Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-10-07
2001-03-20
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S071100, C435S071200, C435S252300, C435S254110, C536S023500
Reexamination Certificate
active
06204017
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mammalian histamine receptors. More particularly, it relates to human histamine receptors, isolated nucleic acids and recombinant vectors encoding the receptors, to methods of making the receptors, to methods of making fragments or fusion proteins of the receptors using recombinant DNA methodology or chemical synthesis, and to methods of using the receptors in screening systems to identify agonists and antagonists of the receptors useful for the treatment of various diseases.
BACKGROUND OF THE INVENTION
Histamines are implicated in a number of medical conditions, including inflammation, asthma, allergy, atopic dermatitis, stroke, myocardial infection, migraine, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis. Histamines regulate the intensity and duration of immune responses and are involved in cell-to-cell communication. Histamines are also involved in leukocyte migration and bronchovasoconstriction. As established by radioligand binding, physiological assays, and molecular cloning, different types of receptors for histamines exist. Furthermore, specific histamine receptor subtypes are involved in specific medical conditions such that drugs with subtype selectivity can be utilized to target individual medical conditions.
In view of the important role that histamines play in many physiological processes and medical conditions, there is a need for materials and methods useful for the identification of agonists and antagonists selective for the specific types of histamine receptors.
SUMMARY OF THE INVENTION
The present invention fills the foregoing need by providing such materials and methods. More particularly, this invention provides a novel mammalian histamine receptor, isolated nucleic acids or recombinant nucleic acids encoding the receptor, and recombinant vectors and host cells comprising such nucleic acids. The histamine receptor can be actively expressed in mammalian cells where it displays active ligand binding and positive intracellular signaling upon ligand activation. This novel receptor has high affinity for histamine. This invention further provides methods for the discovery of selective agonists and antagonists of the receptor that may be useful in the treatment and management of a variety of diseases including, for example, inflammation, asthma, and allergy, atopic dermatitis, stroke, myocardial infection, migraine, COPD, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis.
The isolated or recombinant nucleic acids of the present invention are selected from the group consisting of:
(a) A nucleic acid encoding a polypeptide comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof;
(b) A nucleic acid that hybridizes under moderately stringent conditions to the nucleic acid of (a) and encodes a polypeptide that (i) binds histamine and (ii) is at least 80% identical to a receptor encoded by the nucleic acid of (a); and
(c) A nucleic acid that, due to the degeneracy of the genetic code, encodes a mammalian histamine receptor encoded by a nucleic acid of (a) or (b).
This invention further provides methods of making a polypeptide comprising culturing a host cell comprising a nucleic acid encoding a mammalian histamine receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, under conditions in which the nucleic acid is expressed. In some embodiments, the method further comprises isolation of the polypeptide from the culture.
This invention also provides a recombinant nucleic acid comprising a sequence having at least about 70% identity over a stretch of at least about 30 nucleotides to the nucleic acid sequence of SEQ ID NO: 1, useful, e.g., as a probe or PCR primer for a related gene. Another embodiment further includes a polypeptide comprising at least about 60% identity over a stretch of at least about 20 amino acids to the amino acid sequence of SEQ ID NO: 2.
This invention also provides polypeptides comprising a fragment of a polypeptide having an amino acid sequence corresponding to the sequence of at least about 8 contiguous residues of the amino acid sequence of SEQ ID NO: 2. Preferably, the polypeptides comprise at least about 12, more preferably at least about 20, and most preferably at least about 30 such residues.
Still further, this invention provides fusion proteins comprising a polypeptide defined by SEQ ID NO: 2 or a fragment therefrom covalently linked to a fusion partner. The present invention also provides antibodies, both polyclonal and monoclonal, that specifically bind to one or more of the histamine receptors or to polypeptides therefrom, and anti-idiotypic antibodies, both monoclonal and polyclonal, which specifically bind to the foregoing antibodies.
This invention further provides a method for producing a mammalian histamine receptor comprising culturing a host cell comprising a nucleic acid encoding a mammalian histamine receptor comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, under conditions in which the nucleic acid is expressed. In one embodiment the receptor is isolated from the culture.
The present invention also provides a method for identifying a histamine agonist or antagonist comprising:
(a) Contacting a polypeptide having an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, in the presence of a known amount of labeled histamine or a surrogate thereof with a sample to be tested for the presence of a histamine agonist or antagonist; and
(b) Measuring the amount of labeled histamine specifically bound to the receptor;
whereby a histamine agonist or antagonist in the sample is identified by measuring substantially reduced binding of the labeled histamine to the histamine receptor, compared to what would be measured in the absence of such agonist or antagonist.
In a preferred embodiment, membranes isolated from mammalian cells comprising a nucleic acid encoding the histamine receptor are used as the source of the receptor.
The present invention also provides a method for identifying an agonist or antagonist of a mammalian histamine receptor comprising:
(a) contacting cells expressing a polypeptide comprising an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof, in the presence of a known amount of histamine or surrogate thereof with a sample to be tested for the presence of a mammalian histamine agonist or antagonist; and
(b) measuring at least one cellular function modulated by the binding of a ligand to said receptor present in the cells;
whereby a mammalian histamine receptor agonist or antagonist in the sample is identified by measuring its effect on said cellular function compared to what would be measured in the absence of such agonist or antagonist.
Examples of cellular functions modulated by the binding of a ligand to a mammalian histamine receptor include: intracellular second messenger pathways activated via the histamine receptors (e.g., cyclicAMP, calcium, inositol phosphate and MAP kinase), changes in cell growth rate, secretion of hormones, receptor-stimulated Ca
2++
mobilization, mitogenic effects etc., This invention still further provides a method for treating histamine-mediated medical conditions comprising administering to a mammal afflicted with a medical condition caused or mediated by histamine, an effective amount of an agonist or antagonist of the histamine receptor that specifically binds to a mammalian histamine receptor having an amino acid sequence defined by SEQ ID NO: 2, or a subsequence thereof, and pharmaceutical compositions comprising one or more of such agonist or antagonist and a pharmaceutically acceptable carrier. Preferably, the mammal is a human being.
This invention also provides anti-sense oligonucleotides capable of specifically hybridizing to mRNA encoding a mammalian histamine receptor having an amino acid sequence defined by SEQ ID NO: 2 or a subsequence thereof so as to prevent transl
Behan Jiang X.
Hedrick Joseph A.
Laz Thomas M.
Monsma Frederick J.
Morse Kelley L.
Mertz Prema
Murphy Joseph F.
Schering Corporation
Thampoe Immac J.
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