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Homogeneous assay for enzymatic activity

Homogeneous assay for enzymatic activity description/claims

This application is a Continuation of U.S. patent application Ser. No.10/517,758, filed on Oct. 14, 2005 which was a National Stage Entry of PCT/US03/18912 filed Jun. 16, 2003, which claims priority of U.S. 60/388,792 filed Jun. 14, 2002, now expired, the disclosures of each incorporated by reference in its entirety.

The present invention relates to the assay of enzymatic activity and, more particularly, to the measurement of the activity of kinases, phosphatases, and proteases, as well as other enzymes. More specifically, the present invention relates to measurements of enzymatic activity, which are accomplished in a homogenous assay format utilizing a fluorescence quenching technique employing paramagnetic metal ions.

Enzymes are catalytic proteins that typically act on a substrate to yield an endproduct. Protein kinases and protein phosphatases comprise a class of enzymes that modify protein and/or peptide substrates by catalyzing the attachment or removal, respectively, of a phosphoryl group to sites on certain amino acid side chains of the substrates for these enzymes. Determining the presence or catalytic activity of these enzymes is important since the degree of phosphorylation of a particular protein or peptide has been found to be an important characteristic in regulating cellular functions. Enzymes capable of peptide bond cleavage, known as proteases, are another class of enzymes. Ever increasing emphasis is being placed on discovering what drugs can be used to modulate enzyme activity. This, in turn, has created the demand for the development of improved techniques for the measurement of the activity of enzymes.

Radioactive detection has been used to assay for enzymatic activity; see, for example, U.S. Pat. No. 5,538,858. With respect to kinases, a sample containing the kinase of interest is incubated with activators and a peptide substrate in the presence of radioactive labeled ATP. Then, an aliquot of the incubated mixture (containing phosphorylated and non-phosphorylated peptides) is placed on a filter that binds the substrate and the filter washed to remove excess radioactivity. The amount of radiolabeled phosphate incorporated into the substrate and, in turn, enzyme activity is measured by scintillation counting.

Because of the necessity for the precautions involved in radioactive techniques, non-radioactive assay techniques are also in use. A particularly attractive assay is sold by Pierce Biotechnology, Inc. (formerly known as Pierce Chemical Company) under the SpinZyme™ T brand name and is described in U.S. Pat. No. 5,527,688, issued on Jun. 18, 1996. In this assay, non-radioactive ATP is used in the incubation mixture and the substrate is dye-labeled. After the enzymatic reaction, the incubated mixture is brought into contact with a solid phase containing immobilized Fe+++. The phosphorylated substrate in the mixture binds to the solid phase by chelation with the iron ion. The non-phosphorylated substrate is removed by washing, and the amount of phosphorylated substrate is measured by detection of the dye, which is labeled on the substrate.

The above-described assay techniques are non-homogenous in that they require the phosphorylated and non-phosphorylated substrates to be physically separated between the kinase-initiated phosphorylation and detection. These added steps detract from the use of these techniques in those applications commonly used in drug screening and termed high throughput screening.

Homogenous assay techniques have been developed in a variety of specific areas to overcome the aforementioned drawbacks of non-homogenous assays. One example is radioactive assays, commonly known as scintillation proximity assays. These are described in U.S. Pat. Nos. 4,568,649, 5,665,562 and 5,989,854. Other homogenous assays are described in the April, 2002 issue of Drug Discovery & Development entitled “The Key to Kinases is All in the Kits,” beginning on page 28.

Several other categories of homogenous assays are based on non-radioactive detection methods. Fluorescence techniques, such as those based on fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP), have been introduced; see, for example, U.S. Pat. No. 6,287,774 and US Patent Application 2002/0034766 A1.

In FRET methods, a fluorophore (a light-absorbing dye capable of fluorescence emission) is utilized in combination with another fluorophore (either identical or not) or with a chromophore (a light-absorbing dye not capable of fluorescence emission). A general requirement for FRET is that the two entities of the pair combination (either fluorophore and fluorophore, or fluorophore and chromophore) have an overlapping spectral region. The ability of the FRET technique to be utilized in the construction of an assay relies on the capacity to distinguish, by measurable signal detection, the variation in observed fluorescent emission from the combination pairs employed when they are in close proximity as opposed to spatially separated. Thus, in this technique, a donor fluorophore, such as fluorescein, can be used with the dye, tetramethylrhodamine, as an acceptor fluorophore. When these two fluorophores are in close proximity to each other, excitation of the fluorescein molecule results in energy being transferred to the tetramethylrhodamine acceptor and consequently the normal expected emission from the fluorescein is decreased.

Assays can be constructed using FRET techniques where specific binding events are utilized to bring the two fluorophores into close proximity. Such assays can be quantitated by observing decreased fluorescent emission of the donor fluorophore or by observing increased fluorescent emission of the acceptor fluorophore, both of which are brought about when the binding event occurs. Proteolytic FRET assays utilize the action of a protease to cleave the substrate having the attached fluorophores (for example labeled on the N and C terminus of peptide) to cause the two fluorophores to be more spatially separated and thereby diminishing the FRET event.

In the case of FRET assays utilizing a chromophore, examples of useful chromophores include those commonly known as Black Hole Quenchers and DABCYL (4-(4′-dimethylaminophenylazo)benzoic acid) (See Proc. Natl. Acad. Sci. USA. 1999 May 25; 96 (11): 6394-6399 entitled “Multiplex detection of four pathogenic retroviruses using molecular beacons.” Jacqueline A. M. Vet, Arnit R. Majithia, Salvatore A. E. Marras, Sanjay Tyagi, Syamalima Dube, Bernard J. Poiesz, and Fred Russell Kramer).

Drawbacks of the FRET technique include the requirement of a matched combination pair, which precludes a more universal utility to assay construction, along with other drawbacks, such as increased cost, complications related to assay interpretation via signal breakthrough, and negative assay interactions, such as hydrophobic interactions of the enzyme with the dye molecules, etc.

Turning now to the FP method mentioned above, this technique relies on detecting a measurable change in fluorescent polarization. For example, in FP-based kinase assay, these assays measure the change in fluorescent polarization (FP) that accompanies the kinase catalyzed phosphorylation of a fluorescent dye-labeled substrate. To achieve a measurable change in FP on phosphorylation, a large entity is included in the incubation mixture, which complexes with the phosphoryl group on the derivatized peptides. In such a direct assay format, because of the resulting increase in size of those peptides that have been phosphorylated, their rotational diffusion is significantly less and, in turn, their FP significantly greater, than of those peptides that have not been phosphorylated. Competitive FP formats also have been described. Thus, the difference in FP between the labeled substrate before catalysis with the kinase of interest and after catalysis is indicative of the activity of the enzyme.

FP assays can be run in a homogenous format, which requires no washing and separation steps because both the before and after measurements are of the same parameter, namely, fluorescent polarization; only the change in this parameter is the determinative factor. However, a drawback associated with FP assays is the necessity for using expensive equipment capable of measuring FP. A further drawback of FP assays resides in certain technical limitations associated with its use, such as assay artifacts due to scattered light, viscosity changes, and polarization changes associated with incorporation of small molecular weight fluorophores into large molecular detergent micelles. These limitations are not found in fluorescent assays based on simple measurement of fluorescent intensity.

In accordance with the present invention there is provided a method and associated composition of matter for assaying the activity of an enzyme based on fluorescence quenching. The present invention results from the observation that the fluorescent intensity of a fluorophore label on an enzymatic substrate or endproduct can be quenched by the presence of a paramagnetic metal ion when bound to a target group located in proximity to the label, and the realization that useful enzyme assays are enabled utilizing this observation.

The assays incorporate a paramagnetic multivalent ion that binds specifically to a target group present on a fluorescent dye-labeled enzymatic substrate or endproduct. When bound to the target group, the ion is brought into proximity to the fluorophore and acts as a quencher of the fluorescent dye label by means of intrinsic properties of the metal ion. This specific binding is immediate. Results are quantitated by comparing the observed relative fluorescence units of test samples to blanks containing no enzyme.

More specifically, the method of the present invention involves assaying the activity of an enzyme of choice by contacting the enzyme with a population of fluorophore labeled substrate in an aqueous enzymatic reaction mixture, and allowing the enzymatic reaction to proceed for a selected period of time and temperature as desired. The reaction is then brought into contact with a paramagnetic metal ion to form a complex of the paramagnetic metal ion with a target group on either the enzyme substrate or endproduct. This complex, when in proximity to the fluorophore label, causes specific quenching of the fluorescence from the fluorophore. By measuring the intensity of the observed fluorescent emission from the mixture and relating the observed fluorescence to that of an external reference, a differential fluorescent signal, if any, can be identified and quantitated to ascribe a specific value to the differential. This ascribed differential value in fluorescent signal of the sample is indicative of the final state (i.e., post-enzymatic reaction) of the fluorophore labeled substrate population and, in turn, reflects enzymatic activity.

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