WO2006015797A2 - Procédé de criblage de rendement élevé de liaison de molécules de test avec des molécules cibles - Google Patents

Procédé de criblage de rendement élevé de liaison de molécules de test avec des molécules cibles Download PDF

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WO2006015797A2
WO2006015797A2 PCT/EP2005/008464 EP2005008464W WO2006015797A2 WO 2006015797 A2 WO2006015797 A2 WO 2006015797A2 EP 2005008464 W EP2005008464 W EP 2005008464W WO 2006015797 A2 WO2006015797 A2 WO 2006015797A2
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molecule
binding
molecules
test
target molecule
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PCT/EP2005/008464
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WO2006015797A3 (fr
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Sascha Sauer
Konrad BÜSSOW
Hans Lehrach
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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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
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • 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
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention relates to a method for identifying one or more molecules binding to a target molecule in a mixture of one or more test molecules, each test molecule having at least one known characteristic property, wherein the method comprises the following steps: (a) bringing said one or more test molecules into contact with said target molecule under conditions allowing formation of a complex comprising said target molecule and the molecule(s) binding to said target molecule; (b) separating said complex or said target molecule from non-binding test molecule(s); (c) detecting said non-binding test molecule(s), if any, via their known characteristic property/ies; and (d) identifying said molecule(s) binding to said target molecule, if any, being the test molecule(s) not detected in step (c).
  • detection is effected by mass spectrometry.
  • separation is effected by size exclusion chromatography.
  • Fluorescent assays have shown to be highly sensitive, in comparison to colorimetric assays, suitable for miniaturisation, and robust. However, they require costly labelling of molecules. Fluorescent polarisation (FP, fluorescent anisotropy) readout is advantageous for such assays, since this procedure allows for "mix and read” screening. FP requires only one fluorescently labelled probe molecule in contrast to energy transfer approaches. FP is sensitive to the rotational correlation time of a probe fluorophore. To use FP in HTS assays, a fluorophore is attached to a ligand or an enzyme substrate.
  • FP Fluorescent polarisation
  • FP enzyme assays involve a size change of a fluorescently labelled substrate induced by the enzymatic reaction.
  • a disadvantage of FP screening is that potential binders could bind at a different site than the labelled ligand. No change in FP would be detected but the additional binding might influence activity in a cellular context.
  • SPR surface plasmon resonance
  • biophysical techniques such as analytical centrifugation, spectroscopic methods (circular dichroism, light scattering, and fluorescence), differential scanning calorimetry, isothermal calorimetry, x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy can be used. All of these technologies either lack throughput, accuracy, sensitivity or multiplex detection capability on the analysis side.
  • Mass spectrometry appears to be the method of choice for screening assays that monitor binding of compounds and target proteins (Chan et al., 2002; Benkestock et al., 2002).
  • the main advantage of mass spectrometry over other mentioned detection methods is the speed, accuracy, automation capability and the fact that mass spectrometers, owing to the multiple detection channels in a mass spectrum, are multichannel detectors.
  • Electrospray ionisation mass spectrometry has become an important method in life science (Loo, 1997). It has successfully been used for protein and peptide analysis.
  • ESI is currently one of the gentlest mass spectrometric methods to ionise biomolecules.
  • charged droplets are generated at atmospheric pressure by spraying the sample solution in an electric field.
  • the analyte molecules are ionised in these droplets and are leaving the ion source largely intact. Due to soft ionisation, ESI can be used to directly detect non-covalent complexes, which would be much more difficult by other ionisation techniques such as matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS).
  • MALDI-MS matrix-assisted laser desorption/ionisation mass spectrometry
  • ESI-MS analysis of protein complexes in a mass range lower than about 35 kDa is feasible using modern quadrupole, ion trap or time-of-flight mass spectrometers.
  • Successful analyses of a variety of non- covalent interactions have been shown including protein-protein, protein-ligand, and protein-metal interaction. Complexes observed in solution could be monitored by ESI-MS. It might, however, occur that false positives are observed due to non ⁇ specific aggregation in the gas-phase. Faise negative results were rarely observed.
  • most gas-phase complexes are relatively fragile.
  • the ESI source parameters such as a proper orifice potential and sample solution conditions like pH and organic co-solvents play a major role for the identification of binding partners (WO9856028).
  • the dissociation constant, K 0 is used to characterise the strength of binding (the lower the value, the stronger the binding).
  • the nature of the interaction influences the results of the mass spectrometric experiment depending on the non-covalent forces involved in binding of proteins and ligands. If experimental parameters are optimised for comprehensive analysis of libraries of compounds, ESI-MS can be used for the rapid screening of potential binding partners over a wide range of KD values.
  • this technique should also be suitable for combinatorial chemistry libaries, but in some cases there might remain a low discrimination of conditions (for example the solvent or the mass spectrometer voltage) leading to either desolvation of complexes during ESI or dissociation of the complexes.
  • conditions for example the solvent or the mass spectrometer voltage
  • Frontal affinity chromatography in combination with ESI-MS detection is complementary to the direct mass spectrometric measurement of protein ligand complexes (US6627453, US6613575, US6607921, US6355163, WO9950669, WO9743301).
  • the target protein is immobilised on a column and continuous flow of a compound mixture is passed through it. The flow through is continuously monitored by ESI-MS, which allows for simultaneous measurement of a larger number of compounds in a mixture. Retention of compounds occurs when they bind to the immobilised protein. The retention of the compounds is recorded and used for calculation of binding parameters.
  • the two complementary mass spectrometric methods described can be useful for screening experiments at a medium to high throughput, since they allow for compound multiplexing and study of extract fractions containing compound mixtures.
  • Molecular sieving also called gel filtration or size-exclusion chromatography makes use of the different chromatographic behaviour depending on the size of molecules.
  • a number of solid-phase materials have been developed to provide for efficient molecular sieving, for example well-known Sephadex® based on modified dextrans or Bio-Gel® based on polyacrylamide gels.
  • Molecular sieving can be performed in columns or on thin-layers.
  • the extent of molecular exclusion of molecules is determined by the sterical hindrance of analyte molecules due to matrix substance. Small molecules can enter the interior of the matrix, while large molecules more or less pass the matrix.
  • the permeability depends on the size and partly on the shape of the molecules.
  • Neogenesis Pharmaceuticals Inc. at the 2003 annual conference of the ASMS in Montreal (http://www.inmerge.com/asms03pdf/A030831.pdf, WO9935109, WO0022649), where all steps can be performed online.
  • a solution of a protein target is mixed with mass-encoded small molecules, leading to the formation of complexes of the protein with any suitable small molecule.
  • Mass-coding is achieved by attaching n (2 or more) peripheral moieties Y to a scaffold X 1 yielding compounds of the general formula X(Y) n , wherein a set of distinct peripheral moieties is used which fulfils the criterion that at least about 90% of all possible combinations of n peripheral moieties have a molecular mass sum which is distinct from the molecular mass sum of all other combinations of n peripheral moieties.
  • a spread of masses of the compounds of the general formula X(Y) n is achieved which minimizes overlap in the spectrum and allows identification of the different compounds of the general formula X(Y) n by molecular mass.
  • the complexes are separated from non-binding compounds by size exclusion chromatography (SEC).
  • the SEC band containing the complex is then transferred to a capillary reverse-phase chromatography column, which is maintained at 60 0 C and pH 2.0 to dissociate ligands from the complex.
  • the dissociated ligands are eluted into an ESI-TOF mass spectrometer, and automated software algorithms search a database of mass spectral data to identify ligands by their molecular weight.
  • the binding assays described in the prior art, particularly in Muckenschnabel et al., 2004, and by Neogenesis have in common that at least two chromatographic steps have to be performed: the first for the separation of the complex formed by the target protein and the binding ligand from the non-binding compounds, and the second for the separation of the binding ligand from the target protein, wherein the second chromatographic step is effected under denaturing conditions, i.e. under conditions where the complex falls apart. Eventually the binding ligand obtained from the second chromatographic step is detected and identified. If it were possible to identify the binding ligand by a method which employs only one chromatographic step as opposed to two, a further speed-up would be attainable.
  • the technical problem underlying the present invention was therefore the provision of means and methods for identifying molecules binding to a target molecules, wherein said method involves less steps.
  • this invention relates to a method for identifying one or more molecules binding to a target molecule in a mixture of one or more test molecules, each test molecule having at least one known characteristic property, wherein the method comprises the following steps: (a) bringing said one or more test molecules into contact with said target molecule under conditions allowing formation of a complex comprising said target molecule and the molecule(s) binding to said target molecule; (b) separating said complex or said target molecule from non-binding test molecule(s); (c) detecting said non-binding test molecule(s), if any, via their known characteristic property/ies; and (d) identifying said molecule(s) binding to said target molecule, if any, being the test molecule(s) not detected in step (c).
  • identifying a molecule refers to determining at least one known characteristic property of said molecule.
  • Said property may be the molecular formula or the structure of the molecule, but may also be any other characteristic property such as molecular mass, NMR spectrum, IR spectrum, UV or visible light absorbance, fluorescence or luminescence emission wavelength(s), melting point or boiling point.
  • a "molecule binding to a target molecule” is a molecule which under known conditions occurs predominantly bound to the target molecule.
  • the dissociation constant K 0 of the complex formed by said molecule and the target molecule is less than about 10 '5 M. More preferred, KD is less than 10 "6 M, yet more preferred less than 10 "7 M.
  • Binding involves interaction between one or more moieties or functional groups of the test molecule and one or more moieties or functional groups of the target molecule, wherein said interaction may comprise one or more of charge-charge interactions; charge-dipole interactions; dipole-dipole interactions, wherein said dipoles may be permanent, induced or fluctuating; hydrogen bonds; and hydrophobic interactions. Hydrogen bonds and interactions involving a permanent dipole are of particular relevance in the sense that they confer specificity of binding by their directional character.
  • Binding may be unspecific, for example by interaction with a group such as a charge or a dipole, which may be present many times at the surface of the target molecule.
  • binding is specific, i.e., it occurs at a defined site of the target molecule and goes along with the formation of a network of several distinct and specific interactions. Specific binding may occur with hardly any change of the conformation of the molecules involved ("key-in-lock"), or it may involve conformational changes of one or both of the binding partners ("hand-in-glove” paradigm).
  • One or more test molecules may bind to the target molecule. If more than one test molecule binds the target molecule, the binding test molecules may either bind at the same site.
  • the term "complex” embraces binary complexes of the type (target molecule): (test molecule) and ternary complexes of the type (target molecule):(first test molecule):(second test molecule) as well as complexes of a target molecule with more than two test molecules. In cases, where more than one test molecule is capable of binding to the target molecule, both binary and ternary (and higher order) complexes may be formed.
  • a "target molecule” according to the present invention may be any molecule. Although molecules which are targets for therapeutic intervention are of particular interest for the industrial application of the method of the invention, the term “target molecule” is not limited to targets for therapeutic intervention. Molecules of biological origin are envisaged as target molecules as are xenobiotic molecules.
  • Test molecules are provided as mixtures of test molecules, which preferably contain or comprise two or more test molecules, thereby increasing the number of test molecules assessed in a single binding assay and consequently increasing throughput of the method.
  • each test molecule of the mixture i.e., for each molecule which may bind to the target molecule or be detected in step (c)
  • at least one characteristic property is known.
  • the number of test molecules in said mixture is preferably below 100, more preferred below 50. However, also mixtures of more than 100 test molecules are envisaged to be used in the method of the invention.
  • Suitable conditions in step (a) depend on the target molecule, the test molecule(s) and/or the application. Said conditions are well within the skills of the skilled artisan. Naturally occurring target molecules may be assessed under conditions identical or similar to their natural environment, or may be assessed under conditions which deliberately differ from the conditions of the natural environment, for example, if molecules capable of preventing denaturation of a target molecule are to be identified.
  • Incubation times are adjusted to the times needed for binding to essentially reach equilibrium and are in the range between 1ms and 1 day, preferably between 0.1 sec and 2 hours, and most preferred between 1 and 30 sec. Binding may be performed in the dark, or at daylight, or under irradiation with electromagnetic radiation. Preferred wavelengths of said electromagnetic radiation are between 1 and 10000 nm, preferably between 10 and 2000 nm, and most preferred between 200 and 1000 nm.
  • the assay temperature is between 250 and 400 K, preferably between 273 and 313 K, and most preferred in the interval between 290 and 310 K, i.e., comprising ambient temperature.
  • the preferred temperature interval may be centered around the optimal growth temperature of the source organism.
  • Assay temperature is maintained by means of a thermostat.
  • the assay temperature is ambient temperature, fluctuations of the temperature may occur. Said fluctuations, as measured on the timescale of the assay, do not exceed 2 degrees, preferably 0.5 degree, and most preferred 0.1 degree from the average temperature.
  • the assay may be exposed to air, or performed under a protective atmosphere provided by inert gas, such as nitrogen or argon.
  • the assay may be provided with gaseous substances, such as NO or acetylene.
  • the separation step (b) involves the separation of the complex (if at least one binding test molecule was present) or the target molecule (if no binding test molecule was present) from the non-binding test molecules. If all test molecules bind to the target molecule, step (b) will not involve a separation in the usual sense, as zero non- binders are to be separated from the complex.
  • Detecting in step (c) involves the determination of the known characteristic property/ies of the non-binders separated from the complex or from the target molecule in step (b). It is of note that non-binding test molecules are detected by the method of the invention. Accordingly, the identification of binding molecules in step (d) is not a direct identification as in the methods of the prior art, but instead involves inferring the identity of the binding molecule from a comparison of the data set obtained from determining the characteristic properties of the non-binders and the data set of characteristic properties of all test molecules used in a particular assay which is known prior to conducting the assay.
  • detecting in step (c) is not necessarily a all-or-none process, leading to molecules being classified as either "present” or “absent". Signals from binding molecules may still be detectable, but may be considerably weaker than prior to contacting the target molecule.
  • the term "non-binders” is understood to comprise also weak binders which occur predominantly in unbound form. Alternatively, and in case of strong binding, a signal from a binding molecule might be lower than the detection threshold of the method applied in step (c), leading to the molecule being classified as "absent".
  • the indirect approach of the invention offers significant advantages over the prior art.
  • the second chromatographic step effected under denaturing conditions which serves the separation of the target molecule and binding molecules, is not needed any more. This enables the screening of more compounds per time unit and a further reduction of costs associated with, for example, lead identification or optimization projects in pharmaceutical and biotech industry.
  • the method does not require labelling of target and/or test molecules. As compared to frontal affinity chromatography, the method does not require attaching target or test molecules to matrices.
  • Figure 1 illustrates the workflow of the method of the invention.
  • the conditions in step (a) are selected from the group consisting of aqueous solution, buffered solution and solutions representing physiological conditions such as cell extracts.
  • Buffers are well known in the art and the skilled person is aware of appropriate buffers in dependency of the substances being assayed.
  • ionic strength may be adjusted, e.g., by the addition of sodium chloride and/or potassium chloride.
  • concentration of sodium chloride is between 0 and 2 M, preferably between 100 and 200 mM.
  • Examples comprise PBS (phosphate buffered saline) containing 1.37 M NaCI, 27 mM KCI, 43 mM Na 2 HPO 4 TH 2 O and 14 mM KH 2 PO 4 in the 10-fold aqueous stock solution, which is adjusted to pH 7.3; SSC containing 3 M NaCI and 0.3 M sodium citrate in 20-fold aqueous stock solution, which is adjusted to pH 7.0; and STE (Saline Tris EDTA) containing 10 mM Tris base, 10 mM NaCI and 1mM ETA (acid).
  • PBS phosphate buffered saline
  • SSC containing 3 M NaCI and 0.3 M sodium citrate in 20-fold aqueous stock solution, which is adjusted to pH 7.0
  • STE Se
  • sodium chloride is absent from the buffer preparation.
  • Examples for common buffer preparations without sodium or potassium chloride are TAE (Tris acetate EDTA) containing 2 M Tris acetate and 0.1 M EDTA in the 50-fold aqueous stock solution at pH 8.5; TBE (Tris borate EDTA) containing 0.89 M Tris base, 0.89 M Boric acid and 0.02 M EDTA in the 10-fold aqueous stock solution at pH 8.0; and TE (Tris EDTA) containing 10 mM Tris base and 1 mM EDTA (acid) at pH 7.5.
  • TAE Tris acetate EDTA
  • TBE Tris borate EDTA
  • TE Tris EDTA
  • Physiological conditions in accordance with the present invention may vary significantly, for example when comparing the interior of a cell to the extracellular space.
  • Exemplary intracellular conditions comprise 14 mM Na + , 140 mM K + , 10 "7 mM Ca 2+ , 20 mM Mg 2+ , 4 mM Cl “ , 10 mM HCO 3 " , 11 mM HPO 4 2" and H 2 PO 4 " , 1 mM SO 4 2" , 45 mM phosphocreatine, 14 mM carnosine, 8 mM amino acids, 9 mM creatine, 1.5 mM lactate, 5 mM ATP, 3.7 mM hexose monophosphate, 4 mM protein and 4 mM urea.
  • Exemplary interstitial conditions comprise 140 mM Na + , 4 mM K + , 1.2 mM Ca 2+ , 0.7 mM Mg 2+ , 108 mM Cl “ , 28.3 mM HCO 3 " , 2 mM HPO 4 2" and H 2 PO 4 " , 0.5 mM SO 4 2" , 2 mM amino acids, 0.2 mM creatine, 1.2 mM lactate, 5.6 mM glucose, 0.2 mM protein and 4 mM urea.
  • the target molecule is in its natural environment when it is brought into contact with the one or more test molecules.
  • Said natural environment may be established, for example, by the addition of blood plasma.
  • Another example of said natural environment relates to membrane proteins, comprising peripheral and integral membrane proteins.
  • Said membrane proteins, such as G-protein coupled receptors or ion channels, may be reconstituted in membranes or micelles.
  • the indirect detection of binding molecules according to the invention is particularly advantageous, noting that the complex of the target molecule and the bindjng molecule(s) also comprises membrane lipids, thereby rendering the direct approach considerably more intricate as compared to the detection of non-binders.
  • the method of the invention further comprises after step (b) and prior to step (c) one or more of the following steps: (b 1 ) concentrating the solution comprising said non-binding test molecule(s); and (b") fractionating the solution comprising said non-binding test molecules by chromatographic means, thereby reducing the complexity of the mixture of non- binding test molecules to be analyzed in step (c). Fractionation is advantageous in case of complex mixtures such as natural extracts, combinatorial synthesis mixtures or large pools of test compounds. Also, it may occur that different ionisation efficiencies of a few molecules with different chemical properties can lead to mass spectra of poor quality. For example, one test molecule may require harsher conditions for ionisation than an other test molecule.
  • fractionation might be advantageous.
  • a further scenario where fractionation might be advantageous is a mixture of test compounds, wherein the test compounds differ significantly with regard to their hydrophobicity. In that case, after fractionation, the hydrophobic fraction(s) would be taken up in (a) more hydrophobic solvent(s), whereas the hydrophilic fraction(s) would be taken up in more (a) more hydrophilic solvent(s) for the purpose of detecting in step (c).
  • hydrophobic denotes a preference for lipids or for organic or apolar liquids or for liquids with a small dipole moment as compared to water.
  • the mass flux of a molecule at the interface of two immiscible or substantially immiscible solvents is governed by its hydrophobicity. The more hydrophobic a molecule is, the more soluble it is in the hydrophobic organic phase.
  • the partition coefficient of a molecule that is observed between water and n-octanol has been adopted as the standard measure of hydrophobicity.
  • a molecule is ionizable, a plurality of distinct microspecies (ionized and not ionized forms of the molecule) will in principle be present in both phases.
  • LogP values can be determined by experimental means and/or predicted by computational methods known in the art (see for example Sangster, Octanol-water Partition Coeffcients: fundamentals and physical chemistry, John Wiley & Sons, Chichester. (1997)). In practice, logP values will vary according to the conditions under which they are measured.
  • any chromatographic method is envisaged for the purpose of fractionating the non- binding molecules.
  • said chromatographic method is ion exchange chromatography, liquid chromatography or reverse-phase chromatography.
  • the method of the invention further comprises after step (b) the following steps: (e) breaking said complex formed in step (a) and separated from said non-binding test molecule(s) in step (b) by changing the conditions; (f) separating the target molecule from the molecule(s) bound to the target molecule in step (a); and (g) detecting said molecule(s) bound to the target molecule in step (a), thereby confirming the identity of said molecule(s) bound to the target molecule in step (a) and identified in step (d).
  • this embodiment of the present invention performs a direct detection of the binding molecule(s). It is intended as a positive control and serves further substantiating the result obtained in step (d) of the main embodiment.
  • the target molecule can be recovered by close to 100%, a quantitative procedure for a variety of different target molecules is provided, which does not necessitate individual optimization.
  • the change in conditions is a raise of temperature, a change of pH and/or a change of solvent.
  • the change of conditions is such that binding of the one or more test molecules capable of binding the target molecule is no longer maintained.
  • the change of conditions may lead to a partial or complete loss of the native structure of the target molecule, also referred to as denaturation. If denaturation affects the one or more binding sites on the target molecule, specific interaction can not occur any more.
  • the extent of the raise of temperature needed for breaking the complex depends on the target molecule. For example, many proteins originating from homoiothermal organisms are sensitive to rises of temperature, for example of 50, 20, 10 or 5 degrees, above the normal body temperature of, for example, 310 K. Alternatively, a drop of temperature may also be used to effect denaturation in those cases where target molecules are sensitive to cold denaturation.
  • a change of pH according to the invention may either be towards the acidic or the basic regime.
  • a shift of 5 or more, or 4, 3, 2 or 1 pH units in either direction may be used to induce decomposition of the complex.
  • a change of solvent according to the invention may be the addition of a non-aqueous solvent.
  • Said non-aqueous solvents may act to destabilize the hydrophobic interactions stabilizing for example, the hydrophobic core of a protein.
  • the net effect of an organic solvent on protein structure usually depends on the magnitude of its effect on various polar and nonpolar interactions. At low concentration, some organic solvents can stabilize several enzymes against denaturation. At high concentrations, however, virtually all organic solvents cause denaturation of proteins because of their solubilizing effect on nonpolar side chains. Examples of non-aqueous solvents are acetonitril, acetone and alcohols.
  • the change of solvent may embrace the addition of denaturing agents such as urea and/or guanidinium hydrochloride.
  • the change in conditions may be effected by changing one of the above mentioned parameters or by changing more than one parameter simultaneously or subsequently.
  • the change in conditions may primarily affect the target molecule.
  • the change in conditions may affect the binding test molecule(s) or both the target molecule and the binding test molecule(s).
  • the method of the invention further comprises (h) determining at least one characteristic property of each of said test molecule(s); and (i) identifying molecules determined in step (h), but not detected in step (c), thereby confirming the identity of said molecule(s) binding to said target molecule identified in step (d).
  • the main embodiment requires at least characteristic property of each test molecule to be known.
  • the preferred embodiment described above is directed to the actual determination of said characteristic properties.
  • detecting in steps (c) and/or (g) and/or determining in step (h) is effected by mass spectrometry.
  • mass spectrometry is advantageous because of its low detection limit, large dynamic range, speed, accuracy and automation capability.
  • Mass spectrometers are inherently multichannel detectors allowing the simultaneous detection of a plurality of analytes. Furthermore, there is no need for labelling.
  • the ionisation method employed for mass spectrometry is selected from the group consisting of electron impact, chemical ionisation, atmospheric pressure, fast atom bombardment, thermospray, electrospray, laser desorption ionisation (LDI), matrix-assisted laser desorption ionisation (MALDI), desorption ionisation on silicon (DIOS), and electrospray ionisation (ESI).
  • LBI laser desorption ionisation
  • MALDI matrix-assisted laser desorption ionisation
  • DIOS desorption ionisation on silicon
  • ESI electrospray ionisation
  • ESI is particularly preferred as it is currently one of the gentlest mass spectrometric methods to ionise biomolecules.
  • the mass separation method is selected from the group consisting of magnetic sector, cyclotron resonance, ion trap, quadrupole(s), and time-of-flight.
  • said known characteristic property/ies is or comprise the molecular mass, the mass-to-charge ratio (m/z) and/or the mass spectrum.
  • the molecular mass is a molecular property and may differ from molecule to molecule of the same chemical species. This may be a result of the natural occurrence of more than one isotope of an atom type, which may apply to one or more of the atoms constituting the molecule. Alternatively or additionally, this may occur when more than one isotope of one or more constituent atoms was used for synthesis. In such cases the mass spectrum will exhibit more than one molecular peak, and intensities of the peaks will reflect the abundance of the different isotopes of the constituent atoms.
  • the mass-to-charge ratio is the ratio m/z, wherein m is the molecular mass and z is the charge of the molecule when it is analyzed in the mass spectrometer.
  • the m/z ratio is the property separation in the mass spectrometer relies on.
  • the mass spectrum is a set of intensity and m/z data which may be displayed graphically in two dimensions as a plot intensity vs. m/z. Typically, peaks in a mass spectrum are very sharp (Dirac functions).
  • a database of mass spectra and associated further properties is used to assign said further properties to molecules identified by a method according to any of the preceding claims.
  • identifying refers to determining at least one known characteristic property. Accordingly, this preferred embodiment, which is directed to the provision of further characteristic properties, serves a further identification of said molecule(s).
  • the database records of the envisaged database comprise at least one further property in addition to the mass spectrum of each molecule registered in the database. The database look-up may either by effected manually or in a computer- assisted manner.
  • Mass-spectrometric data lend themselves particularly to digitalisation and computer-based comparison in view of the sharp (Dirac-shaped) peaks.
  • Databases and associated software are well known in the art and available from suppliers of mass spectrometers. If compounds not yet stored in these databases are analysed, the skilled person may set up a new database.
  • a known quantity of a known non-binding molecule is added to said mixture comprising one or more test molecules, thereby allowing to determine the concentration and/or amount of a test molecule.
  • This embodiment is directed to an internal standard.
  • the internal standard permits to determine the relation between the absolute quantity and the intensity of a corresponding signal in a detection step. Accordingly, signal intensities of standard molecules and molecules under investigation can be compared to calculate the concentration or amount of molecules of interest in a sample.
  • the method further comprises determining the height of signals in the mass spectrum, thereby allowing to determine binding constant(s).
  • the strength of binding can be characterized by the equilibrium binding constant or the equilibrium dissociation constant KD, which is the inverse of the binding constant.
  • KD is defined for a binary complex as [T] [B] / [T:B], wherein [T] denotes the concentration of the target molecule, [B] the concentration of a binding molecule and [T: B] the concentration of the complex formed by the latter species.
  • separation in step (b) and/or step (f) is effected by size exclusion chromatography.
  • size exclusion chromatography also referred to as gel filtration or molecular sieving, has been described herein above.
  • Size exclusion chromatography may be effected in columns or microtiter plates. Any matrix suitable for the purpose of size exclusion chromatography is envisaged.
  • the matrix is selected from the group consisting of dextran, polyacrylamide polymers, copolymers of divinylbenzene and a hydrophilic monomer carrying a vinyl group such as N-vinylpyrrolidone or a methacrylate, derivatives of said copolymers, reverse phase matrices, such C4, C8 and C18 material, and agarose.
  • the pore size of the matrix used is important for the efficient separation of large molecules (and complexes thereof) from small molecules.
  • Sephadex® G25 (coarse) resins from Pharmacia with a pore size of 100-300 microns can be used to retain molecules with a molecular mass of 100-5000 Da, while larger molecules would directly flow through the resins, as they would not enter the pores of the resins.
  • size exclusion matrices with matrix particles having a hydrophobic or other suitable surface, such that small bound molecules are also purified from salts, detergents and the like, prior to elution, for example with organic solvents.
  • additional properties of the matrix may be conferred by a coating of the SEC matrix or may be inherent properties of the SEC matrix used.
  • Such matrices provide improved efficiency of purification of molecules during molecular sieving.
  • examples for such matrices are copolymers of divinylbenzene and a hydrophilic monomer carrying a vinyl group such as N-vinylpyrroIidone or a methacrylate, or related materials that could be other reverse-phase or ion exchange resins. When such materials are used, separation is effected by both size and hydrophobic or polar/ionic properties of the molecules to be separated.
  • Copolymers of divinylbenzene and N-vinylpyrrolidone provide a matrix exhibiting a hydrophilic-lipophilic balance owing to their constituents shown below.
  • the two vinyl groups on the divinylbenzene molecule may be positioned ortho, meta or para with respect to each other.
  • structurally different copolymers are provided.
  • the envisaged derivatives of copolymers of divinylbenzene and N- vinylpyrrolidone include sulfonic acid derivatives exemplified below
  • derivatives of copolymers of divinylbenzene and N- vinylpyrrolidone wherein the sulfonic acid substituents occupy different positions on the benzene ring.
  • Other acidic groups such as phosphate groups, may be present alternatively to or in addition to sulfonic acid groups.
  • variants of the dimethyl-butylamine derivative wherein the substituents on the quaternary nitrogen are changed to shorter (in case of the butyl group) or longer (in case of both methyl and the butyl groups) linear or branched alkyl groups, are envisaged.
  • Oasis® HLB (WO 03080211) plates (Waters, Eschbom, Germany) are an example of microtiter plates equipped with a copolymer of divinylbenzene and N-vinylpyrrolidone which has been used in Examples 1 and 2 enclosed herewith.
  • Oasis® HLB plates are conventionally used for de-salting of samples. De-salting is important for the method of the invention in so far as it relates to the use of mass spectrometry, as the absence of salt is a prerequisite for mass spectrometry.
  • the Oasis® material when used for the method of the invention, does not only serve the purpose, if necessary, of de-salting, but primarily serves for separating non-binding test molecules from the complex or the target molecule, respectively. It is of note that the latter use of the Oasis® material is not suggested in the art.
  • Copolymers of divinylbenzene and N-vinylpyrrolidone as used in Oasis® HLB plates have the further advantage that they are significantly more robust than for example Sephadex® G25, as they can be stored for months at room temperature, can dry without loosing performance, and can be used several times.
  • copolymers of divinylbenzene and any organic molecule which is hydrophilic as compared to divinylbenzene and carries a vinyl group rendering it suitable for copolymerization is the above described N-vinylpyrrolidone.
  • a methacrylate may be used. Specific examples of methacrylates are glycidyl methacrylate, 2-hydroxy ethyl methacrylate and 2-hydroxy propyl methacrylate.
  • branched or linear alkyl groups may be comprised in a methacrylate to be used as constituent monomer of a copolymer according to the invention.
  • said methacrylates Prior to copolymerization, said methacrylates may be derivatized, for example in case of glycidyl methacrylate by reacting the epoxy group of glycidyl methacrylate with one or more amines, carboxylic acids, anhydrides and/or compounds with hydroxyl groups, thereby, and after copolymerization, obtaining derivatives of the copolymers according to the invention.
  • Non-binding molecules may be eluted using organic solvents such as methanol or acetonitrile or organic solvent/water mixtures such as 70% methanol / 30% water.
  • concentration of the eluent may be increased in one or more jumps, or may be raised continuously by a gradient.
  • the target molecule is selected from the group consisting of proteins, (poly)peptides, nucleic acids, saccharides, and derivatives thereof.
  • the term , protein refers to any protein. It includes soluble proteins, for example enzymes such as kinases, proteases, phosphatases, oxidases and reductases as well as non-enzyme soluble proteins, for example soluble proteins comprising immunoglobulin domains such as soluble antibodies.
  • Membrane proteins comprising peripheral membrane proteins and integral membrane proteins are also included. Examples of membrane proteins include receptors. Receptors include growth factor receptors, G-protein coupled receptors and receptors with seven, but also with less or more than seven transmembrane helices.
  • protein refers to single-domain proteins as well as to multi-domain proteins. Further embraced are embodiments, wherein the protein functions as part of a metabolic pathway and/or a signal transduction pathway.
  • the protein may consist of or comprise protein domains known to function in signal transduction and/or known to be involved in protein- protein interaction.
  • Examples for such domains are Ankyrin repeats; arm, BcI- homology, Bromo, CARD, CH, Chr, C1 , C2, DD, DED, DH, EFh, ENTH, F-box, FHA, FYVE, GEL, GYF, hect, LIM, MH2, PDZ, PB1 , PH, PTB, PX, RGS, RING, SAM, SC, SH2, SH3, SOCS, START, TIR, TPR, TRAF, tsnare, Tubby, UBA, VHS, W 1 WW, and 14-3-3 domains.
  • (poly)peptide as used herein describes fragments of proteins and refers to a group of molecules which comprise the group of peptides, consisting of up to 30 amino acids, as well as the group of polypeptides, consisting of more than 30 amino acids.
  • a nucleic acid according to the invention may be a DNA or an RNA molecule. It may be a genomic DNA, cDNA, mRNA, tRNA or rRNA molecule. Also envisaged are embodiments wherein the RNA molecule is an RNA capable of performing RNA interference (see, e.g. Zamore Nat Struct Biol 2001 , 8(9):746-50 or Tuschl T. CHEMBIOCHEM. 2001, 2:239-245). Furthermore embraced are embodiments, wherein the DNA molecule is a PNA (peptide nucleic acid) molecule, an LNA (locked nucleic acid) or another derivative of a DNA molecule. Also envisaged are chimeric molecules containing one or more DNA portions and one or more PNA portions. Saccharides according to the invention may be monosaccharides, di-, tri-, tetrasaccharides or longer oligo- or polysaccharides.
  • derivatives thereof refers to any known modification of the molecules listed above. Examples include lipid-modified proteins, oligonucleotides carrying a label such as a fluorescent dye, peptides carrying protection groups and the like. It is understood that the term “derivatives thereof” also embraces combinations of proteins, (poly)peptides, nucleic acids and saccharides in one molecule. Examples of such combinations include glycosylated proteins, PNA-peptide chimera and the like. Proteins, (poly)peptides, nucleic acids, saccharides, and derivatives thereof according to the invention, if they refer to naturally occurring molecules are understood to embrace also isoforms and polymorphisms. On the other hand, also not naturally occurring molecules of these classes are envisaged, for example the products of site-directed mutagenesis and protein engineering.
  • said target molecule has a molecular weight greater than about 5000 Da.
  • the test molecule is selected from the group consisting of organic molecules, inorganic molecules, proteins, (poly)peptides, nucleic acids, saccharides, and derivatives thereof.
  • Organic molecule according to the invention include low molecular weight organic molecules.
  • Low molecular weight organic molecules are organic molecules of natural origin or chemically synthesized organic molecules, preferably with a molecular weight between 100 and 1000, more preferred between 200 and 750, and even more preferred between 300 and 600.
  • An upper threshold of about 500 is generally considered critical for drugs or lead compounds which potentially may mature into drugs.
  • the method of the invention also extends to test molecules, wherein said test molecules have a larger molecular weight than the target molecule.
  • the target molecule in case there are no binding test molecules present in the mixture, will elute prior to the test molecules when using SEC according to the invention for separating in step (b).
  • said mixture of one or more test molecules is obtained by pooling of test molecules.
  • Such deliberate pooling of separately available compounds may be envisaged, for example, for the purpose of increasing throughput of a screening campaign.
  • said mixture is a naturally occurring mixture or a fraction thereof, such as a plant extract or an extract of one or more microorganisms.
  • the method of the invention is effected in parallel, thereby allowing the analysis of binding of multiple test molecules to multiple target molecules.
  • Means for performing the method in parallel are the use of multi-well or microtiter plates, for example with 96, 384 or 1536 wells. Handling of the multi-well plates may be accomplished by one or more robotic systems, which may be computer-controlled. The Figures show:
  • Figure 1 The principle of the procedure is shown. Small molecules and a large target molecule (e.g. a protein) are mixed and applied to matrices as is described in the text. Large molecules and their small molecule binders reside in the breakthrough, while non-binding small molecules remain in the matrices. The remaining molecules are washed, eluted and can finally by detected by mass spectrometry (top). The mass spectrum obtained this way can be compared to mass spectra for which small molecules were subjected to the procedure described without adding the large target molecule. Missing peaks or mass signals with significantly reduced intensity of samples for which target molecules were used indicate binding (top of the illustration).
  • a large target molecule e.g. a protein
  • the breakthrough sample described above can be applied to denaturing conditions such as high temperature or low pH. Subsequently, the large target molecule and its separated binder are subjected to the procedure described above. The denatured target molecule resides in the breakthrough and the small molecule remains on the matrix; it can be washed, eluted and detected by mass spectrometry. In this way, mass signals of the binding molecules that are missing or less intensive in the corresponding spectra of the indirect approach described above can be detected. In samples where no large target molecules were used, no mass signals are expected.
  • Figure 2 A mixture of four different oligonucleotides was used; three of them were unmodified, while at the 5'-end of oligonucleotide 2 a biotiri group was attached. The final concentration of each oligonucleotide was 0.4 ⁇ M and the concentration of streptavidin was 0.75 ⁇ g/ ⁇ l. Separation and purification was performed with Oasis ® HLB micro elution plates using a robotic work station. Mass spectrometry was performed with a Bruker Reflex Il MALDI-TOF mass spectrometer. Mass signals deriving from oligonucleotide metal adducts are marked with an asterisk. Upper trace: oligonucleotide and streptavidin mixture, lower trace: oligonucleotide mixture without streptavidin.
  • FIG. 3 Control experiment. We took the break-through of the experiment described_f.or_Eigure 2_and_denatured the complex-.ot streptavidin and oligonucleotide- 2 containing a biotin at the 5'-end by heating at 95 0 C for 10 minutes. We then subjected the sample to Oasis ® HLB thereby separating the oligonucleotide from the denatured protein, washing and eluting it prior to MALDI analysis. From the samples containing streptavidin, we clearly isolated the biotinylated oligonucleotide (bottom trace) while we obtained blank spectra when no streptavidin was in the samples (top trace).
  • Figure 4 16 % Laemmli-Gel.
  • M Kaleidoscope Prestained Standards, Biorad Marker (26OkDa 1 128kDa, 81kDa, 40.3kDa, 31.6kDa, 19.3kDa, 7.8kDa, K: PPAR ⁇ before molecular sieving using Oasis technology.
  • 3, 6, 9 and 12 PPAR ⁇ after molecular sieving with 2-Chloro-5-nitro-N-phenly-benzamide using Oasis technology (breakthrough collected).
  • 1 , 2, 4, 5, 7, 8, 10, and 11 Samples containing 2-Chloro-5- nitro-N-phenyl-benzamide and/or ⁇ -cyano-4-hydroxy cinamic acid methyl ester but no PPAR ⁇ .
  • Figure 5 Typical result of the screening procedure. Interaction screening of 2- Chloro-5-nitro-N-phenyl-benzamide (275 m/z) and ⁇ -cyano-4-hydroxycinnamic acid methyl ester (202 m/z) with PPAR ⁇ . A Thermo Finnigan LTQ ESI mass spectrometer was used, molecules were detected in the negative ion mode. For the experiment shown in a), only ⁇ -cyano-4-hydroxycinnamic acid methyl ester was subjected to the Oasis procedure, while for b) a mixture of ⁇ -cyano-4-hydroxy cinnamic acid methyl ester and PPAR ⁇ was used. No binding was detected.
  • Streptavidin represents the target protein and a set of small oligonucleotides, one of which was biotinylated, represents a pool of compounds.
  • the oligonucleotides were mixed with streptavidin and applied to molecular sieving and desalting using an Oasis ® HLB plate (Waters, Eschborn, Germany). As a negative control, streptavidin was omitted.
  • the solid phase extraction/molecular sieving was performed using the appropriate manifold kit from Waters. Vacuum was adjusted to 507,957 hPa. 96-well micro-elution plates, packed with Oasis ® HLB resins, were used.
  • the resins were wetted with 200 ⁇ l methanol and equilibrated with 200 ⁇ l deionised water.
  • the samples were filled up to a final volume of 40 ⁇ l in 0.1 M triethylamine, pH 7, and applied to the plates. Then the sample was washed with 50 ⁇ l 100 mM triethylamine/5 % methanol and eluted with 60 % methanol, first with 25 ⁇ l and subsequently with 20 ⁇ l.
  • the eluate pool was lyophilised, resuspended in 10 ⁇ l deionised water and analysed by MALDI mass spectrometry.

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Abstract

La présente invention porte sur un procédé d’identification d’une ou de plusieurs molécules de liaison avec une molécule cible dans un mélange d’une ou de plusieurs molécules de test, chaque molécule de test ayant au moins une propriété caractéristique connue, où le procédé comprend les phases suivantes : (a) mettre ladite ou lesdites molécule(s) de test au contact de ladite molécule cible dans des conditions permettant la formation d’un complexe englobant ladite molécule cible et la(les) molécule(s) de liaison avec ladite molécule cible ; (b) séparation dudit complexe ou de ladite molécule cible de la(des) molécule(s) de test sans liaison ; (c) détection de ladite(desdites) molécule(s) de test sans liaison, le cas échéant, par le biais de sa(ses) propriété(s) caractéristique(s) connue(s) ; et (d) l’identification, ainsi, de ladite(lesdites) molécule(s) de liaison avec ladite molécule cible, le cas échéant, étant la(les) molécule(s) de test non détectée(s) dans la phase (c). Selon un mode de réalisation préféré, la détection fait appel à une spectrométrie de masse. Selon un mode de réalisation préféré, la séparation fait appel à la chromatographie par exclusion de taille.
PCT/EP2005/008464 2004-08-09 2005-08-04 Procédé de criblage de rendement élevé de liaison de molécules de test avec des molécules cibles WO2006015797A2 (fr)

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EP1930444A1 (fr) * 2006-12-08 2008-06-11 BioSpring Gesellschaft für Biotechnologie mbH Procédé spectrométrique de masse sur des échantillons contenant des acides nucléiques

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