EP1141419A1 - Methodes et compositions pour identifier des modulateurs de protease - Google Patents

Methodes et compositions pour identifier des modulateurs de protease

Info

Publication number
EP1141419A1
EP1141419A1 EP99966678A EP99966678A EP1141419A1 EP 1141419 A1 EP1141419 A1 EP 1141419A1 EP 99966678 A EP99966678 A EP 99966678A EP 99966678 A EP99966678 A EP 99966678A EP 1141419 A1 EP1141419 A1 EP 1141419A1
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EP
European Patent Office
Prior art keywords
protease
donor
fusion polypeptide
cell
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99966678A
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German (de)
English (en)
Other versions
EP1141419A4 (fr
Inventor
Rolf Menzel
Shaojie Wang
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Small Molecule Therapeutics Inc
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Small Molecule Therapeutics Inc
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Publication of EP1141419A1 publication Critical patent/EP1141419A1/fr
Publication of EP1141419A4 publication Critical patent/EP1141419A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to protease assays. More particularly, this
  • the 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.
  • 15 further encompasses therapeutic compounds, such as antivirals, identified using the screening methods.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention is directed to methods and compositions for identification of modulators of protease activity.
  • the present invention is directed to methods and compositions for identifying modulators of protease activity using assays that detect complementation of ⁇ -galactosidase.
  • the assays of the invention comprise exposing an ⁇ -donor fusion polypeptide to a protease, 0 either within a cell or in a cell-free system, wherein the ⁇ -donor fusion polypeptide comprises an ⁇ -donor in operative association with a protease substrate, for a time sufficient to allow protease cleavage.
  • the assays of the invention comprise an ⁇ -donor 5 fusion polypeptide, either within a cell or in a cell-free system, that comprises an ⁇ -donor in operative association with a protein precursor.
  • the protein precursor exhibits autocatalyic protease activity such that it proteolytically cleaves itself, thereby cleaving the ⁇ -donor fusion polypeptide.
  • Protease cleavage of the protease 0 substrate results in release of a portion of the ⁇ -donor fusion polypeptide containing the ⁇ - donor portion of the fusion polypeptide.
  • protease activity that is, the ability of the protease to cleave its substrate
  • complementation of ⁇ -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
  • 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.
  • the invention provides a method for identifying a 0 compound that modulates the activity of a protease, comprising: (a) contacting a cell comprising an ⁇ -donor fusion polypeptide, a protease, and an ⁇ -acceptor with a test compound, under conditions and for a period of time sufficient for protease cleavage, wherein the ⁇ -donor fusion polypeptide comprises an ⁇ -donor in operative association with a protease substrate, and wherein protease cleavage of the ⁇ -donor fusion polypeptide results in ⁇ -galactosidase activity; (b) measuring the level of ⁇ -galactosidase activity; and
  • the protease is endogenous to the cell.
  • the cell is genetically engineered to contain the protease, i.e., express the protease from a recombinant molecule.
  • such cells can be, e.g., bacterial, fungal, insect or mammalian cells.
  • the cells comprise the ⁇ -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 ⁇ -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 ⁇ -donor fusion polypeptide, a protease, and an ⁇ - acceptor, under conditions and for a period of time sufficient for protease cleavage, wherein the ⁇ -donor fusion polypeptide comprises an ⁇ -donor in operative association with a protease substrate, and wherein protease cleavage of the ⁇ -donor fusion polypeptide results in ⁇ -galactosidase activity; (b) measuring the level of ⁇ -galactosidase activity; and (c) comparing the level of ⁇ -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
  • the protease is in operative association with the protease substrate.
  • the test compound decreases protease activity.
  • the protease is a serine protease, a metalloprotease, an aspartic protease, or a cysteine protease.
  • the protease is selected from the group consisting of neutrophil elastasae, leukocyte elastase, tyrosine carboxypeptidase, lysosomal carboxypeptidase C, thrombin, dipeptidyl peptidase
  • cathepsin B 2 cathepsin B 2 , cathepsin G, cathepsin L and calpain.
  • the protease is involved in infection and/or replication of a virus.
  • the virus is a human immunodeficiency virus, an adenovirus, a hepatitis virus, a rhinovirus, a herpesviruses, or a picomaviruses.
  • the virus is human cytomegalovirus.
  • the virus is hepatitis C virus.
  • the invention further comprises a cell comprising a nucleic acid molecule or molecules that express an ⁇ -donor fusion polypeptide, a protease, and an ⁇ -acceptor, wherein the ⁇ -donor fusion polypeptide comprises an ⁇ -donor in operative association with a protease substrate, and wherein protease cleavage of the ⁇ -donor fusion polypeptide results in ⁇ -galactosidase activity.
  • 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 ⁇ -donor fusion polypeptide, a protease, and an ⁇ -acceptor, wherein protease cleavage of the ⁇ -donor fusion polypeptide results in ⁇ - galactosidase activity.
  • the invention further comprises compounds identified by the methods of the invention.
  • the invention provides a compound that inhibits the activity of the protease identified by the above-described methods.
  • the invention provides a pharmaceutical composition comprising a compound that inhibits the activity of the protease identified using the methods of the invention.
  • 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 ⁇ -donor fusion polypeptides.
  • the ⁇ -donor fusion polypeptides of the invention can comprise an ⁇ -donor domain and a protease substrate or protein precursor domain.
  • the ⁇ -donor domain is present in the ⁇ -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 ⁇ -donor fusion polypeptide, the ⁇ -donor domain does not complement ⁇ -galactosidase activity.
  • the ⁇ -donor domain can be positioned in the molecule either on the amino- or carboxy-terminal side of the protease substrate or protein precursor domain.
  • compositions of the invention further comprise nucleic acid molecules that encode the ⁇ -donor fusion polypeptides of the invention.
  • nucleic acid molecules can be engineered to express the ⁇ -donor fusion polypeptides of the invention in a host cell.
  • nucleic acid molecules encoding the ⁇ -donor fusion polypeptides of the invention can be engineered to be expressed at a medium to low level in cells.
  • nucleic acid molecules encoding the ⁇ -donor fusion polypeptides of the invention are engineered to be expressed in a constitutive or a regulated, e.g., inducible, manner.
  • compositions of the invention still further comprise ⁇ -donor fusion polypeptide systems that express the ⁇ -donor fusion polypeptides of the invention and that can be utilized as part of the methods of the invention.
  • ⁇ -donor fusion polypeptide systems include cell-based systems as well as in vitro, cell-free systems.
  • the compositions of the invention still further comprise cells that express the ⁇ -donor fusion polypeptides of the invention, including cells that can be utilized as part of the methods of the invention.
  • the cells exhibit ⁇ -galactosidase activity (or substantial increase, generally at least a 4-fold increase, in such activity) only upon cleavage of the ⁇ -donor fusion polypeptide to release a portion of the fusion polypeptide containing the ⁇ -donor domain that complements an ⁇ -acceptor.
  • cells in such an embodiment further comprise nucleic acid sequences that encode an ⁇ -acceptor.
  • the cells of the invention can further comprise nucleic acid sequences that express a protease that cleaves the ⁇ -donor fusion polypeptide in a manner that releases a portion of the fusion polypeptide that contains the ⁇ -donor domain.
  • the nucleic acid sequences comprises regulatory sequences that express the protease in the cell.
  • the nucleic acid sequences encoding the protease are expressed at medium to low levels in the cell.
  • the nucleic acid molecules encoding the protease are expressed in a regulatable, e.g., inducible, manner.
  • a regulatable e.g., inducible, manner.
  • such systems comprise ⁇ -donor fusion polypeptides and/or nucleic acid molecules encoding and expressing ⁇ -donor fusion polypeptides, and an ⁇ -acceptor, or a nucleic acid molecule encoding and expressing the ⁇ -acceptor, that can be complemented upon release of the ⁇ -donor from the cleaved ⁇ -donor fusion polypeptide.
  • the in vitro system further comprises a protease, and/or nucleic acid molecules encoding and expressing the protease, that cleaves the ⁇ -donor fusion polypeptide to yield an ⁇ -donor polypeptide capable of complementing the ⁇ -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.
  • the ⁇ -donor fusion polypeptide is present within an ⁇ - donor fusion polypeptide cell system, and the test compound is exposed to or contacted to the cell.
  • the ⁇ -donor fusion polypeptide and the test compound are exposed or contacted together in vitro in a cell-free ⁇ -donor fusion polypeptide system.
  • ⁇ -donor refers to any portion of ⁇ -galactosidase * ⁇ able to interact with an inactive ⁇ -galactosidase mutant (e.g. , deletion mutant) and form a complex having ⁇ -galactosidase activity.
  • the ⁇ -donor represents a portion of the ⁇ -region of ⁇ -galactosidase.
  • ⁇ -acceptor refers to any ⁇ -galactosidase mutant 0 (e.g., a deletion mutant) that is enzymatically inactive, and is capable of interacting with an ⁇ -donor to form a complex having ⁇ -galactosidase activity.
  • the ⁇ - acceptor represents a portion of the ⁇ -region of ⁇ -galactosidase.
  • the term “complementation” refers to the ability of an ⁇ - 5 donor to interact with an ⁇ -acceptor and form a complex having ⁇ -galactosidase activity.
  • the ⁇ -donor is said to "complement” the ⁇ -acceptor, or to "complement” ⁇ -galactosidase activity.
  • FIG. 1 The structure and cleavage sites of HCMV UL80 polypeptide.
  • FIG. 5 Construction of plasmids that express ⁇ -donor fusion polypeptides comprising HCMV protein precursor domains.
  • FIG. 3 A-C ⁇ -complementation via cleavage of ⁇ -donor fusion polypeptides.
  • A ⁇ -UL80wt ⁇ -donor fusion polypeptide encoded by pW2/ cz-HCMV.
  • B H ⁇ UL80-wt ⁇ -donor fusion polypeptide encoded by pW3/ ⁇ cz-HCMV.
  • C UL80 ⁇ -wt ⁇ -donor fusion polypeptide encoded by pW3/ ⁇ cZ-HCMV.
  • FIG. 4A-C ⁇ -galactosidase activity from cells transformed with different constructs: A. ⁇ -UL80wt ⁇ -donor fusion polypeptide encoded by pW2/ ⁇ cz-HCMV. B.
  • the graphic insert in C represents the enlarged scale from pW2/ ⁇ cZ-wt and pW2/ ⁇ cZ-mut.
  • 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 ⁇ -donor fusion polypeptide proteolytic cleavage by detecting complementation of ⁇ -galactosidase activity.
  • the ⁇ -galactosidase ⁇ -complementation assay is based on the non-covalent interaction of two enzymatically inactive fragments of the ⁇ -galactosidase protein resulting in restoration of enzymatic activity.
  • “Complementation,” as used herein, refers to the ability of an ⁇ - donor to interact with an ⁇ -acceptor and form a complex exhibiting ⁇ -galactosidase activity.
  • the ⁇ -donor is said to "complement” an ⁇ -acceptor, or to "complement” ⁇ - galactosidase activity.
  • compositions of the invention comprise ⁇ -donor fusion polypeptides.
  • the ⁇ -donor fusion polypeptides of the invention can comprise an ⁇ -donor domain and a protease substrate domain or protein precursor domain.
  • the ⁇ -donor domain is present in the ⁇ -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 ⁇ -donor fusion polypeptide, the ⁇ -donor domain does not complement ⁇ -galactosidase activity.
  • the ⁇ -donor domain can be placed either ® amino to or carboxy to the protease substrate domain or protein precursor domain. In instances in which the ⁇ -donor is in an N-terminal position, it is preferred that there be a histidine tag present in the ⁇ -donor fusion polypeptide amino to the ⁇ -donor domain.
  • the ⁇ -donor domain can comprise any portion of the ⁇ -region of ⁇ - 5 galactosidase that interacts with an inactive ⁇ -galactosidase mutant (e.g. , deletion mutant) and forms a complex exhibiting ⁇ -galactosidase activity.
  • the inactive ⁇ -galactosidase mutant is referred to herein as an " ⁇ -acceptor.”
  • ⁇ -donor refers to any portion of ⁇ -galactosidase able to interact with an inactive ⁇ -galactosidase mutant (e.g., deletion mutant) and form a complex having ⁇ -galactosidase activity
  • ⁇ -acceptor refers to any ⁇ -galactosidase mutant (e.g., a deletion mutant) in the ⁇ region, is enzymatically inactive, and is capable of interacting with an ⁇ -donor and form a complex having ⁇ -galactosidase activity.
  • the ⁇ -acceptor and/or ⁇ - donor represent a portion of the ⁇ -region of ⁇ -galactosidase.
  • ⁇ -galactosidase amino acid sequences that can be utilized as ⁇ -donor sequences are well known to those of skill in the art, and include, but are not limited to, those described herein.
  • E. coli ⁇ -galactosidase is a tetrameric protein of approximately 540 kd in size.
  • Each of the four identical monomers of ⁇ -galactosidase consists of 1021 amino acids, and are divided into three regions: (1) the N-terminal proximal segment (the ⁇ - region), (2) a middle region, and (3) a C-terminal distal segment (the ⁇ -region).
  • the 11-41 deletion mutant of ⁇ - galactosidase is called the Ml 5 mutant.
  • Small peptides of ⁇ -galactosidase for example, of the ⁇ -region (e.g., amino acids 3 through 92, called the CNBr2 peptide), can interact with inactive mutants of ⁇ -galactosidase complement and restore full enzymatic activity (Langley
  • complementation refers to the ability of an ⁇ -donor to interact with an ⁇ -acceptor and form a complex having ⁇ - galactosidase activity.
  • the CNBr2 peptide can, for example, serve as an ⁇ donor for the Ml 5 mutant, as well as for the Ml 12 ⁇ -galactosidase mutant, a deletion of amino acids 23-41 within ⁇ -galactosidase that act as an ⁇ -acceptor (Zabin, 1982, Mol. Cell Biochem. 49:87-
  • N-terminal ⁇ -galactosidase fragments of various lengths, such as 1-44 and 3-40
  • Chem. 256:6804-10 can also be utilized as ⁇ -donor amino acid sequences.
  • ⁇ -donor 15 as the ⁇ donor
  • ⁇ -acceptor the larger ⁇ -galactosidase deletion peptide
  • Intracistronic complementation also occurs at the C-terminus of beta- 0 galactosidase (the ⁇ region).
  • the best known sequence data available is for the X90 ⁇ - 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,
  • ⁇ -donor and ⁇ -acceptor are used throughout the specification, it is to be understood that ⁇ -donor and ⁇ -acceptor sequences are not to be limited to only ⁇ -region and ⁇ -region-related sequences and/or mutations.
  • ⁇ -donor/ ⁇ -acceptor amino acid sequences can routinely be 0 identified by those of skill in the art via, e.g., ⁇ -galactosidase mutagenesis and assaying for complementation utilizing pairs of test ⁇ -donor/ ⁇ -acceptor sequences. See, e.g., U.S. Patent
  • protease substrate domains of the ⁇ -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 ⁇ -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 ⁇ -donor fusion polypeptide is cleaved.
  • compositions and methods of the present invention are 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.
  • 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.
  • 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
  • metalloproteases metalloproteases
  • protease substrate domains and the protein precursor domains of the ⁇ - 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
  • 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 ⁇ - 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
  • proteases such as 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,
  • proteases involved in viral infection/replication 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.
  • HIV human immunodeficiency virus
  • HCV hepatitis C virus
  • rhinoviruses including human rhinoviruses
  • herpesviruses including human cytomegalovirus (HCMV) and picomaviruses.
  • 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 ⁇ - 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.
  • the entire HCMV precursor amino acid sequence can be utilized as the protease substrate domain of the ⁇ -donor fusion polypeptide.
  • 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 ⁇ -donor fusion polypeptides of the invention
  • ⁇ -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 ⁇ -donor fusion polypeptides containing HCMV protease substrate domains as well as of cells expressing such ⁇ -donor fusion polypeptides.
  • 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,
  • 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.
  • 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 ⁇ -donor fusion polypeptide protease substrate domain.
  • Such a protease substrate domain would act as a substrate for the HCV host-encoded peptidase discussed above.
  • 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 ⁇ -donor fusion polypeptide protein precursor domain
  • ⁇ -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.
  • 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.
  • ⁇ -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.
  • the ⁇ -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).
  • the ⁇ -donor fusion polypeptides of the invention can comprise picornavirus protease substrate domains or protein precursor domains, as described above.
  • proteases are directly involved in the destructive effects of microbial pathogens, acting on the proteins of the host.
  • 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.
  • the ⁇ -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 ⁇ -donor can be present either amino to or carboxy to the protease substrate or protein precursor domain of the ⁇ -donor fusion polypeptide.
  • the ⁇ -donor domain can be engineered to be present within the protease substrate domain or the protein precursor domain. In instances in which the ⁇ -donor domain is in an
  • the ⁇ -donor fusion polypeptide further comprise a cysteine tag N-terminal to the ⁇ -donor domain Prior to cleavage of the ⁇ -donor fusion polypeptide, the ⁇ -donor does not exhibit the ability to complement ⁇ -galactosidase activity.
  • the ⁇ -donor domain comprises an amino acid sequence that, upon cleavage of the ⁇ -donor fusion polypeptide via protease cleavage, complements ⁇ - galactosidase activity via an ⁇ -acceptor.
  • the released ⁇ -donor should generate a ⁇ -galactosidase activity signal at least about 4-fold over background levels of signal.
  • the ⁇ -donor domain is positioned within the ⁇ -donor fusion polypeptide such that the ⁇ -donor fusion polypeptide portion containing the ⁇ -donor after cleavage comprises as few amino acid residues as possible that are still able to complement ⁇ -galactosidase activity.
  • the ⁇ -donor domain should be positioned within the ⁇ - donor fusion polypeptide relative to the core cleavage site such that the maximum distance exists between the ⁇ -donor and the core cleavage site and which yet continues to allow ⁇ - galactosidase complementation upon cleavage.
  • the ⁇ -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 ⁇ -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.
  • the ⁇ -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 ⁇ -donor fusion polypeptide.
  • ⁇ -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.
  • nucleic acid molecules encoding the ⁇ -donor fusion polypeptides of the invention can be synthesized and/or constructed according to such well known techniques and utilized to express the ⁇ -donor fusion polypeptides of the invention. See, e.g., Sambrook et al, 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold
  • nucleotide sequences of the ⁇ -donor domain and protease substrate and protein precursor domains of the ⁇ -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 ⁇ -donor fusion polypeptide of the invention need only be joined or ligated within the nucleotide sequence encoding the ⁇ -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.
  • test ⁇ -donor fusion polypeptides can also routinely be accomplished by those of skill in the art.
  • standard recombinant techniques can be used to generate nucleic acid molecules that encode and express test ⁇ -donor fusion polypeptides.
  • test ⁇ -donor fusion polypeptides can also be assayed, via, e.g., standard complementation assays described herein, for exhibition of features and preferred features of the ⁇ -donor fusion polypeptides of the invention.
  • Such features and preferred features include, for example, the ⁇ -donor' s lack of complemention while present within and as part of the uncleaved ⁇ -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 ⁇ -donor to complement after cleavage of the ⁇ -donor fusion polypeptide and the level of complementation signal generated by the ⁇ -donor after cleavage.
  • Successful optimization of such such ⁇ -donor fusion polypeptide parameters is demonstrated in the Examples presented below.
  • test ⁇ -donor fusion polypeptide in a cell incapable of supporting cleavage of the ⁇ -donor fusion polypeptide facilitates the selection and optimization of placement of ⁇ -donor domains so that said placement within the ⁇ -donor fusion polypeptide inactivates the ⁇ -donor complementation activity while the ⁇ -donor remains within the uncleaved ⁇ -donor fusion polypeptide.
  • the cell may either be normally unable to cleave the ⁇ -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 ⁇ -donor fusion polypeptide.
  • compositions of the invention further comprise nucleic acid molecules that encode the ⁇ -donor fusion polypeptides of the invention. Such nucleic acid molecules can be generated, e.g., as discussed above.
  • compositions of the invention still further comprise cells that express the ⁇ -donor fusion polypeptides of the invention, including cells that can be utilized as part of the methods of the invention.
  • the nucleic acid molecules encoding the ⁇ -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.
  • a nucleotide vector e.g., a plasmid, cosmid, phagemid, bacteriophage, virus, retrovirus, YAC, BAC or HAC vector
  • the nucleic acid molecules of the invention can further comprise nucleic acid sequences required for transcription and translation of the ⁇ -donor fusion polypeptide in vitro or in a host cell of interest.
  • 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.
  • nucleic acid molecules of the invention can additionally comprise a ribosome binding site, an initiator codon or a terminator site.
  • the nucleic acid molecules of the invention comprise nucleotide sequences that encode the ⁇ -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).
  • nucleic acid molecules of the invention encoding the ⁇ -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. •
  • the present invention further relates to cell-based and in vitro, cell-free ⁇ - donor fusion polypeptide systems.
  • Such systems can, e.g., be utilized as part of the methods of the present invention.
  • the ⁇ -donor fusion polypeptide systems of the invention comprise ⁇ -donor fusion polypeptides and/or nucleic acid molecules encoding and expressing such ⁇ -donor fusion polypeptides, and an ⁇ -acceptor polypeptide and/or a nucleic acid molecule that encodes and expresses such an ⁇ -acceptor polypeptide.
  • the ⁇ -acceptor polypeptide is one which can be complemented by the ⁇ -donor upon cleavage of the ⁇ -donor fusion polypeptide.
  • the ⁇ -donor fusion polypeptide systems of the invention further comprise the ability to cleave the ⁇ -donor fusion polypeptides of the systems at the cleavage site or sites contained within the ⁇ -donor fusion polypeptides.
  • the ⁇ -donor fusion polypeptides of the system contain a protease substrate domain
  • the ⁇ -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.
  • the ⁇ -donor fusion polypeptide contains a protein precursor domain
  • the protein precursor domain exhibits autocatalytic self cleavage activity.
  • the systems do not exhibit ⁇ -galactosidase activity, or exhibit substantially lower levels of ⁇ -galactosidase activity than that detectable upon ⁇ -donor fusion polypeptide cleavage.
  • the level of ⁇ -galactosidase detected after cleavage is at least 4 times the level detectable prior to ⁇ - donor fusion polypeptide cleavage.
  • the systems exhibit ⁇ -galactosidase activity that can be measured using standard ⁇ -galactosidase assay techniques described below.
  • the ⁇ -donor fusion polypeptide systems of the invention represent in vitro, cell-free ⁇ -donor fusion polypeptide systems.
  • Such systems comprise ⁇ - donor fusion polypeptides and/or nucleic acid molecules encoding and expressing ⁇ -donor fusion polypeptides, and an ⁇ -acceptor, or a nucleic acid molecule encoding and expressing the ⁇ -acceptor, that can be complemented upon release of the ⁇ -donor from the cleaved ⁇ - donor fusion polypeptide.
  • the in vitro system further comprises a protease, and or nucleic acid molecules encoding and expressing the protease, that cleaves the ⁇ -donor fusion polypeptide to yield an ⁇ -donor polypeptide capable of complementing the ⁇ -acceptor of the system.
  • nucleic acid molecules encoding a protease e.g., an assemlbin protease
  • an ⁇ -donor fusion polypeptide e.g., one comprising an assembly protein protease substrate, and an ⁇ -acceptor
  • 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 ⁇ -donor fusion polypeptide system of the invention.
  • 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.
  • the ⁇ -donor fusion polypeptide systems of the invention are cell systems.
  • Cells utilized as part of the ⁇ -donor fusion polypeptide cell systems of the invention 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.
  • bacterial e.g., Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and Pseudomonas aeuroginosa, with E. coli being preferred
  • fungal e.g
  • the cell-based ⁇ -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 ⁇ -donor fusion polypeptide, ⁇ -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 ⁇ -donor fusion polypeptide expression and ⁇ -galactosidase complementation activities of the cell-based systems of the invention can also routinely be accomplished.
  • the cells express the ⁇ -donor fusion polypeptides at low to medium levels of expression.
  • the nucleic acid molecules encoding such ⁇ -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.
  • nucleic acid sequences encoding such ⁇ -donor fusion polypeptides can be expressed at high, constitutive levels.
  • the ⁇ -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 ⁇ -donor fusion polypeptides within the cell to yield ⁇ -donor-containing fragments that complement ⁇ -galactosidase activity.
  • proteases can be endogenous to the cell or can be introduced, e.g., recombinantly introduced, into the cell.
  • 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).
  • these embodiments are preferred in that high levels of a particular protease may be harmful or toxic to the cell in which they accumulate.
  • nucleic acid sequences encoding such proteases can be expressed at high, constitutive levels.
  • nucleic acid molecules encoding such ⁇ -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 ⁇ -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.
  • nucleic acid sequences encoding such ⁇ -donor fusion polypeptides can be expressed at high, constitutive levels.
  • Nucleic acid sequences encoding ⁇ -acceptors that can be complemented by released ⁇ -donors can be endogenous to the cell of the ⁇ -donor fusion polypeptide system.
  • nucleic acid molecules encoding the ⁇ -acceptor can be introduced, e.g., recombinantly introduced, into the cell.
  • nucleic acid molecules encoding ⁇ -acceptors can be expressed in a high level, constitutive manner in the cell.
  • nucleic acid encoding the ⁇ -acceptor can be expressed at low to medium levels and/or in a regulatable, e.g., inducible, manner.
  • ⁇ -galactosidase activity-deficient strains that are suitable for engineering into the ⁇ -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) ⁇ -galactosidase activity. The strains can be such that they express neither ⁇ -donor nor ⁇ -acceptor sequences.
  • such strains can express an ⁇ -acceptor sequence that can be complemented by the ⁇ -donor domain of the ⁇ -donor fusion polypeptide of interest (that is, once the ⁇ -donor fusion polypeptide is cleaved by the appropriate protease).
  • ⁇ -acceptor sequence that can be complemented by the ⁇ -donor domain of the ⁇ -donor fusion polypeptide of interest (that is, once the ⁇ -donor fusion polypeptide is cleaved by the appropriate protease).
  • any of a number of well known Messing strains that expres ⁇ -acceptor sequences e.g., strain JM109, which contains Ml 5 sequences, but are deficient in ⁇ -galactosidase activity can be utilized.
  • E. coli and Salmonella genetic stock center or other public collections (E. coli stock center (CGSC), Yale University, New Haven, CT; Salmonella
  • NRRL Peoria, IL
  • NRRL Peoria, IL
  • Strains such as those available from the above sources can be engineered into ⁇ -donor fusion polypeptide cell system strains by utilizing recombinant techniques to produce the ⁇ -donor fusion polypeptide nucleic acids of the invention, and standard genetic techniques can be utilized for introducing the ⁇ -donor fusion polypeptide nucleic acids into cells.
  • Neidhardt, supra, and Yarmolinsky & Steinberg, supra. can be utilized for such purposes.
  • the ⁇ -donor fusion polypeptide construct is generated in a cell by the use of a ⁇ -donor fusion polypeptide nucleic acid which is introduced into a bacterial cell and propagated in progenies of the bacterial cell.
  • 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.
  • CaCl 2 a method for treating cells harvested after exponential growth and subsequently treated by the CaCl 2 method using procedures well known in the art.
  • 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 ⁇ -donor fusion polypeptide, protease or ⁇ -acceptor transcripts in the host cell; and (d) detection of the ⁇ -donor fusion polypeptide, protease or ⁇ -acceptor as measured by immunoassay.
  • cells are constmcted to facilitate uptake of externally provided, e.g., test, compounds.
  • 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
  • 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-
  • 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.
  • 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, ⁇ 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).
  • 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-Pe ⁇ on 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.
  • ⁇ vectors such as ⁇ gtl 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.
  • promoters o of weak strengths 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).
  • high strength promoters can be utilized.
  • strong promoters including but not limited to, ⁇ P L (Pirrotta, 1975, Nature 254: 114-117) the trp promoter (Bennett et al., 1976,
  • 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.
  • nucleic acid sequences expressing ⁇ -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.
  • the lad gene and its gratuitous inducer IPTG can be utilized to yield inducible levels of expression of ⁇ -donor fusion polypeptides when sequences encoding such polypeptides are transcribed via the lacOP regulatory sequences.
  • inducible promoter systems are well known to those of skill in the art which can also be utilized.
  • Other regulated expression systems include but are not limited to, the TET system (Geissendorfer M. & Hillen W.,
  • the cell-based ⁇ -donor fusion polypeptide systems of the invention can also comprise mammalian cells.
  • Starting cells for the production of mammalian cell-based ⁇ -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.,
  • ⁇ -galactosidase complementation can be utilized in mammalian cells, and techniques for engineering mammalian cells such that ⁇ -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 inco ⁇ orated herein by reference in its entirety.
  • a mammalian cell ⁇ -donor fusion polypeptide system can be generated by introducing nucleic acid sequence encoding a Ml 5 ⁇ -galactosidase ⁇ -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 ⁇ -acceptor mutant in the mammalian cell.
  • 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 ⁇ -donor fusion polypeptide is also introduced into the mammalian cell.
  • the ⁇ -donor fusion polypeptide coding sequence encodes an ⁇ -donor fusion polypeptide with an ⁇ -donor domain that upon ⁇ -donor fusion polypeptide cleavage, complements the Ml 5 ⁇ -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.
  • the ⁇ -donor fusion polypeptide-coding sequence can be subcloned into a mammalian cell expression vector compatible with the vector associated with the ⁇ -acceptor coding sequence.
  • the ⁇ -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 ⁇ -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
  • the mammalian cell further comprises a nucleic acid molecule that encodes and expresses the protease that cleaves the protease substrate.
  • the protease is expressed in an inducible manner, e.g., as described above, to minimize any possible toxicity to the host cell.
  • 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.
  • sequences encoding ⁇ -donor fusion polypeptide or protease polypeptides be expressed in an inducible manner.
  • Nucleic acid molecules encoding ⁇ -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.
  • the cell-based ⁇ -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.
  • 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
  • expression of at least the ⁇ -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.
  • the cell-based ⁇ -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 inco ⁇ orated herein by reference in its entirety.
  • the ⁇ -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
  • 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).
  • the methods of the invention comprise methods for identifying compounds that modulate protease activity. That is, the methods of the invention comprise methods for
  • 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.,
  • any kind of natural products may be screened using the methods of the invention, including microbial, fungal, plant or animal extracts.
  • test compounds including small molecule test compounds
  • libraries may be commercially obtained from
  • 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-
  • references are inco ⁇ orated 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.
  • compounds have a molecular weight of more than about
  • the methods of the invention comprise: exposing an ⁇ - 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 ⁇ -galactosidase complementation, so that if a differential level of ⁇ -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 ⁇ -galactosidase activity indicates identfication of a putative antagonist of protease activity, while a relative increase in ⁇ -galactosidase activity indicates identification of a putative agonist of protease activity.
  • the methods of the invention comprise: exposing an ⁇ -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 ⁇ -galactosidase complementation, so that if a differential level of ⁇ -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 ⁇ -galactosidase activity indicates identfication of a putative antagonist of protease activity, while a relative increase in ⁇ -galactosidase activity indicates identification of a putative agonist of protease activity.
  • the ⁇ -donor fusion polypeptide is present within an ⁇ - donor fusion polypeptide cell system, and the test compound is exposed to or contacted to the cell.
  • the ⁇ -donor fusion polypeptide and the test compound are exposed or contacted together in vitro in a cell-free ⁇ -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.
  • the methods of the invention can be performed as described above, but can be modified such that the ⁇ -donor fusion polypeptide comprises an ⁇ -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
  • 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 ⁇ -donor fusion polypeptide system further comprises a protease, such a protease is an inactive one (i.e., does not exhibit protease activity).
  • 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.
  • 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.
  • test cells can be cultured and assayed in an ordered array, such as multi-well plates.
  • ordered array such as multi-well plates.
  • 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.
  • the methods of the present invention assay protease activity via detection and measurement of ⁇ -galactosidase complementation. That is, the amount of ⁇ - donor fusion polypeptide proteolysis in a sample is measured as a direct function of the activity of ⁇ -galactosidase brought about by the release of the ⁇ -donor domain from the ⁇ - donor fusion polypeptide.
  • ⁇ -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 ⁇ - galactosidase that are suitable for spectrophotometric or fluorometric analysis include, but are not limited to: p-aminophenyl- ⁇ -D-galactopyranoside; 2'-N-(hexadecanol)-N-amino-4'- nitrophenyl)- ⁇ -D-galactopyranoside; 4-methylumbel-liferyl- ⁇ -D-galactopyranoside; napthyl-AS-B 1 - ⁇ -D-galactopyranoside; 1 -napthyl- ⁇ -D-galactopyranoside; 2-napthyl- ⁇ -D- galactopyranoside monohydrate; O-nitrophenyl- ⁇ -D-galactopyranoside; m-nitrophenyl- ⁇ -D- galactopyranoside; p-nitrophenyl- ⁇ -D-galactopyranoside; and phenyl- ⁇ -
  • 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.
  • 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.
  • 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.
  • 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.
  • a protease inhibitor or agonist agent which modulates protease activity in the vims or infectious agent as determined by the assays of the invention.
  • agents can be administered by any method known to those skilled in the art, for example, by topical application or by systemic administration.
  • 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.
  • 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.
  • 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) are determined using the same method.
  • 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.,
  • 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.
  • the compound may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions.
  • an appropriate buffer for example, phosphate buffered saline or other physiologically compatible solutions.
  • the resulting complex may be formulated with a non-ionic surfactant such as Tween, polyethylene glycol or glycerine.
  • 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.
  • 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).
  • 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
  • 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).
  • 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
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • 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.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, 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.
  • 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.
  • 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
  • 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.
  • 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.
  • compositions for parenteral dosage form such as intravenous injection or infusion
  • similar pharmaceutical media e.g., water, glycols, oils, buffers, sugar, preservatives and the like know to those skilled in the art.
  • parenteral compositions include, but are not limited to Dextrose 5%w/v, normal saline or other solutions.
  • compositions 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.
  • 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).
  • 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 adso ⁇ tion 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 abso ⁇ tion refers to the accumulation of agents in the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic abso ⁇ tion include: oral, intravenous, subcutaneous, intraperitoneal, intranasal, intrathecal and ocular. Each of these administration routes exposes the agent to an accessible target.
  • Example presented in this Section describes the successful construction of an ⁇ -donor fusion polypeptide and cell-based ⁇ -donor fusion polypeptide systems comprising a human cytomegalovims (HCMV) protein precursor domain.
  • HCMV human cytomegalovims
  • 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 ⁇ -donor fusion polypeptide and an ⁇ -donor fusion polypeptide cell system,
  • HCMV ⁇ -donor fusion polypeptides and ⁇ -donor fusion polypeptide cell-based systems Two expression vectors were constmcted in which the designated protease substrate was inserted in-frame upstream and in-frame downstream of the ⁇ -donor (FIG. 2).
  • a PCR fragment of 51 residues (4-55) of ⁇ - galactosidase from pcDNA3.1/.Hygro// ⁇ cZ (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
  • the resulting plasmid, pW3/ ⁇ cZ can be used for N-terminal ⁇ -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/ ⁇ cZ, in which the lacZ gene is downstream of the multiple cloning site.
  • pW2/ ⁇ cZ can be used for C- terminal ⁇ - donor fusion to any protease substrate.
  • AD 169 and that contains the UL80 gene was utilized for constmction of the protein precursor of the ⁇ -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/ ⁇ cZto form p ⁇ 3lacZ-wt for in-frame expression of the N-terminal ⁇ -fragment fusion protease, ⁇ UL8Owt.
  • 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.
  • Sse8387-Sacl fragment was then subcloned into p ⁇ llacZ to form pW2/ ⁇ cZ-wt to create a constmct for in-frame expression of the C-terminal ⁇ -fragment fusion protease, UL80 ⁇ wt.
  • 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/ ⁇ cZ, which was used to produce an ⁇ -donor fusion polypeptide protease H ⁇ UL80wt.
  • N-terminal and C-terminal fusion proteins were 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 ChameleonTM 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 ⁇ -donor fusion polypeptides were ⁇ UL8Omut, H ⁇ UL80mut, and UL80 ⁇ mut, respectively.
  • 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- ⁇ -galactopyranoside (CPRG) as the substrate. Briefly, single colonies containing different constmcts were grown in a 96 well microtiter plate with or without 20 ⁇ M IPTG. Cell density was measured by OD 600 reading. 200 ⁇ l cells were lysed by adding 30 ⁇ l of chloroform and then 75 ⁇ l of lysed cells were transfened to a new plate containing 150 ⁇ l of 1 mg/ml CPRG in Z buffer. The plate was placed in a plate reader (Molecular Device) and ⁇ -galactosidase activity was monitored at OD J75 over 15 minutes.
  • CPRG chlorophenolred- ⁇ -galactopyranoside
  • ⁇ UL ⁇ Owt Two N-terminal ⁇ -donor fusion polypeptides, ⁇ UL ⁇ Owt and H ⁇ UL ⁇ Owt, were constmcted as described in Sectin 6.1, above.
  • ⁇ UL8Owt 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 ⁇ -galactosidase (the ⁇ -donor domain), 3 linker amino acids and the 708 amino acids of the UL80 polypeptide (the protein precursor domain).
  • H ⁇ UL ⁇ Owt has six additional histidines prior to the ⁇ -fragment ( Figure 3B).
  • HCMV protease self-cleaves at multiple sites, with cleavage at position 143 releasing a 205 amino acid product, named ⁇ UL8o fragment, which is the smallest possible cleavage product and is likely to be most efficient at ⁇ -complementation (FIG. 3A).
  • UL80 ⁇ -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 ⁇ -galactosidase ( ⁇ -donor domain). Autocleavage at position 643 will release an 152 amino-acid residue peptide, UL8 ⁇ fragment, which can participate in ⁇ -complementation (Figure 3C).
  • Colonies from all constructs showed either dark or light blue phenotypes, indicating ⁇ -complementation took place.
  • FIG. 4A-C results are shown in FIG. 4A-C for each of the constmcts: ⁇ -UL80wt ⁇ -donor fusion polypeptide encoded by pW2/ ⁇ cz-HCMV (FIG. 4A); H ⁇ UL80-wt ⁇ -donor fusion polypeptide encoded by pW3/ cz-HCMV (FIG. 4B); UL ⁇ O ⁇ -wt ⁇ -donor fusion polypeptide encoded by pW3/ ⁇ cZ-HCMV (FIG. 4C).
  • Example presented in this Section describes the construction of a hepatitis C vims (HCV) polyprotein-containing ⁇ -donor fusion polypeptide and ⁇ -donor fusion polypeptide cell system.
  • HCV hepatitis C vims
  • a fragment of ⁇ -galactosidase consisting of amino acids 3 through 92 of the ⁇ -region of ⁇ -galactosidase is used as the ⁇ -donor, and the Ml 5 mutant serves as the ⁇ - acceptor.
  • a DNA sequence coding for ⁇ -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 ⁇ -fragment/N5SA fusion proteins assayed for its ability to complement the Ml 5 mutant.
  • the optimum site for insertion of the ⁇ -fragment into the N5SA protein is routinely chosen by identifying that fusion protein with the ⁇ - fragment sequence farthest from the cleavage junctions, yet still able to complement the Ml 5 mutant.
  • the ⁇ -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 ⁇ -donor fusion polypeptide is checked for lack of ability to complement the M15 mutant. Additionally, the ⁇ -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 ⁇ -galactosidase assays and assays for identifying test compounds that modulate HCV protease activity.
  • the ⁇ -donor fusion polypeptide described above is expressed in a cell known or expected to support cleavage of the HCV polyprotein into its component peptides.
  • the ⁇ -fragment/HCV polyprotein is cleaved, and the ⁇ -fragment/NS5A fusion protein is released.
  • the presence of free ⁇ -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.

Abstract

La présente invention concerne des dosages de protéase et, plus particulièrement, des composés et des méthodes utiles pour un dosage visant à déceler une activité protéasique. L'invention concerne également des cribles ciblés, efficaces et à haut rendement, qui permettent d'identifier des composés à petites molécules, des peptides, etc, qui modulent (c'est-à-dire interfèrent avec ou améliorent) l'activité protéasique. L'invention concerne en outre une variété de dosages in vivo ou in vitro, ainsi que des composés thérapeutiques, tels que des composés antiviraux, identifiés par les méthodes de l'invention.
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WO2002038799A2 (fr) * 2000-11-07 2002-05-16 University Of Victoria Innovation And Development Corporation Detection de proteases et criblage d'inhibiteurs de proteases
WO2003035832A2 (fr) * 2001-10-22 2003-05-01 Exelixis, Inc. Ctsls en tant que modificateurs des voies de passage de p53 et de apaf-1 et leurs procedes d'utilisation
EP1474526B1 (fr) * 2002-01-29 2008-06-25 Discoverx, Inc. Analyse de l'activite des proteases
US7452690B2 (en) 2003-01-28 2008-11-18 Discoverx Corporation Protease EFC cell surface fusion protein assay
AU2007225195B2 (en) 2006-03-13 2013-10-24 The Board Of Trustees Of The Leland Stanford Junior University Detection of molecular interactions using a reduced affinity enzyme complementation reporter system
US8101373B2 (en) 2007-10-12 2012-01-24 Discoverx Corporation β-galactosidase donor fragments
US8569057B2 (en) 2011-06-23 2013-10-29 Discoverx Corporation Monitoring protein trafficking using beta-galactosidase reporter fragment complementation
EP3947714A4 (fr) * 2019-03-28 2022-12-14 Eurofins discoverx Products, LLC. Procédés d'analyse de région de promoteur et cellules pour la mise en oeuvre correspondante

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015575A1 (fr) * 1990-04-04 1991-10-17 Chiron Corporation Protease du virus de l'hepatite c
WO1992003559A2 (fr) * 1990-08-28 1992-03-05 Boehringer Ingelheim International Gmbh Systeme d'essai permettant de controler l'activite de proteinases virales
WO1993001291A1 (fr) * 1991-07-05 1993-01-21 The Johns Hopkins University Proteinase du virus de l'herpes et methode de titrage
WO1997027320A1 (fr) * 1996-01-26 1997-07-31 Boehringer Mannheim Corporation DOSAGES RECOURANT A DES FRAGMENTS POLYPEPTIDIQUES RETICULES DE β-GALACTOSIDASE
WO1999006537A1 (fr) * 1997-07-30 1999-02-11 Boehringer Mannheim Corporation Composants de dosage polypetidique reticules

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015575A1 (fr) * 1990-04-04 1991-10-17 Chiron Corporation Protease du virus de l'hepatite c
WO1992003559A2 (fr) * 1990-08-28 1992-03-05 Boehringer Ingelheim International Gmbh Systeme d'essai permettant de controler l'activite de proteinases virales
WO1993001291A1 (fr) * 1991-07-05 1993-01-21 The Johns Hopkins University Proteinase du virus de l'herpes et methode de titrage
WO1997027320A1 (fr) * 1996-01-26 1997-07-31 Boehringer Mannheim Corporation DOSAGES RECOURANT A DES FRAGMENTS POLYPEPTIDIQUES RETICULES DE β-GALACTOSIDASE
WO1999006537A1 (fr) * 1997-07-30 1999-02-11 Boehringer Mannheim Corporation Composants de dosage polypetidique reticules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO0039348A1 *

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