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WO1999005309A1 - Criblage multi-cibles de banques moleculaires par sepctrometrie de masse - Google Patents

Criblage multi-cibles de banques moleculaires par sepctrometrie de masse Download PDF

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Publication number
WO1999005309A1
WO1999005309A1 PCT/US1998/015112 US9815112W WO9905309A1 WO 1999005309 A1 WO1999005309 A1 WO 1999005309A1 US 9815112 W US9815112 W US 9815112W WO 9905309 A1 WO9905309 A1 WO 9905309A1
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Prior art keywords
maldi
library
mass spectrometer
compounds
control
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PCT/US1998/015112
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English (en)
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Yinliang F. Hsieh
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Millennium Pharmaceuticals, Inc.
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Priority to AU84147/98A priority Critical patent/AU8414798A/en
Publication of WO1999005309A1 publication Critical patent/WO1999005309A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66

Definitions

  • the invention relates to methods for high throughput screening of molecular libraries by mass spectrometry . Specifically, the invention relates to methods for simultaneously identifying compounds that inhibit multiple enzyme targets.
  • this method is applicable only to the screening of one receptor at a time. The method would not be suitable if multiple receptors were present, as the determination of which ligand was bound to which receptor prior to elution would be difficult. Furthermore, if the ligand-receptor interaction were instead covalent, it would be difficult to identify any of the ligands because of the much larger molecular weight of the associated receptors.
  • Another screening method is described in Nedved et al . (Anal . Chem . , 8 . :4226-4236 , 1996), in which a mixture is formed that contains an antibody and a library of drug candidates. In this system, the mixture is passed through a protein G immunoaffinity column to remove unbound drugs, and then the non-covalent antibody/drug complexes are eluted from a reverse phase column and subjected to ESI -MS.
  • MS mass spectrometry
  • the new methods can be used to screen for antagonists of enzyme catalyzed reactions.
  • the new methods allow both the drugs and the targets, e.g., enzymes, to be rapidly and accurately identified.
  • the invention features a method of screening a molecular library of compounds for individual compounds that affect (e.g., inhibit) the ability of one or more of a plurality of target molecules (e.g., enzymes or other polypeptides) to catalyze the conversion of corresponding peptide substrates (e.g., peptides, polypeptides, or proteins) to products, or the reaction of a substrate with a target molecule to yield a product .
  • target molecules e.g., enzymes or other polypeptides
  • the method includes the steps of combining each compound in the library with the plurality of target enzymes to form a set of inhibition mixtures; adding to each of the inhibition mixtures a peptide substrate corresponding to each target enzyme to form a set of experimental mixtures (e.g., by mixing the components at the time of use); obtaining a control mixture including the plurality of target enzymes and the corresponding peptide substrates; sampling the control mixture and the set of experimental mixtures at a first time point to obtain control aliquots and experimental aliquots; analyzing the control and experimental aliquots using a mass spectrometer to produce mass spectra (i.e., qualitatively or quantitatively) ; and comparing the mass spectrum of the control aliquot to the mass spectra of the experimental aliquots to detect individual compounds in the library that affect (e.g., inhibit) the ability of one or more of the plurality of target molecules (e.g., enzymes) to catalyze the conversion of corresponding peptide substrates to products.
  • a "corresponding" substrate is a substrate (e.g., a peptide or protein) that normally reacts with, or is converted into a product by, a given target molecule, e.g., an enzyme, under control conditions. Second aliquots of the control and experimental mixtures can be obtained at a second time point.
  • a substrate e.g., a peptide or protein
  • the new method can be used to screen the molecular library of compounds for individual compounds that inhibit the ability of only one of the plurality of target enzymes to catalyze the conversion of a corresponding peptide substrate to a product, and that do not inhibit the ability of the remaining target enzymes to catalyze the conversion of the corresponding peptide substrates to products.
  • Each of the reaction mixtures can include a plurality of synthetic, semi-synthetic, or natural compounds. The aliquots can be chromatographed prior to the analyzing step.
  • the mass spectrometer can be a matrix-assisted laser desorption ionization (MALDI) mass spectrometer, a electrospray ionization (ESI) mass spectrometer, a liquid chromatograph mass spectrometer (LC-MS; can be quantitative), a liquid chromatograph tandem mass spectrometer (LC-MS/MS; can be quantitative) , a capillary electrophoresis mass spectrometer (CE-MS; can employ microchannel chips) , or a capillary electrophoresis tandem mass spectrometer (CE-MS/MS; can employ microchannel chips) .
  • MALDI matrix-assisted laser desorption ionization
  • ESI electrospray ionization
  • the method additionally includes the steps of combining a MALDI matrix with the aliquots from the control mixture to form MALDI control samples; combining a MALDI matrix with the aliquots from the experimental mixtures to form MALDI library samples; and analyzing the MALDI control samples and MALDI library samples using the mass spectrometer to produce mass spectra.
  • the MALDI matrix can be ⁇ -cyano-4- hydroxycinnamic acid ( ⁇ CHCA) or 6-aza-2-thiothymine (ATT) , for example.
  • a "library” is a heterogeneous multiplicity of compounds that can be combined together in a single mixture, divided into several mixtures, or separated into discrete, single- component fractions.
  • a library can include any compounds, including, but not limited to, oligopeptides (e.g., derived from natural or synthetic amino acid monomers, or both) , polypeptides, proteins, peptide nucleic acids (PNA) , oligonucleotides (e.g., natural or synthetic DNA or R A) , small molecules (i.e., including any natural products or synthetic compounds) , or any combination of these components.
  • a library can be dissolved or suspended in a solvent, buffer, or other carrier, or can exist neat.
  • a library can also include a natural product, such as a bodily fluid, organic tissue, or secretion of an organic tissue, including blood, skin, urine, sweat, milk, liver tissue, kidney tissue, hair, thymus tissue, pancreatic tissue, brain tissue, a cell culture (e.g., bacterial, fungal, or yeast; the culture can additionally include a growth medium) , a cell extract, the supernatant of a centrifuged tissue suspension, plant matter, or other organic matter; the tissue can be processed or can be used in a natural state.
  • the components of a library can be soluble or insoluble in a given screening mixture.
  • the library can include 1, 10, 100, or more compounds. Additionally, compressed libraries can be used.
  • the target molecules can be enzymes (e.g., proteases, peptidases, phosphatases, exonucleases, endonucleases, glycosidases, transferases, kinases, and polymerases) , or other proteins or polypeptides.
  • “Multiple target screening” refers to the simultaneous presence of more than one target molecule in an experimental reaction mixture. In each screening reaction, there can be a single compound or a multiplicity of compounds that are screened against the plurality of target molecules.
  • the target molecule can be any molecule (such as an enzyme) that catalyzes the conversion of a corresponding substrate to a product, or that reacts with a corresponding substrate to yield a product .
  • Advantages of the new methods include the ability to determine the specific location on the target where the compound binds, the capacity of the new methods for high throughput screening, and the ability to simultaneously screen a library of compounds against multiple targets. Additionally, because both the substrates and products of enzymatic reactions are detected in the new methods, it is not a problem if multiple compounds from the library inhibit the same target; the library can be deconvoluted later to determine which compounds are acting as inhibitors.
  • Fig. 1 is a mass spectrum of a substrate mixture containing angiotensin I, and substrates for N-myristyl- transferase (NMT) and protein tyrosine kinase (PTPase) .
  • Fig. 2 is a mass spectrum of a reaction mixture that included both the substrate mixture of Fig. 1 and an enzyme mixture containing angiotensin converting enzyme (ACE) , NMT, and PTPase, sampled 15 minutes after mixing.
  • Fig. 3 is a mass spectrum of the reaction mixture of Fig. 2 with ammonium vanadate added, sampled 15 minutes after adding the enzyme/inhibitor mixture to the substrate mixture.
  • Fig. 4 is a mass spectrum of the reaction mixture of Fig. 2 with lisinopril added, sampled 15 minutes after adding the enzyme/inhibitor mixture to the substrate mixture .
  • Fig. 5 is a mass spectrum of the reaction mixture of Fig. 2 with N-myristyltransferase inhibitor (NMTI) added, sampled 15 minutes after adding the enzyme/inhibitor mixture to the substrate mixture.
  • NMTI N-myristyltransferase inhibitor
  • Fig. 6 is a mass spectrum of the reaction mixture of Fig. 2 with ammonium vanadate, lisinopril, and NMTI added, sampled 15 minutes after adding the enzyme/inhibitor mixture to the substrate mixture.
  • the invention is a highly effective mass spectrometric method for the identification and characterization of compounds that bind to a target molecule, such as an enzyme, present in a mixture of targets.
  • the method is based on the detection of substrates and products of an enzymatic reaction catalyzed by the target enzymes, or the substrates and products of any reaction affected by the target molecules, and not necessarily the aforementioned compounds themselves.
  • a mixture of target enzymes and their respective substrates are obtained.
  • the enzymes are combined with the members of a library of compounds in containers (e.g., a combinatorial drug library or a library of natural products, contained, for example, in the wells of a 96-well plate) .
  • the entire mixture of enzymes is added to each container, while the individual members or groups of members of the library are added to distinct containers, in the same or different concentrations.
  • Each well can contain 1, 10, 20, 50, 100, or even 500 or more members.
  • a library of up to 9600 compounds can be screened against an enzyme library in a single 96-well plate by loading each well with 100 library members.
  • reaction mixtures At least one reaction mixture is set up with the target enzyme and substrate mixtures, but without the inclusion of any members of the molecular library. Such a mixture is referred to as a control mixture and is used to monitor the native activity of the enzymes (or other target molecules) in the absence of any compounds other than the substrates .
  • Samples can be drawn from the reaction mixtures at various times (e.g., at 5, 10, and 20 minutes) after the addition of the substrates to the enzymes.
  • the samples can be quenched, e.g., by addition of a low pH solution, and analyzed by mass spectrometric analysis.
  • MALDI MS Matrix-Assisted Laser-Desorption Ionization Mass Spectrometry
  • MALDI MS matrix-assisted laser- desorption ionization
  • the reactions proceeding in the sample are quenched, for example, by addition of a low-pH matrix compound, such as a 10 mg/ml solution of a- cyano-4-hydroxycinnamic acid ( ⁇ CHCA) in a 1:1 mixture of methanol and water or a 1:1 mixture of acetonitrile and water.
  • a low-pH matrix compound such as a 10 mg/ml solution of a- cyano-4-hydroxycinnamic acid ( ⁇ CHCA) in a 1:1 mixture of methanol and water or a 1:1 mixture of acetonitrile and water.
  • the matrix includes a conjugated, amphoteric molecule that not only quenches the reactions, but can also absorb laser energy and transform it into excitation energy (e.g., via electron-phonon coupling) in the molecules trapped within the matrix, disintegrating, and desorbing at least some of the top molecular layers of the sample.
  • excitation energy e.g., via electron-phonon coupling
  • low-pH matrix molecules can also be used, including: sinapinic acid, 2 , 5-dihydroxybenzoic acid (2,5-DHB), 2- (4-hydroxyphenylazo)benzoic acid (HABA) , anthranilic acid, 3-hydroxypicolinic acid, picolinic acid, and nicotinic acid.
  • HABA 2- (4-hydroxyphenylazo)benzoic acid
  • anthranilic acid 3-hydroxypicolinic acid
  • picolinic acid nicotinic acid
  • nicotinic acid nicotinic acid.
  • these matrix molecules are better suited for different ones of peptides, proteins, and oligonucleotides .
  • ⁇ -CHCA is suitable for analysis of peptides.
  • 3-hydroxypicolinic acid and picolinic acid are both satisfactory.
  • Non-covalent complexes held together via hydrogen bonds, van der aals forces, or ionic interactions, dissociate in the presence of the low-pH MALDI matrices.
  • Neutral MALDI matrices such as 6-aza-2-thiothymine (ATT), dithranol, and 2 , 4 , 6-trihydroxyacetophenone (THAP) , however, can be used to analyze non-covalent complexes .
  • a portion of the quenched reaction mixture is transferred to a metal MALDI plate (also called a "MALDI probe") that fits within a MALDI MS instrument. In general, any metal plate adapted to fit into the instrument can be used.
  • the plate can have a number of wells drilled into it to accept the quenched reaction mixture. Plates without wells can also be used; the samples can simply be spotted onto the surface of a flat, polished plate.
  • Each MALDI plate can have from about a hundred, microliter-scale samples to several thousand, picoliter-scale samples. In some cases, the plates are precoated with matrix solution, allowing the quenching and spotting steps to be combined. Precoated plates are commercially available, for example, from Lab Connection Co. (Marlborough, MA) .
  • MALDI has sufficiently high resolution that the isotopic composition of a sample can be monitored. This provides an internal standard that can be used for better mass accuracy in complex sample mixtures.
  • MALDI also promotes high throughput screening, because only 15-20 seconds are typically required per sample for data acquisition, and multiple target enzymes can be screened simultaneously.
  • the laser ionizes the compound and the matrix.
  • the MALDI plate is pulsed with a current (e.g., 20 kV) for a fraction of a second (e.g., about 50 to 100 ns) .
  • the pulse results in the desorption of at least some of the top molecular layers of the sample, which then fly toward a detector.
  • the time of flight is generally about 100 to 200 ⁇ s.
  • the sample is detected by the detector, which sends a signal to a computer for data collection and processing. The entire process takes about one-third of a second.
  • the ionization/desorption process can be repeated 8, 16, 32, 64, 128, or more times for each sample. Accumulation of 32 data points, for example, would take just under 11 seconds at a rate of one scan every third of a second.
  • Electrospray Ionization Mass Spectrometry ESI -MS
  • Other mass spectrometry ionization techniques such as electrospray, ion spray (pneumatically assisted electrospray) , and nanospray allow the formation of multiply charged gas phase macromolecular ions directly from solution at atmospheric pressure via protonation and ion evaporation. These techniques, for example, provide a rapid, straightforward method for the analysis of target molecules with bound drugs.
  • ESI The procedure used for screening with ESI is similar to that used for MALDI. Unlike MALDI, however, ESI does not require the use of a matrix compound.
  • Aliquots from the control and experimental mixtures can be injected directly into the ESI-MS. Although not necessary, it can be desirable to quench the reactions prior to analysis. Quenching can be accomplished, for example, by using a solution of formic acid or trifluoroacetic acid.
  • ESI-MS has the additional advantage of being highly quantitative and ESI data is sufficiently accurate to be used, for example, in FDA drug trials.
  • MALDI is suitable for monitoring molecules having molecular weights over 500; the MALDI matrix molecules currently used all have a molecular weight lower than 500 and thus do not interfere with the mass-to-charge ratio (m/z) of larger molecules.
  • the analyte be larger than m/z 500, nearly all compounds are amenable to analysis with MALDI .
  • examples of such compounds include the substrates of numerous enzymes, such as glycosidases, proteases, nucleases, phosphatases, kinases, and other transferases .
  • Some specific examples of substrates include angiotensin I, PTPase substrates, and NMT substrates.
  • ESI-MS is an excellent quantitation method for monitoring the appearance and disappearance of substrates, products, and even noncovalent intermediates for performing kinetic analyses of enzymatic reactions.
  • the beam of the MALDI laser typically has a diameter of 100 to 200 ⁇ m. Consequently, the sample wells on a MALDI plate can be of a similar size, and are generally in the range of 100 ⁇ m to about 2 mm.
  • MALDI plates having up to 100, 4,000, or even more samples, e.g., on a 5 cm x 5 cm sample area can be used.
  • the sample volume required for this high-density spotting is on the order of microliters ( ⁇ l; e.g., for 100 sample plates) down to picoliters (pi; e.g., for 4,000 well plates) .
  • ⁇ l microliters
  • pi picoliters
  • the spotting of so many of such tiny samples onto a single plate is facilitated by a robot that can transfer the samples from the reaction containers to the MALDI plate.
  • An advantage of spotting several thousand samples onto a single plate is that hundreds of thousands of compounds can be screened against multiple targets in several hours.
  • the rate of throughput is limited only by the rate of automated sample introduction, as the MALDI mass spectrometer can accumulate data points for each sample and then prepare to analyze the next sample, all within just a few seconds.
  • Commercial mass spectrometry software packages to control both lasing and the movement of the laser are available from many of the manufacturers of MALDI mass spectrometers (e.g., VoyagerTM from PerSeptive Biosystems, Inc., Framingham, MA).
  • the use of compressed libraries, wherein each well of the reaction plate could have up to 50 compounds, or more, also enables faster screening.
  • the new methods provide an advantage over other methods in the screening of compressed libraries. For example, the compounds contained in whichever wells yield multiple "hits" can be screened independently to identify exactly which compounds are responsible for the activity. In some cases, several compounds in a single reaction mixture can have activity against the same target . Unlike screening methods that rely on the detection of a ligand after it is eluted away from its receptor, the new methods allow identification of all compounds in a mixture that bind to the target, not just the strongest or the fastest. Robotics
  • a robot can be used to introduce the samples onto the MALDI plate.
  • Suitable robots are available from, for example, Tecan U.S., Inc. (Durham, NC) , and can be controlled using available software and standard techniques. Robots can also be custom made to fit a particular application (e.g., for high density spotting. For low density spotting (i.e., about 100 samples per plate) each sample volume can be about 1 ⁇ l .
  • a Tecan robot (GenesisTM Model 1020) can be used to transfer a solution containing multi-enzyme targets to a 96-well sample mixing plate.
  • a compressed combinatorial library or natural library (e.g., from bacterial or fungal fermentation and extraction) is selected and robotically mixed with the multi-enzyme target solution in the mixing plate.
  • a multi-enzyme substrate mixture is transferred and combined with the enzyme-drug library in the mixing plate by the robot.
  • a sample of the enzyme-library- substrate mixture is taken out and mixed with a MALDI matrix solution in a second 96-well plate by the robot. The robot dispenses a 1 ⁇ L aliquot of the sample mixture with the matrix solution onto a MALDI plate.
  • each sample volume is on the order of one picoliter.
  • a robot capable of spotting several thousand samples onto a MALDI plate for qualitative analysis requires an error tolerance of 100 to 200 ⁇ m.
  • the robot transfers a solution containing multi-enzyme targets to a 96-well sample mixing plate.
  • a compressed combinatorial library or natural library is selected and robotically mixed with the multi-enzyme target solution in the mixing plate.
  • a multi-enzyme substrate mixture is transferred and combined with the enzyme-drug library in the mixing plate by robot.
  • the robot spots a sample of the enzyme-drug-substrate mixture onto a MALDI plate precoated with the matrix solution.
  • the new methods can be combined with enzymatic digestion (e.g., with trypsin, chymotrypsin, or subtilisin) to determine the specific location of the binding site if a drug covalently binds to a target.
  • enzymatic digestion e.g., with trypsin, chymotrypsin, or subtilisin
  • the mass spectrum obtained after trypsin digestion of a target-drug complex can be compared with the corresponding mass spectrum obtained after digestion of the target itself.
  • the peaks that differ between the two spectra indicate the precise segment to which the drug binds .
  • the sample can be analyzed with, for example, ESI liquid chromatography tandem mass spectrometry (LC-MS/MS) . See, for example, Anal . Chem . , 68.:455-462 (1996).
  • LC-MS/MS liquid chromatography tandem mass spectrometry
  • Time course studies can be carried out with the new methods. For example, time course studies can be used to evaluate the product-to-substrate ratio at various time points, relative to a control sample, to provide an understanding of the kinetic inhibition of the enzymes by the drugs. The corresponding inhibition constants can be thereby determined. Time course studies can also be used to ascertain the time required for partial or total inhibition. Finally, the pharmacological kinetic behavior of the drug-enzyme system can be determined via time course studies. Similarly, the new methods can be used in concentration/ effect studies. By collecting mass spectra corresponding to various quantities of a single compound in a series of reaction mixtures, dosage information can be derived.
  • Example 1 Screening of a Molecular Library for Inhibitors of Angiotensin Converting Enzyme (ACE) by MALDI Angiotensin converting enzyme (ACE) is an important target in research aimed at the design of anti- hypertensive drugs.
  • ACE Angiotensin Converting Enzyme
  • This example illustrates a method for identifying an inhibitor of ACE from a molecular library. The procedure employed in this example can also be extended to the simultaneous screening of multiple targets .
  • Sample preparation 1 ⁇ l samples were removed from each well at various time points (i.e., 0, 5, 10, 20, 30, and 50 minutes) .
  • 1 ⁇ l of a MALDI matrix solution (1.0% ⁇ CHCA and 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile/50% water solvent mixture) was added to the sample, thereby quenching the reaction.
  • 1 ⁇ l of the resulting solution was applied with a micropipettor to the surface of a stainless-steel 100-well MALDI plate (PerSeptive Biosystems, Inc., Framingham, MA).
  • the plate was then air-dried and inserted into the sample introduction port of the VoyagerTM Elite BiospectrometryTM MALDI time-of-flight (TOF) mass spectrometer (PerSeptive Biosystems, Inc., Framingham, MA).
  • MALDI -TOF MS analysis The Voyager was operated in reflector mode using the delayed extraction function mode with an accelerating voltage of 20 kV from the ion source; these conditions resulted in better resolution compared with the continuous extraction function mode.
  • the software packaged with the Voyager was used for controlling the laser and for collecting and analyzing the data.
  • the VoyagerTM-Elite BiospectrometryTM Workstation with Voyager-DETM technology can be operated in continuous or delayed extraction (DE) mode.
  • continuous extraction mode accelerating voltage is continuously applied; a potential gradient exists when the sample is ionized; and ions are immediately accelerated.
  • delayed extraction mode a potential gradient does not exist when the sample is ionized (i.e., the sample plate and grid are at similar potentials) ; the energy of the ions is stabilized before acceleration; and accelerating voltage is pulsed after the potential gradient is applied and the ions are accelerated.
  • Voyager-DETM (delayed extraction) technology allows control of field strength and delays in the acceleration of ions, thereby minimizing ion energy loss due to collision and extraction field strength.
  • ions are formed in a weak electrical field, and then extracted by applying a high voltage pulse after a predetermined time delay.
  • Delayed extraction of ions improves performance over that obtained with continuous extraction of ions by improving resolution and mass accuracy. It also improves the quality of MALDI mass spectra by suppressing matrix background, reducing chemical noise, and minimizing the effect of laser intensity on overall performance.
  • the samples were ionized with 128 pulses from a 337 nm nitrogen laser, using the Voyager's oscilloscope to rapidly acquire data.
  • the resulting signal was digitized at 2 GHz frequency (i.e., 0.5 ns resolution) and the data was downloaded to the Voyager's data system.
  • the peptides were isotopically resolved.
  • Results The MS spectrum for the "control" sample in the second well showed the disappearance of the angiotensin I (DRVYIHPFHL; SEQ ID NO:l; MW 1296.68) and the concomitant appearance of the product, angiotensin II (DRVYIHPF; SEQ ID NO : 2 ; MW 1046.52).
  • the predominant species was the angiotensin I, at m/z 1296.68. Smaller, but still finite, quantities of other species were also present at m/z 931.47, 1046.52, and 1181.65.
  • the peak at 1181.65 corresponds to an impurity present in the angiotensin I solution (RV ⁇ IHPFHL; SEQ ID NO: 3) ; the impurity is identical to angiotensin I (SEQ ID NO:l) except that it lacks the N-terminal aspartic acid residue.
  • the peak at 1046.52 is angiotensin II (SEQ ID N0:2), as described above.
  • the peak at 931.47 (RVYIHPF; SEQ ID NO: 4) is the conversion product of the impurity; it is thus identical to angiotensin II (SEQ ID NO: 2), except that it lacks the N-terminal aspartic acid residue.
  • Aliquots were taken from the control well at 0, 5, 10, 20, 30, and 50 minutes. In the mass spectra of these aliquots, the intensity of the angiotensin II (SEQ ID NO: 2) peak increased over the first 20 minutes as the intensity of the angiotensin I (SEQ ID NO:l) peak declined. At 20 minutes, nearly all of the starting material had been converted. At 30 and 50 minutes, there was little change from the 20 minute time point. In the well containing the experimental library, the transformation was slowed significantly.
  • the inhibitors were screened individually against ACE.
  • the transformation was significantly slowed by the captopril relative to the reaction in the control well.
  • the intensity of the angiotensin I peak decreased and the intensity of the product peaks increased over the 30 minute period, but approximately 50% of the starting material remained after 30 minutes.
  • the reaction was partially inhibited by captopril relative to the control sample .
  • Example 2 Simultaneous Screening of Multiple Targets by MALDI MS
  • ACE N-myristyltransferase
  • PTPase protein tyrosine kinase
  • Buffer A buffer solution was prepared (pH 7.4), containing 20 mM Tris HC1 and 3 mM dithiothreitol .
  • Enzyme mixture A 2:1:1 mixture of ACE (final concentration, 305 fmol/ ⁇ l) , PTPase (0.39 fm/ ⁇ l) , and NMT (1.98 pmol/ ⁇ l) was prepared in the buffer solution.
  • Fig. 1 is a mass spectrum (MS profile) of the mixture of the three substrates (myristyl-CoA is not a substrate, but a cofactor of NMT) , at mass-to-charge ratios (m/z) of 801.35 (peptide; SEQ ID NO:6), 1296.68 (angiotensin I; SEQ ID NO:l), and 1702.88 (PTPase substrate; SEQ ID N0:5).
  • the peaks at m/z 1181.66 and 1587.81 corresponded, respectively, to impurities introduced along with the angiotensin I (SEQ ID NO: 3) and the PTPase substrate (TRDIYETYYRK; SEQ ID NO: 8) .
  • Fig. 2 is the MS profile of the reaction mixture of well 1 after 15 minutes. All three substrates appeared to have been fully consumed. Four new peaks developed, corresponding to the products formed by reaction of the enzymes with the substrates and impurities, at m/z 1011.58 (N-myristyl-GNAASARR-NH 2 ; SEQ ID NO:9), 931.45 (SEQ ID NO : 4 ; product formed by reaction of the impurity in the angiotensin I), 1046.51 (SEQ ID NO:2; angiotensin II), and 1622 (TRDIYETDYYRK; SEQ ID NO:10; dephosphorylated tyrosine-bearing peptide). It was also noted that not all of the angiotensin I had been consumed within the 15 minutes.
  • each of the substrates was independently analyzed by MS before and after addition of the corresponding enzymes.
  • the NMT substrate was analyzed by MS before (m/z 801.55) and after addition of NMT. Addition of NMT caused the substrate to be converted to a product of MW 1011.78 (SEQ ID NO: 9) .
  • the PTPase substrate was analyzed before (m/z 1703.05, impurities at 1588.00 and 1608.40) and after addition of PTPase.
  • the starting materials in this case became products having molecular weights of 1623.11 (SEQ ID NO:10), 1508.04, and 1606.41, respectively.
  • angiotensin I was analyzed before (m/z 1296.68, impurity at 1181.63) and after (m/z 1046.49 and 931.44, respectively) addition of ACE.
  • Fig. 6 indicates that there was no conversion of the ACE and NMT substrates (m/z 801, 1296, and 1181) , whereas the PTPase substrates (m/z 1702) reacted completely.
  • the impurity introduced with the PTPase substrate (m/z 1587) also reacted completely, producing a product of m/z 1507 upon loss of a phosphate group .
  • LC-MS Liquid chromatography followed by mass spectrometry
  • a calibration curve of each free substrate is constructed by injecting various concentrations of the substrates into the LC-MS, and then calculating the area under the total ion chromatographic peaks resulting from the LC-MS analysis.
  • the calibration curve is used to determine the concentration of each component substrate in the experimental reaction mixtures.
  • the substrate concentration remains much higher with NMTI than after screening with other drug libraries.
  • the concentrations of all of the substrates and products are monitored simultaneously and quantitated.
  • reaction mixtures Five hundred reaction mixtures are prepared, each containing NMT, ACE, and PTPase, the respective substrates of these enzymes (SEQ ID NOS:6, 1, and 5), and a library of 500 sets of compressed mixtures with 50 test drug candidates in each set (i.e., a total of 25,000 compounds are screened) .
  • a control mixture is also prepared without any drug candidates.
  • the drug library reaction mixtures, as well as the control mixture, are sampled and analyzed after 30 minutes. Typically, if none of the test drug candidates are inhibitors, the substrates will have been completely converted to the corresponding products (SEQ ID NOS : 9 , 2, and 10) thirty minutes after mixing the target/library mixture with the substrate mixture.
  • test reaction mixtures in which no angiotensin II or PTPase product is observable at the half hour time point, but in which the NMT substrate has been completely converted, contain one or more test drug candidates that each inhibit ACE and/or PTPase, but not NMT.
  • reaction mixtures are prepared with the three enzymes, their substrates, and the individual members of the successful mixture.
  • a second control mixture is prepared. This time, most of the reaction mixtures yield results that are the same as in the control mixture.
  • reaction mixtures that contain the PTPase and NMT conversion products, but no angiotensin II contain a drug candidate that inhibits only ACE. If only some angiotensin II is observed, e.g., if about 60% of the angiotensin I remains, then the ACE inhibitor is a weak inhibitor. Other reaction mixtures will give additional information. Because the identity of the individual compounds in each reaction mixture are known, compounds that weakly and strongly inhibit ACE and a compound that moderately inhibits PTPase are identified from the molecular library.
  • one of the binding partners can be immobilized on the MALDI plate by coating the plate with a layer of gold and covalently attaching the binding partner to the gold surface.
  • an antibody, antigen, ligand, oligonucleotide, or oligopeptide can be immobilized.
  • a similar technique can also be employed for analysis of non-covalent compound-receptor interactions .

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Abstract

L'invention est basée sur la découverte que l'on peut utiliser la spectrométrie de masse (SM) dans le criblage d'une banque de médicaments potentiels afin de déterminer l'activité contre des cibles multiples simultanément. Les nouveaux procédés permettent d'identifier rapidement et avec précision à la fois les médicaments et les cibles impliquées.
PCT/US1998/015112 1997-07-23 1998-07-22 Criblage multi-cibles de banques moleculaires par sepctrometrie de masse WO1999005309A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU84147/98A AU8414798A (en) 1997-07-23 1998-07-22 Multiple target screening of molecular libraries by mass spectrometry

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5347797P 1997-07-23 1997-07-23
US60/053.477 1997-07-23
US2272698A 1998-02-12 1998-02-12
US09/022,726 1998-02-12

Publications (1)

Publication Number Publication Date
WO1999005309A1 true WO1999005309A1 (fr) 1999-02-04

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/015112 WO1999005309A1 (fr) 1997-07-23 1998-07-22 Criblage multi-cibles de banques moleculaires par sepctrometrie de masse

Country Status (2)

Country Link
AU (1) AU8414798A (fr)
WO (1) WO1999005309A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1220283A2 (fr) * 2000-07-20 2002-07-03 Pfizer Products Inc. Prédiction de la polarité de composés pour rendre efficace la spectrometrie de masse
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
WO2024151979A3 (fr) * 2023-01-12 2024-11-14 Exvivo Labs Inc. Approches de conception et de réalisation d'expériences biologiques compressées et systèmes de mise en œuvre de celles-ci

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995025737A1 (fr) * 1994-03-23 1995-09-28 The Penn State Research Foundation Procede d'identification d'elements de bibliotheque combinatoire
US5583000A (en) * 1991-09-03 1996-12-10 The Regents Of The University Of California Protease-binding compounds and methods of use
US5811241A (en) * 1995-09-13 1998-09-22 Cortech, Inc. Method for preparing and identifying N-substitued 1,4-piperazines and N-substituted 1,4-piperazinediones

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5583000A (en) * 1991-09-03 1996-12-10 The Regents Of The University Of California Protease-binding compounds and methods of use
WO1995025737A1 (fr) * 1994-03-23 1995-09-28 The Penn State Research Foundation Procede d'identification d'elements de bibliotheque combinatoire
US5811241A (en) * 1995-09-13 1998-09-22 Cortech, Inc. Method for preparing and identifying N-substitued 1,4-piperazines and N-substituted 1,4-piperazinediones

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BRUMMEL C. L., ET AL.: "A MASS SPECTROMETRIC SOLUTION TO THE ADDRESS PROBLEM OF COMBINATORIAL LIBRARIES.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 264., 15 April 1994 (1994-04-15), US, pages 399 - 402., XP002912852, ISSN: 0036-8075, DOI: 10.1126/science.8153627 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1220283A2 (fr) * 2000-07-20 2002-07-03 Pfizer Products Inc. Prédiction de la polarité de composés pour rendre efficace la spectrometrie de masse
EP1220283A3 (fr) * 2000-07-20 2004-07-14 Pfizer Products Inc. Prédiction de la polarité de composés pour rendre efficace la spectrometrie de masse
US7074334B2 (en) 2001-05-23 2006-07-11 Klaus Wanner Method for determining the binding behavior of ligands which specifically bind to target molecules
WO2024151979A3 (fr) * 2023-01-12 2024-11-14 Exvivo Labs Inc. Approches de conception et de réalisation d'expériences biologiques compressées et systèmes de mise en œuvre de celles-ci

Also Published As

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