Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-06-28
2004-05-04
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S007210, C435S007200
Reexamination Certificate
active
06730472
ABSTRACT:
TECHNICAL FIELD
This invention relates to thyroid hormone receptors, and more particularly to non-nuclear effects of thyroid hormone.
BACKGROUND
Thyroid hormone (TH) has diverse effects on mammals, including effects on the neonatal and adult brain. In the developing animal, thyroid hormone regulates various events such as neuronal processing, glial cell proliferation, myelination, and neurotransmitter enzyme synthesis. The metabolically active form of thyroid hormone, 3,5,3′-triiodothyronine (T3), acts by binding to two receptors; TR&agr;1 and TR&bgr;1. These two receptors are encoded by the c-erbA&agr; locus. Two truncated receptor transcripts have been identified that are also transcribed from the c-erbA&agr; locus; &Dgr;TR&agr;1 and &Dgr;TR&agr;2 (Chassande et al., 1997, Mol. Endocrinol. 11: 1278-1290). Neither truncated receptor has a DNA binding region and &Dgr;TR&agr;1 has been shown to antagonize T3-induced transcriptional activation.
Type II iodothyronine 5′-deiodinase (D2) is the key enzyme in the pathway that mediates the conversion of intracellular thyroxine (T4) to 3,5,3′-triiodothyronine (T3). D2 concentration can be mediated by thyroid hormone concentration and is regulated by enzyme inactivation. The D2 activity appears to be more sensitive to T4 than T3. The degradation of the enzyme is energy-dependent and apparently requires the structural integrity of the actin cytoskeleton, i.e., is regulated at least in part by actin-based endocytosis. p29 is the substrate binding subunit of D2. T4 induces inactivation of D2 and initiates the binding of p29 to F-actin. The bound p29 is transported to an endosomal pool followed by dissociation of the F-actin-p29 complex (Farwell et al., 1993, J. Biol. Chem. 268: 5055-5062).
SUMMARY
The present invention relates to the effects of thyroid hormone that are mediated by non-nuclear mechanisms. In particular, the invention relates to methods of identifying compounds that interact with a &Dgr;TR&agr;1 polypeptide and/or &Dgr;TR&agr;2 polypeptide (&Dgr;TR&agr;1; &Dgr;TR&agr;2). The invention also includes transgenic animals with altered or missing &Dgr;TR&agr;1 and &Dgr;TR&agr;2. Such animals are useful for identifying new targets for drug discovery.
Although &Dgr;TR&agr;1 and &Dgr;TR&agr;2 do not bind to 3,5,3′-triiodothyronine (T3), they do bind with high affinity to 3,3′,5′-triiodothyronine (reverse T3; rT3) and that binding can be displaced by thyroxine (T4) and rT3 (Example 2). The invention relates to the discovery that the association of myosin V with p29 vesicles is dependent on thyroid hormone, e.g., T4 and rT3, bound to a &Dgr;TR&agr;2, and that stable complexes between p29, several synaptic vesicle proteins and myosin V can be isolated on actin fibers. Thus, T4, rT3, and certain analogs are useful for regulating actin-based endocytosis, especially movement of synaptic vesicles.
The invention features a method of assaying the functionality of a translation product of a mutant &Dgr;TR&agr;2 gene in a cell. The method includes binding a labeled ligand for a &Dgr;TR&agr;2 polypeptide to the translation product in a cell and measuring the amount, location, or rate of transit of the ligand in the cell. An increase in the amount, location, or rate of transit of the ligand in the cell compared to that in a cell that does not comprise a mutant &Dgr;TR&agr;2 gene indicates an increase in functionality of the translation product. A decrease in the amount location, or rate of transit of the ligand in the cell compared to a cell that does not comprise a mutant &Dgr;TR&agr;2 gene indicates a decrease in the functionality of the translation product. The ligand can be, e.g., a flavone, an aurone, or a T4 analog.
The invention includes an inhibitor of &Dgr;TR&agr;2 expression or activity. The inhibitor can be, e.g., a flavone, an aurone, or a T4 analog.
The invention also features a method of identifying a candidate compound that modulates &Dgr;TR&agr;2 activity by obtaining a &Dgr;TR&agr;2 polypeptide, contacting the &Dgr;TR&agr;2 with a test compound, assaying for binding of the test compound to &Dgr;TR&agr;2, such that binding indicates that the test compound that binds to the &Dgr;TR&agr;2 polypeptide is a candidate compound that modulates &Dgr;TR&agr;2 activity. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
In another aspect, the invention provides a method of identifying a candidate compound that modulates &Dgr;TR&agr;2 activity. This method includes obtaining a &Dgr;TR&agr;2 polypeptide bound to a &Dgr;TR&agr;2 ligand, contacting the &Dgr;TR&agr;2 bound to the &Dgr;TR&agr;2 ligand with a test compound, and measuring the displacement of the &Dgr;TR&agr;2 ligand from the &Dgr;TR&agr;2 polypeptide, such that displacement indicates that the a test compound is a candidate compound that modulates &Dgr;TR&agr;2 activity. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
The invention also includes a method of identifying a candidate compound that modulates &Dgr;TR&agr;2 activity. This method includes the steps of obtaining a test sample containing a &Dgr;TR&agr;2, incubating the test sample with a test compound, and assaying the test sample containing the test compound for an alteration in type II 5′ deiodinase (D2) activity, such that a test compound that alters D2 activity when compared to a test sample that was not incubated with the test compound is a candidate compound. In this method, the test compound may decrease the amount of D2 activity. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
The invention also features a method of identifying a candidate compound that modulates &Dgr;TR&agr;2 activity which includes the steps of obtaining a test sample containing a &Dgr;TR&agr;2, performing an actin binding assay with the test sample in the presence of a test compound, such that a test compound that alters the binding of p29 vesicles to F-actin when compared to a test sample that was not incubated with the test compound is a candidate compound. The test compound can be, e.g., a flavone, an aurone, or a T4 analog.
The invention includes a compound identified by the any of the methods described above. The invention also includes an inhibitor of &Dgr;TR&agr;2 expression or activity.
Other aspects of the invention are methods of treating a subject who has a neurologic disorder or a psychiatric disorder (e.g., a mood disorder or depression) by administering to the subject a therapeutically effective amount of a &Dgr;TR&agr;2 ligand.
The invention also features an isolated nucleic acid molecule that includes a &Dgr;TR&agr;2 targeting construct that contains a DNA sequence homologous to sequences encoding a mouse &Dgr;TR&agr;2, such that when the construct is introduced into a non-human animal (e.g., a mouse) cell or an ancestor of the animal cell at an embryonic stage, and the construct-derived sequences are incorporated into an endogenous TR&agr; gene, the cell does not express &Dgr;TR&agr;2 in significant amounts (e.g., not more than 75%, 50%, 25%, 10%, or 5% of the level of expression in a cell or animal having a wild type gene). The invention includes a vector containing this nucleic acid. The construct can contain a nucleic acid sequence that is homologous to intron 7 of a mouse TR&agr; gene or a nucleic acid sequence that is homologous to exon 10 of a mouse TR&agr; DNA sequence. In some aspects of the invention, introduction of the construct into the cell disrupts the AP1, ctf, GR, SP1, or ets1 sequence of intron 7. The isolated nucleic acid molecule can also include a gene selection cassette.
The invention features a transgenic, non-human animal whose germ cells and somatic cells include a mutated TR&agr; gene, the mutation being sufficient to inhibit binding of thyroxine (T4) to &Dgr;TR&agr;2 transcribed from the gene. The mutated gene is introduced into the non-human animal or an ancestor of the animal at an embryonic stage, such that the animal, if homozygous for the mutation, has impaired motor function. The non-human anima
Farwell Alan P.
Leonard Jack L.
Fish & Richardson P.C.
Kemmerer Elizabeth
University of Massachusetts
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