Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2003-05-23
2004-09-14
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007930, C435S007940, C435S007950, C436S518000, C530S388100, C530S388220
Reexamination Certificate
active
06790632
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally concerns reagents and methods useful for membrane receptor-ligand binding assays.
2. Description of the Related Art
Much of modern pharmacology and biochemistry is focused on the interactions between different types of biological signaling molecules and their corresponding membrane receptors. Structurally, these membrane receptors are often G-Protein Coupled Receptors (GPCR) from the 7-transmembrane (7tm) protein family, and other types of related 7-transmembrane proteins such as ion channels.
Biological signaling molecules and membrane receptors are present in many physiological processes, and are particularly important for nervous system function. Indeed, nervous system membrane receptor systems, such as the dopamine, serotonin, and opioid receptor families, have been found to be involved in many mental disorders, such as anxiety, depression, and drug abuse. Furthermore, these membrane receptors have been found to be excellent drug targets. GPCR reactive drugs are involved in many other biological processes as well. Kenakin,
Annu Rev Pharmacol Toxicol
2002;42:349-79).
To date, however, only a small number of the many possible interactions between the millions of potential candidate drug ligands, and the thousands of different membrane receptors, have been well characterized. With the recent advances in both genomics and nucleic acid microarray technology, the cellular distribution and sequence of these membrane receptors and receptor families are now readily available. As a result, one of the key challenges going forward is to utilize this new knowledge to aid in the development of next generation drugs.
Modern drug discovery and development is a multi-step process. Usually, one or more medically important target receptors are identified, a large number of prototype lead drugs are synthesized, and appropriate High Throughput Screening (HTS) assays are conducted to assess the proper differential binding to an initial set of selected target and non-target receptors. Those lead candidates that survive the process are then subjected to progressively more expensive and stringent assays; whole cell assays, animal studies, Absorption, Distribution, Metabolism, Excretion, Toxicity (ADMET) studies, and finally human Phase I, II, and III studies. The expense and time involved in the later stages, typically hundreds of millions of dollars and many years, are such that it is enormously important that optimal leads be found as early in the process as possible.
One of the best ways to exploit the recent advances in genomics is to use the information to clone and express these membrane receptors in a pure state, and use these cloned receptors for HTS lead identification and optimization. In theory, the later stages of drug development could be significantly streamlined if low-cost and efficient HTS methods were developed to initially optimize a lead's specific binding to its target receptor, and to detect any unwanted cross-reaction with non-target receptors.
At present, however, membrane HTS screening methods are sub-optimal. Membrane proteins are hydrophobic, and typically only assume their correct physiological conformation in a lipid bilayer membrane environment. Moreover, many membrane receptor proteins rely upon certain aspects of lipid bilayers, such as receptor lateral mobility, association with lipid rafts, association with other proteins, small molecule cofactors etc., for proper function. Such faithful recreations of native membrane structures are difficult to reproduce in a synthetic, in-vitro, environment.
As a result, present membrane HTS screening methods usually rely upon the binding of radioactively labeled ligands to natural membrane receptors (e.g. receptors obtained from natural sources such as cultured cells). These natural membrane receptors are often bound to a solid phase, such as a filter or microwell plate. Radioactive ligand is applied to the sample, followed by one or more washing steps. The bound radioactive ligand is then detected by its radioactive scintillation signal. Thus relatively large quantities of membrane proteins and candidate drug ligands are required for each assay. For example, even the optimized Packard Bioscience FlashPlate™ system requires 25 to 50 ul of fluid for each assay point.
Although the use of natural membranes has strong physiological merit, it is slow, expensive, and cumbersome. An alternate membrane receptor HTS methodology that could return physiologically useful data with cloned membrane receptors would be highly advantageous, as it would enable the many recent genomic insights to be easily and rapidly used.
An ideal membrane-receptor HTS methodology would have a number of other characteristics as well. At present, HTS methods are typically restricted to large, well-financed, commercial organizations. This is because the present methodologies require the use of large quantities of expensive membrane receptors, expensive synthetic drug candidates, and expensive automation. If alternative methods could be devised to reduce the quantities of receptors and synthetic drug candidates by several orders of magnitude, the financial and logistical burden of HTS studies would be greatly eased. This would enable a much larger number of receptors and drug candidates to be screened, and could also make HTS methods feasible in smaller scale settings, such as academic laboratories.
One good way to accomplish this goal is by the development of suitable membrane receptor microarray technology.
Microarray technology: In recent years, microarrays have become widely used for genomic and proteomic biotechnology, biomedical research, and biomedical diagnosis. In particular, microarray methods have become widely used for nucleic acid research, and a large number of nucleic acid microarrays are commercially available from Affymetrix Inc., Incyte Pharmaceuticals, Inc., and many other companies. These methods (reviewed in Schena,
Microarray Biochip Technology
(2000) Eaton Publishing. Natick, Mass.) generally work by binding a large number of nucleic acid microsamples to the surface of a flat support. Samples containing one or more unknown complementary nucleic acids are then exposed to the nucleic acid microarray, and the sample is allowed to hybridize to the microarray. Hybridized nucleic acids are then detected by various means, and the overall nucleic acid composition of the unknown sample is assessed.
The general principle has been that to detect two biologically interacting elements that form a pair, such as complementary nucleic acid strands, the microarray will contain one-half of the pair, the unknown analyte will contain the other half of the pair, and the interaction between the two elements will generate a detectable signal.
A good overview of protein microarray technology in general, as it exists at the time of this patent application, can be found in the article by Mitchell, “
A perspective on protein microarrays”, Nature biotechnology
(20), 2002, 225-229, the contents of which are incorporated herein by reference.
There have been some previous attempts to produce membrane receptor microarrays, such as U.S. Pat. No. 6,228,326; and Salafsky et. al.,
Biochemistry
(1996) 35: 14773-14781. One approach, pioneered by Boxer and coworkers (Groves et. al.,
Science
(1997), 275: 651-653) relies upon the formation of an artificial planar lipid membrane parallel to a microarray surface. The microarray uses a series of mechanical barriers to separate one lipid region from another, enabling multiple regions to be patterned. The microarray is analyzed by fluorescence recovery after photobleaching (FRAP) techniques. Here, fluorescent membrane components are exposed to a localized region of intense laser irradiation. Following irradiation, a bleached region forms, which can be observed by a fluorescence microscope. This gradually disappears as unbleached membrane components from surrounding regions gradually migrate into the bleached region.
Although useful for demonstra
Le Long V.
Yang Nelson
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