Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1999-03-04
2001-07-17
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S193000, C435S004000, C436S063000, C436S064000, C530S350000
Reexamination Certificate
active
06261793
ABSTRACT:
TECHNICAL FIELD
The present invention relates to human ras converting endoprotease (RCE). More particularly, it relates to methods of using human RCE in screening systems to identify inhibitors of human RCE useful for the treatment of various medical conditions. This invention further relates to inhibitors of human RCE.
BACKGROUND OF THE INVENTION
Mutations in Ras proteins are found in more than 30% of all human cancers, including greater than 90% of pancreatic cancers, 50% of colon cancers and 30% of lung adenocarcinoma. Ras genes encode a family of guanine nucleotide-binding proteins that, to be functional, must be associated with the inner surface of the plasma membrane. Ras proteins lack the conventional transmembrane or hydrophobic sequences associated with other membrane-associated proteins and are initially synthesized as soluble, cytoplasmic proteins. Their membrane association is triggered by a series of post-translational processing steps involving a carboxy terminal motif referred to as the CaaX box. The CaaX box consists of a conserved cysteine residue, two aliphatic amino acids, and a carboxy-terminal amino acid residue. The post-translational steps required for the attachment of Ras proteins to the inner plasma membrane are: (i) the transfer of farnesyl pyrophosphate (a 15-carbon isoprene lipid) or geranylgeranyl moiety (a four-isoprene unit molecule) onto the cysteine in the carboxy terminal “CaaX” motif by a prenyl transferase i.e., farnesyl protein transferase (FPT) or geranylgeranyl protein transferase (GGPT); (ii) proteolytic cleavage of the three carboxy terminal amino acid residues by Ras Converting Endoprotease (RCE); and(iii) methylation of the resulting carboxy terminal prenyl cysteine residue by prenyl cysteine specific Carboxymethyltransferase (PC-CMT).
Cleavage by RCE modulates Ras function in yeast, therefore it may be a novel target to modulate oncogenic Ras in human tumors. Null mutations in RCE cause no obvious growth or viable defects, whereas mutations in FPT cause cells to be either growth defective or dead. If these results in yeast translate to human cells, inhibitors of RCE may be safer therapeutic agents than inhibitors of FPT.
Accordingly, the identification of human RCE will provide a critical tool necessary for the development of inhibitors of RCE which represent novel therapeutic agents for the treatment of human cancers.
SUMMARY OF THE INVENTION
The present invention provides human ras converting endopeptidase (RCE) having the amino acid sequence of SEQ ID NO: 2. Also provided are isolated nucleic acids encoding human RCE.
In addition, this invention provides methods of identifying inhibitors of human RCE comprising:
(a) contacting a prenylated protein, wherein one or more of the three carboxy terminal amino acid residues of the prenylated protein is radiolabeled, with a sample to be tested for the presence of a RCE inhibitor, and with human RCE having the amino acid sequence of SEQ ID NO: 2; and
(b) measuring the amount of labeled tripeptide released.
whereby the human RCE inhibitor in the sample is identified by measuring the amount of labeled tripeptide released, compared to what would be measured in the absence of such inhibitor.
Also provided are methods of identifying inhibitors of human RCE comprising:
(a) contacting a prenylated peptide with: (i) a sample to be tested for the presence of a RCE inhibitor; (ii) human RCE having the amino acid sequence of SEQ ID NO: 2; (iii) prenyl cysteine specific carboxymethyltransferase; and (iv) a radiolabeled methyl group donor; and
(b) measuring the amount of the radiolabeled methyl group incorporated into the carboxy terminal of the prenylated peptide
whereby the human RCE inhibitor in the sample is identified by measuring the reduction in the amount of radiolabeled methyl group incorporated into the prenylated peptide, compared to what would be measured in the absence of such inhibitor.
In a preferred embodiment, membranes isolated from cells expressing a nucleic acid encoding human RCE are used as the source of human RCE.
The present invention also contemplates molecules that specifically inhibit the activity of human RCE. Such molecules include small organic molecules, peptides, and antibodies or antigen binding fragments of antibodies which specifically inhibit human RCE activity. In a preferred embodiment the human RCE inhibitors are orally active, small organic molecules.
This invention also contemplates pharmaceutical compositions, for use in treating human cancers, comprising: (a) an effective amount of a human RCE inhibitor; and (b) a pharmaceutically acceptable carrier.
This invention further provides a method for treating human cancers comprising administering to a subject afflicted with cancer a pharmaceutical composition comprising: (a) an effective amount of a human RCE inhibitor; and (b) a pharmaceutically acceptable carrier.
This invention also provides anti-sense oligonucleotides capable of specifically hybridizing to mRNA encoding human RCE having the amino acid sequence defined by SEQ ID NO: 2 so as to prevent translation of the mRNA. Additionally, this invention provides anti-sense oligonucleotides capable of specifically hybridizing to the genomic DNA molecule encoding human RCE having an amino acid sequence defined by SEQ ID NO: 2.
DESCRIPTION OF THE INVENTION
All references cited herein are hereby incorporated in their entirety by reference.
Cloning of Human Ras Converting Endoprotease
Yeast RCE1 amino acid sequence was used to query dbEST databases. Partial cDNAs for the human homolog of yeast RCE was found from dbEST [W96411 (p=2e-19); human and AA168614 (p=2e-20); mouse]. We obtained a human RCE homolog clone (358628) from the IMAGE Consortium, and sequenced it to confirm its homology to yeast RCE.
Northern analysis was performed to determine tissue distribution of human RCE using IMAGE clone 358628 as the probe. RCE expression appeared highest in placenta, with a transcript size of approximately 2.0 Kb. A human placenta phage library was screened to obtain a full length clone of human RCE. Six positive clones were found. Sequencing of these clones confirmed their homology to yeast RCE, IMAGE clone 358628, and to each other.
One of the positive clones, designated hRCE-7, was chosen for use in cloning and expression studies due to the presence of a start codon at the 5′ end, a coding region containing a stop codon, and a 3′ untranslated region including a poly A
+
tail. To demonstrate expression of human RCE, hRCE-7 was cloned into mammalian, bacterial, and yeast expression vectors. Epitope tags were cloned onto the amino and carboxy terminals of hRCE-7 for Western detection. The construct pDD1-hRCE containing the coding region of hRCE-7 with a 5′ S-tag in a yeast expression vector was used to transform a yeast Rce/Afc double knock out, and the resulting yeast clone (136B) was positive in both the Halo and biochemical assays.
The nucleotide sequence of the complete open reading frame and the corresponding amino acid sequence of human RCE cDNA cloned from the human placental cDNA library are defined in the Sequence Listing by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
Protein Purification
The proteins, polypeptides and fragments of this invention can be purified by standard methods, including but not limited to salt or alcohol precipitation, preparative disc-gel electrophoresis, isoelectric focusing, high pressure liquid chromatography (HPLC), reversed-phase HPLC, gel filtration, cation and anion exchange and partition chromatography, and countercurrent distribution. Such purification methods are well known in the art and are disclosed, e.g., in
Guide to Protein Purification, Methods in Enzymology
, Vol. 182, M. Deutscher, Ed., 1990, Academic Press, New York, N.Y.
Nucleic Acids and Expression Vectors
As used herein, the term “isolated nucleic acid” means a nucleic acid such as an RNA or DNA molecule, or a mixed polymer, which is substantially separated from other components that are normally found in cells or in r
Hockenberry Tish
McGuirk Marnie
Nuñez-Oliva Irma
Pai James
Whyte David
Caputa Anthony C.
Holleran Anne L.
McLaughlin Jaye
Schering Corporation
Thampoe Immac J.
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