Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-18
2002-11-05
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
Reexamination Certificate
active
06475744
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention relates to the regulation of circadian rhythms.
BACKGROUND OF THE INVENTION
Circadian rhythms in mammals are regulated by a master clock located in the suprachiasmatic nucleus (SCN) of the brain (Klein et al., Suprachiasmatic nucleus: The Mind's Clock, Oxford University Press, New York, 1991; Reppert and Weaver,
Cell
89:487-490, 1997). Environmental light-dark cycles entrain the SCN clock to the 24-hr day via direct and indirect retinal projections. The timekeeping capability of the SCN is expressed at the level of single neurons (Welsh et al.,
Neuron
14:697-706, 1995).
The SCN clock mechanism is cell-autonomous, possibly based on transcriptional and translational negative feedback loops (Reppert,
Neuron
21:1-4, 1998). Precedent for such a mechanism has been described for circadian clocks in the fly
Drosophila melanogaster.
In the fly, autoregulatory transcriptional loops occur in which protein products of clock genes periodically enter the nucleus to suppress their own transcription. This feedback loop involves dynamic regulation of the clock genes period (per) and timeless (Tim). As the levels of PER and TIM rise, they are phosphorylated, form heterodimers, and are then translocated to the nucleus where they negatively regulate their own transcription (Saez and Young,
Neuron
17:1-920, 1996; Darlington et al.,
Science
280:1599-1603, 1998). Negative transcriptional regulation appears to involve interference with drosophila CLOCK:drosophila dBMAL-1 (dCLOCK:dBMAL-1) and may be mediated by direct interaction of PER and TIM with dCLOCK. dCLOCK and dBMAL-1 are positive factors which drive Per and Tim transcriptional activation by binding to CACGTG E-box enhancers in the promoters of Per and Tim (Allada et al.,
Cell
93:791-804, 1998; Rutila et al.,
Cell
93:805-814, 1998; Darlington et al., supra; Hao et al.,
Mol. Cell Biol.
17:3687-3693, 1997). The temporal phosphorylation of PER provides at least part of the time delay between transcription and PER-TIM negative feedback necessary to sustain a 24-hr molecular oscillation in drosophila (Price et al.,
Cell
94:83-95, 1998).
SUMMARY OF THE INVENTION
The invention is based, in part, on the discovery that the core clockwork in the SCN is comprised of interacting feedback loops. It was discovered that cryptochrome (CRY) proteins are critical players in the negative limb of the mammalian clock feedback loop and Period 2 (PER2) protein is a critical regulator of the Bmal-1 loop. The CRY proteins and PER2 protein therefore function as important modulators of mammalian circadian rhythm.
It was discovered that mammalian CRY proteins can translocate from the cytoplasm to the nucleus of a cell and inhibit CLOCK:BMAL-1 induced transcription. It was also discovered that CRY proteins can homodimerize or heterodimerize with other circadian proteins. The ability of CRY to heterodimerize with other proteins provides a mechanism whereby CRY can modulate the activity of other circadian proteins. For example, mouse CRY proteins can function as dimeric and potentially trimeric partners for mouse PER proteins; these interactions lead to the nuclear translocation of PER. Once in the nucleus, PER can inhibit CLOCK:BMAL-1 induced transcription. In addition, it was discovered that mouse CRY can form heterodimeric complexes with mouse TIM. The interaction of TIM with CRY may have a role in modulating the negative feedback of mouse PER and/or mouse CRY rhythms. Thus, the compounds which can disrupt the interaction of CRY with itself and other circadian proteins can be used to reset the circadian clock.
In addition, it was discovered that PER2 positively regulates transcription of the Bmal-1 gene. The ability of PER2 to positively regulate the transcription of Bmal-1 indicates that PER2 controls the rhythmic regulation of Bmal-1. The availability of BMAL-1 is critical for restarting the circadian clock loop. When BMAL-1 is available, it heterodimerizes with CLOCK, thereby driving the transcription of Per genes (e.g., in the mouse(m), mPER1-3) and Cryptochrome genes (e.g., mouse mCry1 and mCry2). Compounds which can disrupt the ability of PER2 to positively activate Bmal-1, or compounds which can modulate transcription of Bmal-1, can be used to reset the circadian clock.
Accordingly, the invention includes a method for identifying a compound which binds to a mammalian CRY protein. The method, which is useful as a quick initial screen for CRY agonists and antagonists, includes contacting the CRY protein with a test compound and determining whether the latter binds to the CRY protein. Binding by the test compound to the CRY protein indicates that the test compound is a CRY protein binding compound. For ease of detection, the test compound can be labeled, e.g., radiolabeled. The CRY protein can any mammalian CRY protein such as a CRY from a mouse, rat, rabbit, goat, horse, cow, pig, dog, cat, sheep, pig, non-human, primate, or human. In particular, the CRY protein is a mouse CRY1 or CRY2 or human CRY1 or CRY2.
The method may further include contacting the test compound with: a CRY protein in the presence of a PER protein; a CRY protein in the presence of a TIM protein; a CRY protein in the presence of a CLOCK:BMAL-1 complex; or a CRY protein in the presence of a BMAL-1 protein; and determining whether the test compound disrupts the association of the CRY protein with the PER, TIM, CLOCK:BMAL-1, or BMAL-1 protein, as the case may be; wherein a decrease in the association in the presence of the test compound compared to the association in the absence of the test compound indicates that the test compound disrupts the association of the CRY protein with the indicated binding partner. The PER protein can any mammalian PER protein such as mouse, rat, rabbit, goat, horse, cow, pig, dog, cat or human. For example, the PER protein may be mouse or human PER1, PER2 or PER3.
The method can further include contacting the test compound with the first CRY protein in the presence of a second CRY protein and determining whether the test compound disrupts the association of the first CRY protein with the second CRY protein, wherein the second CRY protein has an amino acid sequence the same as or different than the first CRY protein, and wherein a decrease in the association in the presence of the test compound compared to the association in the absence of the test compound indicates that the test compound disrupts the association of the first CRY protein and the second CRY protein. The first and second CRY proteins can be any mammalian CRY protein such as a CRY from a mouse, rat, rabbit, goat, horse, cow, pig, dog, cat, sheep, non-human, primate or human. For example, each CRY protein can be a mouse or human CRY1 or CRY2 and the second CRY protein is a mouse CRY1 or CRY2.
The method can further include providing a cell or cell-free system which includes a CRY protein, a CLOCK:BMAL-1 complex, and a DNA comprising an E-box operatively linked to a reporter gene. The method includes introducing the test compound into the cell or cell-free system and assaying for transcription of the reporter gene, wherein an increase in transcription in the presence of the compound compared to transcription in the absence of the compound indicates that the compound blocks CRY-induced inhibition of CLOCK:BMAL-1-mediated transcription in a cell. The cell can be any cell type, such as a cultured mammalian cell, e.g., a NIH3T3 cell, a COS7 cell, or a clock neuron. The reporter gene can be a gene that encodes a detectable marker, e.g., luciferase.
The invention further includes a method for identifying a compound which disrupts the association of a CRY protein and a second protein or protein complex, which can be any of the following: a PER protein, a TIM protein, a BMAL-1 protein, a second CRY protein, or a CLOCK:BMAL-1 complex. The method includes contacting a test compound with the CRY protein in the presence of the second protein (or protein complex) and determining whether the test compound disrupts the association of the CRY prote
Jin Xiaowei
Kume Kazuhiko
Reppert Steven M.
Sathyanarayanan Sriram
Shearman Lauren
Carlson Karen Cochrane
Fish & Richardson P.C.
The General Hospital Corporation
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