Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Tripeptides – e.g. – tripeptide thyroliberin – etc.
Reexamination Certificate
2001-05-24
2003-07-15
Weber, Jon P. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Tripeptides, e.g., tripeptide thyroliberin , etc.
C514S018700
Reexamination Certificate
active
06593454
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention generally relates to assays for enzyme activity and for enzyme inhibitors. More specifically, the invention relates to assays for detecting methionine aminopeptidases and inhibitors of methionine aminopeptidases.
(2) Description of the Related Art
In all living cells, protein synthesis is initiated with an AUG codon, specifying methionine as the N-terminal amino acid in nascent proteins. In both prokaryotes and eukaryotes, this N-terminal methionine will be removed by a methionine aminopeptidase (MAP) (EC 3.4.11.18) if the penultimate amino acid residue is small and uncharged, e.g., Gly, Ala, Ser, Cys, Thr, Pro, and Val, although methionine cleavage activity by MA is reduced when the N-terminal three amino acids are Met-Thr-Pro or Met-Val-Pro (Moerschell et al., 1990,
J. Biol. Chem.
265, 19638-19643; Tsunasawa et al., 1985,
J. Biol. Chem.
260, 5382-5391). Removal of the N-terminal methionine is essential for certain proteins to function normally in vivo. For example, the removal of the initiator methionine is often required for subsequent N-terminal modifications, such as N-myristoylation, which is essential for the normal function of various signal transduction proteins, cancer cells, protein targeting moieties, and enzymes (Gordon et al., 1991,
J. Biol. Chem.
266, 8647-8650; Duronio et al., 1989,
Science
243, 796-800).
Methionine aminopeptidases have been isolated and cloned from several organisms, including
E. coli
and several other eubacteria, yeast, rat, and various archaea. Currently discovered MAPs have been categorized into two types, type 1 MAP and type 2 MAP, based on structural and sequence similarities. Eubacteria have type 1, archaea have type 2, and eukaryotes have both types. In eukaryotes, null mutants in either type are viable but slow growing, but null mutants of both MAP types are nonviable (Li and Chang, 1995,
Proc. Natl. Acad. Sci. USA
92, 12357-12361; Li and Chang, 1996,
Biochem. Biophys. Res. Commun.
227, 152-159; Bradshaw et al., 1998,
Trends Biochem. Sci.
21, 285-286). Similarly, knockouts of the bacterial MAP1 gene are lethal (Ben-Bassat et al., 1987,
H. Bacteriol.
169, 751-757). Thus, MAP activity is essential for normal functioning of prokaryotic and eukaryotic cells.
Aside from their role in cleaving the initiator methionine of proteins, MAPs affect other cellular functions. For example, human type 2 MAP also serves as eukaryotic initiation factor-2, which regulates protein synthesis (U.S. Pat. No. 5,885,820). Also, the mode of action of fumagillin-type angiogenesis inhibitors is the irreversible inhibition of type 2 MAP (Griffith et al., 1997,
Chem. Biol.
4, 461-471; Liu et al., 1998,
Science
282, 1324-1327; Lowther et al.,
Proc. Natl. Acad. Sci. USA
95, 12153-12157; Sin et al., 1997,
Proc. Natl. Acad. Sci. USA
94, 6099-6103) thus indicating an essential role of type 2 MAP in angiogenesis.
Because of the crucial role of MAPs in prokaryotic and eukaryotic functions, there is an interest in the discovery of additional inhibitors of these enzymes, which may serve as antibiotics or as chemotheraputic agents which inhibit angiogenesis in tumors. However, current methods for monitoring MAP activity are inadequate for this task. These methods include the colorimetric ninhydrin method (Doi et al., 1981,
Anal. Biochem.
118, 173-184); amino acid oxidase treatment followed by peroxidase reaction and o-dianisidine color development (Carter and Miller, 1984,
J. Bacteriol.
159, 453-459); amino acid analysis via ion exchange chromatography followed by postcolumn derivatization with ninhydrin (Moore et al., 1958,
Anal. Biochem.
30, 1185-1190), fluorescamine (Stein et al., 1973,
Arch. Biochem. Biophys.
155, 202-212), or o-phthalaldehyde/&bgr;-mermercaptoethanol (Roth, 1971,
Anal. Chem
43, 880-882); precolumn derivatization of amino acids followed by reverse phase HPLC chromatography (Cohen and Strydom, 1988,
Anal. Biochem.
174, 1-16; Zuo et al., 1994,
Anal. Biochem.
222, 514-516); and separation of substrate peptides and products by reverse phase HPLC with on-line UV detection of each separated compound (Larrabee et al., 1999,
Anal. Biochem.
269, 194-198; Walker et al., 1999,
Biotechnol. Appl. Biochem.
29, 157-163). These methods are either not sensitive or accurate enough for quantitative assays, or are not rapid enough for high-throughput screening procedures. Thus, there is a need for new assays for MAP activity which are rapid and quantitative enough for use in procedures requiring high-throughput MAP analysis, such as screening for MAP inhibitors.
SUMMARY OF THE INVENTION
Accordingly, the inventor has succeeded in inventing a novel MAP assay which is rapid, quantitative, and suitable for automated procedures. The assay employs a second peptidase and a peptide which comprises an N-terminal methionine which can be cleaved by MAP, along with a C-terminal detection moiety which can be released from the peptide by the second peptidase only if the N-terminal methionine has been cleaved from the peptide.
Thus, one embodiment of the invention is directed to methods for detecting methionine aminopeptidase (MAP) activity in a composition. These methods comprise (a) combining the composition with a peptide comprising an N-terminal methionine under conditions that the N-terminal methionine can be cleaved from the peptide by a MAP to produce a cleaved peptide, wherein the peptide contains a C-terminal detection moiety which is released by a second peptidase only if the N-terminal methionine has been cleaved from the peptide; (b) reacting any cleaved peptide produced in (a) with the second peptidase to release the detection moiety; and (c) detecting any detection moiety released. A preferred second peptidase is dipeptidyl peptidase IV. When dipeptidyl peptidase IV is utilized, a preferred peptide comprises Met-X
aa
-Pro, wherein X
aa
is Ala, Cys, Gly, or Ser; a most preferred peptide is Met-Gly-Pro-p-nitroanilide.
The present invention is also directed to methods for determining whether a substance inhibits a MAP. The methods comprise (a) combining the substance, the MAP, and a peptide comprising an N-terminal methionine under conditions that the N-terminal methionine can be cleaved from the peptide by the MAP to produce a cleaved peptide, wherein the peptide contains a C-terminal detection moiety which is released by a second peptidase only if the N-terminal methionine has been cleaved from the peptide; (b) reacting any cleaved peptide produced in (a) with the second peptidase to release the detection moiety; and (c) detecting any detection moiety released. As with the previously described method, a preferred second peptidase is dipeptidyl peptidase IV, and a preferred peptide is Met-Gly-Pro-p-nitroanilide. This method also preferably comprises quantitating the amount of detection moiety released, in order to more accurately detect MAP inhibitors.
In another embodiment, the present invention is directed to reaction mixtures suitable for use in the methods described above. The reaction mixture comprises (a) a peptide comprising an N-terminal methionine which can be cleaved by the methionine aminopeptidase, and a C-terminal detection moiety which can be released by a second peptidase only if the N-terminal methionine has been cleaved from the peptide; and (b) the second peptidase. A MAP may also be included in the mixture.
In an additional embodiment, the present invention is directed to a kit for performing the methods described above, comprising the second peptidase and the peptide described in those methods, along with instructions for performing the methods. Preferably, the kit also comprises a MAP.
The present invention is also directed to the peptides described in the above embodiments.
Among the several advantages achieved by the present invention, therefore, may be noted the provision of methods and reagents for rapidly detecting and quantifying MAP activity and MAP inhibitor activity. These methods are more suitable than previously known method
Saint Louis University
Thompson & Coburn LLP
Weber Jon P.
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