EP0724650A1 - Selection et utilisation de peptides antiviraux - Google Patents

Selection et utilisation de peptides antiviraux

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Publication number
EP0724650A1
EP0724650A1 EP94923428A EP94923428A EP0724650A1 EP 0724650 A1 EP0724650 A1 EP 0724650A1 EP 94923428 A EP94923428 A EP 94923428A EP 94923428 A EP94923428 A EP 94923428A EP 0724650 A1 EP0724650 A1 EP 0724650A1
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EP
European Patent Office
Prior art keywords
icp4
protein
complex
dna
tfiid
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EP94923428A
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German (de)
English (en)
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EP0724650A4 (fr
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Neal A. Deluca
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University of Pittsburgh
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University of Pittsburgh
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Publication of EP0724650A4 publication Critical patent/EP0724650A4/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus

Definitions

  • the present invention relates to methods of screening and selecting antiviral compounds or peptides.
  • a potential antiviral compound or peptide mimics an essential surface of a wild-type viral protein in an in vitro reconstitution assay.
  • the compound or peptide is incubated with a wild-type viral protein, host transactivation factors which interact with the wild-type viral protein and an appropriate DNA promoter fragment.
  • Potential antiviral compounds and peptides are detected by formation of altered protein.protein and/or DNA.protein transcription complexes as compared to transcriptional complexes formed in the absence of such exogenous compounds or peptides.
  • Viruses that infect eukaryotic cells often encode transcriptional regulatory proteins to ensure the appropriate expression of genes during the viral life cycle.
  • Disclosed examples include the Ela protein of adenovirus (Berk, 1986, Annu. Rev. Genet. 20: 45-79; Flint and Shenk, 1989, Annu. Rev. Genet. 23: 141-161); large T antigen of simian virus 40 (Keller and Alwine, 1984, Cell 36: 381-389); and the E2 protein of papiloma virus (Phelps and Howley, 1987, J. Virol. 61: 1630-1638).
  • Herpes simplex virus type-1 contains a double-stranded, linear DNA genome comprised of approximately 152 kbp of nucleotide sequence, which encodes approximately 75 genes.
  • the viral genes are transcribed by cellular RNA polymerase II and are temporally regulated, resulting in transcription and subsequent synthesis of gene products in roughly three discernable phases. These phases, or kinetic classes of genes, are referred to as the immediate-early, early and late genes.
  • HSV-1 gene expression can be summarized as follows.
  • five immediate-early genes are transcriptionally activated through the agency of VP16, a 65kD protein present in the incoming virus particle (Patterson and Roizman, 1983, J. Virol. 46: 371-377; Campbell, et al., 1984, J. Mol. Biol. 180: 1-9) to yield infected cell polypeptides 0, 4, 22, 27 and 47 (Periera, et al., 1977, Virology 27: 733-749). Transcription of these five genes does not require prior viral protein synthesis (Clements, et al., 1977, Cell 12: 275-285).
  • the products of immediate-early genes are required to activate transcription and regulate the remainder of the HSV genome.
  • Late genes specify virion structural proteins and can be further divided into two subclasses, ⁇ l and ⁇ 2. Transcription of ⁇ l genes from input genomes occurs at low levels in the absence of viral DNA synthesis but is maximal when progeny viral DNA is synthesized (Holland, et al., 1980, Virology 101: 10-24; O'Hare and Hayward, 1985, J. Virol. 53: 751-760).
  • ICP4 infected cell polypeptide 4
  • ⁇ 4 or Vmwl75 is an essential immediate-early protein localized in the nucleus of the host cell which can be purified as a homodimer (Metzler and Wilcox, 1985, J. Virol. 55: 329-337).
  • ICP4 is multifunctional, including being involved in the enhanced expression of early and late HSV-1 genes (Dixon and Schaffer, 1980, J. Virol. 36:189-203), as well as autoregulation at the transcriptional level.
  • Transient expression assays performed in the presence and absence of wild type ICP4 with ICP4 promoter-reporter gene e.g., chloramphenicol acetyl transferase [CAT]
  • ICP4 promoter-reporter gene e.g., chloramphenicol acetyl transferase [CAT]
  • CAT chloramphenicol acetyl transferase
  • the ICP4 gene has been dissected via mutation analysis so as to locate and define functional domains.
  • DeLuca and Schaffer (1987, Nucleic Acids Res. 11: 4491-4511) generated nonsense mutations by inserting synthetic oligonucleotides in all three reading frames throughout the ICP4 coding region. Domains involved in both transactivation and autoregulation domain were found to reside within the amino terminal 60% of the ICP4 gene as shown by an ability to induce an early gene promoter - CAT chimeric construction and an ability to negatively regulate an ICP4-CAT chimeric construction, respectively.
  • a number of additional nonsense and deletion mutations were introduced into both ICP4 genomic copies of HSV-1 such that the resulting mutants expressed only defined subsets of the wild-type ICP4 amino acid sequence.
  • Amino acid residues responsible for transactivation were further localized to an amino acid domain between positions 143 and 210 (Shepard, et al., 1989, J. Virol. 63: 3714-3728), or possibly from positions 275 to 490 and positions 840 to 1100 (Paterson and Everett, 1988, Virology 166: 186-191). Regions involved in dimerization, phosphorylation and nuclear localization have also been delineated (DeLuca and Schaffer, 1988, J. Virol. 62: 732-743; Shepard, et al., 1989, J. Virol. 63: 3714-3728; Wu and Wilcox, 1990, Nucleic Acids Res. 18: 531-538).
  • ICP4 deletion mutant was constructed which retained primary structural domains for DNA binding and autoregulation, but lacks the domain required to confer transactivation (Shepard, et al., 1990, J. Virol. 64: 3916-3926).
  • This ICP4 mutant, X25 forms an ICP4 heterodimer containing one wild-type and one mutant ICP4 subunit.
  • the ICP4 mutant subunit lacks both transactivation domains yet retains the ability to bind with DNA as well as retaining dimerization activity (e.g., the ability to form a heterodimer with the wild type subunit). Heterodimer formation resulted in a dominant inhibitory phenotype with regard to HSV-1 growth.
  • ICP4 binds DNA non-specifically (Freeman and Powell, 1982, J. Virol. 44: 1084-1087) but it but also exhibits a preference for a consensus sequence. When this consensus sequence overlaps the start site of mRNA synthesis, ICP4 can inhibit transcription when provided in trans (Roberts, et al., 1988; J. Virol. 62: 4307-4320; DeLuca and Schaffer, 1985, Mol. Cell. Biol. 5: 1997-2008; O'Hare and Hayward, 1987, J. Virol. 56: 723-733).
  • ICP4 serves to induce a gene
  • deletion of sites for which ICP4 shows relatively high affinity does not affect activation by ICP4 (Smiley, et al., 1992, J. Virol. 66: 623-631).
  • exhaustive studies have failed to reveal evidence supporting the existence of ICP4-specific induction sequences (Coen, et al., 1986, Science 234: 53-59; Eisenberg, et al., 1985, Mol. Cell. Biol. 5: 1940-1947).
  • mutant ICP4 mutant molecules have been isolated which exhibit reduced affinity for DNA in vitro, but which still retain the ability to stimulate gene expression in the context of the infected cell (Imbalzano, et al., 1990, J. Virol.
  • thymidine kinase (tk) is transactivated by ICP4 (DeLuca, et al., 1984, J. Virol. 52: 767-776; DeLuca and Schaffer, 1985, Mol. Cell. Biol. 5: 1997-2008; O'Hare and Hayward, 1985, J. Virol. 53: 751-760).
  • Thymidine kinase is transcribed by RNA polymerase II and contains a CCAAT box and two Spl sites upstream of a TATA box (Jones, et al., 1985, Cell 42: 559-572; McKnight and Kingsbury, 1982, Science 217: 316-324).
  • Studies of the tk promoter indicate that only cis sequences interacting with cellular transcription factors are required for expression during viral infection, no induction-specific sequences have been identified (Boni and Coen, 1989, J. Virol. 63: 4008-4092; Coen, et al., 1986, Science 234: 53-59; Eisenberg, et al., 1985, Mol. Cell. Biol. 5: 1940-1947).
  • TFIID The general transcription factor (GTF's): TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH and TFID assemble on the DNA template in a very defined and ordered fashion along with RNA polymerase II during transcription initiation.
  • the first factors to assem ⁇ ble are TFIID, TFIIA and TFIIB (Buratowski, et al., 1989, Cell 56: 549-561; Maldonado, et al., 1990, Mol. and Cell Biol. 10: 6335-6347).
  • TFIID consists of many different proteins nucleated in a complex containing the TATA-binding protein (TBP), a 43 kD protein that specifically binds TATA boxes (Kao, et al., 1990, Science 248: 1646-1650; Peterson, et al., 1990, Science 248: 1625-1630; Hoffmann, et al., 1990, Nature 346: 387-390).
  • TFIIB consists of a single protein (33kD) which binds to TFIID as well as TBP to form a DNA-protein complex termed "DB" (Ha, et al., 1991, Nature 352: 689-695; Peterson, et al., 1990, Science 248: 1625-1630).
  • TFIIA is known to participate in the formation of "DA” and "DAB” complexes.
  • Chimeric promoters combining the identified cis elements (CCAAT and Spl elements) of the early tk promoter and late gC promoter were constructed and recombined in place of the wild-type tk promoter in an ICP deficient virus.
  • ICP4 is influenced inversely by the apparent affinity of the TATA box for TBP.
  • the literature has documented that ICP4 is an essential immediate-early HSV-1 viral protein that is involved, among other things, in the transactivation of early and late HSV-1 viral genes. Certain ICP4 mutants have shown an ability to form a heterodimer with wild-type ICP4 in vitro, which may or may not effect the ability of ICP4 to induce early or late gene expression. In addition, studies indicate a relation between induction by ICP4 and the apparent affinity of a specific TATA box for cellular transcription factors.
  • the present invention relates to methods of screening for and selecting compounds or peptides which possess antiviral activity.
  • the invention is based on the interaction between viral and host cellular factors acting in trans to regulate expression of essential viral genes subsequent to host infection.
  • a wild-type viral protein and at least one host transcription factor and a nucleic acid -s-acting sequence are incubated with a potential antiviral compound or peptide in conditions conducive to formation of a protein :protein and/or DNA:protein transcription complex.
  • a potential antiviral agent forms unique complexes in comparison to protein: protein and DNA:protein interactions involving the wild type-viral protein.
  • the potential antiviral compound or peptide can be any inorganic or organic compound or peptide sequence able to interfere with formation of a wild-type viral-host transcription complex.
  • the peptides may be mutants of a wild-type protein or biologically active fragments thereof involved in transactivation of a viral gene.
  • mutant derivatives generated for screening may be chosen at random by site-directed mutagenesis of the gene encoding the target protein, by a rational peptide design mechanism, or by utilization of a microbial based selection scheme to generate a pool of mutants for entry into the antiviral screening method of the present invention.
  • Antibodies to essential surface regions of a wild-type viral transacting factor may also be candidates for entry into the in vitro assay of the present invention.
  • potential antiviral compounds may be naturally occurring or synthetic compounds or a population of such compounds, any or all of which may be candidates for entry into the antiviral screening assay of the present invention. All of these, and additional methods of generating screening candidates, are available to the skilled artisan and have been described in the literature. For example; deletion, addition, nonsense or point mutations as well as biologically active fragments of a wild-type viral protein with trans-acting capability, such as ICP4, is a candidate for antiviral screening.
  • the present invention provides in part for formation of a general viral-host transcription complex by mixing one or more known cellular transcription factors prior to or simultaneously upon incubation with a potential antiviral peptide and nucleic acid promoter fragment.
  • the host transcription complex may comprise, but is not solely limited to, at least one the eukaryotic transcription factors selected from the group consisting of TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH and TFIU.
  • the present invention also provides for a nucleic acid promoter sequence containing cis sequences binding a viral-host protein :protein complex.
  • a nucleic acid fragment known to interact with the protein:protein complex in vivo or in an in vitro reconstitution assay would be a nucleic acid fragment containing cis sequences that bind the viral-host transcription complex.
  • a synthetic nucleic acid fragment containing an appropriate cis sequence such as a TATA sequence and/or an ICP4-binding site, can comprise a portion of the nucleic acid sequence in the assay.
  • ICP4 is essential for expression of early and late HSV-1 gene expression.
  • the nucleic acid fragment chosen for use in the assay contains cis sequences known to bind the ICP4-host transcription complex. It is against the protein:protein and DNA:protein binding characteristic in a control assay that potential antiviral peptides are compared and detected as inhibitors of wild-type viral gene expression, and hence, as antiviral compounds.
  • HSV herpes simplex virus
  • the wild-type trans-acting factor is a trans-acting factor targeted for inhibition chosen from the group consisting of the immediate-early genes of HSV, including but not limited to ICP4, ICP0 and ICP27.
  • the wild-type trans-acting factor targeted for inhibition is ICP4. Deletion, addition, nonsense and point mutations of ICP4, as well as generation of biologically active fragments of ICP4 are candidates for antiviral screening.
  • the nucleic acid sequence utilized in the in vitro reconstitution assay is any HSV gene regulated by ICP4, including but not limited to regulatory sequences involved in controlling self-expression of ICP4, expression of the HSV early gene, thymidine kinase, or the late HSV gene, gC.
  • the host transcription complex includes, but is not solely limited to, at least one of the eukaryotic general transcription factors selected from the group consisting of TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH and TFIU.
  • the host transcription complex includes TFIIB and TFIID, which form the eukaryotic host transcription complex for use in the in vitro reconstitution assay.
  • the host transcription complex comprises the TATA Binding Protein (TBP) and TFIIB to form the general transcription complex for use in the in vitro reconstitution assay.
  • This preferred embodiment of the invention is based on the present disclosure that regulation of ICP4 gene expression is correlated with its ability to form a tripartite complex with the two general transcription factors, TFIID and TBP.
  • TFIID and TBP The formation of this tripartite complex allows for the selection of antiviral compounds and peptides which interfere with the formation of this essential complex and its ability to act in trans with the ICP4 regulatory sequence.
  • the host transcription complex comprises use of either TBP and TFIIB alone as the host transcription factor for use in the in vitro reconstitution assay.
  • the nucleic acid sequence utilized is a fragment from the regulatory region controlling expression of thymidine kinase. Any of the embodiments disclosed in this specification in regard to utilizing a DNA fragment from the ICP4 regulatory region may be utilized with the HSV thymidine kinase promoter.
  • the nucleic acid sequence utilized is a fragment from the regulatory region controlling expression of the late HSV gC promoter. Any of the embodiments disclosed in this specification in regard to utilizing a DNA fragment from the ICP4 regulatory region may be utilized with the late HSV gC promoter.
  • the reconstitution assay selects for potential antiviral peptides by identifying peptides which promote protein :protein and/or DNA:protein complexes distinct from the documented wild-type formation of these transcription complexes.
  • a pharmaceutically effective amount of the antiviral peptide will be targeted to HSV infected cells of the patient such that formation of the viral-host mediated transcription complex is blocked, thus inhibiting further HSV gene expression and viral growth within the patient.
  • a pharmaceutically effective amount of the antiviral peptide will be targeted to HSV infected cells of the patient such that formation of the ICP4 mediated tripartite complex is blocked, thus inhibiting further HSV gene expression and viral growth within the patient.
  • FIGURES Figure 1 illustrates activities of rTFIIB and rTBP purified from E. coH.
  • Part A depicts the activity of transcription factors disclosed in Example Section 6.
  • Fractionated HeLa cell factors and purified recombinant rTBP and rTFIIB were combined to reconstitute transcription from the adenovirus major late promoter in a G-less cassette assay as described in Example Section 6.
  • Part B depicts a gel shift assay using rTBP and rTFIIB proteins.
  • Part C is a diagram of the 130 bp probe derived from the ICP4 promoter. It contains an ICP4-binding site overlapping the start site of transcription, a TATA box with the sequence TATATGA, and a Spl site with the sequence GGGCGGG. Figure 2 illustrates that rTFIIB helps rTBP bind the TATA box.
  • Part A shows that the 130 bp ICP4 probe labeled on the coding strand was incubated with the indicated mixture of proteins, treated with DNase I and then run on a sequencing gel.
  • rTBP Varying amounts of rTBP (400, 200, 100 and 50 ng) were reacted with the probe alone (lanes 3-6) or with the probe in combination with a constant amount of rTFIIB (0.3 ⁇ g, lanes 7-10). As controls, the probe was incubated with buffer alone (lane 2) or with rTFIIB alone (lane 11). For sizing, a G-ladder of the probe was included (lane 1). The position of the TATA box is indicated; the numbers refer to positions relative to the start site of transcription. Part B shows the same experiment as Part A performed using probe labeled on the non-coding strand.
  • FIG 3 shows that ICP4 helps DNA-binding of the DB complex.
  • Part A depicts a DNase 1 protection assay as performed and described in Figure 2 using the 130 bp ICP4 probe labeled on the coding strand. Varying amounts of rTBP (100, 50, 25, and 12.5 ng) and a constant amount of rTFIIB (0.3 ⁇ g) were reacted with the probe alone (lanes 3-6) or with the probe in combination with a constant amount of ICP4 (0.25 ⁇ g, lanes 7-10). As controls, the probe was incubated with buffer alone (lane 2) or with rTFIIB alone (lane 11). For sizing, a G-ladder of the probe was included (lane 1).
  • Part B depicts results from the same experiment as shown in Part A except the assay was performed using probe labeled on the non-coding strand.
  • Part C depicts a DNase I protection assay using HeLa TFIID in place of rTBP. The same assay as in part A was performed substituting various amounts of Hela TFIID (DB fraction) for rTBP.
  • Figure 4 depicts formation of a novel complex upon the simultaneous additions of TBP, TFIIB, and ICP4.
  • ICP4 60 ng
  • rTFIIB 0.5 ⁇ g
  • rTBP 50 ng
  • ICP4 60 ng
  • rTFIIB 0.5 ⁇ g
  • rTBP 50 ng
  • the 58S antibody reacts with ICP4 near the carboxy terminus (DeLuca and Schaffer, 1988, J. Virol. 62: 732-743).
  • Part C shows that the tripartite complex and the DB complex respond similarly in response to decreasing concentrations of rTFIIB.
  • rTBP 60 ng
  • ICP4 12 ng
  • rTFIIB 0.5, 0.25, 0.125, 0.063, and 0.032 ⁇ g
  • Figure 5 shows DNase I footprinting of the complexes isolated by gel shift. DNase I footprinting of the complexes isolated by gel shift was performed as described in the Example section. Part A represents the preparative gels run to isolate the indicated protein-DNA complexes.
  • Part B DNA from these complexes were run on the 8% sequencing gel shown in Part B.
  • the G-ladder used to size the DNase I cleavage products; the accompanying numbers are relative to the transcriptional start site.
  • the TATA homology and the sequence ATCGTC which has been shown to be important for specific DNA-binding by the ICP4 protein.
  • FIG. 6 shows that ICP4 DNA binding alone is not sufficient to form the tripartite complex.
  • Part A shows a gel shift assay in which about 12 ng of purified ICP4, n208, X25, nd3-8 or nd8-10 was used in conjunction with the indicated proteins in a standard gel shift assay using the 130 bp BamHI-EcoRI fragment spanning the ICP4 transcriptional start site. For rTBP, 60 ng was used; for rTFIIB, 250 ng was used. The intensity of this complex was used to obtain the value for the DB4 complex in Table 2.
  • Part B is a diagram of ICP4 and the mutant proteins along with their associated activities. The domains of ICP4 are those described previously.
  • FIG. 7 shows that ICP4 forms a tripartite complex with rTBP and rTFIIB on the thymidine kinase (TK) promoter, an inducible template.
  • Constant amounts of ICP4 (12 ng) and rTBP (0.06 ⁇ g) were titrated against varying amounts of rTFIIB.
  • the EcoRI-Bglll fragment spanning the TK transcriptional start site was used as probe.
  • the same fragment but bearing a mutated TATA box (LS -18/-29) was used as probe.
  • Chros refers to an anti-lCP4 monoclonal antibody.
  • Figure 8 is a model of how ICP4 interacts with its own promoter and with the TK promoter.
  • the arrows indicate interactions as identified either by DNase I footprinting assays or by cooperativity in DNA-binding.
  • the arrows beneath the templates indicate the start sites of transcription.
  • the checkered boxes represent TATA boxes.
  • the other boxes depict ICP4 binding sites. High affinity ICP4 binding sites positioning ICP4 over the start of transcription may result in repression, whereas ICP4's potential interaction with many lower affinity sites throughout the genome may result in transactivation by recruiting the GTFs without tightly binding to the start of transcription.
  • the present invention relates to methods of screening for and selecting compounds or peptides which possess antiviral activity.
  • the invention is based on the interaction between viral and host cellular factors acting in trans to regulate expression of essential viral genes subsequent to host infection.
  • a wild-type viral protein and at least one host transcription factor and a nucleic acid cw-containing sequence are incubated with a potential antiviral compound or peptide in conditions conducive to formation of a protein:protein and/or DNA:protein transcription complex.
  • a potential antiviral agent i.e., any such compound or peptide
  • the in vitro reconstitution assay is based on a potential antiviral agent forming unique complexes in comparison to interactions involving a wild type viral protein. Pools of potential antiviral compounds or peptides can be screened using the in vitro reconstitution assay disclosed in the present invention. Potential antiviral compounds or peptides generated from this screening process may then be subjected to additional assays confirming antiviral activity and selecting for those with the greatest therapeutic potential.
  • the potential antiviral peptide can be any peptide sequence able to interact with a viral-host transcription complex.
  • the peptide may be a mutant of a wild-type protein or biologically active fragments thereof.
  • deletion, addition, nonsense or point mutations as well as biologically active fragments of a wild-type viral protein with trans-acting capability is a candidate for antiviral screening.
  • addition, deletion, nonsense or point mutations as well as biologically active peptide fragments of the wild type viral protein ICP4 are candidates for antiviral screening.
  • One such series of potential antiviral peptides may comprise ICP4 transactivation domains.
  • Potential antiviral peptides may be generated for presentation in the selection scheme of the present invention by any one of a number of methods described previously in the literature.
  • peptides presented for antiviral peptide screening may be chosen as a result of a rational design mechanism to generate one or several potential antiviral peptide; or by mutagenesis of cloned DNA, the cloned DNA sequence encoding the target wild-type gene or a portion thereof. Any number of DNA mutagenesis techniques which have been described in detail in available literature may be utilized (see Ausabel, et al, 1991, Cu ⁇ ent Protocols in Molecular Biology, "Mutagenesis of Cloned DNA”:Chapter 8). Additionally, potential antiviral compounds may be naturally occurring or synthetic compounds or a population of such compounds, any or all of which may be candidates for entry into the antiviral screening assay of the present invention. All of these, and additional methods of generating screening candidates are available to the skilled artisan.
  • the present invention provides in part for formation of a general viral-host transcription complex by mixing one or more known cellular transcription factors prior to or simultaneously upon incubation with a potential antiviral compound or peptide and nucleic acid promoter fragment.
  • the host transcription complex may comprise, but is not solely limited to, at least one of the eukaryotic general transcription factors selected from the group consisting of TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH and TFIU.
  • Proteinaceous components of the general transcription complex may be isolated and substantially purified for use in the assay by any means known to one skilled in the art. Methods of isolation include, but are not limited to, isolation and purification of the protein directly from its in vivo source.
  • the gene encoding the protein of interest may be cloned into an expression vehicle (such as an expression plasmid) via recombinant DNA techniques and transformed into a recombinant host cell.
  • the recombinant host cell may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila derived cell lines.
  • Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL1651), CHO-K1 (ATCC CCL61), 3T3 (ATCC CCL92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL2),
  • the bacterial cell most used for expression of recombinant protein is Escherichia coli. There are various strains of E. coli available and are well known in the art.
  • the literature discloses a variety of expression vector host systems suitable for bacterial and fungal hosts, including plasmids, bacteriophages, cosmids and derivatives thereof. Examples of these systems in regard to bacterial expression are pBR322, pCRl, col Fl, phage lambda, M13 and filamentous viruses.
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
  • human TATA binding protein is obtained by the TBP expression plasmid, pETHIID, transformed into the E. coli strain BL21; and recombinant TFIIB is obtained by the TFIIB expression plasmid, phllB, also transformed into the E. coti strain BL21.
  • TBP TBP expression plasmid
  • pETHIID transformed into the E. coli strain BL21
  • recombinant TFIIB is obtained by the TFIIB expression plasmid, phllB, also transformed into the E. coti strain BL21.
  • the skilled artisan will be prompted by the teachings of this disclosure to utilize any of one or a combination thereof of host transcriptions factor(s) interacting to form a protein:protein complex with the wild-type viral trans-acting factor(s); this protein:protein complex prompting formation of an active DNA: protein transcription complex in the presence of an appropriate nucleic acid sequence.
  • an in vitro reconstitution assay is devised so as to select for compounds or peptides which interfere with the ICP4-host nuclear transcription complex.
  • Any compound or peptide which interferes with this wild type characteristic is by definition an antiviral peptide by way of inhibiting the role of ICP4 as a transactivator of viral gene expression. This is so because a loss of ICP4 induced transactivating activity for ICP4 equates to an inability for continued viral growth.
  • a deletion mutant of ICP4 was tested as an antiviral compound.
  • the present invention also provides for a nucleic acid promoter sequence containing cis sequences binding a viral-host protein: protein complex.
  • the nucleic acid fragment of choice is one known to interact with the protein-protein complex in vivo or in an in vitro reconstitution assay.
  • a synthetic nucleic acid fragment containing an appropriate cw-acting sequence, such as a TATA sequence can be utilized as the nucleic acid fragment in the assay.
  • the appropriate nucleic acid fragment will contain cw-acting sequences (such as a TATA box element) known to interact with the wild-type general transcription complex either in vivo or in an in vitro reconstitution experiment.
  • cw-acting sequences such as a TATA box element
  • an assay which targets ICP4 as the wild-type viral protein would utilize any HSV-1 viral promoter fragment that ICP4 is known to interact with in vivo. Any such regulatory sequence or portion thereof which results in a stable viral-host transcription complex would be a potential template for use in the assay.
  • Such a promoter fragment might be, by way of example and not by limitation, the promoter region upstream of the gene encoding ICP4, since ICP4 is known to interact with this promoter in the autoregulation its own expression.
  • Another promoter might be, by way of example and not by limitation, the promoter region upstream of an early or late HSV-1 gene known to be transactivated by ICP4.
  • a specific example of such a promoter is the promoter fragment upstream of the early HSV-1 gene encoding thymidine kinase.
  • Another specific example of such a promoter is the promoter fragment upstream of the late HSV-1 gene encoding gC.
  • alterna- tive promoter fragments to those known to interact with ICP4 in vivo on the basis of sequence homology in regards to cw-acting elements known to interact with ICP4 mediated transcription complexes.
  • These alternative promoter fragments may be purified from existing cloned DNA sequences or may be produced by synthetic means known to one skilled in the art. Therefore, the choice of a wild type target (such as ICP4) determines the strategy of choosing an appropriate promoter fragment, as well as the general transcription factors to be utilized in the assay. The general patterns of the wild-type protein :protein and DNA:protein interactions are then elucidated.
  • the interaction may be detected through a gel retardation experiment and/or DNA footprint analysis, both of which are discussed in detail in Example Section 6.
  • a potential antiviral compound or peptide is added to for the experimental assay once the characteristics have been determined for the basic wild type viral-host factor/nucleic acid fragment interaction. If this addition causes a different result by way of, for example, mobility in a gel retardation assay or a difference in the ability to bind to the nucleic acid fragment (whether that difference be location on the template or the strength of binding), the peptide or compound is singled out as a potential antiviral agent.
  • nucleic acid fragment known to interact an essential surface of a wild-type viral protein so as to alter formation of a protein :protein or DNA:protein complex in vitro.
  • An appropriate viral promoter would be a nucleic acid fragment containing cis sequences that bind a viral-host transcription complex.
  • a synthetic nucleic acid fragment containing an appropriate cis sequence such as a TATA sequence, can be utilized as the nucleic acid sequence in the assay.
  • ICP4 is essential for expression of early and late HSV-1 gene expression. Therefore, the nucleic acid fragment chosen for use in the assay contains cis sequences known to bind the ICP4-host transcription complex. It is against the protein :protein and DNA:protein binding characteristic in a control assay that potential antiviral compounds or peptides are compared and detected as inhibitors of viral gene expression, and hence, as antiviral compounds.
  • HSV herpes simplex virus
  • the wild type trans-acting factor is a trans-acting factor targeted for inhibition selected from the group consisting of ICP4, ICPO, and ICP27.
  • the wild-type trans-acting factor targeted for inhibition is ICP4. Deletion, addition, nonsense and point mutations of ICP4, as well as generation of biologically active fragments of ICP4 are candidates for antiviral screening. Additionally, chemical compounds which inhibit essential surface interactions between ICP4 and host transcription complexes or the formation of a competent DNA:protein complex are candidates for antiviral screening.
  • the nucleic acid sequence utilized in the in vitro reconstitution assay is an HSV gene regulated by ICP4, including but not limited to regulatory sequences involved in controlling self-expression of ICP4, expression of the early HSV gene, thymidine kinase, or expression of the late HSV gene, gC.
  • the host transcription complex includes, but is not solely limited to, at least one of the eukaryotic transcription factors selected from the group consisting of TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH and TFIU.
  • the host transcription complex includes TFIIB and TFIID, which form the eukaryotic host transcription complex for use in the in vitro reconstitution assay.
  • the host transcription complex comprises the TATA Binding Protein (TBP) and TFIIB to form the general transcription complex for use in the in vitro reconstitution assay.
  • TBP TATA Binding Protein
  • TFIIB TATA Binding Protein
  • This prefe ⁇ ed embodiment of the invention is based on the present disclosure that regulation of ICP4 gene expression is co ⁇ elated with its ability to form a tripartite complex with the two general transcription factors, TFIID and TBP. The formation of this tripartite complex allows for the selection of antiviral compounds or peptides which interfere with the formation of this essential complex and its ability to act in trans with the ICP4 regulatory sequence.
  • the host transcription complex comprises use of either TBP and TFIIB alone as the host transcription factor for use in the in vitro reconstitution assay.
  • the nucleic acid sequence utilized is a fragment from the regulatory region controlling expression of thymidine kinase. Any of the embodiments disclosed in this specification in regard to utilizing a DNA fragment from the ICP4 regulatory region may be utilized with the HSV thymidine kinase promoter.
  • the nucleic acid sequence utilized is a fragment from the regulatory region controlling expression of the late HSV gC gene. Any of the embodiments disclosed in this specification in regard to utilizing a DNA fragment from the ICP4 regulatory region may be utilized with the gC promoter.
  • potential antiviral agents can comprise any inorganic or organic compound, natural or synthetic, as well as any amino acid containing entity.
  • the basis for inclusion as a potential antiviral agent is based in part on the ability to interact and effect an essential surface of the wild-type viral protein involved in transactivation of viral genes.
  • Wild-type ICP4 was obtained from IO 8 Vero cells infected with HSV-1 (strain
  • the truncated mutant, n208 (DeLuca and Schaffer, 1988, J. Virol. 62: 732-743) was obtained from n208-infected Vero cells.
  • the mutant X25 was obtained following infection of X25 cells with KOS.
  • X25 cells harbor the gene expressing the X25 mutant. Since this gene retains the IE promoter/regulatory sequences, it can be induced to high levels by VP16 supplied by viral infection.
  • X25 cells, their induction upon infection, and the subsequent purification of X25 homodimers were previously described (Shepard, et al. 1990, J. Virol. 64: 3916-3926).
  • the viruses expressing the nd3-8 (£30-142, £774-1298) , nd8-10 (£ 142-210, £774-1298), and d8-10 (£ 142-210) ICP4 mutant proteins were constructed as described previously (DeLuca and Schaffer, 1988, J. Virol. 62: 732-743; Shepard, et al. 1990, J. Virol. 64: 3916-3926), using the previously characterized plasmids pndi3-i8, pndi8-il0, and pdi8-il0, respectively (Shepard, et al. 1990, J. Virol. 64: 3916-3926).
  • hTBP Human TATA binding protein
  • TFIIB Recombinant TFIIB was generated by the TFIIB expression plasmid, phllB, obtained from Dr. Danny Reinberg and described previously (Ha, et al., 1991, Nature 353: 689-695). The procedures for bacterial expression, lysis and purification of TFIIB through the phosphocellulose column were as previously described (Ha, et al., 1991, Nature 353: 689-695 ). The TFIIB eluting from the phosphocellulose column at 0.5 M KCl was further fractionated on an FPLC superose 12 column.
  • TFIIA activity in fraction A was eluted from a DEAE-sephacel column with 0.3 M KCl buffer (AB).
  • TFIIB, TFIIE/F, and RNA Pol II activities were eluted from the DEAE-sephacel column with 0.1 M KCl (fraction CA), 0.25 M KCl (fraction CB) and 1 M KCl (fraction CC), respectively.
  • Fraction D, containing TFIID activity was loaded on a DEAE-sephacel column, and TFIID was eluted with 0.25 M KCl buffer (fraction DB).
  • the activity and relative requirements of the fractionated general transcription factors were assessed by performing transcription reactions using the adenovirus major later promoter hooked to the G-less cassette.
  • In vitro transcription reactions contained 0.2 mg of template, 1 ⁇ l fraction AB (TFIIA), 4 ⁇ l fraction CB (TFIIE/F), 2 ⁇ l fraction CC (Pol II), 4 ⁇ l fraction DB (TFIID), 0.017 ⁇ g rTFIIB, 15 units RNAse Tl, 15 units RNasin, 7mM MgCl, 60 uM ATP and CTP, 25 ⁇ M UTP, 0.1 mM OMe GTP and 5 ⁇ Ci and [ ⁇ - 32 P] UTP.
  • RNA transcripts were separated from free nucleotides through a G-50 spin column. The RNA was then precipitated and resuspended in loading buffer and applied on a 4% denaturing mini gel. 6.1.2. GEL RETARD ATION ASSAYS
  • DNA-binding reactions were performed as follows. One nanogram of an end-labeled probe (3 x IO 4 to 6 x IO 4 cpm/ng) and the indicated mixture of proteins were incubated together at 30 °C for 40 minutes in a buffer consisting of 10 mM N-2-hydroxyethylpiperzine-N'- 2-ethanesulfonic acid (HEPES, pH 7.9), 5 mM ammonium sulfate, 8% (vol/vol) glycerol, 2% (wt/vol) polyethylene glycol 8000, 50 mM KCl, 5 mM ⁇ -mercaptoethanol, 0.2 mM ethylenediamine-tetraacetic acid (EDTA), and 25 ⁇ g of poly(dG)-poly(dC) per ml in a total volume of 30 ⁇ l.
  • HEPES N-2-hydroxyethylpiperzine-N'- 2-ethanesulfonic acid
  • EDTA
  • the reactions were then electrophoretically separated at 200 V on a native 4% polyacrylamide gel containing TBE buffer (89 mM Tris, 89 mM boric acid, 0.1 mM EDTA, pH 8.2).
  • TBE buffer 89 mM Tris, 89 mM boric acid, 0.1 mM EDTA, pH 8.2
  • the gels were then dried and exposed to Kodak XAR-5 film with intensifying screens. Densitometric scans of the gel tracks were done by using a Hoefer scanning densitometer and compatible data processing software for the Macintosh.
  • DNASE I FOOTPRINTING OF ISOLATED DNA:PROTEIN COMPLEXES the DNA-binding reactions were scaled-up 20 times. After the 40 minute incubation, 300 ⁇ l of DNase I buffer plus 20 ⁇ l of DNase I (50 ng) were added at room temperature. After 1 minute, 12 ⁇ l of 0.5 M EDTA was added. The reactions were then loaded and run on a native TBE gel as described in Example Section 6.1.3. The wet gels were exposed overnight at 4°C and the resulting autoradiogram was used to cut out the indicated bands.
  • the bands were diced using a razor blade and the resulting pieces were put into an eppendorf tube containing 0.5 mis of 0.25 M ammonium acetate, 1 mM EDTA in order to elute the DNA. After an overnight incubation at 37°C, the eluted DNA was purified over a G-50 Sephadex spin column, ethanol precipitated and resuspended in 95% formamide. The samples were then appropriately normalized such that there were equivalent cpm per ⁇ l and then loaded on a 8% denaturing sequencing gel.
  • AdMLP recombinant TBP
  • rTBP recombinant TBP
  • rTFIIB will substitute for mammalian TFIIB.
  • the results in Figure 1A demonstrate that preparations of the recombinant proteins substitute for their natural analogs as well, thus demonstrating their functional integrity.
  • TFIIB and rTBP interact to form a higher order complex with DNA substrates containing TATA boxes in gel retardation assays (Peterson et al., 1990, Science 248: 1625-1630).
  • FIG. 1C A 130 bp EcoRI-BamHI fragment spanning the ICP4 transcriptional start site was used as a probe. As diagrammed in Figure 1C, this fragment contains a TATA box and an ICP4 binding site. As shown in Figure IB, rTFIIB failed to retard the probe to any significant extent when used alone. This was expected since it has been documented that rTFIIB lacks any specific DNA-binding capacity (Ha, et al. 1991, Nature 352: 689-695). In contrast to rTFIIB, rTBP did bind the probe when used alone, albeit at very low levels.
  • the probe used in the above experiments is derived from the ICP4 promoter and contains an ICP4 binding site located at the start of ICP4 transcription (Figure IC).
  • ICP4 was added to reactions similar to those that generated the footprints in Figure 2 to ascertain its effect on the formation of DB-TATA box complexes.
  • rTFIIB and rTBP were reacted in the presence or absence of ICP4 with the 130 bp probe labeled on the coding strand.
  • the amount of ICP4 used in this experiment is sufficient to give only a very weak footprint. Larger amounts of ICP4 results in a unique footprint extending form -10 to about +15 with respect to the start of transcription.
  • This stretch contains the strong consensus site for specific binding by ICP4 (Faber and Wilcox, 1986, Nucleic Acids Res. 14: 6067-6083; Kristie and Roizman, 1986, Proc. Natl. Acad. Sci. 83: 3218-3222; Muller, 1987, J. Virol. 61: 858-865). More significantly, the inclusion of ICP4 enhanced the DB footprint as evidenced by the fact that at low concentrations of rTBP and rTFIIB where hardly any TATA-protection was detected in the absence of ICP4 a very strong footprint was obtained in the presence of ICP4. The enhancement was approximately 5-fold.
  • DNase I footprint analysis was conducted on the individual DNA-protein complexes to further identify the proteins and nucleic acid sequences involved (Figure 5).
  • Preparative gel retardation reactions were performed with the protein mixtures as indicated for Figure 5 A and the 130 bp probe labeled on the coding strand. The reactions were treated with DNase I, run on native TBE gels, and exposed to film. The bands marked A through F were cut out, and their DNA was eluted. The eluted DNA was then run on a denaturing polyacrylamide gel to determine which sequences were protected from DNase I cleavage. DNA from band A represents nonretarded probe and was susceptible to DNase I cleavage throughout its entire length. DNA from band B represents probe complexed with ICP4 alone.
  • sequences from about -10 to at least + 10 with respect to the transcriptional start site were protected.
  • This stretch includes the sequence ATCGTC known to be involved in ICP4-DNA interactions (Faber and Wilcox, 1986, Nucleic Acids Res. 14: 6067-6083).
  • DNA from band D represents retarded probe resulting after reaction with ICP4 and rTBP.
  • ICP4 footprint obtained, but also some very weak protection, covering the area extending from about -29 to -18, an area that contains the TATA box, was observed. This result indicates that a low level of co-occupancy of the DNA template by rTBP and ICP4.
  • DNA from band C represents retarded probe resulting after reaction with ICP4 and rTFIIB.
  • DNA from band E represents the supershifted probe resulting from the reaction of ICP4, rTBP and rTFIIB altogether.
  • both the TATA box and the ICP4 biding site were strongly protected, consistent with the supposition that the supershifted complex disclosed in Figure 4A represents a high-order complex containing ICP4, rTBP, and probably rTFIIB.
  • ICP4 mutant proteins purified from mutant virus-infected cells were assayed for the ability to form tripartite complexes (Figure 6A). The primary structures of these proteins are shown relative to that of the wild type protein in Figure 6B. All five mutant proteins contain the DNA-binding domain, bind DNA and yield wild type footprints at least when assayed on the ICP4 promoter (Shepard and DeLuca, 1991, J. Virol. 65: 299-307). Densitometry data from Figure 6A are shown in Table 2. For wild type ICP4, n208 and nd3-8, the proportion of the probe found in tripartite complexes was significantly greater that the product of the proportions bound by DB and the ICP4 proteins when assayed individually.
  • ICP4 binding site overlapping the region where transcription initiates.
  • TK was chosen to determine if ICP4 could form a tripartite complex using a template whose transcription is induced by ICP4.
  • This fragment spanning -75 to +54 with respect to the start of transcription, contains a TATA box, an Spl site, and a low affinity ICP4 binding site located approximately 40 base pairs downstream of the transcriptional start site (Imbalzano et al, 1990, J. Virol. 64: 2620-2631).
  • ICP4 alone retards the probe and the amount of complex 4 is increased by the addition of TFIIB.
  • TBP TBP
  • TFIIB resulted in a complex of even lower mobility than complex 4 (marked with open circles).
  • the anti-ICP4 monoclonal antibody 58S further retarded the novel complex formed in the presence of rTFIIB, rTBP and ICP4 (marked with a dash and open circle).
  • the experiment in Figure 7B recapitulates the one in Figure 7A expect that one linker-scanning mutant of the probe was used.
  • This mutant designated LS-29/-18, harbors a defective TATA box (McNight and Kingsbury, 1982, Science 217: 316-324).
  • This mutation reduced the ability of rTFIIB and rTBP to form the rTFIIB-rTBP complex.
  • DB the complex labeled DB in Figure 4 and BD in Figure 6A, having the mobility of that generated by the simultaneous addition of rTBP and rTFIIB;
  • ICP4 the complex having the mobility of that generated by the sole addition of the purified ICP4 peptide;
  • DB the novel complex appearing only after the simultaneous addition of theICP4 peptide, rTBP, and rTFIIB; unbound, the band having the same mobility as that generated by the probe run alone on the gel.
  • DB the complex labeled DB in Figure 4 and BD in Figure 6A, having the mobility of that generated by the simultaneous addition of rTBP and rTFIIB
  • ICP4 the complex having the mobility of that generated by the sole addition of the purified ICP4 peptide
  • DB the novel complex appearing only after the simultaneous addition of theICP4 peptide, rTBP, and rTFIIB; unbound, the band having the same mobility as that generated by the probe run alone on the gel.
  • Example Section 6 The data presented throughout Example Section 6 show that ICP4 can participate in the formation of transcriptional complexes at this very early stage of assembly.
  • the simultaneous addition of ICP4, rTFIIB and rTBP to a DNA template containing an ICP4 binding site and a TATA box resulted in a unique complex lower in electrophoretic mobility than the complexes obtained with any of the proteins used singularly or in dual combinations.
  • ICP4 resides in this unique complex, as evidenced by the fact that the complex exhibited a characteristic ICP4 footprint ( Figure 5) and by the fact that it was further shifted by an anti-ICP4 antibody ( Figure 4B and 7).
  • rTBP resides in the complex, as evidenced by the fact that the complex protected the TATA box from DNase I cleavage ( Figure 5).
  • the question of whether rTFIIB resides in the complex is implicated by the fact that in its absence the supershift complex does not form ( Figure 4A).
  • its presence facilitated the formation of the tripartite complex and the DB complex with the same concentration dependence. Consequently, it is concluded that ICP4, rTBP and rTFIIB form tripartite complexes on suitable templates.
  • tripartite complexes may form with varying degrees of efficiency, depending on the relative affinities of the TATA box and ICP4 binding sites for their respective proteins and on the degree of separation between the participating cw-acting sites. Additionally, if the ICP4 binding site in the ICP4 promoter is mutated such that ICP4 no longer binds to the residual sequence, tripartite complexes do not form.
  • ICP4 Within amino acids 142-274 of ICP4 is a serine rich tract that is highly conserved among the ICP4 analogs of a variety of herpes viruses (McGoech, et al., 1986, Nuc. Acids Res. 14:1727-1744), which has been genetically implicated as a site of phosphorylation (DeLuca and Schaffer, 1988, J. Virol. 62: 732-743), and is a target for cellular kinases A and C. It is conceivable that the phosphorylation state of this tract modulates ICP4's interaction with the DB complex. This area has also been previously identified as being involved in transactivation (Shepard and DeLuca, 1991, J. Virol. 65: 787-794).
  • n208 and nd3-8 are capable of activating transactivation and are able to form the complex, while X25 and nd8-10 are deficient for both transactivation and complex formation. Therefore, the data presented in Example Section 6 indicates that the tripartite complex has implications for the events involved in ICP4 function. However, interaction with the DB complex cannot be the sole mechanism by which ICP4 stimulates gene expression. As shown in Figure 6, the d8-10 and d8-10 like mutations (Paterson and Everett, 1990, Nucleic Acids Res.
  • FIG 8 is a pictograph summarizing the interactions disclosed in the present invention. With respect to the question of transactivation, it is important to note that the results reported herein repeatedly underscore the fact that ICP4 increase the DNA-affinity of the DB complex. This is most dramatically seen in Figure 3. As depicted in Figure 3, ICP4 increases the DNA affinity of the DB complex by fivefold. This is also true in the case of human TFIID ( Figure 3C). Consequently, the data indicates that ICP4 enhances gene expression by recruiting DB to the DNA template, an acknowledged rate limiting step in the assembly of the transcriptional preinitiation complex (Lin and Green, 1991, Cell 64: 971-981).
  • ICP4 transactivates by substituting, or by serving as a bridging agent, for one or more of the other cofactors among the myriad participating in the preinitiation complex.
  • ICP4 acts as a nucleating agent by binding DNA and stabilizing the DNA association of the proteins which it contacts, then DNA binding is predicted to play a critical role in ICP4 functioning by this mechanism.
  • the specific binding site mediating the formation of the tripartite complexes on the TK promoter shown in Figure 7 is not necessary for ICP4 induction in the context of the viral chromosome (Halpern, et al., 1984, J. Virol. 50: 733-738). No single site or collection of sites that uniquely specify induction by ICP4 has been identified (Smiley, et al, 1992, J. Virol.
  • ICP4 interacts with the transcriptional preinitiation complex and this interaction may have physiological significance for viral replication.
  • similar scenarios for screening and selecting for antiviral compounds will exist for other viral tr ⁇ /w-acting factors, including but not limited to (1) the 80kDa IE protein of human cytomegalovirus, which interacts directly with the evolutionarily conserved carboxy-terminal domain of rTBP (Hagemeier, et al, 1992, J. Virol. 66: 4454-4462); (2) VP16, a transactivator of HSV which functions at a different stage of in the viral transcription program than does ICP4, also interacts with rTBP (Smiley, et al, 1992, J. Virol.

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Abstract

La présente invention se rapporte au criblage et à la sélection de peptides ou de composés antiviraux potentiels. Des peptides ou composés antiviraux potentiels sont choisis en fonction de leur aptitude à interagir et à interférer avec une fonction essentielle d'une protéine virale de phénotype sauvage suite à une infection d'un hôte. Des peptides ou des composés antiviraux potentiels ou amas de ceux-ci sont criblés au cours d'un dosage de reconstitution in vitro comprenant une protéine virale de phénotype sauvagae, des facteurs cellulaires d'hôte agissant en trans ainsi qu'un fragment promoteur d'ADN approprié. Un agent antiviral potentiel présente l'aptitude à former un complexe unique protéine:protéine ou ADN:protéine par rapport à des interactions impliquant la protéine virale de phénotype sauvage en l'absence de l'agent antiviral.
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WO2011007230A2 (fr) 2009-07-14 2011-01-20 Hetero Research Foundation Dérivés de triterpène de type lupéol comme antiviraux
WO2011061590A1 (fr) 2009-11-17 2011-05-26 Hetero Research Foundation Nouveaux dérivés carboxamides comme inhibiteurs du vih
WO2011080562A1 (fr) 2009-12-29 2011-07-07 Hetero Research Foundation Nouveau aza-peptides contenant du cyclobutyl 2,2-disubstitué et/ou des dérivés alcoxy benzyle substitués comme agents antiviraux
WO2012095705A1 (fr) 2011-01-10 2012-07-19 Hetero Research Foundation Sels de qualité pharmaceutique de nouveaux dérivés de l'acide bétulinique
WO2014105926A1 (fr) 2012-12-31 2014-07-03 Hetero Research Foundation Nouveaux dérivés proline de l'acide bétulinique utilisés comme inhibiteurs du vih
WO2016178092A2 (fr) 2015-02-09 2016-11-10 Hetero Research Foundation Nouveau triterpénone en c-3 avec des dérivés d'amide inverse en c-28 en tant qu'inhibiteurs du vih
WO2020165741A1 (fr) 2019-02-11 2020-08-20 Hetero Labs Limited Nouveaux dérivés de triterpène en tant qu'inhibiteurs du vih

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US5869234A (en) * 1996-01-05 1999-02-09 President And Fellows Of Harvard College Method of identifying compounds which modulate herpesvirus infection
CA2223032A1 (fr) * 1997-02-21 1998-08-21 Smithkline Beecham Corporation Utilisation de ul-15 et de vp5 du hsv-1 pour la detection d'agents antiviraux
US20170129916A1 (en) 2014-06-26 2017-05-11 Hetero Research Foundation Novel betulinic proline imidazole derivatives as hiv inhibitors
WO2016147099A2 (fr) 2015-03-16 2016-09-22 Hetero Research Foundation Nouveaux triterpénone c-3 avec des dérivés amide c-28 servant d'inhibiteurs de vih

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GB8917029D0 (en) * 1989-07-25 1989-09-13 Marie Curie Memorial Foundatio Polypeptide inhibitor of viral replication

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CHEMICAL ABSTRACTS, vol. 118, no. 3, 18 January 1993 Columbus, Ohio, US; abstract no. 20214, XP002077758 & C.A. SMITH ET AL.: "Transdominant inhibition of herpes simplex virus growth in transgenic mice." VIROLOGY, vol. 191, no. 2, 1992, pages 581-588, Chicago IL USA *
See also references of WO9502071A1 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011007230A2 (fr) 2009-07-14 2011-01-20 Hetero Research Foundation Dérivés de triterpène de type lupéol comme antiviraux
WO2011061590A1 (fr) 2009-11-17 2011-05-26 Hetero Research Foundation Nouveaux dérivés carboxamides comme inhibiteurs du vih
WO2011080562A1 (fr) 2009-12-29 2011-07-07 Hetero Research Foundation Nouveau aza-peptides contenant du cyclobutyl 2,2-disubstitué et/ou des dérivés alcoxy benzyle substitués comme agents antiviraux
WO2012095705A1 (fr) 2011-01-10 2012-07-19 Hetero Research Foundation Sels de qualité pharmaceutique de nouveaux dérivés de l'acide bétulinique
WO2014105926A1 (fr) 2012-12-31 2014-07-03 Hetero Research Foundation Nouveaux dérivés proline de l'acide bétulinique utilisés comme inhibiteurs du vih
WO2016178092A2 (fr) 2015-02-09 2016-11-10 Hetero Research Foundation Nouveau triterpénone en c-3 avec des dérivés d'amide inverse en c-28 en tant qu'inhibiteurs du vih
WO2020165741A1 (fr) 2019-02-11 2020-08-20 Hetero Labs Limited Nouveaux dérivés de triterpène en tant qu'inhibiteurs du vih
EP4248960A2 (fr) 2019-02-11 2023-09-27 Hetero Labs Limited Nouveaux dérivés de triterpène en tant qu'inhibiteurs du vih

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Withdrawal date: 20000406