Human phosphodiesterase type IVC, and its production and use

Details Human phosphodiesterase type IVC, and its production and use Human phosphodiesterase type IVC, and its production and use

1998-05-15

2001-09-18

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S069100, C435S196000, C536S023100, C536S023200, C536S023500, C536S024300

Reexamination Certificate

active

06291199

ABSTRACT:

This invention relates to human phosphodiesterase type IVC and its production, to conformers, analogues and fragments thereof, to nucleic acids encoding the enzyme, and to the use of the enzyme in drug screening and as an immunogen.
The role of cyclic AMP (CAMP) as a second messenger is well recognised. It is responsible for transducing the effects of a variety of extracellular signals, including hormones and neurotransmitters. The level of intracellular CAMP is regulated through both its synthesis by adenyl cyclases and degradation by cyclic nucleotide phosphodiesterases (PDE).
PDEs form a family of at least seven enzyme isotypes (I-VII) which differ in their affinity for cAMP and/or cGMP, subcellular localisation and regulation (Beavo J. A. and Reifsnyder D. H. (1990) Trends Pharmacol. Sci. 11 150-155; Conti M. et al. (1991) Endocrine Rev. 12 218-234). In the same way that receptors controlling the synthesis of CAMP have offered opportunities for developing selective therapeutic agents, the PDEs may afford similar possibilities for drug development. In fact the clinical effects of a number of drugs can be.rationalised on the basis of their selectivity for a particular POE isctype. For example, the cardiotonic drugs milrinone and zaprinast are PDE III and POE V inhibitors respectively. (Harrison S. A. et al. (1986) Mol. Pharmacol. 22 506-514; Gillespie P. G. and Beavo J. (1989) Mol. Pharmacol. 36 773-781). The anti-depressant drug, rolipram functions as a selective PDE IV inhibitor. (Schneider H. H. et aL (1986) Eur. J. Pharmacol. 127 105-115.).
The availability of POE isotype selective inhibitors has enabled the role of PDEs in a variety of cell types to be investigated. In particular it has been established that PDE IV controls the breakdown of CAMP in many inflammatory cells, for example basophils (Peachell P. T. et al. (1992) J. Immunol. 148 2603-2510 ) and eosinophils (Dent G. et al. (1991) Br. J. Pharmacol. 103 1339-1346) and that inhibition of this isotype is associated with the inhibition of cell activation. Consequently PDE IV inhibitors are currently being developed as potential anti-inflammatory drugs, particularly for the treatment of asthma in which the non-selective PDE inhibitor, theophylline, has been shown to have a therapeutic effect.
The application of molecular cloning to the study of PDEs has revealed that for each isotype there may be one or more isoforms. For PDE IV, it is has been shown that there are four isoforms (A, B, C and D) each coded for by a separate gene in both rodents (Swinnen J. V. et al. (1989) Proc. Natl. Acad. Sci. USA 86 5325-5329) and man (Bolger G. et al. (1993) Mol. Cell Biol. 13 6558-6571).
The existence of multiple PDE IVs raises the prospect of obtaining inhibitors that are selective for individual isoforms, thus increasing the specificity of action of such inhibitors. This assumes that the different PDE IV isoforms are functionally distinct. Indirect evidence in support of this comes from the selective distribution of these isoforms in different tissues (Swinnen et al. 1989; Bolger et al 1993; Obernolte R. et al. (1993) Gene 129 239-247, ibid) and the high degree of sequence conservation amongst isoforms of different species. To pursue the development of isoform selective inhibitors requires the availability of each enzyme type for evaluation.
To date full length cDNAs for human PDE IVA, B and D (Bolger et al. 1993 ibid; Obemolte et al. 1993 ibid; Mclaughlin M. et al. (1993) J. Biol. Chem. 268 6470-6476) and rat PDE IVA, B and D (Davis R. et al. (1989) Proc. Natl. Acad. Sci. USA 86 3604-3608; Swinnen J. V. et aL, (1991) J. Biol. Chem. 26 18370-18377), have been reported, enabling functional recombinant enzymes to be produced by expression of the cDNAs in an appropriate host cell. These cDNAs have been isolated by conventional hybridisation methods. However using this approach, only partial cDNAs for both human and rat PDE IVC have been obtained. (Bolger etal ibid. 1993 and Swinnen et al. ibid 1989 and International Patent Specification No. WO 91/16457.). These sequences are insufficient for producing a functional enzyme.
Although it might be expected that human PDE IVC cDNA could be fairly readily obtained by using conventional hybridisation approaches, this has not been the case, possibly due to the lower abundance of its mRNAs compared to the other three isoforms (Bolger et al. 1993 ibid). To overcome this problem we have devised a novel strategy for cloning the human PDE IVC mRNA (based on the approach -to primer design and described more particularly in the experimental section hereinafter) which has allowed us to obtain a functional enzyme by expression of the cDNA in mammalian, yeast and insect cells. This has enabled the properties of this enzyme to be compared to the A, B and D isoforms in terms of substrate kinetics and inhibition by PDE IV selective inhibitors.
Thus according to one aspect of the invention we provide an isolated nucleic acid molecule which encodes a human phosphodiesterase type IVC [PDE IVC].
Particular nucleic acids according to the invention comprise the nucleotide sequence depicted in
FIG. 1
hereinafter, (SEQ ID No: 31) analogues and fragments thereof. The term “analogue” is meant to include all those DNA molecules which have the sequence shown in
FIG. 1
but in which one or more nucleotides has been changed or one or more extra nucleotides is present. The term “fragment” is meant to include DNA molecules again having the sequence depicted in
FIG. 1
but in which one or more nucleotides has been deleted. The term is also meant to include analogues in which one or more nucleotides has been deleted. It will be immediately understood that for an analogue or fragment to qualify as a DNA molecule according to the invention it must be able to encode a functional (catalytically active) PDE IVC. The DNA may comprise genomic DNA, cDNA or a combination of both.
The nucleic acids according to the invention may be obtained from any suitable human source using an appropriate probe as described herein. Once obtained, a nucleic acid may be modified by standard molecular biology and/or chemistry techniques, e.g. by use of oligonucleotide directed mutagenesis or oligonucleotide directed synthesis techniques, enzymatic cleavage or enzymatic filling in of gapped oligonucleotides, to obtain nucleic acid analogues or fragments of the invention. Alternatively, the nucleic acid may itself be used as a probe to obtain complementary copies of genomic DNA, cDNA or RNA from other human sources, using conventional genomic, cDNA and/or PCR cloning techniques.
The PDE IVC nucleic acid accor ding to the invention may be of use in therapy, for example where it is desired to modify the production of PDE IVC in vivo and the invention extends to such a use.
Knowledge of the nucleic acid according to the invention also provides the ability to regulate its activity in vivo by for example the use of antisense DNA or RNA. Thus, according to a further aspect of the invention we provide an antisense DNA or an antisense RNA of a gene coding for human phosphodiesterase type IVC, said gene containing nucleic acid comprising the nucleotide sequence of
FIG. 1
herein, or an analogue or fragment thereof.
The antisense DNA or RNA can be produced using conventional means, by standard molecular biology techniques and/or by chemical synthesis. If desired, the antisense DNA and antisense RNA may be chemically modified so as to prevent degradation in vivo or to facilitate passage through a cell membrane, and/or a substance capable of inactivating mRNA, for example ribosyme, may be linked thereto, and the invention extends to such constructs.
The antisense DNA or RNA may be of use in the treatment of diseases or disorders in which the over- or unregulated production of PDE IVC has been implicated, for example in inflammatory diseases.
In particular, however, the nucleic acids according to the invention may be used to produce human PDE IVC or an analogue or fragment thereof. Thus, according to a further aspect of the invention we provide

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