Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2002-11-07
2003-10-07
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S006120, C435S007210, C530S350000
Reexamination Certificate
active
06630297
ABSTRACT:
BACKGROUND OF THE INVENTION
The superfamily of small (21 kDa) GTP binding proteins (small G proteins) comprises 5 subfamilies: Ras, Rho, ADP ribosylation factors (ARFs), Rab, and Ran, which act as molecular switches to regulate numerous cellular responses. Members of the Rho family of GTPases, include RhoA, -B, and -C, Rac1 and −2, and Cdc42. Guanine nucleotide exchange factors (GEFs) activate Rho proteins by catalyzing the replacement of bound GDP with GTP. The GTP-bound form of Rho proteins specifically interact with their effectors or targets and transmit signals to downstream molecules. Rho proteins are inactivated through the hydrolysis of bound GTP to GDP by intrinsic GTPase activity, assisted by GTPase activating proteins (GAPs). The Rho family of GTPases participate in regulation of the actin cytoskeleton and cell adhesion, and are also involved in regulation of smooth muscle contraction, cell morphology, cell motility, neurite retraction, cytokinesis, and cell transformation (Hall, A. Science (1998) 279:509-514).
Ect2, a transforming protein with sequence similarity to the dbl homology (DH) domain proteins, is a GEF that associates with a subset of the Rho family proteins: RhoA, Cdc42, and Rac1. Ect2 phosphorylation, which is required for its exchange activity, occurs during G2 and M phases. Human Ect2 is involved in the regulation of cytokinesis. The human ect2 gene is located on the long arm of chromosome 3, at 3q26 (Takai S, et al., Genomics (1995) 27(1):220-222), a region of increased copy number and expression in a large number of cancers (Bitter M A, et al., Blood (1985) 66(6):1362-1370; Kim D H, et al., Int J Cancer. (1995) 60(6):812-819; Brzoska P M, et al., Cancer Res. (1995) 55(14):3055-3059; Balsara B R, et al., Cancer Res. (1997) 57(11):2116-2120; Heselmeyer K, et al., Genes Chromosomes Cancer (1997) 19(4):233-240; Sonoda G, et al., Genes Chromosomes Cancer. (1997) 20(4):320-8). Data available from the National Cancer Institute (website at ncbi.nlm.nih.gov
cicgap) indicates that human ect2 is overexpressed in cancers of the ovary, uterus, parathyroid, testis, brain, and colon.
The ect2 gene is conserved at the sequence and functional levels in mammals and insects. The pebble gene in Drosoplila (GenBank ID # (GI) 5817603) is the orthologue of mouse (GI293331) and human ect2, and is required for initiation of cytokinesis (Lehner C F, J. Cell Sci. (1992) 103: 1021-1030; Prokopenko S N, et al., Genes Dev (1999) 13(17):2301-2314).
SUMMARY OF THE INVENTION
The invention provides isolated human Ect2 protein and its splice variant as well as fragments and derivatives thereof. Vectors and host cells expressing Ect2 molecules, as well as methods of production of Ect2 and methods of production of cells for expressing Ect2 are also described.
The invention further provides methods of screening for agents that modulate the interaction of an Ect2 polypeptide with an Ect2 binding target. In one aspect, the screening method comprises the steps of expressing a recombinant Ect2 polypeptide, incubating the polypeptide and the Ect2 binding target with a candidate agent and determining whether the candidate agent modulates the binding of the Ect2 polypeptide with the Ect2 binding target. Preferred modulating agents include Ect2-specific antibodies and small molecules identified in high throughput screens.
The invention further provides novel high throughput assays to measure Ect2 activity.
DETAILED DESCRIPTION OF THE INVENTION
The ability to screen or manipulate the genomes of model organisms provides a powerful means to analyze complex genetic pathways. In particular, overexpression screens in Drosophila enable quick identification of genes involved in the same or overlapping pathways as human genetic pathways (Rorth P., et al., Development (1998) 125:1049-1057; WO0015843). We performed an overexpression screen in Drosophila to identify genes that interact with the cyclin dependent kinase inhibitor, p21 (Boume H R, et al., Nature (1990) 348(6297):125-132; Marshall C J, Trends Genet (1991) 7(3):91-95) Pebble, the Drosophila orthologue of human Ect2, was identified as a suppressor of p21 overexpression. To our knowledge, there are no prior reports in the literature of a link between Ect2 and the G1 phase of the cell cycle, or any evidence that suggests that overexpression of Ect2 can overcome a block in the cell cycle. Our identification of an Ect2 orthologue in the Drosophila p21 screen supports both conclusions. Thus, Ect2 is a valuable “target” that can be used to identify compounds and other agents that modulate its function, and thus have utility in treatment of disease or disorders associated with defective cell cycle progression at G1 phase, and in particular, defective p21 function.
Ect2 Nucleic Acids and Polypeptides
We identified cDNA sequences of human ect2 and a splice variant (SEQ ID NO:1 and SEQ ID NO:3, respectively) through bioinformatic analysis of public databases and “contigging” several incomplete EST sequences (AW965920, AI916675, AW504786, BE080710, AW504433, BE080860, AW970802, AA279942, AA206473, AA313301). Northern Blot analysis of mRNA from tumor samples, using full or partial ect2 cDNA (SEQ ID Nos:1 and 3) sequences as probes (Current Protocol in Molecular Biology, Eds. Asubel, et al., Wiley Interscience, NY), can identify tumors that overexpress Ect2, and that, therefore, are amenable to treatment by inhibition of Ect2 function. Alternatively, quantitative PCR, such as the TaqMan® procedure (PE Applied Biosystems) is used for analysis of Ect2 expression in tumor samples.
The term “Ect2 polypeptide” refers to a full-length Ect2 protein or a fragment or derivative thereof. A preferred Ect2 polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:2 or 4, or a fragment or derivative thereof. Compositions comprising Ect2 polypeptides may consist essentially of the Ect2 protein, fragment, or derivative, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, etc.).
Ect2 protein derivatives typically share a certain degree of sequence identity or sequence similarity with SEQ ID NOs:2 or 4, or a fragment thereof. As used herein, “percent (%) sequence identity” with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410; website at blast.wustl.edu/blast/README.html) with search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A “% identity value” is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. “Percent (%) amino acid sequence similarity” is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation. A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid
Joly Alison
Keegan Kevin Patrick
Matthews David
Ollmann Michael Martin
Stout Thomas J.
Brunelle Jan
Exelixis Inc.
Kemmerer Elizabeth
Li Ruixiang
Shayesteh Laleh
LandOfFree
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