Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody – Through a gel
Reexamination Certificate
2000-06-21
2003-02-25
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
Through a gel
C204S166000, C204S451000, C204S452000, C204S453000, C204S298070, C204S407000, C204S600000, C204S601000, C204S602000, C204S603000, C204S604000, C204S605000, C204S606000, C204S607000, C204S608000, C204S610000, C204S612000, C204S615000, C204S616000, C204S617000, C204S618000, C204S621000, C435S004000, C435S005000, C435S006120, C435S007100, C435S007200, C435S007800, C435S007900, C435S007920, C356S344000, C210S198200, C436S514000, C436S515000, C436S536000, C436S538000, C436S540000
Reexamination Certificate
active
06524866
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the discovery of new regulatory compounds, drugs, and/or diagnostic agents. In particular, it relates to capillary electrophoretic methods for detecting ligands binding to a known target molecule, and for determining the relative stabilities of ligand/target complexes, thereby allowing the ranking of different ligands according to their relative binding strengths to a common target. The invention encompasses methods of detecting moderate-to-strong binding ligands in mixtures that also have much higher concentrations of competing, weaker-binding ligands.
BACKGROUND OF THE INVENTION
Developing screens to identify new biologically active compounds can present unique and difficult challenges, especially when screening complex materials, particularly “complex biological materials”: any material that may have an effect in a biological system. Examples include, but are not limited to: naturally occurring samples, products or extracts; various biological preparations; chemical mixtures; libraries of pure compounds; and combinatorial libraries. Examples of major screening problems include: detecting potential hit compounds that bind to a target of interest, especially ligands present at low concentrations in screened samples; accounting for unknown components that can interfere with screening agents; and determining the relative value of screened samples for further investigative efforts. As well, high concentrations of a weak or several weak, competing binder(s) can mask the signal from a moderate-to-strong hit compound occurring at a lower concentration in the same sample.
Recently, the use of capillary electrophoresis techniques has facilitated and improved the process of screening for unknown, biologically active compounds. For example, WO 97/22000 encompasses four broad embodiments of a capillary electrophoretic screening method, 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 completely solve the screening problems listed previously.
Therefore, there remains a need for rapid and cost-effective screening tools for discovering new bioactive compounds and potential regulatory compounds that bind to essential molecules of key metabolic pathways. Also needed is a way of prioritizing candidate ligands and samples of material for further characterization. The present invention addresses these needs, by providing: a means of detecting unknown ligands that may be candidate, new, bioactive compounds; a means of ranking screened samples detected to contain candidate hit compounds or ligands, according to their relative binding strengths and value as potential sources of regulatory and diagnostic compounds; and a means of identifying effective and valuable, strong or moderate, target-binding ligands in the presence of weaker, competitive binders. Identifying and ranking those ligand-containing samples that form the most stable complexes with the selected target, saves time and resources spent on further isolation and characterization of hit compounds. The most stable ligands are potentially more effective and valuable as therapeutic, regulatory and/or diagnostic compounds and drugs.
SUMMARY OF THE INVENTION
The present invention provides: (1) a means to screen for target-binding ligands of a desired binding strength, in complex biological and other materials and mixtures; (2) a means to screen for the ligands or hit compounds of the desired affinity, even in the presence of weak-binding ligands in mixtures; and (3) a means to rank hit compounds according to their relative affinities. All aspects of the method can be performed on samples of pure hit compounds, partially purified hit compounds, and hit compounds present in complex biological mixtures.
The present invention provides simple and rapid, capillary electrophoretic methods for screening for and ranking ligands that do not require knowledge of a particular ligand's structure or concentration within the screened sample. In fact, it can be practiced on samples of complex materials, such as natural samples, that potentially contain one or more unidentified ligand(s) or hit compound(s) that bind to a selected target of interest. The migration of both the target and the target/hit compound complex must be detectable during capillary electrophoresis (CE). The hit compound should be detectable by its ability to alter the CE profile of the detectable target.
The present capillary electrophoretic screening methods allow one to detect hit compounds of any desired affinity or binding strength. Detection of weak, moderate and/or strong binding ligands is possible because the screened sample, once mixed and incubated with a select target, is injected into a CE capillary and electrophoresed. Electrophoretic conditions are optimized to detect hit compounds having a desired affinity for a selected target: e.g., ligands of any binding affinity (weak, moderate, or strong), only
Dunayevskiy Yuriy M.
Hughes Dallas E.
Waters James L.
Cetek Corporation
Le Long V.
Padmanabhan Kartic
Weingarten Schurgin, Gagnebin & Lebovici LLP
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