Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1999-02-18
2003-03-25
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S002600, C514S381000, C514S382000, C549S289000, C546S153000, C546S079000
Reexamination Certificate
active
06538022
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to determining the three-dimensional structure of phospholamban (PLB) using NMR data of sufficiently high resolution for three dimensional structure determination. The invention also relates to methods for rational drug design enabling the design of compounds for deactivating phospholamban based on using the three-dimensional structure data provided on computer readable media, as analyzed on a computer system having suitable computer algorithms. The invention also relates to phospholamban deactivating compounds with structural, physicochemical and spacial characteristics that allow for the interaction of said compounds with specific residues of phospholamban. The interaction prevents the inhibiting effect of phospholamban on Ca-ATPase, making these compounds useful for treating diseases where the Ca-pumping activity of the Ca-ATPase may be diminished, such as congestive heart disease.
BACKGROUND OF THE INVENTION
Phospholamban (PLB) is a low molecular weight protein (52 amino acids), present in cardiac, slow-twitch and smooth muscle, which can be phosphorylated by both cAMP- and Ca
2+
/calmodulin-dependent phosphokinases. The amino acid sequences of phospholamban from different species are shown in FIG.
1
. The phosphorylation/ dephosphorylation of phospholamban has been shown to regulate the Ca
2+
-ATPase of the sarco/endoplasmic reticulum in myocytes (SERCA
—
2). It has been shown that phospholamban, in its non-phosphorylated form, binds to a specific region of the large loop in the cytoplasmic domain of SERCA
—
2 and inhibits this pump by lowering its affinity for Ca
2+
, while the phosphorylated form does not inhibit SERCA
—
2.
It has been proposed that a region essential for functional association of phospholamban with Ca
2+
-ATPase lies in the cytoplasmic domain of phospholamban, while the transmembrane region anchors PLB to the sarcoplasmic membrane.
During the last decade, efforts have been made to elucidate, at least partially, the secondary structure of PLB either by means of cross-linking experiments or by reconstitution of SERCA
—
2 with point-mutated PLB, or, finally, by obtaining direct structural information by circular dichroism, laser light scattering photometry—FTIR spectroscopy and NMR spectroscopy). Molecular modelling has been used to formulate hypotheses on the quaternary structure of the transmembrane region in the PLB pentamer. The structural information obtained has been recently reviewed (Arkin, I. T. et al. (1997) Annu. Rev. Biophys. Biomol. Struct., 26, 157-179).
Since PLB i) is an amphiphatic oligopeptide, ii) contains three cysteins, and iii) is prone to pentamerization also in vitro, it is not straightforward to find good conditions to study its structure and, in particular, an appropriate solvent system which prevents unspecific aggregation. Therefore, until now NMR studies have been carried out either on short PLB fragments or in organic solvents. In no cases has evidence of a tertiary structure for the cytosolic domain of PLB been found.
Inhibition of CaATPases may play a causative role in cardiac disorders where the calcium levels of myocytes are high. As phospholamban inhibits SR CaATPase this inhibition may be harmful in such disorders. A compound capable of relieving the inhibitory effects of phospholamban on cardiac SR Ca
2+
-ATPase, e.g. by interrupting phospholamban-Ca
2+
-ATPase interaction, would be potentially useful in the treatment of such disorders. There have been very few examples on compounds which can prevent the inhibition of CaATPase by phospholamban in the literature. Such compounds include anti-phospholamban antibodies, some large polyanionic oligopeptides and tannins. No small molecules with specific interactions with phospholamban has been reported.
In the present invention it has been found that phospholamban can assume a well characterized conformation in which it can bind a broad series of small compounds with common structural, physicochemical and spacial characteristics that allow an interaction of the said compounds with specific residues of phospholamban in the defined conformation. This interaction deactivates phospholamban and prevents its inhibiting effect on Ca-ATPase. The phospholamban deactivating compounds are potentially useful in the treatment of cardiac disorders, where the activation of the SR CaATPase is beneficial.
SUMMARY OF THE INVENTION
The present invention is based on our complete resolution of the three-dimensional structure of the entire cytosolic domain of phospholamban and the ligand binding site therein.
In one aspect the present invention provides compounds capable of relieving the inhibitory effects of phospholamban on cardiac SR Ca
2+
-ATPase, such compounds thus acting as phospholamban deactivators through direct binding to the phospholamban protein. These componds have common structural, physicochemical and spacial characteristics that allow for the interaction of said compounds with specific residues of the ligand binding site of phospholamban.
In another aspect the present invention provides a method of deactivating phospholamban which comprises administering to a mammal in need thereof a compound of the invention, as well as a pharmaceutical preparation comprising a compound of the invention together with a pharmaceutically acceptable carrier.
In another aspect, the present invention provides methods for rational drug design enabling the design of phospholamban deactivators based on using the three-dimensional structure data of phospholamban cytosolic domain provided on computer readable media, as analyzed on a computer system having suitable computer algorithms.
In still another aspect, the present invention provides the three-dimensional structure of phospholamban cytosolic domain provided on computer readable media.
Other aspects of the present invention will be apparent to one of ordinary skill in the art from the following detailed description and examples relating to the present invention.
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Kohara et al., Synthesis and Angiotensin II Receptor Anagonistic Activities of Benzimidazole Derivatives Bearing Acidic Heterocycles as Novel Tetrazole Bioisosteres, J.Med. Chem., vol. 39, pp 5228-5235, 1996.*
Liu, L.H., et al., “Defective Endothelium-dependent Relaxation of Vascular Smooth Muscle and Endothelial Cell Ca2+Signaling in Mice Lacking Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 3,”J. Biol. Chem. 272:30538-30545, American Society for Biochemistry and Molecular Biology, Inc. (Nov. 1997).
O'Neil, K.T., and DeGrado, W.F., “How calmodulin binds its targets: sequence independent recognition of amphiphilic &agr;-helices,”TIBS 15:59-64, Elsevier Science Publishers Ltd. (UK) (1990).
Sutliff, R.L., et al., “Functional and Biochemical Evidence for Modulation of Endothelial Cell Function by Phospholamban,”FASEB J. 12:A957, Abstract No. 5546, Federation of American Societies for Experimental Biology (1998).
Ferguson, D.G., et al., “Localization of Phospholamban in Smooth Muscle Using Immunogold Electron Microscopy,”J. Cell Biol. 107:555-562, The Rockefeller University Press (1988).
Gao, Y., et al., “Interaction of calmodulin with phospholamban and caldesmon: comparative studies by1H-NMR spectroscopy,”Biochim. Biophys. Acta 1160:22-34, Elsevier Science Publishers B.V. (1992).
Lalli, J., et al., “Targeted Ablation of the Phospholamban Gene Is Associated With a Marked Decrease in Sensitivity in Aortic Smooth Muscle,”Circulation Res. 80:506-513, American Heart Association, Inc. (Apr. 1997).
Lindemann, J.P., et al., “&bgr;-Adrenergic Stimulation of Phospholamban Phosphorylation and Ca2+-ATPase Activity in Guinea Pig Ventricles,”J. Biol. Chem. 258:464-471, American Society of Biological Chemists, Inc. (1983).
Arkin, I.T.,
Kaivola Juha
Lotta Timo
Ovaska Martti
Pollesello Piero
Tenhunen Jukka
Low Christopher S. F.
Lukton David
Orion Corporation
Sterne Kessler Goldstein & Fox PLLC
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