Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-05
2002-06-18
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007200, C435S069100, C435S320100, C435S325000
Reexamination Certificate
active
06406859
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced by partial citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Since the purification of a pressor substance in blood serum termed serotonin (Rapport et al., 1947) and later identified as 5-hydroxytryptamine (5-HT) (Rapport, 1949), there has been a plethora of reports demonstrating that this indoleamine not only plays a role in the functioning of peripheral tissues but, indeed, performs a key role in the brain as a neurotransmitter. Certainly, the anatomical localization of serotonin and serotonergic neurons in both the peripheral and central nervous systems supports its role in such diverse physiologic and behavioral functions as pain perception, sleep, aggression, sexual activity, hormone secretion, thermoregulation, motor activity, cardiovascular function, food intake and renal regulation (For review see Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka, 1991). Taken together, it appears that serotonin plays an important role in homeostasis and in modulating responsiveness to environmental stimuli. Accordingly, studies demonstrating that abnormalities in the serotonergic system may be associated with disease states has created a drug development effort towards agents which may selectively modulate the function of serotonin (Glennon, 1990).
In relation to the characterization of physiologic or biochemical responses resulting from the release of serotonin are simultaneous investigation examining the receptor sites responsible for the actions elicited by the indoleamine transmitter. Following early in vitro pharmacological assays describing the existence of two different serotonin receptors, designated as D and M, in the guinea pig ileum (Gaddum and Picarelli, 1957), the advent of receptor binding technique in the 1970's has brought to light during the last decade the diversity of 5-HT receptors existing in both the brain and peripheral tissues. Thus, although the concept of D and M receptors has not been invalidated, serotonin receptors not fitting either category have been identified using radioligand methods. To date using this technique, there appears to be four classes of serotonin receptors found in the brain: 5-HT
1
, 5-HT
2
, 5-HT
3
and, putatively, 5-HT
4
(Peroutka, 1991). Furthermore, 5-HT
1
sites have been subclassified as: 5-HT
1A
, 5-HT
1B
, 5-HT
1C
, 5-HT
1D
(Hamon et al., 1990) and 5-HT
1E
(Leonhardt et al., 1989). Although a detailed characterization of the 5-HT
1F
binding site is lacking, extensive pharmacologic, biochemical and functional properties have clearly shown that the other four subtypes of 5-HT
1
sites are receptors according to classical criteria.
During the last few years, the field of molecular biology has provided an important facet to receptor research by cloning these proteins and allowing more precise characterizations in isolated systems (Hartig et al,1990). This has been accomplished for the 5-HT
1A
(Fargin et al., 1988), 5-HT
1C
(Julius et al., 1988), 5-HT
1D
(Branchek et al., 1990) and 5-HT
2
receptors (Pritchett et al., 1988). Thus, there is no doubt that these binding sites represent “true” functional receptors. Indeed, the pharmacological characterization of serotonin receptors involved in various physiological or biochemical functions is a key component of drug development for the serotonergic system. As one can deduce from the diversity of serotonin binding sites, many targets are available for advancement in selective drug design. The coupling of molecular biological methods to pharmacological characterization particularly for cloned human receptors will open new avenues for pharmaceutical development which has not been previously explored.
This study is a pharmacological characterization of a serotonergic receptor clone with a binding profile different from that of any serotonergic receptor to date. In keeping with the nomenclature presently accepted for serotonin receptors, this novel site will be termed a 5-HT
1F
receptor based upon the fact that it possesses high affinity for the endogenous neurotransmitter, 5-HT.
SUMMARY OF THE INVENTION
This invention provides an isolated nucleic acid molecule encoding a human 5-HT
1F
receptor (Seq. I.D. No. 1).
This invention also provides an isolated protein which is a human 5-HT
1F
receptor (Seq. I.D. Nos. 2, 7).
This invention provides a vector comprising an isolated nucleic acid molecule encoding a human 5-HT
1F
receptor.
This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human 5-HT
1F
receptor, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, or mammalian cells so located relative to the DNA encoding the 5-HT
1F
receptor as to permit expression thereof.
This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT
1F
receptor.
This invention provides a method for determining whether a ligand not known to be capable of binding to a human 5-HT
1F
receptor can bind to a human 5-HT
1F
receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT
1F
receptor with the ligand under conditions permitting binding of ligands known to bind to a 5-HT
1F
receptor, detecting the presence of any of the ligand bound to a human 5-HT
1F
receptor, and thereby determining whether the ligand binds to a human 5-HT
1F
receptor.
This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT
1F
receptor can functionally activate its activity or prevent the action of a ligand which does so. This comprises contacting a mammalian cell comprising an isolated DNA molecule which encodes a human 5-HT
1F
receptor with the ligand under conditions permitting the activation of blockade of a functional response, detected by means of bioassay from the mammalian cell such as a second messenger response, and thereby determining whether the ligand activates or prevents the activation of the human 5-HT
1F
receptor functional output.
This invention further provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT
1F
receptor on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT
1F
receptor with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a human 5-HT
1F
receptor.
This invention also provides a method of screening drugs to identify drugs which interact with, and activate or block the activation of, the human 5-HT
1F
receptor on the surface of a cell which comprises contacting the mammalian cell comprising an isolated DNA molecule encoding and expressing a human 5-HT
1F
receptor with a plurality of drugs, determining those drugs which activate or block the activation of the receptor in the mammalian cell using a bioassay such as a second messenger assays, and thereby identifying drugs which specifically interact with, and activate or block the activation of, a human 5-HT
1F
receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT
1F
receptor.
This invention also provides a method of detecting expression of the 5-HT
1F
receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT
1F
receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at le
Branchek Theresa
Hartig Paul R.
Weinshank Richard L.
Allen Marianne P.
Cooper & Dunham LLP
Synaptic Pharmaceutical Corporation
White John P.
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