WO2000075160A1 - Assays for apoptosis modulators - Google Patents
Assays for apoptosis modulators Download PDFInfo
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- WO2000075160A1 WO2000075160A1 PCT/US2000/015142 US0015142W WO0075160A1 WO 2000075160 A1 WO2000075160 A1 WO 2000075160A1 US 0015142 W US0015142 W US 0015142W WO 0075160 A1 WO0075160 A1 WO 0075160A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
Definitions
- the invention relates generally to predictive assays for identifying and selecting compounds exerting effects on programmed cell death.
- Apoptosis involves mechanisms that are distinguishable from necrotic cell death (1-4).
- Apoptosis is inducible by interaction of ligands with cell surface proteins, e.g., Fas ligand/Fas, TNFR1/TNF, Death Receptor 3/Apo 3 ligand and glycocorticoids, but also by destruction of cellular DNA and structures by radiation; or by removal of life- sustaining agents such as serum, glucose and/or growth factors.
- Induction of apoptosis is accompanied by intracellular changes in a cascade-like fashion, including changes in heterodimeric cysteine proteases.
- Apoptosis proteases reportedly hydrolyze a variety of life-critical intracellular proteins (2).
- Compounds that selectively affect apoptotic processes constitute valuable molecular probes useful in unraveling the multiple interactions leading up to the commitment of a cell to die.
- Inhibitors of cell death also find potential pharmaceutical uses in treating traumatic disease, stroke and acute diseases associated with premature apoptotic death of cells in tissues. Promoters of cell death find potential use in treatments for cancer.
- screening assays capable of identifying compounds modulating apoptosis constitute valuable scientific and pharmaceutical tools.
- Fluorescence resonance energy transfer involves a distance-dependent interaction between the electronic excited states of two dye molecules in which the excited emission energy from a donor fluorophore is coupled to excitation of an acceptor fluorophore, without emission of a photon. Coupling of the two fluorophores results in both decreased emission from the donor and increased emission from the acceptor. The distance at which energy transfer occurs between the two fluorophores in 50% of excited molecules is defined by the F ⁇ rster radius, which is typically about 10-lO ⁇ A. FRET between linked green fluorescence protein (GFP) mutants as a reporter system was suggested by Cubitt et al. (6) and Tsien et al. U.S. Patent Serial No.5,439,797.
- Heim and Tsien (7) reportedly linked, two GFP mutants, Y66H/Y145F (BFP) and S65C, through a trypsin-cleava ' ble spacer, and reported disruption of FRET following trypsin treatment.
- Activation of members of the caspase family of cysteinyl proteases is a feature of apoptosis.
- the activities of these caspases commonly assayed in cell extracts, have been used to measure apoptotic processes.
- extracellular assays of protease activity do not appear -to be reflective of actual intracellular apoptotic events.
- Cell-based assays for apoptosis involving colorimetric or fluorescent substrates are also problematic since entry of substrate compounds into cells can be variable and/or toxic. Recently Xu et al.
- FRET-based screening assays useful for identifying compounds that modulate apoptosis. Summary of the Invention The invention provides recombinant apoptosis reporter cells expressing FRET reporter polypeptides useful in screening assays for identifying and selecting compounds that modulate apoptosis.
- the screening assays are capable of detecting intracellular apoptotic effects induced by compounds applied extracellularly in a microtiter assay format, with as few as 40-80,000 cells and within just 16-24 hrs after addition of an apoptosis inducer agent.
- the assays are specific, sensitive, stable and reproducible. Apoptotic changes in cells can be monitored without lysing or extracting cells, and in certain preferred embodiments more than 80% of the apoptotic cells remain adherent at the time of assay thereby reducing light scatter and background in the assay.
- Assay validation was achieved by screening more than 24,000 random compounds and identifying and selecting 19 acting to modulate apoptosis. Two of these compounds were subsequently shown to selectively inhibit apoptosis.
- FIGURE 1 depicts the 75 nucleotide, and corresponding 25 amino acid sequences, linking fluorophores in FRET-reporters.
- FIGURE 1A is depicted a GFP-BFP linker region disclosed in the prior art (PRIOR ART).
- FIGURE IB is depicted the sequences of a trypsin-cleavable linker region disclosed in the EXAMPLES section below and termed "SAT”.
- FIGURE 1C is depicted the sequences of a linker region in the FRET apoptosis reporter termed "SgAT”, as disclosed further in the EXAMPLES section below.
- FIGURE ID is depicted the sequences of a linker region in the control FRET reporter termed "SnAT”, as disclosed further in the EXAMPLES section below.
- FIGURE 2A depicts the change in fluorescence resonance energy transfer, as a function of time, from FRET reporter polypeptides expressed in an F9-1-13 FRET reporter cell undergoing apoptosis induced by anti-Fas antibody, as described further in the EXAMPLES section, below.
- FIGURE 2B depicts the change in caspase 3 enzyme activity measured in the FRET reporter cells of FIGURE 2A as a function of time following induction of apoptosis with anti-Fas antibody.
- Xu et al. (5) report transient transfection of cells with GFP-BFP plasmid DNA where the fluorophores were coupled through an 18 amino acid polyGly-Ser linker with an embedded DEVD sequence.
- DEVD is a potential substrate site cleavable by caspase-3.
- apoptosis was induced by transient transfection witb a Rip plasmid, i.e., again resulting in a non-homogeneous population of live and dying cells. Dying apoptotic cells eventually release their contents and these toxic byproducts can exert necrotic effects on adjacent non-transfected cells adding to assay variability unless this is controlled, e.g., as set forth below;
- assay sensitivity was found (herein) to be key to achieving success in a screening assay (i.e., see the EXAMPLES section, below).
- Xu et al. does not disclose assay sensitivity or detection of FRET changes induced by an extracellular apoptosis inducer, e.g., TNF. Instead, possibly to increase signal strength, Xu et al. harvested floating (non-adherent) cells and analyzed cleavage of GFP-BFP by SDS-PAGE and western blotting.
- Xu et al. fail to disclose the number of cells, or the assay volume, or the container size (e.g., wells, plates, flasks), or number of containers necessary to obtain a positive assay result. Since screening is preferably conducted in microplates using robotic pipettor and readers, relatively small numbers must produce a signal having a strength that is significantly greater than background, or the assay is not functional; and
- Screening assays necessarily must be able to reproducibly assay tens of thousands of compounds with specificity, sensitivity and precision if they are to be used successfully to identify those few positive "hits" that may result in identification of a lead compound. Uncontrolled assay variations resulting from uncontrolled changes in cellular physiology can lead to identification of thousands of false-hits.
- FRET fluorescence resonance energy transfer
- kDa kilodaltons
- ATP adenosine triphosphate
- LDH lactate dehydrogenase
- TNF tumor necrosis factor
- IL-1 interleukin 1
- EGF epidermal growth factor
- PDGF platelet derived growth factor
- TGF transforming growth factor
- HTS high throughput screening
- NGF nerve growth factor
- BDNF brain derived neurotrophic factor
- CNTF cilliary neurotrophic factor
- PC positive control
- NC negative control.
- Apoptosis is intended to mean the cascade of energy (ATP) dependent events triggered by an apoptosis inducer agent and leading to programmed cell death through mechanisms commonly involving intracellular caspase enzymes; commonly requiring about 12 to about 24 hrs.; and commonly involving cell death.
- the invention provides methods for assessing apoptosis prior to cell swelling, fragmentation and/or lysis. Mechanistically, during apoptosis dying cells fragment their DNA and become fragmented themselves into membrane- bounded apoptotic bodies. The released apoptotic bodies are ultimately subject to phagocytosis by immune cells.
- the instant methods provide assays for assessing apoptosis in a dying population of cells where fewer than about 30% have undergone lysis and DNA release, and most preferably, where fewer than about 10% of the cells have undergone lysis and DNA release.
- Apoptosis is most definitively proven to have taken place by rescuing dying cells and bringing them back to a condition of growth by addition of an apoptosis inhibiting agent.
- the disclosure provided herein indicates that caspase-3 activity in dying cells in a high throughput screening assay, i.e., employing the methods of the invention, can be decreased through addition of certain selective non-toxic compounds present in a large library of random small organic molecules.
- Apoptosis is recognized to play a fundamental role in cell development, tissue renewal; generating and regulating immune responses; and, preventing, malignant transformation.
- Apoptosis has been implicated in the-pathogenesis of an increasing number of diseases and may contribute to neuronal loss resulting from acute insults, such as ischemia, trauma or seizures, infarcts, and certain chronic neurodegenerative diseases including Alzheimer's disease. Aspects of neuronal apoptosis and necrosis are discussed by Choi, 1996 (1 1).
- Necrosis is intended to mean a process of cell death which is not energy dependent; not involving the caspase enzyme system; and, commonly involves cellular swelling and lysis with release of cellular constituents into the extracellular medium, e.g., LDH, in a relatively rapid manner, e.g., on the order of minutes or hours. Often, rapid release of necrotic materials from dead and dying cells results in death of adjacent cells. Necrosis is recognized to play a fundamental role in inflammation; acute tissue destruction during inflammation; and excitotoxicity, i.e., neuronal cell death resulting from over stimulation of cells through cell-surface receptors such as glutamate receptors.
- Apoptosis induction is intended to mean the process of triggering changes in a cell that, once initiated, will lead to programmed cell death.
- the subject process may be triggered for example either as result of the interaction of an "apoptosis inducer agent" with a cell surface protein molecule, or damage to cellular DNA or structures (e.g., mitochondria) e.g., by radiation, or by the removal of one or more critical growth factor from the cell environment.
- apoptosis inducer agents include: anti-Fas antibody or Fas ligand (binding to Fas); staurosporin; interleukins (e.g., DL-l binding to IL-IR ⁇ ; TNF ⁇ binding to TNFR1 and TNFR2); Apo 3 ligand (binding to Death Receptor 3); glycocorticoids (binding to cell surface glycocorticoid receptors); TRAIL, TRANCE, okadaic acid, NOC18 and the like.
- apoptosis inducers include physical inducer agents, e.g., doses of ultrasound or radiation (e.g., UV, gamma-ray, X-ray and the like) effective to damage nucleic acid and/or life-sustaining cellular DNA and intracellular structures, e.g., mitochondria.
- physical inducer agents e.g., doses of ultrasound or radiation (e.g., UV, gamma-ray, X-ray and the like) effective to damage nucleic acid and/or life-sustaining cellular DNA and intracellular structures, e.g., mitochondria.
- ways in which apoptosis may be induced include removal of life-sustaining agents such as serum, glucose and/or growth factors.
- Representative examples of critical growth factors include, but are not limited to, glucose, and in growth factor-dependent cell lines, EGF, PDGF, TGF ⁇ , BDNF, NGF and the like.
- Representative indicators for induction of apoptosis in a cell include internucleosom
- Proapoptotic agent is intended to mean an intracellular gene product effective to promote one or more apoptotic events in a cell undergoing apoptosis.
- Representative examples of genes whose products promote apoptosis include Bax, Bid, Cifa, Btf and the like.
- Modulating where used to characterize an effect of a compound on apoptosis induced in a population of cells, as measured in one or more assays, is intended to mean that the subject compound is effective to promote, or alternatively, to inhibit, one or more ongoing biochemical apoptotic events in a cell, wherein the subject apoptotic event is triggered by an apoptosis inducer agent.
- the resultant promoting, or inhibiting may be indicated by an increase or decrease, respectively, in one or more of the following: namely, the number or percentage of cells within the population that are committed to cell death; the rate at which cells in the population are committed to die; or, the measured activity of one or more indicators of apoptosis.
- Representative indicators of apoptosis include, but are not limited to, an increase in the measurable enzymatic activity of one or more of caspase enzymes; an increase in internucleosomal DNA fragmentation; a decrease in mitochondrial oxidative energy metabolism; and/or, a decrease in the activity of biosynthetic enzymes, and the like.
- Modulate apoptosis when used with respect to characterizing a measured activity of one or more of the instant test compounds in the instant assay, is intended to mean any of the following: namely, (i) that the subject test compound attenuates the ability of an apoptosis inducer agent to trigger apoptosis in a cell population, e.g., as measured pretreating the cells with the subject test compound, adding an apoptosis inducer agent and then by determining the percentage of cells in a cell culture which are apoptotic about 16-24 hrs.
- apoptosis inducer agent after addition of an apoptosis inducer agent; (ii) that the subject test compound alters the rate of progression of apoptosis in a population of cells, e.g., as determined by making measurements with time of the extent of apoptosis in a cell population; and/or (iii) that the subject test compound alters the extent of apoptosis in a population of cells," e.g., as determined by evaluating the number of cells surviving about 16-24 hrs. after adding an apoptosis inducer agent.
- Interleukin is intended to mean an agent released by a first cell, e.g., a cell of the immune system, that exerts an effect on a biological activity in a second cell, e.g., a neural cell.
- Representative interleukins include cytokines such as tumor necrosis factors (e.g., TNF ⁇ and TNF ⁇ ), IL-1 and the like.
- Growth factor is intended to mean an agent capable of stimulating progression of cells through the cell cycle and resulting in an increase in cell number in a population of cells.
- growth factor is intended to encompass polypeptide factors required to maintain non-dividing differentiated cells (e.g., neurons) in a viable state.
- Representative growth factors include nerve growth factor (NGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF) and the like.
- Linker polypeptide is used to refer to a polypeptide resident within a FRET reporter polypeptide that is bonded to both an exciter fluorophore polypeptide and a emitter fluorophore polypeptide.
- Preferred linker polypeptides are composed of a serial array of amino acid residues wherein each amino acid is peptide bonded to its neighboring amino acid, and wherein the total length of the subject polypeptide (a) falls within the Forster distance of an exciter and a emitter fluorophore; and, (b) maximizes the opportunity for binding and cleavage by one or more apoptosis proteases.
- linker polypeptides are composed of about 18 to about 40 amino acids, preferably about 18 to about 30, and most preferably, about 18 to about 25 amino acids.
- the subject linker polypeptide consists of the following amino acid domains: namely, (1) an end-cap domain containing end-cap amino acids; next, in serial array, (2) an acidic domain containing acidic amino acids; next, (3) a substrate domain containing a substrate for a protease; next, (4) a non-interfering domain, preferably containing acidic, neutral and/or non-polar amino acids but, overall, when assembled not comprising net hydrophilic or hydrophobic properties to the subject domain; and, last (5) a second end-cap domain containing end-cap amino acids.
- End-cap amino acids also referred to herein as “helix-breaking” and “helix end cap” amino acids, when used in regard to the instant "linker” polypeptide, is intended to mean those natural and unnatural amino acids that introduce bends into a polypeptide chain and, in nature, are found in regions of sequence adjacent to, but not in, alpha helices.
- Representative examples of end cap amino acids include Pro, Gly and Tyr.
- Helix-forming amino acids are disclosed by Chou and Fasman (Advances in Enzymology 47: 45-148, 1978; Table 1, page 51), and subsequently by O'Neil and DeGrado (Science 250: 646-651; Table 2, page 650), as amino acids commonly participating in the formation of polypeptide alpha helix.
- helix forming is intended to mean strong "H( ⁇ " helix formers as set forth in Chou and Fasman, supra, and amino acids having P values >1.4 at set forth in O'Neil and DeGrado (Table 2, page 650); Ala, Leu, Phe and Met are representative helix- forming amino acids.
- Non-interfering amino acids when used in regard to the instant "linker" polypeptide, is intended to mean that the subject amino acids do not interfere with cleavage of a substrate domain located within the subject linker polypeptide.
- the subject non-interfering amino acid is either a natural (i.e., product of nature) or an unnatural amino acid commonly having an alkyl side chain, i.e., a side chain composed of hydrogen and carbon, and preferably, about about 1 to about 5 carbons; representative examples include combinations of Ala, Val, De, Leu, Gly, Ser, Thr, Cys, Tyr, Asn and Gin that are effective (in toto) not to interfere with cleavage of a substrate domain, and preferably, in toto are not hydrophilic or hydrophobic.
- Acidic amino acids when used in regard to the instant "linker” polypeptide, is intended to mean a natural or an unnatural amino acid having a side chain containing a carboxylic acid residue; representative examples include Asp and Glu.
- “Hydrophilic” when used in regard to the "non-interfering" domain of the instant "linker” polypeptide is intended to mean that the subject domain consists of amino acid residues that, when assembled into a peptide having about 3 to about 7 amino acids, do not confer hydrophilic properties on the subject peptide, e.g., water solubility at high concentrations of the subject peptide, e.g., solubility at >100mg/ml in water.
- “Hydrophobic” when used in regard to the "non-interfering" domain of the instant "linker” polypeptide is intended to mean that the subject domain consists of amino acid residues that, when assembled into a peptide having about 3 to about 7 amino acids, do not do not confer hydrophobic properties on the subject peptide, e.g., water insolubility at low concentrations, e.g., insolubility at >lmg/ml in water.
- “Cassette” when used in reference to a FRET reporter plasmid capable of encoding a FRET reporter polypeptide, is intended to mean that region of the subject plasmid which is capable of encoding the instant linker polypeptide, and preferably, that region within the subject plasmid that is capable of encoding the instant "substrate domain" portion of the instant linker polypeptide.
- the cassette region of the plasmid is bounded on each side by restriction endonuclease sites, i.e., regions of nucleotide sequence cleavable by one or more restriction endonuclease, and preferably the subject restriction endonuclease sites are useful for orienting the direction (5' to 3') of an ohgonucleotide that is to be inserted at the cassette site.
- restriction endonuclease sites i.e., regions of nucleotide sequence cleavable by one or more restriction endonuclease, and preferably the subject restriction endonuclease sites are useful for orienting the direction (5' to 3') of an ohgonucleotide that is to be inserted at the cassette site.
- In vitro cell-based assay method is intended to mean an assay preferably conducted using a population of adherent cells to which an apoptosis inducer agent is added, or alternatively, conducted using a population of non-adherent cells to which an apoptosis inducer is added. Specifically excluded are assays in which non- adherent dead and dying cells are collected from a culture of initially adherent cells.
- “Expression” and “expressing” when used herein in regard to the instant stable FRET reporter cell lines, and the instant FRET reporter polypeptide synthesized therein, is intended to mean that the subject cells constituting the cell line are capable of transcribing, translating, processing and retaining in intracellular stores the instant FRET reporter polypeptide in a form that comprises a peptide bond in a substrate site that is hydrolyzable by an intracellular enzyme induced during apoptosis, e.g., a caspase. That a cell line is so capable of expression may be determined empirically e.g., according to illustrative methods set forth in the EXAMPLES section, below.
- Identifying when used in regard to one or more of the instant assay methods, is intended to refer to the process by which a random test compound, i.e., not known to modulate apoptosis, when subject to the conditions of evaluation imposed in the instant assay is observed to alter a FRET reporter activity, thereby determining that the compound is a "modulator of apoptosis", as defined supra.
- "Identifying" within the context of the invention is envisaged to include assays involving both analyses of single test compounds as well as analyses in which more than one compound are included in an assay well, e.g., combinatorial approaches, wherein comparison of results obtained with different combinations yields the identity of a test compound effective as a modulator of apoptosis.
- “Selecting” when used in regard to one or more of the instant assay methods is intended to refer to the process by which a test compound, having been subject to, and resulting from a process of "identifying", supra, is subsequently picked from within a group of test compounds for further study as a candidate compound.
- Candidate compound is intended to mean a compound “identified” and “selected”, supra, for its activity in the instant assay.
- Candidate compounds are commonly subject to additional analysis, e.g. in secondary assays formats, to confirm that that they are active in modulating apoptosis, as defined supra.
- Exciter fluorophore when used in regard to the instant FRET reporter, is intended to mean that the subject fluorophore is capable of both (i) being excited by a fluorescent light and (ii) generating a resonance energy capable of exciting an emitter fluorophore located in the same FRET reporter molecule;
- emitter fluorophore when used in regard to the instant FRET reporter, is intended to mean that the subject fluorophore is both (i) capable of receiving resonance energy generated by an exciter fluorophore and (ii) generating a fluorescent emission signal in response to the received resonance energy.
- FRET signal is intended to mean the ratio of fluorescence emission signals recorded, after subtracting background, e.g., at 530 nm and 500 nm (i.e., abbreviated 530:500 nm) or at 530 nm and 495 nm (i.e., abbreviated 530:495 nm).
- the subject recorded emission signals being produced by the instant FRET-reporter emitter and exciter fluorophores as expressed in vitro in a recombinant cell after that cell is exposed to a fluorescent light source capable of activating the subject exciter fluorophore.
- Embodiments of the invention solve the problems in the art by providing assays capable of detecting and quantifying intracellular apoptotic events that are induced following interaction " of an apoptosis inducer agent with a cell, and assays in which:
- the instant stable recombinant FRET reporter cell lines were selected expressing sufficiently high levels of FRET reporter polypeptide on a per cell basis, and on a per cell population basis, that a FRET signal is detected and changes in the FRET signal are reproducibly induced following interaction of apoptosis inducer agents with the cell surface;
- Assay devices for maintaining the instant FRET reporter cells as adherent cells during the course of the instant assay are preferably produced by treating the surface with cell adhesive ligands, e.g., collagen (i.e., gelatin), fibronectin and the like.
- cell adhesive ligands e.g., collagen (i.e., gelatin), fibronectin and the like.
- the instant FRET reporter cells remain adherent during the course of the assay and the instant assay duration is sufficiently short in time that many cells are still (marginally) alive and early enough in the apoptotic process that changes in cell microfilaments, microtubules, and adhesive molecules contributing to maintenance of cell shape are minimized, and rounding and release of cells from the substata is also minimized.
- the instant assay may be conducted with minimal intra-assay, inter-assay and day-to-day variation in signal using the instant stable FRET reporter cells;
- the observed decrease in FRET signal following induction of apoptosis is about 25% to about 50%, and the choice of apoptosis inducer agent and the time at which the FRET signal is measured are adjusted to achieve these final results in control experiments.
- a significant inhibition of apoptosis was evidenced by a difference of as little as 7% between the FRET signal of positive control (apoptosis-induced) and experimental (apoptosis-induced + test compound).
- apoptosis-induced control FRET signal is 50% then a decrease to a value of 43% (i.e., a 14% change) in experimental is commonly reproducible and sufficient to select a compound as a modulator of apoptosis; and, e.g., if the apoptosis-induced positive control FRET signal is 35% then a decrease to a value of 28% in experimental (i.e., a 20% change) is reproducible and sufficient to select a compound.
- greater than about a 10% change i.e., an increase or decrease relative to the positive control, preferably greater than about a 20% change, is sufficient to select compounds capable of modulating apoptosis.
- Embodiments of the invention provide an in vitro cell-based functional assay method for identifying and selecting candidate compounds capable of modulating apoptosis.
- an extrinsically applied apoptosis inducer agent in the instant assays more closely mimics, (i.e., than other available assays), the circumstances in which apoptosis is induced in ischemia, trauma, stroke and the like.
- the invention provides methods for preparation of a variety of different stable FRET reporter cell lines.
- the instant method involves the steps of: (i) inserting the instant FRET reporter plasmid containing a selectable marker into cells; (ii) selecting for the selectable marker, thereby selecting cells having incorporated the instant FRET reporter plasmid; (iii) testing the selected cells to insure that FRET reporter polypeptide is (a) synthesized by the cell, and (b) capable of producing a FRET signal that is at least about 3 to about 5-fold greater than the background signal produced in a control cell lacking the FRET reporter; (iv) further testing the selected cells to determine that (a) the FRET reporter polypeptide is cleaved following addition of an apoptosis inducer agent to a culture of the cells; and, (b) that FRET signal resident in the selected cells decreases by at least about 10% to about 30% within 24 hours after induction of apoptosis.
- the instant assay methods involve the use of the instant stable recombinant FRET reporter cell lines that are determined empirically to be capable of expressing the subject FRET reporter polypeptide in an intracellular form that constitutes an effective substrate for intracellular proteases induced during apoptosis, e.g., caspases. Selection of the instant stable recombinant FRET reporter cell lines is based on the determination that addition of an apoptosis inducer agent to a culture consisting of about 120,000 to about 250,000 test cells per square centimeter results in a measurable decrease of at least 10%, preferably about 15% to about 50% in a FRET fluorescent signal produced by the test culture.
- the instant selection method empirically discards cell lines that do not properly transcribe, translate, process and place FRET reporter polypeptide into the cytosol or widely dispersed intracellular storage compartments that are accessible to proteases induced during apoptosis. Microscopic examination preferably reveals that the instant FRET reporter is relatively evenly distributed throughout the cytoplasm of the instant FRET reporter cell, and the instant reporter is not discretely localized in intracellular vacuoles or vesicles.
- the invention provides expression plasmids capable of encoding intracellular FRET polypeptide substrates that consist of nucleic acid capable of encoding all of: (i) an exciter fluorophore, (ii) a linker polypeptide and (iii) an emitter fluorophore.
- the latter fluorescence exciter fluorophore consists of a fluorophore having an emission wavelength maximum that overlaps with the excitation wavelength profile of the emitter fluorophore.
- the exciter and the emitter fluorophores have wavelength maxima for emission and excitation, respectively, which are within about 0 nm to about 30 nm of one another.
- exciter and emitter fluorophores so active include the mutant fluorophores disclosed according to methods disclosed in Tsien et al. U.S. Patent Serial No. 5,625,048 ('048) and also in published PCT/US96/01457 (WO97/28261; the '457 application).
- exciter and emitter fluorophores include enhanced blue fluorescent protein (EBFP) and enhanced green fluorescent protein (EGFP), respectively, (Clontech, Palo Alto, CA); and "Emerald” and “Topaz” (Packard Instrument Co., Meriden, CT).
- EBFP enhanced blue fluorescent protein
- EGFP enhanced green fluorescent protein
- the subject exciter fluorophores are preferably selected from among fluorescent polypeptides having fluorescence absorbance maxima of about 370 nm to about 490 and emission maxima of about 440 nm to about 514 nm, while the subject emitter fluorophores are selcted from among fluorescent polypeptides having fluorescence absorption maxima of about 470 nm to about 515 nm and emission maxima of about 500 nm to about 530 nm.
- Coupling of FRET signal between the exciter and emitter is achieved by selecting fluorescent polypeptides having properties as follows: namely, the emission wavelength maximum of the instant exciter fluorophore is within about 0 nm to about 50 nm, preferably, about 0 nm to about 30 nm, and most preferably, within about 0 nm to about 10 nm of the excitation wavelength of the instant emitter fluorophore; and also, the emission wavelength maximum of the emitter fluorophore is at least 10 nm to 20 nm greater than the emission wavelength of the exciter fluorophore.
- Illustrative combinations of exciter and emitter fluorophore combinations are set forth in TABLE ⁇ , on the following page.
- the invention provides FRET reporter plasmids encoding, and stable FRET reporter cell lines expressing, FRET reporter polypeptides that have an exciter fluorophore joined to a emitter fluorophore through a linker polypeptide.
- the linker polypeptide consists of about 18 to about 40 amino acids, preferably about 18 to about 30, and most preferably, about 18 to about 25 amino acids.
- the length of the instant linker polypeptide and the choice of its constituent linker amino acids are determined according to two requirements: namely, (1) that the linker join the exciter and emitter fluorophores at a distance which allows fluorescence resonance energy transfer from the exciter to the emitter, i.e., the length of the linker falls within the F ⁇ rster radius of the exciter and emitter fluorophores; and, (2) that the linker contain a substrate domain amino acid sequence sufficiently long, and sufficiently optimized in amino acid sequence, for binding and cleavage by one or more apoptosis proteases.
- Apoptosis proteases exhibit a lesser apparent binding affinity (e.g., higher Km values) for small synthetic peptides than for larger, and a lesser binding affinity for amino acid sequences that are more divergent from native substrates, than those peptides which are more similar to native substrates.
- amino acid composition in and around the instant substrate domain is important for recognition by an apoptosis protease.
- the instant linker polypeptide comprises a serial array of domains selected to favor binding and cleavage by proteases induced during apoptosis.
- the linker polypeptide consists of a first end-cap amino acid domain, followed in order by, an acidic domain, a substrate domain, a non-interfering domain, and a second end-cap domain.
- the preferred length of the instant linker consists of about 18 to about 40 amino acids, preferably about 18 to about 30, and most preferably, about 18 to about 25 amino acids, as detailed further below.
- Embodiments of the invention provide in vitro cell-based assays useful for screening unknown compounds to identify those which modulate apoptosis induced by an extracellular apoptosis inducer agent, or apoptosis induced by removal of a critical factor required for cell survival.
- the instant methods provide cell-based assays for determining the structure resident within a test compound which is required for modulating apoptosis, i.e., structure activity response determinations of drug candidates useful for improving potency and advancing a drug candidate to the status of a clinical candiate.
- the instant methods are useful in screening for novel apoptosis inducer agents, e.g., compounds that may be useful anti-cancel **" agents. The instant assays are conducted by comparing the results obtained in three separate cultures, as described further below.
- PC positive control cell cultures
- each well of the subject 96-well microtiter culture preferably contains about 40,000 to about 100,000 cells, the cells are capable of expressing intracellular FRET reporter polypeptide, and at this point the PC cultures are ready for induction of apoptosis.
- Apoptosis is induced by adding an extrinsic apoptosis inducer agent such as staurosporin, or anti-Fas antibody, or by removing a critical component, e.g., glucose, from the cell's growth medium.
- NC normal negative control cell culture
- test compound a compound that is to be tested for its ability to modulate apoptosis.
- test compounds are added at a final concentration of about 1 nM to about 300 ⁇ M, and most preferably, at a final concentration of about 10 ⁇ M to about 50 ⁇ M.
- the PC, NC and Experimental cultures are cultured under conditions conducive to, and for a time sufficient to, induce apoptosis in the PC culture, e.g., as detected measuring DNA fragmentation or by measuring increased activity of one or more intracellular apoptotic proteases.
- this process involves incubating cells in the PC, NC and Experimental cultures at 37°C, in a humidified atmosphere of 5% CO 2 /95% air, in tissue culture medium containing growth factors and nutrients, such as, 5% fetal bovine serum, non-essential amino acids, glutamine and the like, for about 10 hours to about 24 hours, preferably about 16 to about 24 hrs. and most preferably about 16 to about 18 hrs.
- the selection of the subject assay duration is made after consideration of the rate at which apoptosis progresses after addition of a particular desired apoptosis inducer agent, i.e., as commonly determined by measuring a decrease in FRET signal according to the methods of the invention.
- microscopic observation will preferably show that greater than about 85% to about 95% of the cells in the subject cultures are adherent, and if a large number of non-adherent (floating) cells are observed the assay is discarded and conditions are altered, e.g., by setting up new assay cultures and using either a shorter incubation time or a lower concentration of the subject test compound.
- each culture is exposed to a fluorescent light source capable of exciting the endogenous exciter fluorophore and emission from both the exciter and the emitter fluorophore are measured, i.e., preferably sequentially within about 1 to about 3 minutes, and also preferably at two different selected wavelengths where emission from the exciter and emitter can be distinguished.
- FRET signal (defined supra) is calculated from the relative measured fluorescence units as the ratio of the emission of the emitter divided by the emission of the exciter.
- the instant linker polypeptide in the intracellular FRET reporter polypeptide has been cleaved by a protease whose expression was induced, or whose enzyme activity was activated, during apoptosis, then the reporter polypeptide does not properly transmit resonance energy from the exciter to the emitter and the signal is decreased.
- FRET reporter emission or FRET signal ratio
- the difference in the FRET signal (ratio) recorded from the PC culture is at least 20% to 50%, and preferably about 30% to about 50%, less than that in the NC culture, and preferably the NC culture measured fluorescence emission is at least about 3-fold to about 5-fold greater than the emission recorded in wells lacking cells, or wells containing cells that do not contain a FRET reporter (i.e., NC emission is at least 3-5 fold greater than background). If these conditions are met, then the result recorded in the experimental cultures is next examined.
- Apoptosis modulators may promote or inhibit events triggered following induction of apoptosis.
- a test compound which inhibits apoptosis may be identified by a FRET signal (ratio) in an Experimental culture which is greater than that recorded in a PC culture; and a test compound which promotes apoptosis may be identified by a FRET signal (ratio) in an Experimental culture which is less than that recorded in the NC culture, provided these cultures do not exhibit evidence of necrotic/toxic cell death or fluorescence emission from the test compound.
- test compound is a "candidate compound", i.e., one selected as being worthy of further study as a probable apoptosis modulator, is based on the foregoing comparisons of emission signals recorded in the PC, NC and- Experimental cultures, and the findings of an increase or decrease in fluorescent FRET emission signal recorded from the Experimental culture relative to the PC or NC culture, i.e., as described supra.
- the results obtained with replicate PC and NC cultures in an assay may differ by as little as about 20% to about 50%, but preferably by about 30% to about 50%.
- the instant FRET reporter plasmids and recombinant FRET reporter cell lines are preferably quite stable and uniform in their expression of FRET reporter polypeptide, and the FRET signal (ratio) produced by PC and NC in different assays on different days preferably varies by less than about 10% to about 20%, preferably less than about 15%, e.g., PC from about 0.95 to about 1.05 and NCfrom about 1.27 to about 1.45.
- Embodiments of the invention provide plasmid constructs encoding FRET reporter polypeptides having a variety of different linker polypeptides containing different substrates for different apoptosis proteases.
- the instant methods provide for the preparation of a variety of different stable FRET reporter cell lines.
- Representative examples of presently preferred substrate domains which may be incorporated within the instant linker polypeptide, i.e., at the substrate domain "cassette" site, supra, are set forth in TABLE Dl, below.
- Embodiments of the invention provide stable recombinant FRET reporter cell lines for identifying and selecting compounds capable of modulating apoptosis.
- Certain special requirements are placed on instant cells.
- the preferred instant cells are empirically determined to have all of the following properties: namely, (i) they are responsive to induction of apoptosis by extrinsic agents, preferably an apoptosis-inducing ligand, and most preferably anti-Fas antibody, which binds to a cell surface protein in the cells; (ii) they produce a measurable changes in FRET reporter signal within about 10 to about 24 hrs; (iii) the subject change in FRET reporter signal is accomplished with relatively low numbers of the cells in an assay, preferably about 40-100,000 cells in the assay, and most preferably, about 50-80,000 cells in the assay.
- the preferred instant FRET reporter cells express levels of FRET reporter polypeptide that is about 5 ⁇ g to about 50 ⁇ g of the FRET reporter polypeptide per mg total cell protein, preferably about 20 ⁇ g/mg to about 40 ⁇ g/mg and most preferably about 30 ⁇ g/mg to about 40 ⁇ g/mg.
- Embodiments of the invention provide plasmids encoding, and cells expressing, a FRET reporter polypeptide having a linker polypeptide optimized for binding apoptotic proteases.
- the instant linker polypeptide consists of a maximal length about 18 to about 40 amino acids, preferably about 18 to about 30, and most preferably, about 18 to about 25 amino acids.
- the presently preferred linker polypeptides consist of all of the following substituent domains linked in a serial array: namely, (i) an amino-terminal end-cap domain, located toward the amino terminus of the linker polypeptide and composed of about 3 to about 5 amino acids, wherein about 1 to about 3 of said amino acids are "end-cap" amino acids as defined supra; (ii) an acidic domain composed of about 3 to about 7 amino acids, wherein about 2 to about 5 of said amino acids are acidic amino acids as defined supra; (iii) a substrate domain composed of about 4 to about 7 amino acids selected for their ability to be cleaved by a protease induced during apoptosis, e.g., a caspase substrate site selected from TABLE m, above; (iv) a
- the invention provides nucleotide and amino acid sequences useful for constructing plasmids capable of expressing FRET reporters.
- the instant sequences are inserted, e.g., by ligation, site directed mutagenesis and the like, into a linker region consisting of about 75 nucleotides, located between and connected to nucleotide sequences encoding both an exciter and an emitter fluorophore.
- the instant exciter-linker-emitter nucleotide sequence is, in turn, introduced into any one of a variety of commonly available expression plasmids, as illustrated below.
- the instant exciter-linker-emitter plasmids may contain nucleotide sequences allowing their propagation in prokaryotic cells, but will always contain nucleotide sequences providing for expression of the instant FRET reporter polypeptide in a mammalian cell.
- DNA composing the instant plasmid consists of promoter and 5' regulatory region elements operably linked to a FRET reporter nucleic acid in such manner that expression of the plasmid in a mammalian cell results in the expression of a FRET reporter polypeptide.
- apoptosis inducer agents and cell lines have been described, however relatively little is still known about apoptotic responses triggered in a cell population, i.e., in microtiter culture, and in particular, in a high throughput screening type assay format.
- To achieve uniformity of signal in H-TS ⁇ ii was considered important that after addition of an apoptosis inducer agent a relatively large proportion of the cells within any given cell population should commit to undergo apoptosis, and at relatively the same rates.
- apoptosis inducer agents and conditions were tested in microtiter plate formats, i.e.
- a FRET reporter plasmid was constructed encoding a poly-His-tagged fusion protein containing the GFP mutants "Sapphire" and "Topaz" (Tsien et al. US Patent Serial No. 5,625,048) linked by a polypeptide containing an apopain (caspase-3) substrate domain (i.e., DEVD). Briefly, the constructions utilized green fluorescent proteins (GFP), enhanced by random mutagenesis and selection, as derived according to methods substantially the same as those disclosed by Tsien et al. U.S. Patent Serial No. 5,625,048 and Pollack et al. (9), both of which references are incorporated herein by reference.
- GFP green fluorescent proteins
- GFP-encoding plasmids are also commercially available from Clontech.
- GEM green enhanced mutant
- FIG. 1 A plasmid constructs were used to transform (i) E. coli DH5 ⁇ for preparation of bacterial and DNA stocks; and (ii) E. coli BL21(DE3) (Novagen) for production of FRET-reporter fusion protein.
- the GEM coding sequences were subcloned into pcDNA3 (Invitrogen) to produce mammalian expression plasmid vectors. Expression of the recombinant FRET-reporter fusion protein was evaluated by SDS-PAGE and fluorescence measurements.
- Fusion proteins containing the FRET reporter were isolated and purified with the pRSET XpressTM Kit (Invitrogen) according to the manufacturer's instructions. Briefly, a lysate was prepared from bacteria expressing the FRET reporter-fusion protein encoded by plasmid vector pRSET(A). The lysate was applied to a nickel resin column that binds to a poly-histidine tract in the fusion protein. The column was washed and the fusion protein was eluted with imidazole for fluorescent analysis and analysis by SDS-PAGE.
- FRET reporter proteins For FRET reporter proteins to detect apoptotic proteases they must necessarily not be subject to cleavage by other proteases expressed in normal or apoptotic cells e.g., serine proteases.
- FRET reporter proteins For the FRET reporter proteins to be useful as substrates for apoptotic proteases also requires that cleavage occur at a specific substrate site and not at some other non-specific site in a fluorophore. The possibility existed that reporters containing Emerald, Sapphire or Topaz GFPs might be susceptible to nonspecific cleavage by proteases (6).
- nucleotide sequences encoding the GEMs "Sapphire” and “Topaz” were used for construction of two FRET test reporters, i.e., Sapphire-Lmfcer-r-Topaz (STT; containing a trypsin substrate domain) and Sapphire-L ⁇ r- ⁇ -Topaz (SAT; containing an apopaincaspase-3 substrate domain; see the Materials and Methods section, below).
- STT Sapphire-Lmfcer-r-Topaz
- SAT Sapphire-L ⁇ r- ⁇ -Topaz
- BFP-GFP construct (7) as follows: namely, (i) to facilitate assembly of the construct, mutagenesis was used to introduce a restriction site into the 3' terminus of SAT- and
- STT-linker nucleotide sequences (ii) a substrate domain specific for apopain/caspase-3 was produced by mutation of two codons within the SAT linker; and, (iii) an enterokinase substrate site within the disclosed construct to produce the disclosed linker was eliminated by mutation in SAT.
- the resulting modifications produced a "cassette" linker region bounded by restriction sites.
- the trypsin substrate site was retained in the SAT linker so that trypsin could be used as a comparative control.
- a comparison of the sequence of the STT and SAT linkers with the sequence of the disclosed BFP-GFP linker (7) appears in TABLE IV.
- STT and SAT FRET reporter fusion proteins were expressed in cultures of E. coli BL21(DE3) transformed with pRSET(A)-SAT or pRSET(A)-STT and induced with 0.5 mM PTG for 4 hr.
- Approximately 1 mg of SAT and 0.4 mg of STT fusion proteins were partially purified using Ni-NTA Superflow nickel resin (Qiagen) and FPLC (see Methods). The STT protein was fluorescent but the SAT protein was not (excitation 395 nm, emission 530 nm).
- the fluorescent bacterial STT reporter protein, above, and a Sapphire fusion protein control were treated with trypsin and analyzed to detect cleavage by both western blot analysis, i.e., using a monoclonal GFP antibody (Clontech), and fluorescence measurements in the microplate assay described above. Aliquots of purified reporter protein were assayed for fluorescence in a 24 microtiter plate format using a Cytofluor ll plate reader (PerSeptive Biosystems) (6 reads/well, gain 70).
- the major species detected by western blot analysis was approximately 60 kDa, i.e., consistent with the predicted size of a full-length STT fusion protein, although smaller species, i.e., in the 30-40 kDa size range, were also detected suggesting either cleavage or incomplete synthesis of the STT reporter protein.
- the only detectable bands were in the 30-40 kDa range, i.e., the size predicted for cleavage products.
- Fluorescence studies further revealed about a 15% decrease in FRET signal, i.e., as expressed by the 530:495 ratio, within the first 15 min with no further decrease over the next 30 min, suggesting that, under these conditions, reporter cleavage was complete within 15 minutes.
- the 530:495 ratio of untreated STT controls remained relatively constant over the entire time course, indicating the intrinsic stability of the STT reporter protein.
- the control experiments with purified protein indicated that the maximal FRET signal change resulting from complete trypsin cleavage of linkers (similar to those disclosed in the prior art) elicited only about a 15% change in FRET signal.
- a test system was constructed to evaluate SAT reporter expression and feasibility of detecting FRET signal changes in mammalian cells in microtiter assays.
- the human embryonic kidney-derived HEK293 cell line was chosen as a host because of its relatively high transfection efficiency, rapid growth, easy maintenance and suitability for microplate assay formats. .
- HEK293 cells were transfected with pcDN A3 -Emerald, transformed cells were fluorescent and there was no loss of fluorescence, or obvious detrimental effects on cell mo ⁇ hology or growth, in a selected pool of cells (i.e., mixed populations of cells) over the course of one month. It was also possible to isolate individual stable colonies from within these selected pools of transfected HEK293 cells, in this case, clones of cells having FRET reporter DNA integrated into their genome, derived from cells being found to be present in mixed populations of selected cells after about
- SAT-reporter-transformed HEK293 cultures were treated with 100- 200 ⁇ M etoposide or 0.5-10 ⁇ M staurosporin for 4, 5, 8 or 24 hrs. and assayed for caspase-3 (Materials and Methods, below). After 8 hrs. treatment, caspase-3 activity was increased six- to 10-fold over background in 5 ⁇ M staurosporin-induced HEK293 cells, but no change was observed in etoposide-induced cells.
- cleavage of the FRET reporter by proteases requires correct folding and accessibility of the linker to the protease.
- the SAT reporter protein was sensitive to trypsin, i.e., FRET signal decreased and western blot analysis showed cleavage, and the Sapphire GFP expressed in control cells was not cleaved.
- HEK293 FRET Reporter Cell Lines In an attempt to overcome some of the difficulties encountered in use of pools of selected transfected cells (i.e., after 2-4 weeks selection, above), HEK293 cells were transfected (i.e., 6 ⁇ g pcDNA3-SAT plasmid DNA; LIPOFECTAMINETM) and selected in G418 (500 ⁇ g/ml) for 2 weeks, at which time 12 individual fluorescent colonies, i.e.,. Fl-1 to Fl-12, were isolated and expression of reporter fluorophore was confirmed by western blot analysis. (A pool of transfected cells was also maintained under selection.) Of the 12 parental cultures, Fl-1 and Fl-2 displayed the strongest fluorescent signals.
- SAT reporter fluorophore in 12 selected cell lines was confirmed by western blot analysis (i.e., using monoclonal GFP antibody) of cell lysates. Each of the cell lysates showed strong bands at -60 kDa and -30 kDa, i.e., the expected sizes for full- length SAT trypsin-cleavable reporter protein and for the single GFP fluorophore molecules, respectively.
- cells in the selected FI pool expressed an apparent greater percentage of full-length (60 kDa) SAT reporter than was expressed in a culture of transiently transfected HEK293 cells, i.e., -90% full length vs. -50%, respectively.
- Fl-1 and Fl-2 were subcloned by limiting dilution and reporter fluorescence assayed in four of the subclones was shown to be stable through at least 13 passages.
- Subclone Fl-1 -42 (ATCC No. PTA-83) was selected for initial testing of reporter cleavage and apoptosis-induced FRET changes.
- the fluorescence signal produced in Fl-1-42 cells was found to be stable through at least 21 passages, i.e., conducted over 10 ⁇ z weeks.
- apoptosis was induced by adding staurosporin to a final concentration of 5 ⁇ M, and using vehicle (0.25% DMSO) as a negative control.
- Assays for caspase-3 were used to confirm that apoptosis was induced.
- Final analysis of the data revealed caspase-3 ' activation, cleavage of the SAT reporter by western analysis and a concomitant decrease in FRET signal. Subsequent testing determined that significant changes in FRET signal were first detectable at about 8 hours after addition of staurosporin and were maximal by 24 hrs post-induction.
- HEK293 cells were helpful in establishing that a GFP-based FRET reporter could be expressed stably in a mammalian cell line and at levels sufficient to detect FRET signal changes in living cells after induction of apoptosis.
- human embryonic kidney cells were thought to be relatively useful in screening assays for identifying inhibitors of apoptosis in cells derived from embyonic mesenchymal tissues, they were believed not to be a good system for modeling apoptotic changes in differentiated cells of the central nervous system. Experiments were therefore initiated to evaluate expression of FRET reporters in CNS-derived cells in hopes that development of FRET reporter cell lines from those sources might be useful in screening assays designed to select and identify compounds useful in treating neurological diseases.
- CNS FRET-Reporter Host Cells Criteria considered requisite for selecting an acceptable CNS-like host cell line useful in high throughput screening included: (1) relative hardiness in the presence of potential toxic test agents; (2) rapid growth and undemanding culture requirements (i.e., media, growth factors and substrate); (3) an acceptable >5% transfection efficiency (i.e., >5% of cells compared with about >50%-80% needed for transiently transfected cells); (4) the ability, on the part of the cells, to synthesize, process and fold a functional FRET reporter protein; (5) the ability of the cells to express the FRET reporter protein at levels effective to produce FRET signal changes detectable in apoptotic cultures in a micotiter assay format; (6) induction of apoptosis in more than 50% of the cells in an assay well; (7) induction of apoptosis in the cells by at least one, and preferably two, apoptosis inducer agents; and (8) adherence, i.e., in apop
- IMR-32, NB41A3 and Neuro2A Three CNS derived cell lines, IMR-32, NB41A3 and Neuro2A, were initially evaluated for growth and transfection characteristics, inducibility of apoptosis in a large percentage of cells, and a inducible apoptosis protease activity. From among the three cell lines, only the IMR-32 cell line showed detectable levels of caspase-3 activity by western analysis and therefore it was the only one initially selected for further examination.
- the SYTO 13 cell survival assay and Cell Death Detection ELISA PLUS were used to optimize conditions for induction of apoptosis.
- IMR-32 cells were treated with Actinomycin D (ActD), cycloheximide (CHX), etoposide or staurosporin for 4-8 hrs.
- ActD Actinomycin D
- CHX cycloheximide
- etoposide etoposide or staurosporin
- programmed cell death induced by ActD, CHX and etoposide, i.e., as measured by Cell Death Detection at 4-8hrs. was only about 30% with ActD and about 50% with CHX and etoposide.
- DNA fragmentation as determined by ELISA showed dose- dependent changes only for etoposide, and not for CHX or ActD, i.e., suggesting nonspecific toxic effects of the latter two agents in this test cell system.
- IMR-32 cells were treated with 50 ⁇ M etoposide and monitored over 24 hours for cell survival, internucleosomal DNA fragmentation and mo ⁇ hological changes indicative of apoptosis.
- the SYTO 13 assay showed 55% of cells surviving at 8 hr and just 30% surviving at 24 hrs. In agreement, DNA fragmentation reached a maximal value at 8 hrs.
- the IMR-32 appeared to be a possible host cell for a cell-based FRET assay, but subsequently the following significant drawbacks were observed: namely, (1) an unacceptable level of assay variability, i.e., determined to be due to a constantly changing proportion of cells floating at different distances from the fluorescence detector; and (2) an unacceptable loss of cells and FRET signal, when cells were subject to washing, i.e. to remove media and test compounds which might contribute to background auto-fluorescence.
- EMR-32 apparently exhibited decreased cellular adherence to substrates when apoptosis was induced.
- IMR-32 Host Cells IMR-32 cells were transfected with 6 ⁇ g pcDNA3-SAT using LIPOFECTAMINETM (Life Technologies) according to standard procedures, but colonies lifted off the plate during selection (G418, 500 ⁇ g/ml). Transfection conditions were modified, i.e., using plates coated with Matrigel (Collaborative Biomedical Products) to improve cell adherence and reducing the selection pressure to 100 ⁇ g/ml G418, but these changes still resulted in no improvement: i.e., no colonies survived.
- U-138 Glioblastoma Host Cells The U-138 MG human glioblastoma cell line (ATCC #HTB 16) was evaluated a potential host. This cell line was found to be hardy, strongly adherent and relatively easily cultured and transfected. The cells were maintained in 2MM media (DMEM with L-glutamine, 6% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin) and passaged 1:5 or 1:10 every 3-4 days.
- 2MM media DMEM with L-glutamine, 6% fetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin
- the SAT reporter protein as expressed from pcDNA3-SAT, should theoretically comprise a -60 kDa protein made up of GFP mutants Sapphire and Topaz (Aurora Biosciences) linked together through a linker polypeptide containing both a potential apopain/caspase-3 site (i.e., DEVD) and trypsin cleavage sites (i.e., K and R).
- linker polypeptide containing both a potential apopain/caspase-3 site (i.e., DEVD) and trypsin cleavage sites (i.e., K and R).
- DEVD potential apopain/caspase-3 site
- K and R trypsin cleavage sites
- At least three different alternatives were considered: namely, (1) problems relating to expression of full-length reporter; (2) problems intrinsic within the mutant GFP polypeptides (Sapphire and/or Topaz); or, (3) problems resulting from intracellular cleavage.
- experiments were conducted to evaluate expression of SAT reporter in the HEK293 cells.
- the SAT coding sequence was inserted into mammalian expression vector pcDNA3J/V5-His (Invitrogen) resulting in pcDNA3VH-SAT.
- the signal produced by either the new pcDNA3VH- SAT, or the old pcDNA3-SAT were similar.
- SnAT znsi/oos ⁇ /iDd 09ISZ./00 O ⁇ V
- Both of the linkers also lacked potential cleavage sites for trypsin or other serine proteases (i.e., K and R).
- the SgAT and SnAT nucleotide coding sequences were assembled 5' to a C-terminal His 6 tag in vector pcDNA3J/V5-His (Invitrogen).
- the FRET signal expressed in F9- 1-13 decreased in response to both staurosporin and anti-Fas antibody treatment of cells, while the FRET signal expressed by the F10-24-2 control cells, (i.e., lacking a caspase substrate domain), was unaffected following staurosporin or anti-Fas antibody treatment.
- Kinetics of changes in FRET signal and caspase-3 activity were appropriate for induction of apoptosis, i.e., changes in FRET signal were significantly different from vehicle-treated induced cultures at 8 hrs. and the differences were maximally different at about 16-24 hrs.
- Parameters evaluated and optimized included the following: namely, number of cells per assay, number of wells per assay, time, type and amount of apoptosis inducer agent, duration of fluorescence measurements, type of fluorescence measuring device, inter-well/intra-plate variability, inter-plate variability, culture conditions and the like.
- a 96-well FRET assay was evaluated for inter-assay well variability and inter- plate variability. Possible signal drift during measurement of the FRET fluorescence signal was evaluated using one of two different robotics-compatible rapid fluorescence plate readers (SpeedReader or Cytofluor II). After plating at an initial density of about 60,000 cells/well, and overnight culture F9-1-13 cells were treated with 5 ⁇ M staurosporin, or vehicle. After an overnight incubation in staurosporin (i.e., 16-24 hrs.), the plates were either washed, or not washed, and then assayed repeatedly over the next 1.5 hours using either of the plate readers.
- SpeedReader or Cytofluor II robotics-compatible rapid fluorescence plate readers
- F9-1-13 cells were found to produce measurable changes in FRET signal (using either fluorescence reader) within about 4 hours when treated with 5 ⁇ M staurosporine, i.e. as compared with vehicle- treated wells.
- FRET signal using either fluorescence reader
- the SpeedReader showed no significant differences between experimental and vehicle control cultures; and, large inter-well variability was observed within plates that tended to obscure apoptosis- induced changes in the FRET signal.
- cells of the F9-1-13 stable FRET reporter cell line have been found to produce reliable and consistent results over a relatively broad window of time when a plate reader, i.e., when an appropriate optical fluorescence reader is used.
- the Cytofluor II was selected as the preferred plate reader for a high-throughput FRET assay.
- the FRET signals within the different respective groups were tightly clustered, and the Z-VAD-fmk group mean was significantly (P ⁇ 0.001) higher than the mean FRET signal recorded in the staurosporine treatment group, i.e., -65% greater indicative of Z-VAD-fmk inhibition of the apoptosis-induced decrease in FRET signal.
- Proteases induced during apoptosis may be distantly related to earlier cellular events triggered by apoptosis inducers. Theoretically, interfering with any cellular component of an induced apoptosis pathway, at or following apoptosis induction and prior to caspase-3 activation, will inhibit the apoptosis-induced change in FRET signal in F9-1-13 cells.
- apoptosis modulators identified using F9-1-13 cells may include inhibitors of signal transduction events, inhibitors of co- factor-procaspase complexes (i.e., required for activation of caspase-8 or -9), and modulators of pro- or anti-apoptotic regulatory factors (e.g., Bax or Bcl-2).
- the cell-based FRET reporter assays developed above are capable of detecting signal transduction inhibitors, e.g., MAP kinase inhibitors, inhibitors of post-translational protein processing, and the like. Because the assay is cell-based any inhibitor so detected is not a priori a protease inhibitor.
- 6-fold concentrated (6X) solutions of test compounds i.e., about 300 ⁇ M
- 2SM 1 media containing, (in the 6X solution), 1.5% DMSO then diluted step-wise into the assay to achieve a final concentration of about 50 ⁇ M and 0.25% DMSO in the assay.
- microtiter plates were commonly incubated at 37°C in 5%CO 2 /95% air atmosphere overnight, i.e., about 16-20 hrs., although time-course studies indicated that statistically significant inhibitory effects of certain test compounds could be seen within as little as about 8 to 10 hrs.
- fluorescence of the FRET reporter polypeptide in these cells was determined, as described above.
- wells were gently washed about 2 to about 4 times, about 200 ⁇ l per well, with PBS (0JM phosphate-buffered 0J4M saline, pH 7.4).
- PBS 0.JM phosphate-buffered 0J4M saline, pH 7.4
- Cytofluor II plate reader (Perseptive Biosystems), using a fluorescence source filtered for excitation at 395/40 nm, and detectors set for detecting emission sequentially at
- test wells which yielded RFU that were ⁇ 80% of the mean values recorded in the apoptosis control wells were flagged as being potentially due to toxic effects on the test cells, and test wells that yielded RFU values >200% of mean values recorded in the vehicle control wells were flagged as being potentially due to auto-fluorescence or uncontrolled interference. After certain initial selected flagged compounds were confirmed to be false positive results, all subsequent flagged compounds were just omitted from consideration.
- the results presented in TABLE VI show that the FRET reporter assay identified 40 small chemical entities in a library of 24,276 compounds, i.e., 0.16%, that were capable of inhibiting apoptosis. Subsequent studies suggest that 48% of the compounds identified exert some effect on anti-FRET antibody-induced loss of FRET signal.
- 2SM1 Selection Media 2MM media additionally containing 400 ⁇ g/ml G418 (Gibco #1 181 1-031).
- Gelatin-coated plates 0.1 % gelatin in sterile H 2 O was added to each well for 0.5-4 hr., aspirated and then air-dried.
- Z-VAD-fmk (Enzyme Systems Products #FK-009) was prepared as a 40 mM stock in DMSO.
- Anti-Fas Antibody Anti-human Fas monoclonal antibody was purchased having a concentration of 0.5 mg/ml (Upstate Biotechnology #05-201).
- Apoptosis was induced by irradiation with UV light, 254nM, delivered from a Strategene 1800 source.
- SYTO 13 Cell Viability Assays Cells were seeded into 96-well plates, incubated overnight to adhere cells to the plastic, and then treated in quadruplicate for various times with extrinsically applied apoptosis-inducing agents, i.e., staurosporin or etoposide. Cell viability was determined using 5 ⁇ M SYTO 13 (Molecular Probes) and or 5 ⁇ g/ml propidium iodide (PI, Molecular Probes). As controls, four wells of untreated cells were stained with SYTO 13 and PI, i.e., to measure the maximum signal (max), and four wells of untreated cells were stained with only PI, i.e., to measure the minimum signal (min).
- apoptosis-inducing agents i.e., staurosporin or etoposide.
- PI propidium iodide
- Morphologic Markers of Apoptosis Microscopic markers of apoptosis include membrane blebbing, cell shrinkage (i.e., cytoplasmic condensation) and condensed (dense) chromosomal nuclear materials (i.e., pyknosis).
- DNA Fragmentation ELISA Apoptosis-induced inter-nucleosomal fragmentation of chromosomal DNA was quantified using the "Cell Death Detection ELISA P US " (Boehringer Mannheim) according to the manufacturer's instructions. Briefly, -10 4 cells/well were plated on a poly-D-lysine-coated 96-well plate and treated for various periods of times with different apoptosis-inducing agents.
- the cells were lysed and the nuclei and membranes were pelleted. An aliquot of the cytoplasm-containing supernatant was then transferred to a streptavidin-coated microtiter plate and incubated with anti-histone-biotin and anti-DNA-peroxidase, resulting in the formation of "sandwich" antibody complexes, i.e., (anti-DNA- peroxidase)-(nucleosomaI-DNA-containing-histone)-(anti-histone-biotin)- (streptavidin-microtiter plate). Unbound antibody was removed by washing. Peroxidase substrate (i.e., ABTS) was added and nucleosomal DNA complexes quantified by spectrophotometric analysis of the colored peroxidase reaction product.
- Peroxidase substrate i.e., ABTS
- Apoptotic Membrane Assay Apoptotic membrane changes (i.e., translocation of phosphatidyl serine from the inner to the outer face of the plasma membrane) were assayed with the ApoAlert AnnexinV kit (Clontech) according to the manufacturer's instructions. Briefly, cells plated and treated as above were washed in IX PBS, incubated with annexinV-FITC (1 ⁇ g/ml) and propidium iodide (50 ⁇ g/ml) for 5-15 min, and evaluated by fluorescence microscopy.
- Apoptotic membrane changes i.e., translocation of phosphatidyl serine from the inner to the outer face of the plasma membrane
- Caspase-3 Assay Caspase-3 activity was assayed according to the following protocol. Cells plated and treated as above were washed in IX PBS and lysed in IX assay buffer (1 mM DTT, 1 % Triton X-100, 0.5% Tween-20 in IX PBS, pH 1.2). Lysates were combined with fluorogenic substrate, Ac-DEVD-AMC (200 mM), ⁇ inhibitor, Ac-DEVD-CHO (10 ⁇ M) (Bachem) in IX assay buffer and incubated in the dark for 1 hr. at room temperature. Fluorescence readings were taken on the Cytofluor II plate reader (excitation 360 nm, emission 460 nm, 6 reads/well, gain 50).
- Plasmids encoding the Trypsin (T) linker polypeptide (SAT) were constructed using PCR to amplify portions of pRSET(A), i.e., from the T7 promoter to the polylinker, and to introduce a Ncol site at the 3' end of the product, i.e., using ohgonucleotide primers T7 and KE76.
- Plasmids encoding the Apopain (A) linker polypeptide were constructed using synthetic PCR with overlapping oligonucleotides KE76-KE79 to generate the desired linker sequence with an Nhe ⁇ site at the 5' end and an Ncol site at the 3' end.
- the Sapphire coding sequence with an Xbal site replacing the stop codon was amplified by PCR from pRSET(A)-Sapphire using oligonucleotide primers T7 and KE81.
- An NheVNcol restriction fragment of each spacer PCR product was ligated to a 3.5-kb NheVNcol fragment of pRSET(A)-Topaz.
- the resulting intermediate constructs, i.e., pRSET(A)-TT and pRSET(A)-AT contained, respectively, the T or A spacer 5' to the Topaz coding sequence.
- a 0.8-kb NheVXbal restriction fragment of the Sapphire PCR product was ligated into the ⁇ hel site of pRSET(A)-AT to create pRSET(A)- SAT.
- the SAT coding sequence was subcloned into vectors pcD ⁇ A3 and pcDNA3J/V5-His (Invitrogen) for expression in mammalian cells.
- the STT reporter links Sapphire and Topaz with a 25-residue trypsin-cleavable spacer similar to the BFP-GFP reporter of Heim and Tsien, 1996.
- the pRSET(A)-SAT reporter plasmid constructed above contained a trypsin-cleavable spacer linked to Topaz to form an STT precursor, pRSET(A)-TT, described above.
- the STT coding sequence was subcloned into vector pcDNA3 (Invitrogen) for mammalian expression.
- a calcium phosphate DNA precipitate was prepared for transfection by rapidly adding about l-20 ⁇ g of a plasmid DNA solution in 0.27M CaCl 2 /distilled water to an air bubbling solution of 1.4 mM Na 2 HPO 4 /NaH 2 PO 4 buffer containing 0.27 M NaCl and 42 mM HEPES (free acid; Calbiochem), resulting in a final pH of about 6.9.
- the precipitate formed at room temperature over 20-40 minutes was collected and added dropwise to the plate of cells. After 5-7 hrs. treatment, cell were washed twice with PBS (5 ml each) and fresh media was then added.
- FRET-Reporter Polypeptides in Mammalian Cells: Cell lysates were prepared for western blot analysis by homogenizing cells in 50 mM Tris-HCl, pH 7.4. SDS-PAGE was performed using a 4-20% TRIS-glycine gel (Novex) and Laemmli SDS conditions, according to the manufacturer's instructions. Western blots consisted of protein transfer to Hybond ECL membranes (Amersham) according to the manufacturer's instructions and GFP polypeptide fluorophores were detected using GFP-specific polyclonal or monoclonal antibodies (Clontech).
- Trypsin Treatment of STT That cleavage of the STT reporter produced proteolytic cleavage products of the expected size was determined using fractions obtained from FPLC purification of STT fusion protein. The fractions were treated with trypsin (0.2 mg/ml, 20 min, room temperature) and analyzed by western blot using a monoclonal GFP antibody (Clontech). Partially-purified Sapphire fusion protein (Xpress System, Invitrogen) was included for comparison.
- Etoposide-Induced Cell Death and Apoptotic DNA Fragmentation in IMR-32 Cells Confluent IMR-32 cells were treated with 50 ⁇ M etoposide phosphate for 2, 8, 17 or 24 hrs. To evaluate apoptosis cells were either: i) stained with SYTO 13 and assayed for fluorescence using a Cytofluor II plate reader (excitation 485/20 nm, emission 530/30 nm), or ii) nucleosome-associated DNA fragments were detected using the Cell Death Detection ELISA PLUS (Boehringer Mannheim) according to the manufacturer's instructions. Etoposide caused a) a decrease in cell survival over 24 hrs., as determined by staining, and in comparison with untreated controls; and, b) an increase in measured apoptotic DNA fragmentation.
- Staurosporin-Induced Caspase-3 Activation in HEK293 and IMR-32 Cells Confluent HEK293 cells were treated with 100-200 ⁇ M etoposide tor 5, 8 or 24 hrs. or with 1-5 ⁇ M staurosporin Jp ⁇ 8 hrs. or 24 hrs. Confluent IMR-32 cells were treated with 100 ⁇ M etoposide for 8 hrs. Cell lysates were collected and assayed for caspase-3 activity. HEK293 cells treated with 1-5 ⁇ M staurosporin for 8 hrs. showed a 6- to 10-fold increase in caspase-3 activity over background but etoposide showed no effect. An 8 hr. etoposide treatment increased caspase-3 activity -5-fold in IMR- 32 cells.
Abstract
Description
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CA002375708A CA2375708A1 (en) | 1999-06-04 | 2000-06-01 | Assays for apoptosis modulators |
EP00938044A EP1189919A4 (en) | 1999-06-04 | 2000-06-01 | Assays for apoptosis modulators |
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US09/326,472 US20020177120A1 (en) | 1999-06-04 | 1999-06-04 | Assays for apotosis modulators |
US09/326,472 | 1999-06-04 |
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WO2000075160A1 true WO2000075160A1 (en) | 2000-12-14 |
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PCT/US2000/015142 WO2000075160A1 (en) | 1999-06-04 | 2000-06-01 | Assays for apoptosis modulators |
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US (1) | US20020177120A1 (en) |
EP (1) | EP1189919A4 (en) |
CA (1) | CA2375708A1 (en) |
WO (1) | WO2000075160A1 (en) |
Cited By (2)
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EP1507456A1 (en) * | 2002-01-29 | 2005-02-23 | Oncoimmunin, Inc. | Visualization and quantitiation of cellular cytotoxicity using cell-permeable fluorogenic protease substrates and caspase activity indicator markers |
US6979530B2 (en) | 2001-05-21 | 2005-12-27 | Applera Corporation | Peptide conjugates and fluorescence detection methods for intracellular caspase assay |
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EP1271133B1 (en) * | 2000-02-28 | 2008-05-28 | Daiichi Pure Chemicals Co., Ltd. | Method of measurement based on fluorescence energy transfer using a long-lived fluorescence donor |
WO2004005917A1 (en) * | 2002-07-08 | 2004-01-15 | Daiichi Pure Chemicals Co., Ltd. | Fluorescent probe |
JP4206381B2 (en) * | 2002-10-16 | 2009-01-07 | 哲雄 長野 | Reagent for peroxynitrite measurement |
US7696245B2 (en) * | 2003-03-28 | 2010-04-13 | Sekisui Medical Co., Ltd. | Fluorescent probe for zinc |
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US20100331833A1 (en) * | 2009-06-26 | 2010-12-30 | Michael Maschke | In-vitro device monitoring during minimally invasive ablation therapy |
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US11214610B2 (en) | 2010-12-01 | 2022-01-04 | H. Lundbeck A/S | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris |
KR20190112175A (en) | 2010-12-01 | 2019-10-02 | 앨더바이오 홀딩스 엘엘씨 | Anti-ngf compositions and use thereof |
US9078878B2 (en) | 2010-12-01 | 2015-07-14 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
US9884909B2 (en) | 2010-12-01 | 2018-02-06 | Alderbio Holdings Llc | Anti-NGF compositions and use thereof |
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US6803188B1 (en) * | 1996-01-31 | 2004-10-12 | The Regents Of The University Of California | Tandem fluorescent protein constructs |
-
1999
- 1999-06-04 US US09/326,472 patent/US20020177120A1/en not_active Abandoned
-
2000
- 2000-06-01 WO PCT/US2000/015142 patent/WO2000075160A1/en not_active Application Discontinuation
- 2000-06-01 CA CA002375708A patent/CA2375708A1/en not_active Abandoned
- 2000-06-01 EP EP00938044A patent/EP1189919A4/en not_active Withdrawn
Non-Patent Citations (3)
Title |
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POLLOK ET AL.: "Using GFP in FRET-based applications", CELL BIOLOGY,, vol. 9, February 1999 (1999-02-01), pages 57 - 60, XP002930810 * |
See also references of EP1189919A4 * |
XU ET AL.: "Detection of programmed cell death using fluorescence energy transfer", NUCLEIC ACIDS RESEARCH,, vol. 26, no. 8, 1998, pages 2034 - 2035, XP002930809 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6979530B2 (en) | 2001-05-21 | 2005-12-27 | Applera Corporation | Peptide conjugates and fluorescence detection methods for intracellular caspase assay |
US8623995B2 (en) | 2001-05-21 | 2014-01-07 | Applied Biosystems, Llc | Peptide conjugates and fluorescence detection methods for intracellular caspase assay |
EP1507456A1 (en) * | 2002-01-29 | 2005-02-23 | Oncoimmunin, Inc. | Visualization and quantitiation of cellular cytotoxicity using cell-permeable fluorogenic protease substrates and caspase activity indicator markers |
JP2006503550A (en) * | 2002-01-29 | 2006-02-02 | オンコイミューニン,インコーポレイティド | Visualization and quantification of cellular cytotoxicity using proteolytic enzyme substrate, cell-permeable fluorogenic substrate and caspase activity indicator marker |
EP1507456A4 (en) * | 2002-01-29 | 2007-06-13 | Oncoimmunin Inc | Visualization and quantitiation of cellular cytotoxicity using cell-permeable fluorogenic protease substrates and caspase activity indicator markers |
Also Published As
Publication number | Publication date |
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EP1189919A4 (en) | 2005-01-05 |
EP1189919A1 (en) | 2002-03-27 |
CA2375708A1 (en) | 2000-12-14 |
US20020177120A1 (en) | 2002-11-28 |
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