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Compositions and assays utilizing adp or phosphate for detecting protein modulators

Compositions and assays utilizing adp or phosphate for detecting protein modulators description/claims

This invention is related to the use of adenosine diphosphate (ADP) or phosphate in assays for identifying compounds which bind to or modulate the binding characteristics or biological activity of a protein.

Drugs and other compounds intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease in man or other animal or for use in the agricultural arena, have made a significant impact on the practice of modern medicine and on the agricultural arena. In some cases, such as in the development of vaccines, drugs have essentially eradicated once untreatable diseases. In the case of the agriculture, compounds have been developed which both extend the life and/or volume of produce as well as kill unwanted plants where desirable. Therefore, the development of these compounds is of great interest.

Many useful compounds modulate the physical interaction of proteins. Traditionally, these protein-protein interactions have been evaluated using biochemical techniques, including chemical cross-linking, co-immunoprecipitation, co-fractionation and co-purification. Recently genetic systems have been invented to detect protein-protein interactions. The first work was done in yeast systems, and was termed the “yeast two-hybrid” system. The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene. Subsequent work was done in mammalian cells. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578 (1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463.

In another approach to drug discovery, studies are designed to determine the biological activity of a protein. For example, the conditions such as the specific substrate or stimulator required for an enzymatic reaction are investigated. Moreover, there are a number of studies designed specifically for aide in the detection step in these assays. For example, one study discloses a spectrophotometric assay for inorganic phosphate (Pi) to probe the kinetics of Pi release from biological systems such as GTPases and ATPases. Webb, PNAS, 89:4884-4887 (1992). Another study reports on an enzymatic assay of inorganic phosphate in serum using nucleoside phosphorylase and xanthine oxidase. Ungerer, et al., Elsevier Clinica Chimica Act, 223:149-157 (1993). A continuous spectrophotometric assay for aspartate transcarbamylase and ATPases is reported on in Rieger, et al., Anal. Biochem., 246:86-95 (1997). There is also a study which reports on the measurement of inorganic phosphate release using fluorescent probes and its application to actomysin subfragment 1 ATPase. Brune, et al., Biochem., 33:8262-8271 (1994). U.S. Pat. No. 4,923,796 discloses a method for quantitative enzymatic determination of ADP. Microtubule-stimulated adenosine triphosphate (ATP) hydrolysis by kinesin is discussed in Hackney, J. Biol. Chem., 269(23):16508-16511 (1994). Furthermore, enzymatic fluorimetry and fluorimetric assays for ATPase activity are reported on in Greengard, Nature, 178:632-634 (1956) and Utpal and Siddhrtha, Biochem. J., 266:611-614 (1990), respectively.

In a different approach, modulators of an enzymatic reaction are investigated, wherein the conditions which allow the enzymatic reaction to occur are already known. For example, U.S. Pat. No. 5,759,795 discloses an assay for identifying an inhibitor of a Hepatitis C Virus NS3 protein ATPase which involves a luciferase reaction. Luciferase reactions are known in the art. In the case of an ATPase inhibitor, the presence of an ATPase inhibitor is indicated when ATP is available to drive the oxidation of luciferon by luciferase. This approach requires ATP but does not re-generate ATP.

Thus, while efforts have been made toward drug discovery, more efficient means are desirable. In particular, there is a need for an efficient system which can distinguish between a compound directly binding to a second component, or whether the compound modulates the binding between two other components, or whether the compound modulates the biological activity of a known enzymatic reaction. Accordingly, it is an object of the present invention to provide methods of identifying compounds which either bind to or which modulate the binding characteristics or the biological activity of a target protein. It is also an object to provide compositions for use in the assays provided herein.

The present invention provides methods which identify candidate agents that bind to a a protein or act as a modulator of the binding characteristics or biological activity of a protein. In one embodiment, the method is performed in plurality simultaneously. For example, the method can be performed at the same time on multiple assay mixtures in a multi-well screening plate as further described below. Furthermore, in a preferred embodiment, fluorescence or absorbance readouts are utilized to determine enzymatic activity. Thus, in one aspect, the invention provides a high throughput screening system.

In one embodiment, the present invention provides a method of identifying a candidate agent as a modulator of the activity of a target protein. The method comprises adding a candidate agent to a mixture comprising a target protein which directly or indirectly produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate. The method further comprises subjecting the mixture to an enzymatic reaction which uses said ADP or phosphate as a substrate under conditions which normally allow the ADP or phosphate to be utilized and determining the level of activity of the enzymatic reaction. A change in the level between the presence and absence of the candidate agent indicates a modulator of the target protein.

In one aspect, the target protein indirectly produces the ADP or phosphate by producing a substrate for a reaction which produces the ADP or phosphate. In another aspect, the target protein indirectly produces phosphate or ADP or phosphate by regulating an enzyme which produces ADP or phosphate. In yet a further aspect, the target protein directly produces phosphate or ADP.

In another aspect, the invention provides a method of identifying a candidate agent as a modulator of the activity of a target protein wherein the target protein uses ADP or phosphate directly or indirectly. The method comprises adding a candidate agent to a mixture comprising the target protein under conditions which normally allow the utilization of ADP or phosphate. The method further comprises determining the level of utilization wherein a change in the level between the presence and absence of the candidate agent indicates a modulator of the target protein.

In another embodiment provided herein is a method for identifying whether any two target proteins interact. The method comprises providing a first target chimera comprising a functional molecular motor binding domain and a first target protein. The method further comprises providing a second target chimera comprising a functional microtubule stimulated ATPase domain and a second target protein. Additionally, the method comprises combining the first and second target chimeras under conditions which normally allow activity of a motor protein which comprises a molecular motor binding domain and a microtubule stimulated ATPase domain, wherein an increase in motor protein activity indicates interaction between the two target proteins.

In a further embodiment a method is provided for identifying whether a candidate agent is a modulator of at least one of any two target proteins. The method comprises providing a first target chimera comprising a functional molecular motor binding domain and a first target protein and further providing a second target chimera comprising a functional microtubule stimulated ATPase domain and a second target protein. Additionally, the method comprises combining the first and second target chimeras in the presence and absence of a candidate, wherein a change in motor protein activity, which requires both a molecular motor binding domain and a microtubule stimulated ATPase, between the presence and absence of a candidate agent indicates the candidate agent is a modulator of at least one of the target proteins.

Additionally, provided herein is a chimeric protein comprising a functional molecular motor binding domain and a target binding domain wherein the chimeric protein is independent of a functional microtubule stimulated ATPase domain. Also provided herein is a chimeric protein comprising a functional microtubule stimulated ATPase domain and a target binding domain, wherein the chimeric protein is independent of a functional molecular motor binding domain.

In one aspect, a nucleic acid comprising a nucleic acid encoding a chimeric protein in accordance with the present invention is provided. In another aspect a cell comprising a nucleic acid or a chimeric protein in accordance with the present invention is provided.

The present invention provides methods for the identification of candidate agents that bind to a target protein or serve as modulators of the biological activity of a target protein. These assays utilize various methods to measure, in ways amenable to high throughput screening, the generation or consumption of ADP or phosphate. That is, target proteins that either directly or indirectly produce or consume ADP or phosphate may be screened in the present invention. Thus, by providing assay systems that rapidly, efficiently and inexpensively assay ADP or phosphate, modulators (including both antagonists and agonists) of any test protein that directly or indirectly produces ADP or phosphate may be found. The present invention thus utilizes high throughput assays that obviate the traditional cumbersome steps of using gels or radioactive materials.

Accordingly, the present invention provides methods of screening of target proteins. By “target protein” herein is meant a protein that directly or indirectly produces ADP or phosphate. The target proteins can be from eukaryotes or procaryotes, including mammals, fungi, bacteria, insects, and plants, as well as viruses. In a preferred embodiment, the target proteins are from mammalian cells, with rodents (mice, rats, hamsters, guinea pigs and gerbils being preferred), primates and humans being preferred, and humans being particularly preferred.

“Protein” in this context means a compound that comprises at least two covalently attached amino acids and includes proteins, polypeptides, oligopeptides and peptides. The proteins may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.

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