Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
• • 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
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the invention is a method for the direct detection of the change in a molecule carrying a fluorescent dye by measuring the fluorescence lifetime.
• luminescence processes that are accompanied by an emission of radiation when an excited molecule is in its energetic ground state are referred to as luminescence and are generally divided into fluorescence and phosphorescence.
• excitation energy can be released through various non-radiative processes.
• Fluorescence occurs during the transition from the lowest vibration level of the excited singlet state Si to a vibration level of the singlet ground state S 0 .
• the transition rate k f is in the range from 10 7 to 10 12 s "1.
• the fluorescence excitation takes place at a shorter wavelength than the fluorescence emission, since energy is lost through radiationless processes between absorption and emission of the radiation energy.
• the fluorescence lifetime (FLT) is a measure of the time that a molecule remains on average in the excited state before the fluorescence emission takes place.
• the radiation lifetime ⁇ f corresponds to the inverse fluorescence transition rate k f .
• acceptor dyes have a similar effect, absorbing the excitation energy of the donor dye in a resonance phenomenon without radiation and releasing the absorbed energy either without radiation or as fluorescence. This also lowers the FLT of the donor dye.
• TCSPC time correlated single photon counting
• the alternative to FLT measurements in the time domain are measurements in the frequency domain, which are also called phase-modulated.
• the sample is excited with a continuous laser, the light intensity of which is modulated with a sine curve. Frequencies in the order of magnitude of the fluorescence transition rates are usually used. If a fluorescent dye is excited in this way, the emission is forced to follow this modulation.
• the emission is delayed relatively for excitation. This delay is measured as a phase shift from which the FLT can be calculated.
• the maximum difference between the maximum and minimum of the modulated emission signal decreases with increasing FLT, so that the FLT can also be calculated therefrom.
• the measurement of the fluorescence intensity can, for example, be used to measure the increase in fluorescence of a protease reaction with a fluorogenic peptide substrate from which fluorescent aminocoumarin (AMC) is split off. Large signals are usually measured, but the auto-fluorescence of screening substances could interfere.
• the fluorescence intensity signal is susceptible to the so-called “inner filter effect” if there is an absorbing substance in the solution. Dynamic fluorescence quenching by molecular collision as well as light scattering in cloudy solutions can be as disruptive as the fading of the fluorescent dye or volume / meniscus
• the fluorescence signal is dependent on the concentration of the fluorescent dye and on the temperature.
• FRET fluorescence resonance energy transfer
• the fluorescence lifetime (FLT) is • considerably more robust. It is only disturbed in some cases by strongly autofluorescent substances with comparable FLT. But neither "inner filter effect” nor KoUision quencher, photobleaching, volume effects or concentration influence the FLT. These properties predestine this robust method for use in screening.
• no screening assays have been established for FLT, as has been the case up to now Mainly due to the low throughput and high costs for the instrumentation. Modern developments of powerful and stable lasers as well as detection systems have recently enabled FLT measurements on microtiter plates and thus substance screening. Fi ⁇ na Tecan has ended with the Ultra Evolution In 2002, a commercial device for reading microtiter plates was launched for the first time.
• FLT applications The measurement of FLT was applied to a wide variety of biological issues. Either fluorescent probe molecules were used, which bind cations such as Ca + (Schoutteten L., Denjean P., Joliff-Botrel G., Bernard C, Pansu D., Pansu RB, Photochem. Photobiol. 70, 701-709 (1999)), Mg 2+ (Szmacinski K, Lakowicz JR, J. Fluoresc. 6, 83-95 (1996)), H + (Zz / z HJ., Szamacinski, Anal. Biochem.
• the change in fluorescence lifetime is achieved by a binding reaction with a molecule, which either leads to a smaller FLT for the donor dye through resonance energy transfer (quench or FRET), or in rare cases results in a larger FLT.
• FRET resonance energy transfer
• Binding of a Cy3-labeled anti-phosphotyrosine antibody for example, the activity of a receptor tyrosine kinase was measured (FS Wouters, PIH Bastiaens, Current Biology 9, 1127-1130, 1999).
• Protein (de) phosphorylation is a general regulatory mechanism by which cells selectively modify proteins that transmit regulatory signals from outside to the cell nucleus.
• the proteins that carry out these biochemical modifications belong to the group of kinases or phosphatases.
• Phosphodiesterases hydrolyze the secondary messenger cAMP or cGMP and in this way also influence cellular signal transduction pathways. Therefore, these enzymes serve as highly interesting target molecules in pharmaceutical and crop protection research.
• Newer methods replace radioactive immunoassays with ELISAs (enzyme-linked immunosorbent assay). These methods use purified substrate proteins or synthetic peptide substrates that are immobilized on a substrate surface. After exposure to a kinase, the extent of phosphorylation is quantified by using anti-phosphotyrosine antibodies, which are linked to an enhancing enzyme such as e.g. Peroxidases are coupled, to which phosphorylated immobilized substrates bind.
• an enhancing enzyme such as e.g. Peroxidases are coupled, to which phosphorylated immobilized substrates bind.
• Epps. et al. (US 6203994) describe a fluorescence-based HTS assay for protein kinases and phosphatases which uses fluorescence-labeled phosphorylated reporter molecules and antibodies which specifically bind the phosphorylated reporter molecules. The binding is measured by means of fluorescence polarization, fluorescence quench or fluorescence correlation spectroscopy (FCS).
• FCS fluorescence correlation spectroscopy
• the company Molecular Devices recently offers nanoparticles with charged metal cations on the surface as a generic binding reagent that is suitable for phosphorylation reactions on tyrosine as well as on serine and threonine.
• the binding reaction is carried out at a strongly acidic pH of approx. 5 and at high ionic strength. Therefore, for binding Nanoparticles require a strong dilution of the reaction in the target buffer, which is problematic for total assay volumes of 10 ⁇ l in 1536 format in the uHTS.
• the binding is also measured here by means of fluorescence polarization.
• Fluorescence polarization is a relatively complex measurement method and currently does not allow parallel measurement of a microtiter plate (MTP). Therefore, the measurement times for a 1536-MTP would be very long and the parallel measurement of enzyme kinetics would not be possible. In addition, fluorescence polarization as a method is limited to very small fluorescent substrates.
• the kinase activity can be measured by using ATP using Firefly luciferase or by forming ADP using a downstream enzyme cascade.
• the disadvantage of these assay formats is that, due to the indirect measurement method, they not only produce more scattering measurement values, but also have problems with substances that inhibit the cascade enzymes.
• fiuorogenic substrates with C-terminal dyes such as aminocoumarin can be used. Endoproteases that cut in the middle of peptide sequences can usually be measured well in FRET assays, with the donor (eg EDANS) and acceptor dyes (eg dabcyl) being at the ends of the substrate. The substrate cleavage increases the fluorescence intensity because the acceptor dye can no longer quench the donor dye.
• FRET assays FRET assays
• the enzyme reaction must be measured indirectly either by means of complex chemical analysis (eg HPLC / MS, GC / MS) or by chemical reaction or enzyme cascades.
• complex chemical analysis eg HPLC / MS, GC / MS
• chemical reaction or enzyme cascades eg HPLC / MS, GC / MS
• all disadvantages with regard to the stability of the assay and non-specific reactions of screening substances with the detection reaction have to be accepted.
• the complex analysis is not suitable for high-throughput screening.
• Enzymes whose reactions - in the required throughput - cannot be measured directly include those which, for example, make the following modifications to substrates: phosphorylation / dephosphorylation, sulfation / desulfation, methylation / demethylation, oxidations / reductions, acetylation / deacetylation, Amidation / deamidation, cyclization / ring cleavage, conformational ent, cleavage of amino acids / peptides / coupling of amino acids / peptides, ring expansion / ring reduction, rearrangements, substitutions, eliminations, addition reactions etc.
• FLT fluorescence lifetime
• a suitably coupled dye should indicate this molecular modification by changing the FLT.
• Such a method has the potential to be generically applicable to tyrosine as well as serine / threonine kinases and phosphatases.
• the principle should also be applicable to other modification reactions such as sulfation / desulfation, methylation / demethylation, oxidation / reduction, acetylation / deacetylation, amidation / deamidation, cyclization / ring cleavage, conformational changes, cleavage of amino acids / peptides / coupling of amino acids / Peptides, ring expansion / ring reduction, rearrangements, substitutions, eliminations, addition reactions etc.
• FLT measurements are currently possible very quickly (sometimes 50 ms or less per well), so that the method is suitable for high-throughput screening.
• the high robustness against interference such as inner filter effect, autofluorescence, light scattering, photobleaching, volume / meniscus effects, concentration of the fluorescent substrate is particularly advantageous for HTS applications.
• the application shows that only 2 components, substrate and enzyme, have to be mixed to start and measure the reaction.
• Conventional assay methods usually require the addition of other reagents, such as cascade enzymes, so that the reaction can be measured.
• a pipetting error and thus an additional error for the measurement result is caused, which is also called error propagation. This propagated errors lead 'to an increased variance of the measurement results.
• the homogeneous assay method according to the invention or the method according to the invention for the direct quantitative measurement of molecule modifications is characterized in that the molecule carries a fluorescent dye and in that the fluorescence lifetime of the molecule differs from the fluorescence lifetime of the modified molecule.
• the fluorescence lifetime of the modified molecule can be longer than that of the unmodified molecule.
• the invention also includes an assay method according to the invention in which the fluorescence lifetime of the modified molecule is shorter than that of the unmodified molecule.
• the molecule can be, for example, an organic, in particular a peptide or peptidomimetic, or inorganic molecule.
• the fluorescent dye can be, for example, a coumarin, a fluorescein, a kardamine, an oxazine or a cyanine dye.
• the fluorescent dye used can be covalently or non-covalently coupled to the molecule.
• a spacer molecule can be located between the fluorescent dye and the molecule.
• the assay method according to the invention or the method according to the invention can be used to quantify biochemical assays in which enzymes can carry out the following modification reactions, for example: phosphorylation / dephosphorylation, sulfation / desulfationation, methylation / demethylation, oxidation / reduction, acetylation / deacetylation, amidation / Deamidation, cyclization / ring cleavage, conformational changes, cleavage of amino acids / peptides, coupling of amino acids / peptides, ring expansion / ring downsizing, rearrangements, substitutions, eliminations, addition reactions etc.
• the assay method according to the invention or the method according to the invention can be used to advantage in high-throughput screening - in particular in high-throughput screening for identifying active pharmaceutical ingredients.
• the invention furthermore includes a reagent kit which contains fluorescence-dye-molecule conjugates and other reagents which are required for carrying out the assay method according to the invention or the method according to the invention.
• Fig. 1 Logarithmic temporal fluorescence decay curve of 15 nM of a fluorescein-peptide conjugate. Measured on Ultra FLT prototype (TECAN) using TCSPC.
• Fig. 3 The course of the fluorescence lifetime (FLT in ps) is plotted against the reaction time (time in s).
• Fig. 5 For a potential educt (FJ23, hatched) and its product (FJ24, black) of the reaction with the TAFI enzyme, the fluorescence lifetimes are under different conditions (1: water, 2: pH 6, 3: pH 7 , 4: pH 8.5, pH 9.5, 6: OOmM NaCl, 7: 2 M NaCl) was measured. 'The fluorescence lifetimes are practically independent of the conditions examined. But the fluorescence lifetimes of FJ23 (552 ps) and FJ23 (2194 ps) are very different.
• Fl-P 1 Fluorescein-C6-TEGQYpQPQP-COOH, Eurogentec, phosphorylated
• FLT fluorescence lifetime
• the fluorescence lifetime of Fl-Pl is 3880 ps and the FLT of Fl-1 is 3600 ps. Since the standard deviations are very small with ⁇ 25 ps at a measuring time of 1 s, both molecules can be distinguished very well (see Fig. 2). From the standard deviations and the mean fluorescence lifetimes of Fl-Pl and Fl-1 one can calculate a z'-factor of approx. 0.5 for the performance of a potential biological test with FLT-measurement windows spanned by Fl-Pl and Fl-1. which would be enough for a screening campaign. The z 'factor was developed by Zhang et al. Introduced in 1999 to evaluate the performance of HTS assays (Zhang JH, Chung TDY, Oldenburg KR, J. Biomol. Screen 4, 67-73 (1999)). The activity of a kinase such as p60 src , which would phosphorylate Fl-1, should be very easy to measure using FLT measurements.
• Fl-P kemptide fluorescein-C6-LRRApSLGCONH 2 , Eurogentec, phosphorylated
• Fl-kemptide fluorescein-C6-LRRASLGCONH 2 , Eurogentec, nonphosphorylated
• it should be possible to increase the FLT difference obtained initially e.g. by selecting and combining different parameters such as Fluorescent dye, spacer molecule between dye and substrate molecule, or polarity, pH, ionic strength of the solvent or other additives.
• This example shows how a significant increase in the FLT difference between a phosphorylated and a non-phosphorylated variant of a fluorescein-kemptide-peptide conjugate (Fl-P-kemptide, Fl-kemptide) was achieved by increasing the pH .
• Fl-P-kemptide fluorescein-kemptide-peptide conjugate
• 50 nM Fl-P-Kemptid and Fl-Kemptid were dissolved in the solutions described under Material and their FLT's measured using a modified Nanoscan device (IOM GmbH, Berlin), which transmitted the signals to a transient recorder. 16 decay curves were averaged for each measurement point. The falling part of the logarithmic curve was evaluated using linear regression and the negative slope was converted into the FLT.
• Fl-cAMP 8-Fluo-cAMP, BIOLOG Life Science Institute
• PDElb Phosphodiesterase lb (Laboratory Dr. A. Tersteegen, Bayer AG)
• phosphodiesterases represent a very important class of targets, among others. in the indication areas cardiovascular, metabolic diseases, central nervous system, cancer and respiratory diseases. It is therefore of great interest to have a generic assay format that can measure the conversion of cAMP or cGMP into the respective monophosphates. Detection enzyme cascades are usually used. This example shows that it is possible to measure the phosphodiesterase reaction directly.
• 1 ⁇ M Fl-cAMP and a 1: 360 dilution of PDElb were first mixed in the presence of different concentrations of the inhibitor BAY 383045. The kinetics of the enzyme reaction was measured using an Ultra FLT prototype (Tecan) at room temperature.
• the FLT of Fl-cAMP changes - without inhibitor - in the course of the reaction to Fl-AMP within 100 minutes from approx. 3500 ps to approx. 3350 ps.
• Increasing concentrations of BAY 383045 increasingly inhibit the enzyme reaction (see FIG. 3).
• the clear concentration dependence of the inhibition of the phosphodiesterase reaction has shown that the change in Fluorescence lifetime of the Fl-cAMP is clearly related to enzyme activity. This proves that this method can in principle be used to search (screen) for substances that inhibit phosphodiesterases.
• the measuring principle should also be expandable to kinase and phosphatase and other enzyme assays if a measurable FLT change occurs during the enzymatic modification of the substrate.
• a phosphodiesterase assay with direct FLT detection of the substrate modification should be very robust due to the interference-free measurement signal and few pipetting steps.
• the described assay method one could rule out that substances interfere with detection enzymes.
• the following generally applies to the described assay method based on fluorescence lifetime measurements: The incubation times of phosphodiesterase, kinase and phosphatase assays as well as other enzyme assays can be easily measured in one experiment by the direct and immediate measurement of the enzyme kinetics and set it up exactly for a robot high throughput screening campaign.
• FJ24 Evoblue30-Ttds (spacer) -IFT-COOH, Jerini Peptide Technologies
• TAFI thrombih activatable fibrinolysis inhibitor
• the fluorescence lifetimes of the conjugates FJ23 and FJ24 were measured, both of which carry a fluorescent dye that can be excited at 630 nm (Evoblue30, Mobitec) and differ only in that the C-terminal arginine is missing in the FJ24 conjugate.
• FJ23 represents a potential starting material for the TAFI reaction, while FJ24 would be the corresponding reaction product.
• the conjugates FJ23 and FJ24 were dissolved in a concentration of 60 nM in different buffers with pH values 6, 7, 8 and 9.5, as well as in the presence of 200 mM and 2 M NaCl. Result:

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• 2004
  • 2004-10-05 US US10/575,026 patent/US20070042500A1/en not_active Abandoned
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  • 2004-10-05 WO PCT/EP2004/011100 patent/WO2005043137A1/de active Application Filing
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