Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-10-05
2004-02-10
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200
Reexamination Certificate
active
06689574
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods of identifying novel agonists and antagonists of nuclear receptors utilizing the agonist-dependent interaction of such receptors with CREB-binding protein (CBP) or other nuclear receptor co-activators in which this interaction is detected by fluorescence resonance energy transfer.
BACKGROUND OF THE INVENTION
Nuclear receptors are a superfamily of ligand-activated transcription factors that bind as homodimers or heterodimers to their cognate DNA elements in gene promoters. The superfamily, with more than 150 members, can be divided into subfamilies (e.g. the steroid, retinoid, thyroid hormone, and peroxisome proliferator-activated [PPAR]subfamilies). Each subfamily may consist of several members which are encoded by individual genes (e.g. PPAR&agr;, PPAR&ggr;, and PPAR&dgr;). In addition, alternative mRNA splicing can result in more than one isoform of these genes as in the case of specific PPARs (e.g. PPAR&ggr;1 and PPAR&ggr;2). The nuclear receptor superfamily is involved in a wide variety of physiological functions in mammalian cells: e.g., differentiation, proliferation, and metabolic homeostasis. Dysfunction or altered expression of specific nuclear receptors has been found to be involved in disease pathogenesis.
The PPAR subfamily of nuclear receptors consists of three members: PPAR&agr;, PPAR&ggr;, and PPAR&dgr;. PPAR&agr; is highly expressed in liver and kidney. Activation of PPAR&agr; by peroxisome proliferators (including hypolipidimic reagents such as fibrates) or medium and long-chain fatty acids is responsible for the induction of acyl-CoA oxidase and hydratase-dehydrogenase (enzymes required for peroxisomal &bgr;-oxidation), as well as cytochrome P450 4A6 (an enzyme required for fatty acid o-hydroxylase). Thus, PPAR&agr; has an important role in the regulation of lipid metabolism and is part of the mechanism through which hypolipidimic compounds such as fibrates exert their effects. PPAR&ggr; is predominantly expressed in adipose tissue. Recently, a prostaglandin J2 metabolite, 15-Deoxy-D12,14-prostaglandin J2, has been identified as a potential physiological ligand of PPAR&ggr;. Both 15-Deoxy-D12,14-prostaglandin J2 treatment of preadipocytes or retroviral expression of PPAR&ggr;2 in fibroblasts induced adipocyte differentiation, demonstrating the role of PPAR&ggr;in adipocyte differentiation and lipid storage. The demonstration that anti-diabetic and lipid-lowering insulin sensitizing compounds known as thiazolidinediones are high affinity ligands for PPAR&ggr;suggests a broad therapeutic role for PPAR&ggr; ligands in the treatment of diabetes and disorders associated with insulin resistance (e.g. obesity and cardiovascular disease).
Nuclear receptor proteins contain a central DNA binding domain (DBD) and a COOH-terminal ligand binding domain (LBD). The DBD is composed of two highly conserved zinc fingers that target the receptor to specific promoter/enhancer DNA sequences known as hormone response elements (HREs). The LBD is about 200-300 amino acids in length and is less well conserved than the DBD. There are at least three functions for the LBD: dimerization, ligand binding, and transactivation. The transactivation function can be viewed as a molecular switch between a transcriptionally inactive and a transcriptionally active state of the receptor. Binding of a ligand which is an agonist flips the switch from the inactive state to the active state. The COOH-terminal portion of the LBD contains an activation function domain (AF2) that is required for the switch.
The ligand-induced nuclear receptor molecular switch is mediated through interactions with members of a family of nuclear receptor co-activators (e.g., CBP/p300, SRC-1/NcoA-1, TIF2/GRIP-lJNcoA-2, and p/CIP). Upon binding of agonist to its cognate receptor LBD, a conformational change in the receptor protein creates a co-activator binding surface and results in recruitment of co-activator(s) to the receptor and subsequent transcriptional activation. The binding of antagonist ligands to nuclear receptors will not induce the required conformational change and prevents recruitment of co-activator and subsequent induction of transcription. The co-activators CREB-binding protein (CBP) and p300 are two closely related proteins that were originally discovered by virtue of their ability to interact with the transcription factor CREB. These two proteins share extensive amino acid sequence homology. CBP can form a bridge between nuclear receptors and the basic transcriptional machinery (Kamei et al., 1996, Cell 85:403-414; Chakravarti et al., 1996, Nature 383:99-103; Hanstein et al., 1996, Proc. Natl. Acad. Sci. USA 93:11540-11545; Heery et al., 1997, Nature 387:733-736). CBP also contains intrinsic histone acetyltransferase activity which could result in local chromatin rearrangement and further activation of transcription. Ligand- and AF2-dependent interaction between certain nuclear receptors and CBP has been demonstrated in in vitro pull down assays and far-western assays. This interaction is both necessary and sufficient for the transcriptional activation that is mediated by these nuclear receptors. Thus, an AF2 mutant of the estrogen receptor (ER) which abolishes the transcriptonal function of the receptor is incapable of interacting with CBP.
The N-termini of CBP and p300 have been shown to interact with the ligand-binding domains of some nuclear receptors (Kamei et al., 1996, Cell 85:403-414, hereinafter “Kamei”). Kamei was able to demonstrate direct interaction of CBP and p300 with nuclear receptors by several different methods:
(1) Kamei produced GST fusion proteins of the first 100 amino acids of the N-terminus of CBP. These fusion proteins were run out on a polyacrylamide gel, transferred to a membrane, and the membrane was exposed to
32
P-labeled ligand-binding domains of nuclear receptors. In the presence of ligand, a specific binding interaction between the CBP and nuclear receptor fragments was detected in that the
32
P-labeled ligand-binding domains were observed to bind to the bands on the membrane containing the GST-CBP fusion proteins.
(2) Kamei also utilized the yeast two-hybrid system. The ligand-binding domain of the nuclear receptor fused to the DNA-binding domain of the LexA protein was used as bait. The amino terminal domain of CBP fused to the gal4 transactivation domain was used as prey. In the presence of ligand, a specific binding interaction (occurring in vivo, i.e., within the yeast) was observed between the CBP and nuclear receptor fragments.
(3) Kamei observed ligand-induced binding between CBP and nuclear receptors via a gel-shift assay. This assay is based on the observation that, in the presence of ligand, nuclear receptors will bind to oligonucleotides containing their target recognition sequence. Such binding results in the formation of a nuclear receptor-ligand-oligonucleotide complex having a higher molecular weight than the oligonucleotide alone. This difference in molecular weight is detected via a shift in position of the
32
P-labeled oligonucleotide when it is run out on a polyacrylamide gel. Kamei found that a fragment of CBP (the N-terminal 100 amino acids) was capable of binding to the nuclear receptor-ligand-oligonucleotide complex and shifting the complex's position on the gel to an even higher molecular weight.
(4) Kamei was able to co-immunoprecipitate CBP using antibodies to nuclear receptors in extracts from a variety of cells in the presence of ligand.
(5) By the use of transcriptional activation assays, Kamei was able to demonstrate that nuclear receptors and CBP interact in a functional manner. Such transcriptional activation assays can indicate that two proteins are involved in a pathway that results in transcriptional activation but these assays do not prove that the interaction between the proteins is one of direct binding.
By the above-described methods, Kamei was able to demonstrate specific binding interactions between CBP and the retinoic acid receptor (RAR), glucocorticoid r
Cummings Richard T.
Hermes Jeffrey D.
Moller David E.
Zhou Gaochao
Merck & Co. , Inc.
Pak Michael
Tribble Jack L.
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