Structure-based identification of candidate compounds using...

Details Structure-based identification of candidate compounds using... Structure-based identification of candidate compounds using...

1999-02-05

2004-01-06

Borin, Michael (Department: 1631)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Chemical analysis

C702S019000, C530S350000

Reexamination Certificate

active

06675105

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to three dimensional structures of Fc receptors (FcR), including crystalline Fc&ggr;RIIa, crystalline Fc&egr;RI, three dimensional coordinates of Fc&ggr;RIIa protein, a three dimensional structure of Fc&ggr;RIIa, three dimensional structures of FcR, and particularly Fc&egr;RI and Fc&ggr;RIIIb, derived from the structure of Fc&ggr;RIIa, models thereof, and uses of such structures and models.
BACKGROUND OF THE INVENTION
Fc receptors (FcR) are a family of highly related receptors that are specific for the Fc portion of immunoglobulin (Ig). These receptors have major roles in normal immunity and resistance to infection and provide the humoral immune system with a cellular effector arm. Receptors have been defined for each of the immunoglobulin classes and as such are defined by the class of Ig of which they bind (i.e. Fc gamma receptor (Fc&ggr;R) bind gamma immunoglobulin (IgG), Fc epsilon receptor (Fc&egr;R) bind epsilon immunoglobulin (IgE), Fc alpha receptor (Fc&agr;R) bind alpha immunoglobulin (IgA)). Among the Fc&ggr;R receptors, three subfamily members have been defined; Fc&ggr;RI, which is a high a affinity receptor for IgG; Fc&ggr;RII, which are low affinity receptors for IgG that avidly bind to aggregates immune complexes; and Fc&ggr;RIII, which are low affinity receptors that bind to immune complexes. These receptors are highly related structurally but perform different functions. The structure and function of Fc&ggr;RII is of interest because of its interaction with immune complexes and its association with disease.
Fc&ggr;R are expressed on most hematopoietic cells, and through the binding of IgG play a key role in homeostasis of the immune system and host protection against infection. Fc&ggr;RII is a low affinity receptor for IgG that essentially binds only to IgG immune complexes and is expressed on a variety of cell types including, for example monocytes, macrophages, neutrophils, eosinophils, platelets and B lymphocytes. Fc&ggr;RII is involved in various immune and inflammatory responses including antibody-dependent cell-mediated cytotoxicity, clearance of immune complexes, release of inflammatory mediators and regulation of antibody production. The binding of IgG to an Fc&ggr;R can lead to disease indications that involve regulation by Fc&ggr;R. For example, the autoimmune disease thrombocytopenia purpura involves tissue (platelet) damage resulting from Fc&ggr;R-dependent IgG immune complex activation of platelets or their destruction by Fc&ggr;R+ phagocytes. In addition, various inflammatory disease are known to involve IgG immune complexes (e.g. rheumatoid arthritis, systemic lupus erythematosus), including type II and type III hypersensitivity reactions. Type II and type III hypersensitivity reactions are mediated by IgG, which can activate either complement-mediated or phagocytic effector mechanisms, leading to tissue damage.
The elucidation of the protein structure of Fc&ggr;RIIa, Fc&egr;RI, or indeed any FcR is of importance in the formulation of therapeutic and diagnostic reagents for disease management. Until the discovery of the present invention, the structure and resulting mechanism by which Fc&ggr;RIIa regulates immune responses was unknown. Thus, despite the general multifunctional role of Fc&ggr;RIIa, development of useful reagents for treatment or diagnosis of disease was hindered by lack of structural information of the receptor. The linear nucleic acid and amino acid sequence of Fc&ggr;RIIa have been previously reported (Hibbs et al.
Proc. Natl. Acad. Sci. USA,
vol. 85, pp. 2240-2244, 1988). Mutagenesis studies to identify regions of human Fc&ggr;RIIa (Hulett et al.,
Eur. J Immunol.
, vol. 23, pp. 40-645, 1993; Hulett et al.,
J. Biol. Chem.
, vol. 69, pp. 15287-15293 1994; and Hulett et al.,
J. Biol. Chem.,
vol. 270, pp. 21188-21194, 1995), human Fc&ggr;RIIIb (Hibbs et al.,
J. Immunol.
, vol. 152, p. 4466, 1994; and Tamm et al.,
J. Biol. Chem.
, vol. 271, p. 3659, 1996) and mouse Fc&ggr;RI (Hulett et al.,
J. Immunol.
, vol. 148, pp. 1863-1868, 1991) have defined important regions of IgG binding to the Fc&ggr;R. Information based on linear sequences, however, cannot accurately predict three dimensional structure of the protein and its functional domains. Huber et al. (
J. Mol. Biol.
, vol. 230, pp. 1077-1083, 1993) have described crystal formation of neonatal rat Fc receptor protein (FcRn). Burmeister et al. (
Nature
, vol. 372, pp. 336-343, 1994; and
Nature
, vol. 372, pp. 379-383, 1994) have described the structure of FcRn crystals. FcRn, however, is closely related to major histocompatability protein complex and not related to the leukocyte Fc&ggr;R family by function or structure. Thus, the protein structure of FcRn is not predictive of the FcR structure of the present invention.
Fc&egr;R are expressed on mast cells, and through the binding of IgE, trigger an inflammatory immune response which is primarily due to the release of inflammatory mediators upon degranulation of the mast cell (e.g., histamine and serotonin). Release of these mediators causes localized vascular permeability and increase in fluids in the local tissues, including an influx of polymorphonuclear cells into the site. Thus, binding of IgE to an Fc&egr;RI can lead to disease indications that involve discharge of fluids from the gut and increased mucus secretion and bronchial contraction, such indications typically being associated with diseases involving allergic inflammation. Therefore, the elucidation of protein structure of Fc&egr;RI is of importance in the formulation of therapeutic and diagnostic reagents for disease management, and in particular, for the management of diseases related to allergic inflammation and other Th2-based immune responses. As for the Fc&ggr;R described above, the linear nucleic acid and amino acid sequences of human Fc&egr;RI have been previously reported (Kochan et al., 1998,
Nuc. Acid. Res.
16:3584). Until the discovery of the present invention, however, the structure and resulting mechanism by which Fc&egr;R regulates immune responses was unknown. Thus, despite the knowledge of the general action of Fc&egr;RI, the development of useful reagents for treatment or diagnosis of disease, such as diseases associated with allergic inflammation, was hindered by lack of structural information of the receptor.
Therefore, there is a need in the art to elucidate the three dimensional structures and models of the Fc receptors, and to use such structures and models in therapeutic strategies, such as drug design.
SUMMARY OF THE INVENTION
The present invention relates to crystalline Fc&ggr;RIIa and crystalline Fc&egr;RI, three dimensional coordinates of Fc&ggr;RIIa protein, the three dimensional structure of Fc&ggr;RIIa, three dimensional structures and models of Fc receptors (FcR) derived from the structure of Fc&ggr;RIIa, including Fc&egr;RI and Fc&ggr;RIIIb, and uses of such structures and models. Obtaining such crystals is an unexpected result. It is well known in the protein crystallographic art that obtaining crystals of quality sufficient for determining the structure of a protein is unpredictable. In particular, obtaining crystals of quality sufficient for determining the three dimensional (3-D) structure of Fc&ggr;RIIa has not been achievable until the crystallization of Fc&ggr;RIIa as disclosed in the present application. As such, determination of the three dimensional structure of Fc&ggr;RIIa has not been possible until the discovery of the present invention. Additionally, until the discovery of the present invention, derivation of the three dimensional structure and models of other Fc receptor (FcR) proteins has not been possible. The present inventors are also the first to define the three dimensional structure and provide three dimensional models for drug design for Fc&egr;RI and Fc&ggr;RIIIb.
Accordingly, one object of the present invention is to provide crystals of sufficient quality to obtain a determination of the three dimensional structure of Fc&ggr;RIIa to high resolut

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