Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2000-02-08
2004-08-03
Ponnaluri, Padmashri (Department: 1639)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C435S006120, C435S007100, C435S091200
Reexamination Certificate
active
06770486
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to mass spectrometry methods for detecting binding interactions of ligands to substrates and in particular to methods for determining the mode of binding interaction of ligands to substrates.
BACKGROUND OF THE INVENTION
Drug discovery has evolved from the random screening of natural products into a combinatorial approach of designing large numbers of synthetic molecules as potential bioactive agents (ligands, agonists, antagonists, and inhibitors). Traditionally, drug discovery and optimization have involved the expensive and time-consuming process of synthesis and evaluation of single compounds bearing incremental structural changes. For natural products, the individual components of extracts had to be painstakingly separated into pure constituent compounds prior to biological evaluation. Further, all compounds had to be analyzed and characterized prior to in vitro screening. These screens typically included the evaluation of candidate compounds for binding affinity to their target, competition for the ligand binding site, or efficacy at the target as determined via inhibition, cell proliferation, activation or antagonism end points. Considering all these facets of drug design and screening that slow the process of drug discovery, a number of approaches to alleviate or remedy these matters, have been implemented by those involved in discovery efforts.
The development and use of combinatorial chemistry has radically changed the way diverse chemical compounds are synthesized as potential drug candidates. The high-throughput screening of hundreds of thousands of small molecules against a biological target has become the norm in many pharmaceutical companies. The screening of a combinatorial library of compounds requires the subsequent identification of the active component, which can be difficult and time consuming. In addition, compounds are usually tested as mixtures to efficiently screen large numbers of molecules.
A shortcoming of existing assays relates to the problem of “false positives.” In a typical functional assay, a false positive is a compound that triggers the assay but which compound is not effective in eliciting the desired physiological response. In a typical physical assay, a false positive is a compound that attaches itself to the target but in a non-specific manner (e.g. non-specific binding). False positives are particularly prevalent and problematic when screening higher concentrations of putative ligands because many compounds have non-specific affects at those concentrations. Methods for directly identifying compounds that bind to macromolecules in the presence of those that do not bind to the target could significantly reduce the number of “false positives” and eliminate the need for deconvoluting active mixtures.
In a similar fashion, existing assays are also plagued by the problem of “false negatives,” which result when a compound gives a negative response in the assay but the compound is actually a ligand for the target. False negatives typically occur in assays that use concentrations of test compounds that are either too high (resulting in toxicity) or too low relative to the binding or dissociation constant of the compound to the target.
When a drug discovery scientist screens combinatorial mixtures of compounds, the scientist will conventionally identify an active pool, deconvolute it into its individual members, and identify the active members via re-synthesis and analysis of the discrete compounds. In addition to false positives and false negative, current techniques and protocols for the study of combinatorial libraries against a variety of biologically relevant targets have other shortcomings. These include the tedious nature, high cost, multi-step character, and low sensitivity of many screening technologies. These techniques do not always afford the most relevant structural and binding information, for example, the structure of a target in solution and the nature and the mode of the binding of the ligand with the receptor site. Further, they do not give relevant information as to whether a ligand is a competitive, noncompetitive, concurrent or a cooperative binder of the biological target's binding site.
The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development that has the potential to accelerate the identification of lead compounds in drug discovery. Rapid and direct methods have been developed to identify lead compounds in drug discovery involving affinity selection and mass spectrometry. In this strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to develop a structural activity relationship (SAR) in order to optimize the binding characteristics of the ligand with the target receptor.
One step in the identification of bioactive compounds involves the determination of binding affinity and binding mode of test compounds for a desired biopolymeric or other receptor. For combinatorial chemistry, with its ability to synthesize, or isolate from natural sources, large numbers of compounds for in vitro biological screening, this challenge is greatly magnified. Since combinatorial chemistry generates large numbers of compounds, often isolated as mixtures, there is a need for methods which allow rapid determination of those members of the library or mixture that are most active, those which bind with the highest affinity, and the nature and the mode of the binding of a ligand to a receptor target.
An analysis of the nature and strength of the interaction between a ligand (agonist, antagonist, or inhibitor) and its target can be performed by ELISA (Kemeny and Challacombe, in
ELISA and other Solid Phase Immunoassays: Theoretical and Practical Aspects
; Wiley, New York, 1988), radioligand binding assays (Berson and Yalow, Clin. Chim. Acta, 1968, 22, 51-60; Chard, in
“An Introduction to Radioimmunoassay and Related Techniques
,” Elsevier press, Amsterdam/New York, 1982), surface-plasmon resonance (Karlsson, Michaelsson and Mattson,
J. Immunol. Methods,
1991, 145, 229; Jonsson et al.,
Biotechniques,
1991, 11, 620), or scintillation proximity assays (Udenfriend, Gerber and Nelson,
Anal. Biochem.,
1987, 161, 494-500). Radio-ligand binding assays are typically useful only when assessing the competitive binding of the unknown at the binding site for that of the radio-ligand and also require the use of radioactivity. The surface-plasmon resonance technique is more straightforward to use, but is also quite costly. Conventional biochemical assays of binding kinetics, and dissociation and association constants are also helpful in elucidating the nature of the target-ligand interactions but are limited to the analysis of a few discrete compounds.
A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands (Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W.
Science,
1996, 274, 5252, 1531). The approach is called SAR by NMR because structure-activity relationships (SAR) are obtained from NMR. This technique has several drawbacks for routine screening of a library of compounds. For example, the biological target is required to incorporate a
15
N label. Typically the nitrogen atom of the label is part of amide moiety within the molecule. Because this technique requires deshielding between nuclei of proximal atoms, the
15
N label must also be in close proximity to a biological target's binding site to identify ligands that
Drader Jared J.
Griffey Richard
Hofstadler Steven
Lowery Kristin S.
Mohan Venkatraman
Grumbling Matthew V.
ISIS Pharmaceuticals Inc.
Ponnaluri Padmashri
Rosch Mark P.
Tarbet Kenneth H.
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