WO2000047999A1 - High throughput size-exclusive method of screening complex biological materials for affinity ligands - Google Patents
High throughput size-exclusive method of screening complex biological materials for affinity ligands Download PDFInfo
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- WO2000047999A1 WO2000047999A1 PCT/US2000/003562 US0003562W WO0047999A1 WO 2000047999 A1 WO2000047999 A1 WO 2000047999A1 US 0003562 W US0003562 W US 0003562W WO 0047999 A1 WO0047999 A1 WO 0047999A1
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00702—Processes involving means for analysing and characterising the products
- B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/468—Flow patterns using more than one column involving switching between different column configurations
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
Definitions
- the present invention relates to screening complex biological materials, such as natural products and combinatorial libraries, for affinity ligands, using size-exclusion separation, ultrafiltration, and mass spectrometry .
- Natural extracts represent a highly chemically diverse collection of compounds that make it very difficult to isolate any single active compound. As most successful drug compounds are of small molecular weight (less than 2,000 daltons) , separations based on size can be a useful tool in assisting the isolation of active components .
- Combinatorial chemistry offers the means to generate a large number of different chemicals simultaneously.
- Modern analytical methods allow the screening of such combinatorial libraries to select those compounds possessing desirable properties.
- these methods have their limitations, so that a need remains for a successful analytical methodology that provides high throughput screening of combinatorial libraries against biological targets for identification of active ligands.
- Screening a library generally involves a binding or a functional assay to determine the extent of ligand- receptor interaction. Often, either the ligand or the receptor is immobilized on a solid surface (e.g. polymer bead or plate) and, after detection of the binding or the functional activity, the ligand is released and identified by a different means, for example, by mass spectrometry. Solid-phase screening assays offer faster isolation and identification of active analytes compared to the solution-based methods. On the other hand, the shift of combinatorial research to the creation of soluble non-peptide libraries, and limitations associated with heterogeneous assays, create a demand for a breakthrough technology for rapid and efficient screening of combinatorial libraries in solution.
- a solid surface e.g. polymer bead or plate
- Solution-phase assays are desirable to increase screening specificity, but current methodologies involve iterative processes that are long and laborious.
- electrospray ionization mass spectrometry (ESI-MS) has also been used in screening, directly or in conjunction with a solution-based screening method.
- the direct screening of combinatorial libraries by ESI-MS relies on its ability to characterize non-covalent complexes of proteins bound to ligands (i.e., to distinguish between a protein target and its ligand, even a small one) .
- mass spectrometry ⁇ can be coupled, on-line or off-line, with solution-based screening methodologies, as a final dimension for structure determination of biologically active compounds.
- Hummel-Dreyer method recently used for binding studies of small molecules to proteins, is based on size separation of the receptor protein and its ligand by gel filtration.
- RP-HPLC reversed-phase high-performance liquid chromatography
- the present method combines affinity interactions with size exclusion methods to enable rapid isolation and characterization of small molecule compounds from highly complex mixtures such as natural samples .
- the advantage of the present method's combination of techniques is that it allows one to screen pools of compounds simultaneously, instead of one compound at a time.
- the present invention provides an improved method of rapidly screening complex biological materials for affinity ligands using a unique combination of both size-exclusion separation (e.g., by gel filtration) and ultrafiltration steps, as well as mass spectrometry analysis.
- One advantage of the present method is that allows sustained, high-throughput screening, without having to replace HPLC columns for at least 7 days.
- the rapid screening method of the invention comprises the following main steps in the order given:
- step (iii) subjecting the size-excluded reaction mixture, now containing only large molecular weight materials like unbound proteins and bound L/TG complexes, to conditions conducive to L/TG complex dissociation, to yield free, small molecular weight ligand (L) and target (TG) in solution, and using a second size-exclusion medium, e.g., an ultrafiltration membrane, to isolate the free ligand from the protein target and other large molecules remaining in the size-excluded reaction mixture from step (ii) ; and
- a second size-exclusion medium e.g., an ultrafiltration membrane
- Figures 2A, 2B, and 2C show a schematic representation of an apparatus set-up for practicing an on-line embodiment of the screening method of the invention, at different timepoints in the method;
- Figures 3A-B show liquid chromatography-mass spectrometry (LC-MS) chromatograms from using an off-line embodiment of the method to screen for a ligand, acetazolamide, which binds to human carbonic anhydrase
- the invention provides an improved, high-throughput, size-exlusive method of screening complex biological material, using size-exclusion separation, ultrafiltration, and mass spectrometry. It allows the screening of several pools of compounds at one time, as well as complex mixtures and natural samples containing molecules of very different MW sizes.
- This invention uses a unique combination of steps: allowing binding of a small molecular weight, affinity ligand (L) t+ a protein target (TG) in solution; size- exclusion separation of the ligand/protein (L/TG) complex from unbound, target-inactive material; subsquent dissociation of the L/TG complex and isolation of the target-binding ligand through another size-based separation (ultrafiltration) of the small ligand from large molecules (i.e., the target and other proteins); and ligand identification using mass spectrometry (MS) alone or in conjunction with liquid chromatography (LC- MS) .
- MS mass spectrometry
- 'Small molecules or 'small molecular weight molecules are understood in the art to refer to compounds with a molecular weight of about 2,000 Daltons or less.
- the method can detect and isolate from a screened complex biological sample, target-binding ligands of about 2,000 Daltons or less, more preferably 1000 Daltons or less.
- target-binding ligands of about 2,000 Daltons or less, more preferably 1000 Daltons or less.
- small molecular weight ligands that may be identified by the present method include but are not limited to compounds within such complex biological materials as: combinatorial libraries (e.g., of peptides and the like); natural products, samples, or extracts; and mixtures of pure compounds.
- Large molecular weight compounds are generally those having a molecular weight of about 8,000, more typically 10,000 Daltons or higher. Examples include proteins as well as compexes of proteins noncovalently bound to small oelcule ligands.
- the invention provides a method of screening a complex biological sample for an affinity ligand that binds to a protein target, comprising, preferably in the order given: (1) mixing a protein target and a complex biological sample in solution to form a reaction mixture;
- the reaction mixture passes through a first size-exclusion medium that removes from the reaction mixture any small molecular weight compound having a molecular weight less than a first preset value (e.g., the first preset value may lie in the range of 5000 Daltons or less, with the cut-off being about 3000 Da)
- a first preset value e.g., the first preset value may lie in the range of 5000 Daltons or less, with the cut-off being about 3000 Da
- step (3) (4) subjecting the size-excluded reaction mixture from step (3) to conditions promoting dissociation of any ligand/target complex into free ligand and free target;
- step (4) passing the reaction mixture resulting from step (4) through a second size exclusion medium that removes from the reaction mixture any molecule larger than a second preset value;
- step (6) subjecting the reaction mixture resulting from step (5), to one of the following analyses: (a) mass spectrometry analysis or (b) liquid chromatography coupled on-line with mass spectrometry, thereby characterizing any small molecular weight ligand remaining in the reaction mixture resulting from step (5) .
- the method of the invention further comprises comparing the analytical results of step (6) with a reference standard.
- the reference standard preferably comprises the analytical results (MS or LC-MS results) of subjecting either a sample of the protein target alone or a mixture of the protein target with a non-target-binding complex biological material sample, to steps (2) -(6) of the method.
- the rapid screening method of the invention can be performed in an on-line or off-line format. Additionally, the method can further include a competitive-binding embodiment, as a control to determine whether ligand detected by the method binds to the selected protein target specifically (at the same site as a known competitive ligand) or non-specifically (at another site, e.g., by hydrophobic interactions).
- a competitive-binding embodiment as a control to determine whether ligand detected by the method binds to the selected protein target specifically (at the same site as a known competitive ligand) or non-specifically (at another site, e.g., by hydrophobic interactions).
- the target protein TG is initially incubated with a biological mixture in solution for a time period sufficient to reach equilibrium or near equilibrium binding of any ligand to LC/MS or the target (e.g., about 5-60 minutes, preferably 5-10 minutes) . Then the physical separation of an L/TG complex from unbound small molecule compounds is achieved on a size-exclusion medium, such as a gel filtration column (e.g., Pharmacia HR 10/10 columns) , which provides separation of large molecules (e.g, target protein (TG) and other proteins in the sample) and large complexes (i.e., L/TG complex) from small molecular weight compounds. Non-binding small molecules in the target/sample mixture are retained by the gel-filtration column.
- a size-exclusion medium such as a gel filtration column (e.g., Pharmacia HR 10/10 columns) , which provides separation of large molecules (e.g, target protein (TG) and other proteins in the sample) and large complexes (i.e., L/TG complex
- the size-excluded reaction mixture contains unbound TG and L/TG complex, as well as other large molecules initially present in the biological sample, all of which eluted in the void column volume.
- a solution comprising at least one organic solvent and at least one organic acid (e.g., 100 ⁇ L acetonitrite ACN solution containing 0.1% trifluoroacetic acid (TEA) ) , is added to the size-excluded reaction mixture eluted from the gel filtration column.
- TAA trifluoroacetic acid
- a second size- separation medium such as an ultrafiltration membrane having a small molecular weight cut-off, e.g., in the range of about 1,000-5000 Daltons (Da), preferably about 2,000-4,000, most preferably about 3,000 Da. Only molecules smaller than the ultrafiltration membrane's cut-off can pass through.
- this second size- separation step provides isolation of the dissociated ligand molecules from all large molecules in the screened biological sample. Small molecules remaining in the mixture after the first size-exclusion step (i.e., an affinity ligand having the desired affinity to the protein target) , pass through the ultrafiltration membrane, while the membrane surface retains all large molecules (i.e., target and other large MW molecules originating from the sample) .
- the remaining, ultrafiltered reaction mixture or material is subjected to MS alone or by liquid chromatography coupled on-line with MS (LC-MS), to analyze any target-binding ligand isolated by the ultrafiltration step.
- MS liquid chromatography coupled on-line with MS
- Figures 2A, 2B, and 2C depict the method and instruments at different time points .
- Figure 2A shows the isolation and trapping of large molecular weight biomolecules in the loop.
- Figure 2A shows the transfer of the isolated large molecular weight biomolecules onto an ultrafiltration membrane, together with application of conditions conducive to ligand/target complex dissociation, the separation of any dissociated ligand from the target, and deposition of the isolated ligand on a reverse-phase LC column.
- Fig. 2C shows the characterization of the ligand, after ultrafiltration, by LC-MS analysis.
- the mixture is injected into the ' SEC, and large molecules come out first, non-retained by the SEC stationary phase of the SEC. Small molecules are retained in the pores of the SEC stationary phase.
- the chromatographic peak corresponding to large molecules is forwarded into a sample loop ( Figure 2a) , and the remaining flow from the SEC is then diverted into waste.
- the solution containing organic solvent (e.g., acetonitrite (ACN) ) acid (TFA) ) and organic acid (e.g., acetic acid or trifluoroacetic acid (TFA) ) is then added to the sample in the loop, in order to provide conditions for L/TG complex dissociation.
- organic solvent e.g., acetonitrite (ACN)
- organic acid e.g., acetic acid or trifluoroacetic acid (TFA)
- sample is transferred into the chamber of the ultrafiltration membrane ( Figure 2b) .
- the sample is pumped through the ultrafiltration membrane directly into an MS or into an LC column for further LC-MS analysis of the released ligand (Figure 2c) . Only the released small molecule ligand is able to pass through the membrane, and is subsequently identified by MS or LC-MS.
- Another embodiment of the invention combines the use of competitive binding along with the described screening assay.
- Utilization of a known competitive ligand (CL) that binds to a selected target allows determination of whether the active ligand extracted from a biological mixture is bound to a specific binding site or known site of the target protein.
- a CL that binds to a known S.H. is added to the reaction mixture of target and biological sample containing a small molecular weight affinity ligand at the first, mixing and incubation stage, and during mass spectrometry, both L and CL signals are monitored.
- the reference standard for determining whether a screened sample contains a specific target-binding ligand comprises the MS or LC-MS results of subjecting a mixture of the protein target and the known competitive ligand, in the absence of any other target-binding ligand, to steps (2) - (6) .
- Stock solutions of candidate compounds used for assaying their binding to a protein target e.g., human carbonic anhydrase II (CAII)
- a protein target e.g., human carbonic anhydrase II (CAII)
- CAII human carbonic anhydrase II
- the target protein stock solution can be prepared by dissolving and diluting the target in buffer A.
- Binding mixtures consisted of 50 ⁇ L target stock with 5 ⁇ L of the candidate compounds stock. The reaction mixtures are incubated at room temperature for 1 hr, after gentle mixing by pipette.
- the gel filtration columns e.g., Sephadex G25 fast-desalting spin columns or Pharmacia HR 10/10 columns, in an off-line format of the method) are handled according to the manufacturer' s instructions.
- G25 columns are first washed with 100 ⁇ L water and centrifuged at 3000 rpm (-900 g) at 4°C for 5 min. The reaction mixtures are then loaded on the columns and centrifuged as before. Unbound compounds should be retained on the columns, while any protein, bound or unbound to low molecular weight candidate compounds, should flow through the column. Protein complexes in the flow-through of spin columns are collected. The protein-ligand complex in the eluted fraction is then denatured by adding 100 ⁇ L ACN solution containing 0.1% trifluoroacetic acid (TFA) (Sigma Chemical CO.), incubating for 5 min at room temperature, and vortexing for 15 seconds. The denatured protein is removed by centrifugation at 10 000 g for about 30 seconds.
- TFA trifluoroacetic acid
- the supernatant liquid resulting from the gel filtration protocol is then applied to the second size- exclusion separation medium, preferably an ultrafiltration membrane.
- ultrafiltration membranes having a 20,000, 10,000, or 3,000 Dalton cutoff sold as Microcon-3, by Amicon, Beverly, MA, USA. Preferred are membranes with a cut-off of about 10,000 Da (which lets pass about 1% of molecules over 30,000 Da), or less. A membrane with a 3,000 Da cut-off lets pass only about 0.1% of materials over 30,000 Da.
- Most preferred are ultrafiltration membranes that can withstand 100% organic solvents (e.g., ACN) and organic acids (e.g., TFA or acetic acid) .
- Ultrafiltration membranes having other ⁇ small molecular weight cut-offs e.g., within the range of about 1000-5000 Da, preferably 2000-4000 Da, are also available commercially (e.g., All Filtron Co.,
- the ultrafiltered liquid (containing any candidate ligand that bound to the target) is then analyzed by mass spectrometry alone or by liquid chromatography coupled to mass spectrometry, for the presence of released small molecular weight compounds, after lyophilization and re-suspension in small volumes.
- Mass spectrometry Analysis can be performed on, e.g., ion trap triple guadrupole mass spectrometers LCQ (Thermo-Quert Corporation, San Jose, CA, USA) or APCI.
- the electrospray voltage is generally maintained in the range of about +4.5-4.75 kV.
- Ion optics settings are optimized on the day of the analysis to provide the maximum efficiency of ion to the detector.
- the effective mass range is generally from m/z 150 to m/z 700 at a rate of about 1 s/scan.
- Liquid chromatography For example, samples can be introduced through a HP1100 (Hewlett Packard Paulo Alto, CA USA) chromatography operating in the gradient mode at a flow rate of 1 ml/min.
- An Inertsil C18 base-deactivated microbore column (4.6 mm x 10 cm) from MetaChem Technologies (Torrance, CA, USA) is used for sample separation.
- the mobile phase gradient is Milli-Q H 2 0 + ACN 90/10 (v/v) to a H 2 0 + ACN 0/100 (v/v) in 15 minutes.
- Samples are introduced through a Rheodyne Model 7125 injector (Cotati, CA, USA) with a 10 ⁇ L external loop.
- the sample injection volumes are generally 1-19 ⁇ L.
- CAII human carbonic anhydrase II
- NS natural sample
- CAII e.g., inert fungal extract
- AZ acetazolamide
- Size- exclusion chromatography was performed using a Pharmacia HR 10/10 column at 4 ml/min using a mobile phase of 200 mM ammonium acetate, pH 7.0.
- the excluded volume containing the target protein (CAII) with bound ligand (AZ) elutes from the column in about 0.6-0.7 minutes under such conditions.
- Figure 3A shows an LC-MS chromatogram of a blank experiment, where about 20 uM CAII alone was passed through the entire method of the invention.
- Figure 3B depicts the LC-MS chromatogram from performing the same with that amount of CAII incubated with the inactive natural sample (NS) alone. There are no additional peaks detected, compared to the blank experiment, which indicates the absence of targe-binding ligands from the inactive biological sample.
- Figure 3C [Orig. Fig. lc] shows the LC-MS results from performing the same screening process with a natural (NS) sample now containing about 10 uM of the CAII-binding ligand, acetazolamide (AZ) .
- target CAII (TG) peaks are a series of chromatographic peaks having retention times of 10.5 min. and longer.
- the ligand AZ peak has a retention time of 5.9 min.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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CA002362052A CA2362052A1 (en) | 1999-02-12 | 2000-02-11 | High throughput size-exclusive method of screening complex biological materials for affinity ligands |
EP00908605A EP1151301A4 (en) | 1999-02-12 | 2000-02-11 | High throughput size-exclusive method of screening complex biological materials for affinity ligands |
JP2000598857A JP3788909B2 (en) | 1999-02-12 | 2000-02-11 | A high-throughput size exclusion method for screening complex physiological materials for affinity ligands |
US09/920,435 US7179592B2 (en) | 1999-02-12 | 2001-08-01 | Size-exclusion-based extraction of affinity ligands and active compounds from natural samples |
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US11996699P | 1999-02-12 | 1999-02-12 | |
US60/119,966 | 1999-02-12 |
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US09/920,435 Continuation-In-Part US7179592B2 (en) | 1999-02-12 | 2001-08-01 | Size-exclusion-based extraction of affinity ligands and active compounds from natural samples |
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WO2000047999A1 true WO2000047999A1 (en) | 2000-08-17 |
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JP (1) | JP3788909B2 (en) |
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WO (1) | WO2000047999A1 (en) |
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US11071927B2 (en) | 2017-11-15 | 2021-07-27 | Arup Laboratories, Inc. | Separating and quantifying unbound target analytes from biological samples |
CN112649517B (en) * | 2019-10-12 | 2021-10-15 | 中国科学院大连化学物理研究所 | Method for screening target protein ligand from organism metabolite |
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- 2000-02-11 CA CA002362052A patent/CA2362052A1/en not_active Abandoned
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WO2001062688A3 (en) * | 2000-02-25 | 2002-03-14 | American Home Prod | Methods of structure-based drug design using ms/nmr |
WO2001062688A2 (en) * | 2000-02-25 | 2001-08-30 | Wyeth | Methods of structure-based drug design using ms/nmr |
EP1344060A2 (en) * | 2000-11-17 | 2003-09-17 | Alfred E. Slanetz | Process for determining target function and identifying drug leads |
EP1344060A4 (en) * | 2000-11-17 | 2004-12-22 | Alfred E Slanetz | Process for determining target function and identifying drug leads |
DE10125258A1 (en) * | 2001-05-23 | 2003-01-09 | November Ag Molekulare Medizin | Method for determining the binding behavior of ligands that specifically bind to target molecules |
WO2002095403A3 (en) * | 2001-05-23 | 2003-02-13 | November Ag Molekulare Medizin | Method for determining the binding behaviour of ligands which specifically bind to target molecules |
WO2002095403A2 (en) * | 2001-05-23 | 2002-11-28 | november Aktiengesellschaft Gesellschaft für Molekulare Medizin | Method for determining the binding behaviour of ligands which specifically bind to target molecules |
US7074334B2 (en) | 2001-05-23 | 2006-07-11 | Klaus Wanner | Method for determining the binding behavior of ligands which specifically bind to target molecules |
EP1578781A2 (en) * | 2002-05-17 | 2005-09-28 | Slanetz, Alfred E. | Process for determining target function and identifying drug leads |
EP1578781A4 (en) * | 2002-05-17 | 2007-05-30 | Alfred E Slanetz | Process for determining target function and identifying drug leads |
WO2006015796A1 (en) * | 2004-08-09 | 2006-02-16 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for high-throughput screening of test molecules binding to target molecules |
CN102818868A (en) * | 2012-08-27 | 2012-12-12 | 浙江大学 | Screening method of active ingredients in complex natural product and its application |
CN102818868B (en) * | 2012-08-27 | 2013-11-20 | 浙江大学 | Screening method of active ingredients in complex natural product and application thereof |
Also Published As
Publication number | Publication date |
---|---|
US7179592B2 (en) | 2007-02-20 |
EP1151301A4 (en) | 2004-07-14 |
JP2002541435A (en) | 2002-12-03 |
US20020052006A1 (en) | 2002-05-02 |
CA2362052A1 (en) | 2000-08-17 |
JP3788909B2 (en) | 2006-06-21 |
EP1151301A1 (en) | 2001-11-07 |
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