Optical probes and assays

Details Optical probes and assays Optical probes and assays

1999-05-05

2002-06-25

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S021000

Reexamination Certificate

active

06410255

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the fields of chemistry and biology. More particularly, the present invention relates to optical probes for post translational type modification activities, such as phosphorylation, and methods for their use.
INTRODUCTION
Systems and methods for rapidly identifying chemicals with biological activity in samples, especially small liquid samples, is of particular relevance to the agrochemical and pharmaceutical fields. Various strategies are typically used to reduce processing times and associated costs of screening large numbers of chemical entities, including simplified assay design, automation, robotics and miniaturization of sample size. The advent of high throughput analysis and increasing use of miniaturized formats has led to the development of high density plate formats. For example, containing 384, 864 and 3456 wells as described in U.S. patent application Ser. No. 08/868,049 Entitled “
Low Background Multi
-
Well Plates with greater than
864
Wells for Fluorescence Measurements of Biological and Biochemical Samples
,” filed Jul. 3, 1997, now pending. Even higher density sample processing systems, for example using chips that contain miniaturized microfluidic devices are being developed (see, for example, R & D Magazine, November 1998, pages 38 to 43 entitled “
Lab
-
on
-
a Chip: Biotech's next California Gold Rush
”).
Higher density plates enable faster analysis and handling of large sample or chemical libraries, such as in automated screening systems, but place considerable constraints on the assays that can be successfully employed within them. In particular, there is a need to develop assays that are compatible with miniaturized systems and which give accurate and reproducible assay results. Central to this need is a requirement for high sensitivity assays based on optical analysis, such as fluorescence or luminescence that do not require wash steps (e.g. “addition only assays”).
One of the largest and most important classes of intracellular activities for which drugs may be particularly valuable are those involved in post-translational modification activities. These activities are typically directed to the modification of proteins and nucleic acids within living cells to effect changes in the biological activity and function of these molecules. The major methods of protein or polypeptide, post-translational modification include protein phosphorylation, methylation, prenylation, glycosylation, ubiquitination sulfation and proteolysis (see generally Cells.
A Laboratory Manual
, Cold Spring Harbor Laboratory Press (1998) review). Major methods of nucleic acid modification include methylation, ADP-ribsoylation and restriction digestion. A variety of environmental stimuli such as the presence of growth factors, hormones, changes in the cell cycle and toxins can transiently modulate the post-translational state of many intracellular components. The rapid development of specific, and effective inhibitors for a particular post-translational activity requires the development of suitable assays that can reliably and -sensitively detect these activities in a high throughput screening system.
In spite of their great potential importance however, there are few existing methods of measuring such activities that are homogenous, non radioactive and sensitive enough to accurately and reproducible work in high throughput, or ultra high throughput screening systems. Such assays, by reducing the time required to identify and develop useful chemicals, can dramatically increase the value of a new drug by enabling its patentability and increasing it's period of exclusivity in the market.
Examples of such post-translational activities include, amongst others, protein methylation and prenylation. Protein prenylation involves the addition of isoprenoid moieties such as farnesyl and geranylgeranyl to proteins, and is a major mechanism of post-translational modification for many membrane-associated proteins. (Clark, 1992
Protein isoprenylation and methylation at carboxyl
-
terminal cysteine residues
. Annu. Rev. Biochem. 61 355-386). In most cases, the amino acid derivatized with the isoprenoid is a cysteine, or cysteines close to the carboxyl-terminus of the protein. Present methods of measuring protein prenylation and methylation typically involve labeling cells with radioactive precursors such as [
3
H]-mevalonate or [
3
H]-S-adenosylmethionine, isolation of the protein of interest and measurement of radioactive incorporation. There is thus a need for assays for these activities that are sensitive, simple to use, non-radioactive and adaptable to high throughput screening methods.
Another important example of post-translational modification is protein glycosylation, which plays an extremely important role in the function of a significant number of proteins (Varki, 1993
, Biological roles of oligosaccharides
. Glycobiology 3 97-130). Protein glycosylation, unlike most other types of post-translational modification provides a wide diversity in the oligosaccharides added to a protein because of the potential for branching after the addition of the first sugar residue. Present methods of measuring glycosylation typically involve determining radioactive incorporation of a precursor oligosaccharide into a protein, isolating the protein and then measuring specific radioactive incorporation into a protein. There is thus a need for fluorescence or luminescence based assays for these activities that are adaptable to high throughput screening methods. It is one objective of the present invention to provide optical probes and methods of use that meet this need.
Protein kinases and phosphatases are generally recognized as one of the more important general mechanisms of regulating protein function. A recent review and analysis of diseases associated with genetic defects in protein kinases lists over 400 specific disease states associated with these activities alone. Protein kinases act on proteins via the addition of phosphate groups (phosphorylation) primarily on the amino acids, tyrosine, serine or threonine. Protein phosphatases in contrast, act to remove these phosphate groups thereby reversing the effects of phosphorylation. Changes in the phosphorylation state of proteins, can regulate the enzymatic activity, protein localization and protein-protein interactions of a particular protein within a cell. Such changes can subsequently modulate virtually every aspect of cellular metabolism, regulation, growth and differentiation. The overall balance of kinase and phosphatase activities in a cell is a primary determinant of the phosphorylation state of a protein at any one time.
However, current methods of measuring protein kinases, have many disadvantages, which prevents or hampers the ability to rapidly screen for drugs using miniaturized automated formats of many thousands of compounds.
For example, many current methods of measuring their activity rely on the incorporation and measurement of
32
P into the protein substrates of interest. In whole cells, this necessitates the use of high levels of radioactivity to efficiently label the cellular ATP pool and to ensure that the target protein is efficiently labeled with radioactivity. After incubation with test drugs, the cells must be lysed and the protein of interest purified to determine its relative degree of phosphorylation. This method requires large numbers of cells, long preincubation times, careful manipulation, and washing steps to avoid artifactual phosphorylation or dephosphorylation. Furthermore, this kinase assay approach requires purification of the target protein, and final radioactive incorporation into target proteins is usually very low giving the assay poor sensitivity. In high throughput screening operations, this approach requires large amounts of radioactivity, which can be an environmental and health hazard.
Alternative kinase assay methods, such as those based on phosphorylation-specific antibodies using ELISA-type approaches, involv

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