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WO1996023899A1 - Procedes de selection d'un peptide aleatoire se liant a une proteine cible - Google Patents

Procedes de selection d'un peptide aleatoire se liant a une proteine cible Download PDF

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Publication number
WO1996023899A1
WO1996023899A1 PCT/US1995/009589 US9509589W WO9623899A1 WO 1996023899 A1 WO1996023899 A1 WO 1996023899A1 US 9509589 W US9509589 W US 9509589W WO 9623899 A1 WO9623899 A1 WO 9623899A1
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peptide
protein
rev
cell
hiv
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Christian C. Fritz
Michael R. Green
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UMass Memorial Medical Center
University of Massachusetts Chan Medical School
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UMass Memorial Medical Center
University of Massachusetts Chan Medical School
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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
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • the invention relates to a method of selecting a fusion protein which binds to a target protein of interest within a host cell.
  • the invention also relates to a polypeptide that binds to the human immunodeficiency virus (HIV)-l Rev protein, and methods of inhibiting HIV- 1 production using the polypeptide.
  • HIV human immunodeficiency virus
  • Protein-protein interactions within a cell are the basis for countless physiological control mechanisms. Viral control mechanisms are the targets of searches for the cellular host proteins with which a viral protein interacts as part of its function in the cell.
  • the M two-hybrid" assay is a genetic screen originally described by Fields et al. (Fields S. et al. (1989) Nature , 340:245-246; Fields, S.
  • the "two-hybrid" selection system utilizes the interaction between two proteins of interest to reconstitute an active transcription factor from two fusion proteins; one containing a DNA binding domain fused to the protein of interest, and the other containing a transcriptional activation region fused to a cDNA library (Chien C. T. et al. (1991) P.N.A.S. USA,
  • Test protein-target protein interaction is indicated by expression of a marker gene operably linked to a promoter activated by the reconstituted transcriptional factor. Though useful, the two hybrid system cannot assay all protein-protein interactions. According to Fields, S. and Sternglanz, R. (1994), supra f proteins that cannot fold correctly within the cell are not suitable for selection by this system.
  • Viruses are obligate intracellular parasites and depend on protein-protein interactions between viral and host cell proteins to carry out functions vital to their life cycle. Blocking or inhibiting these critical interactions is a therapeutic strategy that may have profound effects on the viral pathogen and little effect on the host cell. Furthermore, as mutation of the viral protein may be constrained by the requirement for functional interaction with host factors, such a strategy may be resistent to viral escape mutations.
  • Rev was first identified in proviral mutants that were unable to replicate because they did not produce detectable levels of Env and Gag proteins (Sodroski et al. (1986) Nature , 321:412-417: Feinberg et al. (1986) Cell , 16:807-817). It is now clear that Rev induces the synthesis of these structural proteins by allowing the appearance of their mR ⁇ As in the cytoplasm (Malim, M. et al. (1989) Nature, 338:254-257: Emer an, M. et al. (1989) Cell , 5.7:1155-1165).
  • mR ⁇ As coding for Gag, Env, and Pol contain introns with sequences to which the Rev protein specifically binds, these mR ⁇ As are normally translocated to the cytoplasm for translation to their respective proteins during HIV infection.
  • Rev is a small 116 amino acid phosphoprotein that is located predominantly in the nucleus/nucleolus of human cells infected with HIV-l (Cullen, B. R. et al.
  • Rev contains at least two domains that are necessary for function: 1) a basic RNA binding domain at the N-terminus that binds to a specific RNA sequence (RRE, Rev Response Element) present in all unspliced HIV mRNAs, and 2) a C-terminal effector domain (Malim, M. et al. (1989) Cell , 58:205-214; Olsen, H.S. et al. (1990) Genes and Dev. , 4.:1357-1364) necessary for function.
  • RRE Rev Response Element
  • the effector domain was defined by mutations, e.g., M10, that not only abolish Rev function but at the same time create transdominant negative mutants, i.e., mutants able to repress wild type Rev function when added in a molar excess (Malim, M. et al. (1989) , supra) .
  • the M10 mutation does not affect the normal multimerization of Rev nor the ability of Rev to bind RNA, suggesting that the region of Rev containing the M10 mutation interacts with one or more cellular proteins (Malim, M. et al. (1991) J. Virol . , 65:4248- 4254) and that the interaction with a putative cellular target is essential for Rev function.
  • the invention features a method of selecting a random amino acid sequence that binds to a target protein in a host cell, e.g., a yeast cell.
  • a host cell e.g., a yeast cell.
  • the invention is based on the unexpected discovery that a stably folded polypeptide domain (a "scaffold") fused to the random amino acid sequence enhances such selection.
  • the invention features a random peptide detected by the method of the invention, which is not naturally expressed in the cell from which its transcript was isolated, and which unexpectedly exhibited specific binding to the Rev protein effector domain.
  • This fusion protein is expressed from a nucleic acid sequence encoding a random sequence of amino acids covalently linked to a scaffold peptide.
  • the stably folded structure of the scaffold enhances and/or enables binding of the random peptide to the target protein. Binding of the random peptide- scaffold peptide fusion to the target protein of interest is monitored by a two-hybrid selection method (Fields, S. et al. (1989) , supra) .
  • the scaffold is a peptide sequence that stably folds into a 3-dimensional structure and which, when fused to a random amino acid sequence of the invention, enhances presentation of the random sequence to the target protein.
  • Specific embodiments of the scaffold peptide include nucleotide sequences which encode and allow translation of an Adh C-terminus (SEQ ID NO:3); the Bl domain of G protein (Gronenborn, A.M. (1991) Science , 253:657-661) ; a zinc finger domain (Lee, M.S. (1989), Science , 245:635) : the B domain of protein A from S. aureus (Gouda et al.
  • the random peptide can be linked at either end, or preferably in the middle, and most preferably in external loops of the scaffold, making the random peptide accessible to the target protein for binding.
  • the invention further features a non-natural fusion protein, RIP (Rev interacting p_ rote -i- n , SEQ ID NO:2), isolated by expressing a non-naturally translated peptide fused to a scaffold peptide domain, which fusion peptide binds to the HIV-1 Rev protein and inhibits its function.
  • RIP Rev interacting p_ rote -i- n , SEQ ID NO:2
  • the invention features a substantially pure DNA sequence (SEQ ID NO:4), referred to herein as the "R32 nucleotide sequence,” and the deduced amino acid sequence (SEQ ID NO:5). This sequence does not include any polylinker or scaffold sequences.
  • the invention also features a Rev protein binding protein encoded by a DNA sequence of SEQ ID NO:4 linked to a scaffold peptide-encoding sequence.
  • the invention also features a vector containing, in the order of transcription, a nucleotide sequence encoding one member of a two-hybrid binding peptide pair (i.e. a transcriptional activation domain (Gal4 or Herpes virus VP16, for example) ; a nucleotide sequence encoding a random peptide sequence to be tested; and a nucleotide sequence encoding a scaffold peptide domain.
  • a nucleotide sequence encoding one member of a two-hybrid binding peptide pair
  • a transcriptional activation domain Gal4 or Herpes virus VP16
  • the invention features a second vector containing, in the order of transcription, a nucleotide sequence encoding a DNA binding domain (Gal4 or LexA, for example) ; and a nucleotide sequence encoding a target protein.
  • a nucleotide sequence encoding a DNA binding domain Gal4 or LexA, for example
  • a nucleotide sequence encoding a target protein Gal4 or LexA, for example
  • vectors of the two-hybrid system contains a nuclear localization signal to allow translocation of the complex of interacting fusion proteins into the nucleus for interaction with the transcriptional activation sequences of the marker gene. It is also noted that vectors used in selecting a random peptide in the context of a scaffold peptide domain can encode the random peptide 5' to the scaffold peptide domain (in the order of transcription) , or the random peptide-encoding sequence can be inserted into the coding sequence of the scaffold peptide domain.
  • the invention also features an antibody to the RIP protein.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the anti-RIP antibody, or the RIP itself may also be labeled for detection such as with a fluorescent moiety covalently attached to the antibody, or peptide, without interfering with antibody binding to the RIP.
  • the invention features an assay for the qualitative or quantitative detection of Rev protein in a cellular lysate, by inducing a population of cells containing or suspected of containing HIV to proliferate, thereby inducing HIV to replicate and produce Rev protein.
  • the cells are lysed and the proteins of the lysate are contacted with RIP, e.g., immobilized on a solid support, to allow binding of Rev.
  • the presence of the RIP-Rev complex is indicated by a decrease in the detection signal observed when Rev is present compared to a control sample having no Rev protein.
  • the invention further features a method for screening candidate agents that inhibit HIV replication by inhibiting Rev production.
  • Human cells containing actively replicating HIV are contacted with the candidate agent in an appropriate culture medium.
  • a decrease in the cellular concentration of Rev in treated cells compared to a control without the candidate agent indicates that the agent is potentially useful for inhibiting HIV replication in human cells.
  • the invention also features a method of inhibiting HIV reproduction in lymphocytes of a mammal (preferably a human) by introducing DNA encoding RIP into the lymphocytes where the DNA is expressed and RIP is produced in amounts sufficient to inhibit HIV reproduction.
  • the invention features a method of inhibiting the production of HIV-l in human cells, e.g., therapeutically or prophylactically, by administering the RIP protein to the cells using gene therapy techniques.
  • random peptide or “random amino acid sequence,” is meant any peptide that does, or does not, naturally exist in a cell.
  • a random peptide according to the invention can be generated by the translation of a nucleotide sequence that does not encode a naturally occurring full-length protein, or by synthetic methods.
  • the nucleotide sequence encoding a random peptide is encoded, for example, by a cDNA sequence isolated by reverse transcription of a cellular sense or antisense mRNA transcript, or by a synthetic oligonucleotide containing amino acid codons linked in random order.
  • nucleotide sequence encoding a random peptide examples include, but are not limited to: 1) cellular antisense mRNA reverse transcribed into cDNA then expressed as a polypeptide which binds to a target protein of interest, and 2) a polypeptide generated by the expression of a nucleic acid sequence encoding a randomly generated amino acid sequence.
  • a "stably folded peptide” is a peptide that is resistant to denaturation, e.g., thermal denaturation, in a cell.
  • reduced HIV-1 protein production in cells expressing the virus is meant a decrease in the expression of, for example, essential proteins Gag, Env, and Pol in RIP-containing cells compared to the normal expression in the same cells lacking RIP.
  • sufficient concentration of HIV-1 Rev binding peptide is meant a concentration of RIP in a cell above the threshold necessary to inhibit translocation the Gag, Env, and Pol mRNAs from the nucleus into the cytoplasm.
  • substantially pure random peptide-scaffold peptide fusion protein is meant that the random peptide- scaffold peptide fusion protein provided by the invention is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, by weight, random peptide-scaffold peptide fusion protein.
  • a substantially pure random peptide-scaffold peptide fusion protein may be obtained, for example, by expression of the recombinant nucleic acid of the library test vector encoding it, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacryla ide gel electrophoresis, or HPLC analysis.
  • substantially identical amino acid sequence is meant an amino acid sequence that differs from another sequence only by conservative amino acid substitutions. for example, substitution of one amino acid for another of the same class (e.g., valine for glycine or arginine for lysine) , or by one or more non-conservative amino acid substitutions, deletions, or insertions at positions of the amino acid sequence that do not destroy the biological activity of the random peptide-scaffold peptide fusion protein.
  • conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for glycine or arginine for lysine)
  • non-conservative amino acid substitutions, deletions, or insertions at positions of the amino acid sequence that do not destroy the biological activity of the random peptide-scaffold peptide fusion protein.
  • operably linked means that selected DNA is in proximity with transcriptional and translational regulatory sequences to allow these sequences to regulate expression of the selected DNA.
  • Fig. 1 is a diagram of the results of HIV antigen production and Rev binding experiments comparing characteristics of wild type Rev protein and various mutant Rev proteins. Data for HIV-1 gtat and p24 antigen production were obtained from Malim, et al. (1991) , supra . RIP-Rev binding experiments were performed by the method of the invention.
  • Fig. 2 is a diagram of R32 clones used to evaluate regions necessary for Rev binding activity.
  • Fig. 3A is a diagram showing various N-terminal deletions of the R32 nucleotide sequence starting at the JScoRI site.
  • the Xhol site was filled in with ⁇ - thionucleotides to achieve unidirectional deletions according to standard methods (Henikoff (1984) Gene, 21:351-359) .
  • Fig. 3B is a diagram showing the structure of the initial test vector construct and of active test vector constructs after bidirectional exonuclease III deletions. The deletions were started from the jal site.
  • Fig. 4 is a representation of the nucleotide and deduced amino acid sequences of the Rev interacting protein, RIP.
  • the RIP coding sequence consists of the downstream 175 nucleotides of the originally isolated R32 sequence (from the human MCM2 homologue in antisense orientation) linked to DNA linker sequences from the cloning vector cDNA, 15 nucleotides from the yeast ADH gene, and 108 nucleotides encoding 35 amino acids plus a stop codon (SEQ ID NO:3) from the Adh C-terminus.
  • the shaded portions have been found to be dispensable for binding to Rev in C-terminal deletion experiments, but were included for cloning convenience in the experiments expressing RIP in mammalian cells.
  • Fig. 5 is a graph showing the inhibition of HIV-1 gag p24 antigen production by increasing amounts of RIP- encoding DNA introduced into HeLa cells by cotransfection with HIV-1 infectious molecular clone, pNL4-3 (Adachi, A. et al. (1986) J. Virol . 12:284-291).
  • Figs. 6A and 6B are graphs showing the inhibition of HIV production in CEM lymphocytes by RIP protein (Fig. 6A) ; and showing that RIP-induced inhibition is specific to HIV-l Rev since RIP does not affect expression of another protein, alkaline phosphatase, in the CEM lymphocytes (Fig. 6B) . The results also show that inhibition of HIV-1 production by RIP is not caused by non-specific cell toxicity of RIP since the expression of a control gene, alkaline phosphatase,is unaffected.
  • Fig. 7 is a graph showing the specific and dosage dependent inhibition of HIV-1 production in H9 cells by RIP.
  • Fig. 8 is a diagram of a method of assaying for the presence of Rev in a cell lysate by contacting the cellular and viral proteins of the cell lysate with RIP immobilized on a solid support. Detection is accomplished by the relative amounts of anti-RIP antibody that is excluded from binding to the support due to the presence of the Rev-RIP complex.
  • the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA-binding site that regulates the expression of an adjacent reporter rene (Fields, S. and Song, O.-K. (1989) Nature , 340:245-246) .
  • the two-hybrid assay is performed in yeast cells and requires the construction of hybrid genes to encode 1) a D ⁇ A binding domain fused to a protein X; and 2) an activation domain fused to a protein Y.
  • a scaffold peptide domain fused to a random peptide enhances binding selection of the random peptide in a host cell, e.g., a yeast cell.
  • Example 1 A Method of Isolating a Random Peptide- Scaffold Fusion Protein That Binds a Specific Target protein
  • the Adh transcription termination sequence was expressed as a read-through sequence which accompanied transcription of the random amino acid sequence to be selected by the method.
  • the observed binding requirement for the Adh transcription termination sequence indicated that the Adh sequence was necessary as a molecular scaffold that stabilized the entire fusion protein, and thus the random peptide.
  • LVKSPIKWGLSTLPEIYEKMEKGQIVRYWDTSK (SEQ ID NO:3) from the Adh transcription termination sequence are required to be present in the expressed fusion protein to serve as the scaffold for the purpose of structural presentation of a fused random peptide in a two hybrid selection system.
  • Another scaffold protein sequence useful in the method of the invention is the Bl domain of the Streptococcal protein G.
  • This protein domain is a single immunoglobulin binding domain, and the folded structure has been extensively studied (Gronenborn, A.M. et al. (1991) Science , 253:657-661) , but never used in the two- hybrid system.
  • the Bl protein domain forms a stably folded structure which is not denatured below a temperature of 87°C, exhibits reversible thermal denaturation, and maintains its native conformation in aqueous solution containing up to approximately 8 M urea.
  • DNA encoding a random peptide is preferably inserted between amino acids 9 and 12 of the Bl domain at the site of a loop between two beta sheets (see Gronenborn, A.M. (1991) , supra, for Bl structural analysis) , thereby providing optimal presentation of the random peptide for binding to the target protein.
  • the transcriptional activation domain is fused to the N- terminus of the Bl domain.
  • each vector contains, in the direction of transcription, a transcriptional activation domain, a DNA sequence encoding a random amino acid sequence, and a DNA sequence encoding a scaffold peptide such as the Adh C-terminal sequence (SEQ ID NO:3) or the Bl domain of Streptococcal protein G (Gronenborn, A.M. (1991) Science, 253:657-661) .
  • a target vector is also constructed which contains, in the order of transcription, the Gal4 DNA binding domain linked to the coding sequence of a target protein of interest, e.g., the entire Rev coding sequence.
  • the relative position of the target domain and the Gal4-DNA binding domain can be reversed and still generate a functional target fusion protein.
  • other binding domains such as LexA can be used in the assay.
  • the yeast expression vectors of the invention contain appropriate transcriptional and translational regulatory sequences and nuclear localization signal coding sequences for expression and nuclear localization of the test and target fusion proteins.
  • the interaction of the test random amino acid sequence and the target domain in a cell provides a functional GAL4 transcriptional activator resulting in the expression of a marker gene (e.g. lacZ) under the transcriptional control of a GAL4 promoter.
  • a marker gene e.g. lacZ
  • a positive interaction is detected as blue yeast colonies on yeast culture solid medium containing X-Gal.
  • a random 20 amino acid sequence can have any one of 20 naturally occurring amino acids at each position, or 20 20 different sequences.
  • the Rev effector domain as an example of a target domain is likely to bind multiple sequences, increasing the probability of detecting a binding reaction in the millions of independent clones screened in each assay.
  • the HeLa cDNA library vector, pCF128, was constructed as follows.
  • the BamHI fragment for DB20 (Becker et al. (1991) P.N.A.S. USA, 18:1968-1972) containing an ADH promoter and transcription terminator expression cassette was inserted into YEPlacl ⁇ l (Gietz and Sugino (1988) G ⁇ n ⁇ , 21:527-534), which had ben digested with Hindlll and EcoRI and filled in with Klenow enzyme, creating intermediate plasmid pCF124.
  • a PCR product encoding VP16 amino acids 411-500 (Triezenberg et al. (1988), Genes and Dev.
  • HeLa cDNA was prepared using the Riboclone cDNA synthesis kit (Promega Corp. , Madison, WI) using random hexamer primers. Blunt end cDNA, ligated to a BstXI adapter, was inserted into the BstXI sites of pCF128 to generate the test library.
  • the library contained approximately 4xl0 6 independent clones. This library was transformed into the yeast strain GGY1 (Gill, G. et al. (1987) Cell
  • Candidate plasmids were retransformed into GGY1 together with constructs expressing either the Gal4 DNA binding domain Gal4 (1-147) alone, the Gal4 fusion Gal4-
  • the R32 plasmid contains a 377 bp cDNA fragment inserted into vector pCF128 (supra) .
  • the expressed fusion protein contains the VP16 activation domain, the R32-encoded peptide, and the Adh scaffold peptide and was determined to be a Rev interacting protein in subsequent binding studies.
  • the R32 plasmid was transformed into yeast together with Gal4 fusions to a number of different Rev mutants that have previously been described (Malim et al. (1991) , supra) . Binding of RIP to Rev wild-type and mutants was assayed by the method of the invention.
  • the production of HIV-1 gtat and HIV-1 gag p24 assayed by Malim et al. (Malim et al. (1991) , supra) is tabulated in Fig. 1 to illustrate the activity of the Rev mutants relative to wild type Rev. All of the mutants tested contain mutations in the Rev effector domain between amino acids 78 and 90, but not all of these mutations abolish Rev function (Fig. 1) .
  • the functional Rev interaction protein consisted of R32 sequences (derived from hMCM2 antisense transcript) fused to 51 amino acids from polylinker and Adh sequences (see Fig. 4) .
  • the fact that vector sequences were required for the interaction between R32- encoded amino acids and Rev indicates that R32 was not the physiological Rev target but actually represents a peptide sequence selectable when fused to a scaffold peptide domain, the Adh C-terminal sequence.
  • N- and C-terminal ExoIII deletions were performed using construct #5 (Fig. 2) which encoded a functional Rev interaction protein (containing a R32-Adh scaffold fusion) . Plasmid pools of different time points from unidirectional ExoIII deletion experiments were cotransformed into yeast with the Gal4- Rev target vector. Plasmids were rescued from positive yeast colonies. The shortest functional peptide recovered from an N-terminal deletion series had 203 of the 377 bp of the original R32 sequence deleted. The construct was transformed into yeast strains expressing Gal4(1-147), Gal4-Rev, and Gal4-RevM10 to assess binding specificity.
  • the N-terminal deletion peptide not only retained full discrimination between Rev and RevMlO but, judging from the levels of 3-Gal produced by these strains, the interaction between the N-terminal deleted Rev interaction peptide and Rev was stronger than the interaction detected with the original Rev interaction protein isolate.
  • a similar strategy of ExoIII deletions was used. These manipulations were again performed with clone #5 (Fig. 2) . Surprisingly, the active constructs from late deletion time points all showed the same structure: the polylinker and the Adh domain upstream of the stop codon had been deleted.
  • the original R32 clone was 377 nucleotide base pairs in length and encoded 125 amino acids.
  • the two- hybrid library test vector polylinker added 16 amino acids and the last 35 amino acids were contributed from the Adh terminator in the expression cassette. Deletion analysis showed that 67 of the unique 125 amino acids as well as the polylinker sequences could be deleted from the construct without losing Rev binding ability.
  • the minimal protein sequence therefore consists of 58 amino acids encoded by the hMCM2 antisense fragment fused to the Adh scaffold of 35 amino acids (SEQ ID NO:3) of the Adh C-terminus.
  • Example 6 RIP Expression Inhibits HIV Viral Protein Expression Rev is essential for HIV replication in human cells and the Rev effector domain is required for its function (Malim, M. et al. (1989), supra) .
  • the RIP peptide containing a portion of a cDNA isolated from the random pool of normally untranslated messages fused to the Adh-derived scaffold, was tested to determine if its Rev binding ability correlated with an ability to inhibit Rev function.
  • HIV-1 infectious molecular clone pNL4-3 (Adachi, A. et al. (1986) J. Virol . 59:284- 291) , which directs the expression of infectious HIV-1 virions following transfection into a variety of cell types, was used as the source of HIV in human cells.
  • Preliminary experiments were performed in HeLa cells (HeLa S3 ATCC #CCL 2.2). HeLa cells were grown in monolayers in DMEM media (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum.
  • Cells were cotransfected with pNL4-3 and either a CMV promoter-driven expression vector (pcDNAl, Invitrogen, San Diego, CA) or the identical expression vector containing the RIP cDNA (SEQ ID NO:l) (pcRIP) cloned into the ffindlll and J?coRI sites of pcDNAl.
  • pcDNAl CMV promoter-driven expression vector
  • pcRIP the identical expression vector containing the RIP cDNA
  • Transfections were performed on monolayer cells by introducing 20 ⁇ g of total plasmid DNA by the calcium phosphate procedure (Chen c. et al. (1988) Biotechnigues, 1:632-638) using 5 ⁇ g of LTR-CAT (chloramphenicol acetyltransferase) reporter construct, 5 to 15 ⁇ g of test plasmid DNA, and carrier DNA.
  • LTR-CAT chloramphenicol acetyltransferase reporter construct
  • test plasmid DNA and carrier DNA.
  • Suspension cultures were transfected with DEAE-dextran (Margolis et al., (1993) J. Virology, 192:370-374) using 4 ⁇ g of pNL4-3 and 4-12 ⁇ g of test plasmid DNA.
  • HIV-1 replication To assay HIV-1 replication, aliquots of culture media were sampled for detection of HIV-l gag p24 protein (Veronese, et al. (1985) P.N.A.S . USA, 12.:5199-5202) . Assays were performed using an antigen-capture ELISA kit (Coulter, Hialeah, FL) .
  • Example 7 RIP Inhibits HIV Production in Lymphocytes As lymphocytes are a primary target of HIV-l infection in vivo, further experiments were performed in lymphocyte cell lines. In CEM cells, cotransfection of RIP-encoding DNA at a transfection ratio of 2:1 (4 ⁇ g pNL4-3 and 8 ⁇ g pcRIP) resulted in inhibition of HIV-l antigen production up to twenty-fold greater than that mediated by cotransfection of the dominant negative Rev mutant M10 (Fig. 6A) . Previous studies demonstrating potent inhibition by Rev M10 in similar cotransfection studies have used transfection ratios of 10:1 (Malim et al. (1989) , supra) .
  • RIP is a more potent inhibitor requiring a two-fold excess (2:1) of RIP to inhibit whereas a 10-fold excess (10:1) of M10 is required for inhibition of HIV-l antigen production.
  • Cell viability 5 and cell growth were unaffected by RIP expression as tested using well known techniques of cell counting and trypan blue assay.
  • Cotransfection of a secreted alkaline phosphatase reporter gene (pCMU/SEAP) Tropix Inc. , Bedford, MA; Cullen, B. et al. (1992) Meth . Enzyme ,
  • T cell lines, CEM and H9 were maintained in suspension culture in RPMI 1640 media (ICN Biochemicals,
  • Example 8 RIP Inhibition of HIV Is Not Due to Antisense-Mediated Inhibition of MCM2 Expression
  • Example 9 Assay to Detect the Presence and/or Quantity of Rev in a Cell Lvsate Using RIP and Anti-RIP Antibody
  • the RIP of the invention as well as anti-RIP antibodies are useful in an assay for the presence of Rev in cells of a patient.
  • the assay is useful as a diagnostic tool to determine whether cells (e.g. lymphocytes) of a patient contain the HIV virus.
  • lymphocytes of a human suspected of being infected with HIV-l are collected, suspended in an appropriate culture medium to induce cellular and viral replication and induce expression of viral proteins such as Rev.
  • the cells are then lysed and the supernatant containing Rev is contacted with RIP immobilized on the surface of the well of a standard cell culture dish as shown in Fig. 8 (Steps A and B) .
  • the remaining, unbound proteins of the lysate are removed by washing (Fig. 8, Step C) .
  • an anti-RIP antibody is contacted with the proteins in the cell culture well (Fig. 8, Step D) .
  • the anti-RIP antibody binds to the remaining free RIP peptides.
  • Rev protein When Rev protein is not present in the lysate, the signal is high.
  • the lysate contains Rev, the signal is reduced due to a decrease in the number of accessible RIP proteins in the well.
  • the anti-RIP antibody can be directly labelled with a detectable tag such as radiolabelling with 125 I or a covalently bound fluorescent moiety, e.g., fluorescein.
  • a detectable tag such as radiolabelling with 125 I or a covalently bound fluorescent moiety, e.g., fluorescein.
  • unlabeled anti-RIP antibodies can be used, and the signal generated by the secondary binding of a labeled antibody to anti-RIP antibody.
  • the anti-RIP antibody binds RIP with reduced affinity relative to the binding of Rev to RIP.
  • the Rev protein remains bound to RIP during the assay for reliable qualitative or quantitative determination of the presence of Rev in cells.
  • the assay is also useful in screening candidate agents that inhibit viral production by inhibiting the expression of essential viral proteins such as Rev.
  • a cell culture containing actively replicating HIV is contacted with a candidate agent in an appropriate culture medium.
  • the cells are assayed as above for Rev protein.
  • Successful agents are those which reduce the amount of Rev protein by at least approximately 90% in the treated cells relative to untreated control cells.
  • Standard protocols are followed to carry out an assay method of the invention. For example, after bringing the immobilized RIP into contact with a cell lysate, the surface is washed to remove unbound material. Further, standard blocking protocols are generally used prior to contacting the washed surface with labeled antibodies.
  • An alternative to using cells infected with HIV is to provide acutely virus infected cells. For example, lymphocytes are obtained from uninfected persons and infected with a defined amount of HIV. The cells are then treated to remove input virus and cultured. Isolation of RIP of other selected random peptide- scaffold fusion proteins and production of antibodies to them are performed using standard techniques well known to those of ordinary skill in the art.
  • the selected random peptide-scaffold peptide of the invention can be produced by first transforming a suitable host cell with the nucleotide sequence encoding the random peptide- scaffold fusion protein cloned into a suitable expression vehicle followed by expression of the desired protein or polypeptide.
  • a suitable expression vehicle e.g., E. coli
  • a eukaryotic host e.g., Saccharomyces cerevisiae
  • the method of transformation of the cells and the choice of expression vehicle will depend on the host system selected. Methods described herein provide sufficient guidance to successfully carry out the production, purification and identification of RIP or other selected random peptide- scaffold fusion protein.
  • the RIP or other selected random peptide- scaffold fusion protein is expressed, it is isolated, e.g., using immunoaffinity chromatography.
  • an anti-RIP antibody can be attached to a column and used to isolate RIP. Lysis and fractionation of RIP- containing host cells prior to affinity chromatography can be performed by standard methods.
  • the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g.. Fisher, Laboratory Technicrues In Biochemistry and Molecular Biology, eds.. Work and Burdon, Elsevier, (1980)).
  • RIP or other peptides can also be produced by chemical synthesis by standard solution or solid phase peptide synthesis techniques.
  • Substantially pure RIP or other random peptide- scaffold fusion protein can be used to raise antibodies.
  • Antibodies directed to the polypeptide of interest are produced as follows. Peptides corresponding to all or part of the polypeptide of interest are produced using a peptide synthesizer by standard techniques, or are isolated and purified as described above. The peptides are coupled to KLH with m-maleimide benzoic acid N- hydroxysuccinimide ester. The KLH-peptide is mixed with Freund's adjuvant and injected into animals, e.g. guinea pigs or goats, to produce polyclonal antibodies.
  • Monoclonal antibodies can be prepared using the polypeptide of interest described above and standard hybridoma technology (see, e.g., Kohler et al.. Nature (1975) 256:495: Kohler et al. , Eur. J. Immunol. (1976) 6:292; Kohler et al., Eur. J. Immunol. (1976) 1:511; Hammerling et al. , in Monoclonal Antibodies and T Cell Hybridomas. Elsevier, NY, (1981) , which are incorporated herein by reference) . Antibodies are purified by peptide antigen affinity chromatography.
  • antibodies to RIP are tested for specific RIP binding by Western blot or immunoprecipitation analysis by standard techniques.
  • the method of isolating a random peptide-scaffold peptide fusion protein is useful to discover peptides that bind specifically to selected target proteins. As described above, the binding of the random peptide is enhanced by the 3-dimensional structure assumed in the context of the stably folded scaffold peptide.
  • the fused scaffold structure is also useful to determine the 3-dimensional structure of the random peptide such that small organic molecules can be designed to mimic some of its structural features. These small organic molecules are useful as potential therapeutic agents where binding to the target protein affects its function, e.g., inhibition of Rev protein.
  • the isolated random peptide-scaffold fusion protein RIP is useful as an inhibitor of HIV production in cells, such as human lymphocyte cells. RIP and antibodies to RIP are useful in an assay for the presence of Rev in a lysate of cells suspected of being infected with HIV.
  • RIP can be used to treat or prevent HIV infection by introducing a DNA sequence (SEQ ID N0:1) encoding RIP (SEQ ID NO:2), or Rev interacting analogues of the original R32-scaffold fusion, into the genome of a human cell infected with, or likely to be infected with, HIV.
  • RIP is expressed in the cell at sufficient concentration within the cell to inhibit Rev function and, thereby inhibit HIV replication.
  • Cells which are transfected with RIP-encoding DNA sequence include cells that can be infected by HIV such as CD4 + expressing cells.
  • bone marrow stem cells from an infected patient are removed from the patient and then infected with a an appropriate viral vector containing RIP-encoding DNA sequence by standard gene therapy techniques well known in the art (see e.g., Morsy, M.A. et al. (1993) JAMA 270:2338-2345..
  • the patient's remaining bone marrow is preferably destroyed by irradiation or chemical ablation to eliminate any HIV- infected stem cells that do not express RIP.
  • the transfected stem cells are reintroduced into the host from which they were isolated (autologous transplantation) by injection into a vein of the host (see. Biron, P. et al. (1993) in Autologous Bone Marrow Transplantation: Proceedings of the First International Symposium. Dicke, K.A. et al. (eds.), p. 203 for relevant techniques) .
  • the transfected stem cells are allowed to repopulate the bone marrow of the patient and provide CD4 + cells expressing RIP which inhibits HIV replication in these cells when infected with the virus. This results in simultaneously treating and protecting the host from disease resulting from HIV infection and replication. Allogenic transplantation of stem cells expressing RIP from an uninfected host is another embodiment of the invention and provides the same protection to transfected cells and their progeny from subsequent HIV replication.
  • Another use for a random peptide isolated by the method of the invention involves analyzing the structure of the random peptide in the context of the scaffold peptide for designing small molecule therapeutic agents.
  • DNA encoding a random peptide is inserted into the coding sequence of the scaffold peptide, e.g., the Bl domain of G protein.
  • the random peptide is displayed on the surface of a stable protein and therefore is less likely to be degraded.
  • the structural constraints of the scaffold may maintain the peptide in an active conformation.
  • the structure of the random peptide necessary to interact with the target protein is deduced by comparing the 3-dimensional structure of the scaffold peptide domain with and without the inserted random peptide sequence, e.g., by using NMR analysis (Heinz et al. (1994) J. Mol . Biol . 236:869-886.. Such an analysis would not be possible without the structural stabilizing effects of the scaffold peptide domain in combination with the previously solved NMR analysis of the scaffold structure.
  • Peptides that are not constrained by a scaffold peptide domain may not have a defined structure in solution. They assume a specific structure only when binding to their target protein, i.e., an induced fit. Structural analysis of the random peptide inserted in a scaffold peptide domain obviates the need for analysis of the target protein-random peptide complex. Three-dimensional information of an effective binding structure provides useful information for designing small organic molecules that can also bind the target protein. For example, a small organic molecule which mimics an aspect of the 3-dimensional structure of RIP can be designed and tested for its ability to inhibit HIV production in cells.
  • MOLECULE TYPE DNA 10
  • SEQUENCE DESCRIPTION SEQ ID NO:4:
  • MOLECULE TYPE DNA

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Abstract

Cette invention concerne un procédé d'isolation d'une protéine de fusion du type peptide existant naturellement ou non dans une cellule (aléatoire) /peptide résistant à la dénaturation (scaffold), laquelle protéine se lie à une protéine cible prédéterminée. On génère tout d'abord une banque de séquences de nucléotides codant des séquences aléatoires d'acides aminés, et qui sont ensuite clonées en un premier vecteur d'expression de sorte qu'un vecteur de test de la banque soit produit. La séquence de nucléotides est exprimée à l'intérieur d'une cellule de levure en tant protéine de fusion test comportant un domaine d'activation de transcription, un domaine peptidique aléatoire et un domaine de peptide résistant à la dénaturation. La séquence d'ADN codant une protéine cible clonée en un second vecteur d'expression permet d'obtenir un vecteur cible. La protéine de fusion cible exprimée comprend un domaine de liaison d'ADN, ainsi qu'une protéine cible. On détecte l'interaction protéine de fusion test/protéine de fusion cible selon un procédé d'analyse d'interaction entre deux protéines hybrides. Cette invention concerne également un peptide de liaison de la protéine Rev du VIH-1, lequel peptide a été isolé par le procédé décrit dans cette invention, ainsi que des procédés d'utilisation de la protéine d'interaction Rev.
PCT/US1995/009589 1995-02-01 1995-07-27 Procedes de selection d'un peptide aleatoire se liant a une proteine cible Ceased WO1996023899A1 (fr)

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Cited By (15)

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WO1997027212A1 (fr) * 1996-01-23 1997-07-31 Rigel Pharmaceuticals, Inc. Methodes de criblage de peptides effecteurs intracellulaires transdominants et de molecules d'arn
WO1999041407A1 (fr) * 1998-02-11 1999-08-19 Otogene Aktiengesellschaft Procede de developpement d'un principe actif pharmaceutique
EP0972780A1 (fr) * 1998-05-18 2000-01-19 Applied Research Systems ARS Holding N.V. Peptides antagonistes de l'Il-6
US6365344B1 (en) 1996-01-23 2002-04-02 The Board Of Trustees Of The Leland Stanford Junior University Methods for screening for transdominant effector peptides and RNA molecules
WO2001083525A3 (fr) * 2000-05-03 2002-07-18 Amgen Inc Peptides modifies utilises comme agents therapeutiques
EP1352959A1 (fr) * 1997-01-24 2003-10-15 Bioinvent International AB Procédé d'évolution moléculaire in vitro de la fonction protéinique
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
WO2005033147A1 (fr) * 2003-09-30 2005-04-14 Centre National De La Recherche Scentifique Polypeptide d’interaction comprenant un motif heptapeptidique et un domaine de penetration cellulaire
US7442778B2 (en) 2004-09-24 2008-10-28 Amgen Inc. Modified Fc molecules
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
US11485736B2 (en) 2018-12-20 2022-11-01 KSQ Therapeutics, Inc. Substituted pyrazolopyrimidines and substituted purines and their use as ubiquitin-specific-processing protease 1 (USP1) inhibitors
WO2023066881A1 (fr) 2021-10-18 2023-04-27 Astrazeneca Ab Inhibition de map3k15 pour le traitement et la prévention du diabète
US11718624B2 (en) 2020-10-30 2023-08-08 KSQ Therapeutics, Inc. Solid state forms of substituted pyrazolopyrimidines and uses thereof

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Cited By (33)

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US6737241B2 (en) 1996-01-23 2004-05-18 Rigel Pharmaceuticals, Inc. Methods for screening for transdominant intracellular effector peptides and RNA molecules
WO1997027213A1 (fr) * 1996-01-23 1997-07-31 The Board Of Trustees Of The Leland Stanford Junior University Procedes servant a rechercher par criblage des peptides effecteurs et des molecules d'arn transdominants
US6153380A (en) * 1996-01-23 2000-11-28 Rigel Pharmaceuticals, Inc. Methods for screening for transdominant intracellular effector peptides and RNA molecules
US6365344B1 (en) 1996-01-23 2002-04-02 The Board Of Trustees Of The Leland Stanford Junior University Methods for screening for transdominant effector peptides and RNA molecules
WO1997027212A1 (fr) * 1996-01-23 1997-07-31 Rigel Pharmaceuticals, Inc. Methodes de criblage de peptides effecteurs intracellulaires transdominants et de molecules d'arn
EP1295952A3 (fr) * 1996-01-23 2003-07-09 The Board of Trustees of The Leland Stanford Junior University Méthodes de criblage de peptides effecteurs transdominantes et des molécules ARN
US6833245B2 (en) 1996-01-23 2004-12-21 Rigel Pharmaceuticals, Inc. Methods for screening for transdominant effector peptides and RNA molecules
EP1352959A1 (fr) * 1997-01-24 2003-10-15 Bioinvent International AB Procédé d'évolution moléculaire in vitro de la fonction protéinique
WO1999041407A1 (fr) * 1998-02-11 1999-08-19 Otogene Aktiengesellschaft Procede de developpement d'un principe actif pharmaceutique
EP0972780A1 (fr) * 1998-05-18 2000-01-19 Applied Research Systems ARS Holding N.V. Peptides antagonistes de l'Il-6
WO1999060013A3 (fr) * 1998-05-18 2000-03-09 Applied Research Systems Peptides antagonistes de il-6
US6599875B1 (en) 1998-05-18 2003-07-29 Applied Research Systems Ars Holding N.V. IL-6 antagonist peptides
US7169905B2 (en) 1998-10-23 2007-01-30 Amgen Inc. Modified peptides as therapeutic agents
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
US9534032B2 (en) 1998-10-23 2017-01-03 Amgen Inc. Thrombopoietic compounds
US7166707B2 (en) 1998-10-23 2007-01-23 Amgen Inc. Modified peptides as therapeutic agents
US7186810B2 (en) 1998-10-23 2007-03-06 Amgen Inc. Modified peptides as therapeutic agents
US7189827B2 (en) 1998-10-23 2007-03-13 Amgen Inc. Modified peptides as therapeutic agents
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
WO2001083525A3 (fr) * 2000-05-03 2002-07-18 Amgen Inc Peptides modifies utilises comme agents therapeutiques
JP4927546B2 (ja) * 2003-09-30 2012-05-09 サントル・ナショナル・ドゥ・ラ・レシェルシュ・サイエンティフィーク−セ・エン・エール・エス− ヘプタペプチドパターン及び細胞透過ドメインを含む相互作用ポリペプチド
US7709606B2 (en) 2003-09-30 2010-05-04 Ecole Normale Superieure De Lyon Interacting polypeptide comprising a heptapeptide pattern and a cellular penetration domain
JP2008500267A (ja) * 2003-09-30 2008-01-10 サントル・ナショナル・ドゥ・ラ・レシェルシュ・サイエンティフィーク−セ・エン・エール・エス− ヘプタペプチドパターン及び細胞透過ドメインを含む相互作用ポリペプチド
WO2005033147A1 (fr) * 2003-09-30 2005-04-14 Centre National De La Recherche Scentifique Polypeptide d’interaction comprenant un motif heptapeptidique et un domaine de penetration cellulaire
US7442778B2 (en) 2004-09-24 2008-10-28 Amgen Inc. Modified Fc molecules
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US10188740B2 (en) 2005-08-12 2019-01-29 Amgen Inc. Modified Fc molecules
US11266744B2 (en) 2005-08-12 2022-03-08 Amgen Inc. Modified Fc molecules
US11485736B2 (en) 2018-12-20 2022-11-01 KSQ Therapeutics, Inc. Substituted pyrazolopyrimidines and substituted purines and their use as ubiquitin-specific-processing protease 1 (USP1) inhibitors
US11787813B2 (en) 2018-12-20 2023-10-17 KSQ Therapeutics, Inc. Substituted pyrazolopyrimidines and substituted purines and their use as ubiquitin-specific-processing protease 1 (USP1) inhibitors
US11718624B2 (en) 2020-10-30 2023-08-08 KSQ Therapeutics, Inc. Solid state forms of substituted pyrazolopyrimidines and uses thereof
WO2023066881A1 (fr) 2021-10-18 2023-04-27 Astrazeneca Ab Inhibition de map3k15 pour le traitement et la prévention du diabète

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