Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
1995-06-07
2001-11-27
Fredman, Jeffrey (Department: 1655)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C424S085100, C435S007100, C435S004000, C435S019000, C435S183000, C435S173300, C435S196000, C435S091500, C536S023200, C536S023100
Reexamination Certificate
active
06323041
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to novel nucleic acid sequences encoding three novel human phosphodiesterase IV (hPDE IV) isozymes.
Cyclic nucleotide phosphodiesterases (PDE's) are a family of enzymes that catalyze the degradation of cyclic nucleotides. Cyclic nucleotides, particularly cAMP, are important intracellular second messengers, and PDEs are one cellular component that regulates their concentration. In recent years, five PDE enzymes (PDE I-PDE V), as well as many subtypes of these enzymes, have been defined based on substrate affinity and cofactor requirements (Beavo J A and Reifsnyder D H,
Trends Pharmacol. Sci.
11:150 [1990]; Beavo J, in:
Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action
. Beavo J and Housley M D (Eds.). Wiley: Chichester, pp. 3-15 [1990]).
Theophylline, a general PDE inhibitor, has been widely used in the treatment of asthma. It has been speculated that selective inhibitors of PDE isozymes and their subtypes (particularly the cAMP-specific PDE IV) will lead to more effective therapy with fewer side effects (for reviews, see Wieshaar R E et al.,
J. Med. Chem
., 28:537 [1985] and Giembycz M A,
Biochem. Pharm
., 43:2041 [1992], Lowe J A and Cheng J B,
Drugs of the Future
, 17:799-807 [1992]). However, even PDE IV selective drugs such as rolipram suffer from emetic side effects that limit their use. An even more selective approach is to inhibit individual subtypes of PDE IV, each one of which is expected to have its own tissue distribution. If the PDE IV isozyme responsible for efficacy is different from that causing side effects, an isozyme selective drug could separate therapeutic and side effects. The cloning and expression of the human PDE IVs would greatly aid the discovery of isozyme-selective inhibitors by providing purified isoenzymes to incorporate into drug assays.
Mammalian PDE IV, the homologue of the Drosophila Dunce gene (Chen C N et al.,
Proc. Nat. Acad. Sci
. (USA) 83:9313 [1986]), is known to have four isoforms in the rat (Swinnen J V et al.,
Proc. Nat. Acad. Sci
. (USA) 86:5325 [1989]). The cloning of one human isoform of PDE IV from monocytes was reported in 1990 (Livi G P et al.,
Mol. Cell. Bio
., 10:2678 [1990]). From Southern blot data, the authors concluded that this enzyme was probably the only PDE IV gene in humans, with the possible exception of one other isozyme. The same group has recently published the sequence of a second human isoform isolated from brain that they designate hPDE IV-B to distinguish it from the monocyte form, which they designate as hPDE IV-A (McLaughlin M M et al.,
J. Biol. Chem
. 268:6470 [1993]). For clarity, we will use this nomenclature as well.
Our invention relates to the nucleic acid sequences encoding three novel human PDE IV isozymes generated by differential splicing from a single gene. We designate these isoforms as hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. The hPDE IV-B2 sequence encodes a polypeptide nearly identical to that reported for hPDE IV-B (McLaughlin M M et al.,
J. Biol. Chem
. 268:6470 [1993]), and the hPDE IV-B2 splice variant represents the unspliced genomic sequence with respect to the differential splice site. Of the two other splice variants, hPDE IV-B1 encodes the longest polypeptide chain, as well as the N-terminal sequence homologous to its rat homologue, DPD (Colicelli J, et al.,
Proc. Nat. Acad. Sci
. (USA) 86:3599 [1989]).
The novel human PDE IV DNA sequences and their encoded peptides may be used to screen for drugs that are selective for a particular human PDE IV isozyme. Such novel DNA sequences may also be used in assays to detect the presence of a particular PDE IV isozyme in human cell lines, thus providing information regarding the tissue distribution of each isozyme and its biological relevance with respect to particular disease states.
The following abbreviations are used throughout this patent:
BAL
bronchoalveolar lavage
bp
base pair(s)
cAMP
cyclic adenosine 3′,5′-monophosphate
dNTP
2′-deoxynucloside-5′-triphosphate
dATP
2′-deoxyadenosine-5′-triphosphate
dCTP
2′-deoxycytidine-5′-triphosphate
dGTP
2′-deoxyguanidine-5′-triphosphate
dTTP
2′-deoxythymidine-5′-triphosphate
hPDE IV-A
human monocyte PDE IV
hPDE IV-B
human brain PDE IV
hPDE IV-B1
human brain PDE IV, splice variant 1
hPDE IV-B2
human brain PDE IV, splice variant 2
hPDE IV-B3
human brain PDE IV, splice variant 3
kb
kilobase(s)
PCR
polymerase chain reaction
PDE
cyclic nucleotide phosphodiesterase
PDE I
Ca
2+
/ Calmodulin-dependent PDE
PDE II
cGMP stimulated PDE
PDE III
cGMP inhibited PDE
PDE IV
high affinity cAMP-specific PDE
PDE V
cGMP specific PDE
RACE
Rapid Amplification of cDNA Ends
RT
avian myeloblastosis virus (AMV) reverse transcriptase
RT-PCR
PCR of RT-transcribed mRNA
SSC
1X SSC = 0.15M NaCl, 0.015 Na
3
citrate pH 7.0
The nucleotides and amino acids represented in the various sequences contained herein have their usual single letter designations used routinely in the art.
SUMMARY OF THE INVENTION
This invention relates to novel nucleic acid sequences encoding the novel hPDE IV isozymes hPDE IV-B1, hPDE IV-B2 and hPDE IV-B3. More specifically, it relates to DNA segments comprising, respectively, the DNA sequences of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 and SEQUENCE ID NO. 3, as defined below, or alleleic variations of such sequences. It also relates to polypeptides produced by expression in a host cell into which has been incorporated one of the foregoing DNA sequences or an alleleic variation of such sequence.
This invention also relates to an isolated polypeptide comprising the amino acid sequence of SEQUENCE ID NO. 4, SEQUENCE ID NO. 5 or SEQUENCE ID NO. 6.
This invention also relates to recombinant DNA comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variations of such sequence.
This invention also relates to an isolated DNA segment comprising the genomic promoter region that regulates transcription or translation of the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation of such sequence.
This invention also relates to an assay method for detecting the presence of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3 in human cells, comprising: (a) performing a reverse transcriptase-polymerase chain reaction on total RNA from such cells using a pair of polymerase chain reaction primers that are specific for, respectively, hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, as determined from, respectively: (i) the DNA sequence of SEQUENCE ID NO. 1 or an alleleic variation thereof; (ii) the DNA sequence of SEQUENCE ID NO. 2 or an alleleic variation thereof; or (iii) the DNA sequence of SEQUENCE ID NO. 3 or an alleleic variation thereof; and (b) assaying the appearance of an appropriately sized PCR fragment by agarose gel electrophoresis.
This invention also relates to a method of identifying compounds or other substances that inhibit or modify the activity of hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, comprising measuring the activity of, respectively, hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, in: (a) a cell line into which has been incorporated recombinant DNA comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, or (b) a cell line that naturally selectively expresses hPDE IV-B1, hPDE IV-B2 or hPDE IV-B3, as determined by the assay method described above.
This invention also relates to an isolated DNA segment comprising a DNA sequence that is a subset of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, and that is capable of hybridizing to, respectively, SEQUENCE ID NO. 1, SEQUENCE ID NO. 2 or SEQUENCE ID NO. 3, or an alleleic variation thereof, when used as a probe, or of amplifying all or part of such sequence when used as a polymerase chain reaction primer.
As used
Fisher Douglas A.
Robbins Michael D.
Fredman Jeffrey
Ginsburg Paul H.
Myers Jeffrey N.
Pfizer Inc.
Richardson Peter C.
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