Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-09-14
2002-12-31
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S021000
Reexamination Certificate
active
06500631
ABSTRACT:
The invention relates to methods for detecting soluble guanylate cyclase whose heme-complexed iron is oxidized or which contains no heme group, to screening methods for identifying compounds able to activate soluble guanylate cyclase with oxidized heme iron, and to diagnostic aids for detecting a soluble guanylate cyclase having a trivalent heme iron.
Soluble guanylate cyclases are heterodimeric proteins. They consist in each case of an &agr; subunit and a &bgr; subunit, and contain heme as a prosthetic group. Binding of the signal molecule nitric oxide (“NO”) to the heme group activates the enzyme. The cGMP formed through the enzymatic activity of soluble guanylate cyclase is involved, inter alia, in the activation of cGMP-dependent protein kinases and in the regulation of phosphodiesterases or of ion channels. Four isoforms of the subunits have been described. These differ in their sequence and in their tissue-specific and development-specific expression. Subtypes &agr;
1
and &bgr;
1
are found mainly in lung, kidney and brain. The &bgr;
2
chain is expressed mainly in liver and kidney, and the &agr;
2
subunit is expressed mainly in placenta. Dimerization of the subunits is a precondition for a catalytically active soluble guanylate cyclase. The heterodimers &agr;
1
/&bgr;
1
, &agr;
2
/&bgr;
1
, and &agr;
1
/&bgr;
2
are known. The region of the catalytic domain in all the subunits shows a high degree of homology.
Soluble guanylate cyclase (sGC) contains one heme group per heterodimer. Binding of the heme takes place via His-105 in the &bgr;
1
chain. Mutants no longer containing His-105 in the N terminus of the &bgr;
1
subunit cannot be stimulated by NO. The heme group in soluble guanylate cyclase consists of an organic part of the molecule and an iron atom. The organic part, protoporphyrin IX, contains four pyrrole rings which are linked by methine bridges to form a tetrapyrrole system. The iron atom in the heme group is bound to four nitrogen atoms in the center of the protoporphyrin ring. In addition, it is able to engage in two other linkages. The oxidation state of the iron in the heme may be +2 (ferrous form) or +3 (ferric form; oxidized form). The oxidation state of the heme group iron has a crucial effect on the enzymatic function of soluble guanylate cyclase. The enzyme with a trivalent form heme iron shows only basal enzymatic activity, like a soluble guanylate cyclase without a heme group, and cannot be stimulated by NO. The preparation of a heme-free soluble guanylate cyclase is described in
Eur. J. Biochem.
240, 380-386 (1996).
Soluble guanylate cyclase (sGC) catalyzes the conversion of GTP into cyclic guanosine monophosphate (cGMP) and pyrophosphate. cGMP acts as an intracellular messenger (second messenger). Second messengers are produced inside the cell in cascade-like reactions. Their level is controlled by extracellular signals such as hormones, neurotransmitters, growth factors, odorous substances, peptides, or light. Formation of second messengers serves to enhance signals. The second messenger transmits signals inside the cell to particular target proteins (kinases, phosphatases, ion channels, and others) which depend on the cell type. Modulation of soluble guanylate cyclase therefore leads, via the influence on the cGMP levels and target proteins controlled thereby, to a number of pharmacological effects. Examples of mechanisms influenced in this way are the relaxation of smooth muscles (for example, in the walls of blood vessels), the inhibition of platelet activation, the inhibition of proliferation of smooth muscle cells, and the adhesion of leukocytes.
Soluble guanylate cyclase is detectable in organs such as, for example, the heart, lung, liver, kidney, and brain of all mammals, including humans. In pathological processes or in processes relevant for pathological events, the oxidation state of the heme group iron in soluble guanylate cyclase may play an essential part. A higher proportion of soluble guanylate cyclase with oxidized heme group iron would result in the possibility of diminishing activation of soluble guanylate cyclase by endogenous NO. This might lead, inter alia, to an increase in blood pressure, activation of platelets, increased proliferation of cells or enhanced adhesion of cells to permanent high blood pressure, stable or unstable angina pectoris, thromboses, myocardial infarct, strokes, pulmonary edemas, erectile dysfunction, uncontrolled tissue growth with tumor formation, diabetes, renal dysfunction, hepatic dysfunctions, or vascular dysfunction. The endothelial cells of vessel walls secrete NO as paracrine hormone inter alia for activating soluble guanylate cyclase. Compounds frequently used for pharmacological stimulation of soluble guanylate cyclase act as NO donors via intermediate NO release. Examples of NO donors are the organic nitrates. In addition, various compounds which do not act via NO release, but which modify the activity of soluble guanylate cyclase, have been described.
Activation of soluble guanylate cyclase by NO donors or free NO takes place exclusively in the reduced, i.e., (Fe
2+
)-containing, state of heme iron. This is evident from experiments carried out by A. Schrammel et al. in
Mol. Pharmacol.
50, 1 (1996) with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (“ODQ”). ODQ is a specific and highly effective inhibitor of soluble guanylate cyclase. ODQ interacts with the prosthetic heme group. In vitro, the substance causes irreversible oxidation of the heme iron of soluble guanylate cyclase. Treatment of soluble guanylate cyclase with ODQ results in the stimulating effect of NO on the enzyme being lost. Oxidation of the heme iron in soluble guanylate cyclase can also be brought about with oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (Olesen et al.,
British Journal of Pharmacology
123, 299-309 (1998)) or potassium ferricyanide (Koesling et al., in “Reviews of Physiology Biochemistry and Pharmacology,” pp. 41-65, Springer Verlag (1999)). There are also activators of soluble guanylate cyclase which do not act via NO release. A description thereof has been given by, for example, Vesely et al. in
Eur. J. Clin. Invest.
15, 258 (1985). Stimulation of heme-free soluble guanylate cyclase by protoporphyrin IX has been demonstrated by Ignarro et al. in
Adv. Pharmacol
26, 35 (1994). The effect of protoporphyrin IX cannot be inhibited by ODQ, but is instead enhanced (Koesling and Friebe,
Physiol. Biochem. Pharmacol.
135, 41 (1999)). The activators disclosed to date for soluble guanylate cyclase stimulate the enzymatic activity thereof only if the heme iron is in the reduced, i.e. Fe
2+
-containing, state.
Recently, another class of chemical compounds has been described (WO 00/02851), the sulfur-substituted sulfonylamino carboxylic acid N-arylamides, whose representatives are able to activate soluble guanylate cyclase.
The activity of soluble guanylate cyclase to date has been detected by enzymatic methods for detecting cGMP and cAMP, or by photometric methods for detecting the heme group. However, for establishing the dependence of a condition with a pathological change on the state of soluble guanylate cyclase, it is insufficient merely to detect the protein by immunological detection methods or by labeling techniques. It is just as important to know the redox state of the complexed iron in the heme group. Until now, it has only been possible to find information about the functionality of the heme in soluble guanylate cyclase by determining the redox state of the complexed heme iron by ESR measurements or by photometry. These determinations have the disadvantage that they depend on the availability of technically complex equipment. Moreover, the methods have only limited suitability for specific measurements because impurities from other heme-containing proteins readily interfere.
One object of the present invention was therefore to provide simplified and specific methods for detecting an oxidation state of soluble guanylate cyclase. Another object of the invention
Muelsch Alexander
Schindler Peter
Schindler Ursula
Strobel Hartmut
Aventis Pharma Deutschland GmbH
Finnegan Henderson Farabow Garrett & Dunner LLP
Gitomer Ralph
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