Patent Application
20190359686
The present invention pertains to the biotechnological field, particularly to a fluorescent fusion polypeptide, a biosensor comprising said polypeptide and uses thereof.
Bioluminescence resonance energy transfer (BRET) is a powerful and increasingly popular technique for studying protein-protein interactions in live cells and real time. In particular, there has been considerable interest in the ability to monitor interactions between G protein-coupled receptors (GPCRs) and proteins that serve as key regulators of receptor function, such as beta-arrestin. The BRET methodology involves heterologous co-expression of genetically fused proteins that link one protein of interest (e.g., a GPCR) to a bioluminescent donor enzyme and a second protein of interest (e.g., beta-arrestin) to an acceptor fluorophore. If the fusion proteins are in close proximity, resonance energy will be transferred from the donor to the acceptor molecule and subsequent fluorescence from the acceptor can be detected at a characteristic wavelength. Such fluorescence is therefore indicative of the proteins of interest linked to the donor and the acceptor interacting directly or as part of a complex. In addition to monitoring protein-protein interactions to elucidate cellular function, BRET also has the potential to become an important technique for live cell high-throughput screening for drugs targeting GPCRs, utilizing ligand-induced interactions with beta-arrestins.
However, these types of biosensors are highly dependent on the distance between (e.g. <10 nm for CFP/YFP) and the relative orientation of donor and acceptor fluorophore. In addition, BRET or FRET-based biosensors in the context of high content screening methods requires of a detection equipment of at least four filters, two for the excitation and two for the emission. In addition, due to the low intensity of the detection signal, the detection signal range and the screening sensibility are low. Lastly, the use of more than one fluorescence emission signal requires the use of more algorithms in order to correctly analyse the final signal.
Thus, there is still a need to develop improved methods or products for real time measurement of protein-protein interactions in live cells such as interactions between G protein-coupled receptors (GPCRs) and proteins that serve as key regulators of receptor function, such as beta-arrestin.
The present invention confronts the problem of providing tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible that are capable of monitoring a variation in the translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane in vivo by using High-content screening (HCS) in cell-based systems, wherein these tools do not have the disadvantages of FRET-based biosensors.
In order to solve the above problem, the authors of the present invention designed a new fluorescent fusion polypeptide comprising a membrane localization peptide, a peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors by GRKs or RTKs, a vesicularization peptide and a fluorescent peptide. This biosensor is formed by two peptides targeted to two different cellular compartments, allowing the detection of the translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane in vivo by monitoring the distribution of the fluorescent polypeptide within the cellular cytoplasm. In this sense, the biosensor translocation within the cell shall be due to a change in its 3D conformation that hides or exposes the location signals in both ends of the polypeptide triggered by the binding of the peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors to the phosphorylated G-protein or tyrosine receptors.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.
Unless expressly specified otherwise, the term “comprising” is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as a specific embodiment of the present invention that the term “comprising” encompasses the possibility of no further members being present, i.e. for the purpose of this embodiment “comprising” is to be understood as having the meaning of “consisting of”.
In the context of the present invention, the term “fusion polypeptide” refers to a hybrid polypeptide comprising a combination of at least four peptides from different proteins that are combined into the same polypeptide structure.
In the context of the present invention, the term “membrane localization peptide” is intended to mean a peptide whose natural intracellular localization is in the plasma membrane.
As used herein, the term “peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors by GRKs or RTKs” is intended to mean a peptide or protein domain that has the ability to interact with the G-protein or tyrosine receptors only when these are phosphorylated. A clear example of such peptide is the phosphorylated receptor binding peptide from b-arrestin of SEQ ID 2.
It is noted that the nucleotide sequence coding for the phosphorylated receptor binding peptide of SEQ ID NO 2 is as follows:
As used herein, the term “vesicularization peptide” is intended to mean a short peptide chain that directs the transport of the polypeptide to the retention vesicles. Preferably such vesicularization peptide is the Clathrin and beta-adaptin peptide from arrestin. More preferably, the peptide sequence for the Clathrin and beta-adaptin peptide from arrestin is as follows (from hereinafter SEQ ID NO 4):
As used herein, the term “fluorescent peptide” is intended to mean a fluorescent peptide that has fluorescent capacities. Fluorescent peptide domains are characterized by having a specific excitation spectrum and emission spectrum.
In the context of the present invention, the linker has at least one amino acid residue, preferably at least two consecutive amino acid residues.
As used herein, the term “biosensor” is intended to mean a molecular tool or entity that is sensitive to, and can respond to, a physical or chemical stimulus and transmit information about cellular status.
As used herein, the term “drug” is intended to mean a molecule that potentially acts as an agonist or antagonist or modulator of a signalling pathway.
As used herein “stable cell line” is intended to mean a cell line that has been transfected or infected with a foreign piece of DNA that has incorporated itself into the genome of the cell.
The present invention confronts the problem of providing tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible that are capable of monitoring a variation in the translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane in vivo by using High-content screening (HCS) in cell-based systems, wherein these tools do not have the disadvantages of FRET-based biosensors.
In order to solve the above problem, the authors of the present invention designed a new fluorescent fusion polypeptide comprising a membrane localization peptide, a peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors by GRKs or RTKs, a vesicularization peptide and a fluorescent peptide. This biosensor is formed by two peptides targeted to two different cellular compartments, allowing the detection of the translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane in vivo by monitoring the distribution of the fluorescent polypeptide within the cellular cytoplasm. In this sense, the biosensor translocation within the cell shall be due to a change in its 3D conformation that hides or exposes the location signals in both ends of the polypeptide triggered by the binding of the peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors to the phosphorylated G-protein or tyrosine receptors.
In the basal state, the biosensor is located in one of the compartments; this means that the location peptide directed to the other cellular compartment is hidden by the 3D conformation. When there is a translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane due to a cellular stimulation, the peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors binds to the phosphorylated G-protein or tyrosine receptors causing a conformational change in the biosensor. At this point the spatial distribution of the different structural elements in the biosensor is modified and the vesicularization peptide directed to the other cellular compartment is exposed by the new 3D conformation so that the whole biosensor is transported to its new location at the new cellular compartment. All this process can be traced in living cells due to the presence of the fluorescent protein in the biosensor.
It is further noted, that the authors of the present invention realized that the order of the peptides within the above mentioned fluorescent fusion polypeptide could not be placed arbitrarily within the polypeptide. This is the case since after numerous experiments the authors concluded that only one combination of elements provided the technical effect of transporting the biosensor to the other cellular compartment, such combination was:
The authors tested whether such biosensor having the above structure could be employ for detecting and quantifying the translocation of beta-arrestin caused by substance P. As illustrated in the examples disclosed herein, the authors of the present invention constructed a fluorescent fusion polypeptide, capable of detecting and quantifying beta-arrestin successfully, comprising the phosphorylated receptor binding peptide from b-arrestin of SEQ ID 2. Surprisingly, the authors identified that by using as a vesicularization peptide the Clathrin and beta-adaptin peptide from arrestin, the detection signal was significantly better.
The results shown in the examples and drawings presented herein by using the above fusion polypeptide indicate that an increased in the translocation of beta-arrestin induced a conformational change in the biosensor which promoted a redistribution of the fluorescent biosensor. The activity was calculated as an increment of the granularity of the cells transfected with the biosensors of the invention. The fluorescence redistribution of the biosensor was detected by fluorescence using image analysis algorithms. Consequently, the variations in the location of the beta arrestin can be monitored through this “hiding and exposition” process of location signals and the final localization of the biosensor.
Thus, a first aspect of the present invention refers to a fluorescent fusion polypeptide capable of changing its localization within the cell from the cell cytoplasmic membrane to the retention vesicles, upon an increase in the translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane, comprising a membrane localization peptide, a peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors by GRKs or RTKs, a vesicularization peptide and a fluorescent peptide wherein:
Preferably, such fluorescent fusion polypeptide is capable of changing its localization within the cell from the cell cytoplasmic membrane to the retention vesicles, upon an increase in the translocation of GRKs (G-protein receptor kinases) and beta-arrestin from the cell cytoplasm to the cell cytoplasmic membrane.
More preferably the fluorescent fusion polypeptide of the first aspect of the invention is characterized by:
Amino acidic SEQ ID No 3 is as follows:
More preferably the fluorescent fusion polypeptide of the first aspect of the invention is characterized by:
More preferably the fluorescent fusion polypeptide of the first aspect of the invention is characterized by:
In yet another preferred embodiment of the first aspect of the invention, such polypeptide is capable of changing its localization within the cell from the cell cytoplasmic membrane to the retention vesicles, upon an increase in the translocation of Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane.
More preferably the fluorescent fusion polypeptide of the above mentioned preferred embodiment of the invention (the biosensor capable of detecting an increased an increase in the translocation of Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane) is characterized by:
More preferably the fluorescent fusion polypeptide of the above mentioned preferred embodiment of the invention (the biosensor capable of detecting an increased an increase in the translocation of Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane) is characterized by:
More preferably the fluorescent fusion polypeptide of the above mentioned preferred embodiment of the invention (the biosensor capable of detecting an increased an increase in the translocation of Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane) is characterized by:
It is noted that such peptide capable of recognizing the phosphorylated Receptor tyrosine protein, may comprised a SH2 domain and/or a domain designed in silico, any of these capable of recognizing phosphorylated tyrosin-kinase receptors. Such domains are well known to the skilled person.
In another preferred embodiment of the first aspect of the invention, the fluorescent fusion polypeptide may be selected from any of the following list of compounds consisting of GFP, YFP, turboGFP, turboRFP, turboRFP602 and turboFP650.
A second aspect of the invention refers to a nucleic acid molecule comprising a polynucleotide sequence coding for a polypeptide as defined in any of the previous aspects of the invention.
A third aspect of the invention refers to a biosensor comprising the fusion polypeptide as defined in the first aspect of the invention.
A fourth aspect of the invention refers to a cell comprising the fluorescent fusion polypeptide as defined in the first aspect of the invention, wherein preferably said cell is cell line U2OS (see examples).
In a further aspect, the present invention relates to several uses for the fluorescent fusion polypeptide as defined in the first aspect of the invention. A first use of the biosensor according to the present invention is for detecting and quantifying compounds capable of the translocation of GRKs (G-protein receptor kinases) and/or beta-arrestin or Receptor tyrosine kinases (RTKs) from the cell cytoplasm to the cell cytoplasmic membrane including, but not limited thereto, substance P. As already stated, binding the peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors binds to the phosphorylated G-protein or tyrosine receptors results in a substantial change in the spatial conformation that leads to a change in the intracellular fluorescence localization. This fluorescence translocation can be harnessed for compound quantification by fluorescence. In addition, all this process can be traced in living cells due to the presence of the fluorescent protein in the biosensor.
The employment of the fluorescent fusion polypeptide as defined in the first aspect of the invention further involves its use as a tool for drug screening.
In addition, the fluorescent fusion polypeptide as defined in the first aspect of the invention is useful in the practice of essentially any application for which readout of second messenger transduction is obtained. Such applications are well known in the art. However, more exemplary applications of the present invention include but are not limited to:
In a preferred embodiment of the invention, the fluorescent fusion polypeptide as defined in first aspect of the invention can be used to generate stable cell lines which allow studying G-protein coupled receptors (GPCR), and the activity of others proteins in living cells. The rapid translocation of the biosensor of the invention allows the quantification of GPCR and ion channel stimulation.
The fluorescent fusion polypeptide and the corresponding biosensor of the present invention can be made by techniques well known by those skilled in the art but as a way of example, they can be constructed as follows. The coding sequences corresponding to the membrane localization peptide, the fluorescent peptide, the peptide capable of binding G-protein or tyrosine receptors following phosphorylation of these receptors by GRKs or RTKs, and the vesicularization peptide can be easily amplified by PCR and cloned into a shuttle plasmid. These coding sequences can be then easily cloned into the final fusion plasmid in the specific order presented herein using the restriction enzyme sites that flanked each sequence.
The following examples merely serve to illustrate the present invention.
Human bone osteosarcoma cell line (DSMZ), derived from ATCC (Catalog No. HTB-96), was grown in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 HAM (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich), MEM non-essential amino acids (Sigma-Aldrich), gentamicin (Sigma-Aldrich), Geneticin 500 μg/ml (Sigma-Aldrich) and Puromycin 10 μg/ml (Sigma) at 37° C. in a humidified atmosphere supplemented with 5% CO2.
Red ß-ARRNomad biosensor plasmid was created by subsequent cloning of the Tac cDNA, turboFP602 protein cDNA, (Evrogen), phosphorylated receptor binding peptide from b-arrestin, and the vesicularization peptide (Clathrin and beta-adaptin peptide from arrestin) into the geneticin resistant CMV-ptNL vector designed in our lab. Tachykinin receptor 1 (NK1) receptor's cDNA (cDNA.org) was subcloned in a Puromycin resistant vector, CMV-pPuro, designed in our lab using NheI and XhoI restriction enzymes.
All the clonings were confirmed by DNA sequencing. Plasmid containing Red R ARRNomad biosensor was transfected into U2OS cells using Lipofectamine LTX (Invitrogen) and positive transfected cells were selected using 500 μg/ml Geneticin (Sigma-Aldrich) the positive clone named C1 was transfected with the puromycin resistant plasmid containing Tachykinin receptor 1 (NK1-pPuro) and the positive clones were selected using 10 μg/ml puromicyn (Sigma-Aldrich) and 500 μg/ml geneticin (Sigma-Aldrich). Resistant clones were obtained by limit dilution. Double stable cell line, termed “NK1-Red ß-ARRNomad biosensor cell line” was then tested using Substance P (Sigma-Aldrich) for functional Red ß-ARRNomad biosensor response.
Cells were seeded at 18,000 cells/plate in 96-well black, clear-bottom imaging plates (BD Biosciences) for ß-arrestin assays. “NK1-Red ß-ARRNomad biosensor” cell line was treated with 8 log dilution (n=6) of Substance P (Sigma-Aldrich) ranging from 0 to 1 μM in Optimem media (Gibco) for 24 hours at 37° C., 5% CO2, 95% relative humidity before data acquisition.
For fluorescence intensity determinations, changes in the distribution of Red ß-ARRNomad biosensor were measured using “Synergy 2” microplate reader (Biotek). Optimem media with the agonists was removed and replaced by PBS 1× without calcium and magnesium (Sigma-Aldrich) before the data acquisition. TurboFP602 data were acquired with ex/em 590/620 filters.
The image analysis routine of the redistribution of Red ß-ARRNomad biosensor was developed with a BD Pathway 855 High-Content automated image platform (BD Biosciences) using image analysis algorithms with the Attovision bioimaging software (BD). Rhodamine excitation/emission filters used for image acquiring were 548/20 and 570LP. Calculated Nomad biosensor number of vesicles were normalized as percentage of activity compared to the negative control (vehicle H2O).
Cells were seeded at 18,000 cells/plate in 96-well plates for inhibition assay. “NK1-Red ß-ARRNomad biosensor” cell line was co-treated with SubstanceP 100 nM (Sigma-Aldrich) and log dilution of L733,060 hydrochloride (Tocris) ranging from 100 μM to 3 μM in Optimem media (Gibco) for 24 hours at 37° C., 5% CO2, 95% relative humidity before data acquisition.
The signal to background (S/B) parameter was calculated as μc+−μc−, and Z′factor was calculated using the following formula: Z′=1-[(3σc++3σc−)/(μc+−μc−)].
“NK1-Red ß-ARRNomad biosensor” cell line stably expressing red ß-ARRNomad biosensor and Neurokinin receptor 1 has been designed to assay compounds or analyze its capability to modulate NK1 receptor. Before the stimulation mediated by the agonist of interest, the fluorescent biosensor is localized in the cellular membrane, the union of Substance P to Neurokinin 1 receptor leads to a change in the structural folding of Red ß-ARRNomad Biosensor that promotes its cellular relocation in the vesicular trafficking of the cells.
The increase in the fluorescence was detected and analyzed using “Synergy 2” microplate reader from Biotek. The Ec50 for the Substance P was approx. 5.20×10−9 M after a treatment of 24 h with the agonist. The assay was validated with an average of Z′=0.70+/−0.02 (n=6).
Activation and Red ß-ARRNomad biosensor change of localization processes were detected and analyzed using “BD Pathway 855” High-Content Bioimager from BD Biosciences. The Ec50 for the Substance P was approx. 2.99×10−9M after a treatment of 24 h with the agonist. The assay was validated with an average of Z′=0.79+/−0.02 (n=6).
Inhibition Assay
“NK1-Red ß-ARRNomad biosensor” cell line was co-treated with Substance P 100 nM and increasing concentrations of L733,060 (NKR1 inhibitor) during 24 h. After the treatment, nuclei were stained with DAPI and inhibition of NK1 receptor was detected analyzing Red R-ARRNomad biosensor change of localization using “BD Pathway 855” High-Content Bioimager from BD Biosciences. The Ic50 for L733,060 was Approx. 1.84×10-7M after a treatment of 24 h with the agonist. The assay was validated with an average of Z′=0.66+/−0.02.
“EP4 receptor Red ArrestinNomad biosensor” HEK293 cell line transiently expressing red ArrestinNomad biosensor and prostaglandin E receptor 4 has been designed to assay compounds or analyze its capability to modulate EP4 receptor. Before the stimulation mediated by the agonist of interest, the fluorescent biosensor is localized in the cellular membrane, the union of PGE2 to EP4 rececptor leads to a change in the structural folding of Red ArrestinNomad biosensor that promotes its cellular relocation in the vesicular trafficking of the cells. The Ec50 for the PGE2 was 8.64×10−9M after a treatment of 24 h with the agonist. The assay was validated with an average of Z′=0.67+/−0.04 (n=6).
HEK293 cells derived from ATCC (Catalog No. CRL-1573), were grown in Dulbecco's Modified Eagle's Medium-high glucose (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich), MEM non-essential amino acids (Sigma-Aldrich) and gentamicin (Sigma-Aldrich) at 37° C. in a humidified atmosphere supplemented with 5% CO2.
Red ArrestinNomad biosensor plasmid was created by subsequent cloning of the Tac cDNA, turboFP602 protein cDNA, (Evrogen), phosphorylated receptor binding peptide from b-arrestin, and the vesicularization peptide (Clathrin and beta-adaptin peptide from arrestin) into the geneticin resistant CMV-ptNL vector designed in our lab. EP4 receptor (EP4) receptor's cDNA (cDNA.org) was subcloned in a Puromycin resistant vector, CMV-pPuro, designed in our lab using NheI and XhoI restriction enzymes.
Transient transfection was performed using phosphate calcium method and the transiently transfected cell line, termed “EP4-Red ArrestinNomad biosensor cell line” was then tested using PGE2 (Sigma-Aldrich) for functional Red ArrestinNomad biosensor response.
Cells were seeded at 20,000 cells/plate in 96-well black, clear-bottom imaging plates (BD Biosciences) for ß-arrestin assays. “EP4-Red ArrestinNomad biosensor” cell line was treated with 8 log dilution (n=6) of PGE2 (Sigma-Aldrich) ranging from 0 to 10 μM in Optimem media (Gibco) for 24 hours at 37° C., 5% CO2, 95% relative humidity before data acquisition. Fluorescent images were acquired in the BD Pathway 855 High-Content automated image platform with a ×20 dry objective.
For fluorescence intensity determinations, changes in the distribution of Red ß-ARRNomad biosensor were measured using “Synergy 2” microplate reader (Biotek). Optimem media with the agonists was removed and replaced by PBS 1× without calcium and magnesium (Sigma-Aldrich) before the data acquisition. TurboFP602 data were acquired with ex/em 590/620 filters.
The signal to background (S/B) parameter was calculated as μc+−μc−, and Z′factor was calculated using the following formula: Z′=1−[(3σc++3σc−)/(μc+−μc−)]
Ca2+-Arrestin Multiplex “PAR2 Arres-Ca2+Nomad biosensor” in the U2OS cell line stably expressing green Ca2+Nomad biosensor, red ArrestinNomad biosensor and F2R like trypsin receptor 1 has been designed to assay compounds or analyze its capability to modulate PAR2. Before the stimulation mediated by the agonists of interest, the fluorescent biosensor is localized in the cellular membrane, the union of SLIGKV or SLIGRL-NH2 to PAR2 leads to a change in the structural folding of both Nomad biosensors that promotes their cellular relocation in the vesicular trafficking of the cells.
The increase in the fluorescence was detected and analyzed using the “Synergy 2” microplate reader from Biotek. The Ec50 for the agonists was measured after a treatment of 24 h. The assay was validated with Z′>0.6 for each agonist.
Human bone osteosarcoma cell line (DSMZ), derived from ATCC (Catalog No. HTB-96), was grown in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 HAM (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich), MEM non-essential amino acids (Sigma-Aldrich), gentamicin (Sigma-Aldrich) at 37° C. in a humidified atmosphere supplemented with 5% CO2.
The plasmid encoding the green Ca2+Nomad biosensor was transfected into U2OS cells using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, Mass.). Resistant clones were obtained by limit dilution, and positively transfected cells were selected using 10 μg/ml puromycin (Thermo Fisher Scientific, Waltham, Mass.). Once this stable cell line was ready, a positive clone was transfected with the plasmid encoding the red ArrestinNomad biosensor, and the positive clones were selected by limit dilution using 10 μg/ml puromycin (Thermo Fisher Scientific, Waltham, Mass.) and 500 μg/ml geneticin (Sigma-Aldrich, St. Louis, Mo.). The cell line co-expressing both biosensors, named Arres-Ca2+Nomad cell line, was subsequently transfected with the PAR2-CMV-pHygro construction, and resistant clones were obtained by limit dilution and selected using 500 μg/ml geneticin (Sigma-Aldrich, St. Louis, Mo.), 10 μg/ml puromycin (Thermo Fisher Scientific, Waltham, Mass.) and 100 μg/ml hygromycin B (Thermo Fisher Scientific, Waltham, Mass.). These cells constitutively expressed green Ca2+Nomad biosensor, red ArrestinNomad biosensor and the F2R like trypsin receptor 1, and this triple stable cell line was denominated the “PAR2 Arres-Ca2+Nomad cell line”.
Cells were seeded at 20,000 cells/plate in 96-well black, clear-bottom imaging plates (BD Biosciences) for Ca2+ and Arrestin assays. “PAR2 Arres-Ca2+Nomad” cell line was treated with 10 log dilution (n=6) of SLIGKV (Sigma-Aldrich) or SLIGRL-NH2 (Sigma-Aldrich) ranging from 0 to 300 μM in Optimem media (Gibco) for 24 hours at 37° C., 5% CO2, 95% relative humidity before data acquisition. Fluorescent images were acquired in the BD Pathway 855 High-Content automated image platform with a ×20 dry objective.
For fluorescence intensity determinations, changes in the distribution of Red ß-ARRNomad biosensor were measured using “Synergy 2” microplate reader (Biotek). Optimem media with the agonists was removed and replaced by PBS 1× without calcium and magnesium (Sigma-Aldrich) before the data acquisition. TurboFP650 data were acquired with ex/em 590/650 filters and turboGFP lectures were captured with ex/em 485/585 filters.
The signal to background (S/B) parameter was calculated as μc+−μc−, and Z′factor was calculated using the following formula: Z′=1−[(3σc++3σc−)/(μc+−μc−)]
A chemical library comprising 490 compounds was sourced from the Prestwick Chemical Library®, and based on the compounds' chemical and pharmacological diversity and their bioavailability and safety for humans, these compounds were used for the screening assay. The reference compound was Substance P. All compounds from the library were dissolved in dimethyl sulfoxide (DMSO) at 5 mM and the reference compound in water at 1 mM. The NK1R ArrestinNomad U2OS cells were treated with Substance P (positive control) at 10 μM, whereas the compounds of the library were tested at 10 μM for 24 hours in Optimem media before image acquisition.
Image Acquisition and Analysis:
After 24 h of treatment, the cells were fixed with phosphate-buffered saline supplemented with 3.7% formaldehyde for 10 min at room temperature (RT) and permeabilized with 0.3% Triton-X100 in PBS for 3 min at RT. The cell nuclei were then stained with DAPI for 5 min at RT. Fluorescent images were acquired in the BD Pathway 855 High-Content automated image platform with a ×20 dry objective. The changes in the fluorescence patterns of the ArrestinNomad biosensor were processed and quantified using image analysis algorithms with the Attovision bioimaging software in 3×3 subfields of each well (500-1,000 cells analyzed per well). The excitation and emission filters used were the following: for DAPI, 380/10 and 540/20 and for Rhodamine, 548/20 and 570LP. Arrestin activity was measured by counting the red fluorescent granules and normalizing the result to the cell number. The data were normalized as percentages of activity compared to the positive control (Substance P) after subtracting the value of the vehicle control. Cellular viability was determined by counting the nuclei (DAPI). For each condition, 5 replicates were used. The data were processed using Excel and plotted in SigmaPlot 11.0.
The plasmid encoding the green KinNomad biosensor was transfected into U2OS cells using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, Mass.). Resistant clones were obtained by limit dilution, and positively transfected cells were selected using 500 μg/ml geneticin (Sigma-Aldrich, St. Louis, Mo.). These cells constitutively expressed green KinNomad biosensor.
Fluorescent images were acquired in the BD Pathway 855 High-Content automated image platform with a ×20 dry objective.
An anti-TAC inmmunofluorescence assay was performed to trace the localization of ArrestinNomad biosensor. The membrane localization of the biosensor observed in the control (upper panels) is difuminated in the activated cells (lower panels) with the agonist. The biosensor is relocalized from the plasma membrane to cytosolic vesicles.
Cells were seeded at 12,000 cells/plate in 96-well black, clear-bottom imaging plates (BD Biosciences).” NTSR1 ArrestinNomad” cell line was treated with 1 μM of NTS (Sigma-Aldrich) in Optimem media (Gibco) for 24 hours at 37° C., 5% CO2, 95% relative humidity.
After overnight incubation, cell media was removed and it was performed a 15 minutes incubation with FBS (Sigma-Aldrich) at 4° C. Followed by a 90 minutes incubation at 4° C. with FITC anti-human CD25 antibody (Biolegend) at 1:20 concentration. Cells were then washed twice with PBS (Sigma-Aldrich). Fluorescent images were acquired in the BD Pathway 855 High-Content automated image platform (Franklin Lakes, N.J.) with a ×20 dry objective. The excitation and emission filters used were the following: for FITC, 488/10 and 540/20; and for FP650, 548/20 and 570LP.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17382049.9 | Feb 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/052944 | 2/6/2018 | WO | 00 |
