Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-09
2004-02-24
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S254110, C435S325000, C530S300000, C530S350000, C536S023100
Reexamination Certificate
active
06696273
ABSTRACT:
PRIORITY CLAIM
This Application claims the benefit of French Application No. FR00/01628 filed on Feb. 10, 2000 and U.S. Provisional Application No. 60/198,500 filed on Apr. 18, 2000.
Alzheimer's disease (AD) is a neuro-degenerative disease which affects a large proportion of the elderly population. This disease is characterized, clinically speaking, by the loss of cognitive functions, and, neuropathologically speaking, by the presence in the brain of intracellular neurofibrilliary deposits and of extracellular deposits of the &bgr;-amyloid (A&bgr;) peptide, which form amyloid plaques (Yankner, 1996). Amyloid plaques are mainly composed of the A&bgr; peptides having 40 or 42 amino acids, which are generated by a proteolytic process from the precursor protein of the &bgr;-amyloid peptide (APP) (Golde et al., 1992). The extracellular deposits of A&bgr; are specific for AD. They are the early and invariable feature of all the forms of AD, including the hereditary forms. These hereditary forms of the disease appear relatively early on (between 40 and 60 years of age) and are due to mutations in the APP gene and in the presenilin 1 (PS1) and presenilin 2 (PS2) genes. The mutations in these three genes induce changes in the proteolysis of the APP, which lead to an overproduction of A&bgr; and to the early appearance of the pathology and of the symptoms, which are similar to those of the sporadic forms of AD.
Internalization of the membrane APP is a step which is required for the process of proteolysis of the APP (Koo and Squazzo, 1994), which depends on its cytoplasmic domain. Specifically, the deletion of this region of the protein, or the presence of point mutations in the sequence Tyr-Glu-Asn-Pro-Thr-Tyr in the cytoplasmic domain of the APP, induces a considerable decrease in the production of the &bgr;-amyloid peptide (Perez et al., 1999). Several proteins have been identified as interacting with the cytoplasmic domain of the APP; these proteins might thus participate in the regulation of the proteolytic process of the APP and thus in the production of the &bgr;-amyloid peptide. The two protein families FE65 and X11 which interact with the sequence Tyr-Glu-Asn-Pro-Thr-Tyr of the cytoplasmic domain of the APP (Borg et al., 1996, 1998; Bressler et al., 1996; Duilio et al., 1998; Fiore et al., 1995; Guenette et al., 1996; McLoughlin and Miller, 1996; Mercken et al., 1998; Tanahashi and Tabira, 1999a, 1999b and 1999c) should be mentioned. The FE65 protein family consists of three members which are called FE65, COFE65/FE65L1 and FE65L2. The X11 protein family also consists of three members, which are called X11&agr;, X11&bgr;, and X11&ggr;. These two protein (Mercken et al., 1998; Sabo et al., 1999), whereas the overexpression of X11 induces a decrease in the production of the A&bgr; peptide (Borg et al., 1998; Sastre et al., 1998).
Analysis of the primary structure of FE65 indicates that this protein probably plays the role of adapter. Specifically, FE65 contains three protein domains which are involved in protein—protein interactions: a WW domain in the amino-terminal half and two PTB domains (PhosphoTyrosine Binding domain), called PTB1 and PTB2, in the carboxy-terminal half. The construction of deletions has shown that the PTB2 domain of FE65 is involved in the interaction with the cytoplasmic domain of the APP. The WW domain interacts with at least five proteins, of which two have been identified as being the protein Mena (Mammalian homolog of Enabled) (Ermekova et al., 1997). In addition, the PTB1 domain of FE65 interacts with the transcription factor CP2/LSF/LBP1 (Zambrano et al., 1997) and with the receptor LRP (LDL receptor-Related Protein) (Trommsdorff et al., 1998). The role of these proteins in the physiological function of FE65 is not known to date.
The elucidation of the exact role of the FE65 protein in the process of production of the &bgr;-amyloid peptide thus constitutes a major asset for the understanding of, and the therapeutic approach to, Alzheimer's disease and more generally neurodegenerative diseases.
The present invention lies in the identification of partners of the FE65 protein which interact with this protein under physiological conditions. These partners represent novel pharmacological targets for the manufacture or the investigation of compounds which are capable of modulating the activity of FE65, in particular its activity on the production of the &bgr;-amyloid peptide. These proteins, the antibodies, the corresponding nucleic acids and the specific probes or primers can also be used for detecting or for assaying the proteins in biological samples, in particular nervous tissue samples. These proteins or nucleic acids can also be used in therapeutic approaches, to modulate the activity of FE65 and any compound according to the invention which is capable of modulating the interaction between FE65 and the polypeptides of the invention.
The present invention results more particularly from the revelation, by the applicant, of two human proteins which interact with the PTB1 domain of FE65 (represented on the sequence SEQ ID No.:1 and 2). Thus, the present invention shows that the central region of the protein hnRNPL interacts with the PTB1 domain of FE65. It also describes the identification of a novel protein, termed FEBP1 (FE65 Binding PTB1 domain protein), which is capable of interacting with the PTB1 domain of FE65.
The present invention also results from the identification and from the characterization of specific regions of the hnRNPL and FEBP1 proteins above, which are involved in the modulation of the function of the FE65 protein. The demonstration of the existence of these proteins and of regions which are involved in their function makes it possible in particular to prepare novel compounds and/or compositions which can be used as pharmaceutical agents, and to develop industrial methods for screening such compounds.
A first subject of the invention thus relates to compounds which are capable of modulating, at least partially, the interaction of the hnRNPL and/or FEBP1 proteins (or homologs thereof) with the PTB1 domain of FE65, or of interfering with this reaction.
The interference of a compound according to the invention can reveal itself in various ways. The compound according to the invention can slow, inhibit or stimulate, at least partially, the interaction between an hnRNPL and/or FEBP1 protein (or homologs thereof) and the PTB1 domain of FE65. They are preferably compounds which are capable of modulating this interaction in vitro, for example in a system of double-hybrid type or in any acellular system for detecting an interaction between two polypeptides. The compounds according to the invention are preferably compounds which are capable of modulating, at least partially, this interaction, preferably by increasing or inhibiting this interaction by at least 20%, more preferably by at least 50%, with respect to a control in the absence of the compound.
For the purposes of the present invention, the name of the proteins hnRNPL and FEBP1 covers the proteins per se and all homologous forms thereof. “Homologous form” is intended to refer to any proteins which are equivalent to the protein under consideration, of various cellular origin and in particular derived from cells of human origin, or other organisms, and which possess an activity of the same type. Such homologs also comprise the natural variants of the proteins indicated, in particular the polymorphic or splicing variants. The homologous proteins (or polypeptides) can be obtained, for example, by experiments of hybridization between the coding nucleic acids. For the purposes of the invention, a sequence of this type only has to have a significant percentage of identity to lead to a physiological behavior which is comparable to those of the hnRNPL and/or FEBP1 proteins as claimed.
According to a particular embodiment, the compounds of the invention are capable of binding at the level of the domain of interaction between the hnRNPL and/or FEBP1 proteins and the PTB1 domain of FE65
Fournier Alain
Maury Isabelle
Mercken Luc
Aventis Pharma S.A.
Kemmerer Elizabeth
Nichols Christopher James
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