WO2001025421A2 - Materials and method for detecting interaction of cftr polypeptides - Google Patents
Materials and method for detecting interaction of cftr polypeptides Download PDFInfo
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- WO2001025421A2 WO2001025421A2 PCT/US2000/027900 US0027900W WO0125421A2 WO 2001025421 A2 WO2001025421 A2 WO 2001025421A2 US 0027900 W US0027900 W US 0027900W WO 0125421 A2 WO0125421 A2 WO 0125421A2
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- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4712—Cystic fibrosis
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- 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
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- 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
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/20—Detection means characterised by being a gene reporter based analysis
- C12Q2565/201—Two hybrid system
Definitions
- Cystic fibrosis is the most common genetic disease of Caucasians in North America, occurring at a frequency of approximately 1 in 2500 births (Welsh et al, 1995). The disease results from defective function of the gene encoding the Cystic Fibrosis
- Transmembrane Conductance Regulator (CFTR) protein in a variety of tissues, including the pancreas and the lung epithelium.
- Riordan et al. (1989), Rommens et al. (1989) and Kerem et al. (1989) describe the cloning and sequencing of the CFTR gene.
- U.S. Patent No. 5,543,399 to Riordan et al. discloses the purification of CFTR protein.
- Normal CFTR protein is a membrane protein that functions as a cAMP-regulated chloride channel.
- the ⁇ F508 mutation in the CFTR gene which is characterized by a deletion of the phenylalanine amino acid at position 508 of the CFTR protein, is the defect associated with most cases of CF.
- a CFTR protein having the ⁇ F508 mutation does not exit the ER and proceed on to the plasma membrane (Cheng et al, 1990; Gregory et al, 1991). It has been found that the ⁇ F508 mutation causes the temperature-sensitive misprocessing of the mutant protein that prevents the protein from exiting the ER (Denning et al, 1992).
- pancreatic enzymes may be introduced into the diet of CF patients as a means of reversing the effects of pancreatic insufficiency
- CF cells lack CFTR chloride channel activity because they have mutant CFTR genes that encode a defective CFTR protein
- providing a patient with a copy of a normal human CFTR gene by way of gene therapy methods may provide an alternative to conventional therapies for the treatment of CF
- Gene therapy strategies for the treatment of CF thus involve delivery of a normal wildtype human CFTR gene to mutant CF epithelial cells within the lung to restore normal CFTR chloride channel activity
- Gene transfer of the CFTR gene can be accomplished by several different delivery methods
- Recombinant viral vectors containing the wildtype CFTR gene provide one potential means to deliver the CFTR gene to CF cells
- recombinant adenovirus containing the wildtype CFTR gene have been shown to efficiently transfer the wildtype CFTR gene into CF epithelium, and correct the chloride channel defect (Welsh et al , 1994, Zabner et al, 1993)
- high doses of virus are generally required to obtain an efficacious response, which in time can cause inflammation resulting from the immune response to the viral proteins
- Other viruses that might be used for CF gene therapy include AAV (Adeno-associated virus) (Flotte et al, 1994), retrovirus and lentivirus
- AAV Addeno-associated virus
- retrovirus retrovirus
- Gene transfer can also be achieved by transfection of CF cells by lipid-DNA complexes composed of plasmid DNA containing the CFTR cDNA in association with catiomc or neutral
- the subject invention concerns mate ⁇ als and methods for detecting the interaction of CFTR proteins
- the method can be used to determine whether one CFTR NBD1 polypeptide interacts with a second CFTR NBD 1 polypeptide using a yeast dual hybrid assay
- the subject methods can be used to determine whether mutations to the CFTR polypeptide reduce or eliminate dimerization of the CFTR polypeptides
- the present methods can also be used to screen and identify revertant mutations that restore dimerization of a mutant CFTR polypeptide, as well as mutations that enhance dimerization and CFTR activity greater than that of wildtype protein
- the subject invention also provides mate ⁇ als and methods for efficiently identifying and screening for compounds, drugs and other such compositions that facilitate proper dimerization of the CFTR polypeptides
- Compounds identified using materials and methods of the present invention are candidate agents for use in treating patients having CF
- a yeast dual hyb ⁇ d assay is used to identify compounds that can restore dimerization of a protein comprising a region of a CFTR polypeptide having a mutation, such as ⁇ F508, that prevents dimerization
- the assay methods of the present invention can be used to evaluate a large number of compounds in a high throughput format
- the use of a yeast growth bioassav exemplified herein is fast and inexpensive in compa ⁇ son to current screening procedures that involve mammalian cells and assays for CFTR channel activity
- compositions and methods for treating CF The compositions of the invention can be used to restore, promote or enhance the dime ⁇ zation of CFTR protein and/or its exit from the ER and proper localization in the cell
- Figure 1 shows an example of a plate with a positive-testing plant leaf disc on selective media using a yeast two-hybrid assay of the present invention
- Figures 2A-2C show growth of two-hyb ⁇ d yeast strains containing wildtype and mutant CFTR NBD1
- the yeast strain YRG2 was transformed with pADGAL4 and pBDGAL4 constructs containing wildtype and mutant CFTR NBD1 as indicated
- Each strain was grown in synthetic complete media lacking leucine and tryptophan, and equal a quots of cells in 10-fold dilutions were spotted to synthetic complete media lacking leucine, tryptophan and histidine Dilutions for each strain proceed from right to left, with the spots from most dilute cultures corresponding to the tapered end of the triangle
- Figure 3 shows correction of the ⁇ F508 dime ⁇ zation defect by I539T and G550E
- the yeast strain YRG2 (Stratagene) was transformed with pADGAL4 and pBDGAL4 constructs containing wildtype and mutant CFTR NBD1 as indicated
- Each strain was grown in synthetic complete media lacking leucine and tryptophan, and equal ahquots of cells in 10-fold dilutions were spotted to synthetic complete media lacking leucine, tryptophan and histidine Dilutions for each strain proceed from right to left, with the spots from most dilute cultures corresponding to the tapered end of the triangle Plates were incubated at 37°C for 5 days
- Figure 4 shows the effect of the revertant mutations I539T and G550E on CFTR ⁇ F508 chlo ⁇ de channel activity in FRT stable cell lines
- FRT stable cell lines were seeded in permeable Milhcell supports (Milhpore) at a density of 2 5 x 10 5
- SEQ ID NO:l is a primer for PCR amplification of a fragment of a cDNA encoding CFTR
- SEQ ID NO:2 is a p ⁇ mer for PCR amplification of a fragment of a cDNA encoding CFTR
- SEQ ID NO: 3 is a polynucleotide sequence that encodes a wildtype CFTR protein
- SEQ ID NO:4 is an ammo acid sequence of a wildtype CFTR protein
- the subject invention concerns mate ⁇ als and methods for detecting the interaction of cystic fibrosis transmembrane conductance regulator (CFTR) proteins
- the method can be used to determine whether one CFTR polypeptide interacts with a second CFTR polypeptide
- the CFTR polypeptides are mammalian More preferably, the CFTR polypeptides are human CFTR polypeptides
- the methods of the present invention are based on the discover ⁇ that the wildtype CFTR protein forms dimers, and that dime ⁇ zation is essential for the exit of the CFTR protein from the endoplasmic reticulum (ER)
- a method of the present invention for detecting or determining the interaction of a first CFTR polypeptide with a second CFTR polypeptide comprises contacting the CFTR polypeptides and determining whether the polypeptides interact using a system where if interaction does occur then a detectable signal or change is induced in the assay system
- CFTR cystic fibrosis trans
- a method of the invention for detecting or determining the mteraction of a first CFTR polypeptide with a second CFTR polypeptide comprises (a) providing a first fusion protein comp ⁇ sing all or a portion of a first CFTR protein and a
- DNA binding domain of a transcriptional activator that can bind to a site on a detectable reporter gene (b) providing a second fusion protein comprising all or a portion of a second CFTR polypeptide and a transcriptional activation domain of a transcriptional activator that can activate transc ⁇ ption of the detectable reporter gene, (c) contacting the first fusion protein and the second fusion protein under conditions where if the first fusion protein and the second fusion protein interact then the interaction causes the transc ⁇ ptional activation domain to activate transc ⁇ ption of the detectable reporter gene, and (d) detecting transc ⁇ ption of the detectable reporter gene or expression of the detectable reporter gene product
- detectable gene it is meant that expression of the gene or its gene product can be detected
- the detectable gene can be engineered with sequences that b ⁇ ng the gene under control of the transcriptional activator
- the UAS G upstream activation site, galactose
- the interaction of a first CFTR polypeptide with a second CFTR polypeptide can be detected in a host cell by the interaction of signal transduction fusion proteins, or by the interaction of proteins resulting in cleavage of a ubiquitin fusion protein
- SOS recruitment Adsorption protein fusion protein
- split ubiquitin sensor Johnsson and Varshavsky, 1997)
- the preferred host cell for these embodiments is yeast
- the interaction of a first CFTR polypeptide with a second CFTR polypeptide is detected by interaction of signal transduction fusion proteins within a bacte ⁇ al cell
- Methods for detecting protein protein interactions in bactena are also known by those skilled in the art (Ka ⁇ mova et al , 1998)
- the CFTR portion of the fusion protein can contain one or more mutations of the
- wildtype human CFTR used in the subject dual hvb ⁇ d methods interact and result in transc ⁇ ption of the detectable gene in the host cell in the dual hyb ⁇ d embodiment described herein
- Mutations that reduce or prevent dimerization of CFTR proteins can be identified using the methods and mate ⁇ als of the present invention because these mutant CFTR proteins do not interact and, therefore, transc ⁇ ption of the detectable gene in the host cell does not occur
- the ⁇ F508 mutation is shown to interfere with CFTR interaction
- any suitable DNA-binding domain and transcriptional activation domain can be used in the subject invention as long as the domains can be used to activate transc ⁇ ption of the detectable gene when the DNA- binding domain and transcriptional activation domain are brought into sufficiently close proximity to each other
- the DNA-binding domain and transcriptional actuation domain can be derived from the same protein or from different proteins Examples of suitable domains are known m the art and can be obtained from, for example, yeast GAL4 GCN 1 and ADR In an exemplified embodiment of the invention, the domains are derived from yeast GAL4 protein
- Non-yeast DNA-binding and/or transcriptional activation domains are also contemplated for use in the present invention and include, for example, a DNA- binding domain derived from the prokaryotic LexA protein and an 88-res ⁇ due peptide
- the host cells can be any suitable prokaryotic or eukaryotic cell, including bacterial, yeast or mammmalian cells
- the host cell is a yeast cell More preferably, the yeast cell is Saccharomyces
- the interaction of the first hybrid protein and the second hybrid protein in the host cell causes a measurably greater expression of the detectable gene than that observed where the first hybrid protein and the second hybrid protein do not interact or interact at a reduced level
- the detectable gene used in the present invention can be any gene whose transcription can be detected when the detectable gene is expressed as a result of the interaction of the CFTR fusion protein containing the DNA-binding and transc ⁇ ptional activation domains
- expression of the gene is detected directly or indirectly by detecting the expression product of the detectable gene
- the detectable gene may provide for drug resistance or encode an enzyme or other product that can be readily measured or detected Such measurable activity may include providing the host cell with the ability to grow only when the detectable reporter gene is expressed, or providing for the presence of detectable protein or enzyme activity only when the detectable reporter gene is expressed
- Suitable detectable genes are well known in the art Examples of detectable genes include lacZ (which encodes ⁇ -galactosidase), HIS3, LEU2 and the like In an
- the NBDl region of CFTR (containing arnino acids 351-650) was cloned into two plasmids, pBDGAL4 and pADGAL4, (Stratagene) that produce the NBDl -DNA binding domain fusion protein and the NBDl -activation domain fusion protein, respectively, when co-expressed in yeast
- pBDGAL4 and pADGAL4 (Stratagene) that produce the NBDl -DNA binding domain fusion protein and the NBDl -activation domain fusion protein, respectively, when co-expressed in yeast
- pBDGAL4 and pADGAL4 (Stratagene) that produce the NBDl -DNA binding domain fusion protein and the NBDl -activation domain fusion protein, respectively, when co-expressed in yeast
- pBDGAL4 and pADGAL4 (Stratagene) that produce the NBDl -DNA binding domain fusion protein and the NBDl -activation domain fusion protein
- the subject invention also concerns unique host cells that can be used to model wildtype CFTR protein dimerization, and which can also be used to model the effect of
- the host cells are yeast cells, such as Saccharomyces cervistae or other suitable cells
- the host cells are genetically engineered to express a hybrid protein that comprises a first human or other mammalian CFTR protein fused to a DNA binding domain of a transcriptional activator that can bind to a site on a detectable gene in the host cell
- the host cells are also engineered to express a second hyb ⁇ d protein that comp ⁇ ses human or mammalian CFTR protein fused to a transc ⁇ ptional activator domain that can activate transcription of the detectable gene in the host cell when the transc ⁇ ptional activator domain is brought into sufficiently close proximity with the detectable gene in the host cell
- the portion of the CFTR protein expressed in the hyb ⁇ d proteins is the first nucleotide binding domain (NBDl) of a human or other mammalian CFTR protein
- NBDl nucleotide binding domain
- first or second CFTR polypeptides, or both the first and second polypeptides used in the subject method comprise the temperature-sensitive ⁇ F508 mutation
- interaction of the fusion proteins will be reduced when cells are incubated at the nonpermissive temperature
- the polynucleotide encoding the first CFTR polypeptide and the second CFTR polypeptide are therefore expressed in the host cell incubated at the nonpermissive temperature resulting in impaired interaction between the first CFTR polypeptide and the second CFTR polypeptide, and reduced expression of the detectable gene
- a compound is added to the host cell incubated at the nonpermissive temperature, and the expression of the detectable gene is greater than the expression of the detectable gene in the host cell incubated at the nonpermissive temperature in the absence of the compound, then that compound can be used in restoring dimerization of CFTR polypeptides comprising the ⁇ F508 mutation
- the present invention concerns methods for screening chemical compounds for drug candidates with activity to correct the dimeriztion defect
- the YRG2- ⁇ F yeast strain to grow at an increased rate in the proximity of the filter paper disc containing the active compound The enhanced growth of the YRG2- ⁇ F yeast around a filter paper disc thus indicates the presence of an active compound within the test disc
- the compound can then be anaylzed further in secondary assays to determine its activity to restore CFTR ⁇ F508 cAMP-stimulated chloride channel activity in mammalian cells expressing CFTR ⁇ F508 (Sheppard et al , 1994)
- the sample being tested is a complex mixture of chemical compounds in a natural product extract
- the extract can be fractionated by standard techniques and the fractions assayed using the YRG2- ⁇ F yeast as described above to identify fractions with the purified active compound
- media used m screening compounds can contain 3-am ⁇ no-2,3,4-t ⁇ azole at a concentration of about 1 5mM to make the assay more selective for compounds with high activity
- the screening method can contain 3-am ⁇ no-2,3,4-t ⁇
- the present invention also concerns plants and isolated extracts thereof that contain compounds or compositions that facilitate, enhance or restore dime ⁇ zation of CFTR polypeptides Plants that have tested positive for compounds capable of facilitating dime ⁇ zation of CFTR polypeptides include 7) ichiha species
- the present invention also concerns the compounds identified as facilitating, enhancing or restoring CFTR dimerization Active compounds identified using the yeast mating and two-hybrid assays described herein can be purified from plants using standard biochemical function methods known in the art.
- the present invention also concerns methods for screening plants for compounds of interest
- fragments of plant leaves are prepared from a plant to be tested and screened for bioactive compounds using a yeast-based assay of the present invention
- a plant containing a compound that facilitates or enhances dimerization of mutant CFTR polypeptides is indicated by growth of yeast on a selective media
- the methods described herein for screening the plants are efficient because a large number of plants can be tested on one petri dish and the results can be determined within a few days
- plants can be screened for bioactive compounds using a yeast two-hybrid assay according to the present invention
- plants are screened using a yeast strain which contains a CF mutation that prohibits or interferes with the dimerization of CFTR proteins
- the strain cannot grow on a selective media, such as, for example, a histidine-deficient media when the host cells are unable to synthesize histidine
- a selective media such as, for example, a histidine-deficient media when the host cells are unable to synthesize histidine
- a composition that restores the ability of the hybrid proteins containing mutations in the CFTR proteins to interact can then be secondarily tested for activity to restore cAMP-stimulated chloride channel function in mammalian cells expressing CFTR having the same mutation
- Drugs and compounds that restore dimerization and function in vitro can be further evaluated to confirm in vivo efficacy in treating clinical CF disease
- the subject invention also concerns methods for treating CF by providing a drug or other compound that restores, promotes or enhances the dimerization of CFTR protein and/or its exit from ER
- an effective amount of a drug or compound identified using the methods of the present invention is administered to a CF patient
- the amount of the drug or compound to be administered can be readily determined by the ordinarily skilled clinician having the benefit of the subject disclosure.
- the drug or compound can also be provided to a CF patient by gene therapy methods
- a polynucleotide sequence encoding the protein can be delivered to CF cells of a patient either in vivo or ex vivo using standard gene transfer methods and constructs
- the drug or compound is expressed in the CF cell and thereby promotes dimerization of the mutant CFTR protein to enable the CFTR protein to properly localize and function as in a normal, non-CF cell
- the subject invention also concerns drugs, compounds, polypeptides and biologically active fragments thereof, antibodies or antigen binding fragments thereof, polynucleotides and other agents identified using the methods of the invention that restore, promote or enhance the in vivo dimerization of CFTR protein and/or its exit from ER in a cell
- the drugs and compounds of the present invention can be used to treat CF patients according to the methods described herein
- the subject invention also concerns methods for screening for second site mutations that co ⁇ ect the defect in mutant CFTR
- methods for screening for second site mutations that co ⁇ ect the defect in mutant CFTR For example, using the methods of the subject invention, one can screen for mutations that correct a CFTR gene carrying the ⁇ F508 mutation
- the present invention can be used to screen for second site mutations that provide increased expression and function of CFTR that is greater than that observed for normal wildtype CFTR expression
- the present invention also concerns mutant CFTR genes that contain second site mutations that correct the CF defect and provide increased expression and function of CFTR substantially the same as or greater than normal human wildtype CFTR
- YRG2 (genotypes at a, ura3-52, his3-200, ade2-101, lys2-801, trpl-901, le ⁇ -3, 112, gal4-542, gal80-538, LYS2::UAS GAL1 -TATA GAL1 -HIS3 URA::UAS GAL4 17mere(x3) TATA CYC1 -lacZ).
- the plasmids pSWICK-CFTR (obtained from Dr. Michael Welsh, University of Iowa) and pSwick-CFTR ⁇ F508 contain the full length wildtype CFTR cDNA and mutant CFTR cDNA (containing ⁇ F508) respectively. Derivatives of these plasmids
- pSwick-BXWT and pSwick BX ⁇ F respectively that contain a Sma I restriction site at CFTR nucleotide position 1626 and a Xho I site at nucleotide position 1808 were constructed by site-directed mutagenesis.
- a DNA fragment containing CFTR amino acids T351-F492 was produced using pSwick-BXWT plasmid DNA as template and the primers PRNBD1-R1 (5'-
- the resulting 142 bp DNA fragment contained an Eco Rl restriction endonuclease site preceding the CFTR amino acid T351 and a Pst I site following CFTR amino acid S492.
- the fragment was restricted with EcoRl and Pst I restriction endonucleases, and ligated into the unique Eco RI and Pst I restriction sites within pAD-GAL4 to produce pADPRNBDl in which CFTR amino acids are T351-S492 joined in frame to the pAD-GAL4 transcription activation domain.
- a second GAL4- CFTR fusion plasmid was constructed in which a 951 bp Hpal-Taql DNA fragment from pSwick-BXWT (containing CFTR amino acids R334-F650, and with the ends of the fragment made blunt by kenow fragment) was purified from an agarose gel and ligated into the Sma I site of plasmid pBDGAL4 to produce PBD-N.
- the pBD-N plasmid DNA was then cut with Eco RI and Bam HI and the vector molecule purified from an agarose gel.
- the purified Eco RI-Bam HI pBD-N vector molecule was then ligated to an Eco RI- Bam HI restriction fragment from pADPRNBDl (containing amino acids T351-S492 of
- the pBDPN-WT plasmid contains CFTR amino acids T351-F650 fused in frame to the GAL4 DNA binding domain This region contains the predicted cytosolic region that precedes NBDl, the NBDl region, and also a segment that had previously been ascribed to the R domain
- the plasmid pBDPN-WT also contains the TRPl gene of yeast, and replication o ⁇ gin from the yeast 2 ⁇ plasmid
- the Eco Rl-Pst I fragment from pBDPN-WT (containing CFTR amino acids T351-F650) was then cloned into the EcoRI and Pst sites of pADGAL4, producing pADPN-WT pADPN-WT contains CFTR amino acids T351-F650 fused in frame to the GAL4 activation domain
- the pADPN-WT plasmid also contains the LEU2 gene of yeast and the replication origin of the yeast 2 ⁇ circle Both plasmi
- pBDPN ⁇ F was constructed by cutting pBDPN-WT with Bam HI and Xho I and replacing the approximately 180 bp Bam Hl-Xho I fragment (containing the wildtype
- a protein protein interaction between the CFTR NBDl polypeptide segment in each GAL4 fusion protein encoded by pBDPN-WT and pADPN-WT plasmids in YRG2- WT is expected to activate transcription of the HIS3 reporter gene in yeast, resulting in a HIS+ phenotype Accordingly, the YRG2-WT strain was streaked onto agar plates containing synthetic complete media lacking tryptophan, leucine, and histidine (SC-HIS,
- the YRG2-WT strain was phenotypically HIS * at all temperatures tested (21 °C, 30°C, and 34°C), indicating that the two NBDl fusion proteins associated in vivo to activate GAL4 transcription
- the YRG2 strain containing either the pBDPN-WT plasmid or the pADPN-WT plasmid alone was unable to grow on media lacking histidine
- CFTR NBDl segment is thus able to self-associate and form dimers at 21 °C, 30 °C and 34°C
- the YRG2-WT yeast strain had a generation time of approximately 300, 138 and 420 minutes, respectively.
- YRG2- ⁇ F was grown in SC-HIS-LEU-TRP liquid culture media at 21 °C, 30°C and 34°C (Table 2)
- the generation time of YRG2- ⁇ F was observed to be approximately 400 minutes, which is comparable to the generation time of YRG2-WT under the same growth conditions
- the generation time of YRG2- ⁇ F was approximately 1380 minutes, which is substantially longer than the generation time of the YRG2-WT strain (138 minutes) at this temperature
- the generation time of YRG2- ⁇ F incubated at 34°C was also increased substantially as compared to YRG2-WT (420 minutes)
- the effect of the ⁇ F508 mutation on YRG2- ⁇ F growth rate is analogous to the temperature-sensitive effect of ⁇ F508 on the processing of CFTR ⁇ F508, indicating the folding defect of CFTR ⁇ F508 has been faithfully modeled with the YRG2- ⁇ F yeast strain.
- Example 2 Screening of Plants for Compounds Using the Yeast Two-Hybrid Assay
- the YRG- F yeast strain used for this assay expresses two hybrid genes consisting of the N-terminal nucleotide binding domain of CFTR which contains the cystic fibrosis causing mutation ⁇ F508 (NBDl ⁇ F508), fused to the DNA binding domain of the GAL4 transcription activator (GAL4BD) in the first hybrid and to the GAL4 activation domain (GAL4AD) of the second hybrid.
- GAL4BD GAL4 transcription activator
- GAL4AD GAL4 activation domain
- the yeast strain is used as a bioassay tool for the detection of dimerization of the NBDl ⁇ F508 domain of CFTR.
- mutant human CFTR chloride channel containing the ⁇ F508 mutation defective dimerization of the channel is impaired in a temperature- sensitive manner.
- the dimerization of NBDl ⁇ F508 in the YRG2- ⁇ F strain is temperature-sensitive
- the YRG2- ⁇ F strain cannot activate transcription of the HIS3 gene which prevents the strain from growing on selective media lacking histidine at temperatures higher that 21 °C
- the YRG2- ⁇ F strain is not capable of growing in a medium lacking the amino acid histidine as a result of defective NBDl ⁇ F508 dimerization, it can be used in a plate assay to screen for compounds, such as those present in plant leaves, that promote the association of the two NBDl ⁇ F508
- the association of NBDl ⁇ F508 brings the Gal4 activation and DNA binding domain together, thereby activating HIS3 synthesis and permitting growth of the yeast in a medium lacking histidine
- a lawn of the YRG2- ⁇ F strain is spread onto selective media
- This media contains yeast nitrogen base and all amino acids except for histidine, leucine, and tryptophan
- the media also contains 3 -amino 1,2,4-t ⁇ azole at a concentration of 1 5 mM
- the addition of the 3-am ⁇ no 1,2,4-t ⁇ azole to the media inhibits the residual HIS3 enzyme activity present in the YRG2- ⁇ F strain, thereby producing a tighter his- phenotype of the YRG2- ⁇ F strain on media lacking histidine
- the YRG2- ⁇ F strain was added to the selective media, along with leaf discs to be screened and the plates were incubated at 30°C for several days Increased growth of the YRG2- ⁇ F strain around a leaf disc indicates the presence of a compound in the disc that reversed the ⁇ F508 dime ⁇ zation defect
- Figure 1 shows a plate containing a leaf disc
- the two-hyb ⁇ d system of yeast is an effective tool for demonstrating the binding interaction of two protein domains
- the system is based on the well-characterized interaction of the DNA-binding domain (BD) and transc ⁇ ption-activation domain (AD) of the GAL4 transcription factor of yeast
- BD DNA-binding domain
- AD transc ⁇ ption-activation domain
- the association of these two domains within the GAL4 protein results in the specific initiation of transc ⁇ ption of a reporter gene in yeast, but when these domains are expressed in yeast as separate domains, neither is capable of transcriptional activation in the absence of specific interaction with the other
- the two-hybrid system was used to devise a phenotypic assay for the binding of the CFTR NBD
- the DNA sequence encoding wildtype CFTR NBDl T351-F650 was cloned in frame into the carboxy-terminus of the GAL4 DNA-binding domain on a yeast plasmid pBD-GAL4 (Stratagene)
- This plasmid (pBDGAL4-WT), contains a fusion protein consisting of the GAL4 DNA binding domain fused to CFTR NBD 1 expressed under the control of the yeast ADH1 promoter and also contains the yeast selectable marker TRP1 and the 2 ⁇ origin of replication
- the same segment of CFTR NBDl was also cloned in frame into the GAL 4 activation domain on pADGAL4 (with the yeast selectable marker LEU2 and 2 ⁇ o ⁇ gin)
- This plasmid (pADGAL4-WT) contains a fusion protein consisting of the GAL transcription-activation domain fused to NBDl expressed under the regulation of the ADH1 promoter, and also contains the LEU2 gene of
- the two plasmids were transformed into yeast strain YRG2 to produce YRG2- WT
- the YRG2 strain has the endogenous GAL4 transcription factor deleted and has auxotrophies trpl, leu2, and h ⁇ s3 Association of the two fusion proteins mediated by the interaction of NBDl domains on each protein results in the transcriptional activation of the reporter gene, HIS3 that is regulated by GAL4 in YRG2 Activation of the HIS3 gene in yeast allows the YRG strain to grow on media lacking histidine, conferring a HIS+ phenotype As shown m Figures 2A-2C, interaction between the NBDl fusions in YRG- WT results in a HIS+ phenotype at both 21°C and 37°C The wildtype CFTR NBDl segment is thus able to self-associate and form dimers
- the ⁇ F508 mutation was introduced into both pBDGAL4-WT and pADGAL4- WT, creating pBDGAL4- ⁇ F and pADGAL4- ⁇ F, respectively, and both plasmids were used to transform YRG2 (producing YRG- ⁇ F) Unlike the YRG-WT strain the YRG- ⁇ F strain was phenotypically HIS- when tested for growth on selective media lacking histidine at 37°C However, when tested for growth on selective media lacking histidine at 21°C the YRG2 strain was phenotypically HIS+ Thus, the ⁇ F508 mutation conferred a temperature-sensitive HIS+ phenotype to the yeast strain containing pAD ⁇ F and pBD ⁇ F, analogous to the temperature-sensitive processing defect observed for CFTR ⁇ F508 A strain containing pBD ⁇ F and pADWT (YRG- ⁇ F/WT) was also temperature-sensitive, indicating that heterodimers between a mutant
- Second-site revertant mutations in NBDl that restore dimerization of NBDl would constitute a genetic intervention that restores CFTR ⁇ F508 processing and function Revertants of the ⁇ F508 dime ⁇ zation defect were identified using the two- hyb ⁇ d system Revertant mutations would be expected to restore defective dimerization in yeast, and also correct the processing defect of CFTR ⁇ F508 when introduced into a CFTR ⁇ F508 cDNA allele expressed in mammalian cells
- the fact that the formation of dimers between a wildtype NBDl and mutant NBD1 ⁇ F508 is defective at the nonpermissive temperature (Figures 2A-2C), as it is in YRG2- ⁇ F (where both NBDl fusion proteins contain ⁇ F508) was exploited If grown at the permissive temperature however, this "heterozygote strain is HIS+ indicating that the mutant NBDl is able to assume a wild
- CFTR ⁇ F508 cDNA alleles containing either I539T or G550E were constructed for expression in mammalian cells
- the 1539T and G550E mutations were introduced into the plasmid expression vector pSWICK (Swick et al , 1992) (producing pSWICK-CFTR ⁇ F508/I539T and pSWICK- CFTR ⁇ F508/G550E) using oligonucleotide mutagenesis and the polymerase chain reaction
- the pSWICK-CFTR ⁇ F508/I539T plasmid DNA (15 ⁇ g ) and pSWICK- CFTR ⁇ F508/G550E DNA (15 ⁇ g ) were then each mixed with 15 ug of pcDNA3 1 plasmid DNA (Invitrogen), which contains the gene encoding Zeocin (Invitrogen), which contains the gene encoding Zeocin (Invitrogen), which contains the gene encoding Zeocin (
- CFTR ⁇ F508/G550E wildtype CFTR or mutant CFTR ⁇ F508 were then grown as monolayers in Mimcells, and mounted into Ussing chambers to assay for cAMP- stimulated chloride channel activity (Sheppard et al , 1994)
- a control cell line expressing wildtype CFTR produces a cAMP-stimulated chlo ⁇ de current of approximately 67 ⁇ Amps/cm 2
- a cell line expressing CFTR ⁇ F508 produces approximately 1 7 ⁇ Amps/cm 2
- the cell lines expressing CFTR ⁇ F508/I539T and CFTR ⁇ F508/G550E each produced a significantly higher level of cAMP-stimulated chloride current (approximately 26 ⁇ Amps/cm 2 and 17 ⁇ Amps/cm 2 , respectively) as compared to a cell line expressing CFTR ⁇ F508, indicating that both revertant mutations
- Example 5 Screening for molecules to correct the CFTR ⁇ F508 dimenzation defect
- CFTR ⁇ F508 dime ⁇ zation defect the methods of the present invention were used to screen plants for compounds with activity to increase CFTR ⁇ F508 chloride channel processing and function
- the YRG2- ⁇ F strain was used to identify a plant of the genus T ⁇ chilia that produces a compound with activity to reverse the dime ⁇ zation defect in
- YRG2- ⁇ F An extract was prepared from leaf material of the plant and fractionated by standard methods Fractions were assayed using the YRG2- ⁇ F strain to detect activity (i.e., activity to reverse the NBDl dimerization defect resulting from the ⁇ F508 mutation) A compound with activity was purified from the plant extract and designated TS3 The TS3 compound was then assayed for activity to co ⁇ ect the CFTR ⁇ F508 chloride channel defect in mammalian cells
- the TS3 compound was added at a concentration of 40 uM to the cell culture media of FRT cells grown in Millicells for three days Cells were incubated in the presence of TS3 for an additional 72 hours To assay cells for CFTR ⁇ F508 cAMP- stimulated activity, the monolayers were mounted into Ussing chambers, cAMP agonists were added and the resulting peak change in chloride conductance was measured
- the results show that incubation of cells expressing the mutant CFTR ⁇ F508 chloride channel with TS3 results in an approximately 70% increase in cAMP-stimulated chloride channel activity as compared to untreated cells expressing CFTR ⁇ F508
- This data indicates that the TS3 compound has activity to co ⁇ ect the molecular defect of CFTR ⁇ F508 leading to increased functional activity at the plasma membrane
- the data additionally demonstrate that the YRG2- ⁇ F yeast strain is an effective means to identify and purify compounds that have activity to correct the molecular defect causing cystic
Abstract
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AU11939/01A AU1193901A (en) | 1999-10-06 | 2000-10-06 | Materials and method for detecting interaction of cftr polypeptides |
US10/089,875 US7238474B1 (en) | 1999-10-06 | 2000-10-06 | Materials and methods for detecting interaction of CFTR polypeptides |
US11/821,812 US8288107B2 (en) | 1999-10-06 | 2007-06-25 | Materials and methods for detecting interaction of CFTR polypeptides |
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US15799699P | 1999-10-06 | 1999-10-06 | |
US60/157,996 | 1999-10-06 | ||
US18189200P | 2000-02-11 | 2000-02-11 | |
US60/181,892 | 2000-02-11 | ||
US18237300P | 2000-02-14 | 2000-02-14 | |
US60/182,373 | 2000-02-14 |
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US10/089,875 A-371-Of-International US7238474B1 (en) | 1999-10-06 | 2000-10-06 | Materials and methods for detecting interaction of CFTR polypeptides |
US11/821,812 Continuation US8288107B2 (en) | 1999-10-06 | 2007-06-25 | Materials and methods for detecting interaction of CFTR polypeptides |
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EP2968987A1 (en) * | 2013-03-15 | 2016-01-20 | Vertex Pharmaceuticals Inc. | Correctors acting through msd1 of cftr protein |
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WO1994025607A1 (en) * | 1993-04-23 | 1994-11-10 | University Of Iowa Research Foundation | Method of assaying for drugs which restore function of mutant cftr protein |
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US5543399A (en) | 1989-08-22 | 1996-08-06 | Hsc Research & Development Limited Partnership | Cystic fibrosis transmembrane conductance regulator (CFTR) protein |
US5283173A (en) | 1990-01-24 | 1994-02-01 | The Research Foundation Of State University Of New York | System to detect protein-protein interactions |
US5670488A (en) | 1992-12-03 | 1997-09-23 | Genzyme Corporation | Adenovirus vector for gene therapy |
US5939536A (en) | 1990-03-05 | 1999-08-17 | Genzyme Corporation | Methods for purifying cystic fibrosis transmembrane conductance regulation |
US7118911B1 (en) | 1990-03-05 | 2006-10-10 | Genzyme Corporation | DNA molecules stabilized for propagation in bacterial cells that encode cystic fibrosis transmembrane conductance regulator |
CA2037478A1 (en) | 1990-03-05 | 1991-09-06 | Richard Gregory | Diagnostic and treatment methods involving the cystic fibrosis transmembrane regulator |
US5981714A (en) | 1990-03-05 | 1999-11-09 | Genzyme Corporation | Antibodies specific for cystic fibrosis transmembrane conductance regulator and uses therefor |
US5674898A (en) | 1990-03-05 | 1997-10-07 | Genzyme Corporation | Methods and therapeutic compositions for treating cystic fibrosis |
US6270954B1 (en) | 1996-04-10 | 2001-08-07 | The Regents Of The University Of California | Correction of genetic defects using chemical chaperones |
WO1997037645A1 (en) | 1996-04-10 | 1997-10-16 | The Regents Of The University Of California | Correction of genetic defects using chemical chaperones |
US6316223B1 (en) * | 1998-03-30 | 2001-11-13 | Rigel Pharmaceuticals, Inc. | Mammalian protein interaction cloning system |
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Title |
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ANNEREAU ET AL: "Insight into cystic fibrosis by structural modeling of CFTR first nucleotide binding fold (NBF1)" COMPTES RENDUS DES SEANCES DE L'ACADEMIE DES SCIENCES. SERIE III: SCIENCES DE LA VIE,NL,ELSEVIER, AMSTERDAM, vol. 320, no. 2, 1997, pages 113-121, XP002085670 ISSN: 0764-4469 * |
HALLOWS KENNETH R ET AL: "Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase." JOURNAL OF CLINICAL INVESTIGATION, vol. 105, no. 12, June 2000 (2000-06), pages 1711-1721, XP002163368 ISSN: 0021-9738 * |
KUNZELMANN K ET AL: "Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator." FEBS LETTERS, vol. 400, no. 3, 1997, pages 341-344, XP002163366 ISSN: 0014-5793 * |
NEVILLE DAVID C A ET AL: "Expression and characterization of the NBD1-R domain region of CFTR: Evidence for subunit-subunit interactions." BIOCHEMISTRY, vol. 37, no. 8, 24 February 1998 (1998-02-24), pages 2401-2409, XP002163367 ISSN: 0006-2960 * |
THOREAU V ET AL: "Molecular cloning, expression analysis, and chromosomal localization of human Syntaxin 8 (STX8)" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,ACADEMIC PRESS INC. ORLANDO, FL,US, vol. 257, 1999, pages 577-583, XP002102758 ISSN: 0006-291X * |
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EP2968987A1 (en) * | 2013-03-15 | 2016-01-20 | Vertex Pharmaceuticals Inc. | Correctors acting through msd1 of cftr protein |
EP2968987A4 (en) * | 2013-03-15 | 2017-04-26 | Vertex Pharmaceuticals Inc. | Correctors acting through msd1 of cftr protein |
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US20080167259A1 (en) | 2008-07-10 |
WO2001025421B1 (en) | 2001-09-27 |
US8288107B2 (en) | 2012-10-16 |
WO2001025421A3 (en) | 2001-08-30 |
US7238474B1 (en) | 2007-07-03 |
AU1193901A (en) | 2001-05-10 |
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