Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1995-05-22
2001-10-23
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S019000, C435S196000
Reexamination Certificate
active
06306583
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to cDNAs encoding phosphodiesterases (PDEs), their use in the recombinant production of the enzyme and the use of the PDE in drug screening. More specifically this invention relates to a unique subtype of a human, low K
m
, cAMP-specific phosphodiesterase (PDE IV) and its use in the screening of pharmaceutically useful substances.
BACKGROUND OF THE INVENTION
The recent identification and characterization of cDNA clones encoding several different mammalian PDEs has supported the cumulative biochemical evidence for the existence of multiple isozyme families as well as the number and tissue distribution of particular subtypes (Livi, G. P. et al.,
Mol. Cell. Bio
. 10:2678-86 (1990); Colicelli, J. et al.,
Proc. Natl. Acad. Sci
: (
USA
) 86:3599-3603 (1989) and Davis et al.,
Proc. Natl. Acad. Sci
., (
USA
) 86:3604-08 (1989)). A particularly interesting isozyme family with respect to drug discovery is the PDE IV family. There is considerable evidence to suggest that this isozyme family represents a molecular target for a variety of therapeutic agents ranging from anti-depressants (Nicholson, C. D. et al.,
Trends in Pharm. Sci
., 12:14-27 (1991)) to anti-asthmatic and anti-inflammatory agents (Torphy, T. J. and B. J. Undem,
Thorax
46:512-23 (1991)). The cloning, expression and biochemical characteristics of hPDE IV
A
, an enzyme encoded by a cDNA obtained from a human monocyte library has been reported (Livi, G. P. et al. supr (1990) and Torphy T. J. et al.,
J. Biol. Chem
. 267:1798-1804 (1992)). The purpose of this invention is to provide cloned and characterized PDE IV subtypes expressed in human brain.
Cyclic nucleotide phosphodiesterases (PDEs) consist of a family of enzymes that catalyze the hydrolysis of 3′,5′-cyclic nucleotides, resulting in the formation of 5′-nucleotide metabolites. At least five distinct mammalian PDE isozyme families exist, each distinguished on the basis of a number of biochemical properties including 1) enzyme kinetics, 2) substrate selectivity, and 3) selective inhibition by various compounds. These isozyme families are defined as: 1) the Ca
2+
/calmodulin-dependent PDEs; II) the cGMP-stimulated PDEs; III) the cGMP-inhibited PDEs; IV) the cAMP-specific PDEs and V) the cGMP-specific PDEs (Beavo, J. A. et al.,
Trends Pharmacol. Sci
. 11:150-155(1990) and Conti, M. et al.,
Endocrine Rev
. 12:218-234 (1991)).
There is considerable interest in evaluating inhibitors of the low-K
m
, cAMP-specific PDEs (PDE IVs) as potential anti-inflammatory and anti-asthmatic drugs. As mentioned above, the cloning and expression of a cDNA that encodes a human PDE IV subtype expressed in monocytes (hPDE IV
A
) has been reported. This enzyme exhibited significant amino acid sequence homology to PDE IVs from rat brain (Colicelli, supra (1989)) and
Drosophila
(Chen, C-N. et al.,
Proc. Nat'l. Acad. Sci. USA
83:9313-17 (1986)). Furthermore, the recombinant enzyme was overexpressed in both yeast and mammalian cells and defined as a PDE IV based on its kinetic characteristics and sensitivity to isozyme-selective inhibitors. Recombinant hPDE IV
A
possesses a low K
m
for cAMP (K
m
=3.2 &mgr;M), a high K
m
for cGMP, and is inhibited by rolipram (K
i
=0.06 &mgr;M) but not by selective inhibitors of other PDE isozymes.
It has been proposed that the anti-depressant activity of the PDE IV-selective inhibitor rolipram is associated with the inhibition of PDE IVs in the central nervous system. Rolipram binds with high affinity to rat brain homogenates and it has been assumed that this binding site represents either a catalytic or allosteric site within the PDE IV molecule itself Accordingly, it has been recently established that recombinant hPDE IV
A
possesses both catalytic activity and a high affinity (K
d
=2 nM) [
3
H]-rolipram binding site (Torphy T. J. et al.,
J. Biol. Chem
. 267:1798-1804 (1992)). Although the relationship between this high affinity binding site and the catalytic activity of hPDE IV
A
is not clear, it has been proposed that this site may represent either an allosteric site or the catalytic site on one of two distinct catalytic forms of the enzyme. It is of considerable interest to know if an additional PDE IV subtype is expressed in human brain, and if so, whether this subtype has biochemical characteristics similar to hPDE IV
A
. Of particular interest is the determination whether the high affinity rolipram binding site exists on PDE IV subtypes in addition to hPDE IV
A
.
Although the mechanism of action of rolipram can be assessed biochemically using the available recombinant PDE IV enzymes derived from human monocytes and rat brain, a true pharmacological understanding of how PDE IV activity (as well as cellular cAMP content) regulates neurobiochemical processes is limited by lack of knowledge regarding PDE IV subtypes expressed in the human brain, if any. Accordingly, it is a purpose of this invention to provide isolated cDNA clones encoding PDE IV from human brain and to employ this valuable reagent in a screening protocol for the discovery of subtype-specific PDE IV inhibitors. Disclosed herein is the cloning of a cDNA from a human frontal cortex cDNA library that encodes a unique PDE IV subtype. This enzyme is designated as hPDE IV
B
according to the nomenclature of Beavo and Reifsnyder supra (1990). The cDNA product is defined as a type IV PDE based on its comparative amino acid sequence as well as PDE IV-selective inhibitors, the kinetic characteristics and the [
3
H] rolipram binding capacity of the recombinant enzyme. Surprisingly, this PDE IV
B
subtype exhibits a restricted tissue-type pattern of expression.
BRIEF DESCRIPTION OF THE INVENTION
This invention provides an isolated nucleic acid molecule encoding a human PDE IV
B
. This invention also provides a human PDE IV
B
substantially free of other proteins of human origin. The invention also provides molecular reagents such as cloning and expression vectors for the recominbinant production of the PDE IV
B
enzyme. The invention also provides recombinant host cells capable of expressing the enzyme. The invention further includes a method for identifying ligands capable of binding to a PDE IV
B
enzyme comprising: contacting a PDE IV
B
enzyme with a plurality of candidate ligands labeled with an analytically detectable reagent under conditions sufficient for ligand binding and identifying those ligand candidates capable of enzyme binding by detecting the presence of a labeled ligand/enzyme complex. The invention also provides a method of screening compounds to identify those compounds which bind to a human PDE IV
B
enzyme comprising contacting the enzyme with (a) a plurality of drug candidates in the presence of (b) an analytically detectable ligand known to bind to the enzyme, under conditions that permit binding of (a) and (b) to the enzyme, and identifying those candidate compounds capable of enhancing or inhibiting the binding or interaction of the known ligand with the enzyme. The invention also provides for detecting candidate compounds capable of inhibiting the catalytic activity of the enzyme by monitoring the effect of the compound on the ability of the enzyme to hydrolyze substrates (e.g., cAMP). The invention also provides biological screening assay for the detection of PDE IV
B
selective ligands comprising: (a) providing a PDE deficient host cell that exhibits a specific growth arrest phenotype associated with elevated cAMP levels; (b) transforming or transfecting said host cell with the plasmid capable of directing the expression of a PDE IV
B
enzyme and culturing the resultant recombinant host cell under conditions sufficient for the expression of PDE IV
B
enzyme and sufficient to generate a growth arrest response should the expressed PDE IV
B
enzyme be inhibited; (c) contacting the recombinant host cell with a plurality of candidate compounds and (d) identifying those compounds which are capable of inhibiting the enzyme and thereby unmasking the growt
Livi George P.
McLaughlin Megan M.
Torphy Theodore J.
Bugaisky Gabriele E.
Gimmi Edward R.
Hecht Elizabeth J.
Kinzig Charles M.
Low Christopher S. F.
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