Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2002-04-09
2004-09-28
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
Reexamination Certificate
active
06797499
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of dehydrogenases that are related to the retinol dehydrogenase subfamily, recombinant DNA molecules and protein production. The present invention specifically provides novel dehydrogenase polypeptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
BACKGROUND OF THE INVENTION
Dehydrogenases, particularly members of the retinol dehydrogenase subfamilies, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of these subfamily of dehydrogenases. The present invention advances the state of the art by providing a previously unidentified human dehydrogenases that have homology to members of the retinol dehydrogenase subfamilies.
Dehydrogenases
17.beta.-hydroxysteroid dehydrogenase
The enzymes identified as 17.beta.-hydroxysteroid dehydrogenase (HSD) are important in the production of human sex steroids, including androst-5-ene-3.beta.,17.beta.-diol (.DELTA.sup.5-diol), testosterone and estradiol. In humans, several types of 17.beta.-HSD have now been identified and characterized. Each type of 17.beta.-HSD has been found to catalyze specific reactions and to be located in specific tissues. Further information about Types 1, 2 and 3 17.beta.-HSD can be had by reference as follows: Type 1 17.beta.-HSD is described by Luu-The, V. et al., Mol. Endocrinol., 3:1301-1309 (1989) and by Peltoketo, H. et al., FEBSLett, 239:73-77 (1988); Type 2 17.beta.-HSD is described in Wu, L. et al., J. Biol Chem, 268:12964-12969 (1993); Type 3 17.beta.-HSD is described in Geissler, W M, Nature Genetics, 7:34-39 (1994).
Inhibitors of 17.beta.-hydroxysteroid dehydrogenase activity can be used for prophylaxis or treatment of benign prostate hypertrophy (see WO publication 91/100731).
20-alpha-hydroxysteroid dehydroyenase
The enzyme responsible for the ovarian metabolism of progesterone to 20.alpha-hydroxyprogesterone is 20.alpha.-hydroxysteroid dehydrogenase (20.alpha.-HSD). Specifically, 20.alpha.-HSD is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and catalyzes the transfer of hydrogen from NADPH to progesterone.
By metabolizing progesterone to an inactive form, 20.alpha.-HSD plays a central role in inhibiting the maintenance of pregnancy and prevention of implantation [Wiest, Endocrinology 83:1181-184 (1968); Wiest et al., Endocrinology 82:844-859 (1968); Kuhn and Briley, i Biochem. J. 0117:193-200 (1970); Rodway and Kuhn, Biochem. J. 152:433-443 (1975)]. Further supporting this role is the fact that it is the increase in ovarian 20.alpha.-HSD activity rather than a decrease in the synthesis of progesterone that contributes to the lower circulating progesterone levels associated with the termination of pregnancy [Kuhn and Briley, Biochem. J. 117:193-201 (1970)]. Indeed, 20.alpha.-HSD gene expression [Albarracin et al. Endocrinology 134:2453-2460 (1994)] and activity remains repressed throughout pregnancy but are induced before parturition [Wiest et al., Endocrinology 82:844-859 (1968); Kuhn and Briley, Biochem. J. 117:193-200 (1970]. Also, ovarian 20.alpha.-HSD catalyzes the decline in progesterone levels which occur during normal and induced termination of pregnancy and pseudopregnancy [Hashimoto and Wiest, Endocrinology 84:873-885 (1969); Naito et al., Endocrinology Jpn 33(1):43-50 (February 1986)].
While 20.alpha.-HSD is of much interest as a key enzyme in the termination/prevention of pregnancy, it is possible that the enzyme is also of importance in spontaneous abortions. Specifically, it is possible that a significant number of spontaneous abortions are due to early expression of 20.alpha.-HSD. Therefore, detection of early 20.alpha.-HSD expression would be of interest in those susceptible to early spontaneous abortions. If detection is made early enough, progesterone replacement therapy could be initiated to help maintain the pregnancy.
11.beta.-hydroxysteroid dehydrogenase
Corticosteroids, also referred to as glucocorticoids, are steroid hormones, the most common form of which is cortisol. Modulation of glucocorticoid activity is important in regulating physiological processes in a wide range of tissues and organs. Glucocorticoids act within the gonads to directly suppress testosterone production (Monder et al., 1994). High levels of glucocorticoids may also result in excessive salt and water retention by the kidneys, producing high blood pressure.
Glucocorticoid action is mediated via binding of the molecule to a receptor, such as either a mineralocorticoid receptor (MR) or a glucocorticoid receptor (GR). Krozowski et al. (1983) and Beaumont and Fanestil (1983) showed that MR of adrenalectomised rats have an equal affinity for the mineralocorticoid aldosterone and glucocorticoids, for example corticosterone and cortisol. Confirmatory evidence has been found for human MR (Arriza et al., 1988). In patients suffering from the congenital syndrome of Apparent Mineralocorticoid Excess (AME; Ulick et al., 1979), cortisol levels are elevated and bind to and activate MRs normally occupied by aldosterone, the steroid that regulates salt and water balance in the body. Salt and water are retained in AME patients causing severe hypertension.
The enzyme 11.beta.-hydroxysteroid dehydrogenase (11.beta.HSD) converts glucocorticoids into metabolites that are unable to bind to MRs Edwards et al., 1988; Funder et al., 1988), present in mineralocorticoid target tissues, for example kidney, pancreas, small intestine, colon, as well as the hippocampus, placenta and gonads. For example, in aldosterone target tissues 11.beta.HSD inactivates glucocorticoid molecules, allowing the much lower circulating levels of aldosterone to maintain renal homeostasis. When the 11.beta.HSD enzyme is inactivated, for example in AME patients Ulick et al., 1979) or following administration of glycyrrhetinic acid, a component of licorice, severe hypertension results. Further, placental 11.beta.HSD activity may protect the foetus from high circulating levels of glucocorticoid which may predispose to hypertension in later life Edwards et al., 1993).
Biochemical characterisation of 11.beta.HSD activity indicates the presence of at least two isoenzymes (11.beta.HSD1 and 11.beta.HSD2) with different cofactor requirements and substrate affinities. The 11.beta.HSD1 enzyme is a low affinity enzyme that prefers NADP+ as a cofactor (Agarwal et al., 1989). The 11.beta.HSD2 enzyme is a high affinity enzyme (Km for glucocorticoid=10 nM), requiring NAD+, not NADP+ as the preferred cofactor, belonging to a class of glucocorticoid dehydrogenase enzymes hereinafter referred to as “NAD+ dependent glucocorticoid dehydrogenase” enzymes.
Michael et al. (1993) show an inverse correlation between 11.beta.HSD enzyme activity in human granulosa-lutein cells and the success of IVF (in vitro fertilization), and suggest that activity of this enzyme might be related to the success of embryo attachment and implantation following IVF. The measurement of ovarian 11.beta.HSD enzyme activity as a prognostic indicator for the outcome of assisted conception in all species, is the subject of UK Patent Application No 9305984.
3alpha-hydroxysteroid dehydrogenase
Human liver 3alpha-hydroxysteroid plays an important role in the metabolism of steroid hormones and polycyclic aromatic hydrocarbons and in the reduction of ketone-containing drugs (Kume et al., Pharmacogenetics 1999 December; 9(6):763-71). 3alpha-hydroxysteroid is also involved in the metabolism of bile acids (Yamamoto et al., Biol Pharm Bull 1998 November; 21(11):1148-53).
3alpha-hydroxysteroid plays a significant role in 5alpha-dihydrotestosterone metabolism in human liver via 3alpha-hydroxysteroid reduction, followed by subsequent glucuronidation and clearance via the kidney (Pirog et al., J Clin End
Beasley Ellen M.
Di Francesco Valentina
Gong Fangcheng
Yan Chunhua
Applera Corporation
Celera Genomics
Patterson Jr. Charles L.
Sun-Hoffman Lin
LandOfFree
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