Specific inhibitors of NFAT activation by calcineurin and their use in treating immune-related diseases

• US 20040002117A1
• Filed: 02/04/2003
• Published: 01/01/2004
• Est. Priority Date: 02/12/1998
• Status: Abandoned Application

• Current Assignee: CBR Institute for Biomedical Research Inc., President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: CBR Institute for Biomedical Research Inc.
• Inventors: Aramburu, Jose, Kang, Sunghyun, Roehrl, Michael H. A., Wagner, Gerhard, Hogan, Patrick G., Rao, Anjana
• Application Number:
  US10/358,052
• Publication Number:
  US 20040002117A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Abstract

Isolated peptide fragments of the conserved regulatory domain of NFAT protein capable of inhibiting protein-protein interaction between calcineurin and NFAT, or a biologically active analog thereof are described. Isolated polynucleotides and gene therapy vectors encoding such peptide fragments are also described. In addition, methods for treating immune-related diseases or conditions and methods for high throughput screening of candidate agents are described. Pharmaceutical compositions are also provided.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the agent inhibits dephosphorylation of NFAT by calcineurin.

3. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is less than 2500 Da.

4. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is between about 100 and 2000 Da.

5. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is between about 200 and 1500 Da.

6. The pharmaceutical composition of claim 1, wherein the molecular weight of the organic molecule is between about 300 and 1000 Da.

7. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 2×

10⁴ M<sup>−

1</sup>.

8. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 10⁶ M⁻

1.

9. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 10⁷ M⁻

1.

10. The pharmaceutical composition of claim 1, wherein the organic molecule binds calcineurin with an affinity constant of at least about 10⁸ M⁻

1.

11. The pharmaceutical composition of claim 1, wherein the organic molecule is a compound selected from the group consisting of:

formula (I);

wherein;

R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶;

heteroaryl optionally substituted with 1-4 R⁶;

C₂-C₈ alkenyl, or C₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy, trihalomethyl, halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;

R² is C₁-C₂₀ alkyl optionally substituted with 1-20 R₆, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶, C₁-C₆ alkoxy, or hydroxy;

R³ is hydrogen or halogen;

R⁴ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶, or halogen;

R is NR⁷, O or S;

R⁶ is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and

R⁷ is C1-C6 alkyl;

formula (II);

wherein;

R¹ and R² are each independently hydrogen, halogen, amino, C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;

R³ is NR¹¹ or O;

R⁴, R⁵ and R⁸ are each independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a double bond between the carbon atoms to which they are attached;

R⁷ is hydrogen, halogen, or when taken together with R⁶ forms a double bond between the carbon atoms to which they are attached;

R⁹ is OR¹³, or when taken together with R¹⁰ forms a double bond between the carbon and nitrogen atoms to which they are attached;

R¹⁰ is hydrogen, or when taken together with R⁹ forms a double bond between the carbon and nitrogen atoms to which they are attached;

R¹¹ is SO₂R¹²; and

R¹² is aryl optionally substituted with alkyl;

R¹³ is alkyl or aryl; and

formula (III);

wherein,

R¹ and R⁴ are each independently O or NR⁸;

R² and R³ are each independently hydrogen, halogen, or R² and R³ together combine to form aryl optionally substituted with 1-4 R⁹;

R⁵ is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;

R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R⁸ is SO₂R¹⁰; and

R¹⁰ is aryl optionally substituted with alkyl.

12. A method for inhibiting protein-protein interaction between calcineurin and NFAT, comprising:

providing calcineurin and NFAT;

providing the pharmaceutical composition of claim 1;

and

contacting the calcineurin, NFAT, and pharmaceutical composition together, such that the protein-protein interaction between calcineurin and NFAT is inhibited.

13. A method of inhibiting an immune response in an animal, comprising administering to the animal the pharmaceutical composition of claim 1.

14. A method for treating a disease or condition involving hyperactivity or inappropriate activity of the immune system, comprising:

identifying an animal suffering from a disease or condition involving hyperactivity or inappropriate activity of the immune system; and

administering to the animal a therapeutically effective amount of the pharmaceutical composition of claim 1, to thereby treat the disease or condition involving hyperactivity or inappropriate activity of the immune system.

15. The method of claim 14, wherein the organic molecule is a compound selected from the group consisting of:

formula (I);

wherein;

R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶;

heteroaryl optionally substituted with 1-4 R⁶;

C₂-C₈ alkenyl, or C₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy, trihalomethyl, halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;

R² is C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶, C₁-C₆ alkoxy, hydroxy;

R³ is hydrogen or halogen;

R⁴ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶, or halogen;

R⁵ is NR⁷, O or S;

R⁶ is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and

R⁷ is C1-C6 alkyl;

formula (II);

wherein;

R¹ and R² are each independently hydrogen, halogen, amino, C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;

R³ is NR¹¹ or O;

R⁴, R⁵ and R⁸ are each independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a double bond between the carbon atoms to which they are attached;

R⁷ is hydrogen, halogen, or when taken together with R⁶ forms a double bond between the carbon atoms to which they are attached;

R⁹ is OR¹³, or when taken together with R¹⁰ forms a double bond between the carbon and nitrogen atoms to which they are attached;

R¹⁰ is hydrogen, or when taken together with R⁹ forms a double bond between the carbon and nitrogen atoms to which they are attached;

R¹¹ is SO₂R¹²; and

R¹² is aryl optionally substituted with alkyl;

R¹³ is alkyl or aryl; and

formula (III);

wherein,

R¹ and R⁴ are each independently O or NR⁸;

R² and R³ are each independently hydrogen, halogen, or R² and R³ together combine to form aryl optionally substituted with 1-4 R⁹;

R⁵ is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;

R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R⁸ is SO₂R¹⁰; and

R¹⁰ is aryl optionally substituted with alkyl.

16. The method of claim 14 wherein the disease or condition involving hyperactivity or inappropriate activity of the immune system is selected from the group consisting of:

an acute immune disease, a chronic immune disease, and an autoimmune disease.

17. A method for treating a disease involving excessive or inappropriate activation of NFAT, or a molecular target thereof, comprising:

identifying an animal suffering from a disease involving excessive or inappropriate activation of NFAT or a molecular target thereof, and

administering to the animal a therapeutically effective amount of the pharmaceutical composition of claim 1, to thereby treat the disease involving excessive or inappropriate activation of NFAT or molecular target thereof.

18. A process of making an agent that inhibits protein-protein interaction between calcineurin and NFAT, the process comprising:

carrying out a method to identify an agent that inhibits protein-protein interaction between calcineurin and NFAT, wherein the method comprises;

providing a first compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof;

providing a second compound selected from the group consisting of calcineurin or a biologically active derivative thereof, and NFAT or a biologically active derivative thereof, wherein the second compound is different from the first compound, and wherein the second compound is labeled;

providing a candidate agent;

contacting the first compound, the second compound, and the candidate agent with each other; and

determining the amount of label bound to the first compound, wherein a reduction in interaction between the first compound and the second compound as assessed by label bound is indicative of usefulness of the candidate agent in inhibiting protein-protein interaction between calcineurin and NFAT; and

manufacturing the agent, to thereby make an agent that inhibits protein-protein interaction between calcineurin and NFAT.

19. The process of claim 18, wherein the first compound is calcineurin and the second compound is a biologically active derivative of NFAT.

20. The process of claim 18, wherein the biologically active derivative of NFAT comprises the amino acid sequence GPHPVIVITGPHEE.

21. A method of manufacturing an agent that inhibits protein-protein interaction between calcineurin and NFAT, the method comprising:

providing an organic compound capable of inhibiting protein-protein interaction between calcineurin and NFAT;

providing at least one pharmaceutically acceptable carrier; and

combining the organic compound with the pharmaceutically acceptable carrier, to thereby manufacture an agent that inhibits protein-protein interaction between calcineurin and NFAT.

22. The method of claim 21, further comprising the step of manufacturing the agent into a form suitable for administration to an animal via a route selected from a group consisting of:

oral, parenteral, topical, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal.

23. A method for inhibiting protein-protein interaction between calcineurin and NFAT, comprising:

providing calcineurin and NFAT;

providing an organic molecule capable of inhibiting protein-protein interaction between calcineurin and NFAT, wherein the organic molecule is a compound selected from the group consisting of;

formula (I);

wherein;

R¹ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶;

heteroaryl optionally substituted with 1-4 R⁶;

C₂-C₈ alkenyl, or C₂-C₈ alkynyl, cyano, nitro, carboxy, carbo(C₁-C₆)alkoxy, trihalomethyl, halogen, C₁-C₆ alkoxy, hydroxy, aryloxy, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, or ureido;

R² is C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶, C₁-C₆ alkoxy, hydroxy;

R³ is hydrogen or halogen;

R⁴ is hydrogen, C₁-C₂₀ alkyl optionally substituted with 1-20 R⁶, C₃-C₈ cycloalkyl optionally substituted with 1-3 R⁶, aryl optionally substituted with 1-4 R⁶, heterocyclyl optionally substituted with 1-3 R⁶, heteroaryl optionally substituted with 1-4 R⁶, or halogen;

R⁵ is NR⁷, O or S;

R⁶ is halogen, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido; and

R⁷ is C₁-C₆ alkyl;

formula (II);

wherein;

R¹ and R² are each independently hydrogen, halogen, amino, C₁-C₆alkylamino, di(C₁-C₆)alkylamino, arylamino, diarylamino, or 4,4-dimethyl-2,6-dioxocyclohexyl;

R³ is NR¹¹ or O;

R⁴, R⁵ and R⁸ are each independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R⁶ is hydrogen, halogen, or when taken together with R⁷ forms a double bond between the carbon atoms to which they are attached;

R⁷ is hydrogen, halogen, or when taken together with R⁶ forms a double bond between the carbon atoms to which they are attached;

R⁹ is OR¹³, or when taken together with R¹⁰ forms a double bond between the carbon and nitrogen atoms to which they are attached;

R¹⁰ is hydrogen, or when taken together with R⁹ forms a double bond between the carbon and nitrogen atoms to which they are attached;

R¹¹ is SO₂R¹²; and

R¹² is aryl optionally substituted with alkyl;

R¹³ is alkyl or aryl; and

formula (III);

wherein,

R¹ and R⁴ are each independently O or NR⁸;

R² and R³ are each independently hydrogen, halogen, or R² and R³ together combine to form aryl optionally substituted with 1-4 R⁹;

R⁵ is hydrogen, halogen, carboxy, acylamino, alkoxycarbonyl, carboxy, alkylcarbonyl, acyloxy, or cyano;

R⁶, R⁷ and R⁹ are each independently hydrogen, C₁-C₆ alkyl, halogen, hydroxy, nitro, haloalkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkyl amino, dialkylamino, aryl amino, diarylamino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, mercapto or ureido;

R⁸ is SO₂R¹⁰; and

R¹⁰ is aryl optionally substituted with alkyl; and

contacting the calcineurin, NFAT, and organic molecule together such that protein-protein interaction between the calcineurin and the NFAT is inhibited.