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Inhibitors of proteins from the rho-gef familyRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidInhibitors of proteins from the rho-gef family description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060210973, Inhibitors of proteins from the rho-gef family. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present invention relates to inhibitors of proteins from the Rho-GEF family. [0002] The present invention also relates to inhibitors of the protein Trio, and to the corresponding nucleotide sequences. The present invention also relates to the use of these inhibitors for the preparation of drugs. [0003] Rho-GTPases are molecular switches that control actin cytoskeleton modifications during proliferation, transformation, cell migration and morphogenesis (Hall, A., 1998). They cycle between an inactive GDP-bound and an active GTP-bound form. Guanine nucleotide exchange factors (Rho-GEFs) accelerate their GDP/GTP exchange, rendering the GTPase active (Boguski et al., 1993). The large family of Rho-GEFs, like their GTPase targets, has been involved in a variety of cellular processes: several members of the Rho-GEF family were identified on the basis of their oncogenic properties, and numerous reports suggest that they are also involved in neuronal morphology (Stam et al., 1999). [0004] Rho-GEFs usually share a conserved catalytic region termed the DH domain (for Dbl-Homology) in reference to the oncogene Dbl, which was one of the first Rho-GEF characterized (Hart et al., 1994). [0005] A complex protein, named Trio, isolated by its capacity to bind to the transmembrane tyrosine phosphatase LAR, is a protein containing two Rho-GEF domains and one serine kinase domain, spectrin repeats, SH3 and an Immunoglobulin-like domains (Debant et al., 1996). The first Rho-GEF domain (TrioGEFD1) activates RhoG (Blangy et al., 2000), which in turn activates Rac and Cdc42, and promotes anchorage-independent cell growth (Seipel et al., 1999). In contrast, the second Rho-GEF domain (TrioGEFD2) acts specifically on RhoA (Debant et al., 1996), indicating that Trio is a multifunctional protein able to link several Rho-GTPase pathways in vivo (Bellanger et al., 1998). In addition, TrioGEFD1 binds to the actin binding proteins filamin and Tara, providing a direct link between Trio and the actin cytoskeleton (Bellanger et al., 2000; Seipel et al., 2001). [0006] The understanding of Trio function made significant progress with recent data obtained from different studies of Trio family members. The C. Elegans and Drosophila Trio proteins are involved in axon guidance and cell migration (Steven et al., 1998; Bateman et al., 2000; Liebl et al., 2000; Newsome et al., 2000; Awasaki et al., 2000). TrioGEFD1 seems to play a major role in this process, whereas the function of TrioGEFD2 in the context of the full-length protein is unknown. Consistently, the rat Trio-like protein Kalirin is also involved in neuronal function (Penzes et al., 2001). Finally, the knock out of the trio gene in mouse was reported to be lethal, and trio -/- embryos display defects in neuronal organization and in muscle development (O'Brien et al., 2000). [0007] The large Dbl family is involved in numerous physiological processes, and no specific inhibitors exist so far. Rho-GEFs, such as Trio, display multiple catalytic domains, whose relative contribution to the protein function is not always understood. Specific inhibitors of a given catalytic domain would thus represent powerful tools for precise structure-function studies and may be used as dominant negative inhibitors. [0008] The aim of the present invention is to provide specific in vivo inhibitors of a Rho-GEF family member. [0009] Another aim of the invention is to provide new peptides that inhibit a protein of the Rho-GEF family, particularly the protein Trio. [0010] Another aim of the invention is to provide specific in vivo inhibitors of a Rho-GEF family member, blocking specifically the RhoA pathway. [0011] Another aim of the invention is to provide specific inhibitors of the GEFD2 domain of Trio. [0012] Another aim of the present invention is to provide inhibitors of TrioGEF2, in order to develop drugs to protect axons from retraction. [0013] The present invention concerns the use of an aptamer library for determining inhibitors of any one of the proteins from the Rho-GEF family, by an appropriate screening process, said aptamer library being such as defined in Geyer et al. (2000). [0014] The large family of Rho-GEF proteins is involved in numerous physiological processes, but no specific inhibitors exist so far. Crystallographic studies of Rho-GEFs in complex with their GTPase targets indicate that numerous interaction sites between GEF and GTPase exist, rendering the prediction of an efficient inhibitor impossible. This makes the use of the aptamer library containing a large number of peptides, the sequences of which have not been determined, unobvious. [0015] The aptamer library used in the present invention is the one described in Colas et al. (1996) or in Geyer et al. (2000). [0016] It is constituted of random peptides inserted in thioredoxin. [0017] The strategy of the aptamer library developed by P. Colas et al. (1996) is based on the fact that peptide loops that are anchored at both their amino and carboxy termini are capable of specific and high-affinity molecular recognition. The constructed library directs the synthesis in yeast of 20-residue peptides of random sequence inserted in the active site (residue 35) of the E. coli thioredoxin A and fused to a modified set of protein moieties from the original yeast two-hybrid system (Colas et al., 1996; patent no WO 96/02561 by Brent et al.). The library contains 2,9.10.sup.9 members of these constrained random peptides and it can be obtained such as described in the international application WO 96/02561. [0018] The proteins from the Rho-GEF family can be chosen among the Dbl family members, that invariably contain a DH domain (for Dbl-Homology domain, in reference to the first Rho-GEF identified) that harbours the catalytic activity, and a PH domain, thought to be involved mainly in subcellular targeting. More than 40 Rho-GEFs have been identified in mammals. [0019] An appropriate screening process comprises the following steps: [0020] i) the appropriate bait is established, in this case the DH-PH module of the Rho-GEF or possibly the DH domain alone; [0021] ii) the library is then screened with the selected bait to isolate aptamers that specifically bind to the bait; [0022] iii) the selected aptamers are then tested for their capacity to inhibit the catalytic activity of the appropriate RhoGEF to determine inhibitory aptamers; for that purpose, the selected aptamers are expressed as GST-fusion proteins, and their capacity of inhibition is tested using the in vitro GEF assay (see Examples); [0023] iv) the inhibitory aptamers are then tested for their capacity to inhibit in intact cells the activity of the Rho-GEF on the appropriate GTPase, using the GTPase activity assay (Benard et al., 2002); [0024] v) the inhibitory aptamers selected at stage iv) are finally tested for their capacity to inhibit the physiological effect of the RhoGEF in intact cells; for that purpose, the aptamer is expressed as a GFP-fusion protein and transfected into cells, or possibly transduced into cells with fusion peptides or adenovirus infection (see Examples). 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