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WO1993001305A1 - Procede d'identification d'inhibiteurs de proteases - Google Patents

Procede d'identification d'inhibiteurs de proteases Download PDF

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Publication number
WO1993001305A1
WO1993001305A1 PCT/US1992/005745 US9205745W WO9301305A1 WO 1993001305 A1 WO1993001305 A1 WO 1993001305A1 US 9205745 W US9205745 W US 9205745W WO 9301305 A1 WO9301305 A1 WO 9301305A1
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Prior art keywords
protease
nucleic acid
coding sequence
acid sequence
protein
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PCT/US1992/005745
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English (en)
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Robert Balint
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Robert Balint
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • 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
    • C12N2770/34011—Potyviridae
    • C12N2770/34022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the instant invention relates generally to the identification of protease inhibitors. More
  • the invention relates to methods of
  • identifying viral protease inhibitors which can in turn be used to treat or prevent viral infection.
  • Proteases are enzymes that cleave peptide bonds, thereby altering proteins. Besides degrading proteins, these enzymes play a regulatory role in a variety of physiological processes. Proteases fall into four general classes: serine, cysteine, aspartic acid, and metalloproteases. These classes are distinguished primarily by mechanism (Dunn, B.M., in Proteolytic
  • Serine and cysteine proteases have almost identical two-step mechanisms with an acyl-enzyme intermediate. Together they comprise the majority of the known proteases. Aspartic and metallo-proteases catalyze direct hydrolysis of the peptide bond.
  • procaryotic and eucaryotic proteins are synthesized as larger biologically inactive precursors which become activated only when acted upon by
  • endoproteases proteinases
  • proteins proteinsases
  • endoproteases proteinsases
  • proteins proteinsases
  • These enzymes typically recognize specific domains, usually less than ten amino acids in length including the sissile bond, in exposed loops of generally loose secondary structure (Keil, B., in Methods in Protein Sequence Analysis, M. Elzinga, ed., Humana Press, Clifton, N.J., 1982, pp. 291-304).
  • Maturation proteases are responsible for both intracellular and extracellular cleavage of protein precursors and many of the proteolytically processed proteins in turn play key roles in physiological
  • intracellular proteolytic maturation include secreted proteins, lysosomal enzymes, mitochondrial proteins and membrane proteins. These proteins are highly diverse in function, having endocrine, neurological, and immune functions, as well as acting as growth factors and antibiotics. Secreted proteins that undergo
  • extracellular proteolytic processing include the plasma zymogens involved in blood clotting and the immune complement system.
  • Maturation proteases which are indirectly involved in human disease are generally distinguished by their high degree of substrate specificity. However, a host of digestive proteases of lesser specificity are also known and are more directly involved in diseases such as chronic inflammation and tumor metastasis. These enzymes include elastases, collagenases, mast cell proteases, and extracellular matrix-degrading metalloproteases, among others.
  • Plant viruses which encoce proteases for this purpose include the
  • potyviruses comoviruses, nepoviruses, sobemoviruses, and luteoviruses. These viruses cause economically important diseases in all major monocot and dicot families.
  • Similarly equipped animal viruses include the
  • picomaviruses picomaviruses, retroviruses, alphaviruses, flaviviruses, pestiviruses, coronaviruses, and adenoviruses.
  • Diseases caused by these viruses include foot-and-mouth disease, AIDS, the common cold, hepatitis and polio.
  • ZYMV Zika virus
  • picomaviruses (Dougherty, W.G., et al., Virology (1989) 172:302-310: Bazan, J.F., and R.J. Fletterick, Proc.
  • the invention herein is based on the discovery of a unique method for detecting peptide protease inhibitors. These inhibitors can be used directly or indirectly in the treatment of protease-dependent diseases. Alternatively, the inhibitors so identified can be utilized as structural models for the rational design of peptide-mimetics.
  • the subject invention is directed to a method for detecting a protease inhibitor which comprises:
  • the subject invention is directed to a DNA construct comprising:
  • control sequences that are operably linked to the first and second coding sequences whereby the coding sequences can be transcribed and translated in a host cell, and at least one of the control sequences is heterologous to at least one of the coding sequences.
  • the DNA construct further includes a second DNA coding sequence for the protease of interest.
  • the subject invention is directed to host cells stably transformed with these DNA constructs.
  • Figure 1 depicts Protease Inhibitor Selection System I, as applied to ZYMV protease.
  • Figure 2 depicts representative examples of Protease Inhibitor Selection System II.
  • Figure 3 shows the strategy of cDNA synthesis from ZYMV and cloning methods.
  • FIG. 4 shows the nucleotide sequence of the
  • Figure 5A shows the organization of the primary translation products of pZPro ⁇ , pZPro7 and placZ ⁇ -CP.
  • Figure 5B depicts the results of immunoblot analysis of SDS/PAGE separated proteins from E. coli cells harboring these plasmids.
  • Figure 6 depicts the derivation of the pZPro7, pZPro9, pZPro10, pZPro11 and pZPro12 constructs and the organization of the primary translation products.
  • the open boxes denote ZYMV 49 kDa protease (Pro) cleavage sites.
  • Strep R streptomycin-resistant.
  • Amp R
  • ampicillin-resistant i.e., transformed.
  • Cfu colony- forming units.
  • NT not tested.
  • proteases an enzyme that cleaves a peptide bond.
  • the term includes both endopeptidases (also called proteinases) which are proteases that hydrolyze internal peptide bonds, and exopeptidases, which are proteases that cleave either N- or C-terminal peptide bonds .
  • endopeptidases also called proteinases
  • exopeptidases which are proteases that cleave either N- or C-terminal peptide bonds .
  • Some proteases are highly specific , cleaving only between two particular amino acids within a particular protein.
  • Other proteases are less specific, cleaving between more than one amino acid pair and/or cleaving between an amino acid pair in more than one location in the same and/or different proteins.
  • proteases include maturation proteases
  • proteins proteins, lysosomal enzymes, mitochondrial proteins, membrane proteins, plasma zymogens, digestive enzymes, elastases, collagenases, mast cell proteases,
  • extracellular matrix-degrading metalloproteinases such as proteases from potyviruses, comoviruses, nepoviruses, sobemoviruses, and
  • luteoviruses luteoviruses
  • animal viral proteases such as
  • proteases from picomaviruses, retroviruses are proteases from picomaviruses, retroviruses,
  • alphaviruses flaviviruses, pestiviruses, coronaviruses, and adenoviruses.
  • protease inhibitor is meant a molecule capable of altering the activity of a protease such that the protease is unable to completely hydrolyze a peptide bond for which it is specific.
  • Protease inhibitors can be peptides composed solely of genetically encodable amino acids.
  • Protease inhibitor also encompasses synthetic peptide derivatives such as peptide aldehydes and ketones, peptide boronic acids, peptide chloromethyl ketones, azapeptides, peptide hydroxamic acids, and peptide thiols.
  • protee inhibitor also encompasses synthetic nonpeptide compounds such as
  • peptide and "protein” are used in their broadest sense, i.e., any polymer of genetically encodable amino acids (dipeptide or greater) linked through peptide bonds.
  • polymer of genetically encodable amino acids linked through peptide bonds.
  • oligopeptides polypeptides, protein fragments, muteins, fusion proteins and the like.
  • a "host cell” is a cell which has been transformed, or is capable of transformation, by an exogenous nucleotide sequence.
  • host cells for use in the present invention may be either procaryote or eucaryote, depending on the specific protease in question and the selection system desired.
  • bacterial cells either gram-negative or gram-positive
  • Eucaryotic cells can be used, however, in cases where either the protease or its dependent phenotype can be adequately expressed only in such cells, such as cases in which certain types of transport, metabolism, or post-translational modification are required.
  • Eucaryotic cells can also be used to select inhibitors of other types of biological activities which can be expressed only in such cells, such as animal virus replication.
  • One skilled in the art can readily determine an appropriate host cell for use in the present invention.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • double-stranded DNA molecule refers to the polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular
  • this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and
  • chromosomes In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the
  • nontranscribed strand of DNA i.e., the strand having the sequence homologous to the mRNA.
  • a DNA "coding sequence” or a “nucleotide sequence encoding” a particular protein is a DNA
  • a coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bound at the 3' terminus by the translation start codon (ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eucaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Procaryotic promoters contain Shine-Dalgarao sequences in addition to the -10 and -35 consensus sequences.
  • control sequences refers collectively to promoter sequences, ribosome binding sites
  • a coding sequence is "operably linked to" another coding sequence when RNA polymerase will
  • telomere a sequence that is transcribed into mRNA, which is then translated into a chimeric polypeptide encoded by the two coding sequences.
  • the coding sequences need not be contiguous to one another so long as the transcribed sequence is ultimately processed to produce the desired chimeric protein.
  • a control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a cell has been "transformed" by an exogenous nucleotide sequence when the sequence has been introduced inside the cell membrane.
  • An exogenous nucleotide sequence may or may not be integrated (covalently linked) to chromosomal nucleic acid making up the genome of ?the cell.
  • the exogenous nucleotide sequence may be maintained on an episomal element, such as a plasmid.
  • a stably transformed cell is one in which the exogenous nucleotide sequence has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • the heterologous region encodes a bacterial gene
  • the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria.
  • heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • treatment refers to either (i) the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms of the disease of interest (therapy).
  • Described herein is a system which can be used to select effective inhibitors of proteases from large pools of random peptide sequences.
  • the method utilizes a known protease which can be obtained through standard techniques, i.e. direct isolation, synthesis or
  • the nucleotide sequence of the protease can be determined and used to transform a host cell.
  • the host cell is also transformed with a
  • nucleotide sequence encoding a protein that confers a negative phenotype on the cell, such as sensitivity to a given antibiotic, or inability to grow on a given carbon source, which is dependent on the activity of the cloned protease.
  • conferring the dependent phenotype are contained on one or more constructs which have been introduced into the host cell.
  • inhibition of the protease confers a selectable phenotype on the cell (e.g., antibiotic resistance, or the ability to grow on a given carbon source).
  • the particular inhibitor can be isolated, sequenced and further used as described below.
  • the negative phenotype may be expressed by either of two general mechanisms.
  • a gene conferring a dominant negative phenotype is expressed as an inactive precursor protein which is activated by protease-mediated cleavage at a site which has been engineered to resemble a natural substrate of the
  • protease In the second mechanism, a gene conferring a selectable phenotype is inactivated by protease-mediated cleavage at a similarly engineered site.
  • the above-described host cells can then be used to detect effective inhibitors of the protease from large pools of random peptides encoded on another plasmid.
  • This additional plasmid contains a gene which encodes a "carrier" protein in which all or part of an exposed domain has been
  • the randomized domain may range from four to fifteen amino acids in length.
  • the length of the randomized domain may range from four to fifteen amino acids in length.
  • peptides intended for use for the design of peptide mimetics will tend to have shorter sequences than
  • peptides for use in peptide or gene therapy.
  • Such random sequence “libraries” can be constructed by replacing the sequence encoding the exposed domain in the "carrier” protein gene with a set of synthetic oligodeoxynucleotides of random sequence.
  • a natural substrate of the protease in question can be conveniently used for the "carrier” protein.
  • one of the many well-characterized natural protease inhibitors may be used (Proteinase Inhibitors. A.J. Barrett and G. Salvesen, eds. (Elsevier, Amsterdam, 1986), section C), in which the amino acid sequence of the native binding domain has been randomized.
  • flanking domains of the "carrier” may be minimized by flanking the randomized sequence with short "spacers" of polyproline or polyglycine which are highly flexible
  • Some of these "random" peptides will, by chance, have structures which are capable of binding tightly to the active site of the protease, thereby preventing it from either activating or inactivating the negative phenotype-conferring protein, depending on the mechanism employed. This, in turn, will confer the selectable phenotype on the host cell when transformed with these constructs.
  • the structures of effective inhibitors can then be determined by sequencing the appropriate regions of constructs recovered from such phenotype-selected cells.
  • Protease Inhibitor Selection System I Protease Inhibitor Selection System 1 as applied to the ZYMV protease is illustrated in Figure 1.
  • a portion of the ZYMV polyprotein is depicted which includes the protease, replicase (NIb), and coat protein (see U.S. Patent Application Serial No. 07/560,130).
  • the arrows indicate substrate sites at which the protease cleaves the polyprotein.
  • either the replicase or coat protein is replaced with the coding sequence for the protein
  • E. coli ribosomal protein S12 which confers sensitivity to streptomycin on streptomycin- resistant hosts such as E. coli strain MC1009 (Post, L.E., and M. Nomura, J. Biol. Chem. (1980) 255:4660-4666).
  • a transcription repressor protein may also be used such as the lactose, tryptophan, or phage lambda repressors (Lewin, B., Genes IV. Cell Press, Cambridge, 1990, pp. 240-264). These act by repressing expression of antibiotic resistance genes in hosts in which these genes are transcribed from repressible promoters. In either case the negative phenotype is only displayed when the negative phenotype protein is being actively cleaved out of the polyprotein by the protease.
  • protease and the negative phenotype precursor may be convenient to express from separate transcription units.
  • the only requirements are that the negative phenotype protein be linked to an extraneous domain by a peptide sequence which is a natural substrate for the protease in question, and that this precursor be inactive until cleaved by the protease.
  • Figure 2 illustrates the second mechanism wherein the negative phenotype is conferred by protease-mediated inactivation of a protein conferring a
  • Protease Inhibitor Selection System II selectable phenotype
  • proteins include secreted or membrane proteins which confer resistance to the antibiotics ampicillin,
  • protease substrate peptide sequence is inserted between the signal sequence and the mature protein such that cleavage by the protease renders the protein incapable of membrane transport or insertion and thereby inactive.
  • the protease substrate sequence may be inserted into a surface domain of the mature protein such that cleavage by the protease renders the protein
  • a special case of this selection system occurs with proteases which are toxic when expressed in E. coli by virtue of their fortuitous inactivation of one or more host proteins which are required for growth (Baum, E.Z., et al., Proc. Natl. Acad. Sci. USA (1990) 87:5573-5577).
  • the inactivated host protein(s) confer the selectable phenotype in the presence of inhibitors of the protease.
  • the random peptide inhibitor gene library may be delivered to the selector cells by any of several methods, the choice of which will depend to some extent on the size of the library.
  • One skilled in the art can readily determine an acceptable technique to use with a given library. For example, chemical transformation with purified plasmid (Sambrook, J., et al., Molecular
  • bacteriophage-derived vectors can be used for delivery by transduction.
  • a plasmid vector can be converted to a cosmid vector (Sambrook, J., et al., Molecular
  • transductions of the packaged cosmids into selector cells can then be accomplished by established methods.
  • concatemers of the inhibitor gene library can be used instead of "stuffer" DNA in the cosmid to achieve the necessary size for packaging. This reduces, by an order of magnitude, the number of
  • transformants that need to be screened to cover the entire library are transformedants that need to be screened to cover the entire library.
  • the stringency of selection by these systems can be adjusted in a variety of ways.
  • a number of transcriptional promoters and enhancers of varying strengths are available (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989, ch. 17), which can be used with the protease, negative phenotype precursor, and inhibitor genes to raise or lower the inhibitor strength required for selection.
  • Inducible promoters can be used, such that their strengths may be titrated by adjusting the amount of inducer in the growth medium. For example, by having the inhibitor expressed from an inducible promoter, the potency threshold of a pool of selected inhibitors is raised, and the size of the pool reduced, simply by reducing the amount of inducer present during selection.
  • inhibitor inducibility can be used to counterselect stable false positives, such as revertants that have mutated the protease gene, simply by replica plating onto selective medium in the absence of inducer. Only the revertants are able to grow.
  • protease inhibitor can be isolated and chemically characterized, using known techniques. These systems can be used to generate inhibitor peptides for any protease which can be
  • proteases of many important plant and animal viral pathogens inhibitors of the proteases of other types of microbial pathogens as well as cellular proteases which have been implicated in such disorders as rheumatoid arthritis, Alzheimer's disease, and tumor metastasis, can also be identified.
  • the instant invention can be used to identify protease inhibitors which in turn are useful in the treatment of protease-dependent diseases in both plants and animals.
  • the inhibitors can be used directly in peptide therapy or can be encoded in a gene and used in gene therapy.
  • the identified inhibitors can also serve as structural models for the rational design of peptide-mimetics, that is, synthetic compounds that mimic the protease-binding action of the identified protease inhibitors to bring reactive groups into contact with the protease active site. (See, e.g., Demuth, H.-U., J.
  • invention also has a more general application in the construction and use of in vivo systems for the selection of bioactive peptides from peptide libraries.
  • systems may be designed for the selection of peptide inhibitors of hydrolytic enzymes other than proteases.
  • hydrolytic enzymes other than proteases.
  • a number of such enzymes are known which can hydrolyze natural or artificial substrates to produce one or more compounds which are toxic to E. coli (for
  • any phenotype of cultured procaryotic or eucaryotic cells which can be altered by the endogenous expression of appropriate peptides such that cells expressing such peptides can be readily distinguished and isolated from cells which either do not express such peptides or which express peptides which do not alter the phenotype, may provide the basis for establishing an in vivo system for the selection of bioactive peptides from peptide libraries.
  • most tractable medically important phenotypes will be those manifesting susceptibility to microbial
  • the endogenous expression of a random peptide library as an exposed domain of a suitable stable "carrier" protein in a population of cultured mammalian cells of sufficient size to ensure that all or most members of the library are represented in the population may be used to select peptides which interfere with the ability of microbial pathogens such as viruses or bacteria or their toxins to inhibit cell growth.
  • microbial pathogens such as viruses or bacteria or their toxins
  • Such cell populations are challenged by such pathogens or toxins, only those cells expressing inhibitory peptides will grow, allowing the active peptides to be identified by established methods.
  • Such peptides can in turn be used for the development of effective therapies.
  • the identified inhibitors can be used to create transgenic plants.
  • One commonly used method of gene transfer in plants involves the insertion of the gene of interest into the T-DNA region of a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes, respectively.
  • Many control sequences are known which when coupled to a heterologous coding sequence and transformed into a host organism show fidelity in gene expression with respect to tissue/organ specificity of the original coding sequence. See, e.g., Benfey, P.N., and Chua, N.H., Science (1989) 244:174-181.
  • Suitable control sequences for use in these plasmids include promoters for constitutive leaf-specific expression of the desired gene in the various target plants.
  • Other useful control sequences include a
  • NOS nopaline synthase gene
  • vir vir
  • the virulence (vir) gene from either the Ti or Ri plasmid must also be present, either along with the T-DNA portion, or via a binary system where the vir gene is present on a separate vector.
  • Such systems, vectors for use therein, and methods of transforming plant cells are described in U.S. Patent No. 4,658,082, and Simpson, R.B., et al., Plant Mol. Biol. (1986) 6:403-415, incorporated herein by reference in their entirety.
  • these plasmids can be placed into A. rhizogenes or A. tumefaciens and these vectors used to transform cells of plant species which are ordinarily susceptible to the particular plant pathogen.
  • A. tumefaciens or A. rhizogenes will depend on the plant being transformed thereby.
  • A. tumefaciens is the preferred organism for transformation. Most dicotyledons, some gymnosperms, and a few monocotyledons (e.g., certain members of the
  • A. rhizogenes also has a wide host range, embracing most dicots and some gymnosperms, which
  • these cells can be used to regenerate transgenic plants.
  • whole plants can be infected with these vectors by wounding the plant and then introducing the vector into the wound site. Any part of the plant can be wounded, including leaves, stems and roots.
  • plant tissue in the form of an explant, such as cotyledonary tissue or leaf disks, can be inoculated with these vectors and cultured under conditions which promote plant regeneration. Roots or shoots transformed by inoculation of plant tissue with A. rhizogenes or A. tumefaciens. containing the desired gene, can be used as a source of plant tissue to
  • the inhibitors identified by the present method can also be used in gene therapy.
  • HIV-specific protease inhibitor genes in which a natural mammalian protease inhibitor serves as carrier for the HIV protease inhibitor domain, can be used in anti-AIDS gene therapy.
  • Lymphocytes or bone marrow cells from the patient can be transformed with the protease inhibitor gene in vitro and returned to the patient, where they establish an HIV-resistant subpopulation of lymphocytes which can gradually restore cell-mediated immune function as the patient's untransformed lymphocytes are depleted by the virus.
  • proteases active in blood, lymph, or cerebro-spinal fluid which are essential components of disorders such as chronic inflammations, metastatic cancers, and certain viral infections, may be targeted by protease inhibitor gene therapy, in which the inhibitors are secreted by transgenic lymphocytes or other
  • the inhibitors identified by the present method can be altered by established methods to improve their pharmaco-kinetic properties.
  • the inhibitors may be
  • a carrier for example, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin, a statin
  • Suitable carriers are typically large, slowly metabolized macromolecules such as: proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.
  • Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
  • the protein substrates may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine
  • a sulfhydryl group may also be incorporated into the carrier (or inhibitor) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3-(4-dithiopyridyl) propionate.
  • Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides. Methods of coupling peptides to proteins or cells are known to those of skill in the art.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • the active immunogenic ingredient is often mixed with vehicles containing excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents, or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 15th edition, 1975.
  • the composition or formulation to be administered will, in any event, contain a quantity of the inhibitor adequate to achieve the desired effect in the individual being treated.
  • suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations.
  • vehicle composition will include traditional binders and carriers, such as, polyalkylene glycols, or
  • triglycerides Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5V to about 10% (w/w), preferably about 1% to about 2%.
  • Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like. These oral compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules,
  • sustained release formulations or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • Controlled or sustained release formulations are made by incorporating the inhibitor into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel ® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures.
  • carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel ® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures.
  • the inhibitors can also be delivered using implanted mini-pumps, well known in the art.
  • the inhibitors may be formulated into pharmaceutical compositions in either neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for
  • sodium, potassium, ammonium, calcium, or ferric hydroxides examples include sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
  • procaine and the like.
  • the inhibitor of interest is administered parenterally, usually by
  • injectable formulations will contain an effective amount of the active ingredient in a vehicle, the exact amount being readily determined by one skilled in the art.
  • the active ingredient may typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate.
  • the quantity to be administered depends on the animal to be treated and the particular inhibitor used. Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • the subject is treated by administration of the
  • the subject may be administered as many doses as is required to effectively treat the individual.
  • This example describes the construction and expression in E. coli of a gene which encodes a portion of the ZYMV polyprotein.
  • the primary translation product of this gene is a 140 kDa protein which includes the 49 kDa protease and flanking cleavage sites, a portion of the nuclear inclusion 'b' protein (NIb, also referred to as the replicase), including the NIb/coat protein
  • a California isolate of ZYMV was obtained from Professor J.A. Dodds of the University of California at Riverside. The virus was propagated by mechanical inoculation of the cotyledons of ten-day-old Cucurbita pepo cv. early straightneck seedlings. Systemically infected leaves were harvested 3-8 weeks after
  • the virus was quantified by absorbance at 260 nm using an extinction coefficient of 2.8 A 260 /mg/ml.
  • Viral genomic RNA was isolated from purified virions by digestion with protease K in borate buffer (pH 9) containing 1% SDS and 4 mM EDTA for one hour at 37°C, followed by phenol/chloroform extraction and ethanol precipitation. The RNA was redispersed in water,
  • PZRl the largest cDNA clone obtained from the first experiment, had a 2.3 kb insert, the ends of which were sequenced using the Sanger dideoxy chain-terminating method as described in the product literature for the Sequenase 2 sequencing kit (United States Biologicals) with the M13 universal and reverse primers encoded on either end of the multiple cloning site in pBluescript.
  • the orientation of the insert relative to the viral genome and the multiple cloning site was indicated by the appearance of the polyadenylate tract from the 3' end of the genome in the sequence from the reverse primer.
  • the remainder of the clone was sequenced stepwise, 200-300 nucleotides at a time, in both directions from synthetic oligodeoxynucleotide primers complementary to the distal ends of each of the successive sequencing runs. Sequence data were processed and analyzed on a DEC VAX 11/750 minicomputer.
  • the second round of cDNA cloning was accomplished in the same manner as the first except that a synthetic oligodeoxynucleotide complementary to the 5' end of pZRl was used as primer and the cDNAs were ligated directly, without linkers, into the EcoRV site of
  • sequences of adjacent time points to overlap were filled in using synthetic oligodeoxynucleotide primers made from the sequence near the 5' end of the gap.
  • Clone pZB60 was sequenced in both directions from nested deletions in the same manner as for pZB11 except that a unique NcoI site within pZB11 was used as the starting point for deletions in the 5' direction and only those clones with deletions mapping between the 5' end of pZB11 and the 5' end of pZB60 were sequenced.
  • a third round of cDNA cloning was conducted as described above for the preparation of pZB11 and pZB60 except that a synthetic oligomer complementary to the 5' end of pZB60 was used as a primer. From this round, pZF18, having an insert of 3.7 kb was obtained and sequenced as described above for pZB11 and pZB60.
  • the 5' end of the viral RNA sequence was determined by reverse transcription of purified viral RNA using a synthetic oligonucleotide primer complementary to nucleotides 76-99 at the 5' end of pZF18.
  • the Sanger dideoxynucleotide chain-terminating method was used essentially as described in the Promega Gem Seq manual (Promega Corp.).
  • the continuous open reading frame of the viral genome was identified with the aid of a computer as described above.
  • the coding sequences of the functional ZYMV gene products were identified by amino acid sequence homology to those of other potyviruses (Allison, R., et al., Virology (1986) 154:9-20: Domier, L.L., et al.,
  • Figure 4 shows the nucleotide sequence of the ZYMV genome as determined above along with the deduced amino acid sequence.
  • the nucleotide sequence is numbered from the 5' terminus.
  • the 5' non-coding region extends from nucleotide 1 to nucleotide 139.
  • Nucleotides 140-142 initiate the polyprotein coding sequence with a
  • the cleavage site between the 46 kDa protein and the cytoplasmic inclusion protein (CI) is believed to occur between the glutamine at codon 1164 (nucleotides 3629-3631) and the glycine at codon 1165 (nucleotides 3632-3634).
  • the cleavage site between CI and VPg/protease (VPg and protease are probably not separated in ZYMV) is believed to occur between the glutamine at codon 1798 (nucleotides 5531-5533) and the serine at codon 1799 (nucleotides 5534-5536).
  • the cleavage site between VPg/protease and RNA replicase is believed to occur between the glutamine at codon 2284 (nucleotides 6989-6991) and the serine at codon 2285 (nucleotides 6992-6994).
  • the cleavage site between the RNA replicase and the coat protein (CP) is believed to occur between the glutamine at codon 2801 (nucleotides 8540-8542) and the serine at codon 2802 (nucleotides 8543-8545). Termination of the polyprotein coincides with termination of the coat protein and is believed to occur at the stop codon (nucleotides 9380-9382) following the glutamine at codon 3080.
  • the 3' non-coding sequence then extends from nucleotide 9383 to nucleotide 9593 before terminating in a polyadenylate sequence of
  • cDNA clone pZRl contained approximately 80 adenosines at its 3' terminus.
  • pZPro5
  • a restriction fragment of 1666 base pairs (bp) extending between the PvuII and SspI sites of ZYMV cDNA clone pZB11 (described above) was isolated by agarose gel electrophoresis and ligated into the Smal site of plasmid pTZ18U (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989; Mead D.A., et al.,
  • This restriction fragment comprises a portion of the coding sequence of the ZYMV polyprotein which includes part of the
  • cytoplasmic inclusion protein (CI)
  • 6 kDa protein the 6 kDa protein
  • 49 kDa protease the 4 kDa protease
  • NIb protein a portion of the NIb protein. Insertion of this fragment into the Smal site of pTZ18U places the reading frame encoding these proteins in phase with that of the expressible lacZ ⁇ gene of pTZ18U such that expression of this gene from the lac promoter is expected to produce a fusion protein comprised of a small portion of the lacZ ⁇ peptide fused to the amino terminus of the ZYMV polyprotein fragment.
  • This construct was denoted pZPro5 and its structure was confirmed by
  • pZPro6 and pZPro7 The 2280 bp SalI restriction fragment from ZYMV cDNA clone pZR1 (described above), which comprises a portion of the ZYMV genome including part of the NIb protein, CP, the 3' non-coding sequence, and a portion of the polyadenylate sequence, was inserted into the SalI site of pZPro5 to create pZPro6.
  • Dideoxynucleotide sequencing of pZPro6 confirmed that the NIb/CP-encoding reading frame of the inserted fragment was in phase with the open reading frame of pZPro5 such that expression of this construct from the lac promoter is expected to produce a single polypeptide of approximately 140 kDa.
  • strain DH5 ⁇ was transformed with pZPro6 and pZPro7, and transformed clones were identified and isolated by selection for ampicillin resistance.
  • lacZ ⁇ -ZYMV polyprotein gene was monitored by immunoblotting of SDS/PAGE-resolved proteins from these cells using
  • This example describes the construction of expressible genes encoding polyproteins which contain the ZYMV 49 kDa protease and the E. coli ribosomal protein S12. The ability of these gene constructs to confer sensitivity to the antibiotic streptomycin on several streptomycin-resistant E. coli strains by virtue of correct and efficient processing of the polyprotein by the ZYMV 49 kDa protease is demonstrated.
  • Streptomycin lethality in E. coli has been ascribed to its ability to interfere with protein synthesis by binding to the S12 subunit of the 30S component of the ribosome (Gorini, L., in Ribosomes, M. Nomura, A. Tissieres, P. Lengyel, eds., Cold Spring Harbor Laboratory, 1974, pp. 791-803). Streptomycin- resistant mutants have been isolated which express altered forms of S12 which retain the ability to
  • the CP-encoding sequences in pZPro7 were precisely replaced with the coding sequence for S12 to create pZPro9. This was accomplished as follows.
  • the sequence bounded by the Bglll site in NIb and the P1' position of the Nlb/CP cleavage site in pZPro7 was amplified by polymerase chain reaction (PCR, Saiki, R.K., et al., Science (1988) 239:487).
  • the S12 coding sequence from the second amino acid to the end was amplified by PCR from plasmid pNO1523 (Dean, D., Gene
  • the 3' primer contained an MluI site following the stop codon.
  • pZPro9 was then transformed into streptomycin-resistant EL. coli strains MC1009, HB101, and N100
  • the Nlb/CP cleavage site was removed from pZPro9 to create pZPro12, which should produce a polyprotein from which S12 cannot be freed by the protease. This was accomplished by cleaving pZPro9 with EcoRV and Hpal, which removed most of the NIb protein including the Nlb/CP cleavage site , and replacing it with the fragment produced by EcoRV alone, which restored most of the NIb protein down to within 12 amino acids of the Nlb/CP cleavage site (see Figure 6). The structure of pZPro12 was confirmed by dideoxynucleotide sequencing. Transformation with pZPro12 has no
  • the strep-sensitive phenotype produced by pZPro9 is completely dependent on the presence of a substrate cleavage site at which the protease can cleave functional S12 from the polyprotein.
  • pZPro9 polyprotein
  • the expression of the pZPro9 polyprotein places a considerable burden on growing E. coli cells.
  • the pZPro9 was streamlined by removing the EcoRV fragment described above, which contains most of the NIb protein exclusive of the cleavage sites at either end.
  • This construct denoted pZPro10, encodes a
  • strep-resistant E. coli strains displayed a strep-sensitive phenotype identical to that of the pZPro9 transformants.
  • pZPro10 transformants grew considerably more vigorously than pZPro9 transformants, indicating a significant reduction in the metabolic burden on the host cells.
  • inhibitors from peptide libraries or to confirm selected inhibitors on the basis of their ability to restore rapid growth.
  • the protease removes itself from the polyprotein of pZPro10, the 14 kDa S12 is left in a 21.5 kDa precursor until freed by the protease.
  • the NIb/CP cleavage site was removed from pZPro10 in the same manner that the EcoRV-Hpal restriction fragment was removed from pZPro9 to create pZPro12.
  • GATAGTCCTC CGCAAGCCGA GTAAGCAGCG GGTTTTCGCT CGTATCGAGC AGGATGAGGC 480
  • TTACAAGCGC ACATACAAGA AGGAAAGGAA GAAAGTGGCG CAAAAGCAAA TTGTGCAAGC 660
  • AAAATCAACG AGCTTACCTG CACATCTTGC CAAGAAGGGT AAGGTGTTAC TACTCGAACC 3960

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Abstract

L'invention concerne des procédés de détection d'inhibiteurs de protéases. Elle décrit également des constructions d'ADN, et des cellules hôtes transformées à l'aide de ces constructions et utilisées dans les procédés en question. Les procédés utilisent une cellule hôte qui présente un phénotype négatif dépendant de l'activité d'une protéase donnée. Ainsi, l'inhibition de la protéase confère un phénotype sélectionnable à la cellule. Le phénotype négatif peut être conféré soit par activation induite par la protéase soit par l'inactivation d'une protéine conférant un phénotype sélectionnable. L'inhibiteur est détecté par transformation de cellules hôtes exprimant les gènes pour le phénotype sélectionnable et une protéase donnée avec des séquences peptides au hasard. Des inhibiteurs ainsi identifiés peuvent être utilisés soit directement soit indirectement dans le traitement de troubles dépendant de protéases.
PCT/US1992/005745 1991-07-09 1992-07-09 Procede d'identification d'inhibiteurs de proteases WO1993001305A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034976A1 (fr) * 1995-05-01 1996-11-07 Vertex Pharmaceuticals Incorporated Procedes, sequences nucleotidiques et cellules hotes pour evaluer l'activite de protease exogene et endogene
US6117639A (en) * 1998-08-31 2000-09-12 Vertex Pharmaceuticals Incorporated Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
WO2002018588A1 (fr) * 2000-08-29 2002-03-07 Novozymes A/S Procede de criblage de proteases hautement actives et leurs inhibiteurs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0321973A2 (fr) * 1987-12-23 1989-06-28 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Expression de la protéase P2A de HRV2 codée par un virus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0321973A2 (fr) * 1987-12-23 1989-06-28 BOEHRINGER INGELHEIM INTERNATIONAL GmbH Expression de la protéase P2A de HRV2 codée par un virus

Non-Patent Citations (3)

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Title
GENE, Volume 15, issued 1981, D. DEAN, "A Plasmid Cloning Vector for the Direct Selection of Strains Carrying Recombinant Plasmids". *
JOURNAL OF GENERAL VIROLOGY, Volume 71, issued 1990, R. GRUMET et al., "cDNA Cloning and Sequence Analysis of the 3'-Terminal Region of Zucchini Yellow Mosaic Virus RNA", see especially Figure 1. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 87, issued December 1990, E.Z. BAUM et al., "Beta-Galactosidase Containing a Human Immunodeficiency Virus Protease Cleavage Site is Cleaved and Inactivated by Human Immunodeficiency Virus Protease", pages 10023-10027. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034976A1 (fr) * 1995-05-01 1996-11-07 Vertex Pharmaceuticals Incorporated Procedes, sequences nucleotidiques et cellules hotes pour evaluer l'activite de protease exogene et endogene
US5861267A (en) * 1995-05-01 1999-01-19 Vertex Pharmaceuticals Incorporated Methods, nucleotide sequences and host cells for assaying exogenous and endogenous protease activity
US6117639A (en) * 1998-08-31 2000-09-12 Vertex Pharmaceuticals Incorporated Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
US6528276B1 (en) 1998-08-31 2003-03-04 Vertex Pharmaceuticals Inc. Fusion proteins, DNA molecules, vectors, and host cells useful for measuring protease activity
WO2002018588A1 (fr) * 2000-08-29 2002-03-07 Novozymes A/S Procede de criblage de proteases hautement actives et leurs inhibiteurs

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