotic protein is β-galactosidase.

56. The method of claim 54 wherein the first nucleotide sequence and the third nucleotide sequence are in a single expression vector.

57. The method of claim 52 further comprising the step of expressing in the bacterial cell a third nucleotide sequence that encodes a second reporter protein that comprises

58. The method of claim 57 wherein the first, second and third nucleotide sequences are in a single expression vector.

59. The method of claim 44 wherein the host cell is a bacterial cell.

60. A method for determining whether a candidate protease cleaves within or near a protease recognition sequence, comprising a. expressing in a host cell a first nucleotide sequence encoding a first reporter protein comprising an apoptotic protein that contains a protease recognition sequence, wherein the cleavage products of the first reporter protein within or near the recognition sequence have an effect on the vitality of the host cell different from that of the first reporter protein; b. expressing in the host cell a second nucleotide sequence encoding a candidate protease; c. measuring the vitality of the host cell that expresses both the first and the second nucleotide sequences; and d. comparing the vitality of the host cell that expresses both the first and the second nucleotide sequences with that of a host cell that expresses the first nucleotide sequence but not the second nucleotide sequence, wherein the difference between the vitality of the host cell that expresses both the first and the second nucleotide sequences and the vitality of the host cell that expresses only the first nucleotide sequence indicates that the candidate protease cleaves within or near the recognition sequence.

61. The method of claim 60 wherein the apoptotic protein is

Bax.

62. The method of claim 60 wherein the apoptotic protein in

FADD.

63. The method of claim 60 wherein the protease recognition sequence is recognizable by a protease selected from the group consisting of aspartic proteases, serine proteases, cysteine proteases and metallo- proteases.

64. The method of claim 60 wherein the protease recognition sequence is the bait region of an α2-macroglobulin or a fragment thereof.

65. The method of claim 64 wherein the α2-macroglobulin is human α2-macroglobulin.

66. The method of claim 64 wherein the fragment of the bait region of the α2-macroglobulin is 6-8 amino acids in length.

67. The method of claim 60 wherein the protease recognition sequence is recognizable by a viral protease.

68. The method of claim 60 wherein the protease recognition sequence is recognizable by an HIV protease.

69. The method of claim 60 wherein the first and the second nucleotide sequences are in a single expression vector.

70. The method of claim 60 wherein the first and the second nucleotide sequences are under the control of a single promoter.

71. The method of claim 60 further comprising the step of expressing in the bacterial cell that expresses both the first and the second nucleotide sequences a third nucleotide sequence that encodes a second reporter protein that comprises i. a protein other than the apoptotic protein; and ii. a protease recognition sequence that is identical to the protease recognition sequence within the apoptotic protein.

72. The method of claim 71 wherein the first and the third nucleotide sequences are in a single expression vector.

73. The method of claim 69 further comprising the step of expressing in the bacterial cell that expresses both the first and the second nucleotide sequences a third nucleotide sequence that encodes a second reporter protein that comprises i. a protein other than the apoptotic protein; and ii. a protease recognition sequence that is identical to the protease recognition sequence within the apoptotic protein.

74. The method of claim 73 wherein the first, second and third nucleotide sequences are in a single expression vector.

75. The method of claim 60 wherein the host cell is a bacterial cell.

76. A nucleic acid that encodes a reporter protein comprising a protein with an amino acid sequence inserted into the protein, wherein a. the amino acid sequence comprises two or more protease recognition sequences; and b. the cleavage products of the reporter protein by a protease that recognizes at least one of the protease recognition sequences have activities different from that of the reporter protein.

77. The nucleic acid of claim 76 wherein the protein is an apoptotic protein.

78. The nucleic acid of claim 77 wherein the apoptotic protein is a Bax or FADD protein.

79. The nucleic acid of claim 76 wherein the protein is an enzyme or a fluorescent protein.

80. The nucleic acid of claim 79 wherein the protein is selected from the group consisting of β-galactosidase, luciferase, alkaline phosphatase, green fluorescent protein, and the variants of the above proteins.

81. The nucleic acid of claim 76 wherein the amino acid sequence is the bait region of an α2-macroglobulin or a fragment thereof.

82. The nucleic acid of claim 81 wherein the α2-macroglobulin is human α2-macroglobulin.