WO2002050101A1 - Proteine de liaison au retinoblastome - Google Patents

Proteine de liaison au retinoblastome Download PDF

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WO2002050101A1
WO2002050101A1 PCT/AU2001/001635 AU0101635W WO0250101A1 WO 2002050101 A1 WO2002050101 A1 WO 2002050101A1 AU 0101635 W AU0101635 W AU 0101635W WO 0250101 A1 WO0250101 A1 WO 0250101A1
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seq
pai
binding
isolated
residues
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PCT/AU2001/001635
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WO2002050101A8 (fr
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Andreas Suhrbier
Grant Darnell
Toni Marie Antalis
Ricky Wayne Johnstone
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The Council Of The Queensland Institute Of Medical Research
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Priority claimed from AUPR2200A external-priority patent/AUPR220000A0/en
Priority claimed from AUPR6644A external-priority patent/AUPR664401A0/en
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Priority to AU2002221351A priority Critical patent/AU2002221351A1/en
Publication of WO2002050101A1 publication Critical patent/WO2002050101A1/fr
Publication of WO2002050101A8 publication Critical patent/WO2002050101A8/fr

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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/08—Antibacterial agents for leprosy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • THIS INVENTION relates to a novel protein domain that binds the retinoblastoma protein (RB). This domain has been identified in PAI-2 and a number of other proteins that bind the retinoblastoma protein.
  • This invention also relates to agonists and antagonists of binding to the retinoblastoma protein and methods of modulating gene expression using RB-binding domain proteins and agonists or antagonists.
  • This invention also extends to diagnostic methods and therapeutic methods of treating cancers such as those caused by oncogenic viruses and cancers associated with cell cycle disorders, viral infections such as human immunodeficiency virus (HIV) and diseases associated with cell hyperproliferation such as psoriasis, cardiovascular disease and asthma.
  • HIV human immunodeficiency virus
  • PAI-2 was originally characterised as an inhibitor of urokinase- plasminogen activator (Antalis et al, 1988, Proc. Natl. Acad. Sci USA 85 985; Kruithof et al, 1995, Blood 86 4007), an extracellular serine proteinase important for cell migration and invasion (Nassalli et ah, 1991, J. Clin. Invest. 88 1067).
  • PAI-2 was one of the first identified members of a unique and growing subclass of serine proteinase inhibitors (serpins), now called ovalbumin-like serpins (ov-serpins), which lack a typical amino terminal signal sequence, and are often found to reside intracellularly (Remold-O'Donnell, 1993, FEBS Letters 315 105).
  • Serine proteinases are proteins that act essentially as 'molecular scissors' to cleave other proteins within the cell and tissue environment. Serpins are key inhibitors of serine proteinase activities.
  • PAI-2 is very inefficiently secreted, and largely accumulates inside the cell. Indeed, it has been postulated that PAI-2 may play a role in intracellular signaling events.
  • PAI-2 expression confers resistance to cell death induced by the inflammatory factor tumour necrosis factor alpha (T ⁇ F ⁇ ; Dickinson et al, 1995, J. Biol. Chem. 270 27894; Kumar et al,
  • T ⁇ F induced cell death is associated with multiple intracellular effects, and it has been postulated that PAI-2 interferes with one or several of the
  • PAI-2 expressed intracellularly protects cells from the rapid cytopathic effects of alphavirus, and protection was associated with induction of interferon (IFN) ⁇ and ⁇ production and IFN-stimulated gene factor 3
  • ISGF3 activation (ISGF3) activation (Antalis et al, 1998, J. Exp. Med. 187 1799).
  • Retinoblastoma is a childhood cancer that arises when both alleles of the Rb gene (Rb/) are inactivated. Mutations in Rb/ are also associated with osteosarcomas, small lung carcinomas, prostate carcinomas, breast carcinomas, certain leukaemias and cervical carcinomas (reviewed in Herwig &
  • the Rb/ gene product is a protein (RB) comprising 928 amino acids, which undergoes regulatory phosphorylation at one or more of several sites.
  • the "hypophosphorylated" form of RB interacts with SV40 T antigen (DeCaprio et al, 1988, Cell 54 275), adenovirus E1A protein (White et al, 1988, J. Virol. 62
  • the LXCXE motif is also present in many RB-binding cellular proteins such as histone deacetylases 1 and 2 (HDAC1 and HDAC2) and cyclins
  • LXCXE motif-binding domain of RB has been mapped to amino acids within a cleft in the B-half of the RB pocket (Lee et al, 1998, Nature 391 859).
  • RB binds not only E2F but also a large panel of cellular transcription factors and other proteins (Dick et al, 2000. supra), and through these interactions modulates various cellular processes.
  • E2F-mediated transcription Some of the downstream products of E2F-mediated transcription involved in DNA replication include H2 folate reductase, thymidine kinase, DNA polymerase A, histone H2A deacetylase and in cell-cycle progression are cyclins A, D, E, cdc2 and E2F itself (Herwig & Strauss, 1997, supra). Binding of RB by LXCXE-containing viral proteins releases RB-mediated inhibition of E2F- dependent transcription of genes that promote entry into S-phase.
  • viral proteins that do not have an LXCXE motif which nevertheless bind RB.
  • An example of such a protein is the plant geminiviral protein AL1, where a novel RB-binding motif has been identified in a region between amino acid residues 110 and 156 (Kong et al, 2000, EMBO J. 19
  • the present inventors have unexpectedly shown that a mammalian cellular protein, PAI-2, binds mammalian RB via a novel, non-LXCXE RB- binding domain present in PAI-2.
  • the present inventors have also discovered an analagous binding motif in a number of other mammalian, plant and viral RB- binding proteins.
  • the invention is therefore broadly directed to an RB-binding domain, proteins comprising said domain and uses thereof.
  • the RB-binding domain binds mammalian RB.
  • the RB-binding domain is that of a eukaryotic cellular protein or, more preferably, a mammalian cellular protein.
  • the invention resides in an isolated RB-binding domain comprising the amino acid sequence (P/A)Z 2 (F/M/Y/L/H), wherein Z is any intermediate or large polar amino acid.
  • said isolated RB-binding domain comprises an amino acid sequence selected from the group consisting of: PENF (SEQ ID NO:2) PDNM (SEQ ID NO:3) PTNM (SEQ ID NO:4), PDNL (SEQ ID NO:5) PEPF (SEQ ID NO:6) PHDF (SEQ ID NOJ) AHDF (SEQ ID NO:8) PFSF (SEQ ID NO:9) PNLV (SEQ ID NO: 10) PENF (SEQ ID NO:l l) PTIF (SEQ ID NO:12) PEEF (SEQ ID NO:13) PGPM (SEQ ID NO: 14) PEHF (SEQ ID NO: 15)
  • PELF SEQ ID NO: 16
  • PVTH SEQ ID NO: 17
  • PEKY SEQ ID NO: 18
  • PLYF SEQ ID NO: 19
  • PQCF PQCF
  • said isolated RB-binding domain comprises an amino acid sequence Z(X 4 . 5 )J(X 4 )PZZ(F/M/Y/L/H)(X 6 . 7 )Z(X 8 . 9 )JJ with up to five mismatches allowed, wherein Z is an intermediate or large polar residue, J is a hydrophobic amino acid and X is any amino acid.
  • the RB-binding domain has an amino acid sequences as identified hereinafter in any one of the amino acid sequences set forth in Figure 3 (SEQ ID NOS:21-44).
  • the isolated RB-binding domain may further comprises the amino acid sequence LXCXE.
  • the invention also contemplates peptide fragments, variants and derivatives of the isolated RB-binding domain of the first aspect.
  • the invention also contemplates an RB-binding domain having at least 60% sequence identity with an RB-binding domain identified in any one of SEQ JD NOS: 21-44.
  • the invention provides an isolated protein complex comprising RB and the RB-binding domain of the first aspect or an RB- binding protein such as PAI-2.
  • the invention provides use of the RB-binding domain of the first aspect to identify or design an agonist, partial agonist or antagonist of binding to RB.
  • the invention resides in an isolated nucleic acid encoding the RB-binding domain of the first aspect.
  • the invention provides an expression construct comprising the isolated nucleic acid of the fourth aspect.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically-effective amount of an RB-binding domain according to the first aspect, an RB-binding protein or an isolated nucleic acid encoding same, together with a pharmaceutically-acceptable carrier, diluent or excipient.
  • the invention provides a method of modulating gene expression, said method comprising the step of administering to an organism, or cells isolated therefrom, the isolated RB-binding domain of the first aspect, an RB-binding protein; or an isolated nucleic acid encoding same, to thereby modulate expression of one or more genes.
  • E2F-dependent gene expression is modulated by the method of the invention.
  • gene expression is up-regulated, such as by removing RB-mediated repression.
  • gene expression is down-regulated, such as by inducing or augmenting RB-mediated repression.
  • an example of this latter embodiment is down-regulation of HPV 18 E6 and E7 gene expression by administration of PAI-2.
  • down-regulation of HJN gene expression can be achieved by inhibiting binding between cellular PAI-2 and RB.
  • the invention provides a method of modulating RB through PAI-2, an RB- binding domain thereof.
  • RB activity including the step of administering to an organism, or cells isolated therefrom, an isolated RB-binding domain of the first aspect, an RB-binding protein, or an isolated nucleic acid encoding same to thereby modulate RB activity.
  • RB mediated activity is up-regulated.
  • RB mediated activity is down-regulated.
  • HPV E7 is inhibited from binding RB, such that the protein can no longer target RB for degradation.
  • cyclin kinases are inhibited from phosphorylating RB, resulting in enhanced G, arrest.
  • the invention provides a method of cancer prognosis, said method including the step of identifying the absence of a mutation in an RB protein, which mutation would normally reduce or eliminate binding of said RB protein to PAI-2, the absence of said mutation indicating that an individual has a relatively improved cancer prognosis.
  • the method of this aspect further includes the step of measuring PAI-2 expression, wherein a relatively increased level of expression of PAI-2 indicates a relatively improved cancer prognosis.
  • animals include mammals inclusive of domestic companion animals and livestock, fish and other vertebrates as well as animals representative of lower phyla such as crustaceans, worms and molluscs.
  • Preferred animals are mammals.
  • Preferred mammals are humans. Throughout this specification, unless otherwise indicated,
  • FIG. 1 BRIEF DESCRIPTION OF THE FIGURES Figure 1. Recovery of RB, p53, p21 WAFI CIP and c-Jun expression and loss of E7 expression in HeLa cells expressing PAI-2.
  • A Western blot analysis of whole cell protein lysates from the parental HeLa line cells (HeLa), HeLa cells transfected with anti-sense-PAI-2 (A2/7) and HeLa cells lines stably expressing PAI-2 (Sla and Sib). Equal amounts of protein (15 ⁇ g) were loaded in each lane and the blot was probed with antibodies specific for the indicated proteins.
  • B Western blot analysis of HPV E7 expression. As for (A) with 50 ⁇ g of protein loaded per lane. The blot was probed with an antibody specific for HPV 18 E7.
  • C Western blot analysis of HPV E7 expression. As for (A) with 50 ⁇ g of protein loaded per lane. The blot was probed with an antibody specific for HPV 18 E7.
  • Equal ug/ml of 35 S labeled IVT PAI-2 (Input INT PAI-2, lane 3) were incubated with equal ug/ml of glutathione-agarose beads bound to GST (lane 1) and GST- pocket protein (lane 2). Binding reactions were washed, run on an polyacrylamide gel and exposed to X-ray film.
  • Sla cells HeLa derived cells expressing PAI-2 via a transgene
  • KJD cells SN40 transformed keratinocytes that constitutively express PAI-2
  • U937 cells induced to express PAI-2 with 25 ng/ml PMA
  • anti-PAI-2 rhodamine, red
  • anti-RB antibodies FITC, green
  • PAI-2 binding to RB required the C-D interhelical region and was largely independent of the LXCXE-mediated binding.
  • Equal ⁇ g/ml of 35 S IVT PAI-2 (top panel) or PAI-2 C-D interhelical mutant of PAI-2 (bottom panel) were incubated with equal ug/ml of glutathione-agarose beads bound to GST (GST), GST-RB 379"
  • FIG. 4 RB was elevated in PAI-2 expressing Jurkat cells.
  • A Western blot analysis of nuclear and cytoplasmic proteins extracted from the parental Jurkat cell line, a vector control Jurkat clone and Jurkat clones stably expressing PAI-2 (PAI-2 clone 1 and clone 2) using anti-PAI-2, anti-RB and anti-actin antibodies. The low level of the cytoplasmic protein actin in the nuclear extract confirmed the low level of cytoplasmic proteins contaminating this extract. Equal amounts of protein were loaded in each lane.
  • B Quantitative real time RT-PCR expression analysis of RB mRNA levels in the panel of Jurkat cell lines. Data from three experiments is expressed as the mean fold change ( ⁇ SD) compared with parental Jurkat cells.
  • PAI-2 Quantitative real time RT-PCR expression analysis of E2F-1 the dependent genes, dihydrofolate reductase (DHFR) and cyclin A, in mRNA isolated from PAI-2 expressing and control cell lines. Data from three experiments is expressed as the mean fold change mean ⁇ SD compared with parental HeLa cells.
  • DHFR dihydrofolate reductase
  • cyclin A cyclin A
  • FIG. 1 PAI-2 inhibited turnover and calpain-1 -mediated cleavage of RB.
  • A RB turnover was analyzed by pulse chase analysis using the control parental Jurkat cell line (black squares) and the vector control Jurkat cell line (black triangles) and the PAI-2 expressing Jurkat clones 1 (white square) and 2 (white triangle). Cells were 35 S labeled, washed, chased with cold medium, harvested at the indicated times and RB immunoprecipitated, resolved by SDS PAGE and detected by autoradiography.
  • FIG. 7 Summary of proposed new intranuclear activity of PAI-2 (A) PAI-2 activity in Jurkat cells. PAI-2 binds RB via the PENF motif and thereby inhibits calpain-1 mediated cleavage of the N-terminal PEST sequence of RB, which may initiate further degradation of RB. PAI-2 may interact with calpain-1 via the
  • PAI-2 activity in HeLa cells PAI-2 inhibits the accelerated degradation of RB by blocking E7-RB binding and/or by inhibiting calpain-1.
  • E7- mediated degradation of RB may be initiated by cleavage of the PEST sequence (Gonzalez et al, 2001, J. Virol. 75 7583; Jang et al, 1999, Oncogene 18 1789). The resulting recovery of RB appears to result in suppression of E6 E7 transcription and recovery of E6/E7-targeted cellular proteins.
  • FIG. 8 Adenovirus-mediated expression of PAI-2.
  • A HeLa cells were infected with an Adenovirus 5 vector containing cDNA encoding PAI-2. Expression of PAI-2 was monitored by Western Blot using anti-PAI-2 antibodies.
  • B HeLa cells expressing AdPAI-2 48 hr after infection were assayed for sensitivity to TNF-induced apoptosis as measured by CV assay in the presence or absence of TNF (10 ng/mL) + cyclohexamide (CHX; 10 ⁇ g/mL) for 8 hr (Dickenson et al, 1995, supra). Cell survival is expressed as the percent surviving in the presence of TNF + CHX relative to CHX alone.
  • TNF 10 ng/mL
  • CHX cyclohexamide
  • the present invention has arisen, at least in part, from the discovery that PAI-2 binds RB. Furthermore, the domains of PAI-2 which are responsible for binding to RB have been identified. One of these domains comprising but not necessarily limited to the minimal sequence PENF, is hitherto undescribed in any other protein(s). The other domain, an LXCXE domain, has previously been identified in other RB-binding proteins. A non-LXCXE motif has already been identified by Kong et al,
  • helix 3 (NDAAAEA) and helix 4 (KEE-REK) are the key RB-binding elements with the downstream region EKY (159) being critical for AL1/AL1 oligomerisation.
  • the present inventors propose that the PENF motif of PAI-2 is crucially important for RB binding.
  • PAI-2 the analagous helix 3 and helix 4 regions are present in the C-D interhelical mutant which binds RB poorly.
  • RB-binding proteins such as PAI-2 prevent calpain 1 -mediated degradation of RB.
  • the present inventors therefore propose that RB-binding proteins such as PAI-2 and RB-binding domains, may be useful in therapeutic treatments in mammals that target gene expression regulated by RB, and particularly gene expression regulated by mammalian RB and the transcription factor E2F.
  • PAI-2 and/or binding domains or agonist or antagonists based thereupon may be useful in therapeutic treatments that inhibit the binding of RB binding proteins from pathogens and mammals such as HPV E7 and cyclin kinases to RB.
  • Such treatments include potential therapies for diseases such as HIV and papillomavirus-associated diseases including cervical cancer and genital warts, other cancers such as breast cancer, cardiovascular disease, asthma and psoriasis.
  • the term "recombinant” as used herein means artificially produced through human manipulation of genetic material, such as involving techniques generally falling within the scope of "recombinant DNA technology” as is well understood in the art.
  • y isolated is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • Isolated material may be in recombinant or native form.
  • RB-binding proteins, RB-binding domains and RB-containing complexes As used herein a "protein" is an amino acid polymer which includes natural and/or non-natural amino acids as are well known in the art.
  • A' 'polypeptide is a protein having more than fifty (50) amino acids.
  • a “peptide” is a protein having no more than fifty (50) amino acids.
  • Peptides may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled “Peptide Synthesis” by Atherton and Shephard which is included in a publication entitled “Synthetic Vaccines " edited by Nicholson and published by Blackwell
  • peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcal V8-protease.
  • the digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.
  • an "RB-binding protein” is a protein such as PAI-2, although without limitation thereto, that comprises an RB-binding domain of the invention and is thereby capable of binding RB.
  • the RB-binding protein is capable of binding mammalian RB. 14
  • the invention also includes a protein which does not normally bind
  • RB engineered to include an RB-binding domain to thereby confer RB binding to said protein.
  • the invention provides isolated protein complexes comprising an RB-binding domain and RB or an RB-binding protein and RB.
  • a non-limiting example is an isolated protein complex comprising PAI-2 and RB.
  • Isolated protein complexes may be useful such as for crystallographic determination of the fine structure and contact residues involved in RB binding interactions.
  • variants include RB-binding domains in which one or more amino acids have been replaced by different amino acids or have been deleted. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions).
  • sequences set forth in Figure 3 (SEQ ID NOS:21-44) comprise amino acid residues that are highly conserved and residues that may be varied considerably, hence the skilled person is readily able to ascertain the effect of amino acid substitutions and/or deletions. Substantial changes in function are made by selecting substitutions that are less conservative. Other replacements would be non-conservative substitutions and relatively fewer of these may be tolerated. Generally, the 13
  • Non-limiting examples of RB-binding proteins and amino acid sequences of each constituent RB-binding domain are provided in Figure 3B AND 3D.
  • an "RB-binding domain” is a structurally definable region of a protein that is necessary and/or sufficient for binding of said protein to RB.
  • An RB-binding domain at least comprises the aforementioned (P/A)Z 2 (F/MY/LH) minimal consensus sequence, and may further comprise an LXCXE sequence.
  • RB-binding domain specifically excludes a corresponding full-length RB-binding protein.
  • an RB-binding domain of the invention may correspond to a contiguous RB- binding fragment or region of any of the RB-binding proteins set forth in Figure 3 and comprise the aforementioned (P/A)Z 2 (F/M ⁇ 7L/H) consensus sequence alone or together with an LXCXE sequence.
  • Preferred RB-binding domains are selected from the group consisting of:
  • (XV) residues 113to35ofSEQIDNO:35; substitutions which are likely to produce the greatest changes in a polypeptide' s properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser)or no side chain (e.g., Gly).
  • a hydrophilic residue e.g., Ser or Thr
  • derivative proteins are those which have been altered, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art.
  • Such derivatives include amino acid deletions and/or additions to proteins of the invention.
  • Derivative proteins may include fusions of an RB-binding domain with another protein.
  • proteins include Protein A, glutathione S-transferase (GST), green fluorescent protein (GFP) maltose-binding protein (MBP), hexahistidine (HIS 6 ) and epitope tags such as FLAG, haemagglutinin and c-myc tags.
  • derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of non-natural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides, fragments and variants of the invention.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 ; reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; and trinitrobenzylation of amino groups with 2, 4, 6- trinitrobenzene sulphonic acid (TNBS).
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.
  • the guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.
  • Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4- chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4- nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N-bromosuccinimide.
  • Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • the imidazole ring of a histidine residue may be modified by N- carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4- amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.
  • the invention also contemplates RB-binding domains having at least 60%, preferably at least 75% or more preferably at least 90% sequence identity with an RB-binding domain identified in any one of SEQ ID NOS : 21 -44.
  • sequence identity is used herein in its broadest sense to include the number of exact amino acid (or nucleotide) matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid or nucleotide residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity may be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).
  • RB-binding domains and proteins engineered to comprise RB- binding domains are readily made in recombinant form.
  • a recombinant RB-binding domain may be made by truncation or deletion of amino acid residues in an RB-binding protein that lie outside said RB-binding domain.
  • Such recombinant constructs may consist of, or consist essentially of, an RB- binding domain. That is, the recombinant RB-binding domain has no or few amino acids additional to the RB-binding domain itself.
  • a recombinant protein may be engineered to include an RB-binding domain and thereby confer RB-binding activity.
  • Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-1999), incorporated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-1999) which is incorporated by reference herein, in particular Chapters 1, 5, 6 and 7.
  • RB-binding domain or an encoding nucleic acid such as by random mutagenesis or site-directed mutagenesis.
  • nucleic acid mutagenesis methods are provided in in Chapter 8 of CURRENT PROTOCOLS IN MOLECULAR
  • linker-scanning mutagenesis of DNA may be used to introduce clusters of point mutations throughout a sequence of interest that has been cloned into a plasmid vector.
  • linker-scanning mutagenesis of DNA may be used to introduce clusters of point mutations throughout a sequence of interest that has been cloned into a plasmid vector.
  • Region-specific mutagenesis and directed mutagenesis using PCR may also be employed to construct promoter variants according to the invention.
  • site-directed mutagenesis is best performed where knowledge of the amino acid residues that contribute to biological activity is available. In many cases, this information is not available, or can only be inferred by molecular modelling approximations, for example.
  • Random mutagenesis methods include chemical modification of proteins by hydroxylamine (Ruan et al, 1997, Gene 188 35), incorporation of dNTP analogs into nucleic acids (Zaccolo et al, 1996, J. Mol. Biol. 255 589) and PCR-based random mutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91
  • PCR-based random mutagenesis kits are commercially available, such as the DiversifyTM kit (Clontech). Mimetics, Agonists and Antagonists It will be appreciated that the elucidation of the RB-binding domain of the invention will facilitate the identification and/or design of molecules that antagonize binding to RB or mimic binding to RB, as the case may be.
  • Such molecules may be termed “mimetics”, although terms such as “partial agonist” “agonist”, “analogue” and “antagonist” also apply as are well understood in the art.
  • the aforementioned molecules may themselves be peptides or polypeptides, or may be other organic molecules, preferably small organic molecules, with a desired biological activity and half-life.
  • Mimetics may be identified by way of screening libraries of molecules such as phage display libraries, synthetic chemical libraries, including combinatorial libraries, by methods such as described in Nestler & Liu, 1998,
  • libraries of naturally-occurring molecules may be screened by methodology such as reviewed in Kolb, 1998,
  • More rational approaches to designing mimetics may employ computer assisted screening of structural databases, computer-assisted modelling, or more traditional biophysical techniques which detect molecular binding interactions, as are well known in the art.
  • the invention also provides antibodies directed against RB-binding domains, peptide fragments thereof, variants and derivatives.
  • Such antibodies may include any suitable antibodies which bind to or conjugate with RB-binding domains, peptide fragments thereof, variants and derivatives.
  • Such antibodies may be polyclonal, obtained for example by immunizing an animal with a polypeptide or fragment thereof. It is for this purpose that peptides of the invention are particularly useful.
  • Such polyclonal antibodies would be present in blood serum obtained from said animal a suitable time after immunization.
  • said animal could be a mouse, rat, rabbit or goat.
  • monoclonal antibodies may be produced by a standard method such as described in CURRENT PROTOCOLS IN IMMUNOLOGY (1994, Eds. Coligan, Kruisbeek, Marguiles, Shevach and
  • antibody-producing cells such as spleen cells
  • immortalizing said cell such as by fusion with an immortalized fusion partner cell.
  • labels selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule and a radioisotope.
  • Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ -galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzyme label may be used alone or in combination with a second enzyme in solution.
  • Fluorophores may be selected from a group including fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), allophycocyanin (APC), Texas Red (TR), Cy5 or R-Phycoerythrin (RPE). Examples of useful fluorophores may be found, for example, in United States Patent No. 4,520,110 and United States Patent No. 4,542,104 which are herein incorporated by reference. Isolated nucleic acids
  • the invention provides isolated nucleic acids that encode RB- binding domains of the invention.
  • Nucleic acids encoding RB-binding proteins set forth in Figure 3B are well known in the art and the nucleotide sequences can be readily deduced from the amino acid sequence information provided herein.
  • nucleic acid designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.
  • a "polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has up to eighty (80) contiguous nucleotides.
  • a “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.
  • a “primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template” and being extended in a template- dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
  • nucleic acids of the invention which have nucleotide substitutions, deletions or additions which do not substantially alter functional characteristics of proteins encoded thereby.
  • Patent 5,786,464 which provides an example of optimizing protein expression by selecting preferred codons.
  • nucleic acids may be altered so as to introduce "conservative" amino acid changes which, although altering an amino acid sequence, do not affect functional characteristics of proteins encoded thereby.
  • nucleic acid sequence amplification techniques are particularly useful tools for obtaining, mutagenizing and otherwise manipulating nucleic acids of the invention.
  • Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase cain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. supra; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al, 1996, J. Am. Chem. Soc.
  • PCR polymerase chain reaction
  • LCR ligase cain reaction
  • SDA strand displacement amplification
  • RCR rolling circle replication
  • nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et ⁇ /.,1994, Biotechniques 17 1077); ligase chain reaction (LCR) as for example described in International Application WO89/09385; and Q- ⁇ replicase amplification as for example described by Tyagi et al, 1996, Proc. Natl. Acad. Sci. USA 93 5395.
  • amplification product refers to a nucleic acid product generated by nucleic acid amplification techniques.
  • the invention provides an expression construct which comprises an isolated nucleic acid that encodes an RB-binding binding domain or RB- binding protein, as hereinbefore defined, operably linked to one or more regulatory sequences in an expression vector.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.
  • Selectable markers are also incorporated for the purposes of selection in particular types of host cells.
  • transformed bacteria may be selected by inclusion of markers such as bla, kanR and tetR, while transformed mammalian cells may be selected by inclusion of markers such as hygromycin, G418 and puromycin.
  • Suitable host cells for expression include bacteria (eg. E coli.
  • DH5 ⁇ Salmonella sp. Pseudomonas sp.
  • yeast insect cells
  • insect cells eg. Sf9
  • Xenopus oocytes and mammalian cells such as keratinocyte, macrophage/monocyte (eg. MonoMac ⁇ ), T cell, B cell, CHO and COS lines.
  • Both constitutive and inducible promoters may be useful for expression of recombinant proteins according to the invention.
  • inducible promoters are metallothionine-inducible and tetracycline-repressible systems as are well known in the art.
  • An expression construct may also include a fusion partner sequence as hereinbefore defined (usually provided by the expression vector) so that the recombinant polypeptide of the invention is expressed as a fusion polypeptide with said fusion partner.
  • Expression constructs also include gene therapy constructs, which employ specialized gene therapy vectors such as vaccinia, and viral vectors useful in gene therapy.
  • the latter include adenovirus and adenovirus-associated viruses (AAV) such as described in Braun-Falco et ⁇ /.,1999, Gene Ther. 6 432, retroviral and lenti viral vectors such as described in Buchshacher et al, 2000, Blood 95 2499 and vectors derived from herpes simplex virus and cytomegalovirus.
  • a general review of gene therapy vectors may be found in Robbins et al, 1998, Trends in Biotech. 16 35. An overview of viral vectors useful in gene therapy is provided in Stone et al, 2000, J.
  • Expression constructs may comprise operably-linked nucleic acids in 5 -*3 (sense) or 3 -»5 (antisense) orientations.
  • An example of the latter would be antisense expression of a nucleic acid encoding PAI-2 or fragment thereof to reduce PAI-2 expression and thereby reduce the amount of RB-bound PAI-2 in a target cell.
  • Pharmaceutical compositions comprising
  • RB-binding proteins such as PAI-2, an RB-binding domains, fragments thereof, or an isolated nucleic acid encoding same, together with a pharmaceutically- acceptable carrier, diluent or excipient.
  • Pharmaceutical compositions comprising an isolated nucleic acid are preferably in the form of a gene therapy construct.
  • “Pharmaceutically-acceptable carriers, diluents and excipients” generally applicable to pharmaceutical compositions include a solid or liquid filler, diluent or encapsulating substance which may be safely used in systemic administration. Depending upon the particular route of administration, a variety of pharmaceutically-acceptable carriers or diluents, well known in the art may be used.
  • These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, topically administered powders, aerosols and emulsions, transdermal patches, injectable gels, pastes and the like. These dosage forms may also include controlled release devices or other forms of implants modified to act in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
  • compositions of the present invention may be suitable for administration orally or by injection, and in such cases may be presented as discrete units such as capsules, sachets or tablets, as a powder or granules, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsion or a water-in-oil liquid emulsion.
  • IMIQUTMOD an example of a preferred pharmaceutically-acceptable carrier, diluent and excipient particularly suited to topical administration is IMIQUTMOD, such as described in Miller et al, 1999, Int. J. Immunopharmacol. 21 1, which is incorporated herein by reference.
  • Administration of a gene therapy construct to a mammal may include delivery via direct oral intake, systemic injection, or delivery to selected tissue(s) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor.
  • An example of the latter approach would be stem-cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the construct which includes the nucleic acid to be expressed. The stem-cells are cultured for a period and then transferred to the mammal being treated. Delivery of said gene therapy construct to cells or tissues of said mammal or said compatible donor may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g.
  • lipofectin or lipofectamine lipofectin or lipofectamine
  • electroporation calcium phosphate
  • DEAE-dextran-mediated transfection for example.
  • suitable delivery methods may be found in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al;
  • the present invention provides PAI-2 and other RB-binding proteins, RB-binding domains and encoding nucleic acids and pharmaceutical compositions including same useful as therapeutic agents. As will be described in more detail hereinafter, administration of
  • PAI-2 to mammalian cells transformed by papillomavirus oncogenes results (i) recovery of RB and p53, and (ii) down regulation of human papillomavirus E6 and E7 protein expression.
  • PAI-2 a fragment thereof comprising the PENF and perhaps an additional LXCXE motifs or an analagous fragment of another protein which binds RB (or a mimetic or any of these), could be used to suppress papillomavirus E6 and E7 protein activity and/or protein expression and thereby treat cervical cancer and/or genital warts.
  • E2F away from the HIV LTR promoter, where E2F usually plays a repressive role with respect to HIV gene transcription.
  • PAI-2 is normally expressed in cells susceptible to HIV infection such as macrophages.
  • PAI-2 has been observed in some T cells (Reiter et al, 1997, Int. J. Cancer 70 461).
  • the present invention is generally applicable to treatments, either therapeutic or prophylactic, which seek to prevent interaction of viral or cellular RB binding proteins (not limited to E7 or E2F; other binding proteins Dick et al, supra) interacting with RB and as a consequence target gene expression or the cell cycle.
  • viral or cellular RB binding proteins not limited to E7 or E2F; other binding proteins Dick et al, supra
  • cardiovascular diseases such as atherosclerosis.
  • RB and probably other proteins
  • smooth muscle can undergo hyperplasia during atherosclerosis (Chen ⁇ t al, 1997, J. Clin. Invest. 99 2334), or transform to a secretory phenotype to give chronic venous insufficiency (Pappas et al, 1988, J. Surg. Res. 76 149).
  • Inhibition of RB activity could provide a means of inhibiting and/or reversing cardiovascular diseases such as described above.
  • smooth muscle hyperplasia is associated with asthma
  • PAI-2-mediated G arrest of proliferating smooth muscle might be a useful treatment of asthma
  • Another disease associated with cell hyperproliferation is psoriasis.
  • Cyclin overexpression is associated with this disease (Miracco et al, 2000, Br. J. Dermatol. 143 950), and RB is a known negative regulator of cyclin expression. It is therefore proposed by the present inventors that enhanced RB activity, such as by administration of PAI-2 or a drug that mimics PAI-2 binding to RB, could down-regulate cyclin activity and/or expression and thereby treat proriasis.
  • cyclin Dl is over-expressed in many breast cancers (Barbareschi et al, 1997, Int. J. Cancer 74 171), such that down-regulation by RB through administration of PAI-2 or a drug mimicking PAI-2 binding to RB should result in G j arrest of cancerous cells.
  • PAI-2 is a positive prognostic indicator of breast cancer (Duggan et al., 1997, Br. J. Cancer 76 622).
  • Nucleic acids encoding RB-binding domains and/or proteins may be delivered by direct injection of plasmid DNA and/or a through virus based delivery system.
  • An example is the expression of PAI-2 in HeLa cells using an adenovirus based vector as shown in Figure 8A.
  • Expression of AdPAI-2 confers protection against TNF mediated apoptosis ( Figure 8B).
  • Proteins may be expressed as biologically active recombinant protein in eukaryotic cell cultures, e.g. yeast or animal cells.
  • the protein may be encapsulated in a formulation of fatty acids comprising liposomes for cytoplasmic delivery to cells, or administered topically using a suitable carrier. Delivery of mimetics based on RB bindins site of PAI-2
  • Soluble mimetics will be developed, which mimic the specificity and activity of RB-binding proteins, for example based on the RB-binding site of PAI-2.
  • HeLa cells, A2/7, Sla and Sib lines were generated and maintained as described previously (Dickinson et al, 1995 supra).
  • Jurkat cells clone E61 ATCC #TD3- 152 were transfected with pRcCMV-PAI-2 expression plasmid and an empty expression plasmid pRcCMV (Dickinson et al, 1995, supra) via electroporation using a BioRad Gene Pulsar.
  • Cells (10 6 ) and 10 ⁇ g of each plasmid were electroporated in RPMI 1640 at a voltage of 0.40 kV (capacitor strength of 960 ⁇ F), and selected in 800 ⁇ g/ml G418 geneticin (ICN Biochemicals, Costa Mesa, CA) at 48 hrs post transfection. Clonal selection of fransfections was achieved by serial dilution, and expression of PAI-2 was determined by Western blot analysis.
  • NP40 0.5 mM DTT and protease inhibitor cocktail (Roche, Mannheim, Germany) to cell pellets (approximately 10 7 cells). The mixture was incubated on ice for 30 min and the debris pelleted (13,000 x g, 20 min, 4°C) and the supernatant collected. Nuclear and cytoplasmic protein extracts were isolated as described (Janicke et al, 1994, Mol. Cell Biol. 14 5661).
  • Equal amounts of protein as determined by BCA-200 protein assay kit (Pierce, Rockford, IL) were solubilized in 5% SDS and 5% ⁇ -mercaptoethanol (Sigma, St Louis, MI) heated at 100"C for 2 min and separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech).
  • SDS and 5% ⁇ -mercaptoethanol Sigma, St Louis, MI
  • E7 detection 50 ⁇ g total cell protein lysates were separated on 4 to 20% gradient gels (Gradipore, North Ryde, Australia) run at 150 V for 1 hr and proteins transferred immediately.
  • GST fusion proteins comprising the A, B and C pockets of RB (amino acids 379- 928), RB 379"928 -706F, RB 379 928 -del (exon 21), pl07 385 1068 and pl30 414"1135 were expressed in E. coli (XL- 10 Blue) (Stratagene, La Jolla, CA).
  • the suspension was sonicated 3 times 30 seconds and the supernatant mixed with 150 ⁇ L of a 50%) slurry glutathione-agarose beads (Sigma) for 2 hrs rotating at 4°C.
  • the bead slurry was then centrifuged at 5,000 g and washed 5 times with 1 ml of N ⁇ TN buffer (0.5% NP-40, 20 mM Tris pH 8.0, 100 mM NaCl, and ImM ⁇ DTA) and resuspended in 75 ⁇ L N ⁇ TN buffer.
  • N ⁇ TN buffer (0.5% NP-40, 20 mM Tris pH 8.0, 100 mM NaCl, and ImM ⁇ DTA
  • PAI-2, pRcCMV-CD PAI-2 (Dickinson et al, 1995, supra), and pCDNA3- HDAC-3 (Yang et al, 1997, J. Biol. Chem. 272, 28001) were in vitro transcribed (IVT) using TnT Quick coupled transcription translation system (Promega, Madison, WI) in the presence of 35 S-mefhionine (Amersham Pharmacia Biotech) according to manufacture's instructions. INT protein (2 ⁇ l) was incubated with
  • Peptides were synthesized by Mimotopes Pty Ltd. (Clayton, Victoria, Australia).
  • the control peptide had the sequence NAVTPMTFAAKTSCGFMQQ (S ⁇ Q ID NO:45).
  • 150 ⁇ M of each peptide was incubated with GST-RB fusion protein in 200 ⁇ l Licht buffer for 1 hr at 4°C prior to the addition of 2 ⁇ L IVT 35 S-labeled HDAC-3. The sample was incubated for a further 1 hr at 4°C, washed and analyzed as above.
  • oligonucleotide primers Geneset, LaJolla, CA
  • RB sense 5'- GCTAGCCTATCTCCGGCTAAA-3' (SEQ ID NO:46) and antisense 5'-
  • DHFR sense 5'-AGACCTGGTTCTCCATTCC -3' (SEQ ID NO:52) and antisense 5'-TGTGGAGGTTCCTTGAGTTC-3 ' (SEQ ID NO:53).
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense 5 '- GGTCGGTGTGAACGGATTT-3' (SEQ ID NO:54) and antisense 5'- GTCGTTGATGGCAACAATCT-3' (SEQ ID NO:55) primers were used as a standard in all reactions.
  • Amplification reaction mix of 20 ⁇ l contained 0.1 ⁇ g randomly primed cDNA, 0.5 ⁇ M of each primer pair, 2 X Platinum Quantitation PCR supermix-UDG (GibcoBRL) and 10X SYBR green (Molecular Probes, Eugene, OR).
  • the cycling conditions were one cycle of 96°C for 2 min, followed by 35 cycles of 96°C for 15 sec, 56°C for 15 sec and 72°C for 15 sec.
  • Real Time PCR was performed using a Rotogene PCR machine (Corbett Research, Mortlake,
  • HeLa and Jurkat cells expressing PAI-2 were stained with propidium iodide (Sigma), as described (Krishan, 1975, J. Cell. Biol. 66 188) and were analyzed by FACS (FACScalibur, Becton Dickinson) and ModFitLT V2 software (Verity Software House Inc.). 1.8 Pulse chase analysis ofRB turnover
  • Total cell lysates of Jurkat cells were prepared as above (without protease inhibitors). Protein lysates (15 ⁇ g) were incubated at 30°C for 1 hr with combinations of 0.2 U (23 U/mg protein) of calpain-1 (Sigma), 2 mM CaCl 2 , 5 ⁇ g recombinant PAI-2 (Biotech Australia) and/or 300 mM EGTA (Sigma), the reactions were made up to 20 ⁇ L with cleavage buffer (25 mM Tris pH 7.0 and 100 mM NaCl). All treatments were then heated to 90°C for 10 min before treatment with 10 U alkaline phosphotase (New England BioLabs, Beverly, MA). Reaction mixtures were run on 8% SDS PAGE gels (2.5 hrs/130 V), immunoblotted and probed with anti-RB-antibody (G3-245).
  • PAI-2 expression in HeLa cells reverses papilloma E6 and E7 activity
  • HeLa cell lines stably transfected with PAI-2 display a diversity of phenotypes which suggest that PAI-2 can regulate transcription and/or signal transduction (Antalis et al, 1998, supra; Shafren et al, 1999, J. Virol. 73 7193; Dickinson et al, 1995, supra).
  • a search for transcription factors that might be altered in Sla and Sib cells it emerged that these cells expressed significant levels of p53, p21, RB and c-Jun (Fig. 1).
  • the parental HeLa cells and HeLa cells transfected with anti-sense PAI-2 (A2/7 cells) expressed very low to negligible levels of these proteins.
  • Expression of NF-kB and other AP-1 family members was unaffected by PAI-2 expression (data not shown).
  • HeLa cells express the human papilloma 18 oncoproteins, E6 and E7, which normally target p53 and RB, respectively, for accelerated proteolytic degradation, resulting in low level expression of these proteins (Goodwin and DiMaio, 2000, Proc. Natl. Acad. Sci. USA 97 12513).
  • p 21 WAF1 CIP is also normally downregulated in HeLa cells since p53 is required for p21 WAF1 CIP expression.
  • the low level of c-Jun in HeLa cells may be due to the binding of c-Jun by E7 (Antinore et al., 1996,
  • E7 protein and mRNA was analyzed by immunoblotting and quantitative real time RT-PCR, respectively. E7 protein expression was significantly reduced (Fig. IB) and real time RT-PCR showed that mRNA encoding E7 was also substantially reduced in Sla and Sib cells (Fig. 1C). Since E6 and E7 are co-transcribed (Goodwin & DiMaio, 2000, supra) these experiments suggest that PAI-2 expression in HeLa cells resulted in transcriptional inhibition of E6/E7.
  • PAI-2 binds RB andpl30
  • RB is a member of a family of pocket proteins, that includes pi 30 and pi 07, which have distinct but overlapping activities to RB, but also contain LXCXE binding sites (Mulligan &
  • PAI-2 binds RB, pl07 and/or pi 30, the pocket proteins were expressed as glutathione-S-transferase (GST)-fusion proteins and employed in in vitro pull down binding assays in the presence of in vitro transcribed (IVT) PAI-2.
  • GST glutathione-S-transferase
  • IVT in vitro transcribed PAI-2.
  • INT PAI-2 was pulled down by GST-RB and GST-pl30 but not GST-pl07 (Fig. 2 A, lane 2), illustrating that in vitro PAI-2 could be shown to bind RB and pi 30.
  • RB and PAI-2 co-localise in the nucleus Physiological interaction of PAI-2 with RB would require the presence of PAI-2 in the nucleus, where RB is predominantly found. Subcellular localization of RB and PAI-2 was examined using confocal immunofluorescence microscopy and dual labeling for RB and PAI-2.
  • Sla cells which express PAI-2 via a stably integrated plasmid
  • KJD keratinocytes SV40 large T antigen transformed keratinocytes
  • phorbol ester activated U937 cells a leukemia line from the monocyte lineage
  • PAI-2 from its physiological promoter. Whether expressed from a transgene or physiologically, PAI-2 could clearly be detected in the cytoplasm and nucleus of all three cell types (Fig. 2B). In the nucleus, PAI-2 was found to co-localize with RB in dappled or punctate staining patterns (Fig. 2B, overlay, indicated with arrows).
  • the C-D interhelical loop of PAI-2 is important for RB binding
  • the C-D interhelical region contains a novel motif shared by RB-binding proteins
  • RB-binding proteins for which LXCXE-independent binding has not been reported were also found to share the new motif; these were HBRM, cyclin D2 and 3, HDAC-2 (Dick et al, 2000, supra), Elf-1 (Wang et al, 1993, Science 260 1330) and RBP1/2 (Fattaey et al, 1993, Oncogene 8 3149).
  • the homologous PEKY (amino acids 155-158) region in TGMV AL1 lies adjacent to the alpha-helical region that was recently proposed to be involved in plant RB binding (Kong et al, 2000, supra) (Fig. 3B, dashed underline).
  • c-Abl The RB- binding portion of c-Abl has been mapped to amino acids 205-307 (Welch & Wang, 1993, supra) and interestingly, like PAI-2 (Fig. 2 A) c-Abl bound RB but not pi 07, differences that were mapped to sequence variations in the C pocket of the RB family of proteins (Whitaker et al, 1998, Mol. Cell. Biol. 18 4032).
  • HDAC-3 is an RB-binding protein that does not contain an LXCXE motif (Dahiya et al, 2000, supra) and based on the alignments above
  • Fig. 3B was postulated to utilize it's PENF homology motif for RB binding.
  • PAI-2 66"95 peptide underlined in Figs 3B and C substantially inhibited HDAC-3 binding, showing an 80% reduction in the amount of HDAC-3 pulled down by GST-RB relative to the control peptide (Fig. 3C, lane 5 compared to lane 3).
  • PAI-2 expressing Jurkat cells have increased RB
  • PAI-2-mediated recovery of RB may be exaggerated in HeLa cells by the presence of E7, which normally targets RB for accelerated proteolytic degradation.
  • E7 which normally targets RB for accelerated proteolytic degradation.
  • hypophosphorylated RB is the binding of the transcription factor E2F-1 and recruitment of histone deacetylases, causing inhibition of E2F-1 -mediated transcription and cell cycle arrest in Gl (Brehm et al, 1998, Nature 391 597). Consistent with a reduction in E2F-1 -mediated transcription, quantitative real time RT-PCR analysis showed reduced levels of mRNA encoding the E2F-1 -dependent genes, dihydrofolate reductase (DHFR) and cyclin A in both HeLa and Jurkat cell lines expressing PAI-2 compared with control cell lines (Fig. 5A). The percentage of cells in the Gl phase of the cell cycle was also higher in PAI-2 expressing cells compared with control cell lines
  • PAI-2 inhibits RB turnover and calpain-1 -mediated cleavage ofRB
  • PAI-2 containing a mutation in the PI residue of the reactive site loop (RSL), Arg 380 to Ala 380 was unable to confer the ability to protect HeLa cells against TNFa-mediated apoptosis, suggesting that, in addition to the requirement of the C-D interhelical region, intracellular PAI-2 activity was dependent on PAI- 2 proteinase inhibition activity (Dickinson et al, 1995, supra).
  • calpain-1 has recently been shown to be a cell cycle regulated, calcium dependent, nuclear cysteine proteinase that cleaves pi 07, one of the RB family of pocket proteins (Jang et al, 1999, supra).
  • full length RB from a whole cell extract of wild type Jurkat cells was incubated with calpain-1 in the presence and absence of recombinant PAI-2 and calcium, and the RB detected by immunoblotting after dephosphorylation with alkaline phosphatase. Following incubation with calpain-
  • RB showed a slightly higher mobility indicating that a small fragment had been cleaved from RB by calpain-1 (Fig. 6B, lane 1).
  • the cleavage event was calpain and calcium dependent, as the reduction in molecular weight was not seen in the presence of calcium alone (Fig. 6B, lane 5) or when the calcium was chelated by EGTA (Fig. 6B, lane 4).
  • cleavage of RB was inhibited in the presence of PAI-2 (Fig. 6B, lane 2).
  • PAI-2 a target for calpain-1 activity and PAI-2 was able to inhibit calpain-1 -mediated cleavage of RB.
  • PAI-2 C- D interhelical region contains a previously undescribed RB-binding motif, the PENF homology motif, which is shared by many RB-binding proteins.
  • the PENF homology motif was identified in a number of viral and cellular proteins, illustrating that RB binding through this motif is utilized by diverse cellular proteins and several viral pathogens including herpesviruses and geminiviruses (Fig. 3C).
  • AL1 and AL3 proteins (or their equivalents) from many geminiviruses contained the motif, and these proteins might be expected to bind RB (Settlage et al, 2001, supra; Kong et al, 2000, supra).
  • the new RB-binding motif was also present in a number of viral proteins not previously shown to bind
  • RB replication proteins from a number of nanoviruses, for instance the replication initiation protein from banana bunchy top virus, which does not contain an LXCXE motif (Horser et al, 2001, Arch. Virol. 146 71), (ii) human herpesvirus-6 BCLF 1 (Nicholas, 1994, Virology 204 738), (iii) the viral protein R from HIV (amino acids 24-57) and (iv) measles virus RNA polymerase beta subunit (amino acids 1419-1451) (accession number P35975). Interestingly, measles infection has been reported to suppress RB expression (Naniche et al, 1999, J. Virol. 73 1894).
  • replication proteins from a number of nanoviruses for instance the replication initiation protein from banana bunchy top virus, which does not contain an LXCXE motif (Horser et al, 2001, Arch. Virol. 146 71)
  • Bcl-2 and Bcl-xL also contain a PENF homology motif in their large exposed loops at positions 36-69 and 44-77, respectively. This observation may explain the cell cycle retardation affects of Bcl-2 and Bcl-xL, activities found to be independent of the anti- apoptotic functions of these proteins (Vairo et al, 2000, Mol. Cell. Biol. 20 4745; Chang et al, 1997, EMBO J. 16 968).
  • PAI-2 and the papillomavirus oncogene products, E6 and E7 PAI-2 expression in HeLa cells was shown to inhibit E6/E7 mRNA and E7 protein expression and resulted in the restoration of RB, p53, c-Jun (Fig. 1) and ISGF3 (Antalis et al, 1998, supra) expression.
  • the mechanisms behind this PAI- 2-mediated recovery of E6 and E7-targeted proteins may begin with the binding of PAI-2 to RB, which may prevent the E7-mediated degradation of RB, either by directly blocking the LXCXE binding site and/or by inhibiting calpain-1.
  • RB protein levels may then be responsible for transcriptional repression of E6/E7 leading to further increases in RB protein levels and recovery of the other E6/E7 targeted proteins.
  • Salcedo et al, 1995, Arch. Med. Res. 26 157 have previously reported that RB can repress E6/E7 transcription. It might be that RB-mediated repression of E6/E7 transcription involves YY1 (Bauknecht et al, 1995; Pefkova et al., 2001, J. Biol. Chem. 276 7932 and /or C/EBPb (Bauknecht & Shi, 1998, J. Virol.
  • RB is well known as a tumor suppressor and RB function is disrupted in many tumors either through mutations in the RB gene or through disruption of the RB- regulated pathways (Harbour & Dean, 2000, Nat. Cell Biol. 2 65).
  • a PAI-2- mediated increase in RB expression should act to enhance RB's tumor suppressor activity.
  • This view is consistent with the observed association of PAI-2 (but not PAI-1) expression with improved prognosis in a number of human cancers (Yoshino et al, 1998, Br. J. Cancer 78 833; Bouchet et al, 1994, Br. J. Cancer 69 398; Foekens et al, 1995, Cancer Res. 55 1423).
  • the reduced metastasis of tumors expressing PAI-2 (but not PAI-1) observed in murine models
  • E2F-1 strongly induces expression of MAP kinase kinase, kinase 5, also known as apoptosis-regulating kinase 1 (ASK-1) (Muller et al, 2001, Genes Dev. 15 267), a protein required for TNF ⁇ -mediated apoptosis (Ichijo et al, 1997, Science 275 90).
  • ASK-1 apoptosis-regulating kinase 1
  • HIN gene expression is primarily regulated through two tandem ⁇ F-kB sites, however, embedded within these sites is an E2F-1 site.
  • E2F- 1 is able to repress HIV-LTR-driven transcription by binding to this site and interacting with the p50 ⁇ F-kB subunit (Kundu et al, 1997, J. Biol. Chem. 272
  • YY1 is also known to repress the HIV LTR (Coull et al, 2000, J. Virol. 74 6790) and RB inhibits YY1 interaction with D ⁇ A (Petkova et al, 2000, J. Biol. Chem Dec 15) which could also lead to de-repression of the HIN LTR.
  • the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

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Abstract

L'invention concerne un domaine de protéine qui se lie à la protéine du retinoblastome (RB) et qu'on a identifié dans PAI-2 et dans un certain nombre d'autres protéines. On peut utiliser ce domaine de liaison à RB pour produire des agonistes et/ou des antagonistes se liant à la protéine du retinoblastome. Cette invention concerne aussi des méthodes de modulation de l'expression génique par ciblage des gènes régulés par RB, tels que E2F. On peut utiliser le domaine de liaison à RB et lesdites méthodes sur le plan thérapeutique et prognostique dans le traitement des cancers, tels ceux provoqués par des virus oncogènes, des cancers liés aux troubles du cycle cellulaire, des infections virales, telles que celles dues au virus de l'immunodéficience humaine (VIH), et des maladies liées à l'hyperprolifération cellulaire, telle que le psoriasis, les maladies cardio-vasculaires et l'asthme.
PCT/AU2001/001635 2000-12-19 2001-12-18 Proteine de liaison au retinoblastome WO2002050101A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004015072A2 (fr) * 2002-08-07 2004-02-19 Exelixis, Inc. Modificateurs de retinoblastomes (mrb) en tant que modificateurs de la voie des rb et methodes d'utilisation

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023539A1 (fr) * 1992-05-13 1993-11-25 Dana-Farber Cancer Institute, Inc. ADNc DE LA PROTEINE 1 ASSOCIEE AU RETINOBLASTOME
WO1996020207A1 (fr) * 1994-12-23 1996-07-04 Research Development Foundation MUTANTS DES GENES Rb ET p53 ET LEUR EMPLOI
WO1998021228A1 (fr) * 1996-11-15 1998-05-22 Canji, Inc. Expression specifique tissulaire de la proteine du retinoblastome
US6069231A (en) * 1994-08-18 2000-05-30 La Jolla Cancer Research Foundation PR domain peptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023539A1 (fr) * 1992-05-13 1993-11-25 Dana-Farber Cancer Institute, Inc. ADNc DE LA PROTEINE 1 ASSOCIEE AU RETINOBLASTOME
US6069231A (en) * 1994-08-18 2000-05-30 La Jolla Cancer Research Foundation PR domain peptides
WO1996020207A1 (fr) * 1994-12-23 1996-07-04 Research Development Foundation MUTANTS DES GENES Rb ET p53 ET LEUR EMPLOI
WO1998021228A1 (fr) * 1996-11-15 1998-05-22 Canji, Inc. Expression specifique tissulaire de la proteine du retinoblastome

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004015072A2 (fr) * 2002-08-07 2004-02-19 Exelixis, Inc. Modificateurs de retinoblastomes (mrb) en tant que modificateurs de la voie des rb et methodes d'utilisation
WO2004015072A3 (fr) * 2002-08-07 2004-12-29 Exelixis Inc Modificateurs de retinoblastomes (mrb) en tant que modificateurs de la voie des rb et methodes d'utilisation

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