Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-10
2002-08-13
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C436S501000
Reexamination Certificate
active
06432651
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
FIELD OF THE INVENTION
The invention relates generally to methods of screening complex biological materials for effective regulatory, therapeutic, or diagnostic compounds. The invention encompasses using capillary electrophoresis and mass spectrometry together in a method particularly advantageous for detecting and characterizing tight-binding ligands in mixtures that may also include much higher concentrations of competing, weaker-binding ligands. The method also allows ranking of ligands according to their relative binding strengths.
BACKGROUND OF THE INVENTION
Developing screening protocols to identify new, biologically active compounds can present unique and difficult challenges, especially when screening complex materials, particularly a “complex biological sample” (CBS): any sample of material that may have an effect in a biological system. Examples of CBS include but are not limited to: a natural product; a natural extract; a biological preparation; a chemical mixture; a pure compound library; and a combinatorial library.
While capillary electrophoresis has previously been used to detect and/or to analyze known compounds and materials of known composition, this technology has not been widely used, until recently, to screen complex biological samples for target-binding compounds that were previously unknown or unidentified as being ligands to a selected target molecule.
For example, WO 97/22000 encompasses four broad embodiments of a capillary electrophoretic screening method for unknown, biologically active compounds, as follows.
(1) In a non-competitive embodiment of WO 97/22000, a target and complex biological sample are mixed together, then an aliquot of that target/sample mixture is subjected to capillary electrophoresis (CE), and the CE migration of the target is tracked. The target's migration pattern under these conditions are compared against a reference standard, typically the unbound target's migration pattern in the absence of any target-binding ligand.
(2) In a non-competitive, subtractive analysis embodiment of WO 97/22000, a target and sample are mixed together and then subjected to CE. The migration pattern of this mixture is compared to the migration pattern of a sample of the complex biological material alone. Any difference between the two migration patterns suggests the presence in the sample of a hit compound that can bind to the target.
(3) One competitive binding embodiment is provided in WO 97/22000, which tracks known, charged ligand: The target is first mixed with a complex biological material sample and then with a known, charged ligand that binds tightly to the target, to form a sample/target/known ligand mixture. This method uses an essentially equilibrium setting when incubating target and known, tight-binding ligand together, so that the known, tight-binding ligand can displace any weaker-binding hit, prior to CE. This mixture is subjected to capillary electrophoresis and the migration of the known, charged ligand is tracked. (Thus, this method is useful when the target is not easily detected during CE.) Any difference in the known, charged ligand's migration pattern, when in the presence of both the target and a complex biological material sample, from the known ligand's migration pattern when in the presence of the target alone, indicates the presence of a candidate, unidentified target-binding ligand in that sample.
(4) In another competitive binding embodiment of WO 97/22000, the target's migration is tracked and the CE running buffer contains a known, weak-binding, competitive ligand. The target is mixed with a sample, and an aliquot of the mixture is subjected to CE in the presence of a known, relatively weak, target-binding ‘competitor’ ligand in the CE running buffer. The migration of the target is tracked during CE. The reference standard is the migration of a target plug alone in the known ligand-containing CE buffer, its migration being shifted by its weak, reversible binding to the known ligand dispersed in the CE buffer, as compared to the target's migration alone ligand-free buffer. This competitive screening method can detect a tight-binding hit compound in a target
atural sample mixture, because the hit binds up the target for the entire CE run and prevents the target's interaction with the known weak-binding ligand in the buffer. Therefore, the CE migration pattern of the target in the sample/target aliquot would shift back to the target's migration position as it would be in ligand-free running buffer. This method, too, is particularly useful when the unbound target is not easily detected in ligand-free buffer during CE.
While WO 97/22000 provides useful CE screening methods, they do not overcome some common drug-screening problems. A major obstacle to successful and cost-effective drug screening has been the presence of high concentrations of one or several weak, target-binding ligand compound(s) in a screened sample, which can mask the presence of more valuable, moderate-to-tight-binding or tight-binding ligands occurring at a lower concentration within the same sample. Another major obstacle is obtaining structural information about the high affinity ligands, especially when they are present in very complex mixtures.
Therefore, there remains a need for rapid and cost-effective screening tools for discovering new bioactive and/or potential regulatory compounds that bind to molecules involved in disease or essential molecules of key metabolic pathways. Also needed is a way of characterizing those candidate ligands displaying the highest binding strengths to the target.
BRIEF SUMMARY OF THE INVENTION
The present invention answers these needs by providing an improved screening method combining both capillary electrophoresis and mass spectrometry techniques. Capillary electrophoresis, specifically capillary zone electrophoresis, enables rapid and cost-effective separation and identification of compounds in a sample while consuming only minute amounts of the sample. In the particular application taught here, capillary electrophoresis (CE) enables selective identification of particular candidate ligand(s) that bind(s) tightly to a target of interest. The CE steps are optimized to screen out all but those ligands that bind to the target molecule of interest at or above a selected binding strength, as taught in International Application No. PCT/US98/27463, herein incorporated by reference.
Mass spectrometry (MS) enables analysis of biomolecules, such as peptides and proteins, at the molecular level with high mass measurement accuracy. Suitable MS ionization techniques include, but are not limited to electron impact ionization (EI), electrospray ionization (ESI), chemical ionization (CI), atmospheric-pressure chemical ionization (APCI), matrix-assisted-laser-desorption ionization (MALDI), thermospray (TSP), and fast atom bombardment (FAB) ionization. These ionization techniques may be combined with time-of-flight (TOF), single or triple quadrupole, Fourier transform, or ion trap MS analysis to provide additional information of the compounds analyzed. For example, MALDI-TOF mass spectrometry is valued for ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity, and high speed. Ion trap and Fourier transform MS allow re-analysis of the ions, as needed. If desired, the ions may be subjected to fragmentation, such as collision-induced dissociation (CID), during mass spectrometry to provide additional structural or substructural data.
Moreover, since MS allows compounds to be detected and differentiated by their molecular weight and/or size, it may be used to observe selectively the target's migration pattern after undergoing CE either alone or in the presence of a complex biological sample. Therefore, MS allows one to eliminate any separate detector and/or any derivatization of the target, if one so desires.
Alternatively, one may use a separate detector, e.g. an ultraviolet absorbance (U
Dunayevskiy Yuriy M.
Hughes Dallas E.
Karger Barry L.
Waters James L.
Cetek Corporation
Horlick Kenneth R.
Weingarten Schurgin, Gagnebin & Lebovici LLP
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