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Nitrosylation of protein SH groups and amino acid residues as a therapeutic modality

a technology of amino acid residues and proteins, applied in the direction of drug compositions, peptides, metabolic disorders, etc., can solve the problems of life-threatening diseases, oxygen-hemoglobin binding, oxygen transport, etc., and achieve the effect of inhibiting platelet function

Inactive Publication Date: 2006-09-07
THE BRIGHAM & WOMEN S HOSPITAL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0037] The invention is also directed to a method for delivering nitric oxide to specific, targeted sites in the body comprising administering an effective amount of the pharmaceutical compositions of the invention to an animal.
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Problems solved by technology

Hyper-liproteinemias, resulting from excessive lipoprotein (and thus, lipid) uptake, cause life-threatening diseases such as atherosclerosis and pancreatitis.
As a result, methemoglobin is generated, which impairs oxygen-hemoglobin binding, and thus, oxygen transport.
Protein thiols may, under certain pathophysiological conditions, cause a protein to exert a detrimental effect.
However, uncontrolled proteolysis caused by this enzyme leads to tissue damage; specifically lung damage caused by smoking.
The current state of the art lacks chemical methods for modifying the activity and regulating the intermediary cellular metabolism of the amino acids and proteins which play a critical role in biological systems.
Furthermore, because of these differences, it would not be expected that protein thiols could be successfully nitrosylated in the same manner as low molecular weight thiols, or that, if nitrosylated, they would react in the same manner.

Method used

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  • Nitrosylation of protein SH groups and amino acid residues as a therapeutic modality
  • Nitrosylation of protein SH groups and amino acid residues as a therapeutic modality
  • Nitrosylation of protein SH groups and amino acid residues as a therapeutic modality

Examples

Experimental program
Comparison scheme
Effect test

example 1

Synthesis of S-Nitroso-t-PA

A. Nitrosylation of t-PA

[0139] 1. Materials

[0140] t-PA was kindly provided by Genentech, Inc. San Francisco, Calif. Reactivated purified plasminogen activator inhibitor-1 (PAI-1) and a panel of six murine anti-t-PA monoclonal antibodies were kindly provided by Dr. Douglas E. Vaughan. Horse-Radish Peroxidase linked-sheep anti murine antibodies were purchased from Amersham Corp., Arlington, Ill. Sodium nitrite was purchased from Fisher Scientific, Fairlawn, N.J. H-D-isoleucyl-L-prolyl-L-arginyl-p-nitroanilide (S2288) and H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide (S2251) were purchased from Kabi Vitrum, Stockholm, Sweden. Human fibrinogen purified of plasminogen and von Willebrand factor, was obtained from Enzyme Research Laboratories, South Bend, Ind. Epinephrine, ADP and iodoacetamide were purchased from Sigma Chemical Co., St. Louis, Mo. Bovine thrombin was obtained from ICN, ImmunoBiologicals (Lisle, Ill.). Radioimmunoassay kits for the determination ...

example 2

Synthesis of S-Nitroso-BSA

A. Nitrosylation

[0160] In the first method, nitrosylation of BSA was accomplished by incubating BSA (200 mg / ml with NO generated from equimolar NaNO2 in 0.5N HCl (acidified NaNO2) for thirty minutes at room temperature. Solutions were titrrated to pH 7.4 with equal volumes of 1.0 N NaOH and Tris Buffered Saline (TBS), pH 7.4, 0.05 M L-arginine. Dilutions were then made as necessary in TBS.

[0161] In the second method, nitrosylation was achieved in helium-deoxygenated solutions of 0.1 M sodium phosphate (pH 7.4) by exposing the protein solution in dialysis tubing to authentic NO gas bubbled into the dialysate for fifteen minutes. The proteins were then dialyzed against a large excess of 0.01 M phosphate buffer at pH 7.4 to remove excess oxides of nitrogen.

[0162] In the third method, proteins were incubated with bovine aortic endothelial cells stimulated by exposure to high shear forces to secrete EDRF, as in Example 1(A). As a corollary of this method, p...

example 3

Synthesis of S-Nitroso-Cathepsin B

[0166] Nitrosylation of cathepsin, and determination of S-nitrosothiol formation, was accomplished according to the methods described in Example 2. The stoichiometry of S-nitrosothiol / protein molecules for cathepsin is shown in Table 1.

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Abstract

Nitrosylation of proteins and amino acid groups enables selective regulation of protein function, and also endows the proteins and amino acids with additional smooth muscle relaxant and platelet inhibitory capabilities. Thus, the invention relates to novel compounds achieved by nitrosylation of protein thiols. Such compounds include: S-nitroso-t-PA, S-nitroso-cathepsin; S-nitroso-lipoprotein; and S-nitroso-immunoglobulin. The invention also relates to therapeutic use of S-nitroso-protein compounds for regulating protein function, cellular metabolism and effecting vasodilation, platelet inhibition, relaxation of non-vascular smooth muscle, and increasing blood oxygen transport by hemoglobin and myoglobin. The compounds are also used to deliver nitric oxide in its most bioactive form in order to achieve the effects described above, or for in vitro nitrosylation of molecules present in the body. The invention also relates to the nitrosylation of oxygen, carbon and nitrogen moieties present on proteins and amino acids, and the use thereof to achieve the above physiological effects.

Description

RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 216,865 filed Aug. 13, 2002, which is a continuation of U.S. application Ser. No. 08 / 437,884 filed May 9, 1995, issued as U.S. Pat. No. 6,562,344, which is a continuation of U.S. application Ser. No. 08 / 287,830 filed Aug. 9, 1994, issued as U.S. Pat. No. 5,593,876, which is a continuation of U.S. application Ser. No. 08 / 198,854 filed Feb. 17, 1994, abandoned, which is a continuation of U.S. application Ser. No. 07 / 943,835 filed Sep. 14, 1992, abandoned, which is a continuation-in-part of U.S. application Ser. No. 07 / 791,668, filed Nov. 14, 1991, abandoned. [0002] This application is related to U.S. Pat. Nos. 5,863,890, 6,291,424, 6,583,113 and 7,033,999 and U.S. application Ser. No. 11 / 349,178 filed Feb. 8, 2006.[0003] This invention was made with government support under RO1-HL40411, HL43344 and RR04870, awarded by The National Institutes of Health. The government has certain rights in th...

Claims

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Application Information

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IPC IPC(8): A61K38/43A61K38/38A61K38/17C12N9/00C07K14/775C07K14/765A61K35/14A61K38/00A61K38/02A61K38/16A61K38/42A61K38/46A61K38/49A61K39/395A61P9/00C07K1/06C07K1/107C07K14/00C07K14/315C07K14/47C07K14/76C07K14/805C07K16/00C07K16/18C12N9/48C12N9/64C12N9/70C12N9/72
CPCC07K1/1077C07K14/3153C07K14/4702C07K14/76C07K14/765C07K14/805C07K16/00C12N9/6459C12N9/6472A61K38/00C12Y304/21069A61P11/00A61P9/00
Inventor STAMLER, JONATHANLOSCALZO, JOSEPHSIMON, DANIELSINGEL, DAVID
Owner THE BRIGHAM & WOMEN S HOSPITAL INC
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