Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-09-03
2003-06-10
Spector, Lorraine (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C536S023500, C435S069100
Reexamination Certificate
active
06576742
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a cDNA clone encoding a human imidazoline receptive protein, designated as an imidazoline receptor subtype-1 (abbreviated IR
1
), and fragments thereof. Also, the invention relates to an IR
1
polypeptide encoded by the cDNA, as well as fragments containing the receptor binding site(s). The invention also relates to methods for producing such a cDNA clone, methods for expressing the IR
1
protein, and uses thereof.
2. Description of Related Art
It is believed that brainstem imidazoline receptors possess binding site(s) for therapeutically relevant imidazoline compounds, such as clonidine and idazoxan. These drugs represent the first generation of ligands for the binding site(s) of imidazoline receptors. However, clonidine and idazoxan are also known to possess high affinity for &agr;
2
-adrenergic receptors. Second generation ligands, such as moxonidine, possess somewhat improved selectivity for IR
1
over &agr;
2
-adrenergic receptors, but more selective compounds for IR
1
are needed.
An imidazoline receptor clone is of particular interest because of its potential utility in identifying novel pharmaceutical agents having greater potency and/or more selectivity than currently available ligands have for imidazoline receptors. Recent technological advances permit pharmaceutical companies to use combinatorial chemistry techniques to rapidly screen a cloned receptor for ligands (drugs) binding thereto. Thus, a cloned imidazoline receptor would be of significant value to a drug discovery program.
Until now, the molecular nature of imidazoline receptors remains unknown. For instance, no amino acid sequence data for IR
1
, e.g., by N-terminal sequencing, has been reported. Three different techniques have been described in the literature by three different laboratories to visualize imidazoline-selective binding proteins (imidazoline receptor candidates) using gel electrophoresis. Some important consistencies have emerged from these results despite the diversity of the techniques employed. On the other hand, multiple protein bands have been identified, which suggests heterogeneity amongst imidazoline receptors. These reports are discussed below. Some of the abbreviations used hereinbelow, have the following meanings:
&agr;
2
AR
Alpha-2 adrenoceptor
BAC
Bovine adrenal chromaffin
ECL
Enhanced chemiluminescence (protein detection
procedure)
EST
Expressed Sequence Tag
I-site
Any imidazoline-receptive binding site (e.g.,
encoded on IR
1
)
IR
1
Imidazoline receptor subtype
1
IR-Ab
Imidazoline receptor antibody
I
2
Site
Imidazoline binding subtype
2
kDa
Kilodaltons (molecular size)
MAO
monoamine oxidase
MW
molecular weight
NRL
European abbreviation for RVLM (see below)
PC-12
Phaeochromocytoma-12 cells
125
PIC
[
125
I]p-iodoclonidine
PKC
Protein Kinase C
RVLM
Rostral Ventrolateral Medulla in brainstem
SDS
sodium dodecyl sulfate gel electrophoresis
Reis et al. [Wang et al.,
Mol. Pharm
., 42: 792-801 (1992);
Wang et al.,
Mol. Pharm
., 43: 509-515 (1993)] were the first to demonstrate partial purification of an imidazoline-selective binding protein and to characterize it as having MW=70 kDa. This was accomplished using bovine cells (BAC), which lack an &agr;
2
AR [Powis & Baker,
Mol. Pharm
., 29:134-141 (1986)]. The 70 kDa imidazoline-selective protein in those studies had high affinities for both idazoxan and p-aminoclonidine affinity chromatography columns. To date, no one has reported the complete purification of this imidazoline receptor protein. Likewise, no amino acid seguences have been reported for IR
1
.
The partially purified 70 kDa protein was used by Reis and co-workers to raise “I-site binding antiserum”, designated herein as Reis antiserum. The term “I-site” refers to the imidazoline binding site, presumably defined within the imidazoline receptor protein. Reis antiserum was prepared by injecting the purified protein into rabbits [Wang et al, 1992]. The first immunization was done subcutaneously with the protein antigen (10 &mgr;g) emulsified in an equal volume of complete Freund's adjuvant, and the next three booster shots were given at 15-day intervals with incomplete Freund's adjuvant. The polyclonal antiserum has been mostly characterized by immunoblotting, but radioimmunoassays (RIA) and/or conjugated assay procedures, i.e., ELISA assays, are also conceivable [see “Radioimmunoassay of Gut Regulatory Peptides: Methods in Laboratory Medicine,” Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982].
The present inventors and others [Escriba et al.,
Neurosci. Lett
. 178: 81-84 (1994)] have characterized the Reis antiserum in several respects. For instance, the present inventors have discovered that human platelet immunoreactivity with Reis antiserum is mainly confined to a single protein band of MW=33 kDa, although a trace band at 85 kDa was also observed. This 33 kDa band was enriched in plasma membrane fractions as expected for an imidazoline receptor. Furthermore, the intensity of this band was found to be positively correlated with non-adrenergic
125
PIC Bmax values at platelet IR
1
sites in samples from the same subjects, with an almost one-to-one slope factor. In addition, the nonadrenergic
125
PIC binding sites on platelets were discovered by the present inventors to have the same rank order of affinities as IR
1
binding sites in brainstem [Piletz and Sletten,
J.Pharm
. &
Exper. Therap
., 267: 1493-1502 (1993)]. The platelet 33 kDa band may also be a product of a larger protein, since in human megakaryoblastoma cells, which are capable of forming platelets in tissue cultures, an 85 kDa immunoreactive band was found to predominate.
Immunoreactivity with Reis antiserum does not appear to be directed against human &agr;
2
AR and/or MAO A/B. This is significant because &agr;
2
AR and MAO A/B have previously been cloned and also bind to imidazolines. The present inventors have obtained selective antibodies and recombinant preparations for &agr;
2
AR and MAO A/B, and these proteins do not correspond to the 33, 70, or 85 kDa putative IR
1
bands. Thus, there is substantial evidence that, at least in human platelets, the Reis antiserum is IR
1
selective.
Another antiserum was raised by Drs. Dontenwill and Bousquet in France [Greney et al.,
Europ. J. Pharmacol
., 265: R1-R2 (1994); Greney et al.,
Neurochem. Int
., 25: 183-191 (1994); Bennai et al.,
Annals NY Acad. Sci
., 763: 140-148 (1995)] against polyclonal antibodies for idazoxan (designated Dontenwill antiserum). This anti-idiotypic antiserum inhibits
3
H-clonidine but not
3
H-rauwolscine (&agr;
2
-selective) binding sites in the brainstem, suggesting it interacts with IR
1
[Bennai et al., 1995]. As shown in
FIG. 1
, human RVLM (same as NRL) membrane fractions displayed bands of 41 and 44 kDa, as detected by the present inventors using this anti-idiotypic antiserum.
The present inventors have found that the bands of MW=41 and 44 kDa detected by Dontenwill antiserum may be derived from an 85 kDa precursor protein, similar to that occurring in platelet precursor cells. An 85 kDa immunoreactive protein is obtained in fresh rat brain membranes only when a cocktail of 11 protease inhibitors is used. Also, as shown in
FIG. 1
, it is found that Reis antiserum detects the 41 and 44 kDa bands in human brain when fewer protease inhibitors are used.
Additionally, the Dontenwill antiserum weakly detects the platelet 33 kDa band. Thus, the present inventors have hypothesized that the 41 and 44 kDa immunoreactive proteins may be alternative breakdown products of an 85 kDa protein, as opposed to the platelet 33 kDa breakdown product.
In summary, the main conclusion from the above results is that, despite vastly different origins, the Reis and Dontenwill antisera both detect identical bands in human platelets, RVLM, and hippocampus.
Using yet another technique, a photoaffinity imidazoline ligand,
125
AZIPI, has
Ivanov Tina R.
Piletz John E.
Crowell & Moring LLP
Lazar-Wesley Eliane
Spector Lorraine
The University of Mississippi Medical Center
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