METHODS AND COMPOSITIONS FOR IDENTIFYING PROTEASE MODULATORS

Abstract: The present invention relates to protease assays. More particularly, this invention relates to compounds and methods useful for assaying for protease activity. The invention relates to targeted, efficient and high-throughput screens to identify small molecules compounds, peptides, etc. that modulate, i.e, interfere with or enhance, protease activity. The invention encompasses a variety of in vivo and in vitro assays. The invention further encompasses therapeutic compounds, such as antivirals, identified using the screening methods.

Description: METHODS AND COMPOSITIONS FOR IDENTIFYING PROTEASE MODULATORS 1. FIELD OF THE INVENTION The present invention relates to protease assays. More particularly, this * invention relates to compounds and methods useful for assaying for protease activity. The invention relates to targeted, efficient and high-throughput screens to identify small molecules compounds, peptides, etc. that modulate, i.e., interfere with or enhance, protease activity. The invention encompasses a variety of in vivo and in vitro assays. The invention 15 further encompasses therapeutic compounds, such as antivirals, identified using the screening methods. 2. BACKGROUND OF THE INVENTION 0 2.1 Proteases Proteases play critical roles in a number of important human and animal diseases. In some cases, proteases are essential for the replication of microbial pathogens. In other cases they may directly harm target tissues and organs. Proteases are enzymes that are involved in the regulation of homeostatic and diverse pathological processes. Proteases interact with other proteins in many ways, including processing precursors, transporting them to different cellular compartments and regulating their function and destruction. Proteases also interact with cells and tissues in which these proteins are present, affecting their integrity and physiology (Rappay, G., 1989, 0 Prog. Histochem. Cytochem. 18:1-61). Proteases are usually synthesized as inactive precursor molecules. They are activated whenever necessary to execute their biological function and are subsequently eliminated or inactivated by their inhibitors. Uncontrollable protease activity can be extremely harmful to a normal system. Therefore, protease 5 inhibitors play an important role in controlling protease integrity and function. The physiological balance between proteases and inhibitors is important to normal homeostatic processes such as blood clotting and clot lysis, hormonal regulation, and inflammatory responses. < . The functional complexity of proteases suggests that they are also important in the progression of a wide variety of diseases. Proteases are intimately involved in critical physiologic pathways including blood clotting and complement cascade, both of which are involved in the etiology of human disease such as, for example, cancer and inflammation. In addition, proteases are also involved in bacterial, viral, and fungal infection. Despite 10 development of an effective inhibitor of HIV protease that has proven to be successful in the treatment of AIDS, few other successful inhibitors have been developed. 2.2 Assays for Protease Activity Due to the involvement of proteases in many human and animal diseases, it would be desirable to identify potential modulators of the specific proteases involved. Screening assays that can be used to identify such modulators, however, are presently laborious to carry out, relatively insensitive, and/or do not accurately mimic the in vivo situation. A major drawback, for example, of all currently available methods for screening for protease inhibitors is the assays' use of synthetic peptides rather than a protease's natural substrate. Some assays are based upon the electrophoretic separation of products (EP 25 514,830). These assays are impractical for screening large numbers of potential antagonists or agonists. Cleavage of large proteins such as casein and hemoglobin has been assayed by measuring the release of free protons generated by the new hydroxyl group formed by hydrolysis. The amount of TCA soluble peptide generated from the substrate can also be an 30 indicator of protease activity. In addition some large substrates such as azocasine have been chemically modified to produce a substrate molecule with colored dyes attached. Proteases can be assayed by using such substrates by measurement of the TCA soluble peptide generated by digestion of the substrate by the protease. Such assays are undesirable as the solubility of such substrates, coupled with the low detectability of the dyes bound to the substrate often results in an assay with very poor sensitivity. β-galactosidase α-complementation assays utilizing synthetic peptide substrates have been reported (Leibig et al, 1991, Proc. Natl. Acad. Sci. USA 88:5979). In the effort to look for protease-specific inhibitors, various assay systems, both in vitro and in vivo have been attempted. In vitro assays usually are comprised of the purified or partially purified protease and a synthetic peptide substrate. Product formation may be detected by a variety of techniques, including chromatographic, electrophoretic, colorimetric, enzyme-linked immunosorbant, radiometric, and fluorometric methods. Each of these techniques has advantages and disadvantages. Chromatographic and electrophoretic assays are time and labor intensive and hence not suitable for high throughput assay. Enzyme-linked immunosorbant assays often involve multiple reagent additions and extensive washing steps. Radiomeiric assays are expensive and generate radioactive waste. Colorimetric and fluorometric assays allow greater throughput potential and continuous monitoring. Flurorometric assays are typically much more sensitive than colorimetric assays. However, both types of assays suffer from interference due to the presence of absorptive components. In addition, all in vitro assays have similar limitations in that they require large amounts of soluble, active, purified protease. Moreover, all currently available in vitro assays use synthetic peptide as the substrate, and therefore do not screen for candidates that recognize the complex conformation of the natural substrate. Specific inhibitors that interact with the completed substrate may not be able to be identified in these assays. In general, currently available in vivo assays usually start with co-expression of protease and substrate. Detection of product formation, depending on the particular protease, is generally labor intensive, costly, and not suitable for high throughput screening. HCMV protease belongs to a new class of serine proteases. The search for small molecules that inhibit HCMV protease activity has attracted efforts from many groups, however, only modest success has been achieved to date. Several attempts to discover or design HCMV protease inhibitors have used fluorescent substrate assays (Ogilvie et al., 1997, J. Med.Chem. 40:4113-35; Holskin et al., 1995, Anal. Biochem. 227:148-55; Pinko et al., 1995, J. Biol. Chem. 270:23634-40; Flynn et al., 1997, Curr. Opin. Chem. Biol. 1:190-6); Flynn et al., 1997, Drug Des. Discov. 15:3-15). Based on resonance energy transfer, these assays applied internal quenched fluorogenic peptides, such as D ABC YL-ED VANS, which has the conserved maturation site (M-site) of the assembly protein substrate between the fluorescence donor (EDVAN) and acceptor (DABCYL). ,. Upon cleavage at the M-site, the C-terminal fluorogenic peptide-EDVAN was released. A scintillation proximity assay using "phosphorous (Baum et al., 1996, Anal. Biochem. 237:129-34) and a fluorescence polarization assay (Levine et al., 1997, Anal. Biochem. 247:83-88) were also developed for HCMV protease. As discussed above, therefore, high throughput assays that are sensitive and 10 mimic the in vivo situation are desperately needed. 3. SUMMARY OF THE INVENTION The present invention is directed to methods and compositions for identification of modulators of protease activity. In particular, the present invention is directed to methods and compositions for identifying modulators of protease activity using assays that detect complementation of β-galactosidase. Briefly, in one embodiment, the assays of the invention comprise exposing an α-donor fusion polypeptide to a protease, 0 either within a cell or in a cell-free system, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, for a time sufficient to allow protease cleavage. In another embodiment, the assays of the invention comprise an α-donor 5 fusion polypeptide, either within a cell or in a cell-free system, that comprises an α-donor in operative association with a protein precursor. In such an embodiment, the protein precursor exhibits autocatalyic protease activity such that it proteolytically cleaves itself, thereby cleaving the α-donor fusion polypeptide. Protease cleavage of the protease 0 substrate results in release of a portion of the α-donor fusion polypeptide containing the α- donor portion of the fusion polypeptide. Release of the α-donor portion allows the α-donor to activate the α-acceptor of β-galactosidase. Thus, in the assays of the invention, protease activity (that is, the ability of the protease to cleave its substrate) is measured by complementation of β-galactosidase, which can easily be detected via, e.g., chromogenic assays. The assays of the invention can be performed rapidly and without the use of radioactivity. The assays of the present invention represent a major improvement over existing technologies in that they allow for the use of large, more native-like protease substrates, rather than only synthetic peptides, thereby creating an assay system that more - closely mimics endogenous, in vivo situations. The assays according to the invention can be adapted for use in a variety of systems such as bacterial, yeast, insect, mammalian, or cell free systems. Further, prior knowledge of the identity of the protease is unnecessary. In one embodiment, the invention provides a method for identifying a 0 compound that modulates the activity of a protease, comprising: (a) contacting a cell comprising an α-donor fusion polypeptide, a protease, and an α-acceptor with a test compound, under conditions and for a period of time sufficient for protease cleavage, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, and wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity; (b) measuring the level of β-galactosidase activity; and (c) comparing the level of β-galactosidase activity in (b) to the level obtained in the absence of the test compound, such that if the level in (b) differs from that obtained in the absence of u the test compound, a compound that modulates the activity of a protease is identified. In one embodiment of this method the protease is endogenous to the cell. Alternatively, in another embodiment, the cell is genetically engineered to contain the protease, i.e., express the protease from a recombinant molecule. 5 With respect to cell-based systems, such cells can be, e.g., bacterial, fungal, insect or mammalian cells. The cells comprise the α-donor fusion polypeptides of the invention and/or the nucleic acid molecules of the invention in operative association with nucleic acid regulatory sequences that drive expression of the α-donor fusion polypeptide 0 sequences in the cell. The invention further provides a method for identifying a compound that modulates the activity of a protease in vitro. Such method comprises: (a) contacting a test compound with a sample comprising an α-donor fusion polypeptide, a protease, and an α- acceptor, under conditions and for a period of time sufficient for protease cleavage, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, and wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity; (b) measuring the level of β-galactosidase activity; and (c) comparing the level of β-galactosidase activity in (b) to the level obtained in the absence of the test compound, such that if the level in (b) differs from than that obtained in the absence of the test compound, a compound that modulates the activity of a protease is identified. In one embodiment of the methods described above, the protease is in operative association with the protease substrate. In another embodiment, the test compound decreases protease activity. In yet another embodiment, the protease is a serine protease, a metalloprotease, an aspartic protease, or a cysteine protease. In a specific embodiment, the protease is selected from the group consisting of neutrophil elastasae, leukocyte elastase, tyrosine carboxypeptidase, lysosomal carboxypeptidase C, thrombin, dipeptidyl peptidase IV, granzymes, carboxypeptidase A, carboxypeptidaase B, angiotensin converting enzyme, stromelysin, Pseudomonas aeruginosa elastase, renin, cathepsin D, HIV protease, cathepsin B, cathepsin B 2 , cathepsin G, cathepsin L and calpain. In another embodiment, the protease is involved in infection and/or replication of a virus. In one embodiment, the virus is a human immunodeficiency virus, an adenovirus, a hepatitis virus, a rhinovirus, a herpesviruses, or a picomaviruses. In a specific embodiment, the virus is human cytomegalovirus. In another specific embodiment, the virus is hepatitis C virus. The invention further comprises a cell comprising a nucleic acid molecule or molecules that express an α-donor fusion polypeptide, a protease, and an α-acceptor, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, and wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity. In one embodiment, the cell of has been geneically engineered to express the protease. The invention further comprises a kit useful for the identification of compounds that modulate the activity of a protease comprising in one or more containers a nucleic acid molecule or molecules that express an α-donor fusion polypeptide, a protease, and an α-acceptor, wherein protease cleavage of the α-donor fusion polypeptide results in β- galactosidase activity. The invention further comprises compounds identified by the methods of the invention. In one embodiment, the invention provides a compound that inhibits the activity of the protease identified by the above-described methods. In another embodiment, the invention provides a pharmaceutical composition comprising a compound that inhibits the activity of the protease identified using the methods of the invention. In another embodiment, the invention provides a method for treating a patient with an infectious disease comprising administering to the patient a therapeutically effective amount of a compound that inhibits the activity of the ribosomal protein identified using the methods of the invention. The invention provides α-donor fusion polypeptides. The α-donor fusion polypeptides of the invention can comprise an α-donor domain and a protease substrate or protein precursor domain. The α-donor domain is present in the α-donor fusion polypeptide in operative association (that is, the domains are fused, e.g., covalently linked via peptide linkages) with a protease substrate domain or a protein precursor domain such that as part of the α-donor fusion polypeptide, the α-donor domain does not complement β-galactosidase activity. The α-donor domain can be positioned in the molecule either on the amino- or carboxy-terminal side of the protease substrate or protein precursor domain. The compositions of the invention further comprise nucleic acid molecules that encode the α-donor fusion polypeptides of the invention. Such nucleic acid molecules can be engineered to express the α-donor fusion polypeptides of the invention in a host cell. In a preferred embodiment, the nucleic acid molecules encoding the α-donor fusion polypeptides of the invention can be engineered to be expressed at a medium to low level in cells. In another preferred embodiment, the nucleic acid molecules encoding the α-donor fusion polypeptides of the invention are engineered to be expressed in a constitutive or a regulated, e.g., inducible, manner. The compositions of the invention still further comprise α-donor fusion polypeptide systems that express the α-donor fusion polypeptides of the invention and that can be utilized as part of the methods of the invention. Such α-donor fusion polypeptide systems include cell-based systems as well as in vitro, cell-free systems. In one embodiment, the compositions of the invention still further comprise cells that express the α-donor fusion polypeptides of the invention, including cells that can be utilized as part of the methods of the invention. In embodiments wherein the cells can be utilized as part of the methods of the invention, the cells exhibit β-galactosidase activity (or substantial increase, generally at least a 4-fold increase, in such activity) only upon cleavage of the α-donor fusion polypeptide to release a portion of the fusion polypeptide containing the α-donor domain that complements an α-acceptor. Thus, cells in such an embodiment further comprise nucleic acid sequences that encode an α-acceptor. In embodiments wherein the cells express an α-donor fusion polypeptide that comprises a protease substrate domain (that is, the α-donor fusion polypeptide does not exhibit autocatalytic protease activity), the cells of the invention can further comprise nucleic acid sequences that express a protease that cleaves the α-donor fusion polypeptide in a manner that releases a portion of the fusion polypeptide that contains the α-donor domain. The nucleic acid sequences comprises regulatory sequences that express the protease in the cell. In a preferred embodiment, the nucleic acid sequences encoding the protease are expressed at medium to low levels in the cell. In another preferred embodiment, the nucleic acid molecules encoding the protease are expressed in a regulatable, e.g., inducible, manner. With respect to in vitro cell-free α-donor fusion polypeptide systems of the invention, such systems comprise α-donor fusion polypeptides and/or nucleic acid molecules encoding and expressing α-donor fusion polypeptides, and an α-acceptor, or a nucleic acid molecule encoding and expressing the α-acceptor, that can be complemented upon release of the α-donor from the cleaved α-donor fusion polypeptide. In instances wherein the α-donor fusion polypeptide comprises a protease substrate domain, the in vitro system further comprises a protease, and/or nucleic acid molecules encoding and expressing the protease, that cleaves the α-donor fusion polypeptide to yield an α-donor polypeptide capable of complementing the α-acceptor of the system. The methods of the invention comprise methods for identifying compounds that modulate protease activity. That is, the methods of the invention comprise methods for identifying compounds that increase or decrease activity of a protease. In such embodiments, the α-donor fusion polypeptide is present within an α- donor fusion polypeptide cell system, and the test compound is exposed to or contacted to the cell. Alternatively, in such an embodiment, the α-donor fusion polypeptide and the test compound are exposed or contacted together in vitro in a cell-free α-donor fusion polypeptide system. The Examples presented in Sections 6 and 7, below, describe the construction of α-donor fusion polypeptides and α-donor fusion polypeptide cell-based systems that can be used as part of the methods of the invention. As used herein, the term "α-donor" refers to any portion of β-galactosidase * ^ able to interact with an inactive β-galactosidase mutant (e.g. , deletion mutant) and form a complex having β-galactosidase activity. In one embodiment, the α-donor represents a portion of the α-region of β-galactosidase. As used herein, the term "α-acceptor" refers to any β-galactosidase mutant 0 (e.g., a deletion mutant) that is enzymatically inactive, and is capable of interacting with an α-donor to form a complex having β-galactosidase activity. In one embodiment, the α- acceptor represents a portion of the α-region of β-galactosidase. As used herein, the term "complementation" refers to the ability of an α- 5 donor to interact with an α-acceptor and form a complex having β-galactosidase activity. The α-donor is said to "complement" the α-acceptor, or to "complement" β-galactosidase activity. 4. BRIEF DESCRIPTION OF THE FIGURES 0 FIG. 1. The structure and cleavage sites of HCMV UL80 polypeptide. 5 FIG. 2. Construction of plasmids that express α-donor fusion polypeptides comprising HCMV protein precursor domains. FIG. 3 A-C. α-complementation via cleavage of α-donor fusion polypeptides. A. α-UL80wt α-donor fusion polypeptide encoded by pW2/ cz-HCMV. B. HαUL80-wt α-donor fusion polypeptide encoded by pW3/αcz-HCMV. C. UL80α-wt α-donor fusion polypeptide encoded by pW3/αcZ-HCMV. FIG. 4A-C. β-galactosidase activity from cells transformed with different constructs: A. α-UL80wt α-donor fusion polypeptide encoded by pW2/αcz-HCMV. B. HαUL80-wt α-donor fusion polypeptide encoded by pW3/αcz-HCMV. C. UL80α-wt α- donor fusion polypeptide encoded by pW3/αcZ-HCMV. In each case, the cells were treated with OμM or 20μM of IPTG. The graphic insert in C represents the enlarged scale from pW2/αcZ-wt and pW2/αcZ-mut. 5. DETAILED DESCRIPTION OF THE INVENTION The methods and compositions of the invention described herein can be utilized for the identification of compounds that modulate (that is, increase or decrease) protease activity. The assays described herein comprise detecting an α-donor fusion polypeptide proteolytic cleavage by detecting complementation of β-galactosidase activity. The β-galactosidase α-complementation assay is based on the non-covalent interaction of two enzymatically inactive fragments of the β-galactosidase protein resulting in restoration of enzymatic activity. "Complementation," as used herein, refers to the ability of an α- donor to interact with an α-acceptor and form a complex exhibiting β-galactosidase activity. Thus, the α-donor is said to "complement" an α-acceptor, or to "complement" β- galactosidase activity. 5.1 α-Donor Fusion Polypeptides The compositions of the invention comprise α-donor fusion polypeptides. The α-donor fusion polypeptides of the invention can comprise an α-donor domain and a protease substrate domain or protein precursor domain. The α-donor domain is present in the α-donor fusion polypeptide in operative association (that is, the domains are fused, e.g., covalently linked via peptide linkages) with a protease substrate domain or a protein precursor domain such that as part of the α-donor fusion polypeptide, the α-donor domain does not complement β-galactosidase activity. The α-donor domain can be placed either ® amino to or carboxy to the protease substrate domain or protein precursor domain. In instances in which the α-donor is in an N-terminal position, it is preferred that there be a histidine tag present in the α-donor fusion polypeptide amino to the α-donor domain. The α-donor domain can comprise any portion of the α-region of β- 5 galactosidase that interacts with an inactive β-galactosidase mutant (e.g. , deletion mutant) and forms a complex exhibiting β-galactosidase activity. The inactive β-galactosidase mutant is referred to herein as an "α-acceptor." As discussed above, the term "α-donor" refers to any portion of β-galactosidase able to interact with an inactive β-galactosidase mutant (e.g., deletion mutant) and form a complex having β-galactosidase activity, while the term "α-acceptor" refers to any β-galactosidase mutant (e.g., a deletion mutant) in the α region, is enzymatically inactive, and is capable of interacting with an α-donor and form a complex having β-galactosidase activity. In various embodiments, the α-acceptor and/or α- donor represent a portion of the α-region of β-galactosidase. β-galactosidase amino acid sequences that can be utilized as α-donor sequences are well known to those of skill in the art, and include, but are not limited to, those described herein. E. coli β-galactosidase is a tetrameric protein of approximately 540 kd in size. Each of the four identical monomers of β-galactosidase consists of 1021 amino acids, and are divided into three regions: (1) the N-terminal proximal segment (the α- region), (2) a middle region, and (3) a C-terminal distal segment (the ω-region). Mutations, e.g., deletions, of β-galactosidase, for example, deletion of amino acids 11 through 41, prevent formation of the enzymatically active tetramer. The 11-41 deletion mutant of β- galactosidase is called the Ml 5 mutant. Small peptides of β-galactosidase, for example, of the α-region (e.g., amino acids 3 through 92, called the CNBr2 peptide), can interact with inactive mutants of β-galactosidase complement and restore full enzymatic activity (Langley & Zabin, 1976, Biochemistry 15:4866). As used herein, the term "complementation" refers to the ability of an α-donor to interact with an α-acceptor and form a complex having β- galactosidase activity. The CNBr2 peptide can, for example, serve as an α donor for the Ml 5 mutant, as well as for the Ml 12 β-galactosidase mutant, a deletion of amino acids 23-41 within β-galactosidase that act as an α-acceptor (Zabin, 1982, Mol. Cell Biochem. 49:87- 1 96). Other N-terminal β-galactosidase fragments of various lengths, such as 1-44 and 3-40 (Langley et al, 1975, Proc. Natl. Acad. Sci. USA 72:1254-7; Welply et al., 1981, J. Biol. Chem. 256:6804-10) can also be utilized as α-donor amino acid sequences. In general, the smaller α fragment of such a complementary pair is referred to 15 as the α donor, while the larger β-galactosidase deletion peptide is referred to as an α- acceptor. For purposes of the present invention, the terms "α-donor" and "α-acceptor" are, however, as described above. Intracistronic complementation also occurs at the C-terminus of beta- 0 galactosidase (the ω region). The best known sequence data available is for the X90 ω- acceptor peptide that deletes the last 10 amino acids, 1011-1021. The X90 peptide exists as a monomer and can be complemented by CNBr24, a cyanogen bromide digestion product of beta-amino acids 990-1021 to reform enzymatically active tetramer (Welphy, et al., 1980, Biochem. Biophys. Res. Common. 93:223). Thus, although for ease of description, the terms "α-donor" and "α-acceptor" are used throughout the specification, it is to be understood that α-donor and α-acceptor sequences are not to be limited to only α-region and α-region-related sequences and/or mutations. Additional α-donor/α-acceptor amino acid sequences can routinely be 0 identified by those of skill in the art via, e.g., β-galactosidase mutagenesis and assaying for complementation utilizing pairs of test α-donor/α-acceptor sequences. See, e.g., U.S. Patent No.5, 643,734, which is incorporated herein by reference in its entirety. 5 The protease substrate domains of the α-donor fusion polypeptides of the invention can comprise any amino acid sequence tht acts as a substrate for a protease of interest. The protein precursor domain of the α-donor fusion polypeptide of the invention can comprise any amino acid sequence that exhibits autocatalytic proteolytic activity such that (at least in the absence of a test compound) the α-donor fusion polypeptide is cleaved. Any protease substrate or protein precursor exhibiting autocatalytic protease activity can be utilized as part of the protease substrate or protein precursor domains of the α-donor fusion polypeptides of the invention. An advantage of the compositions and methods of the present invention is the ability to utilize natural protease substrates or protein precursors, as opposed to only short synthetic peptides. It is to be noted, however, that embodiments of the compositions and methods of the invention comprise short peptides representing core protease cleavage sites. The protease substrate domains of the invention can, for example, include, but are not limited to, protease substrates for: serine proteases, which can be divided into trypsin-like, chymotrypsin-like and elastase enzymes, based upon their primary substrate specificity; cysteine proteases; aspartic acid proteases; and/or metalloproteases. Examples of the protease substrate domains and the protein precursor domains of the α- donor fusion polypeptides of the invention can include, but are not limited to, proteases involved in such processes as, for example, viral infection/replication, bacterial infection/replication, fungal infection/replication, phagocytosis, fibrinolysis, blood clotting cascades or complement cascades, including, for example, inflammation and arthritis, cell growth, attachment and shape. Specific serine proteases, can include, but are not limited to, neutrophil elastasae, which is involved in pulmonary emphysema, leukocyte elastase, tyrosine carboxypeptidase, lysosomal carboxypeptidase C, thrombin, dipeptidyl peptidase IV, granzymes, and their respective protease substrates and/or protein precursors; specific metalloproteases can include, but are not limited to, carboxypeptidase A, carboxypeptidaase B, angiotensin converting enzyme, which is involved in hypertension, stromelysin, which is involved in inflammatory disorders such as rheumatoid arthritis, Pseudomonas aeruginosa elastase, which is involved in lung infections, particularly in cystic fibrosis and their respective protease substrates and/or protein precursors; specific aspartic proteases can include, but are not limited to, rerun, which is involved in hypertension, cathepsin D, and HIV protease, and their respective protease substrates and/or protein precursors; specific cysteine proteases can include, but are not limited to, lysosomal carboxypeptidase β- galactosidase (cathepsin B 2 ), cathepsin B, which is involved in cell proliferative disorders, cathepsin G, cathepsin L and calpain, which is involved in brain destruction during stroke 10 and related diseases, and their respective protease substrates and/or protein precursors. Such proteases, protease substrates and/or protein precursors, including, for example, those involved in the processes discussed above, are well known to those of skill in the art. See, e.g., Rabay, G., ed., "Proteinases and their Inhibitors in Cells and Tissues, 15 1989, Gustav Fischer Verlag, Stuttgart; Powers, J.C. et al., in "Proteases-Structures, Mechanism and Inhibitors," 1993, Birkhauser Verlag, Basel, pp. 3-17; Patick, A.K. & Potts, K.E., 1998, Clin. Microbiol. Rev. 11:614-627 ; Dery, O. et al., 1998, Am. J. Physiol. 1998, 274:C1429-1452; Kyozuka, S.J. et al., 1998, Cell Calcium 23:123-130; Howells, H.L. et al., 20 1998, Br. J. Haematol. 101:1-9; Hill, J. & Phylip, L.H., 1998, Adv. Exp. Med. Biol. 436:441-444; Kidd, V.J., 1998, Annu. Rev. Physiol. 60:533-573; Matsushita, M. et al., 1998, Curr. Opin. Immunol. 10:29-35; Pallen, M.J. & Wren, B.W., 1997, Mol. Microbiol. 26:209-221; DeClerk, Y.A. et al. , 1998, Adv. Exp. Med. Biol. 425:89-97; Thornberry, N.A., 1997, Br. Med. Bull. 53:478-490, each of which is incorporated herein by reference in its entirety, and the references cited therein. With respect to proteases involved in viral infection/replication, among the protease substrates and/or protein precursors that can be utilized as part of the α-donor fusion polypeptides of the invention are proteases involved in infection and/or replication of 0 retroviruses such as human immunodeficiency virus (HIV)-l and HIV-2, adenoviruses, hepatitis viruses, such as, hepatitis C virus (HCV), rhinoviruses, including human rhinoviruses, herpesviruses, including human cytomegalovirus (HCMV) and picomaviruses. 5 With respect to HCMV, HCMV, like other herpesviruses, expresses a number of enzymes that perform essential roles in DNA metabolism, capsid assembly, and packaging of the genome. One such enzyme, HCMV protease, cleaves the assembly protein precursor and is essential for dissolution of the capsid scaffold and packaging of the viral genome daring capsid maturation. Therefore, in an embodiment utilizing an HCMV protease substrate domain, the HCM assembly precursor can be utilized as part of an α- donor fusion polypeptide of the invention. The sequence of the HCMV assembly protein precursor and the HCMV protease is well known to those of skill in the art. In such an embodiment, the entire HCMV precursor amino acid sequence can be utilized as the protease substrate domain of the α-donor fusion polypeptide. Alternatively, that portion of the HCMV precursor protein containing a core cleavage site that can be cleaved by the HCMV protease can be utilized as the protease substrate domain of the α-donor fusion polypeptides of the invention, α-donor fusion polypeptides comprising HCMV protease substrate domains can, in conjunction with HCMV for example, be utilized as part of the methods of the invention for the identification of compounds that inhibit HCMV infection/replication by interfering with HCMV protease function. The Example presented in Section 6, below, demonstrates the successful construction and utilization of α-donor fusion polypeptides containing HCMV protease substrate domains as well as of cells expressing such α-donor fusion polypeptides. With respect to HCV, the HCV genome contains a 9.4 kb RNA that codes for a single polyprotein of 3010-3033 amino acids (Landro, et al., 1997, Biochemistry, 36:9340). Both host and virus encoded proteases process this peptide into nine distinct peptides: C, El, E2, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The C, El and E2 peptides are excised from the polyprotein by a host encoded peptidase. Hydrolysis at the NS2/NS3 junction is catalyzed by an incompletely characterized protease comprising NS2 and the N-terminus of NS3. The NS3 protein excises the NS5A fragment. Therefore, in an embodiment utilizing HCV, the HCV polyprotein, or a portion thereof, comprising the core cleavage sites that, upon cleavage, generate the C, El and/or E2 peptides can comprise an α-donor fusion polypeptide protease substrate domain. Such a protease substrate domain would act as a substrate for the HCV host-encoded peptidase discussed above. In an alternative embodiment, the HCV polyprotein, or a portion thereof, comprising the autocatalytic protease activity and the core cleavage sites that, upon cleavage, generate the NS2/NS3 junction and/or the NS5A fragment, can be utilized as part of an α-donor fusion polypeptide protein precursor domain, α-donor fusion polypeptides comprising HCV protease substrate domains or protein precursor domains can be utilized as part of the methods of the invention for the identification of compounds that inhibit HCV infection replication by interfering with HCV protease or host encoded peptidase function. The Example presented in Section 7, below, describes the construction and utilization of α-donor fusion polypeptides containing HCV protein precursor domains as well as of cells expressing such α-donor fusion polypeptides. Other herpesviruses also express a large precursor protein that is proteolytically processed. This precursor is autocatalytically cleaved at a specific amino acid sequence known as the "release" site, yielding separate protease and assembly proteins. The assembly protein is cleaved further by the protease at another site termed the "maturation" site. In embodiments of the α-donor fusion polypeptides of the invention, therefore, α-donor fusion polypeptide protein precursor domains can comprise herpesvirus protease activity as well as the core cleavage sites comprising the release and/or maturation sites. HIV-1 and HIV-2 proteases, which are part of the pol gene product, are instrumental in the cleavage of large precursor proteins into structural and functional viral proteins. The sequences of such protease/protease substrate sequences are well known to those of skill in the art. In yet another embodiment, therefore, the α-donor fusion polypeptides of the invention can comprise HIV-1 or HIV-2 protease substrate domains or protein precursor domains. The genomes of the picomaviruses comprise an RNA molecule that encodes a single large polyprotein. This polyprotein is cleaved to produce mature viral proteins. In poliovirus, these cleavages are carried out by two virally encoded proteases designated 2A and 3C. The 2A protease is responsible for the primary cleavage that generates the viral capsid precursor PI (Toyoda et al., 1986, Cell 45:761). The 3C protease, which is itself released from the polyprotein by an autocatalytic cleavage, then carries out all but one of the remaining cleavage reactions (Hanecak et al., 1984, Cell 37:1063). In still another embodiment, therefore, the α-donor fusion polypeptides of the invention can comprise picornavirus protease substrate domains or protein precursor domains, as described above. In some cases, proteases are directly involved in the destructive effects of microbial pathogens, acting on the proteins of the host. For example, Clostridium histolyticum, a bacterium that causes gas gangrene, secretes collagenase, which destroys the connective-tissue barriers of the host. The bacterium itself is unaffected because it is devoid of collagen. Destructive effects of collagenases have also been implicated in disease states that do not involve microbial pathogens. For example, in some forms of arthritis, collagenases are known to destroy the connective tissue collagen in bone joints. In another embodiment, therefore, the α-donor fusion polypeptides of the invention can comprise protease substrate domains that comprise collagenase core cleavage sequences. Such sequences are well known to those of skill in the art. The α-donor can be present either amino to or carboxy to the protease substrate or protein precursor domain of the α-donor fusion polypeptide. In certain instances, the α-donor domain can be engineered to be present within the protease substrate domain or the protein precursor domain. In instances in which the α-donor domain is in an N-terminal position, it is preferred that the α-donor fusion polypeptide further comprise a cysteine tag N-terminal to the α-donor domain Prior to cleavage of the α-donor fusion polypeptide, the α-donor does not exhibit the ability to complement β-galactosidase activity. As discussed above, the α-donor domain comprises an amino acid sequence that, upon cleavage of the α-donor fusion polypeptide via protease cleavage, complements β- galactosidase activity via an α-acceptor. In general, the released α-donor should generate a β-galactosidase activity signal at least about 4-fold over background levels of signal. Preferably, the α-donor domain is positioned within the α-donor fusion polypeptide such that the α-donor fusion polypeptide portion containing the α-donor after cleavage comprises as few amino acid residues as possible that are still able to complement β-galactosidase activity. In addition, the α-donor domain should be positioned within the α- donor fusion polypeptide relative to the core cleavage site such that the maximum distance exists between the α-donor and the core cleavage site and which yet continues to allow β- galactosidase complementation upon cleavage. The α-donor domain is preferably engineered, therefore, to be as far away from a protease core cleavage site as possible while still continuing to be able to complement via an α-acceptor upon cleavage. This placement allows for the most native conformation of the cleavage junction or junctions within the protease substrate domain or the protein cleavage domain. The maximal distance ensures that the protease cleavage site most closely resembles the endogenous protease substrate. In particular, in alternate embodiments, the α-donor domain is present about 5-20, preferably about 20-50 and more preferably about 50-200 amino acid residues amino or carboxy to the protease cleavage site of interest within the α-donor fusion polypeptide. α-donor fusion polypeptides can routinely be generated via, for example, standard recombinant engineering and expression techniques well known to those of skill in the art. For example, nucleic acid molecules encoding the α-donor fusion polypeptides of the invention can be synthesized and/or constructed according to such well known techniques and utilized to express the α-donor fusion polypeptides of the invention. See, e.g., Sambrook et al, 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York, both of which are incorporated herein by reference in their entirety. The nucleotide sequences of the α-donor domain and protease substrate and protein precursor domains of the α-donor fusion polypeptide of the invention are well known to those of skill in the art, e.g., the references discussed, above, earlier in this section. Sequences to be utilized as part of the domains of the α-donor fusion polypeptide of the invention need only be joined or ligated within the nucleotide sequence encoding the α-donor fusion polypeptide in such a manner that the translational reading frame among the domain coding regions is maintained and is uninterupted by termination signals. Testing the activity of an α-donor fusion polypeptide, as well as optimization of position and relative orientation and spacing of the α-donor domain and the protease or protein precursor domain within the α-donor fusion polypeptide can also routinely be accomplished by those of skill in the art. For example, standard recombinant techniques can be used to generate nucleic acid molecules that encode and express test α-donor fusion polypeptides. Such test α-donor fusion polypeptides can also be assayed, via, e.g., standard complementation assays described herein, for exhibition of features and preferred features of the α-donor fusion polypeptides of the invention. Such features and preferred features include, for example, the α-donor' s lack of complemention while present within and as part of the uncleaved α-donor fusion polypeptide, the ability of the protease substrate or protein precursor to be cleaved via its cleavage junction or junctions, the ability of the α-donor to complement after cleavage of the α-donor fusion polypeptide and the level of complementation signal generated by the α-donor after cleavage. Successful optimization of such such α-donor fusion polypeptide parameters is demonstrated in the Examples presented below. Expression of the test α-donor fusion polypeptide in a cell incapable of supporting cleavage of the α-donor fusion polypeptide facilitates the selection and optimization of placement of α-donor domains so that said placement within the α-donor fusion polypeptide inactivates the α-donor complementation activity while the α-donor remains within the uncleaved α-donor fusion polypeptide. The cell may either be normally unable to cleave the α-donor fusion polypeptide, or may be rendered so unable through the use of known protease inhibitors or through recombinant techniques, such as through the expression of anti-sense RNA homologous to any protease capable of cleaving the α-donor fusion polypeptide. The compositions of the invention further comprise nucleic acid molecules that encode the α-donor fusion polypeptides of the invention. Such nucleic acid molecules can be generated, e.g., as discussed above. The compositions of the invention still further comprise cells that express the α-donor fusion polypeptides of the invention, including cells that can be utilized as part of the methods of the invention. In one embodiment, the nucleic acid molecules encoding the α-donor fusion polypeptides may be present as part of a nucleotide vector (e.g., a plasmid, cosmid, phagemid, bacteriophage, virus, retrovirus, YAC, BAC or HAC vector), for example, an expression vector. The nucleic acid molecules of the invention can further comprise nucleic acid sequences required for transcription and translation of the α-donor fusion polypeptide in vitro or in a host cell of interest. With respect to cellular expression, the nucleic acid molecules of the invention can also further comprise nucleotide sequences (e.g., reporter gene sequences) that promote maintenance and/or replication of the nucleic acid molecules of the invention in the host cell. The nucleic acid molecules of the invention can additionally comprise a ribosome binding site, an initiator codon or a terminator site. Hence, in one embodiment, the nucleic acid molecules of the invention comprise nucleotide sequences that encode the α-donor fusion polypeptides of the invention, one or more origins of replication, and one or more selectable markers which allow phenotypic selection of recombinant cells, containing such nucleic acid molecules (in either an integrated or extrachromosomal manner). In instances wherein the nucleic acid molecules of the invention encoding the α-donor fusion polypeptides of the invention are expressed, it is preferable that they be expressed in a manner that results in a low to medium level of expression. Techniques for accomplishing such levels of expression in particular host cells of interest are well known to those of skill in the art. See, e.g., Section 5.2, below. • 5.2 α-Donor Fusion Polypeptide Systems of the Invention The present invention further relates to cell-based and in vitro, cell-free α- donor fusion polypeptide systems. Such systems can, e.g., be utilized as part of the methods of the present invention. The α-donor fusion polypeptide systems of the invention comprise α-donor fusion polypeptides and/or nucleic acid molecules encoding and expressing such α-donor fusion polypeptides, and an α-acceptor polypeptide and/or a nucleic acid molecule that encodes and expresses such an α-acceptor polypeptide. The α-acceptor polypeptide is one which can be complemented by the α-donor upon cleavage of the α-donor fusion polypeptide. The α-donor fusion polypeptide systems of the invention further comprise the ability to cleave the α-donor fusion polypeptides of the systems at the cleavage site or sites contained within the α-donor fusion polypeptides. In instances wherein the α-donor fusion polypeptides of the system contain a protease substrate domain, the α-donor fusion polypeptide systems of the invention further comprise a protease that cleaves the protease substrate and/or a nucleic acid molecule that encodes and expresses such a protease. In instances wherein the α-donor fusion polypeptide contains a protein precursor domain, the protein precursor domain exhibits autocatalytic self cleavage activity. In the absence of α-donor fusion polypeptide cleavage, the systems do not exhibit β-galactosidase activity, or exhibit substantially lower levels of β-galactosidase activity than that detectable upon α-donor fusion polypeptide cleavage. In general, the level of β-galactosidase detected after cleavage is at least 4 times the level detectable prior to α- donor fusion polypeptide cleavage. Upon cleavage, the systems exhibit β-galactosidase activity that can be measured using standard β-galactosidase assay techniques described below. In one embodiment, the α-donor fusion polypeptide systems of the invention represent in vitro, cell-free α-donor fusion polypeptide systems. Such systems comprise α- donor fusion polypeptides and/or nucleic acid molecules encoding and expressing α-donor fusion polypeptides, and an α-acceptor, or a nucleic acid molecule encoding and expressing the α-acceptor, that can be complemented upon release of the α-donor from the cleaved α- donor fusion polypeptide. In instances wherein the α-donor fusion polypeptide comprises a protease substrate domain, the in vitro system further comprises a protease, and or nucleic acid molecules encoding and expressing the protease, that cleaves the α-donor fusion polypeptide to yield an α-donor polypeptide capable of complementing the α-acceptor of the system. In one embodiment of an in vitro α-donor fusion polypeptide system, said embodiment of which is described herein as example, and not by way of limitation, nucleic acid molecules encoding a protease, e.g., an assemlbin protease, an α-donor fusion polypeptide, e.g., one comprising an assembly protein protease substrate, and an α-acceptor, are separately subcloned, expressed and purified. For example, such sequences can be expressed via the expression plasmid pBAD/His (Invitrogen), and purified with metal- chelating chromatography. Proteins expressed in such plasmids are expressed as poly- histidine tagged fusion proteins with N-terminal poly-histidine tags. Proteins which have a succession of six or more histidine residues at their amino or carboxyl terminus have a strong binding affinity to nickel. Poly-histidine-tagged fusion proteins bind specifically to the surface of a solid phase coated with chelated nickel. In one embodiment of such an example, therefore, microtiter plates coated with metal chelates can capture the poly- histidine-tagged fusion proteins (Pierce), thereby producing an in vitro α-donor fusion polypeptide system of the invention. See, also, Janknecht, et al. (1991, Proc. Nail. Acad. Sci. USA 88, 8972-8976) which describes a system for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, the gene or genes of interest (α-donor fusion polypeptide, α-acceptor and/or protease) is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia vims are loaded onto Ni 2+ -nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In another embodiment, the α-donor fusion polypeptide systems of the invention are cell systems. Cells utilized as part of the α-donor fusion polypeptide cell systems of the invention can can include, but are not limited to, bacterial (e.g., Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and Pseudomonas aeuroginosa, with E. coli being preferred), fungal (e.g., yeast cells, such as Saccharomyces or Pichia cells), mammalian (e.g., COS, CHO, BHK, 292, 3T3, C2F3, VERO, HeLa, MDCK and W138) or insect (e.g., baculoviral) cells. The cell-based α-donor fusion polypeptide systems of the invention can be generated utilizing standard techniques well known to those of skill in the art, as discussed below. Likewise, standard techniques well known to those of skill in the art can be utilized, as discussed below, to express the α-donor fusion polypeptide, α-acceptor and/or protease nucleic acid sequences of these systems. Methods for coexpression of two or more constructs in cells are also well known to those of skill in the art. For example, cells containing two separate expression constructs can routinely be selected for and maintained by utilizing vectors comprising appropriately compatible origins of replication and independent selection systems. Testing and optmization of the α-donor fusion polypeptide expression and β-galactosidase complementation activities of the cell-based systems of the invention can also routinely be accomplished. In instances wherein the α-donor fusion polypeptides of the cell systems of the invention comprise protein precursor domains, it is preferable that the cells express the α-donor fusion polypeptides at low to medium levels of expression. The nucleic acid molecules encoding such α-donor fusion polypeptides in cell systems of the invention can also be regulatable (e.g., inducible). These embodiments are preferred in that high levels of a particular protease may be harmful or toxic to the cell in which they accumulate. In instances wherein the protein precursor, or its cleavage products, of the α-donor fusion polypeptide is known or demonstrated to not be harmful or toxic to the cells at high levels, nucleic acid sequences encoding such α-donor fusion polypeptides can be expressed at high, constitutive levels. As discussed above, in instances wherein the α-donor fusion polypeptides of the cell systems of the invention comprise protease substrate domains, such cells can further comprise nucleic acid molecules that encode and express protease polypeptides that cleave the α-donor fusion polypeptides within the cell to yield α-donor-containing fragments that complement β-galactosidase activity. Such proteases can be endogenous to the cell or can be introduced, e.g., recombinantly introduced, into the cell. In instances wherein the protease-coding sequence is introduced into the cell, it is preferable that the cells express the protease polypeptides at low to medium levels of expression. Such introduced nucleic acid molecules can also be regulatable (e.g., inducible). As above, these embodiments are preferred in that high levels of a particular protease may be harmful or toxic to the cell in which they accumulate. In instances wherein the protease is known or demonstrated to not be harmful or toxic to the cells at high levels, nucleic acid sequences encoding such proteases can be expressed at high, constitutive levels. Although α-donor fusion polypeptides of the cell systems of the invention that comprise protease substrate domains do not exhibit autocatalytic protease activity, it is generally preferable that nucleic acid molecules encoding such α-donor fusion polypeptides also be expressed to only low to medium levels within cells. It is also generally preferable that the nucleic acid molecules encoding such α-donor fusion polypeptides in cell systems of the invention should be regulatable (e.g., inducible). These embodiments are preferred in that high levels of a particular protease substrate may be harmful or toxic to the cell in which they accumulate. In instances wherein the protein substrate of the α-donor fusion polypeptide is known or demonstrated to not be harmful or toxic to the cells at high levels, nucleic acid sequences encoding such α-donor fusion polypeptides can be expressed at high, constitutive levels. Nucleic acid sequences encoding α-acceptors that can be complemented by released α-donors can be endogenous to the cell of the α-donor fusion polypeptide system. Alternatively, nucleic acid molecules encoding the α-acceptor can be introduced, e.g., recombinantly introduced, into the cell. In the absence of harmful or toxic effects of accumulation of the α-acceptor in the cell, nucleic acid molecules encoding α-acceptors can be expressed in a high level, constitutive manner in the cell. In the event accumulation of the α-acceptor polypepide does cause harmful or toxic effects on the cell, nucleic acid encoding the α-acceptor can be expressed at low to medium levels and/or in a regulatable, e.g., inducible, manner. With respect to bacterial cells, standard techniques that can be utilized for engineering α-donor fusion polypeptide cell systems of the invention are well known to those of skill in the art. See, e.g., see Miller, J.H., 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, NY; Neidhardt, F.C., ed., 1987, Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington, D.C.; Yarmolinsky, M.B. & Stemberg, N., 1988, pp. 291-438, in Vol. 1 of The Bacteriophages, R. Calendar, ed., Plenum Press, New York, each of which is incoφorated herein by reference in its entirety, as well as Ausubel et al., supra, and Sambrook et al, supra. β-galactosidase activity-deficient strains that are suitable for engineering into the α-donor fusion polypeptide cell systems of the invention are also well known to those of skill in the art. As discussed above, the genotypic backgrounds of such strains can be such that they exhibit no (or substantially reduced) β-galactosidase activity. The strains can be such that they express neither α-donor nor α-acceptor sequences. Alternatively, such strains can express an α-acceptor sequence that can be complemented by the α-donor domain of the α-donor fusion polypeptide of interest (that is, once the α-donor fusion polypeptide is cleaved by the appropriate protease). For example, any of a number of well known Messing strains that expres α-acceptor sequences, e.g., strain JM109, which contains Ml 5 sequences, but are deficient in β-galactosidase activity can be utilized. Available starting strains can be obtained from, e.g., from private laboratories, such as the E. coli and Salmonella genetic stock center or other public collections (E. coli stock center (CGSC), Yale University, New Haven, CT; Salmonella Genetic Stock Center, University of Calgary, Calgary, Alberta, Canada; American Type Culture Collection (ATCC), Manassas, VA; or Northern Regional Research Laboratory (NRRL), Peoria, IL), or from commercial suppliers. It is desirable, but not necessary, to use cells that have been developed for drug screening processes, and for which conditions for growth, maintenance, and manipulations are known. Strains such as those available from the above sources can be engineered into α-donor fusion polypeptide cell system strains by utilizing recombinant techniques to produce the α-donor fusion polypeptide nucleic acids of the invention, and standard genetic techniques can be utilized for introducing the α-donor fusion polypeptide nucleic acids into cells. For example, general gene transfer techniques including transformation, conjugation, including Mu conjugation, transduction, including PI transduction, λ lysogeny, and Mu transposition, which are well known to those of skill in the art (see, e.g., Miller, supra, Neidhardt, supra, and Yarmolinsky & Steinberg, supra.) can be utilized for such purposes. In one convenient approach, for example, the α-donor fusion polypeptide construct is generated in a cell by the use of a α-donor fusion polypeptide nucleic acid which is introduced into a bacterial cell and propagated in progenies of the bacterial cell. For example, where the host is E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl 2 method using procedures well known in the art. Alternatively, MgCl 2 or RbCl could be used. Cells that contain the nucleic acid of interest may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions (e.g., resistance to antibiotics) that can be co-introduced with the nucleic acid of interest; (c) assessing the level of transcription as measured by the expression of α-donor fusion polypeptide, protease or α-acceptor transcripts in the host cell; and (d) detection of the α-donor fusion polypeptide, protease or α-acceptor as measured by immunoassay. In one embodiment, cells are constmcted to facilitate uptake of externally provided, e.g., test, compounds. For example, the cell systems of the invention can further comprise mutations that increase cell permeability to externally provided compounds. Such mutations are well known to those of skill in the art, and can include, for example, a 4312 10 imp (increased /wembrane permeability) mutation (Sampson et al., 1989, Genetics 122, 491- 501). Incorporation of an imp mutation alters the permeability of the bacterial (e.g., E. coli) outer membrane, and results in increased sensitivity to a variety of chemicals. As discussed above, generally a low to medium level of expression is desired 15 for expression of the α-donor fusion polypeptide and protease nucleic acid sequences within the cells. For achieving such expression, nucleic acid molecules can, for example, be designed to reside on a medium or a low-copy plasmid. Medium-copy plasmids are well known in the art, such as pBR322, which has a ColEl derived origin of replication and 15- 20 20 copies per cell (Bolivar et al., 1977, Gene 2:95-113; see Sambrook et al., 1989, supra), or pACYCl 84, one of the pACYClOO series of plasmids, which have a pl5A origin of replication and exist at 10-12 copies per cell (Chang & Cohen, 1978, J. Bacteriol. 134:1141- 56; see also Miller, 1992, pi 0.4- 10.11). Low-copy plasmids are also well known in the art, for example, pSClOl, which has a pSClOl origin, and approximately 5 copies per cell. Both pACYC and pSClOl plasmid vectors have convenient cloning sites and can co-exist in the same cell as pBR and pUC plasmids, since they have compatible origins of replication and unique selective antibiotic markers. When even less expression is desired, the nucleic acids can be inserted into 0 the bacterial chromosome at a single copy level using gene transfer techniques well known in the art (see Miller, 1992, supra). Such insertions can be constructed using gene transfer techniques including transformation, conjugation, or transduction. Useful techniques to insert genes into bacterial chromosomes include, but are not limited to, PI transduction, Mu 5 transposition, λ lysogeny and conjugation (see Miller, 1992, supra; Neidhardt, F.C., ed., 1987, Esherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington, D.C.; Yarmolinsky, M.B. & Stemberg, N., 1988, pp. 291-438, in Vol. 1 of The Bacteriophages, R. Calendar, ed., Plenum Press, New York). As discussed above, in certain instances it is desirable to achieve a high level of expression. For example, when a high level of expression is desired, nucleic acid molecules can be designed to reside on a high-copy plasmid such as a plasmid containing a ColEl -derived origin of replication, examples of which are well known in the art (see Sambrook et al., 1989, supra; see also Miller, 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, NY, and references therein), such as pUC19 and its derivatives (Yanisch-Peπon et al., 1985, Gene 33:103-119). pUC vectors exist at levels of 500-700 copies per cell and have convenient cloning sites for insertion of foreign genes. For very high expression, λ vectors, such as λgtl 1 (Huynh et al., 1984, in "DNA Cloning 5 Techniques:, Vol I: A Practical Approach", D. Glover, ed., pp 49-78, IRL Press, Oxford), or the T7 or SP6 phage promoters in cells containing T7 and Sp6 polymerase expression systems (Studier et al., 1990, Methods Enzvmol.. 185:60-891. Low to medium levels of expression can also be achieved by using promoters o of weak strengths. Weak promoters are well known in the art, for example, maltose, galactose, or other desirable promoter (sequences of such promoters are available from Genebank (Burks et al. 1991, Nucl. Acids Res.19:2227-2230). In instances wherein high level expression is desired, high strength promoters can be utilized. For example commonly used strong promoters, including but not limited to, λP L (Pirrotta, 1975, Nature 254: 114-117) the trp promoter (Bennett et al., 1976, Proc. Natl. Acad. Sci USA 73:2351-55), and the lacUVS promoter (Gilbert & Maxam, 1973, Proc. Natl. Acad. Sci. USA 70:1559-63), that result in an accumulated level of about 1 to 10% of total cellular protein for a protein whose level is controlled by each promoter. If a stronger promoter is desired, the tac promoter is approximately tenfold stronger than lacUV5, but will result in high baseline levels of expression, and should be used only when overexpression is required. As also discussed above, nucleic acid sequences expressing α-donor fusion polypeptides and proteases in cells can be expressed in a regulatable, e.g., inducible manner. This is another way by which potential cell toxicity of protease or protease substrates can be avoided. Inducible expression can be obtained by utilizing a variety of inducible regulatory sequences. In one embodiment, for example, the lad gene and its gratuitous inducer IPTG can be utilized to yield inducible levels of expression of α-donor fusion polypeptides when sequences encoding such polypeptides are transcribed via the lacOP regulatory sequences. A variety of other inducible promoter systems are well known to those of skill in the art which can also be utilized. Other regulated expression systems that can be utilized include but are not limited to, the TET system (Geissendorfer M. & Hillen W., 1990, Appl. Microbiol. Biotechnol. 33:657-663), the p L promoter of phage λ temperature and the inducible lambda repressor CI g57 (Petrenko et al, 1989, Gene 78:85-91) and the trp repressor (Wame et al, 1986, Gene 46:103-112), Ipp (Nokamura et al., et al., 1982, J. Mol. Appl. Gen. 1 :289-299) and the araC promoter which is inducible by arabinose (AraC). Other inducible bacterial promoters that can be used, include but are not limited to, the T7 gene- 10 promoter, phoA (alkaline phosphatase), recA (Horii et al. 1980), and the tac promoter, a tφ-lac fusion promoter, which is inducible by tryptophan (Amann et al., 1983, Gene 25:167-78). As discussed above, the cell-based α-donor fusion polypeptide systems of the invention can also comprise mammalian cells. Starting cells for the production of mammalian cell-based α-donor fusion polypeptide systems of the invention can be obtained, e.g., from the sources listed earlier in this Section. Standard techniques for manipulating and maintaining mammalian cells are well known to those of skill in the art. See, e.g., Ausubel, above; as well as Butler, M., ed., 1991, "Mammalian Cell Biotechnology: A Practical Approach," IRL Press, New York; and Doyle, A. et al., 1993, "Cell and Tissue Culture: Laboratory Procedures," Wiley, New York, each of which is incoφorated herein by reference in its entirety. β-galactosidase complementation can be utilized in mammalian cells, and techniques for engineering mammalian cells such that β-galactosidase complementation can be assayed in such cells are well known to those of skill in the art. See, e.g., Rossi et al., 1997, Proc. Natl. Acad. Sci USA 94:8405-8410; and Mohler & Blau, 1996, Proc. Natl. Acad. Sci USA 93:12423-12427, each of which is incoφorated herein by reference in its entirety. In one embodiment described herein as example and not by way of limitation, a mammalian cell α-donor fusion polypeptide system can be generated by introducing nucleic acid sequence encoding a Ml 5 β-galactosidase α-acceptor mutant into a mammalian cell of interest, using standard techniques well known to those of skill in the art. The nucleic acid is introduced into the cell in operative association with regulatory sequences that drive expression of the α-acceptor mutant in the mammalian cell. For example, the Ml 5 sequences can be subcloned into a eukaryotic expression vector, such as pcDNA3.1 (Invitrogen), which uses the CMV enhancer-promoter to drive a high-level of constitutive expression. The Ml 5 protein is nontoxic and can be constitutively expressed at high levels. A nucleic acid encoding an α-donor fusion polypeptide is also introduced into the mammalian cell. The α-donor fusion polypeptide coding sequence encodes an α-donor fusion polypeptide with an α-donor domain that upon α-donor fusion polypeptide cleavage, complements the Ml 5 α-acceptor. This sequence is also introduced in operative association with regulatory sequences that drive expression (preferably regulated, e.g., inducible expression) in the mammalian cell. For example, the α-donor fusion polypeptide-coding sequence can be subcloned into a mammalian cell expression vector compatible with the vector associated with the α-acceptor coding sequence. For example, the α-donor fusion polypeptide-coding sequence can be cloned into appropriate cloning signals in a pTRE vector (Clontech), which contains an element referred to as a TRE element that can be used to express the α-donor fusion polypeptide in an inducible fashion in an appropriate cell line, e.g., a Tet-on expression (Gossen et al., 1995, Science 268:1766-9) cell line such as Cos7 (Clontech). The low level of the expression observed in the absence of tetracycline inducer minimizes any possible cytotoxicity due to the resident protease. In instances wherein the α-donor fusion polypeptide comprises a protease substrate domain, the mammalian cell further comprises a nucleic acid molecule that encodes and expresses the protease that cleaves the protease substrate. Preferably, the protease is expressed in an inducible manner, e.g., as described above, to minimize any possible toxicity to the host cell. In addition to the regulatory system described in the embodiment presented directly above, a host of inducible and constitutive expression systems exist for expression in mammalian cells and are well known to those of skill in the art. These include but are not limited to, the expression and regulatory sequences and systems described earlier in this Section that can also operate in mammalian cells. Regarding expression, as noted above, it is preferable that sequences encoding α-donor fusion polypeptide or protease polypeptides be expressed in an inducible manner. Nucleic acid molecules encoding α-acceptor polypeptides can generally be expressed in a constitutive manner, unless the polypeptides prove harmful or toxic to the host cell, in which case these nucleic acid molecules can also be expressed in a regulatable manner. As discussed above, the cell-based α-donor fusion polypeptide systems of the invention can comprise insect cells. Starting cells for producing insect cell-based systems of the invention can be obtained, e.g., from the sources listed earlier in this Section, above. In one embodiment of such an insect system, Autographa califomica nuclear polyhedrosis vims (AcNPV) is used as a vector to express the α-donor fusion polypeptide, α-acceptor and or protease polypeptides. The vims grows in Spodoptera frugiperda cells. The coding sequences may be cloned individually into non-essential regions (for example the polyhedrin gene) of the vims and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of coding sequences will result in inactivation of the polyhedrin gene and production of non-occluded recombinant vims (i.e., vims lacking the proteinaceous coat coded for by the polyhedrin gene). These recombinant vimses are then used to infect Spodoptera frugiperda cells in which the inserted coding sequences are expressed. (E.g.. see Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Patent No. 4,215,051). In prefened embodiments, expression of at least the α-donor fusion polypeptide and protease sequences is regulatable, and can be achieved via standard techniques that are well known to those of skill in the art. As discussed above, the cell-based α-donor fusion polypeptide systems of the invention can comprise yeast cells. Starting cells for producing yeast cell-based systems of the invention can be obtained, e.g., from the sources listed earlier in this Section, above. Standard techniques can be utilized for manipulation and maintaining yeast cells. Standard techniques can also be utilized for recombinant expression, including regulatable expression, can also be utilized. See, e.g., Kaiser, C, 1994, "Methods in Yeast Genetics," Cold Spring Harbor Laboratory Press, New York; and Spenser, J.F.T., 1989, "Yeast Genetics," Springer- Verlag, New York, each of which is incoφorated herein by reference in its entirety. Cell-based α-donor fusion polypeptide cell strains, cell cultures, cell lines 10 can be generated, expanded, stored and retrieved by standard techniques known in the art that are appropriate to the particular cell. The α-donor fusion polypeptide cell systems may be cultured under standard conditions of temperature, incubation time, optical density, plating density and media composition conesponding to the nutritional and physiological 15 requirements of the particular cell. Any techniques known in the art may be applied to establish the optimal conditions. In adddition, the cells of the invention can be preserved by stab culture, plate culture, or in glycerol suspensions and cryopreserved in a freezer (at - 20°C to -100°C) or under liquid nitrogen (-176°C to -196°C). 20 5.3 Methods for the Identification of Compounds That Modulate Protease Activity The methods of the invention comprise methods for identifying compounds that modulate protease activity. That is, the methods of the invention comprise methods for 25 identifying compounds that increase or decrease activity of a protease. Compounds that can be tested and identified as modulating protease activity utilizing the methods described herein can include, but are not limited to, compounds obtained from any commercial source, including Aldrich (1001 West St. Paul Ave., 30 Milwaukee, WI 53233), Sigma Chemical (P.O. Box 14508, St. Louis, MO 63178), Fluka Chemie AG (Industriestrasse 25, CH-9471 Buchs, Switzerland (Fluka Chemical Coφ. 980 South 2nd Street, Ronkonkoma, NY 11779)), Eastman Chemical Company, Fine Chemicals (P.O Box 431, Kingsport, TN 37662), Boehringer Mannheim GmbH (Sandhofer Strasse 35 116, D-68298 Mannheim), Takasago (4 Volvo Drive, Rockleigh, NJ 07647), SST Coφoration (635 Brighton Road, Clifton, NJ 07012), Ferro (111 West Irene Road, Zachary, LA 70791), Riedel-deHaen Aktiengesellschaft (P.O. Box D-30918, Seelze, Germany), PPG Industries Inc., Fine Chemicals (One PPG Place, 34th Floor, Pittsburgh, PA 15272). Further any kind of natural products may be screened using the methods of the invention, including microbial, fungal, plant or animal extracts. Furthermore, diversity libraries of test compounds, including small molecule test compounds, may be utilized. For example,s libraries may be commercially obtained from Specs and BioSpecs BN. (Rijswijk, The Netherlands), Chembridge Coφoration (San Diego, CA), Contract Service Company (Dolgopmdny, Moscow Region, Russia), 1° Comgenex USA Inc. (Princeton, NJ), Maybridge Chemicals Ltd. (Cornwall PL34 OHW, United Kingdom), and Asinex (Moscow, Russia). Combinatorial libraries of test compounds, including small molecule test compounds, can be utilized, and may, for example, be generated as disclosed in Eichler & Houghten, 1995, Mol. Med. Today 1:174- 15 180; Dolle, 1997, Mol. Divers. 2:223-236; Lam, 1997, Anticancer Drug Des. 12:145-167. These references are incoφorated hereby by reference in their entirety. It is to be noted that such references also teach additional screening methods which may be employed for the further testing of compounds identified via the methods of the invention and which can aid 0 in identifying and isolating compounds which can represent leads and therapeutic compounds having a desired effect on the physiological activity and/or function on the protease activity of interest. In one embodiment, compounds have a molecular weight of more than about 50, but less than about 3,000, and preferably less than 1,000 daltons. Various predetermined concentrations can be used for screening such as, for example, 0.001 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM. In one embodiment, the methods of the invention comprise: exposing an α- donor fusion polypeptide containing to a protease substrate to a protease substrate and to a 0 test compound for a time sufficient for protease substrate cleavage; and detecting the level of protease cleavage by detecting the amount of β-galactosidase complementation, so that if a differential level of β-galactosidase complementation is observed relative to that observed in the absence of the test compound, a compound that modulates protease activity is 5 identified. A relative decrease in β-galactosidase activity indicates identfication of a putative antagonist of protease activity, while a relative increase in β-galactosidase activity indicates identification of a putative agonist of protease activity. In another embodiment, the methods of the invention comprise: exposing an α-donor fusion polypeptide containing a protein precursor to a test compound for a time sufficient to allow protease autocatalytic cleavage; and detecting the level of protease cleavage by detecting the amount of β-galactosidase complementation, so that if a differential level of β-galactosidase complementation is observed relative to that observed in the absence of the test compound, a compound that modulates protease activity is identified. A relative decrease in β-galactosidase activity indicates identfication of a putative antagonist of protease activity, while a relative increase in β-galactosidase activity indicates identification of a putative agonist of protease activity. In such embodiments, the α-donor fusion polypeptide is present within an α- donor fusion polypeptide cell system, and the test compound is exposed to or contacted to the cell. Alternatively, in such an embodiment, the α-donor fusion polypeptide and the test compound are exposed or contacted together in vitro in a cell-free α-donor fusion polypeptide system. I order to verify or conoborate the specificity of a test compound identified as a putative modulator of protease activity, various controls can also be performed. For example, the methods of the invention can be performed as described above, but can be modified such that the α-donor fusion polypeptide comprises an α-donor domain and either a protease substrate domain or an inactive protein precursor domain. An inactive protease substrate refers to one that cannot be cleaved by the protease that usually cleaves it, and an inactive protein precursor refers to a protein precursor that does not exhibit autocatalytic activity. The method of the invention can also be performed as above, but can be modified such that, in instances wherein the α-donor fusion polypeptide system further comprises a protease, such a protease is an inactive one (i.e., does not exhibit protease activity). Alternatively, the methods of the invention can be performed as above, but can be performed in the presence of a known inhibitor of the protease of interest. In various embodiments of the invention, the contacting of a test compound may be effected in any vessel and by any means, such as wells or disks impregnated with a solution or suspension of a test compound. Standard protocols, such as serial dilution, may be used. The amount of time allowed for the test compound to modulate the activity of a protease in a test cell may be determined empirically, such as by running a time course and monitoring the accumulation of reporter molecule as a function of time. To facilitate high throughput screening, the test cells can be cultured and assayed in an ordered array, such as multi-well plates. Typically, the individual cultures are inoculated and allowed to grow in the wells under the appropriate conditions. Manipulations of the cultures and fluid handling can be done with a multi -channel devices. Most of the transfers and manipulations can be automated and miniaturized and performed by laboratory robots. As discussed above, the methods of the present invention assay protease activity via detection and measurement of β-galactosidase complementation. That is, the amount of α- donor fusion polypeptide proteolysis in a sample is measured as a direct function of the activity of β-galactosidase brought about by the release of the α-donor domain from the α- donor fusion polypeptide. Methods for detection of β-galactosidase activity are well known to those of skill in the art. See, e.g., Miller et al., 1992, A Short Course in Bacterial Genetics, Cold Spring Harbor Press, New York, pp. 92-94. β-galactosidase enzyme activity is monitored by the appearance of a product of the enzymatically-catalyzed reaction or by disappearance of the enzyme substrate. This is the rate of conversion of substrate. Substrates for β- galactosidase that are suitable for spectrophotometric or fluorometric analysis include, but are not limited to: p-aminophenyl-β-D-galactopyranoside; 2'-N-(hexadecanol)-N-amino-4'- nitrophenyl)-β-D-galactopyranoside; 4-methylumbel-liferyl-β-D-galactopyranoside; napthyl-AS-B 1 -β-D-galactopyranoside; 1 -napthyl-β-D-galactopyranoside; 2-napthyl-β-D- galactopyranoside monohydrate; O-nitrophenyl-β-D-galactopyranoside; m-nitrophenyl-β-D- galactopyranoside; p-nitrophenyl-β-D-galactopyranoside; and phenyl-β-D-galacto- pyranoside, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, resorufin-β-D- galactopyranoside, 7-hydroxy-4-trifluoromethyl coumarin, ω-nitrostyryl-β-D- galactopyranoside, and flourescein-β-D-galactopyranoside. The Examples presented in Sections 6 and 7, below, desribe the constmction of α-donor fusion polypeptides and α-donor fusion polypeptide cell-based systems that can be utilized as part of the methods of the invention. Test compounds that score positive in the screening assays of the invention are agents that interfere with activity of a protease, and are useful, for example, as leads for the development of therapeutic agents for the treatment of viral and infectious diseases. Once a compound has been identified via the methods of the invention, the compound can further be assayed in native, e.g., mammalian, systems to test the compound's effects on the protease of interest within the protease's usual biological context. 5.4 Protease Modulators Identified by the Assays In yet another embodiment, the invention provides novel protease agonist and antagonist agents discovered by the methods described above. These agents are capable of modulating n vitro heterogeneous assay using recombinant protease activity associated with diseases, such as viral infections. These agents may, for example, act by inhibiting a protease required for the production of proteins required by a vims during infection. Such compounds may also be used as therapeutic agents against other diseases, including, but not limited to cancer, inflammation, bacterial, and fungal infection. The invention also includes novel pharmaceutical compositions which comprise protease inhibitors and agonist agents discovered as described above formulated in pharmaceutically acceptable formulations. In another embodiment, the invention features a method for treating a subject infected with a viral or infectious agent by administering to that subject a therapeutically effective amount of a protease inhibitor or agonist agent which modulates protease activity in the vims or infectious agent as determined by the assays of the invention. Such agents can be administered by any method known to those skilled in the art, for example, by topical application or by systemic administration. In yet another embodiment, protease inhibitors or agonists of the present invention can be used to treat contaminated items, such as crops, wood, metal or plastic and the like, by methods such as, but not limited to, spraying or dusting of that agent onto the contaminated item, or impregnating that agent into the item. By "therapeutically effective amount" is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the patient. Additionally, by "therapeutically effective amount" is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a viral or infectious disease or condition. ° 5.4.1 Determination of MIC The minimum inhibitory concentration (MIC) against infectious agents is determined for each test compound that is positive in the assay. Methods known in the art may be used such as broth microdilution testing, using a range of concentrations of each test 5 compound (1993, National Committee for Clinical Laboratory Standards). Methods for Dilution Antimicrobial Susceptibility Tests For Bacteria That Grow Aerobically - Third Edition: Approved Standard, M7-A3). The MIC against a variety of pathogens are determined using the same method. 5.4.2 Cytotoxicity Testing Cytotoxicity can be measured by methods known in the art. One such method is assessing growth of mammalian cells in the presence of the test compound, using a protein binding dye, sulforhodamine B (SRB). SRB binds electrostatically to basic amino acids. Binding and solubilization of the dye can be controlled by changes in pH. SRB binds stoichiometrically to proteins in one pH range but can be solubilized and extracted for measurement in another. An increase in total protein is correlated to cell growth. Cell growth in the presence of compound is compared to growth without added compound to establish a growth inhibitory concentration (GI 50 ) (Skehan et al., 1990, J. Natl. Cancer. Inst., 82:1107-1112). Another method of measuring cytoxicity which may be used in an assay containing 3 [4,5-dimethylthiazol-2-y 1 ]-2,5,-diphenyltetrazolium bromide/2,3-bis[2- methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt ("MTT/XTT") as described in Mosmann T., 1983, J. Immunol. Methods, 65:55-63, which is incoφorated by reference in its entirety for all purposes. 5.4.3 Formulation Therapeutic compounds effective at modulating protease activity identified by methods of the invention may be formulated into pharmaceutical preparations for administration to animals for treatment of a variety of viral and infectious diseases. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, packaged, labeled for treatment of and used for the treatment of viral and infectious diseases, such as those listed infra. If the compound is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, polyethylene glycol or glycerine. Thus, the compounds and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, dermal, vaginal, rectal administration and drug delivery device, e.g., porous or viscous material, such as lipofoam. For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, sy ps or suspensions, or may be presented as a dmg product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. , sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The therapeutic compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. , containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the therapeutic compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic dmgs. The therapeutic compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pharmaceutical compositions of the present invention comprise an antiviral agent as the active ingredient, or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients, for example antibiotics. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases. The pharmaceutical compositions include compositions suitable for oral, rectal, mucosal routes, transdermal, parenteral (including subcutaneous, intramuscular, intrathecal and intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated. I practical use, an antiviral agent can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including tablets, capsules, powders, intravenous injections or infusions). In preparing the compositions for oral dosage form any of the usual pharmaceutical media may be employed, e.g., water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like; in the case of oral liquid preparations, e.g., suspensions, solutions, elixirs, liposomes and aerosols; starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations e.g., powders, capsules, and tablets. In preparing the compositions for parenteral dosage form, such as intravenous injection or infusion, similar pharmaceutical media may be employed, e.g., water, glycols, oils, buffers, sugar, preservatives and the like know to those skilled in the art. Examples of such parenteral compositions include, but are not limited to Dextrose 5%w/v, normal saline or other solutions. 5.4.4 Administration For administration to subjects, therapeutic compounds discovered by using the assays of the invention are formulated in pharmaceutically acceptable compositions. The compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These compositions can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro. In employing them in vivo, the compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonially, rectally, vaginally, nasally, orally, transdermally, topically, ocularly, or intraperitoneally. As will be readily apparent to one skilled in the art, the magnitude of a therapeutic dose of an antiviral compound in the acute or chronic management of an infectious disease will vary with the severity of the condition to be treated, the particular composition employed, and the route of administration. The dose, and perhaps dose frequency, will also vary according to the species of the animal, the age, body weight, condition and response of the individual subject. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art. Desirable blood levels may be maintained by a continuous infusion of an antiviral compound as ascertained by plasma levels. It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity. Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects). In selected cases, d g delivery vehicles may be employed for systemic or topical administration. They can be designated to serve as a slow release reservoir, or to deliver their contents directly to the target cell. Such vehicles have been shown to also increase the circulation half-life of dmgs which would otherwise be rapidly cleared from the blood stream. Some examples of such specialized dmg delivery vehicles which fall into this category are liposomes, hydrogels, cyclodextrins, and bioadhesive microspheres. These vehicles have been developed for chemotherapeutic agents. Topical administration of agents is advantageous when localized concentration at the site of administration with minimal systemic adsoφtion is desired. This simplifies the delivery strategy of the agent to the disease site and reduces the extent of toxicological characterization. Furthermore, the amount of material to be administered is far less than that required for other administration routes. Antiviral agents may also be systemically administered. Systemic absoφtion refers to the accumulation of agents in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absoφtion include: oral, intravenous, subcutaneous, intraperitoneal, intranasal, intrathecal and ocular. Each of these administration routes exposes the agent to an accessible target. 6. EXAMPLE: Construction of a Human Cytomegalovirus Protease α-Fragment Fusion Polypeptide The Example presented in this Section describes the successful construction of an α-donor fusion polypeptide and cell-based α-donor fusion polypeptide systems comprising a human cytomegalovims (HCMV) protein precursor domain. The HCMV UL8O gene product, a 708 amino acid polypeptide (FIG. 1), is the HCMV assembly protein precursor. Because HCMV protease performs self-proteolytic cleavage of the assembly protein and the protease and substrate are in the same open reading frame of 708 amino acids, the HCMV UL80 product was utilized in the constmction of an α-donor fusion polypeptide and an α-donor fusion polypeptide cell system, 6.1 Materials and Methods Construction and expression of HCMV α-donor fusion polypeptides and α-donor fusion polypeptide cell-based systems. Although the α-donor can be inserted into any position adjacent to the designated cleavage site, two expression vectors were constmcted in which the designated protease substrate was inserted in-frame upstream and in-frame downstream of the α-donor (FIG. 2). A PCR fragment of 51 residues (4-55) of β- galactosidase from pcDNA3.1/.Hygro//αcZ (Invitrogen) with a Hindlll site at the 5 '-end and a Sse8387 site at the 3'-end was made using standard amplification techniques. This PCR product was inserted into HindIII-Sse8387 of pW3 (FIG. 2), a derivative of plasnid pMS421 (Maloy et al., 1989, "Genetic Analysis of Pathogenic Bacteria," Cold Spring Harbor Press, New York). The resulting plasmid, pW3/αcZ, can be used for N-terminal α-donor fusion to any protease substrate or protein precursor. A Sapl-Drdl (blunt) fragment from pUC19 was used to replace the Sapl-Xbal (blunt) fragment of pW3 to form pW2/αcZ, in which the lacZ gene is downstream of the multiple cloning site. pW2/αcZ can be used for C- terminal α- donor fusion to any protease substrate. A cosmid, pi 61, that contains a Hindlll fragment subcloned from HCMV AD 169 and that contains the UL80 gene was utilized for constmction of the protein precursor of the α-donor fusion polypeptide. A 5' primer having an Sse8387 site before the start codon of UL8O and a 3 ' primer having a Xbal site after the stop codon of UL8O were used to amplify the UL8O gene from cosmid pi 61, using standard techniques. The Sse8387-Xbal fragment was subcloned using these sites in pW3/αcZto form pΨ3lacZ-wt for in-frame expression of the N-terminal α-fragment fusion protease, αUL8Owt. The same 5 ' primer was used with a 3' primer that contained a Sad site and removed the stop codon of UL8O, to amplify the UL8O gene from cosmid pi 61. The Sse8387-Sacl fragment was then subcloned into pΨllacZ to form pW2/αcZ-wt to create a constmct for in-frame expression of the C-terminal α-fragment fusion protease, UL80αwt. Both PCR fragment clones were sequenced to confirm that no mutations were introduced during PCR amplification. A six-histidine tag was placed into the N- terminal portion of the fusion protein immediately following the Hindlll site to form a plasmid named pW3H/αcZ, which was used to produce an α-donor fusion polypeptide protease HαUL80wt. Expression of both N-terminal and C-terminal fusion proteins was under control of an inducible lac operator/promoter and lacP repressor. This system was chosen because of its low basal level of transcription in the non-induced state. A single mutation was also made, replacing His63 with Ala by site-directed mutagenesis using Chameleon™ double-stranded site-directed mutagenesis kit (Stratagene). The Sse8387-BamHI fragment bearing the point mutation was used to replace the same fragment in the wild-type gene. The mutant α-donor fusion polypeptides were αUL8Omut, HαUL80mut, and UL80αmut, respectively. All wild type and mutant constructs were transformed into JM109 cells. Cells were plated on LB agar with 200 μg/ml X-gal (5-bromo-4-chloro-3-indolyl-β- galactoside), 100 μM IPTG (isopropyl-β-thiogalactoside) and 40 μg/ml spectinomycin. Assay of β-galactosidase Activity. Beta-galactosidase activity was measured according to the method described by Miller (Miller et al., 1992, supra, pp. 72-74) with the modification of using chlorophenolred-β-galactopyranoside (CPRG) as the substrate. Briefly, single colonies containing different constmcts were grown in a 96 well microtiter plate with or without 20 μM IPTG. Cell density was measured by OD 600 reading. 200μl cells were lysed by adding 30 μl of chloroform and then 75 μl of lysed cells were transfened to a new plate containing 150 μl of 1 mg/ml CPRG in Z buffer. The plate was placed in a plate reader (Molecular Device) and β-galactosidase activity was monitored at OD J75 over 15 minutes. 6.2 Results Two N-terminal α-donor fusion polypeptides, αULδOwt and HαULδOwt, were constmcted as described in Sectin 6.1, above. αUL8Owt is a 770 amino acid residue polypeptide. It includes the first 8 amino acids from the vector, 51 amino acids from residues 4-55 of β-galactosidase (the α-donor domain), 3 linker amino acids and the 708 amino acids of the UL80 polypeptide (the protein precursor domain). HαULδOwt has six additional histidines prior to the α-fragment (Figure 3B). HCMV protease self-cleaves at multiple sites, with cleavage at position 143 releasing a 205 amino acid product, named αUL8o fragment, which is the smallest possible cleavage product and is likely to be most efficient at α-complementation (FIG. 3A). UL80α-wt is an 806 amino acid residue polypeptide. It includes the first 11 amino acids expressed from the vector, 708 amino acids of the UL8O polypeptide (the protein precursor domain), a 7 amino-acid linker, and 80 amino acid residues from the plasmid pUC19, of which the first 51 amino acids represent residues 4-55 of β-galactosidase (α-donor domain). Autocleavage at position 643 will release an 152 amino-acid residue peptide, UL8α fragment, which can participate in α-complementation (Figure 3C). Colonies from all constructs showed either dark or light blue phenotypes, indicating α-complementation took place. It was anticipated that α-donor ability to complement β-galactosidase was due to HCMV protease activity, in that it was unlikely that α-donor within the α-donor fusion polypeptide could function to complement. To test this hypothesis, an α-donor fusion polypeptide site-directed mutation was constructed in which the HCMV His63 (an amino acid residue believed to be essential for HCMV protease activity) was replaced with Ala, thereby inhibit HCMV protease activity. It was expected that inactive HCMV protease would block the α- complementation. The blue color from the wild type HCMV fusion protease was much darker than that of the mutant fusion polypeptide proteases, thereby confirming that the ability of the α-donor fusion polypeptide α-donor to complement is greatly dependent upon HCMV protease activity. In order to measure differences among constmcts, β-galactosidase assays were performed. The results are shown in FIG. 4A-C for each of the constmcts: α-UL80wt α-donor fusion polypeptide encoded by pW2/αcz-HCMV (FIG. 4A); HαUL80-wt α-donor fusion polypeptide encoded by pW3/ cz-HCMV (FIG. 4B); ULδOα-wt α-donor fusion polypeptide encoded by pW3/αcZ-HCMV (FIG. 4C). Consistent with their blue/white phenotypes, all three wild type fusion polypeptide proteases showed higher β-galactosidase activity than the same fusion with mutant protease. The activity from αUL8O-wt was more than 10 fold higher than that from αUL8O-mut, strongly suggesting that the protease- derived α-complementation took place. In general, β-galactosidase activity obtained with plasmid wild-type fusion polypeptide proteases was lower than that obtained with plasmid containing the small minimal lacZ α-fragment (except for pW3lacZ). This was probably because the cleavage products were much larger than the original minimal lacZ α-fragment, and therefore had weaker α-complementation. Activity differences were also observed among plasmids with the minimal lacZ α-fragment alone and plasmids with wild-type fusion polypeptide proteases. This was probably due to the addition of amino acid residues in the cleavage products and the position of the α-fragment. Previous reports have suggested that residues 26-31 of β-galactosidase play a key role in α-complementation (Welply et al., 1981, J. Biol. Chem. 256:6811-6) and additional amino acids placed either N-terminal or C-terminal of α-fragment will affect the lacZ phenotype (Zeilstra-Ryalls & Somerville, 1992, Curr. Top. Cell. Regul. 33:81-104.). After cleavage, the UL8Oα fragment was sandwiched in by two non-lacZ polypeptides, not likely to be the most favorable condition for productive α-complementation. The alternative C-terminal fusion was more favorable. For example, the α-fragment could be placed a few residues after the M-site. In addition, it was noted that the His-tag placed prior to the α-fragment enhanced α- complementation. To further confirm whether the observed α-complementation was due to the HCMV protease activity, Western blot analysis was performed with equal amounts of total protein from cells transformed with pW3H/ cZ, pW3H/αcZ-wt, and pW3UlacZ-mut. Nothing was detected in cells transformed with pW3H/ cZeven using high percentage SDS-PAGE gels, most likely due to instability of small size polypeptides. A polypeptide of approximately 86 kDa was observed in cells transformed with the mutant fusion polypeptide protease, which is consistent with the intact HαUL80 (see FIG. 1). However, the -23 kDa polypeptide, HαULδO fragment, cleaved from I-site at position 143, was not detected. Instead, a polypeptide at MW 7δ kD was observed, which probably conesponded to a product of cleavage at the M-site. Due to its large size, it is possible that this product was performing α-complementation. 7. EXAMPLE: Construction of a Hepatitis C Viral Polyprotein a- Fragment Fusion Polypeptide The Example presented in this Section describes the construction of a hepatitis C vims (HCV) polyprotein-containing α-donor fusion polypeptide and α-donor fusion polypeptide cell system. A fragment of β-galactosidase consisting of amino acids 3 through 92 of the α-region of β-galactosidase is used as the α-donor, and the Ml 5 mutant serves as the α- acceptor. A DNA sequence coding for α-fragment is placed, in frame, at various positions within a DNA coding for the NSFA protein of the hepatitis C viral polyprotein. This constmct is expressed and the resulting α-fragment/N5SA fusion proteins assayed for its ability to complement the Ml 5 mutant. The optimum site for insertion of the α-fragment into the N5SA protein is routinely chosen by identifying that fusion protein with the α- fragment sequence farthest from the cleavage junctions, yet still able to complement the Ml 5 mutant. Once this site is determined, the α-donor is inserted, in frame, into the corresponding site within a DNA sequence coding for the entire HCV polyprotein. This sequence is expressed in a cell unable to support cleavage of the polyprotein, and the HCV polypeptide-containing α-donor fusion polypeptide is checked for lack of ability to complement the M15 mutant. Additionally, the α-fragment is inserted into a mutant HCV polyprotein that is unable to autocatalytically cleave. This is expressed in a cell lacking proteases able to cleave the HCV polypeptide, and the resulting fusion protein tested for a lack of ability to complement the M15 mutant. Both the normal and autocatalytic deficient polypeptides are used in β-galactosidase assays and assays for identifying test compounds that modulate HCV protease activity. Next, the α-donor fusion polypeptide described above is expressed in a cell known or expected to support cleavage of the HCV polyprotein into its component peptides. In such cells, the α-fragment/HCV polyprotein is cleaved, and the α-fragment/NS5A fusion protein is released. The presence of free α-fragment/NS5A fusion protein is assayed by testing for the ability of the expressed protein to complement the Ml 5 mutant. Since only cleaved fusion proteins are able to complement the Ml 5 mutant, complementation is indicative of cleavage. Various test compounds are then tested for their ability to inhibit cleavage of the HCV polyprotein. Such inhibition is indicated by a reduction or loss of the ability to complement the Ml 5 mutant.

Claims: WHAT IS CLAIMED IS: 1. A method for identifying a compound that modulates the activity of a protease, comprising: a) contacting a cell comprising an α-donor fusion polypeptide, a protease, and an α-acceptor with a test compound, under conditions and for a period of time sufficient for protease cleavage, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, and wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity; b) measuring the level of β-galactosidase activity; and c) comparing the level of β-galactosidase activity in (b) to the level obtained in the absence of the test compound, such that if the level in (b) differs from that obtained in the absence of the test compound, a compound that modulates the activity of a protease is identified. 2. The method of Claim 1, wherein the cell is genetically engineered to contain the protease. 3. The method of Claim 1, wherein the cell is a bacterial cell, a yeast cell, or insect cell, or a mammalian cell. 4. A method for identifying a compound that modulates the activity of a protease, comprising: a) contacting a test compound with a sample comprising an α-donor fusion polypeptide, a protease, and an α-acceptor, under conditions and for a period of time sufficient for protease cleavage, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, and wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity; b) measuring the level of β-galactosidase activity; and c) comparing the level of β-galactosidase activity in (b) to the level obtained in the absence of the test compound, such that if the level in (b) differs from than that obtained in the absence of the test compound, a compound that modulates the activity of a protease is identified. 5. The method of Claim 1 or 4, wherein the protease is in operative association with the protease substrate. 6. The method of Claim 1 or 4, wherein the test compound decreases protease activity. 7. The method of Claim 1 or 4, wherein the protease is a serine protease, a metalloprotease, an aspartic protease, or a cysteine protease. 8. The method of Claim 1 or 4, wherein the protease is selected from the group consisting of neutrophil elastasae, leukocyte elastase, tyrosine carboxypeptidase, lysosomal carboxypeptidase C, thrombin, dipeptidyl peptidase IV, granzymes, carboxypeptidase A, carboxypeptidaase B, angiotensin converting enzyme, stromelysin, Pseudomonas aeruginosa elastase, renin, cathepsin D, HIV protease, cathepsin B, cathepsin B 2 , cathepsin G, cathepsin L and calpain. 9. The method of Claim 1 or 4, wherein the protease is involved in infection or replication of a vims. 10. The method of Claim 9, wherein the vims is a human immunodeficiency vims, an adenovims, a hepatitis vims, a rhinovims, a heφesviruses, or a picomavimses. 11. The method of Claim 9, wherein the vims is human cytomegalovims. 12. The method of Claim 9, wherein the vims is hepatitis C vims. 13. A cell comprising a nucleic acid molecule or molecules that express an α- donor fusion polypeptide, a protease, and an α-acceptor, wherein the α-donor fusion polypeptide comprises an α-donor in operative association with a protease substrate, and wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity. 14. The cell of Claim 13 which has been genetically engineered to express the protease. 15. The cell of Claim 13 which is a bacterial cell, a yeast cell, or insect cell, or a mammalian cell. 16. The cell of Claim 13 wherein the protease is in operative association with the protease substrate. 17. An α-donor fusion polypeptide comprising an α-donor in operative association with a protease substrate. 18. An α-donor fusion polypeptide of Claim 17, wherein the protease substrate is a substrate for a hepatitis C vims protease or a human cytomegalovirus protease. 19. An α-donor fusion polypeptide of Claim 17, further comprising a protease in operative association with the protease substrate. 20. An α-donor fusion polypeptide of Claim 19, wherein the protease is a hepatitis C vims protease or a human cytomegalovims protease. 21. A kit useful for the identification of compounds that modulate the activity of a protease comprising in one or more containers a nucleic acid molecule or molecules that express an α-donor fusion polypeptide, a protease, and an α-acceptor, wherein protease cleavage of the α-donor fusion polypeptide results in β-galactosidase activity. 22. A compound that inhibits the activity of the protease identified using the method of Claim 1 or 4. 23. A pharmaceutical composition comprising a compound that inhibits the activity of the protease identified using the method of Claim 1 or 4. 24. A method for treating a patient with an infectious disease comprising administering to the patient a therapeutically effective amount of a compound that inhibits the activity of the ribosomal protein identified using the method of Claim 1 or 4.

Inventors: MENZEL ROLF, WANG SHAOJIE, MENZEL, ROLF, WANG, SHAOJIE
Applicants: SMALL MOLECULE THERAPEUTICS IN, SMALL MOLECULE THERAPEUTICS, INC.
Application year: 1999
Main classification: C12Q3
IPC codes: C07K14, C12Q1, G01N33