Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-04-01
2002-06-25
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S069100, C436S518000, C424S141100, C536S023100, C536S023500
Reexamination Certificate
active
06410245
ABSTRACT:
TECHNICAL FIELD
The invention relates to the fields of molecular genetics and pharmacology. The invention provides methods and compositions for determining the capability of a compound, a macromolecular species, or of a stimulatory effector to produce a conformational change in a predetermined nuclear receptor and/or its accessory protein(s), typically to agonize or antagonize a ligand-induced activation of the nuclear receptor. An aspect of the invention can provide a means for identifying agents which are pharmacological agonists or antagonists for one or more predetermined nuclear receptor species. An aspect of the invention relates to a method of rank-ordering a set of compounds with respect to each compound's ability to affect an interaction between one or more nuclear receptor species and a plurality of coactivator and/or corepressor species or similar interfaces; the rank-ordering provides a dataset for identifying pharmacologically active agonists, antagonists, partial agonists, potentiators, and the like.
BACKGROUND OF THE INVENTION
A variety of nuclear receptors exist in animal cells and generally function to effectuate transcriptional regulation on one or more subsets of regulable genes. Most often, the nuclear receptor exhibits a high-affinity binding interaction with one or more species of hydrophobic ligand. These ligand binding interactions can produce a conformational change in the nuclear receptor. The conformational change induced by ligand binding modifies the ability of the nuclear receptor to interact with certain specific receptor-binding DNA sequences and/or to interact with other nuclear proteins (e.g., transcription factors, coactivators, corepressors), so as to modulate the transcription of genes having the specific receptor-binding DNA sequence(s) located so as to influence the transcription of the gene. In one model of steroid receptor action, a ligand-induced conformational change in a ligand-binding domain unmasks a DNA-binding activity in another structural domain of the steroid receptor protein. In the absence of this ligand-induced conformational change, the ligand-binding domain represses the DNA binding activity of the linked structural domain. It has been recently shown that one or more superfamily of proteins, termed “coactivators”, and “corepressors”, respectively interact with nuclear receptors in a ligand-dependent fashion so as to effect transcriptional activation (coactivators) or so as to inhibit or silence transcription (corepressors) of genes which are transcriptionally modulated by nuclear receptors.
Nuclear hormone receptors comprise a superfamily of over 40 transcription actors. About half of them are classical receptors for lipophilic ligands such as steroids and vitamins. The nuclear hormone receptor gene superfamily encodes structurally related proteins that regulate transcription of target genes. These macromolecules include receptors for steroid and thyroid hormones, vitamins, retinoids, fatty acids, and other nuclear receptor proteins for which no ligands have been found, so-called “orphan receptors”. These receptors have modular domains with readily identifiable structural features and sequence motifs. The DNA-binding domain (“DBD”) directs the receptors to bind specific DNA sequences as monomers, homodimers, or heterodimers. The ligand-binding domain (“LBD”) responds to binding of the cognate hormone; this domain and the amino terminal domain interact with other transcription factors, and with the coactivators and/or corepressors. Nuclear receptor-specific actions are derived from a combination of diverse elements, including availability of ligand, receptors, and nonreceptor factors; target-site structure; interactions with other proteins, such as the general transcription factors and very importantly with the coactivator and/or corepressor proteins.
The steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors encompasses not only the receptors for steroids, thyroid hormone, retinoids and vitamin D, but also a large number of proteins whose functions and/or ligands are unknown and which are thus termed orphan receptors. This family of transcription factors integrates signals from ligands as well as from signal transduction pathways, resulting in alterations in mRNA and protein expression that are unique to the complex signals received. These nuclear receptors are implicated in the control of a wide range of physiological responses and homeostatic conditions, including cell differentiation, neoplasia, control of cellular metabolism, and neurological function. For a review of the steroid hormone receptor superfamily, see Ribiero R C (1995) Annu. Rev. Med. 46: 443-453.
There has been substantial interest in identifying ligands which interact with nuclear receptors and modulate the biological effects mediated by these nuclear receptors. Such ligands, whether agonistic or antagonistic to natural physiological ligands of the receptors, would serve as candidate pharmaceuticals for controlling the biological effects of nuclear receptor-mediated transcriptional control and the attendant physiological effects produced thereby. Unfortunately, most conventional assays for identifying potential ligands rely upon the use of libraries of radiolabeled compounds which are tested for their binding coefficient (e.g., via Scatchard analysis) to a purified nuclear receptor species. It is difficult and labor-intensive to obtain such libaries of radiolabeled compounds and then screen the library using binding assays. Furthermore, it has been found that a compound's binding constant is not necessarily predictive of its biological activity as a ligand. As a better proxy for ligand function, transcriptional assays have been developed to assay for ligand-induced transcriptional activation of a nuclear receptor as detected by transcription of a reporter sequence operably linked to a nuclear receptor response element and promoter.
Unfortunately, many of the transcriptional responses generated by ligand-activated nuclear receptors can be subtle and are frequently difficult to detect and/or quantify by conventional transcriptional assay procedures, which are relatively insensitive for monitoring expression of genes which are not abundantly transcribed. Furthermore, many of the conventional transcription assay procedures are difficult to perform and entail problematic steps, such as requiring lysis of the cells being assayed. It is desirable to have a method for detecting ligands of predetermined nuclear receptors with high specificity, sensitivity, and selectivity. In particular, methods to reduce readout background noise that can obscure legitimate signals would find great use in the art for identifying novel pharmaceutical agents that are nuclear receptor ligands, as well as providing sensitive assays for detection and quantitation of ligands which are environmental pollutants that activate nuclear receptors (e.g., TCDD).
Moreover, many nuclear receptor ligands, particularly steroids and steroid-like compounds, often exhibit pleiotropic biological effects through nuclear receptors. For example, both estradiol and tamoxiphen bind to the estrogen receptor, but each compound can produce different biological effects and transcriptional profiles (i.e., the set of genes which are transcriptionally modulated by ligand presence) depending upon the tissue and cell-type involved.
Thus, there exists a need in the art for methods to efficiently identify agents which modulate nuclear receptor function. It is important that such methods have the necessary levels of sensitivity and specificity for identifying bona fide nuclear receptor agonists and/or antagonists. The present invention fulfills these and other needs in the art.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
SUMMARY OF THE INVENTION
In acco
Hart Charles P.
Northrop Jeffrey P.
Schatz Peter J.
Affymax, Inc.
Celsa Bennett
Townsend and Townsend / and Crew LLP
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