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WO1996012007A1 - Protease de recombinaison stable du cmv humain - Google Patents

Protease de recombinaison stable du cmv humain Download PDF

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Publication number
WO1996012007A1
WO1996012007A1 PCT/US1995/012987 US9512987W WO9612007A1 WO 1996012007 A1 WO1996012007 A1 WO 1996012007A1 US 9512987 W US9512987 W US 9512987W WO 9612007 A1 WO9612007 A1 WO 9612007A1
Authority
WO
WIPO (PCT)
Prior art keywords
hcmv
protease
dna
recombinant
pcr
Prior art date
Application number
PCT/US1995/012987
Other languages
English (en)
Inventor
Robert L. Lafemina
Vinod V. Sardana
Charlotte A. Veloski
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU39528/95A priority Critical patent/AU3952895A/en
Publication of WO1996012007A1 publication Critical patent/WO1996012007A1/fr

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Classifications

    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/04—Varicella-zoster virus
    • G01N2333/045—Cytomegalovirus

Definitions

  • HCMV human cytomegalovirus
  • the HCMV encodes a protease that participates in the maturation of the viral capsid.
  • the enzyme processes the viral assembly protein within the capsid core by mediating cleavage between the ala-ser peptide bond at residue positions 308/309. This results in the linked extrusion of the assembly protein and the encapsidation of the viral genomic DNA.
  • the association of the individual assembly proteins into the capsid likely results from specific intermolecular protein interactions.
  • the presence of the protease at the N-terminus of a 80kD precursor that also contains the assembly protein assures localization of the enzyme in the capsid as a consequence of interactions mediated by the assembly protein portion.
  • a mutant of the herpes simplex virus type 1 (HSV-1 ) which expresses temperature-sensitive alterations in the protease, is incapable of processing the assembly protein and encapsidating genomic DNA at the non-permissive temperature. This result indicates that a specific potent inhibitor of the viral enzyme would be useful as a therapeutic agent.
  • V 141G, V207G and V207G were discovered that V 141G, V207G and V207G.
  • V254G substitutions in the HCMV protease increase stability.
  • the resulting stable recombinant HCMV protease is useful as a screening tool for HCMV antivirals in the potency range of about 100-200 nM, as well as a diagnostic tool for diseases resulting from HCMV infection.
  • HCMV cytomegalovirus
  • substitutions are glycine for valine at position 141 , glycine for valine at position 207, or glycine for valine at position 254.
  • recombinant HCMV protease is useful as a screening tool for HCMV antivirals, as well as a diagnostic tool for diseases resulting from HCMV infection.
  • One embodiment of the present invention are the sequences for stable HCMV protease with the double substitution V 141G and V207G (SEQ. ID NOs: 5 & 6).
  • One utility for stable HCMV proteases is a screening assay for the detection of compounds that inhibit HCMV protease. This assay has a procedure comprising the steps of:
  • Also encompassed in the present invention are compounds that substantially inhibit the stable HCMV protease, i.e., those with an IC 50 value of ⁇ 200 nM.
  • Such cells act as hosts and include
  • E. coli, B. subtilis, yeasts, fungi, plant cells or animal cells E. coli, B. subtilis, yeasts, fungi, plant cells or animal cells. Expression vectors for many of these host cells have been isolated and
  • expression vector for the expression of a desired foreign DNA largely depends on availability of the host cell and how fastidious it is, whether the host cell will support the replication of the expression vector, and other factors readily appreciated by those of ordinary skill in the art.
  • the technology for recombinant procaryotic expression systems is now old and conventional.
  • the typical host cell is E. coli.
  • the technology is illustrated by treatises such as Wu, R (ed) Meth.
  • the foreign DNA insert of interest comprises and DNA sequence coding for HCMV (or stable mutant thereof) of the present invention, including any synthetic sequence with this coding capacity or any such cloned sequence or combination thereof.
  • HCMV peptides coded and expressed by an entirely recombinant DNA sequence is encompassed by this invention.
  • ribosome binding sites appropriate ribosome binding sites, termination codons, enhancers, terminators, or replicon elements.
  • additional features can be inserted into the vector at the appropriate site or sites by conventional splicing techniques such as restriction endonuclease digestion and ligation.
  • Yeast expression systems which are one variety of recombinant eukaryotic expression systems, generally employ
  • Saccharomyces cerevisiae as the species of choice for expressing recombinant proteins.
  • S. cerevisiae and similar yeasts possess well known promoters useful in the construction of yeast expression systems, including but not limited to GAP491 , GAL10, ADH2, and alpha mating factor.
  • Yeast vectors useful for constructing recombinant yeast expression systems for expressing HCMV include, but are not limited to, shuttle vectors, cosmids, chimeric plasmids, and those having sequences derived from 2-micron circle plasmids.
  • Insertion of the appropriate DNA sequence coding for HCMV or stable mutant thereof, into these vectors will, in principle, result in a useful recombinant yeast expression system for HCMV where the modified vector is inserted into the appropriate host cell, by transformation or other means.
  • One preferred expression system is with baculovirus, under the control of the polyhedrin promoter. See. e.g., D.R. O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual W.H. Freeman 1992, for a background description of this expression technology.
  • a host mammalian cell can be any cell that has been efficiently cloned in cell culture.
  • Host mammalian cells useful for the purposes of constructing a recombinant mammalian expression system include, but are not limited to, Vero cells, NIH3T3, GH3, COS, murine Cl27 or mouse L cells.
  • Mammalian expression vectors can be based on virus vectors, plasmid vectors which may have SV40, BPV or other viral replicons, or vectors without a replicon for animal cells. Detailed discussions on mammalian expression vectors can be found in the treatises of Glover, D.M. (ed.) "DNA Cloning: A Practical
  • Recombinant HCMV may possess additional and desirable structural modifications not shared with the same organically
  • synthesized peptide such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristoylation.
  • synthesized peptide such as adenylation, carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristoylation.
  • PCR polymerase chain reaction
  • the primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers.
  • Taq DNA Polymerase catalyzes primer extension in the amplification process.
  • the enzyme is a thermostable DNA polymerase isolated from Thermus aquaticus. Because it stays active through repeated elevations to high denaturation temperatures, it needs to be added only once. Deoxynucleotide triphosphates provide the building blocks for primer extension.
  • telomere sequences are generated as the cycle is repeated.
  • the coding domain and any additional primer-encoded information such as restriction sites or translation signals (signal sequences, start codons and/or stop codons) is obtained.
  • PCR protocols are often performed at the 100 ⁇ L scale in 0.5 ml microcentrifuge tubes.
  • the PCR sample may be single- or double-stranded DNA or RNA. If the starting material is RN A, reverse transcriptase is used to prepare first strand cDNA prior to PCR.
  • PCR primers are oligonucleotides, typically 15 to 50 bases long, and are complementary to sequences defining the 5' ends of the complementary template strands. Non-template complementary 5' extensions may be added to primers to allow a variety of useful post amplification operations on the PCR product without significant perturbation of the amplification itself. It is important that the two PCR primers not contain more than two bases complementary with each other, especially at their 3' ends. Internal secondary structure should be avoided in primers.
  • PCR buffer is composed of about 50 mM potassium chloride, 10.0 mM Tris-HCl (pH 8.3 at room temperature), 1.5 mM magnesium chloride, and 0.001 % w/v gelatin.
  • a titration series in small increments over the 1.5- to 4-mM range will locate the magnesium concentration producing the highest yield of a specific product. Too little free magnesium will result in no PCR product and too much free magnesium may produce a variety of unwanted products.
  • a typical PCR protocol for amplification of the DNA template includes an initial 8 minute 94°C denaturation step, followed by 30 cycles of 30 seconds at 94°C
  • Primer annealing is usually performed first at 55°C, and the specificity of the product is evaluated. If unwanted bands are observed, the annealing temperature should be raised in subsequent optimization runs. While the primer annealing temperature range is often 37-55°C, it may be raised as high as the extension temperature in some cases. Merging of the primer annealing and primer extension steps results in a two-step PCR process. Primer extension, in most applications, occurs effectively at a temperature of 72°C and seldom needs optimization. In the two-temperature PCR process the temperature range may be 65-70°C. In situations where enzyme concentration limits amplification in late cycles, the extension is preferably increased linearly with cyclic number. Usually, 25 to 45 cycles are required for extensive
  • amplification i.e., 1 ,000,000 fold of a specific target.
  • HCMV strain AD 169 DNA was prepared from supernate virions as previously described [LaFemina, R.L., et al., J. Gen Vir., 64. 373 ( 1983)].
  • the N-terminal 256 amino acid protease domain was PCR amplified using primers derived from the DNA seguence as described by Chee, M.S., et al., Curr. Top Microbiol. Immunol., 154, 125 ( 1990), Genbank accession number X17403.
  • the seguence of the N-terminal primer was 5'GCTAGGCTCATATGACGATGGACGAGCAGCAG (SEQ ID NO: 7), while the seguence of the C-terminal primer was 5'GCTAGGCTAGATCTTTACGCCTTGACGTATGACTCGC (SEQ ID NO: 8).
  • PCR conditions consisted of: 6 cycles with 0.5 min
  • the amplified DNA was digested with Ndel and Bglll prior to ligation into the Ndel and BamHI sites of the T7 expression vector pET3c.
  • the resulting plasmid, pT7CMVPr-4, was introduced into E. coli BL21 DE3 for expression. Expression was induced by standard IPTG induction for 2 hr.
  • microfluidizer at 25 psi.
  • the lysate (20 ml) was centrifuged 30 min at 8,000 ⁇ g and the pellet washed twice with 10 ml of lysis buffer containing 0.1 % NP40.
  • the pellet was incubated at 40°C under N 2 in 4.5 ml of 6.0
  • the enzyme was loaded in Buffer A (25 mM Tris-HCl, pH 7.5, 1 mM DTT and 1 mM EDTA) at 0.3 ml/min and eluted with a 0-1.0 M NaCl salt gradient at 1 ml/min.
  • the gradient used was Buffer A for 10 min, linear increase to 0.3 M NaCl in Buffer A for 30 min, followed by linear increase to 1.0 M NaCl in 15 min.
  • the active protease eluted at 0.15 to 0.20 M NaCl.
  • the fractions were analyzed by SDS-PAGE and by native gel electrophoresis as well as by two HPLC sizing columns attached in series (300 ⁇ 7.5 mm) in PBS.
  • Absorbance of the eluate was monitored at 210 nm using a photodiode array detector.
  • the enzyme concentration used in the assay varied from 150 to 1500 nM depending on the substrate used.
  • Each substrate peptide was titrated from 50 ⁇ M to 5.0 mM.
  • Kinetic parameters (kcat and km) were determined by fitting the velocity (initial rates at ⁇ 5.0% of total substrate hydrolysis) versus substrate concentration data to the Michaelis-Menton equation (hyperbolic). The initial velocity and steady-state conditions for the enzyme reaction were established for each peptide substrate.
  • the recombinant human cytomegalovirus protease (with V 141G and V207G substitutions, 256 amino acids, 20 nM) cleaves the peptide substrate 3 H-Acetyl Gly Val Val Asn Ala Ser Cys Arg Leu Arg Arg amide ( 1 mM) (SEQ ID NO. 9) at the Ala Ser bond.
  • the assay is performed in 100 mM Hepes (pH 7.5), 1 mM EDTA .05% BSA, 25 mM NaCl (50 ⁇ l total volume) and quenched by adding 50 ⁇ l of 5% phosphoric acid.
  • the assay mix is transferred to a tube containing Dowex ion exchange, the tubes are rinsed with water (2 ⁇ 200 ⁇ l).
  • the cleaved radioactive peptide in the supernatant is quantitated by a scintillation counter. Reduction in radioactivity in presence of compounds gives the measure of inhibition, and is determined as the concentration of inhibitory compound giving 50% inhibition or IC 50 .
  • Highly potent compounds generally have IC 50 less than or equal to about 200 nM.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La protéase du cytomégalovirus (CMV) humain est rendue stable par certaines mutations découvertes par les déposants, et est utile en tant qu'outil de dosage d'agents antiviraux anti-CMV humain ainsi qu'en tant qu'outil de diagnostic pour les maladies induites par une infection à CMV humain.
PCT/US1995/012987 1994-10-17 1995-10-13 Protease de recombinaison stable du cmv humain WO1996012007A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39528/95A AU3952895A (en) 1994-10-17 1995-10-13 Stable recombinant hcmv protease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32395394A 1994-10-17 1994-10-17
US323,953 1994-10-17

Publications (1)

Publication Number Publication Date
WO1996012007A1 true WO1996012007A1 (fr) 1996-04-25

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PCT/US1995/012987 WO1996012007A1 (fr) 1994-10-17 1995-10-13 Protease de recombinaison stable du cmv humain

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2311605A (en) * 1996-03-26 1997-10-01 Merck & Co Inc Assay for inhibitors of human cytomegalovirus (HCMV) protease
WO1998004286A3 (fr) * 1996-07-26 1998-05-14 Searle & Co Vaccin a base d'herpesvirus a deficience d'assemblage

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5434074A (en) * 1991-07-05 1995-07-18 Gibson; D. Wade Cytomegalovirus proteinase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5434074A (en) * 1991-07-05 1995-07-18 Gibson; D. Wade Cytomegalovirus proteinase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAUM ET AL: "Expression and Analysis of the Human Cytomegalovirus UL80-Encoded Protease: Identification of Autoproteolytic Sites.", JOURNAL OF VIROLOGY, vol. 67, no. 1, January 1993 (1993-01-01), pages 497 - 506 *
JONES ET AL: "Proteolytic Activity of Human Cytomegalovirus UL80 Protease Cleavage Site Mutants.", JOURNAL OF VIROLOGY, vol. 68, no. 6, January 1993 (1993-01-01), pages 3742 - 3752 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2311605A (en) * 1996-03-26 1997-10-01 Merck & Co Inc Assay for inhibitors of human cytomegalovirus (HCMV) protease
WO1998004286A3 (fr) * 1996-07-26 1998-05-14 Searle & Co Vaccin a base d'herpesvirus a deficience d'assemblage

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Publication number Publication date
AU3952895A (en) 1996-05-06

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