WO2006084131A2 - Inhibiteurs de la furine et alpha-defensines destines au traitement ou a la prevention d'une infection a papillomavirus - Google Patents

Inhibiteurs de la furine et alpha-defensines destines au traitement ou a la prevention d'une infection a papillomavirus Download PDF

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WO2006084131A2
WO2006084131A2 PCT/US2006/003844 US2006003844W WO2006084131A2 WO 2006084131 A2 WO2006084131 A2 WO 2006084131A2 US 2006003844 W US2006003844 W US 2006003844W WO 2006084131 A2 WO2006084131 A2 WO 2006084131A2
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furin
alpha
defensin
inhibitor
amino acid
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PCT/US2006/003844
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WO2006084131A3 (fr
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Patricia M. Day
Rebecca Richards
Christopher Buck
John T. Schiller
Douglas R. Lowy
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • the present invention relates to the treatment or prevention of papillomavirus infection using furin inhibitors and alpha-defensins.
  • Viral capsids have evolved to fulfill numerous roles which are critical to the establishment of viral infection.
  • the proteinaceous coat encases and protects the viral nucleic acid, and provides the initial interaction of the viral particle with the host cell. Following receptor engagement, the virus is internalized and its coat is disassembled to allow the encapsidated genome access to the cellular transcription and replication machinery.
  • DNA viruses excluding the poxviruses, this process necessitates the navigation of the genome into the nucleus.
  • PV papillomaviruses
  • the PV capsid is composed of two structural proteins; the major capsid protein, Ll, which can self-assemble into icosahedral virus-like particles in the absence of L2, the minor capsid protein, which is nonetheless necessary for establishment of infection. Most PVs appear to enter the host cell via clathrin-dependent receptor-mediated endocytosis (Day, P.
  • L2 The localization of the genome to the transcriptionally-active NDlO domain, which is critical to the efficient establishment of infection, is dependent on L2. It has also been demonstrated that a carboxyl terminal region of L2 mediates endosomal escape after viral uncoating (Kaemper, N. et al. 2006 J Virol 80:759-768). Additionally, a recent report showed that L2 interacts with syntaxin 18 during entry, possibly using this resident ER protein as a tether for transport towards the nucleus (Bossis, I. et al. 2005 J Virol 79:6723-6731).
  • Furin is a cell-encoded PC present in the Golgi complex, at the plasma membrane, and within endosomes (Seidah, N. G. & Chretien, M. 1997 Curr Opin Biotechnol 8:602-607; Thomas, G. 2002 Nat Rev MoI Cell Biol 3:753-766). Furin cleavage is known to be required for endosome escape by several bacterial toxins, including anthrax toxin and Pseudomonas exotoxin A (Gordon, V. M. & Leppla, S. H. 1994 Infect Immun 62:333-340). However, this is the first example linking furin to a viral entry process. Antimicrobial Peptides
  • Vertebrates have evolved a variety of innate defenses against viral infections. These defenses include sophisticated detection systems such as those involving the toll-like receptors (TLRs), which detect common microbial structures, as well as effector signaling molecules such as the interferons, which stimulate cellular defenses directly targeting the common molecular functions of viruses (Janeway, C. A., Jr. & Medzhitov, R. 2002 Annu Rev Immunol 20:197-216) and (Platanias, L. C. 2005 Nat Rev Immunol 5:375-386).
  • TLRs toll-like receptors
  • Papillomaviruses are a diverse group of non-enveloped DNA tumor viruses that infect the epithelial tissues of a range of different vertebrate species. While many of the more than 100 known human papillomavirus (HPV) species are adapted to infection of non-genital skin surfaces, where they may cause benign warts (papillomas), a subset of HPV species tend to infect genital and mucosal sites.
  • HPV human papillomavirus
  • Adaptive cell mediated immunity is thought to be involved in control and elimination of HPV infection.
  • a possible role for antimicrobial peptide-based innate immunity in inhibiting initial HPV transmission or subsequent viral spread has not been investigated.
  • Recent meta-analyses have suggested that, in contrast to other types of sexually- transmitted infections, condoms probably afford relatively little protection against the initial sexual transmission of HPVs, perhaps due to the fact that mucosotropic HPVs also infect cornified ano-genital skin that is not covered by condoms (Manhart, L. E. & Koutsky, L. A. (2002) Sex Transm Dis 29:725-735; Holmes, K. K. et al. 2004 Bull World Health Organ 82:454-461).
  • HPVs are difficult to propagate in culture.
  • the limited availability of a tractable cell culture model for HPV infection has constrained past efforts to identify compounds that might block HPV transmission (Sokal, D. C. & Hermonat, P. L. 1995 Sex Transm Dis 22:22-24; Christensen, N. D. et al. 2001 Antimicrob Agents Chemother 45:3427-3432; Howett, M. K. et al. 1999 Antimicrob Agents Chemother 43:314-321; Hermonat, P. L. et al. 1992 Sex Transm Dis 19:203-205; Bousarghin, L. et al. 2004 J Med Virol 73:474-480; Drobni, P. et al.
  • the present invention includes a method for inhibiting papillomavirus infection in a human comprising administering to a human at risk thereof a furin inhibitor or alpha- defensin.
  • the invention also includes use of a furin inhibitor or alpha-defensin for the manufacture of a medicament for inhibiting papillomavirus infection in a human.
  • the invention provides a method for inhibiting papillomavirus infection comprising identifying an individual at risk of papillomavirus infection, and administering to said individual a furin inhibitor or alpha-defensin.
  • the invention provides a method for inhibiting papillomavirus infection comprising administering to an individual a furin inhibitor or alpha-defensin, and measuring in said individual an indicia of success against papillomavirus infection.
  • the invention provides a method of inhibiting papillomavirus infection in a human comprising transfecting a cell in vivo with an nucleic acid comprising a nucleotide sequence encoding a furin inhibitor or alpha-defensin.
  • the invention provides a method for determining whether a compound modulates furin inhibitor or alpha-defensin activity in a cell comprising contacting a furin inhibitor or alpha-defensin producing cell with the compound; and determining the functional effect of the compound on furin inhibitor or alpha-defensin activity.
  • the invention further provides a method for inhibiting papillomavirus infection in a human comprising administering to the human an identified compound to augment furin inhibitor or alpha-defensin activity.
  • FIG. 1 Effect of protease inhibition and expression on PV infectivity.
  • A HeLa cells were incubated with various protease inhibitors for the duration of infection with HPVl 6 pseudovirus containing a GFP expression plasmid. Infection was quantified by flow cytometric analysis of GFP expression. Two concentrations were evaluated for each inhibitor. The lightly shaded bar illustrates the higher concentration and the darker bar shows the lower concentration.
  • the inhibitors are as follows: A) N-Acetyl-Leu-Leu-Met (200 nM, 100 nM), B) N-Acetyl-Leu-Leu-Nle-CHO (200 nM, 100 nM), inhibitors of calpain I and II and cathepsins B and L, C) Ca-074 (20 ⁇ M, 10 ⁇ M), an inhibitor of intracellular cathepsin B, D) calpeptin (1.0 ⁇ M, 0.5 ⁇ M), a calpain inhibitor, E) cathepsin L inhibitor (10 ⁇ M, 1 ⁇ M), F) chymostatin (50 ⁇ M, 10 ⁇ M) , an inhibitor of chymotrypsin, papain and cysteine proteases, G) pepstatin A (50 ⁇ M, 10 ⁇ M), an inhibitor of aspartic proteinases, H) decanoyl-RVKR-cmk (100 ⁇ M, 25 ⁇ M
  • C HPV16 pseudovirus infection of HeLa cells, FDI l cells and the FDl l+furin cells.
  • D A comparison of the infectivity of HPVl 6 pseudoviruses containing either wild type L2 or the R12S L2 mutation.
  • E A comparison of the infectivity of BPV 1 pseudoviruses containing either wild type L2 or the R9S L2 mutation.
  • FIG. 3 In vitro cleavage of BPV L2 proteins.
  • the wild type BPVl or BR9S L2 cDNAs fused, at either the N- or the C- termini, with the sequence encoding the HA epitope were transfected into HeLa cells.
  • the L2 proteins were partially purified and digested with furin or left untreated as indicated. Each sample was divided into two samples and processed for immunoblotting with either an anti-HA antibody (upper panel) or an anti-L2 antibody (lower panel).
  • FIG. 4 Uncoating and nuclear trafficking of HA-epitope-tagged wild-type L2 BPVl pseudovirions or B-R9S-L2 pseudovirions.
  • HeLa cells were allowed to internalize pseudovirions assembled in the presence of 20 ⁇ M BrdU. At 24 hours post-entry, the cells were fixed and processed for either L2-HA detection or BrdU detection.
  • Panels A and B show the detection of L2 proteins with an anti-HA antibody; A, C-terminal HA-L2 (CHA- L2); B, N-terminal HA-L2 (NHA-L2).
  • Panels C through F show the detection of the encapsidated pseudogenome with anti-BrdU antibody; C, CHA-L2; D, NHA-L2; E, CHA- B-R9S-L2; F, NHA-B-R9S-L2.
  • FIG. 1 Localization of de novo synthesized L2 proteins.
  • HeLa cells were transfected with (A) L2-CHA, (B) L2-NHA, (C) R9S-CHA, or (D) R9S-NHA and processed for immunofluorescent staining at 24 hours post-transfection as previously described. The HA epitope was detected with an anti-HA antibody.
  • FIG. 7 Localization of L2 and the viral pseudogenome in cells treated with furin inhibitor.
  • C 127 cells a murine line commonly used to evaluate BPVl infection, as detection of the Lamp-1 protein in these cells was most compatible with our staining protocol, although we have observed a similar colocalization in HeLa cells.
  • C 127 cells were allowed to internalize BPV-CHA pseudo virions assembled in the presence of 20 ⁇ M BrdU. After 24 hours, cells were fixed and processed.
  • A-C show the same field, processed for co-detection of the HA epitope and Lamp-1.
  • Mouse anti-HA staining is shown in the green channel (A) and rat anti-Lamp- 1 (clone 1D4B) is shown in the red channel (B).
  • the merge is shown in panel C.
  • Panels D-F show the same cells, with the green channel (D) representing the mouse anti-BrdU staining and the red channel (E) showing the rat anti-Lamp- 1.
  • the merge is shown in (F).
  • Lam ⁇ -1 is detectible in a ring pattern at the perimeters of late endosomes and lysosomes. Localization of the L2-CHA and pseudogenome within the boundaries defined by Lamp-1 staining is evident, indicating entry of the uncoated virions into the late endosomal/lysosomal compartments.
  • Figure 8 Inhibition of various papillomavirus types by HNP-I and HD-5. Inhibition curves are shown for genital (A) and cutaneous (B) papillomavirus types. Panel C shows the IC50 for HNP-I or HD-5 inhibition curves fitted to combined data points for genital or cutaneous types. Error bars represent the 95% confidence intervals for the combined curves.
  • FIG. 9 HD-5 time course experiments. The percent inhibition of HPVl 6 PsV transduction of HeLa cells treated with 5 ⁇ g/ml HD-5 is shown. In panel A, HD-5 was added or removed at different time points. In panel B, cells were treated with HD-5 for overlapping four hour intervals (represented by horizontal bars).
  • FIG. 10 Microscopic analysis of HNP-I and HD-5 inhibition.
  • HeLa cells were mock treated (panel A) or treated with BrdU-labeled HPVl 6 PsV (panels B-D) in the presence of HNP-I (panel C) or HD-5 (panel D). 24 hours after virus inoculation, the cells were stained for BrdU to reveal uncoated viral DNA (green). Cells were counter-stained with the DNA stain DAPI (blue).
  • the present invention provides methods of increasing levels of exogenous furin inhibitors and/or alpha-defensins in individuals by administering therapeutic compounds, e.g., furin inhibitors and/or alpha-defensins (synthetic, recombinant or naturally occurring), furin inhibitor and/or alpha-defensin nucleic acids, and pharmaceutical compositions comprising one or more furin inhibitors and/or alpha-defensins, to individuals infected with papillomavirus or at risk of papillomavirus infection.
  • therapeutic compounds e.g., furin inhibitors and/or alpha-defensins (synthetic, recombinant or naturally occurring), furin inhibitor and/or alpha-defensin nucleic acids, and pharmaceutical compositions comprising one or more furin inhibitors and/or alpha-defensins, to individuals infected with papillomavirus or at risk of papillomavirus
  • the present invention also provides methods of inhibiting papillomavirus infection by providing methods of increasing endogenous furin inhibitor and/or alpha-defensin activity.
  • the present invention provides assays for the identification of modulators of furin inhibitor and/or alpha-defensin activity. Such modulators of activity are useful for inhibiting papillomavirus infection by increasing levels of endogenous furin inhibitor and/or alpha-defensin in the body.
  • the present invention also provides compositions comprising a furin inhibitor and/or alpha-defensin and a pharmaceutically acceptable carrier for delivery to individuals infected with papillomavirus or at risk of papillomavirus infection. Such compositions can be used to treat or prevent papillomavirus infection in an individual.
  • a person is a "at risk" of papillomavirus infection if that person belongs to a group whose risk of papillomavirus infection is higher than the risk of the population as a whole.
  • a "small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about
  • a "small organic molecule" as described herein is distinct from a peptide.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site).
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about a few amino acids or nucleotides in length, or more preferably over a region that is more amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman 1981 Adv Appl Math 2:482-489, by the homology alignment algorithm of Needleman & Wunsch 1970 J MoI Biol 48:443-453, by the search for similarity method of Pearson & Lipman 1988 Proc Natl Acad Sd USA 85:2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995, supplement).
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups ⁇ e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • L and D amino acids are nonsuperimposable mirror images of each other, with L being found in living matter and D not found in natural proteins.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (I), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • Secondary structure refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains.
  • Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity, e.g., a kinase domain. Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -helices. "Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • "Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells may express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • Recombinant cells may also express native genes whose expression has been turned on by the insertion of regulatory DNA sequences, such as promoters or enhancers.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes. “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a deemed ionic strength pH.
  • T m thermal melting point
  • the Tm is the temperature (under deemed ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the 25 nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous references (e.g., Current Protocols in Molecular Biology, ed. Ausubel, et al.).
  • a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length.
  • a .temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50 0 C 5 to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 9O 0 C - 95°C for 30 seconds-2 minutes, an annealing phase lasting 30 seconds-2 minutes, and an extension phase of about 72°C for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
  • terapéuticaally or prophylactically effective dose herein is meant a dose that produces the desired effects for which it is administered.
  • the exact dose will depend on the purpose of the treatment or prophylaxis, and will be ascertainable by one skilled in the art using known techniques ⁇ see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,
  • a therapeutically or prophylactically effective amount of a furin inhibitor or alpha-defensin for use as an anti- papillomavirus agent will be an amount necessary to achieve any indicia of success in the treatment or prophylaxis of papillomavirus infection in an individual, including any objective or subjective criteria such as papillomavirus viral inhibition, diminishing of symptoms associated with papillomavirus infection, or improvement of a patient's physical or mental well-being.
  • a therapeutically or prophylactically effective dose will be from 1 microgram per kg of body weight to 10 milligrams per kg of body weight.
  • proteinase inhibitor In vivo cellular function of many proteases is regulated among others by the action of endogenous physiological inhibitors.
  • An endogenous protein called proteinase inhibitor
  • al-PI Alpha 1 protease inhibitor
  • AT antitrypsin
  • OMTKYS turkey ovomucoid third domain, a2-M, alpha 2 macrogolobulin.
  • ⁇ l- Antitrypsin is a physiological protein generated in endoplasmic reticulum (ER) as a 394 amino-acid (aa)-long protein that acts as an inhibitor of neutrophil elastase. Consistent with other serpins, it is first cleaved by the enzyme at the active site A-I-P-M 358 ⁇ S (SEQ ID NO: 46), after which the enzyme gets entrapped within the newly generated cavity of the serpin and the nearby multiparallel sheets. During this process, the enzyme becomes fully degraded, leading to the destruction of its enzymatic activity.
  • ⁇ l-PIT A second mutation in ( ⁇ l-PIT that gave rise to the sequence R 355 -I-P-R 358 (SEQ ID NO: 42) led to the generation of ⁇ l-AT-Portland ( ⁇ l-PDX), which strongly inhibits furin and other proprotein convertases (PCs).
  • Turkey ovomucoid third domain (0MTKY3) Turkey ovomucoid contains three homologous tandem kazal family inhibitory domains.
  • ⁇ 2-Macroglobulin ( ⁇ 2-M) This homotetrameric glycoprotein of 1,474 aa, present in high concentration in the bloodstream, contains an internal S-ester. This unique bond is primarily responsible for its inhibitory action against furin. It has a flexible and surface-accessible peptide stretch called bait region that contains specific cleavage sites for different proteinases.
  • proteolytic enzymes are synthesized as inactive zymogen forms and their pro domains act as intramolecular chaperone by catalyzing the protein-folding process, hi addition, they also serve as regulator to activity and cellular trafficking of the enzyme.
  • This unique property of prosegment has been described as an additional means for delaying onset of enzyme activity until the moment when the enzyme needs to be fully functional.
  • the prodomains of all PCs inhibit cognate enzymes with Kj in nanomolar to even picomolar range. In some cases, these inhibitory prodomains were constructed with an additional N- or C-terminal extension of His 6 , V 5 tags, extra peptide sequence, or a fusion peptide. Boudreault et al.
  • Small molecule inhibitors are always more attractive than large macromolecule protein inhibitors, because of their long-lasting metabolic and proteolytic stabilities. Moreover, such inhibitors are generally bioavailable and are easily accessible by synthetic routes. In addition, structural modifications of these molecules can be achieved for further structure-activity relation analysis.
  • small inhibitors in many cases are not soluble in aqueous phase at the physiological pH, and they have poor cell permeability.
  • Peptides derived from specific regions of prodomain of furin inhibit its activity with varying degrees of efficiency depending on size and location. These peptides range from 5 to 40 aa long and are usually less potent than the full-length prodomain polypeptides, which are usually 80-120 aa long. Studies revealed that for furin the presence of both primary and secondary activation sites in the peptide sequence is crucial for maximum furin inhibitory activity. The presence of P1-P2 basic residues (Lys/Arg-Arg) of primary prodomain processing site is highly crucial for enzyme inactivation. Depending on their nature and location, synthetic prodomain derived peptides exhibit competitive, noncompetitive, or mixed-type inhibition meaning interactions with either catalytic, noncatalytic, or both domains of the enzyme. Polyarginine approach
  • Histidine-rich antibacterial human salivary peptides histatin 3 (32 aa, 22% His) and histatin 5 (24 aa, 33% His) were both shown to inhibit furin.
  • Other antibicrobial peptides, particularly those containing basic histidine or other residues, may display similar bioactivity towards furin. Cyclic peptide inhibitors
  • a constrained disulfide-bridged cyclic peptide mimicking the reactive site loop structure of a large protein inhibitor of furin is constructed as a potential inhibitor.
  • This approach has been successfully adopted to develop a cyclic octadecapeptide inhibitor encompassing the active site loop structure of barley serine proteinase inhibitor 2 (BSPI-2), often referred to as chymotrypsin inhibitor 2 (CI-2), where an RXKR (SEQ ID NO: 50) recognition motif of PC has been inserted.
  • BSPI-2 barley serine proteinase inhibitor 2
  • CI-2 chymotrypsin inhibitor 2
  • RXKR SEQ ID NO: 50
  • decanoyl-RVKR-cmk SEQ ID NO: 51
  • decanoyl-RVKR-cmk SEQ ID NO: 51
  • Peptidyl semicarbazones Several short peptide semicarbazones (SCs) containing furin recognition sequence
  • peptidyl oxime like peptidyl chloromethyl ketone provides an alternate approach for design of small compound inhibitors of furin even though they are less potent.
  • appropriate C-terminal functionalization of peptides containing minimal furin recognition motif is an effective method for development of furin inhibitors.
  • Table B Kinetic parameters for inhibition of proprotein convertases, including form, by some potent small compound inhibitors of peptide, pseudopeptide, and nonpeptide type.
  • This strategy involves the replacement of scissile peptide bond (-CO-NH-) of a substrate by a noncleavable pseudo peptide bond such as keto methylene (-COCH 2 -). It has been successfully adopted to generate inhibitors of furin.
  • a typical example is decanoyl- Arg-Val-Lys-Arg-( ⁇ COCH 2 )-Ala- VaI-GIy-NH 2 (SEQ ID NO: 66), where the Arg-Ala peptide bond has been replaced by nonscissile ketomethylene group ( ⁇ CO-CH 2 ).
  • the best example is the pseudo aminomethylene bond ( ⁇ CH 2 NH-) containing 18-mer cyclic peptide derived from barley serine protease inhibitor- 2 (BSPI-2), where P2, P4 and/or P6 aa have been mutated to match PC consensus motif.
  • BSPI-2 barley serine protease inhibitor- 2
  • P2, P4 and/or P6 aa have been mutated to match PC consensus motif.
  • a nonpeptide furin inhibitor is based on a study on the medicinal plant Andrographis Paniculata.
  • Several diterpene constituents of this plant including its major component andrographolide and its 3-glycosylated derivative known as neoandrographolide, inhibit furin with Kj or IC 50 values in the low micromolar range.
  • derivatization of andrographolide via O-succinoylation led to a compound with enhanced enzyme inhibitory property.
  • the finest inhibitor in this category is succinoyl ester of andrographolide (SEA), which had a K; of 14 ⁇ M towards furin, many orders of magnitude less than the known most potent peptide inhibitor.
  • SEA succinoyl ester of andrographolide
  • Mammalian defensins are a family of cationic and Cys-rich antimicrobial mini- proteins expressed predominantly in leukocytes and epithelial cells. They form part of the first line of defense in the innate immune arsenal against pathogens, killing a broad range of microbes primarily through membrane disruption.
  • defensins function as effective immune modulators in adaptive immunity by activating certain types of immune cells via receptor-mediated signaling pathways. Based on the connectivity of their six cysteine residues, mammalian defensins are classified into ⁇ , ⁇ , and ⁇ families. Despite the divergent sequences and different disulfide pairings, the tertiary structures of defensins from ⁇ and ⁇ families are quite similar.
  • Characteristic of the overall fold is a three- stranded anti-parallel ⁇ -sheet constrained by three disulfide bonds.
  • a sequence alignment of known ⁇ -defensins from human, mouse, rhesus macaque, rabbit, guinea pig, and rat (Fig. 1) reveals seven conserved residues, the six Cys residues and the glycyl residue located within the second ⁇ -strand of the molecule.
  • Defensins a group of small cationic proteins featuring a ⁇ -sheet-rich fold and a framework of three disulfide bonds, have attracted considerable interest in recent years.
  • Defensins function not only in the innate immune system against infectious microbes, but also in adaptive immunity by activating certain types of immune cells.
  • ⁇ - and ⁇ -defensins two classes of defensins, termed ⁇ - and ⁇ -defensins, have been identified, which differ with respect to their tissue specificities, amino acid composition and linkage pattern of six conserved cysteine residues.
  • HNP1-4 human neutrophil peptide 1-4
  • HNP1-3 accounting for 5-7% of the total neutrophil protein, are two orders of magnitude more abundant than HNP4.
  • the other two human ⁇ -defensins (HD5-6) were initially found in Paneth cells in the small intestine and secreted into the gut lumen in response to bacterial stimulation.
  • HNP1-3 differ only at the N-terminal residue.
  • HNP4, HD5, and HD6, show less sequence similarity with HNP1-3 or each other.
  • the biological functions of human ⁇ -defensins are diverse. Members of the family exhibit broad antimicrobial activities at micromolar concentrations against bacteria, fungi, and certain enveloped viruses.
  • HNP1-3 have been reported to suppress HIV-I infection in vitro.
  • HNP4 has anti-adrenocorticotropic hormone (ACTH) activity at nanomolar concentrations.
  • ACTH anti-adrenocorticotropic hormone
  • Antimicrobial peptides are a well-recognized component of the innate immune repertoire at mucosal surfaces.
  • the defensins are a major family of antimicrobial peptides with two subfamilies, the alpha- and beta- defensins, which differ by the pairing arrangement of their six invariant cysteines into three disulfide bridges.
  • defensins exert multiple additional activities, including chemoattraction for cells of the adaptive immune system and the initiation of cytokine release.
  • the alpha-defensins are made by neutrophils during hematopoiesis (human neutrophil peptides HNPl to -4) and by select epithelial cells (human defensins 5 and 6).
  • HD5 is constitutively produced by Paneth cells in the small intestine and the squamous epithelium of the vagina and ectocervix, but expression is regulated in the endocervix and endometrium both by female hormones and by infection. Recently, HD5 has also been demonstrated in testis.
  • Defensins like other antimicrobial peptides, are synthesized as preproproteins, which must be proteolytically cleaved to liberate potent antimicrobial peptides.
  • the mature peptides have a broad spectrum of antimicrobial activity via disruption of microbial target membranes, and their N-terminal processing is an important factor in their functional activity.
  • processing of the neutrophil defensin prepropeptides only occurs intracellularly by an as-yet-unidentified enzyme, some epithelial defensins are modified after their secretion.
  • Paneth cell-derived HD5 is stored as a propeptide and is only cleaved upon secretion by Paneth cell-derived trypsin.
  • ⁇ -defensins include polypeptides, pre- proproteins, processed proteins, polymorphic variants, alleles, and mutants that have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 15, 20, 25, 30 or more amino acids, to an amino acid sequence of a human ⁇ -defensin as described herein, e.g., ⁇ -defensins 1-6, preferably ⁇ -defensins 1, 2, 3, 4, and 5.
  • ⁇ -defensins include polypeptides, preproproteins, processed proteins, polymorphic variants, alleles, mutants and interspecies homologs that have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 15, 20, 25, 30 or more amino acids, to an amino acid sequence of an ⁇ -defensin from humans and other animals as described herein, e.g., mouse defensins, macaque defensins, rabbit defensins, guinea pig defensins, and rat defensins.
  • defensins of the present invention can be further characterized by their ability to (1) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding an alpha-defensin protein, and conservatively modified variants thereof; or (2) have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200,
  • nucleic acid 500, 1000, or more nucleotides, to an alpha-defensin nucleic acid.
  • Naturally occurring furin inhibitors and alpha-defensins can be purified from any furin inhibitor or alpha-defensin source, e.g., from myeloid, airway, and intestinal cells, from human tissue such as neutrophils, pholmorphonuclear leukocytes, PBMC, or other phagocytic cells; and from epithelial tissue and other furin inhibitor or alpha-defensin- expressing tissue as well as from furin inhibitor or defensin-expressing tissue of other mammals, such as mouse, macaque, rabbit, guinea pig and rat.
  • naturally occurring alpha-defensins are purified from neutrophil and macrophage myeloid cells.
  • Such purification methods include large scale purification, e.g., isolation of neutrophils from whole blood, e.g., obtained from a blood bank or medical waste products such as blast-relief apharesis from patients having acute myelogenous leukemia, and purification using known peptide purification methods for positively charged peptides.
  • the polypeptides may be purified using chromatographic procedures, such as reverse phase HPLC, gel permeation, ion exchange, e.g., anionic exchange, size exclusion, affinity, partition, or countercurrent distribution.
  • Methods of purifying defensins are described in Raj et al. 2000 Biochem J 347:633-641, and Ganz et al.
  • Furin inhibitors and alpha-defensins can also be obtained using known cell culturing techniques to culture alpha-defensin-producing cells and to isolate the alpha-defensin molecules using the methods described herein.
  • Suitable defensin- producing cell lines include, but are not limited to, CD8+ T lymphocytes, neutrophil and myeloid cell lines for human alpha-defensins 1-4 and intestinal cell lines for human alpha-defensins 5-6.
  • Furin inhibitors and alpha-defensin molecules can also be obtained from commercial sources, such as Calbiochem, American Peptide, and Bachem.
  • This invention relies, in part, on routine techniques used in the field of recombinant genetics.
  • Basic texts disclosing general recombinant methods suitable for use in this invention include, but are not limited to, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).
  • Furin inhibitor and alpha-defensin nucleic acids, polymorphic variants, orthologs, and alleles that are substantially identical to an amino acid sequence described herein can be isolated using furin inhibitor or alpha-defensin nucleic acid probes and oligonucleotides under stringent hybridization conditions, by screening libraries.
  • expression libraries can be used to clone furin inhibitor or alpha-defensin protein, pre-proprotein, polymorphic variants, orthologs, and alleles by detecting expressed homologs immunologically with antisera or purified antibodies made against a furin inhibitor or alpha-defensin or portions thereof.
  • a source that is rich in furin inhibitor or alpha-defensin RNA 5 e.g., PBMCs, polymorphonuclear leukocytes, neutrophils, lymphoblastoid cell lines, and other phagocytic cells.
  • the cells can be primary cells or cell lines.
  • the mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
  • Methods for making and screening cDNA libraries are well known (see, e.g., Gubler et al. 1983 Gene 25:263-269; Sambrook et al., supra; Ausubel et al., supra).
  • the DNA is extracted from the tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb.
  • the fragments are then selected by gradient centrifugation and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro.
  • Recombinant phage are analyzed by plaque hybridization as described in Benton and Davis 1977 Science 196:180-182. Colony hybridization is carried out as generally described in Grunstein et al. 1975 Proc Natl Acad Sci USA 72:3961-3965.
  • Degenerate oligonucleotides can be designed to amplify furin inhibitor or alpha-defensin homologs using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of furin inhibitor or alpha-defensin encoding mRNA in physiological samples, for nucleic acid sequencing, or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • the gene for a furin inhibitor or alpha-defensin polypeptide may typically be cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • These intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors.
  • homologous recombination can be used to activate a native furin inhibitor or alpha-defensin gene.
  • furin inhibitor or alpha-defensin polypeptides of the present invention are relatively short in length, they can be prepared using any of a number of chemical peptide synthesis techniques well known to those of ordinary skill in the art, including both solution methods and solid phase methods, with solid phase synthesis being presently preferred.
  • a suitable approach for chemically synthesizing defensin polypeptides is disclosed by Raj et al. 2000 Biochem J347:633-641.
  • solid phase synthesis in which the C-terminal amino acid of the peptide sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for preparing the furin inhibitor or alpha-defensin polypeptides of the present invention.
  • Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis. Synthesis, Biology (Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284.(1980)); Merrifield et al. 1963 J Am Chem Soc 85:2149-2156; and Stewart et al., Solid Phase Peptide Synthesis (2nd ed., Pierce Chem. Co., Rockford, IL (1984)).
  • Solid phase synthesis is started from the carboxy- terminal end ⁇ i.e., the C-terminus) of the peptide by coupling a protected amino acid via its carboxyl group to a suitable solid support.
  • the solid support used is not a critical feature of the present invention provided that it is capable of binding to the carboxyl group while remaining substantially inert to the reagents utilized in the peptide synthesis procedure.
  • a starting material can be prepared by attaching an amino- protected amino acid via a benzyl ester linkage to a chloromethylated resin or a hydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin.
  • halomethyl resins such as chloromethyl resin or bromomethyl resin
  • hydroxymethyl resins such as phenol resins, such as 4-( ⁇ -[2, 4 dimethoxyphenyl]-Fmoc-aminomethyl) phenoxy resin
  • tert- alkyloxycarbonyl-hydrazidated resins such as tert- alkyloxycarbonyl-hydrazidated resins, and the like.
  • the acid form of the peptides of the present invention may be prepared by the solid phase peptide synthesis procedure using a benzyl ester resin as a solid support.
  • the corresponding amides may be produced by using benzhydrylamine or methylbenz hydrylamine resin as the solid support.
  • BHA or MBHA resin is used, treatment with anhydrous hydrofluoric acid to cleave the polypeptide from the solid support produces a polypeptide having a terminal amide group.
  • the ⁇ -amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive ⁇ -amino function.
  • Certain amino acids also contain reactive side-chain functional groups (e.g., sulfhydryl, amino, carboxyl, hydroxyl, etc.) which must also be protected with appropriate protecting groups to prevent chemical reactions from occurring at those sites during the polypeptide synthesis.
  • Protecting groups are well known to those of skill in the art. See, for example, The Peptides: Analysis, Synthesis, Biology. Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, N. Y. (1981)).
  • the peptide can be cleaved and the protecting groups removed by stirring the insoluble carrier or solid support in anhydrous, liquid hydrogen fluoride (HF) in the presence of anisole and dimethylsulfide at about 0 0 C for about 20 to 90 minutes, preferably 60 minutes; by bubbling hydrogen bromide (HBr) continuously through a 1 mg/10 mL suspension of the resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting groups selected; or, by incubating the solid support inside the reaction column used for the solid phase synthesis with 90% trifluoroacetic acid, 5% water and 5% triethylsilane for about 30 to 60 minutes.
  • HF liquid hydrogen fluoride
  • TFA trifluoroacetic acid
  • Other deprotection methods well known to those of skill in the art may also be used.
  • cysteine residues present in the known naturally occurring alpha- defensins.
  • the disulfide linkages formed by the cysteine residues are between the first and sixth cysteine, the second and fourth cysteine, and the third and fifth cysteine, wherein the cysteines are numbered one through six from the N-termimis to the C terminus of the peptide.
  • disulfide bonds between the cysteine residues present in alpha- defensin molecules can be carried out using known methods. For example, in Raj et al., supra, three distinct thiol protecting groups and a three stage process is used to form the disulfide bonds in order to minimize undesired side-products. In order to provide the proper orientation for bond formation, a disulfide bridge is first formed between a N and C terminal cysteine (See Hill et al. 1991 Science 25:1481-1485 and Pardi et al. 1992 Biochemistry 31:11357-11364).
  • the cationic polypeptides e.g., furin inhibitor and alpha-defensin polypeptides, of the present invention can be isolated and purified from the reaction mixture by means of peptide purification methods well known to those of skill in the art, e.g., purification, methods described above.
  • the furin inhibitor and alpha-defensin polypeptides can be modified to remove protease cleavage sites ⁇ e.g., trypsin and chymotrypsin cleavage sites) ⁇ see, e.g., Selsted et al. 1985 J Clin Invest 76:1436-1439). Typically, this is done by replacing the amino acid recognized by the protease ⁇ e.g., arginine) with an amino acid of like-kind that is not recognized by the protease.
  • modifications ⁇ e.g., additions, deletions and substitutions) fall within the scope of the present invention in that such modified furin inhibitor and alpha-defensin polypeptides can be used in the compositions and methods of the present invention.
  • Site directed mutagenesis is well known in the art and is described in the following references, e.g., Botstein et al. 1985 Science 229:1193-1201 and Kunkel, Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. MJ. eds., Springer Verlag, Berlin (1987)); mutagenesis using uracil containing templates (Kunkel 1985 Proc Natl Acad Sd USA 82:488-492); oligonucleotide-directed mutagenesis (Zoller and Smith 1983 Methods in Enzymol 100:468-500; phosphorothioate-modified DNA mutagenesis (Taylor et al.
  • Oligonucleotide directed mutagenesis can also be used to introduce site-specific mutations in a nucleic acid sequence of interest. Examples of such techniques are described in the references set forth above and in, e.g., Reidhaar-Olson et al. 1988 Science 241:53-57.
  • cassette mutagenesis can be used in a process that replaces a small region of a double stranded DNA molecule with a synthetic oligonucleotide cassette that differs from the native sequence.
  • the oligonucleotide can contain, e.g., completely and/or partially randomized native sequence(s). 5. Purification of Recombinant Furin Inhibitors and A ⁇ pha-Defensins
  • Recombinant furin inhibitors and alpha-defensins can be purified from any suitable expression system, as described below.
  • the furin inhibitor or alpha-defensin protein can be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others ⁇ see, e.g., Scopes, Protein Purification: Principles and Practice (1982); Ausubel et al., supra; and Sambrook et al., supra).
  • furin inhibitor or alpha-defensin protein can be purified using immunoaffnity columns.
  • proteins having established molecular adhesion properties can be reversibly fused to the furin inhibitor or alpha- defensin protein.
  • the appropriate ligand or substrate e.g., anti-furin inhibitor or anti- defensin antibodies or a negatively charged substrate
  • furin inhibitor and alpha-defensin proteins can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity.
  • furin inhibitor or alpha-defensin proteins can be purified using immunoaffnity columns.
  • Recombinant furin inhibitor or alpha-defensin proteins can be purifed from any suitable source, include yeast, insect, bacterial, and mammalian cells.
  • furin inhibitors and alpha-defensins, nucleic acids encoding furin inhibitors and alpha-defensin proteins, and small organic molecules that increase furin inhibitor or alpha-defensin activity or expression can be used to inhibit papillomavirus, e.g., by blocking the initial stage of papillomavirus infection.
  • Furin inhibitors and alpha- defensins can be used therapeutically or prophylactically in a person.
  • the furin inhibitor and alpha-defensin proteins, nucleic acids encoding furin inhibitor and alpha-defensin proteins, and small organic molecules can also be used to stop disease progression in a papillomavirus infected subject.
  • furin inhibitor and alpha-defensins can also be used prophylactically, e.g., before and after exposure or suspected exposure to papillomavirus to prevent infection or to prevent transmission, e.g., maternal transmission of the virus to the fetus.
  • the compounds of this invention have been found to possess valuable pharmacological properties. They exhibit an antiviral effect against papillomavirus. This effect can be demonstrated, for example, using the psuedotyped papillomavirus infectivity assay.
  • these compounds can be used to treat or prevent papillomavirus infection.
  • the compounds are particularly useful as microbicides.
  • the compounds can be used in in vitro diagnostics to identify whether an individual has a predisposition to papillomavirus infection.
  • the compounds of this invention are generally administered to animals, including but not limited to livestock, household pets, and humans.
  • the pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.
  • The, compounds of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral ⁇ e.g., oral) or topical application which do not deleteriously react with the active compounds.
  • conventional excipients i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral ⁇ e.g., oral) or topical application which do not deleteriously react with the active compounds.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., spermicides.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which
  • injectable, sterile solutions preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories.
  • Ampoules are convenient unit dosages.
  • enteral application particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules.
  • a syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.
  • Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the new compounds and use the lyophilizates obtained, for example, for the preparation of products for injection.
  • viscous to semisolid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water.
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a Freon.
  • Topical administration is preferred.
  • the compounds of this invention are dispensed in unit dosage form comprising 0.1 mg to 1 g in a pharmaceutically acceptable carrier per unit dosage. They are incorporated in topical formulations in concentrations of about a small to a great weight percent.
  • the dosage of the compounds according to this invention generally is 1 ⁇ g to lO mg/kg/day, preferably 10 ⁇ g to 1 mg/kg/day, when administered to patients, e.g., humans to treat or prevent papillomavirus infection analogously to carageenans.
  • the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular situs and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol. 2. Combination Therapies hi numerous embodiments, the furin inhibitors and/or alpha-defensins of the present invention may be administered in combination with one or more additional compounds or therapies.
  • furin inhibitors and/or alpha-defensins can be co- administered, or one or more furin inhibitors or alpha-defensins can be administered in conjunction with one or more therapeutic compounds, hi one embodiment, the first therapeutic agent is one that is used to prevent or treat papillomavirus infection, hi another embodiment, the other therapeutic agent is an agent used to prevent or treat another sexually transmitted disease.
  • Suitable medicinal agents for use in combination with the furin inhibitors and/or alpha-defensins of the present invention include, but are not limited to, protease inhibitors.
  • suitable medicinal agents include zidovudine, didanosine, stavudine, interferon, lamivudine, adefovir, nevirapine, delaviridine, loviride, saquinavir, indinavir and AZT.
  • Other suitable medicinal agents include, but are not limited to, antibiotics or other anti-viral agents, e.g., acyclovir.
  • Warts may appear at any age but are most frequent in older children and uncommon in the elderly. Warts may be single or multiple and may develop by autoinoculation. Appearance and size depend on the location and on the degree of irritation and trauma. The course may be variable. Complete regression after many months is usual, but warts may persist for years and may recur at the same or different sites. Some warts can become malignant (See Table C).
  • HPV27, HPV29, HPV37, HPV38, HPV46, HPV48 and HPV49 are also recovered from skin warts.
  • Mosaic warts are plaques formed by the coalescence of myriad smaller, closely set plantar warts. Filiform warts are long, narrow, frondlike growths usually on the eyelids, face, neck, or lips. This morphologically distinctive variant of the common wart is benign and easy to treat. Flat warts (smooth, flat- topped, yellow-brown papules) are more common in children and young adults, most often on the face and along scratch marks, and develop by autoinoculation. Variants of the common wart that are of unusual shape (e.g., pedunculated, or resembling a cauliflower) are most frequent on the head and neck, especially on the scalp and bearded region.
  • unusual shape e.g., pedunculated, or resembling a cauliflower
  • Anogenital warts caused by HPV types 6, 11, 16, 18, 31, 33, and 35 are transmitted sexually and have an incubation period of 1 to 6 months. Cervical wart infections caused by types 16 or 18 have been implicated as a cause of cervical intraepithelial neoplasia and cervical cancer. Types 16 and 18 HPV generally do not cause external genital warts, which are usually caused by types 6 and 11. The 15 types thought to cause cervical cancer are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68 and HPV73 (see Munoz N. et al. 2004 Int J Cancer 111: 278-285).
  • Genital warts usually appear as soft, moist, minute pink or gray polyps that enlarge, may become pedunculated, and are usually found in clusters.
  • the surfaces resemble the surface of a cauliflower.
  • men they occur most commonly on warm, moist surfaces in the subpreputial area, on the coronal sulcus, within the urethral meatus, and on the penile shaft, hi women, the vulva, the vaginal wall, the cervix, and the perineum may become involved. They are particularly common in the perianal region and rectum in homosexual men and may be more severe and difficult to treat in irnmunosuppressed patients. Growth rates vary, but pregnancy, immunosuppression, or maceration of the skin may accelerate both the growth of individual lesions and their spread.
  • Genital warts usually can be identified by their appearance but must be differentiated from the flat-topped condyloma lata of secondary syphilis. Biopsies of atypical or persistent warts may be necessary to exclude carcinoma. Cervical warts can be detected only at colposcopy and should not be treated until Papanicolaou smear results are available. Cytologic and Histologic Analyses
  • cervical samples for Papanicolaou testing are deposited in sectioning compound that can be hardened for thin sectioning.
  • Thin-layer slides are prepared according to the manufacturer's specifications, screened by cytotechnologists, and reviewed by pathologists at designated cytology laboratories. The results are classified as unsatisfactory if more than 60 percent of the target area of the slide has " no epithelial cells. Cellular changes are classified according to the Bethesda system (Kurman, R. J. et al. 1991 Obset Gynecol 77:779-782). Cervical-biopsy specimens are fixed in 10 percent formalin and embedded in paraffin.
  • the probes can be labeled with radioactive or nonradioactive compounds.
  • Southern hybridization of DNAs extracted from fresh or fresh- frozen tissues serves well. Dot-blot and slot-blot hybridization techniques are another example. Reverse-blot hybridization also serves for HPV detection. In tissue in situ hybridization tests, cells or tissue sections on slides are hybridized using radioactive or nonradioactive probes. This is another example for HPV detection.
  • PCR polymerase chain reaction
  • Cutaneous or genital specimens are prepared for PCR according to standard methods.
  • DNA is amplified with the use of HPV type-specific primers based on HPV Ll, or alternatively E6, and E7 genes.
  • PCR products are identified by hybridization with the use of HPV type- and gene-specific oligonucleotides.
  • a positive result is defined as any signal that exceeds the background level associated with an HPV negative sample of human DNA.
  • Appropriate negative and positive controls are included in each assay. Any sample that tests positive for at least two genes is considered positive. Any sample that tests positive for only one gene is considered positive if, on retesting, it is positive for two or three genes or the same single gene.
  • Serological assays are used to quantify serum HPV antibodies using immunoassays such as competitive assays, non-competitive assays and Western blot analysis.
  • HPV polypeptides are used as capture agents to detect a target antibody.
  • a serological assay based on virus like particles (VLPs) is the most extensively used and validated method for type-specific serodiagnosis of HPV infection (Kirnbauer et al., 1994 J Natl Cancer Inst 86: 494-499).
  • Serum antibodies directed against an HPV of interest can be quantified by a variety of immunoassay methods and the immunoassays can be performed in any of several configurations.
  • Modulators of furin inhibitors or alpha-defensins are useful for inhibiting papillomavirus infection.
  • administration of modulators can be used to treat papillomavirus infected individuals or protect individuals at risk of papillomavirus infection.
  • the compounds tested as modulators of furin inhibitors or alpha-defensins can be any small organic molecule or a biological entity, such as a protein, sugar, nucleic acid or lipid.
  • the method for determining whether a compound modulates furin inhibitor or alpha-defensin activity in a cell comprises a) contacting a furin inhibitor or alpha-defensin producing cell with the compound and b) determining the functional effect of the compound on furin inhibitor or alpha-defensin activity.
  • Compounds that act as modulators of furin inhibitors or alpha-defensins can be used to inhibit papillomavirus infection in a human by administering to the human the compounds to augment furin inhibitor or alpha- defensin activity.
  • Papillomaviruses comprise a large family of non-enveloped DNA viruses that include the oncogenic PV types that are the causative agents of human cervical cancer.
  • papillomaviruses are taken into the cell via endocytosis, and must escape from the endosomal compartment to the cytoplasm to initiate infection.
  • this step is dependent upon the site-specific enzymatic cleavage of the papillomavirus minor virion protein, L2, at a consensus form recognition site.
  • Furin preferentially recognizes the cleavage site sequence R-X-K/R-R (Duckert, P. et al. 2004 Protein EngDes SeI 17:107-112; Krysan, D. J. et al. 1999 J Biol Chem 274 23229-23234).
  • the Pl arginine residue within the putative furin recognition site in HPVl 6 L2 was mutated to a serine residue (16-R12S), abrogating the consensus cleavage site.
  • This mutation reduced the infectivity of HPVl 6 to negligible levels (Fig. 2D).
  • the analogous mutation within the BPVl L2 (B-R9S) produced a similar result (Fig. 2E).
  • the furin binding pocket does not well tolerate a non-arginine residue at the Pl position.
  • the infectivity of the L2 mutant pseudoviruses followed their predicted susceptibility to furin cleavage.
  • Both of the L2 mutant pseudovirions incorporated levels of L2 and encapsidated DNA indistinguishable from wild type, ruling out these alternate possibilities for their loss of infectivity.
  • Furin can cleave L2 in vitro
  • L2 is a furin substrate
  • Wild-type BPVl L2 or B-R9S L2 constructs were made that contained the HA epitope tag at either their C- or N-terminus (designated CHA and NHA, respectively). These L2 proteins were expressed in cells and partially purified. As expected from the location of the cleavage site near the N-terminus, furin treatment of the tagged wild type proteins liberated the N-terminal HA tag (Fig. 3) as shown by the lack of detection with an antibody directed against the HA epitope.
  • the PV entry process has extremely slow kinetics, with disassembly being initially detected at 6-8 hours post-entry (Day, P. M. et al. 2004 Proc Natl Acad Sd U S A 101:14252-14257).
  • the C-terminally tagged L2 protein was predominantly detected in the nucleus, along with the pseudoviral genome (Fig. 4A), as previously described.
  • the N-terminally tagged L2 protein was not detectable in the nucleus (Fig. 4B), although it was seen in cytoplasmic vesicles.
  • the pseudoviral genome was detected in the nucleus at equivalent levels for the CHA- and NHA -containing pseudoviruses (Fig. 4C, D), and both pseudoviruses were equally infectious.
  • the N-terminal tag is removed from the L2 protein in vivo, prior to its entry into the nucleus during the infectious process.
  • furin cleavage of L2 is essential for the correct trafficking of the genome and L2 out of the endocytic compartment prior to their transit into the nucleus. Pre-cleavage of L2 can bypass the requirement for cellular furin.
  • pseudoviruses when pseudoviruses are produced, they initially form as immature particles with a loose conformation and then undergo a maturation process, resulting in particles with a more compact conformation (Buck, C. B. et al.
  • Immature (iPV) or mature (mPV) BPVl pseudoviruses were either treated with furin (furin- tx.) or left untreated (untx.). The infectivity of these preparations was evaluated on untreated HeLa cells (-FI) or HeLa cells treated with furin inhibitor (+FI). The percent of GFP+ cells is shown. Furin does not affect production of infectious PV
  • furin during PV entry contrasts sharply with the previously described role of furin in the production of a variety of infectious enveloped viruses.
  • furin cleavage of an envelope protein must occur within the Golgi complex to yield the mature, fusogenic form of the protein which is expressed on the cell surface prior to particle budding (Nagai, Y. 1993 Trends Microbiol 1:81-87; Nakayama, K. (1997) Biochem J 327:625-635).
  • papillomaviruses are non-enveloped viruses, they would not be expected to utilize furin during virus production. Indeed, furin inhibition during pseudovirus production, in contrast to pseudovirus entry, did not affect the titer of the resultant virus (Fig. 6). Discussion
  • furin as an L2 -processing protease that is critical for the establishment of PV infection. Based upon genetic, biochemical, and cytological evidence, we conclude that furin removes the N ⁇ -terminus of the L2 protein early in the infectious entry pathway, and that this cleavage is essential for processes leading to L2-mediated endosome escape. L2 that is not cleaved by furin does not leave the endocytic compartment, and the accompanying genome is likewise withheld.
  • Furin is a type I membrane protein localized predominantly in the trans-Golgi network (TGN). However, it has also been demonstrated to be present in an active form both on the cell surface and within the endosomal compartment, and thus is present in cellular locations where it could intersect with incoming viral capsids (Thomas, G. 2002 Nat Rev MoI Cell Biol 3:753-766; Mayer, G. et al. 2004 J Histochem Cytochem 52:567- 579; Molloy, S. S. et al. 1999 Trends Cell Biol 9:28-35). These proteases play a role in the endoproteolytic processing of cell-encoded precursor proteins in mammalian cells within the TGN.
  • furin digestion of immature capsids prior to their incubation with cells, overcame the inhibition of cellular furin with dec.-RVKR-cmk.
  • Furin cleavage of L2 could serve several functions. First, it could enable the release of the L2-genome complex from Ll, since Ll does not appear to exit from the endosomal compartment (Day, P. M. et al. 2004 Proc Natl Acad Sd USA 101:14252-14257). Second, it could promote L2 binding to a specific receptor. Interestingly, a syntaxin 18 binding site on L2 has been mapped to a peptide immediately downstream of the cleavage site (Bossis, I. et al. 2005 J Virol 79:6723-6731). It will be interesting to determine if the furin-cleaved form of L2 preferentially interacts with syntaxin during the entry process.
  • L2 immunogens exposing this cleavage site might be particularly effective at inducing broadly cross-neutralizing papillomavirus antibodies.
  • Furin inhibition by dec.-RVKR-cmk prevents the escape of L2 and the genome from the endosome, but does not appear to interfere with virion trafficking or uncoating. It was previously reported that an N-terminally truncated BPV L2 could not produce infectious virions (Roden, R. B. et al. 2001 J Virol 75:10493-10497). Like our results with the R9S mutation, truncated L2 was incorporated into the virion, and the viral genome was encapsidated at wild type levels. The truncated protein was initiated just after the furin cleavage site. A priori, we would expect this mutant to behave like the in vitro digested pseudovirus, and effectively lead to infection.
  • a possible explanation for the discrepancy is that initiation at the ninth amino acid results in a modified residue that cannot participate in downstream events. Either the additional N-terminal methionine residue is not cleaved or the next residue, an alanine, is modified. N-terminal alanines are often acetylated. An alternative explanation is that the truncated L2 protein does not correctly participate in a necessary pre-cleavage conformational change, which is mimicked in the immature capsids.
  • Furin belongs to the family of proprotein convertases (PCs), which are subtilisin- like eukaryotic endoproteases (reviewed in Gensberg, K. et al. 1998 Semin Cell Dev Biol
  • Keratinocytes which are the natural host cell for PVs, are known to express furin, PACE4, PC5/6, and PC7/8 (Pearton, D. J. et al. 2001 Exp Dermatol 10:193-203).
  • CHO cells express PC7/8, but not PACE4 (Pinnix, I. et al. 2001 Faseb J 15:1810-1812), while LoVo express PACE4 (Seidah, N. G. et al. 1994 Biochimie 76:197-209; Nakayama, K. (1997) Biochem J 327:625-635).
  • PC5/6 has not been ruled out as a putative physiologic PC for cleavage of the L2 sequence.
  • furin activity is required for PV infection provides the basis for the use of furin (or furin/PC5/6) inhibitors as topical microbicides to prevent genital infection by HPV.
  • the inhibitors might also inhibit viruses, such as HIV, whose production depends on furin (Zhong, M. et al. 1999 J Biol Chem 274:33913-33920; Kibler, K. V. et al. 2004 J Biol Chem 279:49055-49063).
  • Furin inhibitors are under development, to prevent the activation of bacterial toxins such as anthrax toxin (Peinado, J. R. et al. 2004 Biochem Biophys Res Commun 321:601-605). Results from mice protected against anthrax toxemia by systemic treatment with furin inhibitors suggest that topical application might have acceptable side effects (Sarac, M. S. et al. 2004 Infect Immun 72:602-605).
  • HPVs sexually-transmitted human papillomaviruses
  • pseudoviruses papillomaviral vectors
  • human alpha defensins 1-3 (known as human neutrophil peptides (HNPs) 1-3) and human alpha defensin 5 (HD-5) are potent antagonists of infection by both cutaneous and mucosal papillomavirus types, hi contrast, human beta defensins 1 and 2 displayed little or no anti-HPV activity.
  • HD-5 was particularly active against sexually-transmitted HPV types, with 50% inhibitory doses in the high ng/ml range.
  • Microscopic studies of pseudovirus inhibition by the alpha defensins revealed that they block virion escape from endocytic vesicles, but not virion binding or internalization.
  • HPV inhibition assay Because HPVl 6 is the type most commonly associated with malignant cervical lesions worldwide (reviewed in Bosch, F. X. & de Sanjose, S. 2003 J Natl Cancer Inst Monogr 31:3-13), it was chosen as a model HPV to screen various compounds for their capacity to inhibit infection.
  • alpha defensins 1-3 also known as human neutrophil peptides (HNPs) 1-3
  • HNP -4 was nearly an order of magnitude less active
  • alpha defensin-5 was about an order of magnitude more active relative to HNPs 1-3.
  • HNP-I and HD-5 were selected for follow-up study. They were both found to inhibit HPVl 6 PsV transduction of other cell types, such as the human embryonic kidney line 293TT, the spontaneously immortalized keratinocyte line HaCaT, and murine C127 fibroblast cells (Table 3).
  • a separate set of experiments showed that HNP-I was equally effective against mature and immature (Buck, C. B. et al. 2005 J Virol 79:2839-2846) PsV.
  • the presence of serum has previously been reported to interfere with the ability of HNP-I to inhibit viral infection of some cell types in vitro (Daher, K. A. et al. 1986 J Virol 60:1068-1074; Chang, T. L. et al. 2005 J CHn 115:765-773). Therefore, we performed the PsV inhibition assay using serum-free medium. HNP-I and HD-5 were only slightly more effective against the HPVl 6 PsV in serum-free conditions (Table 3).
  • HNP-I was tested in a standard bovine papillomavirus type 1 (BPVl) focal transformation assay using murine C 127 cells (Dvoretzky, I. et al. 1980 Virology 103:369-375; Pastrana, D. V. et al. 2001 Virology 279:361-369).
  • BPVl bovine papillomavirus type 1
  • HPVs 18 and 31 are mucosotropic types associated with a high risk of cervical cancer.
  • HPV6 is a low-risk sexually-transmitted type that belongs to the same genus as the high-risk types. HPV6 typically infects genital skin, where it can cause genital warts.
  • Several cutaneous PsV types have also been developed.
  • HPV5 commonly infects non-genital skin and does not cause discernable lesions in most individuals.
  • Two animal papillomaviruses, BPVl and cottontail rabbit papillomavirus (CRPV) cause non- genital skin lesions in their natural hosts.
  • Each PsV type was tested for inhibition by serial dilutions of HNP-I or HD-5.
  • the PsVs differed in their susceptibility to inhibition by the two alpha defensins in a manner correlating with the epithelial region they typically infect (Figure 8).
  • the genital HPV types were highly susceptible to inhibition by HD-5, with IC50 values of about 0.6 ⁇ g/ml, but were much less susceptible to inhibition by HNP-I, with IC50 values ranging from 2.9 to 7.2 ⁇ g/ml.
  • the non-genital papillomavirus types showed an intermediate degree of inhibition by both HNP-I and HD-5 ( Figure 8 panel C).
  • HNP-I is thought to prevent some families of enveloped viruses from infecting cultured cells, at least in part by interfering with initial virion binding to cells. We therefore determined, by flow cytometric analysis, whether HNP-I could block the binding of GFP-tagged HPV16 virions to HeLa cells. No impairment of binding of the GFP -tagged virions was observed in the presence of HNP-I.
  • L2 and the viral genome (or reporter plasmid) escape from a vesicular compartment, allowing them to traffic together to a subnuclear site known as an ND-10 domain (Day, P. M. et al. 2004 Proc Natl Acad Sd USA 101:14252-14257).
  • alpha defensins a group of cysteine- and arginine-rich antimicrobial peptides.
  • Defensins have been implicated in the innate antibacterial and antiviral defenses of multicellular organisms ranging from plants to humans (reviewed in Selsted, M. E. & Ouellette, A. J. 2005 Nat Immunol 6:551-557; Ganz, T. 2003 Nat Rev Immunol 3:710-720; Lehrer, R. I. 2004 Nat Rev Microbiol 2:727-738).
  • human defensins are typically expressed in leukocytes and epithelial tissues.
  • the highly homologous HNPs 1-3 are abundant in neutrophil granules, along with the more distantly related and less abundant HNP-4.
  • HNPs can be released into extracellular fluids, such as cervicovaginal secretions, where they can reach effective microbicidal concentrations (Valore, E. V. et al. 2002 Am J Obstet Gynecol 187:561-568; Panyutich, A. V. et al. 1993 J Lab Clin Med 122:202-207; Hein, M. et al 2002Am J Obstet Gynecol 187:137-144).
  • Human alpha defensins 5 and 6 are secreted by specialized
  • HD-5 paneth cells resident in the crypts of the small intestine.
  • HD-5 is also known to be expressed in other epithelial tissues.
  • Other studies have reported HD-5 expression in the testes and male urethra, as well as in breast milk and keratinocytes of the oral and airway mucosa (Com, E. et al. 2003 Biol Reprod 68:95-104; Porter, E. et al.
  • HD-5 is expressed as a pro-peptide that is proteolytically processed to yield a variety of mature HD-5 isoforms.
  • this processing is accomplished by tryptic digestion during or just after secretion into the intestinal lumen (Ghosh, D. et al. 2002 Nat Immunol 3:583-590).
  • a recent report has shown that, in the male urethra, HD-5 processing may depend on proteases secreted by neutrophils (Porter, E. et al. 2005 Infect Immun 73:4823-4833).
  • alternative HD-5 isoforms other than the dominant tryptic form we have studied here, are active against papillomaviruses remains to be determined.
  • HD-5 may function to dampen non-productive infection of more superficial keratinocytes, which could trigger a protective adaptive immune response to the virus.
  • women expressing high levels of alpha defensins (Linzmeier, R. M. & Ganz, T. 2005 Genomics 86:423-430; Aldred, P. M. et al. 2005 Hum MoI Genet 14:2045- 2052) (or defensin-processing proteases) may be preferentially resistant to initial HPV infection and persistence, particularly if persistence requires multiple rounds of autoinoculation (Winer, R. L. et al. 2003 Am J Epidemiol 157:218-226; Winer, R. L. et al.
  • alpha defensins might function as safe, non-immunogenic, and non-inflammatory topical microbicides is bolstered by the fact that the defensins can normally be found at virucidal or nearly virucidal concentrations in the female genital tract, hi contrast, heparin would not be considered an attractive candidate for use as a topical microbicide because of its anticoagulant activity.
  • the ability of alpha defensins to substantially inhibit in vitro HPV infection, even six hours after cell surface binding, provides the basis for their use as a postcoital microbicide.
  • alpha defensins have previously been found to inhibit HIV-I, herpes simplex viruses, Neisseria gonorrhoeae and Chlamydia trachomatis they could potentially function as broad-spectrum topical microbicides to simultaneously block multiple sexually- transmitted pathogens (Zhang, L. et al. 2002 Science 298:995-1000; Daher, K. A. et al. 1986 J Virol 60:1068-1074; Yasin, B. et al. 1996 Infect Immun 64:709-713; Porter, E. et al. 2005 Infect Immun 73:4823-4833).
  • HPV sexual transmission of HPV
  • the high incidence of initial HPV infection in young adults, and its relative resistance to condom prophylaxis (which must be encouraged in clinical trials) might make it possible to determine microbicide efficacy using relatively small populations of study subjects.
  • defensins exert antibacterial effects primarily by interacting with and destabilizing bacterial membranes. Soon after their initial identification as bactericidal effectors, it was found that alpha defensins could also inactivate certain types of enveloped viruses (Lehrer, R. I. et al. 1985 J Virol 54:467-472; Daher, K. A. et al. 1986 J Virol 60:1068-1074). hi contrast, these reports found the defensins to be inactive against two non-enveloped viruses, echovirus type 11 and reovirus type 3. Therefore, it was proposed that defensins exert antimicrobial effects primarily by direct interaction with viral or bacterial membranes.
  • HNP-I can block HIV-I infection by interfering with cellular signaling in some cell types (Chang, T. L. et al. 2005 J Clin Invest 115:765-773).
  • synthetic HNP-I Bastian, A. & Schafer, H. 2001 Regal Pept 101:157-161
  • pro-HD-5 Gropp, R. et al. 1999 Hum Gene Titer 10:957-964
  • HNP-I was found to noncompetitively inhibit the metalloproteinase activity of LeTx, showing that HNP-I can function as a type of protease inhibitor.
  • a panel of metalloproteinase inhibitor drugs failed to inhibit HPVl 6 PsV transduction of HeLa cells, the concept that HNP-I might block papillomavirus infection by reversibly inhibiting a required cellular protease other than furin remains a possibility.
  • One common feature of the infectious pathways of adenoviruses and papillomaviruses is their ability to destabilize cellular membranes following endocytosis.
  • mutant HNPs with reduced antibacterial activity retain the ability to inhibit papillomaviruses could be useful in the setting of a topical microbicide, where preservation of a healthy vaginal bacterial flora would be a desirable goal.
  • Example 1 Cleavage of the Papillomavirus minor capsid protein L2 at a furin consensus site is necessary for infection Pseudovirus production
  • oligonucleotides used for mutagenesis were as follows, in both cases the reverse complement was also used, but not listed: for HPV16 R12S 5'- GCGCCAAGAGGACCAAGAGCG-CCAGCGCCACCCAGC-3' (SEQ ID NO: 74); for BPV R9S, 5'-GCCCGCAAGAGAGTGAAGAGCGCCAGCGCCTACGACCTG-S' (SEQ ID NO: 75). Construction of CHA-L2 has been previously described (Day, P. M. et al. 2004 Proc Natl Acad Sd U S A 101:14252-14257).
  • HeLa cells and C 127 cells were grown in DMEM supplemented with 10% FBS and antibiotics.
  • FDIl and FDll+furin cells were a kind gift of Dr. Stephen Leppla (NIAID, NIH) (Gordon, V. M. et al. 1995 Infect Immun 63:82-87). They were cultured in DMEM supplemented with 10% FBS, antibiotics and proline. Pseudovirus Infection
  • pepstatin A cathepsin L Inhibitor
  • chymostatin calpeptin
  • ALLN ALLM
  • decanoyl-RVKR-cmk D- VFK-cmk
  • cbz-VNSTLQ-cmk H-AAF-cmk
  • HeLa cells were transfected with the cDNA for either the wild-type CHA-L2 or CHA-BPV R9S-L2 or the wild-type NHA-L2 or NHA-BPV R9S-L2.
  • nuclei were isolated by standard methodology and disrupted by sonication.
  • L2 proteins were immunoprecipitated with a polyclonal anti-L2 antiserum (17/28) conjugated to protein A sepharose (Pierce, Rockford, IL).
  • the sepharose-antibody-L2 complexes were resuspended in furin digestion buffer (100 mM Hepes, ImM CaC12, 0.5% Triton-X-100) and each sample was divided into two equal portions one of which was treated with 5U of furin (Alexis Biochemical, San Diego, CA). All samples were incubated at 37 0 C for 16 hours. Each sample was again divided in two equal aliquots. One of them was immunopurified with anti-HA magnetic beads according to the manufacturer's directions (Miltenyi Biotec, Auburn, CA).
  • Immature and mature pseudoviruses were prepared as previously described (Buck, C. B. et al. 2005 J. Virol. 79:2839-2846). Pseudovirus preparations were clarified by high speed centrifugation of cellular debris and used as a crude preparation. The preparations were adjusted to 100 mM Hepes, ImM CaCl 2 , divided into two aliquots and incubated at 37 0 C for 7 hours. One aliquot was incubated in the presence of 3.5 U of furin and the other was incubated without furin. Following this incubation, pseudovirions were added to HeLa cells and the infection was continued for 72 hours in the absence or presence of 10 ⁇ M of decanoyl-RVKR-CMK. GFP expression was quantified by flow cytometry.
  • Alpha-Defensins Block Papillomavirus Infection Cell culture and pseudovirus production
  • PsV pseudovirus
  • HPV5-based PsV the nucleotide sequences of the Ll and L2 genes of a genomic clone of HPV5 were verified by direct sequencing (Genbank accession number DQ080001). The verified Ll and L2 open reading frames were then reconstructed synthetically (Blue Heron Biotechnology) according to a previously-reported "as different as possible" codon modification strategy (constructs p5Llh and p5L2w) (Buck, C. B. et al.
  • GFP -tagged HPVl 6 capsids were produced using a plasmid, puL2f, expressing an HPV16 L2:GFP fusion protein.
  • the L2:GFP fusion protein was incorporated into capsids at an L1:L2 ratio similar to that seen when capsids were produced using wild-type L2.
  • Binding inhibition studies were performed by applying roughly 5000 capsid equivalents of Ll per cell at 37 0 C for two hours in the presence or absence of varying doses of alpha defensin-1 (known as human neutrophil peptide-1 (HNP-I)), followed by flow cytometric analysis.
  • HNP-I human neutrophil peptide-1
  • HeLa cells were pre-plated overnight in 96-well plates at 6000 cells per well in 50 ⁇ l of DMEM-10.
  • the various candidate inhibitors were serially diluted in DMEM- 10 in a separate 96-well plate at 3 times their final dose. 50 ⁇ l of each dilution were then added to the pre-plated HeLa cells, followed by 50 ⁇ l of virus stock diluted in DMEM- 10 supplemented with 3x antibiotic-antimycotic mixture (Invitrogen). Control experiments showed that the antibiotic mixture had no effect on PsV transduction.
  • PsV doses were calibrated such that between 5 and 25% of cells registered as GFP+ in control conditions.
  • the cells were fed by adding 100 ⁇ l of DMEM- 10 24 hours after PsV inoculation. The cells were incubated for a total of 48 to 56 hours after inoculation then trypsinized and subjected to flow cytometric analysis. For HNP-I and alpha defensin-5 (HD-5), similar inhibition curves were observed when cells were analyzed 75 hours after inoculation. Percent inhibition was calculated using the formula 100 x (1 - (net percentage of cells GFP+ in test / net percentage of cells GFP+ in mock)). The IC50 and 95% confidence interval (CI) for inhibition curves were calculated using Prism 4 (GraphPad Software) to fit a variable slope sigmoidal dose-response curve with top and bottom values constrained at 100 and zero, respectively.
  • Cytotoxicity was defined as a >50% reduction in turnover of WST-I metabolic substrate (Roche) at the time of cell harvest. Cytotoxicity was confirmed by microscopic inspection of cell morphology and density, or by the appearance of alterations in forward scatter, side scatter and autofluorescence profile of the cells on the flow cytometer.
  • the inhibition assay was performed using HeLa cells pre-incubated for two hours with standard doses of various drugs. Drugs were removed 24 hours after PsV inoculation.
  • Tested compounds included the G-protein coupled receptor antagonist pertussis toxin (Yang, D. et al. 2000 J Leukoc Biol 68:9-14; Befus, A. D. et al. 1999 J Immunol 163:947-953); the protein kinase C (PKC) agonists bryostatin-I (Chang, T. L. et al.
  • Microscopy hnmunofluorescent microscopy was performed as previously described (Day, P. M. et al. 2004 Proc Natl Acad Sd USA 101:14252-14257). Where noted, HNP-I (30 ⁇ g/ml) or HD-5 (5 ⁇ g/ml) were added together with pseudovirus inoculum for 24 hours. All images were acquired using a Zeiss LSM 510 confocal system.

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Abstract

La présente invention concerne un procédé permettant d'inhiber une infection à papillomavirus chez un humain, consistant à administrer à un humain exposé à une infection à papillomavirus un inhibiteur de la furine ou une alpha-défensine. L'invention concerne également un procédé permettant d'inhiber une infection à papillomavirus chez un humain, consistant à transfecter une cellule in vivo avec un acide nucléique comprenant une séquence nucléotidique codant pour un inhibiteur de la furine ou une alpha-défensine. L'invention concerne également un procédé permettant de déterminer si un composé module l'activité d'un inhibiteur de la furine ou d'une alpha-défensine dans une cellule. L'invention concerne également un procédé permettant d'inhiber une infection à papillomavirus chez un humain, consistant à lui administrer un composé identifié permettant d'augmenter l'activité d'un inhibiteur de la furine ou d'une alpha-défensine.
PCT/US2006/003844 2005-02-01 2006-02-01 Inhibiteurs de la furine et alpha-defensines destines au traitement ou a la prevention d'une infection a papillomavirus WO2006084131A2 (fr)

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