WO2004076646A2 - Methodes et compositions destinees a traiter le cancer du col uterin - Google Patents

Methodes et compositions destinees a traiter le cancer du col uterin Download PDF

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Publication number
WO2004076646A2
WO2004076646A2 PCT/US2004/006001 US2004006001W WO2004076646A2 WO 2004076646 A2 WO2004076646 A2 WO 2004076646A2 US 2004006001 W US2004006001 W US 2004006001W WO 2004076646 A2 WO2004076646 A2 WO 2004076646A2
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Prior art keywords
pdz
protein
peptide
hpv
binding
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PCT/US2004/006001
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English (en)
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WO2004076646A3 (fr
Inventor
Peter S. Lu
Christoph Peter Bagowski
Johannes Schweizer
Chamorro Somoza Diaz-Sarmiento
Jonathan D. Garman
Michael P. Belmares
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Arbor Vita Corporation
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Priority claimed from US10/630,590 external-priority patent/US7312041B2/en
Application filed by Arbor Vita Corporation filed Critical Arbor Vita Corporation
Priority to EP04715735A priority Critical patent/EP1599214A4/fr
Publication of WO2004076646A2 publication Critical patent/WO2004076646A2/fr
Publication of WO2004076646A3 publication Critical patent/WO2004076646A3/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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57411—Specifically defined cancers of cervix
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • the present invention relates to therapeutics for the treatment of pathogenic infections such as human Papillomavirus (HPV) infections, and methods for using such therapeutics to treat cells, tissues, or patients that are infected and may develop cancerous growth or other disorders.
  • pathogenic infections such as human Papillomavirus (HPV) infections
  • HPV Papillomavirus
  • Cervical cancer is the second most common cancer diagnosis in women and is linked to high-risk human papillomavirus infection 99.7% of the time.
  • HPVs Human papillomaviruses
  • HPV high-risk and low-risk subtypes based on their association with cervical carcinomas or with benign cervical lesions or dysplasias.
  • a number of lines of evidence point to HPV infections as the etiological agents of cervical cancers.
  • Multiple studies in the 1980's reported the presence of HPV variants in cervical dysplasias, cancer, and in cell lines derived from cervical cancer.
  • Further research demonstrated that the E6-E7 region of the genome from oncogenic HPV 18 is selectively retained in cervical cancer cells, suggesting that HPV infection could be causative and that continued expression of the E6-E7 region is required for maintenance of the immortalized or cancerous state.
  • Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae.
  • Specific human papillomavirus types including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types such as HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer.
  • HPV human papillomaviruses
  • Papanicolaou tests are a valuable screening tool, but they miss a large proportion of HPV-infected persons due to the unfortunate false positive and false negative test results. In addition, they are not amenable to worldwide testing because interpretation of results requires trained pathologists. Because of the limited use and success rate of the Papanicolaou test, many HPV-infected individuals fail to receive timely diagnosis, a problem that precludes efforts to administer treatment prior to the appearance of clinical symptoms. A significant unmet need exists for early and accurate diagnosis of oncogenic HPV infection as well as for treatments directed at the causative HPV infection, preventing the development of cervical cancer by intervening earlier in disease progression.
  • oncogenicity of HPV has been shown to be protein based, treatments that specifically block the activity of oncogenic strains of HPV protein may provide more effective and less invasive treatments than those currently in use.
  • Administration of antagonistic compounds specific for oncogenic strains of HPV may eliminate the need for expensive surgical procedures by treating the causative HPV infection prior to the appearance of clinical symptoms or early in the disease progression.
  • specificity of an oncogenic HPV antagonist significantly reduces risk of damage to healthy cells, thereby minimizing side effects.
  • the invention provides methods and compositions for treating pathogen infections, particularly human papillomavirus infections. Specifically, the invention provides a method of screening for modulators of protein-protein interactions that involves determining an effect of a candidate agent on binding of an E6 protein from an oncogenic strain of HPV to a polypeptide containing the amino acid sequence of a particular PDZ domain from the cellular protein MAGT1. The invention provides methods to treat diseases associated with expression of pathogen proteins by modulating their interactions with MAGI-1, and a number of isolated peptides useful in such methods. Also provided are kits for performing the subject methods. Accordingly, in one embodiment, the invention provides a method of screening.
  • the subject screening methods generally involve determining an effect of a candidate agent on binding of an oncogenic E6 protein to a polypeptide comprising the amino acid sequence of a second PDZ domain from MAGI-1.
  • a polypeptide comprises the sequence of SEQ ID NO:320, or a oncogenic E6 protein-binding valiant thereof, examples of which are set forth as SEQ ID NOS:321-357.
  • the candidate agent is contacted with such a MAGI-1 PDZ polypeptide, and the effect of binding of the polypeptide to an oncogenic E6 protein in the presence of the agent is determined.
  • the screening methods are done in both the presence and absence of the candidate agent, and any agent that reduces binding between the two molecules may be used as an anti-HPV agent.
  • a library of candidate agents is screened for anti-HPV activity. Binding of the MAGI-1 PDZ domain and the oncogenic E6 protein may be assayed using assays that are well known in the art. For example, binding may be assayed biochemically, or, in other embodiments, the MAGI-1 PDZ domain and the oncogenic E6 protein may produce a signal when bound together. In testing candidate agents, such a signal can be assayed in order to assess binding between the two proteins.
  • the MAGI-1 PDZ domain and the oncogenic E6 protein may form a fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), or colorimetric signal producing system, that could be assayed.
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • colorimetric signal producing system that could be assayed.
  • the screening assays may be extracellular (i.e., biochemical) assays using isolated polypeptides, or, in some embodiments, cellular assays, where binding of the two proteins is assayed in a cell contacted with a candidate agent.
  • agents that disrupt interactions between the two proteins may be tested in HPV oncogenicity assays in vitro, which assays are well known in the art.
  • the invention also provides isolated peptides that can effectively inhibit binding between the second MAGI-1 PDZ domain and an E6 protein from oncogenic strains of HPV.
  • the peptides contain at least two (e.g. 3, 4, 5, 6, 7 or more, usually up to about 10 or 15), contiguous amino acids of the C-terminus of an E6 protein from oncogenic strain of HPV.
  • the peptides contain a sequence that is at the immediate C- terminus (i.e., containing the terminal amino acid) of such an E6 protein, whereas in other embodiments, the peptides contain a sequence that is spaced from the terminu of the E6 protein by 1, 2, or 3 or more amino acids.
  • the at least three contiguous amino acids, when present in a subject peptide, are typically, although not always, at the C-terminus of the isolated peptide.
  • a subject peptide may be linked to a cell permeable peptide carrier moiety that provides for internalization of a subject peptide. Such moieties are well known in the art, and described in greater below.
  • the subject peptides may be used to modulate an interaction between a MAGI-1 protein and an oncogenic HPV E6 protein.
  • this method involves contacting the MAGI-1 protein a subject isolated peptide.
  • the invention also provides a method of reducing the oncogenicity of an oncogenic strain of HPV in a cell.
  • this method involves reducing binding of an E6 protein of said HPV to a MAGI-I protein of the cell.
  • the cell may be present in vitro, e.g., as a cultured cell or the like, or as a cell in vivo, i.e., in a subject.
  • binding between the two polypeptides can be reduced by contacting at least one of the components, usually the MAGI-1 protein, with a subject peptide, or an agent discovered using the subject screening assays.
  • a subject isolated peptide may be present in a pharmaceutical composition containing the peptide and a pharmaceutically acceptable carrier, and such a composition may be used in a method of treating a cancer associated with HPV infection.
  • this method involves administering to a subject in need thereof such a pharmaceutical composition.
  • the subject has one or more of the following HP -related cancers: cervical cancer, uterine cancer, anal cancer, colorectal cancer, penile cancer, oral cancer, skin cancer or esophageal cancer.
  • kit containing a subject peptide is provided.
  • such a kit also contains instructions for using the peptide to treat a cancer associated with HPV infection.
  • the present inventors have identified methods for treating diseases associated with HPV, including but not limited to cervical cancer, anal cancer, penile cancer, tliroat cancer and skin cancers.
  • the methods of the invention involve modulation of interactions between PDZ proteins and HPV PL proteins as listed in Table 3, interactions that play a significant role in the biological function and morphology associated with HPV infection.
  • Methods for determining PDZ-PL interactions are disclosed herein, as well as methods for identifying modulators of those interactions in vitro and in vivo. Administration and optimization of treatment is also disclosed.
  • the methods of the invention provide treatment that is highly specific, targeting cells that are infected with HPV. This specificity significantly reduces or eliminates the negative effects of treatment of uninfected, healthy cells, thereby minimizing side effects. Because the treatments of the invention can be administered prior to the appearance of clinical symptoms, HPV infection can be effectively treated before life-threatening diseases (e.g. cervical cancer) develop. In addition, early and specific treatment eliminates the need for invasive and costly surgical procedures that cause significant damage to healthy tissue and often fail to eliminate all infected cells.
  • the invention provides methods of screening for anti-cancer agents, methods of reducing the oncogenicity of an oncogenic HPV, methods for reducing a cancerous phenotype of a cell infected with an oncogenic HPV, and methods for treating HPV infection or cancer, e.g., cervical cancer.
  • the methods involve disrupting the interaction between a PDZ protein, particularly MAGI-1, and the PDZ ligand found in the E6 proteins of oncogenic strains of HPV.
  • the subject invention involves modulating (i.e., increasing or decreasing) interactions between PTEN and PDZ proteins, e.g., MAGI-1, in order to modulate downstream molecular events that involve cell division.
  • PTEN and PDZ proteins e.g., MAGI-1
  • the subject invention involves blocking JNK, FAK or the transcription factor AP-1 to reduce the oncogenicity of an oncogenic HPV, reduce a cancerous phenotype of a cell infected with an oncogenic HPV, and treat HPV infection or cancer.
  • the invention also provides assays for identifying agents for reducing the oncogenicity of an oncogenic HPV, methods for reducing a cancerous phenotype of a cell infected with an oncogenic HPV, and methods for treating HPV infection or cancer.
  • these methods involve providing a cell that produces MAGI-1 and oncogenc HPV E6 proteins, and testing the ability of agents to E6 activation of FAK, JNK or API, or any other downstream event activated by binding of the E6 protein to MAGI-1.
  • Methods for assessing activity of FAK, JNK and API are well known in the art or are described herein.
  • API activity can be measured using a promoter-reporter fusion, where the promoter is an API promoter or a promoter from a gene activated by API, or a JNK assay, a method for which is provided herein.
  • screening methods using transgenic mice that recombinantly express an oncogenic E6 protein, such as a mouse that is known in the art.
  • Such methods may use a mouse with reduced MAGI-1 expression (e.g., a MAGI-1 "knockout" mouse).
  • a MAGI-1 "knockout” mouse Such E6 and MAGI-1 mice may be crossed with each other, and may be in genetic backgrounds that have altered FAK, JNK or API activity (e.g., they have a knockout in or overexpress on of these genes).
  • the methods of the invention provide a more specific, effective, and cost-efficient alternative to current treatments for oncogenic HPV infection.
  • FIGURE 1A Northern blot analysis of HPV16 E6 and HPV18 E6 expression in various cell lines. Lanes: 1 B-cell (Ramos); 2 No HPV (HTB32); 3 1550 HPV 16+18; 4 1595 HPV18; 5 1594 HPV 18; 6 HTB 35 (HPV 16); 7 RNA marker. HPV18 E6 and HPV16 E6 refer to the radiolabeled probe used to detect expression in each of the cell lines.
  • FIGURE IB Northern blot analysis of Magi- 1 and TIP-1 expression in various cervical cell lines.
  • the expected size for Magi-1 mRNA is 4.5 kb, although alternative splice forms are noted in Genbank.
  • the expected size for Tip-1 mRNA is 1.4 kb.
  • PDZ proteins can specifically recognize oncogenic E6 proteins from human papillomavirus.
  • FIGURE 3 Inhibition of the interaction between HPV E6 16 and TIPl by Tax peptide.
  • OD (A450) is shown on the y-axis, and titrating concentrations of Tax inhibitor (uM) are shown on the x-axis.
  • HPV E6 16 peptide was used at a concentration of lOuM, and TIPl fusion protein was used at a concentration of 5ug/mL. See Example 7 for further details.
  • FIGURE 4 is a complation of four panels of autoradiographs, A), B) and C).
  • Oncogenic HPV E6 16 but not non-oncogenic HPV E6 11, activates c-JUN N-terminal kinase (JNK), a kinase known to be involved in numerous oncogenic pathways.
  • JNK c-JUN N-terminal kinase
  • B) HPV E6 16-dependent activation of JNK can be inhibited by co-injection of peptide corresponding to the C-terminus of oncogenic Tax, but not with the peptide representing the C-terminus of non-oncogenic HPV E6 11.
  • C) HPV E6 16 dependent activation of JNK can be inhibited by peptide representing HPV E6 16 oncoprotein, but not by peptide representing the C-terminus of nononcogenic HPV E6 11.
  • FIGURES 5A, 5B, 5C and 5D show results of mammalian cell migration assays.
  • Cells were transfected with a construct that expresses the E6 protein from HPV 16 or the same protein with a deletion of 3 amino acids at the carboxyl-terminus that abolishes the ability to interact with PDZ domains.
  • E6-transfected cells migrate through a scratch, indicative of cell transformation, while E6 cells with a c-terminal deletion do not migrate to fill in the scratch.
  • FIGURE 6 Examination of cJUN N-terminal Kinase (JNK) activity using a kinase assay for it's ability to phosphorylate a GST-cJUN protein.
  • JNK N-terminal Kinase
  • FIGURE 7 Titration curve showing binding of a 20 amino acid peptide corresponding to the C-terminus of the E6 protein from HPV 16 to a PDZ domain containing protein TIP-1. Assay was performed as described in the specification (G assay). Numbers on the X-axis are micromolar units.
  • FIGURE 8 displays four panels of graphs, A-E, showing effect of small molecule inhibitors on the interaction between E6 protein from HPV 16 and TIP-1.
  • FIGURE 9 A, 9B, 9C and 9B HPV E6 activates JNK in epithelial cells.
  • HEK293 cells were transiently transfected with indicated Ha-tagged constructs. Lysates were used for immunoprecipitation and immunoblot detection with anti-HA antibodies (upper). Lysates from the same experiment were investigated in a GST-Jun pull down in vitro kinase assay for their JNK activity. Shown is the autoradiogram of the JNK assay (lower) (B)
  • Xenopus oocytes were microinjected with bacterial expressed proteins of GST HPV16E6 , GST HPV 18E6 and GST HPV 11 E6 at 1 OOnM final concentration calculated per oocyte. After 3h cells were lysed and lysates were tested for JNK activity (Upper). Oocytes were coinjected with GST HPV16E6 (lOOnM) and a 20 mer peptide corresponding to the C- terminus of HPVE616. The peptide concentrations are indicated and are calculated as final concentration per oocyte. The control is the 20mer C-terminal peptide of HPV11E6 at 10 ⁇ M.
  • HEK293 cells were transfected with pSilencer vectors encoding small interfering RNA's for sequences not present in the human genome (si-control), present in GAPDH (si-GAPDH) (as an additional control) and for a sequence present in MAGI l.(si- MAGI). Protein expression levels of MAGI 1 were significantly reduced compared to the two controls (Upper). JNK activity was measured from lysates of these transfection. Sorbitol treated 293 cells were used for positive control (Similar results were obtained in three independent experiments) .
  • FIGURE 10A, 10B, IOC and 10D Regulation of MAGI 1 expression by HPV16 E6 PL
  • A MAGI 1 and Dlgl protein levels in HPV positive or negative cervical cancer cells. Total cell lysates analyzed by western blot with anti-Magil and anti-Dlgl antibodies
  • B Relative levels of Magi 1 and Dlgl RNA levels in cervical cancer cell lines, as determined by real time PCR #
  • C MAGI 1 and Dlgl protein expression in HEK293 cells expressing E6 and E6 ⁇ PL. Cells were transiently transfected with pmkit-HA-E6, pmkit-HA-E6 ⁇ PL or the control pmkit-HA expression vector. Shown are the MAGI 1 protein expression levels. E6 protein expression levels were determined with anti-HA antibody and were comparable for E6 and -E6 ⁇ PL (not shown)
  • D Magil and Dlgl RNA levels in 293 cells transfected with E6 and E ⁇ PLanalyzed by real time PCR.
  • FIGURES 11 A, 11B, 11C show the structures of various chemical groups used in the subject compositions and methods in panels A through O.
  • biological function in the context of a cell, refers to a detectable biological activity normally carried out by the cell, e.g., a phenotypic change such as cell proliferation, cell activation (e.g., T cell activation, B cell activation, T-B cell conjugate formation), cytokine release, degranulation, tyrosine phosphorylation, ion (e.g., calcium) flux, metabolic activity, apoptosis, changes in gene expression, maintenance of cell structure, cell migration, adherence to a substrate, signal transduction, cell-cell interactions, and others described herein or known in the art.
  • a phenotypic change such as cell proliferation, cell activation (e.g., T cell activation, B cell activation, T-B cell conjugate formation), cytokine release, degranulation, tyrosine phosphorylation, ion (e.g., calcium) flux, metabolic activity, apoptosis, changes in gene expression, maintenance of cell structure, cell migration,
  • a 'marker or “biological marker” as used herein refers to a measurable or detectable entity in a biological sample.
  • markers include nucleic acids, proteins, or chemicals that are present in biological samples.
  • One example of a marker is the presence of viral or pathogen proteins or nucleic acids in a biological sample from a human source.
  • isolated refers to a polynucleotide, a polypeptide, an antibody, or a host cell that is in an environment different from that in which the polynucleotide, the polypeptide, the antibody, or the host cell naturally occurs.
  • a polynucleotide, a polypeptide, an antibody, or a host cell which is isolated is generally substantially purified.
  • a subject "infected" with HPV is a subject having cells that contain HPV.
  • the HPV in the cells may not exhibit any other phenotype (i.e., cells infected with HPV do not have to be cancerous).
  • cells infected with HPV may be pre-cancerous (i.e., not exhibiting any abnormal phenotype, other than those that may be associated with viral infection), or cancerous cells.
  • the term “substantially purified” refers to a compound (e.g., either a polynucleotide or a polypeptide or an antibody) that is removed from its natural environment and is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated.
  • a composition containing A is "substantially free of B when at least 85% by weight of the total A+B in the composition is A.
  • A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95% or even 99% by weight.
  • polypeptide and “protein” are used interchangeably throughout the application and mean at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Peptidominetics will be discussed in greater detail below.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration.
  • amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
  • Naturally occurring amino acids are normally used and the protein is a cellular protein that is either endogenous or expressed recombinantly.
  • polypeptides may be of any length, e.g., greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 300 amino acids, usually up to about 500 or 1000 or more amino acids.
  • “Peptides” are generally greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, usually up to about 3, 4, 5, 10, 30 or 50 amino acids, hi some embodiments, peptides are between 5 and 30 amino acids in length.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes, but is not limited to, the production of a protein from one organism in a different organism or host cell.
  • the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
  • a “fusion protein” or “fusion polypeptide” as used herein refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two (or more) distinct, heterologous polypeptides that are not normally fused together in a single amino acid sequence.
  • a fusion protein can include a single amino acid sequence that contains two entirely distinct amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in the same configuration in a single amino acid sequence found in nature.
  • Fusion proteins can generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a recombinant gene fusion product, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art.
  • a "fusion protein construct" as used herein is a polynucleotide encoding a fusion protein.
  • an “oncogenic HPV strain” is an HPV strain that is known to cause cervical cancer as determined by the National Cancer Institute (NCI,2001).
  • "Oncogenic E6 proteins” are E6 proteins encoded by the above oncogenic HPV strains. Exemplary oncogenic strains are shown in Table 3. Oncogenic strains of HPV not specifically listed here, are known in the art, and may be found at the world wide website of the National Center for Biotechnology Information (NCBI).
  • an "oncogenic E6 protein binding partner” is any molecule that specifically binds to an oncogenic E6 protein.
  • Suitable oncogenic E6 protein binding partners include a PDZ domain (as described below), an antibody against an oncogenic E6 protein; other proteins that recognize oncogenic E6 protein (e.g., p53, E6-AP or E6-BP); DNA (i.e., cruciform DNA); and other partners such as aptamers or single chain antibodies from phage display).
  • binding partner bind E6 with an binding affinity of 10 "5 M or more, e.g., 10 "6 or more, 10 “7 or more, 10 "s M or more (e.g., 10 "9 M, 10 "10 , 10 "11 , etc.).
  • PDZ domain refers to protein sequence (i.e., modular protein domain) of less than approximately 90 amino acids, (i.e., about 80-90, about 70-80, about 60-70 or about 50-60 amino acids), characterized by homology to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein ZOl (ZOl).
  • PDZ domains are also l ⁇ iown as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally appear to maintain a core consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76).
  • PDZ domains are found in diverse membrane-associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin- associated proteins, collectively known as syntrophins.
  • PDZ domain-containing proteins and PDZ domain sequences are shown in TABLE 2 and EXAMPLE 4.
  • the term "PDZ domain” also encompasses variants (e.g., naturally occurring variants) of the sequences (e.g., polymorphic variants, variants with conservative substitutions, and the like) and domains from alternative species (e.g. mouse, rat).
  • PDZ domains are substantially identical to those shown in US PATENT APPLICATION 09/724553, e.g., at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence.
  • PDZ domains can be mutated to give amino acid changes that can strengthen or weaken binding and to alter specificity, yet they remain PDZ domains (Schneider et al,., 1998, Nat. Biotech. 17:170-5).
  • a reference to a particular PDZ domain e.g. a MAGI-1 domain 2 is intended to encompass the particular PDZ domain and HPV E6-binding variants thereof.
  • a reference is also made to variants, of that PDZ domain that bind oncogenic E6 protein of HPV, as described below.
  • the numbering of PDZ domains in a protein may change.
  • the MAGI-1 domain 2 as referenced herein, may be referenced as MAGI-1 domain 1 in other literature.
  • this reference should be understood in view of the sequence of that domain, as described herein, particularly in the sequence listing.
  • PDZ protein refers to a naturally occurring protein containing a PDZ domain.
  • exemplary PDZ proteins include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYNla, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN-4, prILl ⁇ , 41.8kD, KIAA0559, RGS12, KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303, CBP, MINT3, TIP-2, KIAA0561, and TIP-1.
  • PDZ-domain polypeptide refers to a polypeptide containing a PDZ domain, such as a fusion protein including a PDZ domain sequence, a naturally occurring PDZ protein, or an isolated PDZ domain peptide.
  • a PDZ— domain polypeptide may therefore be about 60 amino acids or more in length, about 70 amino acids or more in length, about 80 amino acids or more in length, about 90 amino acids or more in length, about 100 amino acids or more in length, about 200 amino acids or more in length, about 300 amino acids or more in length, about 500 amino acids or more in length, about 800 amino acids or more in length, about 1000 amino acids or more in length, usually up to about 2000 amino acids or more in length.
  • PDZ domain peptides are usually no more than about 100 amino acids (e.g. 50-60 amino acids, 60-70 amino acids, 80-90 amino acids, or 90-100 amino acids), and encode a PDZ domain.
  • PL protein or "PDZ Ligand protein” refers to a polypeptide that may be a naturally-occurring or non-naturally occurring peptide, that forms a molecular complex with a PDZ-domain, or to a protein whose carboxy-terminus, when expressed separately from the full length protein (e.g., as a peptide of 4-25 residues, e.g., 8, 10, 12, 14 or 16 residues), forms such a molecular complex.
  • the molecular complex can be observed in vitro using the "A assay” or "G assay” described infra, or in vivo.
  • Exemplary PL proteins listed in TABLES 2 and 3 are demonstrated to bind specific PDZ proteins. This definition is not intended to include anti-PDZ antibodies and the like.
  • a "PDZ ligand sequence” refers to the amino acid sequence of the C- terminus of a PL protein (e.g., the C-terminal 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25 residues) ("C-terminal PL sequence") or to an internal sequence known to bind a PDZ .. domain (“internal PL sequence”), or variant thereof.
  • a "PDZ ligand peptide” is a peptide of having a sequence from, or based on, the sequence of the C-terminus of a PL protein.
  • Exemplary PL peptides (biotinylated) are listed in TABLE 2.
  • a "PL detector” is a protein that can specifically recognize and bind to a PL sequence.
  • a "PL fusion protein” is a fusion protein that has a PL sequence as one domain, typically as the C-terminal domain of the fusion protein.
  • An exemplary PL fusion protein is a tat-PL sequence fusion.
  • PL inhibitor peptide sequence refers to PL peptide amino acid sequence that (in the form of a peptide or PL fusion protein) inhibits the interaction between a PDZ domain polypeptide and a PL peptide (e.g., in an A assay or a G assay).
  • a "PDZ-domain encoding sequence” means a segment of a polynucleotide encoding a PDZ domain.
  • the polynucleotide is DNA, RNA, single stranded or double stranded.
  • the terms "antagonist” and “inhibitor,” when used in the context of modulating a binding interaction are used interchangeably and refer to an agent that reduces the binding of the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZ protein, PDZ domain peptide).
  • PL sequence e.g., PL peptide
  • PDZ domain sequence e.g., PDZ protein, PDZ domain peptide
  • the terms "agonist” and “enhancer,” when used in the context of modulating a binding interaction are used interchangeably and refer to an agent that increases the binding of the, e.g., PL sequence (e.g., PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZ protein, PDZ domain peptide).
  • PL sequence e.g., PL peptide
  • PDZ domain sequence e.g., PDZ protein, PDZ domain peptide
  • peptide mimetic As used herein, the terms “peptide mimetic, " “peptidomimetic,” and “peptide analog” are used interchangeably and refer to a synthetic chemical compound that has substantially the same structural and or functional characteristics of a PL inhibitory or PL binding peptide of the invention.
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or inhibitory or binding activity.
  • a mimetic composition is within the scope of the invention if it is capable of binding to a PDZ domain and or inhibiting a PL-PDZ interaction.
  • Polypeptide mimetic compositions can contain any combination of nonnatural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a secondary structural mimicry i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • aminomethylene CH 2 -NH
  • ethylene olefin
  • ether CH 2
  • a polypeptide can also be characterized as a mimetic by containing all or some non- natural residues in place of naturally occu ⁇ ing amino acid residues.
  • Nonnatural residues are well described in the scientific and patent literature; a few exemplary nonnatural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
  • Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L- phenylglycine; D- or L-2 thieneylalanine; D- or L-l, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3- pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p- fluorophenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p- methoxybiphenylphenyla
  • Aromatic rings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
  • Carboxyl side groups e.g., aspartyl or glutamyl
  • Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or
  • Nitrile derivative e.g., containing the CN-moiety in place of COOH
  • Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
  • Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
  • Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines, to give carboxymethyl or carboxyamidomethyl derivatives.
  • alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines
  • Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha- bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3- nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2- chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
  • cysteinyl residues e.g., bromo-trifluoroacetone, alpha- bromo-beta-(5-imidozoyl) propionic acid
  • chloroacetyl phosphate N-alkylmaleimides
  • 3- nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
  • Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other . alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate.
  • imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate.
  • Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
  • Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
  • Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para- bromophenacyl bromide.
  • mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution withN-methyl amino acids; or amidation of C-terminal carboxyl groups.
  • a component of a natural polypeptide can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality.
  • an amino acid or peptidomimetic residue of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, generally referred to as the D- amino acid, but which can additionally be referred to as the R- or S- form.
  • the mimetics of the invention can also include compositions that contain a structural mimetic residue, particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like.
  • a structural mimetic residue particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like.
  • substitution of natural amino acid residues with D-amino acids; N-alpha-methyl amino acids; C-alpha-methyl amino acids; or dehydroamino acids within a peptide can induce or stabilize beta turns, gamma turns, beta sheets or alpha helix conformations.
  • Beta turn mimetic structures have been described, e.g., by Nagai (1985) Tet. Lett. 26:647-650; Feigl (1986) J. Amer. Chem. Soc.
  • Beta sheet mimetic structures have been described, e.g., by Smith (1992) J. Amer. Chem. Soc. 114:10672- 10674.
  • a type VI beta turn induced by a cis amide surrogate, 1 ,5-disubstituted tetrazol is described by Beusen (1995) Biopolymers 36: 181-200.
  • peptide variants and “conservative amino acid substitutions” refer to peptides that differ from a reference peptide (e.g., a peptide having the sequence of the carboxy-terminus of a specified PL protein) by substitution of an amino acid residue having similar properties (based on size, polarity, hydrophobicity, and the like).
  • a reference peptide e.g., a peptide having the sequence of the carboxy-terminus of a specified PL protein
  • substitution of an amino acid residue having similar properties based on size, polarity, hydrophobicity, and the like.
  • amino acids may be generally categorized into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses.
  • Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains.
  • Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
  • the definitions of the classes of amino acids as used herein are as follows: "Hydrophobic Amino Acid” refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids include He, Leu and Val. Examples of non- genetically encoded hydrophobic amino acids include t-BuA.
  • Aromatic Amino Acid refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated ⁇ -electron system (aromatic group).
  • aromatic group may be further substituted with groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others.
  • genetically encoded aromatic amino acids include Phe, Tyr and Tip.
  • aromatic amino acids include phenylglycine, 2-naphthylalanine, ⁇ -2- thienylalanine, l,2,3,4-tetrahydroisoquinoline-3 -carboxylic acid, 4-chloro-phenylalanine, 2- fluorophenyl-alanine, 3-fluorophenylalanine and 4-fluorophenylalamne.
  • Apolar Amino Acid refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar.
  • Examples of genetically encoded apolar amino acids include Gly,-Pro and Met.
  • Examples of non-encoded apolar amino acids include Cha.
  • Aliphatic Amino Acid refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
  • genetically encoded aliphatic amino acids include Ala, Leu, Val and He.
  • non- encoded aliphatic amino acids include Nle.
  • Hydrophilic Amino Acid refers to an amino acid having a side chain that is attracted by aqueous solution.
  • examples of genetically encoded hydrophilic amino acids include Ser and Lys.
  • examples of non-encoded hydrophilic amino acids include Cit and hCys.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion.
  • Examples of genetically encoded acidic amino acids include Asp and Glu.
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include Arg, Lys and His.
  • non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4- diaminobutyric acid and homoarginine.
  • Poly Amino Acid refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • genetically encoded polar amino acids include Asx and Glx.
  • non-genetically encoded polar amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
  • Cysteine-Like Amino Acid refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage.
  • cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group.
  • examples of genetically encoded cysteine-like amino acids include Cys.
  • examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine.
  • cysteine has both an aromatic ring and a polar hydroxyl group.
  • cysteine has dual properties and can be included in both the aromatic and polar categories.
  • cysteine in addition to being able to form disulfide linkages, cysteine also has apolar character.
  • cysteine can be used to confer hydrophobicity to a peptide.
  • Certain commonly encountered amino acids which are not genetically encoded of which the peptides and peptide analogues of the invention may be composed include, but are not limited to, ⁇ -alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib); ⁇ -aminohexanoic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (
  • TABLE 1 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues which may comprise the peptides and peptide analogues described herein.
  • Other amino acid residues which are useful for making the peptides and peptide analogues described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein.
  • Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
  • a "HPV E6-binding variant" of a particular PDZ domain is a PDZ domain variant that retains HPV E6 PDZ ligand binding activity.
  • Assays for determining whether a PDZ domain variant binds HPV E6 are described in great detail below, and guidance for identifying which amino acids to change in a specific PDZ domain to make it into a variant may be found in a variety of sources.
  • a PDZ domain may be compared to other PDZ domains described herein and amino acids at corresponding positions may be substituted, for example.
  • sequence a PDZ domain of a particular PDZ protein may be compared to the sequence of an equivalent PDZ domain in an equivalent PDZ protein from another species.
  • sequence a PDZ domain from a human PDZ protein may be compared to the sequence of other known and equivalent PDZ domains from other species (e.g., mouse, rat, etc.) and any amino acids that are variant between the two sequences may be substituted into the human PDZ domain to make a variant of the PDZ domain.
  • sequence of the human MAGI-1 PDZ domain 2 may be compared to equivalent MAGI-1 PDZ domains from other species (e.g.
  • MAGI-1 PDZ domain 2 sequence is provided as SEQ ID NOS:320-328.
  • Particular variants may have 1, up to 5, up to about 10, up to about 15, up to about 20 or up to about 30 or more, usually up to about 50 amino acid changes as compared to a sequence set forth in the sequence listing.
  • Exemplary MAGI-1 PDZ variants include the sequences set forth in SEQ ID NOS: 329-357. In making a variant, if a GFG motif is present in a PDZ domain, in general, it should not be altered in sequence.
  • variant PDZ domain polypeptides have a PDZ domain that has at least about 70 or 80%, usually at least about 90%, and more usually at least about 98% sequence identity with a variant PDZ domain polypeptide described herein, as measured by BLAST 2.0 using default parameters, over a region extending over the entire PDZ domain.
  • a "detectable label” has the ordinary meaning in the art and refers to an atom (e.g., radionuclide), molecule (e.g., fluorescent), or complex, that is or can be used to detect (e.g., due to a physical or chemical property), indicate the presence of a molecule or to enable binding of another molecule to which it is covalently bound or otherwise associated.
  • label also refers to covalently bound or otherwise associated molecules (e.g., a biomolecule such as an enzyme) that act on a substrate to produce a detectable atom, molecule or complex.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Labels useful in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 3 P), enzymes ( e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and others commonly used in ELISAs), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and colorimetric labels such as colloidal gold
  • Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Means of detecting such labels are well known to those of skill in the art.
  • radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence-activated cell sorting).
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal generating system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal generating system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • ligands and anti-ligands can be used.
  • a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
  • any haptenic or antigenic compound can be used in combination with an antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected by observing the color associated with the label. It will be appreciated that when pairs of fluorophores are used in an assay, it is often preferred that they have distinct emission patterns (wavelengths) so that they can be easily distinguished.
  • the term "substantially identical" in the context of comparing amino acid sequences means that the sequences have at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence.
  • An algorithm that is suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm, which is described in Pearson, W.R. & Lipman, D.J., 1988, Proc. Natl Acad. Sci. U.S.A. 85: 2444. See also W. R. Pearson, 1996, Methods Enzymol. 266: 227-258.
  • sandwich As used herein, the terms “sandwich”, “sandwich ELISA”, “Sandwich diagnostic” and “capture ELISA” all refer to the concept of detecting a biological polypeptide with two different test agents.
  • a PDZ protein could be attached to a solid support.
  • Test sample could be passed over the surface and the PDZ protein could bind it's cognate PL protein(s).
  • An antibody with detection reagent could then be used to determine whether a specific PL protein had bound the PDZ protein.
  • test compound or “test agent” are used interchangeably and refer to a candidate agent that may have enhancer/agonist, or inhibitor/antagonist activity, e.g., inhibiting or enhancing an interaction such as PDZ-PL binding.
  • test agents or test compounds may be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies (as broadly defined herein), sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • test agents are prepared from diversity libraries, such as random or combinatorial peptide or non-peptide libraries. Many libraries are known in the art that can be used, e.g.
  • phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al, 1990, Science, 249:404-406; Christian, R.B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated August 18, 1994.
  • In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated April 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad.
  • a benzodiazepine library see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91 :4708-4712
  • Peptoid libraries can also be used.
  • binding refers to binding between two molecules, for example, a ligand and a receptor, characterized by the ability of a molecule (ligand) to associate with another specific molecule (receptor) even in the presence of many other diverse molecules, i.e., to show preferential binding of one molecule for another in a heterogeneous mixture of molecules. Specific binding of a ligand to a receptor is also evidenced by reduced binding of a detectably labeled ligand to the receptor in the presence of excess unlabeled ligand (i.e., a binding competition assay).
  • a "plurality" of PDZ proteins has its usual meaning.
  • the plurality is at least 5, and often at least 25, at least 40, or at least 60 different PDZ proteins.
  • the plurality is selected from the list of PDZ polypeptides listed in TABLE 8.
  • the plurality of different PDZ proteins are from (i.e., expressed in) a particular specified tissue or a particular class or type of cell.
  • the plurality of different PDZ proteins represents a substantial fraction (e.g., typically at least 50%, more often at least 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in lymphocytes or hematopoetic cells.
  • the plurality is at least 50%, usually at least 80%, at least 90% or all of the PDZ proteins disclosed herein as being expressed in a particular cell.
  • a “plurality” may refer to at least 5, at least 10, and often at least 16 PLs such as those specifcally listed herein, or to the classes and percentages set forth supra for PDZ domains.
  • HPV PL protein refers to a protein in the family of human papillomavirus proteins that displays a PDZ-ligand motif on the C-terminus of the protein.
  • Methods and compositions for treating a disease correlated with binding between a PDZ protein and a HPV protein containing a PL motif are also disclosed herein, the method comprising administering a therapeutically effective amount of a modulator as provided herein, wherein the PDZ protein and the PL protein are a binding pair as specified in Table 3.
  • a modulator as provided herein, wherein the PDZ protein and the PL protein are a binding pair as specified in Table 3.
  • such methods can be used to treat a variety of diseases associated with HPV infection, including, but not limited to, cervical cancer, penile cancer, anal cancer, throat cancer, skin cancer and genital warts.
  • Certain methods involve introducing into the cell an agent that alters binding between a PDZ protein and a HPV PL protein in the cell, whereby the biological function is modulated in the cell, and wherein the PDZ protein and PL protein are a binding pair as specified in Table 3.
  • the agent is a polypeptide comprising at least the two, three or four carboxy-terminal residues of the PL protein.
  • Screening methods to identify compounds that modulate binding between PDZ proteins and PL peptides or proteins are also provided. Some screening methods involve contacting under suitable binding conditions (i) a PDZ-domain polypeptide having a sequence from a PDZ protein, and (ii) a PL peptide, wherein the PL peptide comprises a C- terminal sequence of the PL protein, the PDZ -domain polypeptide and the PL peptide are a binding pair as specified in Table 3; and contacting is performed in the presence of the test compound. Presence or absence of complex is then detected.
  • the presence of the complex at a level that is statistically significantly higher in the presence of the test compound than in the absence of test compound is an indication that the test compound is an agonist
  • the presence of the complex at a level that is statistically significantly lower in the presence of the test compound than in the absence of test compound is an indication that the test compound is an antagonist
  • the modulator is (a) a peptide comprising at least 3 residues of a C-terminal sequence of a PL protein, and wherein the PDZ protein and the PL protein are a binding pair as specified in Table 3; or (b) a peptide mimetic of the peptide of section (a); or (c) a small molecule having similar functional activity with respect to the PDZ and PL protein binding pair as the peptide of section (a).
  • the modulator can be either an agonist or antagonist.
  • Such modulators can be formulated as a pharmaceutical composition.
  • the modulator is administered topically, in the form of a cream.
  • TABLE 3 lists PDZ proteins and HPV PL proteins which the current inventors have identified as binding to one another. TABLE 3 is organized into four columns. The columns from left to right show the HPV E6 strain and terminal 4 amino adds of the PL that was tested in the G assay (generally 20 amino acids), followed by the PDZ domains that bound that ligand at high affinity, and then a repetition of additional HPV strains and PDZ domains that bind the E6 PL immediately to the left of the domains. Thus, the first column in each section is labeled "HPV Strain" and lists the names of the various E6 proteins and the carboxy-terminal 4 amino acids (potential PLs) that were examined.
  • the second column labeled "PDZ binding partner” lists PDZ domains that bind the biotinylated peptide at relatively high strength. All ligands are biotinylated at the amino-terminus and partial sequences are presented in TABLE 3.
  • the PDZ protein (or proteins) that interact(s) with HPV E6 - PL peptides are listed in the third column labeled "PDZ binding partner". This column provides the gene name for the PDZ portion of the GST-PDZ fusion that interacts with the PDZ-ligand to the left.
  • PDZ domain-containing proteins with multiple domains the domain number is listed to the right of the PDZ (i.e., in column 4 labeled "PDZ Domain"), and indicates the PDZ domain number when numbered from the amino-terminus to the carboxy-terminus.
  • Column 5 labeled "Classification” lists a measure of the level of binding, as determined in the "G” Assay.
  • absorbance value at 450 nm which indicates the amount of PL peptide bound to the PDZ protein.
  • the following numerical values have the following meanings: '1' - A4 5 onm 0-1; '2' - A 450 nm 1-2; '3'- A 5 onm 2-3; '4' - A ⁇ onm 3-4; '5' - A 45 omn of 4 more than 2X repeated; '0' - A 45 onm 0, i.e., not found to interact.
  • TABLE 2 provides a listing of the amino acid sequences of peptides used in the assays.
  • HPV strain provides the name of the HPV strain, corresponding to the name listed in column 1 of Table 2.
  • E6 C-terminal sequence provides the predicted sequence of the carboxy-terminal 10 amino acids of the E6 protein.
  • PL yes/no designates whether the E6-PL sequence contains sequence elements predicted by the inventors to bind to PDZ domains.
  • PDZ# indicates the Pfam-predicted PDZ domain number, as numbered from the amino-terminus of the gene to the carboxy-terminus.
  • sequence fused to GST construct provides the actual amino acid sequence inserted into the GST-PDZ expression vector as determined by DNA sequencing of the constructs.
  • the PDZ proteins listed in TABLE 3 are naturally occurring proteins containing a PDZ domain. Only significant interactions are presented in this table. Thus, the present invention is particularly directed to the modulation of interactions between a PDZ protein and a HPV PL protein.
  • cellular abnormalities or diseases can be treated through the correction of imbalances in the expression levels of cellular PDZ proteins or PL proteins.
  • PL protein or the PDZ protein in an assay derived from the 'A assay' or 'G assay' one can determine the protein expression levels of binding partners in a normal or abnormal cell. Differences in protein expression levels have been con-elated with a number of diseases.
  • a PDZ protein is used to treat diseases associated with the presence of a PL protein from a pathogenic organism, such as diseases associated with HPV infection, including but not limited to cervical cancer, genital warts, penile cancer, and anal cancer.
  • an antagonist of the interaction is used to block the interaction between a PDZ protein and a PL protein from a pathogenic organism, thus providing treatment for diseases associated with that pathogen.
  • An antagonist may be in the form of a PL peptide, a PL protein, a peptide mimetic, a small molecule, or any other antagonist compound known in the art.
  • ELISA immunoassays can be used to identify peptides that specifically bind PDZ-domain polypeptides.
  • two different, complementary assays were developed to detect PDZ-PL interactions. In each, one binding partner of a PDZ-PL pair is immobilized, and the ability of the second binding partner to bind is determined.
  • These assays which are described infra, can be readily used to screen for hundreds to thousands of potential PDZ-ligand interactions in a few hours. Thus these assays can be used to identify yet more novel PDZ-PL interactions in cells. In addition, they can be used to identify antagonists of PDZ-PL interactions (see infra).
  • fusion proteins are used in the assays and devices of the invention. Methods for constructing and expressing fusion proteins are well known. Fusion proteins generally are described in Ausubel et al., supra, Kroll et al., 1993, DNA Cell. Biol. 12:441, and Imai et al., 1997, Cell 91:521-30. Usually, the fusion protein includes a domain to facilitate immobilization of the protein to a solid substrate ("an immobilization domain").
  • the immobilization domain includes an epitope tag (i.e., a sequence recognized by an antibody, typically a monoclonal antibody) such as polyhistidine (Bush et al, 1991 , J Biol Chem 266:13811-14), SEAP (Berger et al, 1988, Gem 66:1-10), or Ml and M2 flag (see, e.g, U.S. Pat. Nos. 5,011,912; 4,851,341; 4,703,004; 4,782,137).
  • the immobilization domain is a GST coding region.
  • the protein in addition to the PDZ-domain and the particular residues bound by an immobilized antibody, protein A, or otherwise contacted with the surface, the protein (e.g., fusion protein), will contain additional residues.
  • these are residues naturally associated with the PDZ-domain (i.e., in a particular PDZ-protein) but they may include residues of synthetic (e.g., poly(alanine)) or heterologous origin (e.g., spacers of, e.g., between 10 and 300 residues).
  • PDZ domain-containing polypeptide used in the methods of the invention are typically made by (1) constructing a vector (e.g., plasmid, phage or phagemid) comprising a polynucleotide sequence encoding the desired polypeptide, (2) introducing the vector into an suitable expression system (e.g., a prokaryotic, insect, mammalian, or cell free expression system), (3) expressing the fusion protein and (4) optionally purifying the fusion protein.
  • a vector e.g., plasmid, phage or phagemid
  • an suitable expression system e.g., a prokaryotic, insect, mammalian, or cell free expression system
  • expression of the protein comprises inserting the coding sequence into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence required for the expression system employed, e.g., control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence.
  • control elements including enhancers, promoters, transcription terminators, origins of replication, a suitable initiation codon (e.g., methionine), open reading frame, and translational regulatory signals (e.g., a ribosome binding site, a termination codon and a polyadenylation sequence.
  • a suitable initiation codon e.g., methionine
  • open reading frame e.g., open reading frame
  • translational regulatory signals e.g
  • the coding sequence of the fusion protein includes a PDZ domain and an immobilization domain as described elsewhere herein.
  • Polynucleotides encoding the amino acid sequence for each domain can be obtained in a variety of ways known in the art; typically the polynucleotides are obtained by PCR amplification of cloned plasmids, cDNA libraries, and cDNA generated by reverse transcription of RNA, using primers designed based on sequences determined by the practitioner or, more often, publicly available (e.g., through GenBank).
  • the primers include linker regions (e.g., sequences including restriction sites) to facilitate cloning and manipulation in production of the fusion construct.
  • the polynucleotides corresponding to the PDZ and immobilization regions are joined in-frame to produce the fusion protein-encoding sequence.
  • the fusion proteins of the invention may be expressed as secreted proteins (e.g., by including the signal sequence encoding DNA in the fusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-61) or as nonsecreted proteins.
  • secreted proteins e.g., by including the signal sequence encoding DNA in the fusion gene; see, e.g., Lui et al, 1993, PNAS USA, 90:8957-611
  • GST-PDZ domain fusion proteins were prepared for use in the assays of the invention.
  • PCR products containing PDZ encoding domains (as described supra) were subcloned into an expression vector to permit expression of fusion proteins containing a PDZ domain and a heterologous domain (i.e., a glutathione-S transferase sequence, "GST").
  • PCR products i.e., DNA fragments
  • PDZ domain encoding DNA were extracted from agarose gels using the "Sephaglas" gel extraction system (Pharmacia) according to the manufacturer's recommendations. Amino acid sequences for all of the PDZ domains used in the assays of the invention are listed in Table 8.
  • PCR primers were designed to include endonuclease restriction sites to facilitate ligation of PCR fragments into a GST gene fusion vector (pGEX-3X; Pharmacia, GenBank accession no. XXU13852) in-frame with the glutathione-S transferase coding sequence.
  • This vector contains an IPTG inducible lacZ promoter.
  • the pGEX-3X vector was linearized using Bam HI and Eco RI or, in some cases, Eco RI or Sma I, and dephosphorylated. For most cloning approaches, double digestion with Bam HI and Eco RI was performed, so that the ends of the PCR fragments to clone were Bam HI and Eco RI.
  • restriction endonuclease combinations used were Bgl II and Eco RI, Bam HI and Mfe I, or Eco RI only, Sma I only, or BamHI only.
  • the DNA portion cloned represents the PDZ domains and the cDNA portion located between individual domains. Precise locations of cloned fragments used in the assays are indicated in US Patent Application (60/360061). DNA linker sequences between the GST portion and the PDZ domain containing DNA portion vary slightly, dependent on which of the above described cloning sites and approaches were used.
  • the amino acid sequence of the GST-PDZ fusion protein varies in the linker region between GST and PDZ domain.
  • Protein linker sequences corresponding to different cloning sites/approaches are shown below. Linker sequences (vector DNA encoded) are bold, PDZ domain containing gene derived sequences are in italics.
  • Colonies were screened for presence and identity of the cloned PDZ domain containing DNA as well as for correct fusion with the glutathione S -transferase encoding DNA portion by PCR and by sequence analysis. Positive clones were tested in a small-scale assay for expression of the GST PDZ domain fusion protein and, if expressing, these clones were subsequently grown up for large scale preparations of GST/PDZ fusion protein.
  • GST-PDZ domain fusion protein was overexpressed following addition of IPTG to the culture medium and purified.
  • Detailed procedure of small scale and large-scale fusion protein expression and purification are described in "GST Gene Fusion System", (second edition, revision 2; published by Pharmacia).
  • a small culture (50mls) containing a bacterial strain (DH5 ⁇ , BL21 or JM109) with the fusion protein construct was grown overnight in 2xYT media at 37°C with the appropriate antibiotic selection (lOOug/ml ampicillin; a.k.a. 2xYT-amp).
  • the overnight culture was poured into a fresh preparation of 2xYT-amp (typically 1 liter) and grown until the optical density (OD) of the culture was between 0.5 and 0.9 (approximately 2.5 hours).
  • IPTG isopropyl ⁇ -D-thiogalactopyranoside
  • Buffer A- 50mM Tris, pH 8.0, 50mM dextrose, ImM EDTA, 200uM phenylmethylsulfonylfluoride.
  • Buffer A+ Buffer A-, 4mg/ml lysozyme
  • Buffer B 0.5% Tween- 20, 0.5% NP40 (a.k.a.
  • Fusion proteins were assayed for size and quality by SDS gel electrophoresis (PAGE) as described in "Sambrook.” Fusion protein aliquots were stored at minus 80°C and at minus 20°C.
  • Certain PDZ domains are bound by the C-terminal residues of PDZ-binding proteins.
  • To identify PL proteins the C-terminal residues of sequences were visually inspected for sequences that one might predict would bind to PDZ-domain containing proteins (see, e.g., Doyle et al., 1996, Cell 85, 1067; Songyang et al, 1997, Science 275, 73), including the additional consenses for PLs identified at Arbor Vita Corporation (US Patent Application 60/ 360061). TABLE 2 lists some of these proteins, and provides corresponding C-terminal sequences.
  • Synthetic peptides of defined sequence can be synthesized by any standard resin-based method (see, e.g., U. S. Pat. No. 4,108,846; see also, Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215- 223; Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, et al., 1995, Science 269:202).
  • the peptides used in the assays described herein were prepared by the FMOC (see, e.g., Guy and Fields, 1997, Meth. En ⁇ .
  • peptides were labeled with biotin at the amino-terminus by reaction with a four-fold excess of biotin methyl ester in dimethylsulfoxide with a catalytic amount of base.
  • the peptides were cleaved from the resin using a halide containing acid (e.g. trifluoroacetic acid) in the presence of appropriate antioxidants (e.g. ethanedithiol) and excess solvent lyophilized.
  • peptides can be redissolved and purified by reverse phase high performance liquid chromatography (HPLC).
  • HPLC solvent system involves a Vydac C-18 semi-preparative column running at 5 mL per minute with increasing quantities of acetonitrile plus 0.1% trifluoroacetic acid in a base solvent of water plus 0.1% trifluoroacetic acid. After HPLC purification, the identities of the peptides are confirmed by MALDI cation-mode mass spectrometry.
  • a and G Two complementary assays, termed “A” and “G”, were developed to detect binding between a PDZ-domain polypeptide and candidate PDZ ligand.
  • binding is detected between a peptide having a sequence corresponding to the C- terminus of a HPV protein anticipated to bind to one or more PDZ domains (i.e. a candidate HPV PL peptide) and a PDZ-domain polypeptide (typically a fusion protein containing a PDZ domain).
  • the candidate PL peptide is immobilized and binding of a soluble PDZ-domain polypeptide to the immobilized peptide is detected (the "A”' assay is named for the fact that in one embodiment an avidin surface is used to immobilize the peptide).
  • the PDZ-domain polypeptide is immobilized and binding of a soluble PL peptide is detected (The “G” assay is named for the fact that in one embodiment a GST-binding surface is used to immobilize the PDZ-domain polypeptide). Preferred embodiments of these assays are described in detail infra.
  • the PDZ- containing proteins or PL polypeptides are immobilized on a solid surface.
  • the substrate to which the polypeptide is bound may in any of a variety of forms, e.g., a microtiter dish, a test tube, a dipstick, a microcentrifuge tube, a bead, a spinnable disk, a permeable or semi- permeable membrane, and the like.
  • Suitable materials include glass, plastic (e.g., polyethylene, PVC, polypropylene, polystyrene, and the like), protein, paper, carbohydrate, lipid monolayer or supported lipid bilayer, films and other solid supports.
  • plastic e.g., polyethylene, PVC, polypropylene, polystyrene, and the like
  • protein e.g., polyethylene, PVC, polypropylene, polystyrene, and the like
  • protein e.g., polyethylene, PVC, polypropylene, polystyrene, and the like
  • paper e.g., polyethylene, PVC, polypropylene, polystyrene, and the like
  • carbohydrate e.g., polyethylene, PVC, polypropylene, polystyrene, and the like
  • Other materials that may be employed include ceramics, metals, metalloids, semiconductive materials, cements and the like.
  • the PDZ and/or PL fusion proteins are organized as an array.
  • array refers to an ordered arrangement of immobilized fusion proteins, in which particular different fusion proteins (i.e., having different PDZ domains) are located at different predetermined sites on the substrate. Because the location of particular fusion proteins on the array is l ⁇ iown, binding at that location can be correlated with binding to the PDZ domain situated at that location. Immobilization of fusion proteins on beads (individually or in groups) is another particularly useful approach. In one embodiment, individual fusion proteins are immobilized on beads. In one embodiment, mixtures of distinguishable beads are used.
  • Distinguishable beads are beads that can be separated from each other on the basis of a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods). Binding to particular PDZ domain may be determined.
  • a property such as size, magnetic property, color (e.g., using FACS) or affinity tag (e.g., a bead coated with protein A can be separated from a bead not coated with protein A by using IgG affinity methods). Binding to particular PDZ domain may be determined.
  • Methods for immobilizing proteins are known, and include covalent and non-covalent methods.
  • One suitable immobilization method is antibody-mediated immobilization.
  • an antibody specific for the sequence of an "immobilization domain" of the PDZ-domain containing protein is itself immobilized on the substrate (e.g., by adsorption).
  • One advantage of this approach is that a single antibody may be adhered to the substrate and used for immobilization of a number of polypeptides (sharing the same immobilization domain).
  • an immobilization domain consisting of poly- histidine (Bush et al, 1991, J Biol Chem 266:13811-14) can be bound by an anti-histidine monoclonal antibody (R&D Systems, Minneapolis,.
  • an immobilization domain consisting of secreted alkaline phosphatase (“SEAP") (Berger et al, 1988, Gene 66:1-10) can be bound by anti-SEAP (Sigma Chemical Company, St. Louis, MO); an immobilization domain consisting of a FLAG epitope can be bound by anti-FLAG.
  • SEAP secreted alkaline phosphatase
  • anti-SEAP Sigma Chemical Company, St. Louis, MO
  • an immobilization domain consisting of a FLAG epitope can be bound by anti-FLAG.
  • Other ligand-antiligand immobilization methods are also suitable (e.g., an immobilization domain consisting of protein A sequences (Harlow and Lane, 1988, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory; Sigma Chemical Co., St.
  • the immobilization domain is a GST moiety, as described herein.
  • glass and plastic are especially useful substrates.
  • the substrates may be printed with a hydrophobic (e.g., Teflon) mask to form wells.
  • a hydrophobic (e.g., Teflon) mask to form wells.
  • Preprinted glass slides with 3, 10 and 21 wells per 14.5 cm 2 slide "working area" are available from, e.g., SPI Supplies, West Chester, PA; also see U.S. Pat. No. 4,011,350).
  • a large format (12.4 cm x 8.3 cm) glass slide is printed in a 96 well format is used; this format facilitates the use of automated liquid handling equipment and utilization of 96 well format plate readers of various types (fluorescent, colorimetric, scintillation).
  • higher densities may be used (e.g., more than 10 or 100 polypeptides per cm 2 ). See, e.g., MacBeath et al, 2000, Science 289:1760-63.
  • substrates e.g., glass substrates
  • Suitable adsorption conditions include incubation of 0.5- 50ug/ml (e.g., 10 ug/ml) mAb in buffer (e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4°C) to 37°C and from lhr to more than 24 horns.
  • buffer e.g., PBS, or 50 to 300 mM Tris, MOPS, HEPES, PIPES, acetate buffers, pHs 6.5 to 8, at 4°C
  • Proteins may be covalently bound or noncovalently attached through nonspecific bonding. If covalent bonding between the fusion protein and the surface is desired, the surface will usually be polyfunetional or be capable of being polyfunctionalized.
  • Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like.
  • the manner of linking a wide variety of compounds to various surfaces is well l ⁇ iown and is amply illustrated in the literature. i. "A Assay” Detection of PDZ-Ligand Binding Using Immobilized PL
  • the invention provides an assay in which biotinylated candidate PL peptides are immobilized on an avidin-coated surface. The binding of PDZ-domain fusion protein to this surface is then measured.
  • the PDZ-domain fusion protein is a GST/PDZ fusion protein and the assay is carried out as follows:
  • Avidin is bound to a surface, e.g. a protein binding surface.
  • avidin is bound to a polystyrene 96 well plate (e.g., Nunc Polysorb (cat
  • Biotinylated PL peptides are immobilized on the surface of wells of the plate by addition of 50 uL per well of 0.4 uM peptide in PBS/BSA for 30 minutes at 4°C.
  • each different peptide is added to at least eight different wells so that multiple measurements (e.g. duplicates and also measurements using different (GST/PDZ-domain fusion proteins and a GST alone negative control) can be made, and also additional negative control wells are prepared in which no peptide is immobilized.
  • the plate is washed 3 times with PBS.
  • GST/PDZ-domain fusion protein (prepared as described supra) is allowed to react with the surface by addition of 50 uL per well of a solution containing 5 ug/mL GST/PDZ-domain fusion protein in PBS/BSA for 2 hours at 4°C.
  • GST alone i.e. not a fusion protein
  • specified wells generally at least 2 wells (i.e. duplicate measurements) for each immobilized peptide. After the 2 hour reaction, the plate is washed 3 times with PBS to remove unbound fusion protein.
  • the binding of the GST/PDZ-domain fusion protein to the avidin-biotinylated peptide surface can be detected using a variety of methods, and detectors known in the art.
  • 50 uL per well of an anti-GST antibody in PBS/BSA e.g. 2.5 ug/mL of polyclonal goat-anti-GST antibody, Pierce
  • PBS/BSA polyclonal goat-anti-GST antibody, Pierce
  • HRP horseradish peroxidase
  • TMB horseradish peroxidase-conjugated polyclonal rabbit anti-goat immunoglobulin antibody
  • a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal.
  • a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors.
  • a statistical test e.g. a T-test
  • comparing repeated measurements of the signal with repeated measurements of the background will result in a p-value ⁇ 0.05, more typically a p-value ⁇ 0.01, and most typically a p-value ⁇ 0.001 or less.
  • the signal from binding of a GST/PDZ- domain fusion protein to an avidin surface not exposed to (i.e. not covered with) the P ( L peptide is one suitable negative control (sometimes referred to as "B").
  • the signal from binding of GST polypeptide alone (i.e. not a fusion protein) to an avidin-coated surface that has been exposed to (i.e. covered with) the PL peptide is a second suitable negative control (sometimes referred to as "B2"). Because all measurements are done in multiples (i.e.
  • the arithmetic mean (or, equivalently, average) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding.
  • the standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-l).
  • the invention provides an assay in which a GST PDZ fusion protein is immobilized on a surface ("G" assay).
  • G assay
  • the binding of labeled PL peptide (e.g., as listed in TABLE 2) to this surface is then measured.
  • the assay is carried out as follows:
  • a PDZ-domain polypeptide is bound to a surface, e.g. a protein binding surface.
  • a GST/PDZ fusion protein containing one or more PDZ domains is bound to a polystyrene 96-well plate.
  • the GST/PDZ fusion protein can be bound to the plate by any of a variety of standard methods known to one of skill in the art, although some care must be taken that the process of binding the fusion protein to the plate does not alter the ligand-binding properties of the PDZ domain.
  • the GST PDZ fusion protein is bound via an anti-GST antibody that is coated onto the 96-well plate. Adequate binding to the plate can be achieved when: a. 100 uL per well of 5 ug/mL goat anti-GST polyclonal antibody
  • PBS polystyrene 96-well plate
  • Nunc Polysorb a polystyrene 96-well plate
  • the plate is blocked by addition of 200 uL per well of PBS/BSA for 2 hours at 4°C.
  • the plate is washed 3 times with PBS.
  • 50 uL per well of 5 ug/mL GST/PDZ fusion protein) or, as a negative control, GST polypeptide alone (i.e. not a fusion protein) in PBS/BSA is added to the plate for 2 hours at 4°C.
  • the plate is again washed 3 times with PBS.
  • Biotinylated PL peptides are allowed to react with the surface by addition of 50 uL per well of 20 uM solution of the biotinylated peptide in PBS/BSA for 10 minutes at 4°C, followed by an additional 20 minute incubation at 25°C. The plate is washed 3 times with ice cold PBS.
  • Specific binding of a PL peptide and a PDZ domain polypeptide is determined by comparing the signal from the well(s) in which the PL peptide and PDZ domain polypeptide are combined, with the background signal(s).
  • the background signal is the signal found in the negative control(s).
  • a specific or selective reaction will be at least twice background signal, more typically more than 5 times background, and most typically 10 or more times the background signal.
  • a statistically significant reaction will involve multiple measurements of the reaction with the signal and the background differing by at least two standard errors, more typically four standard errors, and most typically six or more standard errors.
  • a statistical test e.g.
  • the signal from binding of a given PL peptide to immobilized (surface bound) GST polypeptide alone is one suitable negative control (sometimes referred to as "B 1 "). Because all measurement are done in multiples (i.e. at least duplicate) the arithmetic mean (or, equivalently, average.) of several measurements is used in determining the binding, and the standard error of the mean is used in determining the probable error in the measurement of the binding.
  • the standard error of the mean of N measurements equals the square root of the following: the sum of the squares of the difference between each measurement and the mean, divided by the product of (N) and (N-l).
  • specific binding of the PDZ protein to the platebound peptide is determined by comparing the mean signal ("mean S") and standard error of the signal ("SE") for a particular PL-PDZ combination with the mean Bl. iii. "G' assay” and "G” assay”
  • G' assay and "G” assay”
  • Two specific modifications of the specific conditions described supra for the "G assay” are particularly useful.
  • the modified assays use lesser quantities of labeled PL peptide and have slightly different biochemical requirements for detection of PDZ-ligand binding compared to the specific assay conditions described supra.
  • the assay conditions described in this section are referred to as the "G' assay” and the “G' ' assay,” with the specific conditions described in the preceding section on G assays being referred to as the “G° assay.”
  • the “G' assay” is identical to the “G° assay” except at step (2) the peptide concentration is 10 uM instead of 20 uM. This results in slightly lower sensitivity for detection of interactions with low affinity and/or rapid dissociation rate. Correspondingly, it slightly increases the certainty that detected interactions are of sufficient affinity and half-life to be of biological importance and useful therapeutic targets.
  • the "G' ' assay” is identical to the “G° assay” except that at step (2) the peptide concentration is 1 uM instead of 20 uM and the incubation is performed for 60 minutes at 25°C (rather than, e.g., 10 minutes at 4°C followed by 20 minutes at 25°C). This results in lower sensitivity for interactions of low affinity, rapid dissociation rate, and/or affinity that is less at 25°C than at 4°C. Interactions will have lower affinity at 25°C than at 4°C if (as we have found to be generally true for PDZ-ligand binding) the reaction entropy is negative (i.e. the entropy of the products is less than the entropy of the reactants).
  • the PDZ-PL binding signal may be similar in the "G” assay” and the “G° assay” for interactions of slow association and dissociation rate, as the PDZ-PL complex will accumulate during the longer incubation of the "G” assay.”
  • comparison of results of the "G” assay” and the “G° assay” can be used to estimate the relative entropies, enthalpies, and kinetics of different PDZ-PL interactions.
  • thermodynamics and kinetics of PDZ-PL interactions can be used in the design of efficient inliibitors of the interactions.
  • a small molecule inhibitor based on the chemical structure of a PL that dissociates slowly from a given PDZ domain may itself dissociate slowly and thus be of high affinity.
  • step (2) of the "G assay” can be used to provide insight into the kinetics and thermodynamics of the PDZ-ligand binding reaction and into design of inhibitors of the reaction.
  • step (2) of the "G assay” can be used to provide insight into the kinetics and thermodynamics of the PDZ-ligand binding reaction and into design of inhibitors of the reaction.
  • step (2) of the "G assay” can be used to provide insight into the kinetics and thermodynamics of the PDZ-ligand binding reaction and into design of inhibitors of the reaction.
  • iv. Assay Variations As discussed supra, it will be appreciated that many of the steps in the above- described assays can be varied, for example, various substrates can be used for binding the PL and PDZ-containing proteins; different types of PDZ containing fusion proteins can be used; different labels for detecting PDZ/PL interactions can be employed; and different ways of detection can be used.
  • the PDZ-PL detection assays can employ a variety of surfaces to bind
  • a surface can be an "assay plate" which is formed from a material (e.g. polystyrene) which optimizes adherence of either the PL protein or PDZ-containing protein thereto.
  • a material e.g. polystyrene
  • the individual wells of the assay plate will have a high surface area to volume ratio and therefore a suitable shape is a flat bottom well (where the proteins of the assays are adherent).
  • Other surfaces include, but are not limited to, polystyrene or glass beads, polystyrene or glass slides, papers, dipsticks, plastics, films and the like.
  • the assay plate can be a "microtiter" plate.
  • microtiter plate when used herein refers to a multiwell assay plate, e.g., having between about 30 to 200 individual wells, usually 96 wells. Alternatively, high-density arrays can be used. Often, the individual wells of the microtiter plate will hold a maximum volume of about 250 ul.
  • the assay plate is a 96 well polystyrene plate (such as that sold by Becton Dickinson Labware, Lincoln Park, N.J.), which allows for automation and high throughput screening.
  • polystyrene microtiter ELISA plates such as that sold by Nunc Maxisorp, Inter Med, Denmark. Often, about 50 ul to 300 ul, more preferably 100 ul to 200 ul, of an aqueous sample comprising buffers suspended therein will be added to each well of the assay plate.
  • the detectable labels of the invention can be any detectable compound or composition which is conjugated directly or indirectly with a molecule (such as described above).
  • the label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze a chemical alteration of a substrate compound or composition which is detectable.
  • the preferred label is an enzymatic one which catalyzes a color change of a non-radioactive color reagent.
  • the label is indirectly conjugated with the antibody.
  • One of skill is aware of various techniques for direct and indirect conjugation.
  • the antibody can be conjugated with biotin and any of the categories of labels mentioned above can be conjugated with avidin, or vice versa (see also "A” and “G” assay above).
  • Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect mamier. See, Ausubel, supra, for a review of techniques involving biotin-avidin conjugation and similar assays.
  • the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody).
  • an anti-hapten antibody e.g. anti-digoxin antibody
  • washing is meant exposing the solid phase to an aqueous solution (usually a buffer or cell culture media) in such a way that unbound material (e.g., non-adhering cells, non-adhering capture agent, unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody) is removed therefrom.
  • a detergent e.g., Triton X
  • the aqueous washing solution is decanted from the wells of the assay plate following washing. Conveniently, washing can be achieved using an automated washing device. Sometimes, several washing steps (e.g., between about 1 to 10 washing steps) can be required.
  • blocking buffer refers to an aqueous, pH buffered solution containing at least one blocking compound which is able to bind to exposed surfaces of the substrate which are not coated with a PL or PDZ- containing protein.
  • the blocking compound is normally a protein such as bovine serum albumin (BSA), gelatin, casein or milk powder and does not cross-react with any of the reagents in the assay.
  • BSA bovine serum albumin
  • the block buffer is generally provided at a pH between about 7 to 7.5 and suitable buffering agents include phosphate and TRIS.
  • enzyme-substrate combinations can also be utilized in detecting PDZ-PL interactions.
  • enzyme-substrate combinations include, for example: (i) Horseradish peroxidase (HRP or HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. orthophenylene diamine [OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]) (as described above).
  • HRP Horseradish peroxidase
  • HRPO horseradish peroxidase
  • a dye precursor e.g. orthophenylene diamine [OPD] or 3,3',5,5'-tetramethyl benzidine hydrochloride [TMB]
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • Beta-D-galactosidase (Beta D-Gal) with a chromogenic substrate (e.g. p- nitrophenyl- Beta-D-galactosidase) or fluorogenic substrate 4-methylumbelliferyl- Beta-D- galactosidase.
  • a chromogenic substrate e.g. p- nitrophenyl- Beta-D-galactosidase
  • fluorogenic substrate 4-methylumbelliferyl- Beta-D- galactosidase.
  • the "A” and “G” assays of the invention can be used to determine the "apparent affinity" of binding of a PDZ ligand peptide to a PDZ-domain polypeptide.
  • Apparent affinity is determined based on the concentration of one molecule required to saturate the binding of a second molecule (e.g., the binding of a ligand to a receptor).
  • Two particularly useful approaches for quantitation of apparent affinity of PDZ-ligand binding are provided infra. These methods can be used to compare the sensitivity and affinity of differing PL constructs. Understanding the sensitivity of the PDZ for pathogen PLs is essential because it helps in the design of a modulator with the appropriate specificity for the interaction, PL, or PDZ.
  • a GST/PDZ fusion protein, as well as GST alone as a negative control, are bound to a surface (e.g., a 96-well plate) and the surface blocked and washed as described supra for the "G" assay.
  • a surface e.g., a 96-well plate
  • the net binding signal is determined by subtracting the binding of the peptide to GST alone from the binding of the peptide to the GST/PDZ fusion protein.
  • the net binding signal is then plotted as a function of ligand concentration and the plot is fit (e.g. by using the Kaleidagraph software package curve fitting algorithm; Synergy Software) to the following equation, where "Signal [ i igand] " is the net binding signal at PL peptide concentration "[ligand]," "Kd” is the apparent affinity of the binding event, and "Saturation Binding” is a constant detennined by the curve fitting algorithm to optimize the fit to the experimental data:
  • a fixed concentration of a PDZ-domain polypeptide and increasing concentrations of a labeled PL peptide are mixed together in solution and allowed to react.
  • preferred peptide concentrations are 0.1 uM, 1 uM, 10 uM, 100 uM, 1 mM.
  • appropriate reaction times can range from 10 minutes to 2 days at temperatures ranging from 4°C to 37°C.
  • the identical reaction can also be carried out using a non-PDZ domain-containing protein as a control (e.g., if the PDZ-domain polypeptide is fusion protein, the fusion partner can be used).
  • (2) PDZ-ligand complexes can be separated from unbound labeled peptide using a variety of methods l ⁇ iown in the art.
  • the complexes can be separated using high performance size-exclusion chromatography (HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity 9:699), affinity chromatography(e.g. using glutathione Sepharose beads), and affinity absorption (e.g., by binding to an anti-GST-coated plate as described supra).
  • HPSEC high performance size-exclusion chromatography
  • affinity chromatography e.g. using glutathione Sepharose beads
  • affinity absorption e.g., by binding to an anti-GST-coated plate as described supra.
  • the PDZ-ligand complex is detected based on presence of the label on the peptide ligand using a variety of methods and detectors l ⁇ iown to one of skill in the art. For example, if the label is fluorescein and the separation is achieved using HPSEC, an in-line fluorescence detector can be used. The binding can also be detected as described supra for the G assay. (4) The PDZ-ligand binding signal is plotted as a function of ligand concentration and the plot is fit.
  • SignalrLigand] Saturation Binding x ([ligand] / ([ligand + Kd]) Measurement of the affinity of a labeled peptide ligand binding to a PDZ-domain polypeptide is useful because knowledge of the affinity (or apparent affinity) of this interaction allows rational design of inhibitors of the interaction with known potency. The potency of inhibitors in inhibition would be similar to (i.e. within one-order of magnitude of) the apparent affinity of the labeled peptide ligand binding to the PDZ-domain.
  • the invention provides a method of determining the apparent affinity of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different concentrations of the ligand, determining the amount of binding of the ligand to the immobilized polypeptide at each of the concentrations of ligand, and calculating the apparent affinity of the binding based on that data.
  • the polypeptide comprising the PDZ domain and a non-PDZ domain is a fusion protein.
  • the e.g., fusion protein is GST-PDZ fusion protein
  • other polypeptides can also be used (e.g., a fusion protein including a PDZ domain and any of a variety of epitope tags, biotinylation signals and the like) so long as the polypeptide can be immobilized In an orientation that does not abolish the ligand binding properties of the PDZ domain, e.g, by tethering the polypeptide to the surface via the non-PDZ domain via an anti-domain antibody and leaving the PDZ domain as the free end. It was discovered, for example, reacting a PDZ- GST fusion polypeptide directly to a plastic plate provided suboptimal results.
  • binding affinity itself can be determined using any suitable equation (e.g., as shown supra; also see Cantor and Schimmel (1980) BIOPHYSICAL CHEMISTRY WH Freeman & Co., San Francisco) or software.
  • the polypeptide is immobilized by binding the polypeptide to an immobilized ⁇ nmunoglobulin that binds the non-PDZ domain (e.g., an anti- GST antibody when a GST-PDZ fusion polypeptide is used).
  • the step of contacting the ligand and PDZ-domain polypeptide is carried out under the conditions provided supra in the description of the "G" assay. It will be appreciated that binding assays are conveniently carried out in multiwell plates (e.g., 24- well, 96-well plates, or 384 well plates).
  • the present method has considerable advantages over other methods for measuring binding affinities PDZ-PL affinities, which typically involve contacting varying concentrations of a GST-PDZ fusion protein to a ligand-coated surface.
  • some previously described methods for determining affinity e.g., using immobilized ligand and GST-PDZ protein in solution
  • an estimate of the relative strength of binding of different PDZ-PL pairs can be made based on the absolute magnitude of the signals observed in the "G assay.” This estimate will reflect several factors, including biologically relevant aspects of the interaction, including the affinity and the dissociation rate. For comparisons of different ligands binding to a given PDZ domain-containing protein, differences in absolute binding signal likely relate primarily to the affinity and/or dissociation rate of the interactions of interest.
  • Another method of increasing the specificity or sensitivity of a PDZ-PL interaction is through mutagenesis and selection of high affinity or high specificity variants.
  • Methods such as UV, chemical (e.g., EMS) or biological mutagenesis (e.g. Molecular shuffling or DNA polymerase mutagenesis) can be applied to create mutations in DNA encoding PDZ domains or PL domains. Proteins can then be made from variants and tested using a number of methods described herein (e.g., 'A' assay, 'G' assay or yeast two hybrid).
  • the present invention provides powerful methods for analysis of PDZ-ligand interactions, including high-throughput methods such as the "G" assay and affinity assays described supra.
  • the affinity is determined for a particular ligand and a plurality of PDZ proteins.
  • the plurality is at least 5, and often at least 25, or at least 40 different PDZ proteins.
  • the plurality of different PDZ proteins are from a particular tissue (e.g., reproductive system) or a particular class or type of cell, (e.g., a cervical cell, a muscular cell, an epithelial cell) and the like.
  • the plurality of different PDZ proteins represents a substantial fraction (e.g., typically a majority, more often at least 80%>) of all of the PDZ proteins l ⁇ iown to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in cervical cells.
  • the plurality is at least 50%, usually at least 80%>, at least 90% or all of the PDZ proteins disclosed herein as being expressed in cervical cells.
  • the binding of a ligand to the plurality of PDZ proteins is determined. Using this method, it is possible to identify a particular PDZ domain bound with particular specificity by the ligand.
  • the binding may be designated as "specific” if the affinity of the ligand to the particular PDZ domain is at least 2-fold that of the binding to other PDZ domains in the plurality (e.g., present in that cell type).
  • the binding is deemed "very specific” if the affinity is at least 10-fold higher than to any other PDZ in the plurality or, alternatively, at least 10-fold higher than to at least 90%, more often 95% of the other PDZs in a defined plurality.
  • the binding is deemed “exceedingly specific” if it is at least 100-fold higher.
  • a ligand could bind to 2 different PDZs with an affinity of 1 uM and to no other PDZs out of a set 40 with an affinity of less than 100 uM. This would constitute specific binding to those 2 PDZs.
  • Similar measures of specificity are used to describe binding of a PDZ to a plurality of PLs. It will be recognized that high specificity PDZ-PL interactions represent potentially more valuable targets for achieving a desired biological effect. The ability of an inhibitor or enhancer to act with high specificity is often desirable. In particular, the most specific PDZ- ligand interactions are also the therapeutic targets, allowing specific disruption of an interaction.
  • the invention provides a method of identifying a high specificity interaction between a particular PDZ domain and a ligand l ⁇ iown or suspected of binding at least one PDZ domain, by providing a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain; determining the affinity of the ligand for each of said polypeptides, and comparing the affinity of binding of the ligand to each of said polypeptides, wherein an interaction between the ligand and a particular PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the particular PDZ domain with at least 2-fold higher affinity than to immobilized polypeptides not comprising the particular PDZ domain.
  • the affinity of binding of a specific PDZ domain to a plurality of ligands is determined.
  • the invention provides a method of identifying a high specificity interaction between a PDZ domain and a particular ligand known or suspected of binding at least one PDZ domain, by providing an immobilized polypeptide comprising the PDZ domain and a non-PDZ domain; determining the affinity of each of a plurality of ligands for the polypeptide, and comparing the affinity of binding of each of the ligands to the polypeptide, wherein an interaction between a particular ligand and the PDZ domain is deemed to have high specificity when the ligand binds an immobilized polypeptide comprising the PDZ domain with at least 2-fold higher affinity than other ligands tested.
  • the binding may be designated as "specific” if the affinity of the PDZ to the particular PL is at least 2-fold that of the binding to other PLs in the plurality (e.g., present in that cell type).
  • the binding is deemed “very specific” if the affinity is at least 10-fold higher than to any other PL in the plurality or, alternatively, at least 10-fold higher than to at least 90%), more often 95% of the other PLs in a defined plurality.
  • the binding is deemed “exceedingly specific” if it is at least 100-fold higher.
  • the plurality is at least 5 different ligands, more often at least 10. VII. Assays for detecting oncogenic E6 proteins
  • Oncogenic E6 proteins can be detected by their ability to bind to PDZ domains. This could be a developed into a single detection stage approach or more favorably as a two-stage or 'sandwich' approach for increased sensitivity and specificity.
  • a 'tagged 5 version of a PDZ domain that specifically recognizes oncogenic E6 proteins can be used to directly probe for the presence of oncogenic E6 protein in a sample.
  • an example of this would be to attach the test sample to a solid support (for example, cervical cells or tissue could be coated on a slide and 'fixed' to permeablize the cell membranes), incubate the sample with a tagged 'PL detector' protein (a PDZ domain fusion) under appropriate conditions, wash away unbound PL detector, and assay for the presence of the 'tag' in the sample.
  • PDZ domains may also bind endogenous cellular proteins. Thus, frequency of binding must be compared to control cells that do not contain E6 oncoproteins or the 'PL detector' should be modified such that it is significantly more specific for the oncogenic E6 proteins (see section X).
  • the PL detector is coupled with a second method of either capturing or detecting captured proteins.
  • the second method could be using an antibody that binds to the E6 oncoprotein or a second compound or protein that can bind to E6 oncoproteins at a location on the E6 protein that does not reduce the availability of the E6 PL.
  • proteins may include, but not be limited to, p53, E6-AP, E6- BP or engineered compounds that bind E6 oncoproteins.
  • various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a peptide.
  • the peptide may be attached to a suitable carrier, such as BSA or KLH, by means of a side chain functional group or linkers attached to a side chain functional group.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum bacilli Calmette-Guerin
  • Monoclonal antibodies to a peptide may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et al. , 1983, Inununology Today 4:72; Cote et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030 and the EBV-hybridoma technique (Cole et al, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)).
  • Antibody fragments containing deletions of specific binding sites may be generated by l ⁇ iown techniques.
  • such fragments include but are not limited to F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al, 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the peptide of interest.
  • the antibody or antibody fragment specific for the desired peptide can be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify peptides of the invention. See, Scopes, 1984, Protein Purification: Principles and Practice, Springer- Verlag New York, Inc., NY, Livingstone, 1974, Methods Enzymology: Immunoaffinity Chromatography of Proteins 34:723-731.
  • Antibodies can also be linked to other solid supports for diagnostic applications, or alternatively labeled with a means of detection such an enzyme that can cleave a colorimetric substrate, a fluorophore, a magnetic particle, or other measurable compositions of matter.
  • E6 proteins have historically been difficult to produce.
  • An example is to prepare the E6 antigen (to raise antibodies against) in the same manner that one would prepare tissue or cell samples for testing.
  • Another method that could be employed is to use peptides corresponding to antigenic regions of the E6 proteins as predicted by Major Histocompatibility Complex (MHC) and T Cell Receptor (TCR) consensus binding.
  • MHC Major Histocompatibility Complex
  • TCR T Cell Receptor
  • detection of HPV strains in a sample can be used for the detection of HPV strains in a sample, facilitating the treatment of HPV infection.
  • Ongoing detection coupled with treatment programs can act as an effective prophylactic to prevent the development of diseases associated with HPV infection.
  • detection of a particular PL motif(as shown in Table 2) in a patient allows for the use of treatments specific for strains containing that PL motif.
  • Certain antagonists may disrupt interactions of one PL more effectively than another different PL motif.
  • Treatments can be designed to target a certain HPV strain with a maximum specificity by using an antagonist that disrupts an interaction of a particular HPV PL with the highest possible efficiency.
  • antibodies specific for the HPV C-terminal PL motif may be used for both detection and treatment of HPV infection.
  • Antibodies against the PL of a HPV strain can not only detect the presence of a particular HPV strain in a sample, they can effectively block the PDZ binding motif of a HPV protein in vivo, preventing interaction with intracellular PDZ proteins and thus blocking the development or progression of HPV-associated diseases.
  • antibodies that block the binding pocket of a particular PDZ protein also prevent interactions between that PDZ protein and a PL protein.
  • Antibodies can also be used to deliver peptide mimetics or small molecules to a specific cell type. Methods for generating human antibodies are well known in the art. VIII. Use of Array for Global Predictions
  • One discovery of the present inventors relates to the important and extensive roles played by interactions between PDZ proteins and PL proteins, particularly in the biological function of cervical cells and other cells involved in the reproductive system. Further, it has been discovered that valuable information can be ascertained by analysis (e.g., simultaneous analysis) of a large number of PDZ-PL interactions.
  • the analysis encompasses all of the PDZ proteins expressed in a particular tissue (e.g., reproductive tissue) or type or class of cell (e.g., cervical cell, muscle cell, epithelial cell and the like).
  • the analysis encompasses at least about 5, or at least about 10, or at least about 12, or at least about 15 and often at least 50 different polypeptides, up to about 60, about 80, about 100, about 150, about 200, or even more different polypeptides; or a substantial fraction (e.g., typically a majority, more often at least 80%) of all of the PDZ proteins l ⁇ iown to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in cervical cells.
  • the arrays and methods of the invention are directed to the analysis of PDZ and PL interactions, and involve selection of such proteins for analysis.
  • the devices and methods of the invention may include or involve a small number of control polypeptides, they typically do not include significant numbers of proteins or fusion proteins that do not include either PDZ or PL domains (e.g., typically, at least about 90%o of the arrayed or immobilized polypeptides in a method or device of the invention is a PDZ or PL sequence protein, more often at least about 95%, or at least about 99%).
  • simultaneous analysis facilitates, for example, the direct comparison of the effect of an agent (e.g., an potential interaction inhibitor) on the interactions between a substantial portion of PDZs and/or PLs in a tissue or cell.
  • an agent e.g., an potential interaction inhibitor
  • the invention provides an array of immobilized polypeptide comprising the PDZ domain and a non-PDZ domain on a surface.
  • the arcay comprises at least about 5, or at least about 10, or at least about 12, or at least about 15 and often at least 50 different polypeptides.
  • the different PDZ proteins are from a particular tissue (e.g., reproductive tissue) or a particular class or type of cell, (e.g., a cervical cell, muscle cell, or epithelial cell) and the like.
  • the plurality of different PDZ proteins represents a substantial fraction (e.g., typically a majority, more often at least 60%, 70% or 80%) of all of the PDZ proteins known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZ proteins known to be present in cervical cells.
  • arrays that include a plurality, usually at least 5, 10, 25, 50 PDZ proteins present in a particular cell of interest.
  • array refers to an ordered series of immobilized polypeptides in which the identity of each polypeptide is associated with its location.
  • the plurality of polypeptides are arrayed in a "common" area such that they can be simultaneously exposed to a solution (e.g., containing a ligand or test agent).
  • the plurality of polypeptides can be on a slide, plate or similar surface, which may be plastic, glass, metal, silica, beads or other surface to which proteins can be immobilized.
  • the different immobilized polypeptides are situated in separate areas, such as different wells of multi-well plate (e.g., a 24-well plate, a 96-well plate, a 384 well plate, and the like). It will be recognized that a similar advantage can be obtained by using multiple arrays in tandem. IX.
  • multi-well plate e.g., a 24-well plate, a 96-well plate, a 384 well plate, and the like.
  • screening can be carried out by contacting the library members with a PDZ-domain polypeptide immobilized on a solid support (e.g. as described supra in the "G" assay) and harvesting those library members that bind to the protein.
  • panning techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al, 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited hereinabove.
  • the two-hybrid system for selecting interacting proteins in yeast can be used to identify molecules that specifically bind to a PDZ domain-containing protein. Furthermore, the identified molecules are further tested for their ability to inhibit transmembrane receptor interactions with a PDZ domain.
  • antagonists of an interaction between a PDZ protein and a PL protein are identified.
  • a modification of the "A” assay described supra is used to identify antagonists.
  • a modification of the "G” assay described supra is used to identify antagonists.
  • screening assays are used to detect molecules that specifically bind to PDZ domains.
  • Such molecules are useful as agonists or antagonists of PDZ-protein- mediated cell function (e.g., cell activation, e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeleton rearrangement, cell movement, chemotaxis, and the like).
  • cell activation e.g., T cell activation, vesicle transport, cytokine release, growth factors, transcriptional changes, cytoskeleton rearrangement, cell movement, chemotaxis, and the like.
  • such assays are performed to screen for leukocyte activation inhibitors for drug development.
  • the invention thus provides assays to detect molecules that specifically bind to PDZ domain-containing proteins.
  • recombinant cells expressing PDZ domain-encoding nucleic acids can be used to produce PDZ domains in these assays and to screen for molecules that bind to the domains.
  • Molecules are contacted with the PDZ domain (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to such domains are identified. Methods that can be used to carry out the foregoing are commonly known in the art.
  • a biological test is used to identify agonists or antagonists of PDZ:PL binding. Examples of this are give in Figures 4, 5, and 6 and their conesponding examples. These assays are demonstrated to be effected by modulation of PDZ:PL interactions.
  • biological assays such as those included herein can be used to examine the biological effect of modulators identified through biochemical assays or other assays described in this disclosure.
  • antagonists are identified by conducting the A or G assays in the presence and absence of a l ⁇ iown or candidate antagonist. When decreased binding is observed in the presence of a compound, that compound is identified as an antagonist. Increased binding in the presence of a compound signifies that the compound is an agonist.
  • a test compound in one assay, can be identified as an inhibitor (antagonist) of binding between a PDZ protein and a PL protein by contacting a PDZ domain polypeptide and a PL peptide in the presence and absence of the test compound, under conditions in which they would (but for the presence of the test compound) form a complex, and detecting the formation of the complex in the presence and absence of the test compound. It will be appreciated that less complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an inhibitor of a PDZ protein -PL protein binding.
  • the "G” assay is used in the presence or absence of a candidate inhibitor. In one embodiment, the "A” assay is used in the presence or absence of a candidate inhibitor.
  • one or more PDZ domain-containing GST-fusion proteins are bound to the surface of wells of a 96-well plate as described supra (with appropriate controls including nonfusion GST protein). All fusion proteins are bound in multiple wells so that appropriate controls and statistical analysis can be done.
  • a test compound in BSA PBS (typically at multiple different concentrations) is added to wells.
  • a detectably labeled (e.g., biotinylated) peptide known to bind to the relevant PDZ domain is added in each of the wells at a final concentration of, e.g., between about 2 uM and about 40 uM, typically 5 uM, 15 uM, or 25 uM.
  • This mixture is then allowed to react with the PDZ fusion protein bound to the surface for 10 minutes at 4°C followed by 20 minutes at 25°C. The surface is washed free of unbound peptide three times with ice cold PBS and the amount of binding of the peptide in the presence and absence of the test compound is determined.
  • the level of binding is measured for each set of replica wells (e.g. duplicates) by subtracting the mean GST alone background from the mean of the raw measurement of peptide binding in these wells.
  • the A assay is carried out in the presence or absence of a test candidate to identify inhibitors of PL-PDZ interactions.
  • a test compound is detennined to be a specific inhibitor of the binding of the PDZ domain (P) and a PL (L) sequence when, at a test compound concentration of less than or equal to 1 mM (e.g., less than or equal to: 500 uM, 100 uM, 10 uM, 1 uM, 100 nM or 1 nM) the binding of P to L in the presence of the test compound less than about 50% of the binding in the absence of the test compound, (in various embodiments, less than about 25%, less than about 10%, or less than about 1%).
  • the net signal of binding of P to L in the presence of the test compound plus six (6) times the standard error of the signal in the presence of the test compound is less than the binding signal in the absence of the test compound.
  • assays for an inliibitor are carried out using a single PDZ protein- PL protein pair (e.g., a PDZ domain fusion protein and a PL peptide).
  • the assays are carried out using a plurality of pairs, such as a plurality of different pairs listed in TABLE 3.
  • These antagonists can be identified by carrying out a series of assays using a candidate inhibitor and different PL-PDZ pairs (e.g., as shown in the matrix of TABLE 3) and comparing the results of the assays. All such pairwise combinations are contemplated by the invention (e.g., test compound inhibits binding of PL] to PDZi to a greater degree than it inhibits binding of PL] to PDZ or PL 2 to PDZ 2 ).
  • test compound inhibits binding of PL] to PDZi to a greater degree than it inhibits binding of PL] to PDZ or PL 2 to PDZ 2 ).
  • the Ki (“potency") of an inhibitor of a PDZ- PL interaction can be determined.
  • Ki is a measure of the concentration of an inhibitor required to have a biological effect.
  • administration of an inhibitor of a PDZ-PL interaction in an amount sufficient to result in an intracellular inhibitor concentration of at least between about 1 and about 100 Ki is expected to inhibit the biological response mediated by the target PDZ-PL interaction.
  • the Kd measurement of PDZ-PL binding as determined using the methods supra is used in determining Ki.
  • the invention provides a method of determining the potency (Ki) of an inhibitor or suspected inhibitor of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and inhibitor, wherein the different mixtures comprise a fixed amount of ligand and different concentrations of the inhibitor, determining the amount of ligand bound at the different concentrations of inhibitor, and calculating the Ki of the binding based on the amount of ligand bound in the presence of different concentrations of the inhibitor.
  • the polypeptide is immobilized by binding the polypeptide to an immobilized immunoglobulin that binds the non-PDZ domain.
  • This method which is based on the "G” assay described supra, is particularly suited for high-throughput analysis of the Ki for inliibitors of PDZ-ligand interactions. Further, using this method, the inhibition of the PDZ- ligand interaction itself is measured, without distortion of measurements by avidity effects. Typically, at least a portion of the ligand is detectably labeled to permit easy quantitation of ligand binding.
  • the concentration of ligand and concentrations of inhibitor are selected to allow meaningful detection of inhibition.
  • the concentration of the ligand whose binding is to be blocked is close to or less than its binding affinity (e.g., preferably less than the 5x Kd of the interaction, more preferably less than 2x Kd, most preferably less than lx Kd).
  • the ligand is typically present at a concentration of less than 2 Kd (e.g., " between about 0.01 Kd and about 2 Kd) and the concentrations of the test inhibitor typically range from 1 nM to 100 uM (e.g. a 4-fold dilution series with highest concentration 10 uM or 1 mM).
  • the Kd is determined using the assay disclosed supra.
  • the Ki of the binding can be calculated by any of a variety of methods routinely used in the art, based on the amount of ligand bound in the presence of different concentrations of the inhibitor.
  • [I] is expressed as a molar concentration.
  • an enhancer (sometimes referred to as, augmentor or agonist) of binding between a PDZ domain and a ligand is identified by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with the ligand in the presence of a test agent and determining the amount of ligand bound, and comparing the amount of ligand bound in the presence of the test agent with the amount of ligand bound by the polypeptide in the absence of the test agent.
  • At least two-fold (often at least 5-fold) greater binding in the presence of the test agent compared to the absence of the test agent indicates that the test agent is an agent that enhances the binding of the PDZ domain to the ligand.
  • agents that enhance PDZ-ligand interactions are useful for disruption (dysregulation) of biological events requiring normal PDZ-ligand function (e.g., cancer cell division and metastasis).
  • the invention also provides methods for determining the "potency" or "K en ha nc er" of an enhancer of a PDZ- ligand interaction.
  • the Ken hance r of an enhancer of a PDZ-PL interaction can be detennined, e.g., using the Kd of PDZ-PL binding as determined using the methods described supra.
  • K en hancer is a measure of the concentration of an enhancer expected to have a biological effect.
  • administration of an enhancer of a PDZ-PL interaction in an amount sufficient to result in an intracellular inhibitor concentration of at least between about 0.1 and about 100 K enhancer (e.g., between about 0.5 and about 50 Ken ancer) is expected to disrupt the biological response mediated by the target PDZ-PL interaction.
  • the invention provides a method of determining the potency ( enhancer) of an enhancer or suspected enhancer of binding between a PDZ domain and a ligand by immobilizing a polypeptide comprising the PDZ domain and a non-PDZ domain on a surface, contacting the immobilized polypeptide with a plurality of different mixtures of the ligand and enhancer, wherein the different mixtures comprise a fixed amount of ligand, at least a portion of which is detectably labeled, and different concentrations of the enhancer, determining the amount of ligand bound at the different concentrations of enhancer, and calculating the potency (K ⁇ n hancer) of the enhancer from the binding based on the amount of ligand bound in the presence of different concentrations of the enhancer.
  • At least a portion of the ligand is detectably labeled to permit easy quantitation of ligand binding.
  • This method which is based on the "G” assay described supra, is particularly suited for high- throughput analysis of the Kenhancer for enhancers of PDZ-ligand interactions.
  • the concentration of ligand and concentrations of enhancer are selected to allow meaningful detection of enhanced binding.
  • the ligand is typically present at a concentration of between about 0.01 Kd and about 0.5 Kd and the concentrations of the test agent/enhancer typically range from 1 nM to 1 mM (e.g. a 4-fold dilution series with highest concentration 10 uM or 1 mM).
  • the Kd is determined using the assay disclosed supra.
  • the potency of the binding can be determined by a variety of standard methods based on the amount of ligand bound in the presence of different concentrations of the enhancer or augmentor. For example, a plot of labeled ligand binding versus enhancer concentration can be fit to the equation:
  • the invention provides a method for determining if a test compound inhibits any PDZ-ligand interaction in large set of PDZ-ligand interactions (e.g., a plurality of the PDZ-ligands interactions described in US PATENT application 09/724553; a majority of the PDZ-ligands identified in a particular cell or tissue as described supra (e.g., cervical tissue) and the like.
  • the PDZ domains of interest are expressed as GST- PDZ fusion proteins and immobilized as described herein. For each PDZ domain, a labeled ligand that binds to the domain with a known affinity is identified as described herein.
  • any known or suspected modulator (e.g., inhibitor) of a PDZ-PL interaction(s) it is useful to know which interactions are inhibited (or augmented). This information could be used to develop a highly specific treatment for a pathogen (e.g., an oncogenic HPV strain).
  • the profile of PDZ interactions inhibited by a particular agent is refened to as the "inl ⁇ ibition profile” for the agent, and is described in detail below.
  • the profile of PDZ interactions enhanced by a particular agent is referred to as the "enhancement profile" for the agent. It will be readily apparent to one of skill guided by the description of the inhibition profile how to determine the enhancement profile for an agent.
  • the present invention provides methods for determining the PDZ interaction (inhibition/enhancement) profile of an agent in a single assay.
  • the invention provides a method for determining the PDZ-PL inhibition profile of a compound by providing (i) a plurality of different immobilized polypeptides, each of said polypeptides comprising a PDZ domain and a non-PDZ domain and (ii) a plurality of corresponding ligands, wherein each ligand binds at least one PDZ domain in (i), then contacting each of said immobilized polypeptides in (i) with a corresponding ligand in (ii) in the presence and absence of a test compound, and determining for each polypeptide-ligand pair whether the test compound inhibits binding between the immobilized polypeptide and the conesponding ligand.
  • the plurality is at least 5, and often at least 25, or at least 40 different PDZ proteins.
  • the plurality of different ligands and the plurality of different PDZ proteins are from the same tissue or a particular class or type of cell, e.g., a cervical cell, an endothelial cell and the like.
  • the plurality of different PDZs represents a substantial fraction (e.g., at least 80%) of all of the PDZs known to be, or suspected of being, expressed in the tissue or cell(s), e.g., all of the PDZs known to be present in cervical cells (for example, at least 80%, at least 90% or all of the PDZs disclosed herein as being expressed in cervical cells).
  • the inhibition profile is determined as follows: A plurality (e.g., all known) PDZ domains expressed in a cell (e.g., cervical cells) are expressed as GST-fusion proteins and immobilized without altering their ligand binding properties as described supra. For each PDZ domain, a labeled ligand that binds to this domain with a l ⁇ iown affinity is identified. If the set of PDZ domains expressed in cervical cells is denoted by ⁇ PI ...Pn) , any given PDZ domain Pi binds a (labeled) ligand Li with affinity K i.
  • the "G” assay can be performed as follows in 96-well plates with rows A-H and columns 1-12. Column 1 is coated with PI and washed. The corresponding ligand LI is added to each washed coated well of column 1 at a concentration 0.5 K d l with (rows B, D, F, H) or without (rows A, C, E, F) between about 1 and about 1000 uM) of test compound X. Column 2 is coated with P2, and L2 (at a concentration 0.5 Kd2) is added with or without inhibitor X. Additional PDZ domains and ligands are similarly tested.
  • Compound X is considered to inhibit the binding of Li to Pi if the average signal in the wells of column i containing X is less than half the signal in the equivalent wells of the column lacking X. Thus, in this single assay one determines the full set of cervical cell PDZs that are inhibited by compound X.
  • the test compound X is a mixture of compounds, such as the product of a combinatorial chemistry synthesis as described supra.
  • the test compound is l ⁇ iown to have a desired biological effect, and the assay is used to determine the mechanism of action (i.e., if the biological effect is due to modulating a PDZ- PL interaction).
  • an agent that modulates only one, or a few PDZ-PL interactions, in a panel is a more specific modulator than an agent that modulate many or most interactions.
  • an agent that modulates less than 20%> of PDZ domains in a panel is deemed a "specific" inhibitor, less than 6% a "very specific” inhibitor, and a single PDZ domain a "maximally specific” inhibitor.
  • compound X may be a composition containing mixture of compounds (e.g., generated using combmatorial chemistry methods) rather than a single compound.
  • the assay above is performed using varying concentrations of the test compound X, rather than fixed concentration. This allows determination of the Ki of the X for each PDZ as described above. Examples of this is shown in Figure 8 for small molecules, and in Figure 3 for peptide inhibition.
  • a mixture of different labeled ligands is created that such that for every PDZ at least one of the ligands in the mixture binds to this PDZ sufficiently to detect the binding in the "G" assa ⁇ . This mixture is then used for every PDZ domain.
  • compound X is known to have a desired biological effect, but the chemical mechanism by which it has that effect is unknown.
  • the assays of the invention can then be used to determine if compound X has its effect by binding to a PDZ domain.
  • PDZ-domain containing proteins are classified in to groups based on their biological function, e.g. into those that regulate chemotaxis versus those that regulate transcription.
  • An optimal inhibitor of a particular function e.g., including but not limited to an anti-chemotactic agent, an anti-T cell activation agent, cell-cycle control, vesicle transport, apoptosis, etc.
  • the assay is used in one embodiment in screening and design of a drug that specifically blocks a particular function.
  • an agent designed to block chemotaxis might be identified because, at a given concentration, the agent inhibits 2 or more PDZs involved in chemotaxis but fewer than 3 other PDZs, or that inhibits PDZs involved in chemotaxis with a Ki > 10-fold better than for other PDZs.
  • the invention provides a method for identifying an agent that inhibits a first selected PDZ-PL interaction or plurality of interactions but does not inhibit a second selected PDZ-PL interaction or plurality of interactions.
  • the two (or more) sets of interactions can be selected on the basis of the l ⁇ iown biological function of the PDZ proteins, the tissue specificity of the PDZ proteins, or any other criteria.
  • the assay can be used to determine effective. doses (i.e., drug concentrations) that result in desired biological effects while avoiding undesirable effects.
  • interactions between PDZ proteins and PL proteins in cells may be disrupted or inliibited by the presence of pathogens.
  • Pathogens can be identified using screening assays described herein. Agonists and antagonists of PDZ- Pathogen PL interactions or PDZ-Cellular PL interactions can be useful in discerning or confirming specific interactions.
  • an agonist will increase the sensitivity of a PDZ-pathogen PL interaction.
  • an antagonist of a PDZ- pathogen PL interaction can be used to verify the specificity of an interaction.
  • the motifs disclosed herein are used to design modulators.
  • the antagonists of the invention have a structure (e.g., peptide sequence) based on the C-terminal residues of PL-domain proteins listed in TABLE 2. In some embodiments, the antagonists of the invention have a structure (e.g., peptide sequence) based on a PL motif disclosed herein or in US PATENT application 09/724553.
  • the PDZ/PL antagonists and antagonists of the invention may be any of a large variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • polymers e.g., oligopeptides, polypeptides, oligonucleotides, and polynucleotides
  • small molecules antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptide mimetics, nucleic acid analogs, and the like), and numerous other compounds.
  • PDZ-PL interaction agonists can also be use
  • the peptides and peptide mimetics or analogues of the invention contain an amino acid sequence that binds a PDZ domain in a cell of interest.
  • the antagonists comprise a peptide that has a sequence corresponding to the carboxy-terminal sequence of a PL protein listed in TABLES 2 or 3, e.g., a peptide listed TABLES 2 or 3.
  • the peptide comprises at least the C-terminal two (3), three (3) or four (4) residues of the PL protein, and often the inhibitory peptide comprises more than three residues (e.g., at least four, five, six, seven, eight, nine, ten, twelve or fifteen residues) from the PL protein C- terminus.
  • the inhibitor is a peptide, e.g., having a sequence of a PL C- terminal protein sequence. An example of this is shown in Figure 3.
  • the antagonist is a fusion protein comprising such a sequence. Fusion proteins containing a transmembrane transporter amino acid sequence are particularly useful.
  • the inhibitor is conserved variant of the PL C-terminal protein sequence having inhibitory activity.
  • the antagonist is a peptide mimetic of a PL C-terminal sequence.
  • the inhibitor is a small molecule (i.e., having a molecular weight less than 1 kD).
  • the antagonists comprise a peptide that has a sequence of a PL protein carboxy-terminus listed in TABLE 2.
  • the peptide comprises at least the C-terminal two (2) residues of the PL protein, and typically, the inhibitory peptide comprises more than two residues (e.g, at least three, four, five, six, seven, eight, nine, ten, twelve or fifteen residues) from the PL protein C-terminus.
  • the peptide may be any of a variety of lengths (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, or at least 20 residues) and may contain additional residues not from the PL protein.
  • the residues shared by the inhibitory peptide with the PL protein are found at the C-terminus of the peptide.
  • the sequence is internal.
  • the inhibitory peptide comprises residues from a PL sequence that is near, but not at the c-terminus of a PL protein (see, Gee et al., 1998, J Biological Chem. 273:21980-87).
  • the PL protein carboxy-terminus sequence is referred to as the "core PDZ motif sequence” referring to the ability of the short sequence to interact with the PDZ domain.
  • the "core PDZ motif sequence” contains the last four C-terminus amino acids.
  • the four amino acid core of a PDZ motif sequence may contain additional amino acids at its amino terminus to further increase its binding affinity and/or stability.
  • the PDZ motif sequence peptide can be from four amino acids up to 15 amino acids. It is preferred that the length of the sequence to be 6-10 amino acids. More preferably, the PDZ motif sequence contains 8 amino acids. Additional amino acids at the amino terminal end of the core sequence may be derived from the natural sequence in each HPV protein or a synthetic linker. The additional amino acids may also be conservatively substituted. When the third residue from the C-terminus is S, T or Y, this residue may be phosphorylated prior to the use of the peptide. •
  • the peptide and nonpeptide inhibitors of the are small, e.g., fewer than ten amino acid residues in length if a peptide.
  • a limited number of ligand amino acids directly contact the PDZ domain (generally less than eight) (Kozlov et al., 2000, Biochemistry 39, 2572; Doyle et al., 1996, Cell 85, 1067) and that peptides as short as the C-terminal three amino acids often retain similar binding properties to longer (> 15) amino acids peptides (Yanagisawa et al., 1997, J. Biol. Chem. 272, 8539).
  • the variants have the same or a different ability to bind a PDZ domain as the parent peptide.
  • amino acid substitutions are conservative, i.e., the amino acid residues are replaced with other amino acid residues having physical and/or chemical properties similar to the residues they are replacing.
  • conservative amino acid substitutions are those wherein an amino acid is replaced with another amino acid encompassed within the same designated class, as shown in Table 1.
  • peptide mimetics can be prepared using routine methods, and the inhibitory activity of the mimetics can be confirmed using the assays of the invention.
  • the agonist or antagonist is a peptide mimetic of a PL C-terminal sequence.
  • individual synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY.
  • Polypeptides incorporating mimetics can also be made using solid phase synthetic procedures, as described, e.g., by Di Marchi, et al., U.S. Pat. No. 5,422,426.
  • Mimetics of the invention can also be synthesized using combinatorial methodologies.
  • Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (19.97)ivlol. Divers.
  • the agonist or antagonist is a small molecule (i.e., having a molecular weight less than 5 kD or 2 kD). Methods for screening small molecules are well known in the art and include those described supra. Small molecules agonists or antagonists can be identified using any of the biochemical PDZ:PL interaction assays disclosed herein. Following identification of small molecule antagonists/agonists, the effects of these compounds can be tested in the biological assays provided herein. An example of the identification of small molecule antagonists of binding between an oncogenic E6 protein and a PDZ protein is shown in Figure 8.
  • the small molecules may be isolated peptide molecules, particularly peptides of no more that 5 amino acids in length and containing two, three or four amino acids corresponding to the amino acids at the C-terminus of an oncogenic E6 protein, may contain certain chemical moieties covalently bonded to the N- and/or C-terminus of the peptide.
  • any subject polypeptide may be modified at the C-terminus, the N-terminus, or both the C- or N- terminus. In cases whfere both the C- and N-termini of a peptide are modified, any of the three C-terminal moieties may be combined with any of the 15 N-terminal moieties.
  • the structures of four different peptides having at least two contiguous amino acids from the C-terminus of an oncogenic E6 protein are shown below.
  • the peptides are named “EV peptide”, “QL peptide”, “TEV peptide” and “TQL peptide”, corresponding to the E6 proteins of HPV strains 16 and 18, and others.
  • the R 2 groups of any of these peptides may be carboxyl, hydroxyl or tetrazole moieties.
  • the R ! groups of any of these peptides may be may be any of the moieties shown in Fig. 11, panels A-O. For example, as shown in Fig.
  • RI may be a substituted N-Phenyl-benzene-1,2- diamine (panel A), a substituted 2,3,4,9-Tetrahydro-lH-b-carboline group (panel B), a substituted 6-Methoxy-2,3,4,9-tetrahydro-lH-b-carboline group (panel C), a Benzo[b]thiophene group (panel D), a linked naphthalene group (panel E), a substituted Naphthalen-2-ol group (panel F), a Naphthalene group (panel G), a Quinoxaline group (panel H), a substituted 2-Phenyl-furan group (panel I), a IH-Indole group (panel J) a substituted 2- methyl-lH-pyrrol-3-yl)-methanol group (panel K) a substituted (2-Methyl-furan-3-yl)- methanol or (2-Methyl-thiophen-3-yl)
  • JNK cJUN N-terminal Kinase
  • PDZ domain-containing proteins are involved in a number of biological functions, including, but not limited to, vesicular trafficking, tumor suppression, protein sorting, establishment of membrane polarity, apoptosis, regulation of immune response and organization of synapse formation, hi general, this family of proteins has a common function of facilitating the assembly of multi-protein complexes, often serving as a bridge between several proteins, or regulating the function of other proteins. Additionally, as also noted supra, these proteins are found in essentially all cell types. Consequently, inappropriate PDZ-PL interactions or abnormal interactions can be targeted for the treatment of a wide variety of biological and physiological conditions. In particular, PL proteins from pathogenic organisms can be targeted using PDZ domains as therapeutics. Examples are given below. A. Chemical Synthesis
  • the peptides of the invention or analogues thereof may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues.
  • the peptides may be prepared in linear form using conventional solution or solid phase peptide syntheses and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures And Molecular Principles, W.H. Freeman and Co., N.Y.). Suitable procedures for synthesizing the peptides described herein are well known in the art.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).
  • analogues and derivatives of the peptides can be chemically synthesized.
  • the linkage between each amino acid of the peptides of the invention may be an amide, a substituted amide or an isostere of amide.
  • Nonclassical amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the sequence.
  • Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, ⁇ - amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N -methyl amino acids, and amino acid analogues in general.
  • the amino acid can be D (dextrorotary) or L (levo
  • the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques.
  • a polynucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, i. e. , a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • the expression vehicle is then transfected into a suitable target cell that will express the peptide.
  • the expressed peptide is then isolated by procedures well-established in the art.
  • host-expression vector systems may be utilized to express the peptides described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g. , Ti plasmid) containing an appropriate coding sequence; or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with re
  • increasing the number of copies of a PL therapeutic may be used to increase the specificity or sensitivity of treatment.
  • An example of this is presented in EXAMPLES 5.
  • the TIP-TIP -IgG vector produces a fusion protein that has duplicated copies of the PDZ domain from TIP-1 and the protein itself should dimerize on the basis of the IgG constant region backbone. Hence, a single protein contains 2-4 copies of the TIP-1 PDZ domain.
  • addition tandem repeats of PL capturdetectors could be fashioned.
  • different PDZ domains from different proteins could be engineered to express as a single protein (e.g., the PDZ domains of TIP-1 and MAGI-1 could be engineered to detect or block oncogenic HPV E6 proteins).
  • a different lg backbone could be used to increase the avidity of a construct. For example, the IgG constant regions will dimerize with itself, but the IgM constant regions will form a complex often monomers.
  • the expression elements of the expression systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used;
  • promoters such as the baculovirus polyhedron promoter may be used;
  • promoters derived from the genome of plant cells e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein
  • plant viruses e.g.
  • the 35S RNA promoter of CaMV; the coat protein promoter of TMV may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of expression product, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter
  • SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.
  • sequences encoding the peptides of the invention may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. , 1984, Nature 310:511 -514), or the coat protein promoter of TMV (Takamatsu et al. , 1987, EMBO J. 3:1311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al, 1984, EMBO J.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes.
  • the virus grows in Spodopterafrugiperda cells.
  • a coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene).
  • recombinant viruses are then used to infect Spodopterafrugiperda cells in which the inserted gene is expressed, (e.g., see Smith et al, 1983, J. Virol. 46:584; Smith, U.S. Patent No. 4,215,051). Further examples of this expression system may be found in Cunent Protocols in Molecular Biology, Vol. 2, Ausubel etal, eds., Greene Publish. Assoc. & Wiley Interscience. In mammalian host cells, a number of viral based expression systems may be utilized.
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. , region El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81 :3655-3659).
  • the vaccinia 7.5 K promoter may be used, (see, e.g. , Mackett et al, 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett et al, 1984, J. Virol. 49:857-864; Panicali et al, 1982, Proc. Natl. Acad. Sci. USA 79:4927-4931).
  • Other expression systems for producing linear peptides of the invention will be apparent to those having skill in the art.
  • Hemagglutinin epitopes Hemagglutinin epitopes, myc epitopes, etc.
  • chemical tags include, but are not limited to, biotin, gold, paramagnetic particles or fluorophores. These examples can be used to deliver therapeutic agents to specific tissues or cells or can be used by those skilled in the art to purify proteins or compounds from complex mixtures. D. Purification of the Peptides and Peptide Analogues
  • the peptides and peptide analogues of the invention can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.
  • the actual conditions used to purify a particular peptide or analogue will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
  • the purified peptides can be identified by assays based on their physical or functional properties, including radioactive labeling followed by gel electrophoresis, radioimmuno-assays, ELISA, bioassays, and the like. XII. Formulation and Route of Administration
  • the PDZ-PL antagonists of the invention are introduced into a cell to modulate (i.e., increase or decrease) a biological function or activity of the cell.
  • Many small organic molecules readily cross the cell membranes (or can be modified by one of skill using routine methods to increase the ability of compounds to enter cells, e.g., by reducing or eliminating charge, increasing lipophilicity, conjugating the molecule to a moiety targeting a cell surface receptor such that after interacting with the receptor).
  • Methods for introducing larger molecules e.g., peptides and fusion proteins are also well known, including, e.g., injection, liposome-mediated fusion, application of a hydrogel, conjugation to a targeting moiety conjugate endocytozed by the cell, electroporation, and the like).
  • the antagonist or agent is a fusion polypeptide or derivatized polypeptide.
  • a fusion or derivatized protein may include a targeting moiety that increases the ability of the polypeptide to traverse a cell membrane or causes the polypeptide to be delivered to a specified cell type (e.g., cancer cells) preferentially or cell compartment (e.g., nuclear compartment) preferentially.
  • targeting moieties include lipid tails, amino acid sequences such as antennapoedia peptide or a nuclear localization signal (NLS; e.g., Xenopus nucleoplasmin Robbins et al., 1991, Cell 64:615).
  • a peptide sequence or peptide analog determined to inhibit a PDZ domain-PL protein binding, in an assay of the invention is introduced into a cell by linking the sequence to an amino acid sequence that facilitates its transport through the plasma membrane (a "transmembrane transporter sequence").
  • the peptides of the invention may be used directly or fused to a transmembrane transporter sequence to facilitate their entry into cells.
  • each peptide may be fused with a heterologous peptide at its amino terminus directly or by using a flexible polylinker such as the pentamer G-G-G-G-S (SEQ ID NO.T) repeated 1 to 3 times.
  • linker has been used in constructing single chain antibodies (scFv) by being inserted between V H and V L (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).
  • the linker is designed to enable the correct interaction between two beta-sheets forming the variable region of the single chain antibody.
  • Other linkers that may be used include Glu-Gly- Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:2) (Chaudhary et al., 1990, Proc. Natl.
  • transmembrane transporter peptides examples include, but are not limited to, tat derived from HIV (Vives et al., 1997, J. Biol. Chem. 272:16010; Nagahara et al., 1998, Nat. Med. 4:1449), antennapedia from Drosophila (Derossi et al., 1994, J. Biol. Chem.
  • a truncated HIV tat peptide having the sequence of GYGRKKRRQRRRG (SEQ ID ⁇ O:4) is used.
  • a transmembrane transporter sequence is fused to a HPV protein carboxyl terminal sequence at its amino-terminus with or without a linker.
  • a linker Generally, the C- terminus of a PDZ motif sequence (PL sequence) must be free in order to interact with a PDZ domain.
  • the transmembrane transporter sequence may be used in whole or in part as long as it is capable of facilitating entry of the peptide into a cell.
  • a HPV protein C-terminal sequence may be used alone when it is delivered in a manner that allows its entry into cells in the absence of a transmembrane transporter sequence.
  • the peptide may be delivered in a liposome formulation or using a gene therapy approach by delivering a coding sequence for the PDZ motif alone or as a fusion molecule into a target cell.
  • the compounds of the of the invention may also be administered via liposomes, which serve to target the conjugates to a particular tissue, such as cervical tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule that binds tooncogenic HPV protein or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired peptide or conjugate of the invention can be directed to the site of transformed cervical cells, where the liposomes then deliver the selected inhibitor compositions.
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired HPV-transformed cervical cells.
  • a liposome suspension containing a peptide or conjugate may be administered intravenously, locally, topically, etc. in a dose which varies according to, the manner of administration, the conjugate being delivered, and the stage of the disease being treated.
  • the peptide may be linked to a cell-specific targeting moiety, which include but are not limited to, ligands for surface molecules that are preferentially presented on the surface of HPV-infected or cancerous cells, such as growth factors, hormones and cytokine receptors, as well as antibodies or antigen-binding fragments thereof. Proteins expressed on the surface of appropriate infected cells should be selected as the homing signal for increasing the concentration of therapeutic at the infected site. Antibodies are the most versatile cell-specific targeting moieties because they can be generated against any cell surface antigen.
  • Monoclonal antibodies have been generated against many cell-surface markers such as CD antigens, ion channels, and signal transduction molecules.
  • Antibody variable region genes can be readily isolated from hybridoma cells by methods well known in the art. However, since antibodies are assembled between two heavy chains and two light chains, it is preferred that a scFv be used as a cell-specific targeting moiety in the present invention.
  • Such scFv are comprised of V H and V L domains linked into a single polypeptide chain by a flexible linker peptide.
  • the PDZ motif sequence may be linked to a transmembrane transporter sequence and a cell-specific targeting moiety to produce a tri-fusion molecule. This molecule can bind to a cervical cell surface molecule, passes through the membrane and targets PDZ domains.
  • a PDZ motif sequence may be linked to a cell-specific targeting moiety that binds to a surface molecule that internalizes the fusion peptide.
  • microspheres of artificial polymers of mixed amino acids may be linked to a transmembrane transporter sequence and a cell-specific targeting moiety to produce a tri-fusion molecule. This molecule can bind to a cervical cell surface molecule, passes through the membrane and targets PDZ domains.
  • a PDZ motif sequence may be linked to a cell-specific targeting moiety that binds to a surface molecule that internalizes the fusion peptide.
  • microspheres of artificial polymers of mixed amino acids may be linked to a cell-specific targeting moiety that binds to
  • proteinoids have been used to deliver pharmaceuticals.
  • U.S. Pat. No. 4,925,673 describes drug-containing proteinoid microsphere carriers as well as methods for their preparation and use. These proteinoid microspheres are useful for the delivery of a number of active agents.
  • a polynucleotide that encodes a PL sequence peptide of the invention is introduced into a cell where it is expressed.
  • a polynucleotide encoding a PDZ domain is introduced into a cell where it is expressed. The expressed peptide then inhibits the interaction of PDZ proteins and PL proteins in the cell.
  • the polypeptides of the invention are expressed in a cell by introducing a nucleic acid (e.g., a DNA expression vector or mRNA) encoding the desired protein or peptide into the cell. Expression may be either constitutive or inducible depending on the vector and choice of promoter. Methods for introduction and expression of nucleic acids into a cell are well known in the art and described herein. In a specific embodiment, nucleic acids comprising a sequence encoding a peptide disclosed herein, are administered to a human subject. In this embodiment of the invention, the nucleic acid produces its encoded product that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • the therapeutic composition comprises a coding sequence that is part of an expression vector.
  • a nucleic acid has a promoter operably linked to the coding sequence, said promoter being inducible or constitutive, and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the coding sequence and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the nucleic acid (Roller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any methods known in the art, e.g. , by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No.
  • a nucleic acid-ligand complex can be fonned in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992; WO 92/22635 dated December 23, 1992; WO92/20316 dated November 26, 1992; W093/14188 dated July 22, 1993; WO 93/20221 dated October 14, 1993).
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
  • adeno viruses as viral vectors can be used in gene therapy.
  • Adenoviruses have the advantage of being capable of infecting non-dividing cells (Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3 :499-
  • adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and Mastrangeli et al., 1993, J. Clin. Invest. 91 :225-234.
  • adenoviral vectors with modified tropism may be used for cell specific targeting (WO98/40508).
  • Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).
  • retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
  • the coding sequence to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, lipofection, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the cell used for gene therapy is autologous to the patient.
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding sequence, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • Oligonucleotides such as anti-sense RNA and DNA molecules, and ribozymes that function to inhibit the translation of a targeted mRNA, especially its C-terminus, are also within the scope of the invention.
  • Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of a nucleotide sequence, are preferred.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimeti ⁇ ylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target RNA sequences.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites that include the following sequences, GUA, GUU and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides conesponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable.
  • the suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
  • RNA molecules and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well l ⁇ iown in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors that contain suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • compositions comprising the compounds of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, - encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of the active peptides or peptide analogues into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. This route of administration may be used to deliver the compounds to the nasal cavity.
  • the compounds can be readily formulated by combining the active peptides or peptide analogues with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
  • the compounds may take the form of tablets, lozenges, etc. formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • Topical compositions and medicated carriers e.g., medicated "tampon" may also be used for such routes of administration.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneousl or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well l ⁇ iown examples of delivery vehicles that may be used to deliver peptides and peptide analogues of the invention.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization may be employed.
  • the compounds of the invention may contain charged side chains or termini, they may be included in any of the above-described formulations as the free acids or bases or as pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are those salts which substantially retain the biologic activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the conesponding free base forms. D. Effective Dosages
  • the compounds of the invention will generally be used in an amount effective to achieve the intended purpose.
  • the compounds of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.
  • therapeutically effective amount is meant an amount effective ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • An "inhibitory amount” or “inhibitory concentration” of a PL-PDZ binding inhibitor is an amount that reduces binding by at least about 40%, preferably at least about 50%, often at least about 70%>, and even as much as at least about 90%). Binding can be measured in vitro (e.g., in an A assay or G assay) or in situ.
  • a therapeutically effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well l ⁇ iown in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data. Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds that are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5 to 1 mg kg/day. Therapeutically effective serum levels may be achieved by administering multiple doses each day.
  • the effective local concentration of the compounds may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • the amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • the therapy may be repeated intermittently while symptoms detectable or even when they are not detectable.
  • the therapy may be provided alone or in combination with other drugs.
  • the drugs that may be used in combination with the compounds of the invention include, but are not limited to, steroid and non-steroid anti-inflammatory agents.
  • a therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity.
  • Toxicity of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or the LD 100 (the dose lethal to 100% of the population).
  • the dose ratio between toxic and therapeutic effect is the therapeutic index.
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the compounds described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • kits thereof for practicing one or more of the above- described methods.
  • the subject reagents and kits thereof may vary greatly.
  • the kits at least include a subject peptide that may or may not contain a cell permeable peptide carrier.
  • the subject kits may also include one or more additional reagents, e.g., reagents employed in administering the peptides, such as diluents, syringes, etc.
  • the subject kits can further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.
  • EXAMPLE 1 SEQUENCE ANALYSIS OF HPV E6 PROTEINS TO DETERMINE ONCOGENIC POTENTIAL PDZ proteins are l ⁇ iown to bind certain carboxyl-terminal sequences of proteins
  • PLs PL sequences that bind PDZ domains are predictable, and have been described in greater detail in US Patent Applications 09/710059, 09/724553 and 09/688017.
  • One of the major classes of PL motifs is the set of proteins terminating in the sequences -X-(S/T)-X- (V/I L).
  • V/I L the set of proteins terminating in the sequences -X-(S/T)-X- (V/I L).
  • Table 3B shows the results of the G assay looking at interactions between the E6 PDZ ligand and PDZ domains. Listed are interactions that gave a signal to noise of around 2 or higher. This demonstrates the extent of PDZ binding and the non-obvious nature of this ligands interaction with cellular PDZ proteins. However, we see a number of interactions that are common to most all PLs from oncogenic E6 proteins that can be specifically targeted to treat HPV induced cancers. TABLE 3A: higher affinity interactions between HPV E6 PLs and PDZ domains
  • HPV strain denotes the strain from which the E6 C-terminal peptide sequence information was taken. Peptides used in the assay varied from 18 to 20 amino acids in length, and the terminal four residues are listed in parenthesis. Names to the right of each HPV E6 variant denote the human PDZ domain(s) (with domain number in parenthesis for proteins with multiple PDZ domains) that saturated binding with the E6 peptide in the G assay (See Description of the Invention). * - denotes that the PDZ domains of hDlgl were not tested against these proteins yet due to limited material, although both have been shown to bind hDlgl in the literature.
  • HPVE6#16 Syntrophin 1 alpha 1 10 5 1.3325 0.442 8.03
  • HPVE6#16 Syntrophin gamma 2 1 10 5 0.7725 0.074 3.13
  • HPVE6#16 Syntrophin 1 alpha 1 10 5 0.5635 0.002 3.70
  • HPVE6#16 Syntrophin gamma 2 1 10 5 0.469 0.028 2.76
  • PL - indicates the PDZ ligand from the E6 protein of HPV 16.
  • Gene Name the name of the gene containing a PDZ domain.
  • Domain the PDZ domain number as assigned from amino terminus, also listed in Table 8.
  • [Peptide] - concentration of peptide in micromolar used for the assay, [protein] - concentration of PDZ domain fusion used for this assay in micromolar.
  • StDev Standard deviation of the two points used to generate the average.
  • OD S N the Absorbance signal to noise ratio versus the GST only well for that specific G Assay plate.
  • Probes corresponding to HPV E6 from strains 16 or 18 were generated using PCR with the oligos listed in Example 4. Probes for TIP-1 and MAGI-1 were generated using PCR with primers listed in Example 5. All probes were radioactively labeled with 32 P using the Ready-To-Go labeling kit (Amersham Pharmacia). Blots were crosslinked, blocked with CHURCH solution (7% SDS, 1%BSA and phosphate buffered), and hybridized with the appropriate probe for several hours at 42°C in Church solution. Hybridized blots were washed several times with lx SSC, 0.2%SDS at 65°C followed by 2-3 stringent washes of 0.2xSSC, 0.1%SDS at 65°C. Washed blots were exposed to film overnight and are shown in Figures 1 A and IB.
  • Figure 1A shows the expression of E6 from HPV 16 or HPV 18 in various cell lines used in these studies. Lanes: 1 B-cell (Ramos); 2 No HPV (HTB32); 3 1550 HPV 16+18; 4 1595 HPV18; 5 1594 HPV 18; 6 HTB 35 (HPV 16); 7 RNA marker.
  • HPV18 E6 and HPV 16 E6 refer to the radiolabeled probe used to detect expression in each of the cell lines.
  • Figure IB shows the expression of TIPl and MAGI1 in various cervical cell lines used in this study. Both genes are expressed in cervical cancers indicating that they could be involved in the mechanism of E6 oncogenicity.
  • Cloning vectors were pGEX-3X (Amersham Pharmacia #27-4803-01), MIE (a derivative of MSCV, containing IRES and EGFP, generated by recombinant DNA technology), pmKit, pcDNA3.1 (Invitrogen, modified to include a HA tag upstream of the cloning site) and pMAL (New England Biolabs Cat# N8076S, polylinker modified in house to include BamHl and EcoRl sites).
  • HPV E6 genes and 3 ' truncated ( ⁇ PL) versions, were subsequently cloned into MIE (MSCV-IRES-EGFP) vector, pcDNA-HA vector, and pmKit vector, using the purified HPV E6-pGEX3x fusion plasmid as the PCR template, and using the same purification protocols as listed above.
  • Truncated versions of HPV E6 have a stop codon inserted after the -3 position amino acid, so as to delete the last three amino acids from the coding region of the gene.
  • AACGTGTTCTTGATG HPV E6 16 ⁇ PL generates a BamHl site.
  • ATC (SEQ ID NO:42) Used for cloning into MIE.
  • TGCACCAAAAGAG HPV E6 16 generates a Sal I site. Used for
  • CTTAATATTATAC HPV E6 generates a Xho I site. Used for
  • CTTAATATTATAC HPV E6 generates a Hind HI site. Used for
  • GTCGTTGAGGTCG HPV E6 18 ⁇ PL generates a Hind III site.
  • Table 4 discloses a list of oligonucleotides used to amplify and clone specific regions E6 proteins from various HPV strains into expression vectors.
  • the first designation is an internal name for the primer.
  • the second column represents the sequence presented 5' to 3'.
  • the third column is a description of directions of the primer, intended construct, and restriction site for cloning.
  • MIE contains IRES and EGFP to the 3' end (downstream) of the cloning site
  • MIE contains IRES and EGFP to the 3' end (downstream) of the cloning site •Construct: HPV E6 18 ⁇ PL-MIE
  • MIE contains IRES and EGFP to the 3' end (downstream) of the cloning site
  • Hind Ill/Hind III pcDNA3.1 (modified) contains HA to the 5'end (upstream) of the cloning site
  • constructs using pGEX-3X expression vector were used to make fusion proteins according to the protocol outlined in the GST Fusion System, Second Edition, Revision 2, Pharmacia Biotech. Method II and was optimized for a IL LgPP.
  • Purified DNA was transformed into E.coli and allowed to grow to an OD of 0.4-0.8 (600 ⁇ ). Protein expression was induced for 1-2 hours by addition of IPTG to cell culture. Cells were harvested and lysed. Lysate was collected and GS4B beads (Pharmacia Cat# 17- 0756-01) were added to bind GST fusion proteins. Beads were isolated and GST fusion proteins were eluted with GEB II. Purified proteins were stored in GEB II at -80°C.
  • Purified proteins were used for ELISA-based assays, functional assays and antibody production.
  • Other vectors encoding portions of HPV proteins or cellular proteins were transfected directly into mammalian cells by various means for testing.
  • E6 and E7 expression constructs from a variety of HPV strains were constructed in a similar manner to those described above.
  • PDZ domain containing genes were into eukaryotic expression vectors in fusion with a number of protein tags, including but not limited to Glutathione S-Transferase (GST), Enhanced Green Fluorescent Protein (EGFP), or Hemagglutinin (HA).
  • GST Glutathione S-Transferase
  • EGFP Enhanced Green Fluorescent Protein
  • HA Hemagglutinin
  • RT-PCR from RNA from a library of individual cell lines (CLONTECH Cat# K4000-1) derived RNA, using random (oligo-nucleotide) primers (Invitrogen Cat.# 48190011).
  • DNA fragments corresponding to PDZ domain containing genes or portions of PDZ domain containing genes were generated by standard PCR, using above purified cDNA fragments and specific primers (see Table 5 for example). Primers used were designed to create restriction nuclease recognition sites at the PCR fragment's ends, to allow cloning of those fragments into appropriate expression vectors.
  • DNA samples were submitted to agarose gel electrophoresis. Bands corresponding to the expected size were excised.
  • DNA was extracted by Sephaglas Band Prep Kit (Amersham Pharmacia Cat# 27- 9285-01) and digested with appropriate restriction endonuclease. Digested DNA samples were purified once more by gel electrophoresis, according to the same protocol used above. Purified DNA fragments were coprecipitated and ligated with the appropriate linearized vector. After transformation into E.coli, bacterial colonies were screened by colony PCR and restriction digest for the presence and correct orientation of insert. Positive clones were innoculated in liquid culture for large scale DNA purification. The insert and flanking vector sites from the purified plasmid DNA were sequenced to ensure correct sequence of fragments and junctions between the vectors and fusion proteins. B. Vectors:
  • PDZ domain-containing genes were cloned into the vector pGEX-3X (Amersham Pharmacia #27-4803-01, Genemed Acc#U13852, GI#595717), containing a tac promoter, GST, Factor Xa, ⁇ -lactamase, and lac repressor.
  • the amino acid sequence of the pGEX-3X coding region including GST, Factor Xa, and the multiple cloning site is listed below. Note that linker sequences between the cloned inserts and GST-Factor Xa vary depending on the restriction endonuclease used for cloning.
  • TIPl TAX Interacting Protein 1
  • CD5 ⁇ CD5 ⁇
  • PEAK10 both provided by the laboratory of Dr. Brian Seed at Harvard University and generated by recombinant DNA technology, containing an IgG region
  • MIN a derivative of MSCV, containing IRES andNGFR, generated by recombinant DNA technology
  • MAGI 1. PDZ domain 2 of 6: Acc#:AB010894 GI#:3370997
  • TIPl TAX Interacting Protein 1
  • Purified DNA was transformed into E.coli and allowed to grow to an OD of 0.4-0.8 (600 ⁇ ). Protein expression was induced for 1-2 hours by addition of IPTG to cell culture. Cells were harvested and lysed. Lysate was collected and GS4B beads (Pharmacia Cat# 17- 0756-01) were added to bind GST fusion proteins. Beads were isolated and GST fusion proteins were eluted with GEB II. Purified proteins were stored in GEB II at -80°C.
  • the constructs using the CD5gamma or PeaklOIgG expression vectors were used to make fusion protein.
  • Purified DNA vectors were transfected into 293 EBNA T cells under standard growth conditions (DMEM +10% FCS) using standard calcium phosphate precipitation methods (Sambrook, Fritsch and Maniatis, Cold Spring Harbor Press) at a ratio of ⁇ 1 ug vector DNA for 1 million cells. This vector results in a fusion protein that is secreted into the growth medium. Transiently transfected cells are tested for peak expression, and growth media containing fusion protein is collected at that maxima (usually 1-2 days). Fusion proteins are either purified using Protein A chromatography or frozen directly in the growth media without addition.
  • GST-HPV E6 fusion proteins were constructed as described in Example 4 corresponding to the full length protein sequence of E6 from HPV 18 (oncogeneic) and HPV11 (non-oncogenic).
  • binding of a TIP-TIP -IgG fusion protein two copies of the TIP-1 PDZ domain fused to the hlgG constant region, purification of fusion protein partially described in Example 5 to these two E6 variants was assessed using the ELISA listed below.
  • AVC phosphate buffered saline 8gm NaCl, 0.29 gm KC1, 1.44 gm Na 2 HP04, 0.24gm KH 2 P04, add H20 to 1 L and pH 7.4; 0.2 micron filter • 2% BSA/PBS (lOg of bovine serum albumin, fraction V (ICN Biomedicals cat#IC15142983) into 500 ml PBS
  • enzyme-conjugated detection Ab (anti-hlgG-HRP, anti- goat-HRP, or anti-mouse-HRP) 100 ul per well on ice, 20 minutes at 4°C
  • TIP-1 a representative PDZ domain that binds most oncogenic E6 PLs (EXAMPLE 2, TABLES 3 A,3B), is able to specifically recognize PLs from full length oncogenic E6 variants (HPV18-E6) without binding to non-oncogenic variants (HPV11-E6; FIGURE 2). Furthermore, even unpurif ⁇ ed TIP-TIP-IgG fusion protein is able to recognize GST- HPV18E6 fusion protein at levels comparable to an antibody generated against HPV18-E6. Antibodies against GST were used to confirm that the GST-HP 18E6 and GST-HP 11E6 were uniformly plated (data not shown).
  • Figure 3 shows the results of inhibition assay with Tax PL peptide. Inhibition was measured by depression of A450 reading compared to positive control (HPV E6 16 + TIPl without Tax PL). As shown in the figure, increasing concentrations of Tax PL peptide decrease binding between TIPl and HPV E6 16 in vitro. These results suggest that peptides, peptide mimetics, or other inhibitory molecules may effectively block HPV PL-PDZ interactions in vivo.
  • Table 6 contains examples of some pathogens that are l ⁇ iown to involve proteins containing a PL motif. These PL proteins may provide valuable therapeutic targets for the treatment of diseases resulting from pathogen infections. As for HPV E6, the C-terminal PL domains of these proteins may be used as an anti-viral therapy.
  • the following example shows the results of assays to determine the rate of migration and proliferation of cells bearing oncogenic HPV E6 16 proteins or fragments thereof.
  • Plasmid constructs of HPV E6 16 wild type and HPV E6 16 ⁇ PL were generated using the vector pmKit, containing an HA tag. Recombinant plasmids were generated by recombinant DNA cloning methods known in the art and outlined in Examples 4 and 5. Primers used to generate HPV DNA fragments are shown in Table 7.
  • Each of the three transfected cell groups (pmKit-HA-HPV E6 16 wt, pmKit-HA-HPV E6 16 ⁇ PL, pmKit-HA control) were seeded onto a 12- well plate, and allowed to adhere and grow to confluent (about 24 hours) in RPMI media with 4% FBS and non-essential amino acids. A sterile pipet tip (about 1mm diameter) was dragged through the cells, creating a gap in the lawn. Cells were monitored and photographed at 48-hour intervals.
  • Figure 5 A shows HPV E6 16 wildtype and ⁇ PL transfections 1 day after scratching.
  • Figure 5B shows HPV E6 16 wildtype and ⁇ PL transfections 3 days after scratching.
  • Figure 5C shows HPV E6 16 wildtype and ⁇ PL transfections 5 days after scratching.
  • Figure 5D shows HPV E6 1 wildtype and ⁇ PL transfections 7 days after scratching.
  • EXAMPLE 10 EXOGENOUS ONCOGENIC E6 PROTEIN ACTIVATES JNK ACTIVITY IN XENOPUS OOCYTES THAT CAN BE BLOCKED BY PEPTIDE INHIBITORS Experimental Design: This experiment was divided into two phases. In the first phase, an MBP-E6 fusion protein (HPV 16; see example 4) was microinjected into Xenopus oocytes at different concetrations and then the oocytes were assayed for JNK activity.
  • MBP-E6 fusion protein HPV 16; see example 4
  • peptides corresponding to the C-termini of non-oncogenic E6 protein HPV 11
  • HPV16 E6 oncogenic
  • Tax shown to block oncogenic E6 binding to PDZ domains, Figure 3
  • Oocyte Xenopus ovarian tissue was surgically removed, and oocytes were defolliculated for 1-1.5 h at room temperature with 2 mg/ml collagenase and 0.5 mg/ml polyvinylpyrrolidone in Ca21-free modified Earth's solution (88 mM NaCl, 1 mM KC1, 0.82 mM MgS04, 2.4 mM aHC03, 10 mM HEPES, pH 7.5). The oocytes were then washed four times with modified Barth's solution.
  • Stage VI oocytes were sorted manually and incubated at 16 °C for at least 10 h in OR2 solution (82.5 mM NaCl, 2.5 mM KC1, 1 mM CaC12, 1 mMNa2HP04 5 5 mM HEPES, pH 7.5) supplemented with 1 mg/ml bovine serum albumin and 50 mg/ml gentamicin.
  • Immature oocytes were microinjected with purified MBP-E6 protein or E6 protein and peptide and transferred to fresh OR2 for the duration of the time course. Five oocytes were collected per time point, frozen on dry ice, and stored at -80 °C.
  • Lysis of Oocytes, Eggs, and Embryos Frozen oocytes, eggs, and embryos were thawed rapidly and lysed by pipetting up and down in 60 ml of ice-cold extraction buffer (EB) (0.25 M sucrose, 0.1 M NaCl, 2.5 mM MgC12, 20 mM HEPES, pH 7.2) containing 10 mM EDTA, protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin, 10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride), and phosphatase inhibitors (50 mM 2-glycerophosphate, 1 mM sodium orthovanadate, 2 mM microcystin). Samples were clarified by centrifugation for 2.5 min in a Beckman E microcentrifuge with a right angle rotor. Crude cytoplasm was collected and processed for immunoblotting
  • Immunoblotting Aliquots of oocyte, egg, or embryo lysates were added to 0.2 volumes of 63 Laemmli sample buffer. Samples were separated on 10% SDS-polyacrylamide gels (bisacrylamide:acrylamide, 100: 1) and the proteins transferred to PVDF blotting membrane (Amersham Pharmacia Biotech). The membrane was blocked with 3% nonfat milk in Tris- buffered saline (150 mM NaCl, 20 mM Tris, pH 7.6) and incubated with primary antibodies. Blots were washed five times with TBS, 0.5% Tween 20 and probed with an peroxidase- conjugated secondary antibody for detection by enhanced chemiluminescence (ECL-Plus, Amersham Pharmacia Biotech). For reprobing, blots were stripped by incubation with 100 mM Tris-HCl, pH 7.4, 100 mM 2-mercaptoethanol, and 2% SDS at 70 °C for 40 min.
  • Jun Kinase Assay Jun kinase assays were performed as described. Crude oocyte, egg, or embryo cytoplasm was diluted 1 : 1 in EB and pre-cleared with 20 ml of glutathione- Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C with moderate shaking. Lysates were incubated with glutathione S'-transferase GST-c-Jun-(l-79) fusion protein (hereafter denoted GST-Jun) immobilized on glutathione- Sepharose beads.
  • GST-Jun glutathione S'-transferase GST-c-Jun-(l-79) fusion protein
  • the beads were washed three times with 50 mM HEPES, pH 7.5, 150 M NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin (24) and once with 0.4 ml of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgC12, 1 mM dithiothreitol, 200 mM sodium orthovanadate).
  • FIG. 4 A shows that oncogenic HPV 16 E6 , but not non-oncogenic HPV11 E6 , activates c-JUN N-terminal kinase (JNK), a kinase known to be involved in numerous oncogenic pathways.
  • Figure 4B demonstrates that HPV 16 E6 - dependent activation of JNK can be inhibited by co-injection of peptide corresponding to the C-terminus of Tax (an independent PDZ ligand that binds similar PDZ domains), but not with peptide representing the C-terminus of non-oncogenic HPV E6 11.
  • JNK c-JUN N-terminal kinase
  • Figure 4C demonstrates that HPV 16 E6 dependent activation of JNK can be inhibited by peptide representing the c-terminus of the HPV 16 E6 oncoprotein, but not by peptide representing the C-terminus of nononcogenic HPV 11 E6 protein.
  • Mammalian 293 cells were transfected by standard Calcim Phosphate methods with pmKIT vectors carrying inserts from the group: A (no insert), HPV 16 E6, HPV16 E6 ⁇ PL (C-terminal 3 amino acids deleted), or HPV16 E7.
  • Transfected cells were collected after 2 days and assayed of JNK activity through the lysates ability to phophorylate GST-cJUN (see Example 10). JNK activity positive controls were treated with EGF or Sorbitol prior to lysis to activate JNK.
  • FIG. 6 shows the results of these experiments.
  • HPV 16 E6 protein alone can activate JNK activity in mammalian 293 cells. This activity is dependent on the PDZ Ligand (PL), as the ⁇ PL construct that is identical to HPV 16 E6 construct except for a deletion of the c- tenninal 3 amino acids fails to activate JNK. This activation is not depedent upon E7 co- transfection.
  • PL PDZ Ligand
  • HPV E6 16 The C-terminal motif of HPV E6 16 is required for cellular transformation in rodent cells. Further cellular assays have demostrated that cell migration of HPV E6 16 transfected cells is PL dependent, where E6 wt cells migrate faster than ⁇ PL cells.
  • a library of FDA approved drugs was tested for potential small molecule inhibitors of the HPV 16 E6/TIP 1 interaction (shown in Figure 7). From this drug screen, five potential drug inhibitors were selected (drugs 43 (benztropine mesylate), 102 (clomipramine hydrochloride), 264 (methotrimeprazine), 276 (mitoxantrone hydrochloride) and 410 (verapamil hydrochloride)) and titrated against the TIP 1/HPV E6 16 interaction as shown in Figure 8 ( Figures 8A-8E respectively). The IC50 for these reactions was on the order of 100-200 ⁇ M. The inhibition reactions were performed using the G assay protocol described supra at a HPV 16 E6 concentration of 2 ⁇ M for the drug screen experiments.
  • MAGI-1 PDZ domain 1 may the same as MAGI PDZ domain 2, as referenced in the rest of this patent application.
  • Anti-JNK antibodies used were mouse monoclonal anti-phospho-JNK (P-Thr 183/P-Tyr 185) (Cell Signaling), rabbit polyclonal anti-JNKl (SC571 ; Santa Cruz
  • Mouse IgG-2a R- phycoerythrin and the isotype control Ab were from Caltag Laboratories.
  • Phycoerythrin- labeled mouse monoclonal Ab to human CD69 were purchased from Caltag laboratories.
  • Anti-CD69 Microbeads were obtained from Miltenyi Biotec.
  • Cell lines-- Human cervical cancer cell lines HeLa, SiHa, Caski and C 33 A, and human embryonic kidney cells (HEK 293) were obtained from the American Type Culture
  • DMEM fetal calf serum
  • FCS fetal calf serum
  • GST-Jun was expressed and purified from BL21 E. coli cells.
  • HEK 293 Human embryonic kidney cells (HEK 293) cells were maintained in culture under Dulbecco's modified Eagle medium containing 10% fetal bovine serum. They were sustained in a 37°C incubator with a 5% C0 2 atmosphere. Immediately before transfection cholorquine at a final concentration of 25 uM was added to the cell culture media. Plasmids were transfected into HEK 293ET cells via calcium phosphate DNA precipitation, using 30 ug DNA per 95% confluent 10cm diameter plate. Cells were incubated at 37 C C for 8 hours, after which the media was changed. Harvesting of the cells took place at 24- and 48-hour post-transfection intervals. Transfection efficiency was checked by analyzing cells that had been transfected in parallel with an eGFP plasmid, transfection efficancies were 85-95%.
  • Lysis of Xenopus Oocytes Frozen oocytes, eggs, and embryos were thawed rapidly and lysed by pipetting up and down in 60 ⁇ l of ice-cold extraction buffer (EB) (0.25 M sucrose, 0.1 M NaCl, 2.5 mM MgC12, 20 mM HEPES, pH 7.2) containing 10 mM EDTA, protease inhibitors (10 ⁇ g/ml leupeptin, 10 ⁇ g/ml pepstatin, 10 ⁇ g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride), and phosphatase inhibitors (50 mM 2-glycerophosphate, 1 mM sodium orthovanadate, 2 ⁇ M microcystin).
  • EB ice-cold extraction buffer
  • protease inhibitors 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml pepstatin, 10 ⁇
  • JNK kinase assay Lysates were obtained as described above, incubated with glutathione S-transferase GST-c-Jun-(l-79) fusion protein (hereafter denoted GST-Jun) immobilized on glutathione-Sepharose beads.
  • the beads were washed three times with 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and 10 ⁇ g/ml aprotinin (24) and once with 0.4 ml of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl 2 , 1 mM dithiothreitol, 200 ⁇ M sodium orthovanadate).
  • the bound JNK activity was detected by the addition of 1 ⁇ Ci of [ ⁇ - 32 P]ATP. The reaction was terminated after 20 min at 30°C, and the products were resolved by SDS-PAGE. The gels were transferred to PVDF membranes (Hybond; Amersham Pharmacia Biotech) and the incorporation of 32 P into GST-Jun was visualized by autoradiography and quantified by Phosphorlmaging. Matrix assays
  • HPV16-E6-PL was usually considered to bind a particular PDZ when (1) the OD signal was greater or equal to 0.5, the relative standard deviation of the measurement was less than 0.25, and the signal to noise ratio was greater than 2 ( [OD measurement OD background (GST alone)] >2) These criteria together with high OD measurement values were used to determine the strongest interactions.
  • HPV E6 proteins have recently been identified as ligands for cellular PDZ domain-containing proteins, including the human homologue of the
  • Drosophila tumor suppressor discs large (3). Removal of a single C-terminal amino acid from the PDZ ligand (PL) sequence of a high-risk HPV E6 protein abolished its ability to transform cell lines or form tumors in nude mice (4). Moreover, K14-HPV16 E6 transgenic mice developed skin tumors and cervical carcinomas dependent on the presence of the PL (29). We examined the C-terminal sequences of the E6 proteins encoded by all high-risk and low-risk HPVs. We found a 100% correlation between the presence of a PL consensus sequence with the classification as high-risk HPV (Table 9).
  • HPV 4 GYCRNCIRKQ (SEQ ID NO:79) No No n.a.
  • HPV 11 WTTCMEDLLP SEQ ID NO:80 No No n.a.
  • HPV 20 GICRLCKHFQ (SEQ ID NO:81) No No n.a.
  • HPV 24 KGLCRQCKQI (SEQ ID NO:82) No No n.a.
  • HPV 28 WLRCTVRIPQ (SEQ ID NO:83) No No n.a.
  • HPV 36 RQCKHFYNDW (SEQ ID NO:84) No No n.a.
  • HPV 48 CRNCISHEGR SEQ ID NO:85 No No n.a.
  • HPV 50 CCRNCYEHEG (SEQ ID NO:86) No No n.a.
  • HPV 35 WKPTRRETEV (SEQ ID NO:90) Yes Yes 18,30,39,45,51, 68,59
  • Table 9 E6 C-terminal sequences and oncogenicity. HPV variants are listed at the left. Sequences were identified from. Genbank sequence records. PL Yes/No was defined by a match or non-match to the consensus X-(S/T)-X-(V/I/L). Oncogenicity data collected from National Cancer Institute.
  • HPVs (Table 9), suggesting a role for a PDZ/PL interaction in cervical cancer development.
  • HPV26 E6 E6
  • HPV34 E6 ATVV
  • HPV53 E6 ESAV
  • HPV E6 proteins bind to a number of cellular proteins, including E6AP (6), PAXILLIN (7), IRF-3 (8), BAK (9), and to the PDZ containing proteins DLG 1 (3), MUPP 1 (10), VARTUL (11), MAGI 1 (12), MAGI 2 (13) and MAGI 3 (13).
  • E6AP (6) PAXILLIN (7)
  • IRF-3 (8) IRF-3 (8)
  • BAK (9) BAK
  • HPV E6 proteins bind to a number of cellular proteins, including E6AP (6), PAXILLIN (7), IRF-3 (8), BAK (9), and to the PDZ containing proteins DLG 1 (3), MUPP 1 (10), VARTUL (11), MAGI 1 (12), MAGI 2 (13) and MAGI 3 (13).
  • MUPP 1 10
  • VARTUL 11
  • MAGI 1 MAGI 1
  • MAGI 2 MAGI 2
  • MAGI 3 MAGI 3
  • the fusion proteins were used in the ELISA-based Matrix TM assay to determine binding of the 215 PDZ domains to a 20mer C-terminal peptide of HPV 16 E6 (Complete binding data see Suppl. 1).
  • the seven high- risk HPV E6 PL peptides tested were chosen because they represent all PL sequence variations (positions 0 and -2 of consensus motif) present in the 15 E6 proteins encoded by known high-risk HPVs (see Table 9).
  • HPV 16 E6 The three low-risk HPV E6 PL (HPV57, HPV63, and HPV77) failed to interact with any PDZ domains tested. Besides confirming the 6 interactions previously described in the literature, we discovered eight novel PDZ- interactions for HPV16 E6 (Table 10). Relative binding affinities for the 14 most significant interactions with different PDZ domains were determined by E6 peptide titrations. A compilation of relative EC50 values for these interactions with HPV 16 E6 is shown in Table 10.
  • MAGI 1 domain 1 was the only PDZ domain tested that bound to each of the 7 high-risk HPVE6 PLs tested.
  • TIPl a small protein with a single PDZ domain , bound each of the high-risk HPV E6 PLs except HPV52 E6 (data not shown).
  • Our data demonstrate that all high-risk HPV E6 proteins tested bound PDZ domains with a rather conserved binding pattern. To narrow down the number of potentially physiologically relevant PDZ protein targets of HPV E6 protein, we tested expression of these PDZ-proteins in cervical cancer cell lines.
  • Table 10 shows the mRNA expression in cervical cancer cell lines of selected PDZ genes: Tipl, SAST1, Vartul (hScrib), MAGI 1, MAGI 3, Synaptojanin 2 binding protein (Syn2bp) and DLG 1.
  • Two of the PDZ genes, Sast 2 and Magi 3 showed no mRNA expression in any of the cell lines tested, and consequently were therefore ruled out as physiological targets of E6.
  • FIG. 10B A comparison of MAGI 1 mRNA expression levels of HPV-negative, C33A cells and the HPV-positive cervical carcinoma cell lines: HeLa (HPV18), SiHa (HPV16), Caski (HPV16), C4-1 (HPV18), ME180 (HPV68), and MS751 (HPV45) is shown in figure 10B.
  • MAGI 1 mRNA expression levels were markedly lower in all HPV positive cell lines compared to the HPV-negative cells ( Figure 10B). All six HPV-positive lines expressed significantly lower levels of MAGI 1 protein compared to the HPV-negative C33A cells or the HEK293 cells ( Figure 10A).
  • MAGI 1 is a membrane-associated protein of the MAGUK family that localizes to tight junctions in epithelial cells (20), where it may function as a scaffold protein.
  • JNK interacting proteins JIP1 and JIP2 can either enhance or inhibit JNK activation dependent on their cellular abundance (21).
  • Overexpression of the MAPK scaffold protein POSH (Plenty of SH-3) also causes JNK activation without external stimuli (22).
  • POSH Plenty of SH-3
  • scaffold recruitment interaction in the yeast MAPK pathway can be replaced by PDZ domain-mediated interactions (23). If MAGI 1 functions as a scaffold protein in the JNK pathway and by sequestering components of the signaling cascade, inhibits JNK activation, then E6 may abrogate this blockade by downregulating MAGI 1 levels. E6 also activates the JNK pathway through interfering with the tumor . suppressor PTEN.
  • E6 PTEN contains a C-terminal PL and has been shown to bind to MAGI 1, MAGI 2 and MAGI 3 (24). PTEN dephosphorylates and thereby inhibits Focal Adhesion Kinase activation (FAK) (25). Importantly, activation of FAK can lead to JNK activation (26).
  • FAK Focal Adhesion Kinase activation
  • JNK activation 26.
  • E6 by disrupting the MAGI/PTEN interaction, prevents PTEN from dephosphorylating and deactivating FAK, thus leading to higher JNK activity. Supportive evidence comes from recent studies on transgenic mice showing that a conditional null mutant for PTEN and an E6 transgene give rise to a similar phenotype.

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Abstract

L'invention concerne des méthodes et des compositions destinées à traiter des infections pathogènes, et notamment des infections par le papillomavirus humain. Plus particulièrement, l'invention concerne une méthode de criblage consistant à déterminer un effet d'un agent candidat sur la liaison d'une protéine E6 provenant d'une souche oncogène du HPV à un polypeptide contenant la séquence d'acides aminés d'un domaine PDZ particulier provenant de la protéine cellulaire MAGI-1. L'invention concerne des méthodes destinées à traiter des maladies associées à l'expression de protéines pathogènes par modulation de leurs interactions avec MAGI-1, ainsi qu'une pluralité de peptides isolés utiles dans ces méthodes. Elle se rapporte en outre à des trousses destinées à la mise en oeuvre de ces méthodes.
PCT/US2004/006001 2003-02-27 2004-02-27 Methodes et compositions destinees a traiter le cancer du col uterin WO2004076646A2 (fr)

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US7432065B2 (en) 1999-05-14 2008-10-07 Arbor Vita Corporation Methods of identifying modulators of interaction between PDZ domain proteins and PL proteins
WO2010102070A1 (fr) * 2009-03-03 2010-09-10 Arbor Vita Corporation Procédés de traitement d'un cancer associé aux hpv au moyen d'inhibiteurs de jnk
WO2015097268A1 (fr) * 2013-12-23 2015-07-02 Universite De Strasbourg Construction chimérique pour le diagnostic et le traitement des cancers induits par le papillomavirus
WO2021155830A1 (fr) * 2020-02-05 2021-08-12 Tcrcure Biopharma Corp. Récepteurs de lymphocytes t anti-hpv et cellules modifiées

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CA2457424A1 (fr) * 2001-08-03 2003-02-20 Arbor Vita Corporation Interactions moleculaires dans les cellules

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7432065B2 (en) 1999-05-14 2008-10-07 Arbor Vita Corporation Methods of identifying modulators of interaction between PDZ domain proteins and PL proteins
US7514224B2 (en) 1999-05-14 2009-04-07 Arbor Vita Corporation Arrays of PDZ domain polypeptides
WO2010102070A1 (fr) * 2009-03-03 2010-09-10 Arbor Vita Corporation Procédés de traitement d'un cancer associé aux hpv au moyen d'inhibiteurs de jnk
WO2015097268A1 (fr) * 2013-12-23 2015-07-02 Universite De Strasbourg Construction chimérique pour le diagnostic et le traitement des cancers induits par le papillomavirus
WO2021155830A1 (fr) * 2020-02-05 2021-08-12 Tcrcure Biopharma Corp. Récepteurs de lymphocytes t anti-hpv et cellules modifiées

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