Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-06-08
2004-05-18
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S069200
Reexamination Certificate
active
06737246
ABSTRACT:
The present invention relates to a method for detecting substances of medical value, which act in a novel and advantageous manner and which are protein inhibitors.
Enzymes are high molecular weight proteins present in all cells. Enzymes are biocatalysts that make the numerous biochemical metabolic pathways of life possible. Each enzyme is specific for a very particular reaction. Enzymes interact with substrates, wherein the intermediate formed is an enzyme/substrate complex. Following chemical conversion, the converted substrate formed is released into the medium.
Simple enzymes carry only one binding site to take up the substrate. More complex systems comprise a plurality of covalently or noncovalently linked enzyme domains with binding sites A, B, C, D, etc., which are frequently assigned to one enzyme domain each. Said binding sites in turn recognize in the substrate one or more structural or chemically functional groups Z which may be identical (Z1, Z2, Z3, etc.) or different, i.e. Y, X, W, etc.
The corresponding more complex enzyme reactions are composed of a plurality of partial reactions. Thus, domain A can interact with substrate group Z1. Domain B can likewise react with Z1 or with Z2 or with one or more sites Y, X, etc.
A catalytic domain of a protein or enzyme is an amino acid sequence which binds and chemically converts a substrate. A binding domain of a protein or enzyme is an amino acid sequence which binds a substrate reversibly. In these enzymatic systems, it is also possible for binding sites to recognize and bind one or more ligands instead of the substrate; however, said ligands neither behave like a substrate, i.e. they are not chemically converted, nor do they always behave like inhibitors.
When searching for inhibitors or ligands of complex proteins, usually inhibitors of the catalytic domain are found, because said inhibitors lead to a reduction in the chemical conversion of the substrate. Absent or reduced conversions of the marker substrate can also indicate the presence of said inhibitors. It is substantially more difficult to find inhibitors for the binding domain.
It is therefore the object of the present invention to find substances which reduce or essentially prevent binding of substrates to the binding domain of a protein. Said substance may act as inhibitor or may be a ligand.
The object is achieved by incubating a protein, the marker substrate and the substrate with a substance and by determining whether the marker substrate is converted by the protein.
The invention therefore relates to a method to determine whether a test substance is an inhibitor or a ligand of a protein, which method comprises
a) using a protein which contains at least one catalytic domain and at least one binding domain,
b) using at least one marker substrate which binds to the catalytic domain and is converted,
c) using at least one substrate which can bind to the catalytic domain and to the binding domain,
d) incubating said protein, the marker substrate and the substrate with the substance or ligand, and
e) determining whether the marker substrate is converted by the protein.
Suitable probes are, for example, enzymes which contain at least one catalytic domain and at least one binding domain. The enzymes may, however, also contain 1 to 8 catalytic domains and 1 to 8 binding domains, with the most common enzymes containing 1, 2, 3 or 4 catalytic domains or binding domains. The substrate is a compound which can bind both to the binding domain and to the catalytic domain and which is chemically converted by the catalytic domain but not chemically modified by the binding domain. The marker substrate is a compound which is chemically different from the substrate, is chemically converted by the catalytic domain and allows monitoring of the conversion reaction.
If a test substance binds essentially reversibly or irreversibly to the binding domain of the protein, it may be regarded as an inhibitor of the binding domain of the protein. Not to be limited by theory, inhibition of the binding domain of the protein decreases competition for the catalytic domain of the protein between the marker substrate and the substrate-favoring conversion of the marker substrate-because the substrate generally binds to the binding domain before it is transformed in the catalytic domain of the protein. A desired inhibitor or ligand, would not significantly prevent chemical conversion of the marker substrate by the catalytic domain of the protein because the marker substrate does not need to bind to the binding domain of the protein to be converted. In summary, when an inhibitor or ligand is present, conversion of the substrate by the protein decreases but no inhibition of marker substrate occurs. Conversely, when the test substance is not an effective inhibitor or ligand, there is competition between the marker substrate and the substrate for the catalytic domain of the protein with the end result that conversion of the marker substrate is reduced.
An example of a suitable protein is the enzyme collagenase which comprises at least two covalently linked enzyme domains, one which binds collagen and another which has proteolytic capabilities and cuts the collagen strand. This catalytic proteolytic domain, whether part of the full-length enzyme, i.e. the naturally occurring active enzyme, or just a proteolytic domain prepared by recombination, has the ability to cleave marker substrates of different types.
Examples of suitable marker substrates for collagenase are peptides provided with fluorescent markers or UV markers. Examples of marker substrates for enzymes of this kind are (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH
2
(SEQ ID NO: 1), (C. G. Knight et al, FEBS Letters 296:263-266 (1992)), Dnp-Pro-&bgr;-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-Nh
2
(SEQ ID NO: 2) (D. M. Bickett et al., Analytical Biochemistry 212:58-64(1993)); Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH
2
(SEQ ID NO: 3) (V. Knäuper et al., JBC 271/3, 1544-1550 (1996)); Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys-Dnp-NH
2
(SEQ ID NO: 4) (H. Nagase et al., JBC 269/33, 20952-20957 (1994)); Dnp-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-NH
2
(SEQ ID NO: 5) (L. Niedzwiecki et al., Biochemistry 31 :12618-12623 (1992)) (D. M. Bickett et al., Analytical Biochemistry 212:58-64 (1993); C. G. Knight, et al, FEBS Letters 296:263-266 (1992); V. Knäuper et al., JBC 271/3, 1544-1550 (1996); H. Nagase et al., JBC 269/33, 20952-20957 (1994); L. Niedzwiecki et al., Biochemistry 31:12618-12623 (1992)), or radiolabeled peptides.
Detection of marker substrates is not limited to fluorescence, UV/Vis, or radioactivity measurements but includes any suitable method known in the art for this purpose, such as HPLC, GC among others.
A suitable substrate for collagenase is collagen. Collagen is a proline-rich structural protein (scleroprotein) and the major component of mesenchymal intercellular supportive substances, which protects against enzymatic attacks. Three protein chains with left-handed helical structure are twisted into a right-handed triple helix (superhelix). 18 collagen types (collagen type I to type XVIII) have been identified, which can be classified according to structure or function into fibrillar, fibril-associated and nonfibrillar collagens.
A particularly suitable substrate for collagenase is type II collagen. This collagen supports the cartilage matrix in joints. In certain diseases such as osteoarthritis and rheumatism the joints are destroyed, in particular due to proteolytic degradation of collagen by collagenases. Inhibitors of enzymes of this kind are known but have the disadvantage of attacking the catalytic domain of the enzyme (K. U. Weithmann et al., Inflamm. Res. 46:246-252 (1997)). Said catalytic domain is part, in a similar structure, of many enzymes and, as a result, the inhibitors act in an unwanted manner upon many enzymes, including those having a vital function (I. Massova et al., The FASEB Journal 12:1075-1095 (1998)). It is the object of the present inven
Aventis Pharma Deutschland GmbH
Gitomer Ralph
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