Cell-protective compounds and their use

Abstract

The present invention is directed to cell-protective, in particular, cardio- and renal-protective organic compounds, preferably to organic compounds that inhibit substrate phosphorylation by the G-protein-coupled receptor kinase 2 (GRK2, ADRBK1). Preferably, the organic compounds inhibit the GRK2-mediated phosphorylation of serine/arginine-rich splicing factor 1 (SRSF1, ASF-1, SF2) and/or phosducin for treating hypertension, heart diseases, heart dysfunction or failure and heart disease-associated pathologies, e.g. cardiomyocyte necrosis, ischemic cardiac disease and/or ischemic heart damage or ageing. Furthermore, the present invention is directed to a method for the identification of inhibitors of the (GRK2)-mediated phosphorylation of (SRSF1) and/or phosducin.

Claims

1. A method for treatment of heart failure in a patient, the method comprising: administering to the patient in need of such treatment a therapeutically effective amount of a compound according to Formula (Ia): ##STR00054## wherein: X is N; a is an integer between 0 and 15; R.sup.1 is selected from the group consisting of (i) hydroxyl, F, Cl, Br and oxo; (ii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether and (C.sub.4-10)carbocyclic ether; (iii) linear or branched, substituted or non-substituted (C.sub.2-10)alkenyl and (C.sub.2-10)alkynyl; (iv) substituted or non-substituted (C.sub.3-10)carbocycle; and (v) non-substituted indazolyl, substituted or non-substituted benzimidazolyl, and substituted or non-substituted benzodioxolyl, (C.sub.7-C.sub.10)carbo-bicycle, and substituted or non-substituted (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S; R.sup.2 is selected from the group consisting of (i) hydrogen, hydroxyl, O—R.sup.14, —O—C(═O)—R.sup.14, F, C.sub.1, Br and oxo wherein R.sup.14 is selected from the group consisting of (aa) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, and (C.sub.2-10)alkynyl; (bb) substituted or non-substituted aromatic or non-aromatic (C.sub.3-10)carbocycle; and (cc) substituted or non-substituted aromatic or non-aromatic (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S; (ii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, (C.sub.2-10)alkynyl, and (C.sub.3-10)carbocycle; (iii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether and (C.sub.4-10)carbocyclic ether; and (iv) substituted or non-substituted (C.sub.3-6)heterocycle and (C.sub.7-C.sub.10)carbo- or heterobicycle having 1 to 3 heteroatoms each independently selected from N, O and S; R.sup.3 is selected from the group consisting of (i) hydroxyl, —O—R.sup.14, —O—C(═O)—R.sup.14, F, C.sub.1, Br and oxo, wherein R.sup.14 is selected from the group consisting of (aa) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, and (C.sub.2-10)alkynyl; (bb) substituted or non-substituted aromatic or non-aromatic (C.sub.3-10)-carbocycle; and (cc) substituted or non-substituted aromatic or non-aromatic (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S; (ii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, (C.sub.2-10)alkynyl, (C.sub.3-10)carbocycle, and (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S; (iii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether and (C.sub.4-10)carbocyclic ether; (iv) ##STR00055##  wherein R.sup.12 is selected from the group consisting of (aa) hydrogen, hydroxyl, non-substituted N, F, Cl and Br; (bb) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl, and (C.sub.2-10)alkynyl; (cc) substituted or non-substituted aromatic or non-aromatic (C.sub.3-10)carbocycle; and (dd) substituted or non-substituted aromatic or non-aromatic (C.sub.3-6) heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S; and (v) ##STR00056##  wherein X is N or C, a is an integer between 0 and 15, and R.sup.13 is selected from the group consisting of (aa) hydrogen, hydroxyl, F, Cl and Br; (bb) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl and (C.sub.2-10)alkynyl; (cc) substituted or non-substituted (C.sub.3-6)cycloalkyl, (C.sub.7-C.sub.10)carbo- or heterobicycle and (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S, optionally, for R.sup.3, R.sup.13 is (C.sub.7)heterobicycle having 2 heteroatoms selected from N and S, substituted or non-substituted indazolyl, benzimidazolyl and benzodioxolyl, and indazolyl, benzimidazolyl and benzodioxolyl connected via position (5) or (6), via position (5) of the indazolyl and benzodioxolyl or position (6) of the benzimidazolyl; and (dd) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether and (C.sub.4-10)carbocyclic ether; wherein, if position (2) is sp.sup.3-hybridized, R.sup.2 is optionally (R)- or (S)-configured; R.sup.4 is hydroxyl; R.sup.5 is selected from the group consisting of (i) hydrogen, hydroxyl, F, C.sub.1, and Br; (ii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether, (C.sub.2-10)alkenyl ether, (C.sub.2-10)alkynyl ether and (C.sub.4-10)carbocyclic ether; (iii) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl and (C.sub.2-10)alkynyl; (iv) substituted or non-substituted (C.sub.3-10)carbocycle; and (v) (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S, substituted or non-substituted imidazolyl and pyrazolyl, and imidazolyl and pyrazolyl connected via imidazolyl-/pyrazolyl-position-(1)-nitrogen to the rings of Formula (Ia); wherein, if position (5) of the ring of Formula (Ia) is sp.sup.3-hybridized, R.sup.5 is optionally (S)- or (R)-configured; wherein one or more of R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are either directly attached to the rings of Formulas (Ia) or are attached to a linker between R.sup.2, R.sup.3, R.sup.4, and R.sup.5 and the rings of Formulas (Ia), wherein the linker is selected from the group consisting of linear or branched, substituted or non-substituted (C.sub.1-10)alkyl ether, (C.sub.2-10) alkenyl ether, (C.sub.2-10)alkynyl ether, (C.sub.4-10)carbocyclic ether, linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.2-10)alkenyl and (C.sub.2-10)alkynyl; and pharmaceutically acceptable salts.

2. The method according to claim 1, wherein at least one of: a is 0 or 1; R.sup.1 is selected from the group consisting of (i) substituted or non-substituted cyclopropyl, unsubstituted phenyl, and phenyl that is mono-substituted in para position by a substituent selected from the group consisting of H, Cl, F, Br, methyl, —(CF.sub.3) and cyclopropyl; and (ii) non-substituted indazolyl, substituted or non-substituted benzimidazolyl and substituted or non-substituted benzodioxolyl, each connected via position (5) or (6); or a combination thereof.

3. The method according to claim 1, wherein: when R.sup.1 is an indazolyl or benzodioxolyl, it is connected via position 6; or when R.sup.1 is a benzimidazolyl, it is connected via position 5.

4. The method according to claim 2, wherein: when R.sup.1 is an indazolyl or benzodioxolyl, it is connected via position 6; or when R.sup.1 is a benzimidazolyl, it is connected via position 5.

5. The method according to claim 1, wherein at least one of: R.sup.2 is selected from the group consisting of (i) hydrogen or oxo; (ii) linear or branched, substituted or non-substituted (C.sub.1-5)alkyl; and (iii) substituted or non-substituted indazolyl, substituted or non-substituted benzimidazolyl, and substituted or non-substituted benzodioxolyl; R.sup.3 is selected from the group consisting of (i) linear or branched, substituted or non-substituted (C.sub.1-5)alkyl; (ii) ##STR00057##  wherein R.sup.12 is selected from the group consisting of (aa) N; and (bb) substituted or non-substituted cyclopropyl, unsubstituted phenyl, and phenyl that is mono-substituted in para position by cyclopropyl or —(CF.sub.3) or di-substituted in meta position by cyclopropyl or —(CF.sub.3) in each meta position; and (iii) ##STR00058##  wherein X is N, a is 1 and R.sup.13 is selected from the group consisting of substituted or non-substituted indazolyl, benzimidazolyl and benzodioxolyl connected via position (6) or (5) of indazolyl, benzimidazolyl and benzodioxolyl, via position (5) of the indazolyl and benzodioxolyl or position (6) of the benzimidazolyl; R.sup.4 is hydroxyl; wherein R.sup.2 is optionally (R)- or (S)-configured; R.sup.5 is selected from the group consisting of (i) hydrogen; (ii) linear or branched, substituted or non-substituted (C.sub.1-5)alkyl; (iii) substituted or non-substituted cyclopropyl, unsubstituted phenyl, and phenyl that is mono-substituted in para position by a substituent selected from the group consisting of H, C.sub.1, F, Br, methyl, —(CF.sub.3) and cyclopropyl; (iv) cyclopenta-2,4-dien-1-yl; and (v) substituted or non-substituted imidazolyl and pyrazolyl connected via the imidazolyl-/pyrazolyl-position-(1)-nitrogen to the ring of Formula (Ia); wherein R.sup.5 is optionally (R)- or (S)-configured; or a combination thereof.

6. The method according to claim 1, wherein when R.sup.2 is a indazolyl, benzimidazolyl and benzodioxolyl, it is connected via position (5) or (6).

7. The method according to claim 5, wherein when R.sup.2 is a indazolyl, benzimidazolyl and benzodioxolyl, it is connected via position (5) or (6).

8. The method according to claim 1, wherein the compound is a compound of Formula (la): ##STR00059## wherein X is N and a is 0, and wherein at least one of: R.sup.1 is selected from the group consisting of non-substituted indazolyl, substituted or non-substituted benzimidazolyl and substituted or non-substituted benzodioxolyl connected via position (6) or (5); R.sup.2 is oxo; R.sup.3 is selected from the group consisting of (i) methyl; and (ii) ##STR00060##  wherein R.sup.12 is selected from the group consisting of (aa) N; and (bb) cyclopropyl and phenyl that is mono-substituted in para position by cyclopropyl or —(CF.sub.3), or di-substituted in meta position by cyclopropyl or —(CF.sub.3) in each meta position; R.sup.4 is hydroxyl; R.sup.5 is selected from the group consisting of (i) hydrogen; (ii) methyl; (iii) cyclopropyl and phenyl that is mono-substituted in para position by a substituent selected from the group consisting of H, Cl, F, Br, methyl, —(CF.sub.3) and cyclopropyl; (iv) cyclopenta-2,4-dien-1-yl; and (v) imidazolyl and pyrazolyl connected via the imidazolyl-/pyrazolyl-position-(1)-nitrogen to the ring of Formula (Ia); wherein R.sup.5 is optionally (R)- or (S)-configured; or a combination thereof.

9. The method according to claim 8, wherein: R.sup.1 is selected from the group consisting of non-substituted indazolyl connected via position (6) of the indazolyl, substituted or non-substituted benzimidazolyl connected via position (5) of the benzimidazolyl and substituted or non-substituted benzodioxolyl connected via position (6) of the benzodioxolyl.

10. The method according to claim 1, wherein at least one of: R.sup.1 is selected from the group consisting of ##STR00061## R.sup.2 is selected from the group consisting of hydrogen, oxo, methyl, ##STR00062## R.sup.3 is selected from the group consisting of methyl, ##STR00063## R.sup.4 is hydroxyl; a combination thereof; wherein at least one of: R.sup.2 is optionally (R)- or (S)-configured R.sup.5 is selected from the group consisting of: hydrogen, methyl, cyclopenta-2,4-dien-1-yl, ##STR00064## wherein R.sup.5 is optionally (R)- or (S)-configured, or a combination thereof.

11. The method according to claim 1, wherein the compound is selected from the group consisting of: a first residue selected from the group consisting of 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-methyl-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-2-cyclopropyl-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-2-(cyclopenta-2,4-dien-1-yl)-5-oxo-3-hydroxy-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-(pyrazol-1-yl)-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-(imidazol-1-yl)-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-phenyl-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-(p-tolyl)-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-2-(4-chlorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-2-(4-fluorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-2-(4-bromophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-2-(4-cyclopropylphenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1,3-benzodioxol-5-yl)-3-hydroxy-5-oxo-2-[4-(trifluoromethyl)phenyl]-2H-pyrrol-4-yl, 3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-[4-(trifluoromethyl)phenyl]-2H-pyrrol-4-yl, 3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-phenyl-2H-pyrrol-4-yl, 3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-(p-tolyl)-2H-pyrrol-4-yl, 2-(4-chlorophenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 2-(4-fluorophenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 2-(4-bromophenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 2-(4-cyclopropylphenyl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 2-cyclopropyl-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-methyl-2H-pyrrol-4-yl, 3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2-(pyrazol-1-yl)-2H-pyrrol-4-yl, 2-(cyclopenta-2,4-dien-1-yl)-3-hydroxy-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 3-hydroxy-2-(imidazol-1-yl)-1-(1H-indazol-6-yl)-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-dimethyl-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-2-cyclopropyl-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-(pyrazol-1-yl)-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-(imidazol-1-yl)-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-2-(cyclopenta-2,4-dien-1-yl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-2-(4-fluorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-[4-(trifluoromethyl)phenyl]-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-phenyl-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-3-hydroxy-5-oxo-2-(p-tolyl)-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-2-(4-chlorophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-2-(4-bromophenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, 1-(1H-benzimidazol-5-yl)-2-(4-cyclopropylphenyl)-3-hydroxy-5-oxo-2H-pyrrol-4-yl, wherein the numbering of the 2H-pyrrole ring is as follows: ##STR00065## covalently bound to a second residue selected from the group consisting of methyl, ##STR00066##

12. The method according to claim 1, wherein the compound is ##STR00067##

13. The method according to claim 1, wherein the compound is administered as a pharmaceutical composition comprising the compound.

14. The method according to claim 1, wherein the treatment of heart failure reduces the risk of one or more of the following (i) cardiovascular mortality and/or morbidity under conditions of essential hypertension and/or chronic hypertension; (ii) cardiovascular disease-induced ageing; (iii) cardiovascular mortality and/or morbidity under conditions of left ventricular dysfunction and signs of heart failure after recent myocardial infarction; (iv) cardiovascular mortality and/or morbidity under conditions of chronic heart failure and left ventricular dysfunction; (v) cardiovascular mortality and/or morbidity under conditions of dilated cardiomyopathy; (vi) cardiovascular mortality and/or morbidity under conditions of left ventricular dysfunction; (vii) cardiovascular mortality and/or morbidity under conditions of cardiomyocyte necrosis; (viii) cardiovascular mortality and/or morbidity under conditions of cardiac fibrosis; (ix) cardiomyocyte necrosis and/or dilated cardiomyopathy, optionally under conditions with increased risk for ischemic cardiac diseases and ischemic heart damage, optionally as a consequence of cardiovascular risk factors selected from the group consisting of hypertension, atherosclerosis, chronic and acute stress, depression, diabetes mellitus, chronic heart failure, angina pectoris, atrial fibrillation, chronic renal failure and aging; and (x) cardiomyocyte necrosis and/or dilated cardiomyopathy in patients with previous events selected from the group consisting of acute cardiovascular disease, myocardial infarction, ischemic heart disease, angina pectoris, atrial fibrillation, decompensated and chronic heart failure, and cerebrovascular stroke, wherein the patient is a mammalian patient.

15. The method according to claim 1, wherein the treatment of heart failure reduces the risk of a nephropathy, a nephropathy caused by at least one of: hypertension, renal artery stenosis, ischemia, diabetes, toxic agents, or a combination thereof.

16. The method of claim 1, wherein the compound has the chemical structure: ##STR00068## wherein: R.sup.4 is selected from the group consisting of hydroxyl, —O—R.sup.14, and —O—C(═O)—R.sup.14; and R.sup.14 is selected from the group consisting of: (aa) linear or branched, substituted or non-substituted (C.sub.1-10)alkyl, (C.sub.1-5)alkyl, methyl, ethyl, propyl, (C.sub.2-10)alkenyl, and (C.sub.2-10)alkynyl; (bb) substituted or non-substituted aromatic or non-aromatic (C.sub.3-10)carbocycle, (C.sub.3-6) Cycloalkyl, (C.sub.3)carbocycle, (C.sub.6)carbocycle, and phenyl that is mono-substituted in para position by (C.sub.3)carbocycle or —(CF.sub.3) or di-substituted in meta position by (C.sub.3)carbocycle or —(CF.sub.3); and (cc) substituted or non-substituted aromatic or non-aromatic (C.sub.3-6)heterocycle having 1 to 3 heteroatoms each independently selected from N, O and S.

17. The method of claim 1, wherein: R.sup.1 is selected from non-substituted indazolyl, substituted or non-substituted benzimidazolyl, and substituted or non-substituted benzodioxolyl; R.sup.2 is selected from non-substituted indazolyl, substituted or non-substituted benzimidazolyl, and substituted or non-substituted benzodioxolyl; or a combination thereof.

Description

(1) FIGS. 1A-E: Identification of SRSF1 as Novel Non-Receptor GRK2 Substrate

(2) A, Left panel: Enrichment of GRK2 by immunoaffinity chromatography from heart biopsy specimens from heart failure patients, and detection of enriched GRK2 and co-enriched SRSF1 in immunoblot. Right panel: Nano-LC-ESI-MS/MS analysis identified SRSF1 (Serine/arginine-rich splicing factor 1, ASF/SF2, SEQ ID NO: 7) as a previously unrecognized GRK2-interacting protein in heart biopsy specimens from heart failure patients. B, GRK2 phosphorylates SRSF1 in an in vitro kinase assay. Nano-LC-ESI-MS/MS analysis Identification of phospho-containing peptides with serine199/201 as GRK2 phosphorylation site(s) (SEQ ID NO: 8 and SEQ ID NO: 9). C, Paroxetine inhibits the GRK2-mediated phosphorylation of SRSF1 with a half maximum inhibitory concentration (IC50 value) of 2.38 μM (n=4). D, Inhibition of GRK2-mediated SRSF1 phosporylation by GRKInh, another cardio-protective GRK2 inhibitor. E, Immunoblot detection of the activating phosphorylation of SRSF1 by GRK2 with phospho-specific SR antibody 1H4 (p-SRSF1).

(3) FIGS. 2A-2L: GRK2 Induces Activating Srsf1 Phosphorylation In Vivo

(4) A, B, Generation of Tg-GRK2 mice with myocardium-specific expression of GRK2 under control of the alpha-MHC promoter (A) and immunoblot detection of increased GRK2 protein in heart lysates from Tg-GRK2 mice relative to non-transgenic B6 controls (B, C; n=4 hearts/group). D, Tg-GRK2 mice have a decreased left ventricular ejection fraction as determined by echocardiography (±s.d., n=6). E-G, Immunoblot detection of increased p-Srsf1 (right) and Camk2d isoforms B and C contents in heart lysates from Tg-GRK2 mice relative to non-transgenic controls (n=4/group). H, Myocardial necrosis in Tg-GRK2 hearts was determined by von Kossa stain (n=6; ±s.d.), bar: 100 micron. I, J, Immunoblot detection of pSrsf1 (I) and Camk2d isoforms B/C (J) in Tg-GRK2 hearts transduced with a control lentivirus or a lentivirus targeting Srsf1 by a miRNA (+miSrsf1). K, Down-regulation of Srsf1 by miSrsf1 retards the development of cardiac dysfunction in Tg-GRK2 mice (n=4, ±s.d.). L, Myocardial necrosis was determined with von Kossa stain (n=6; ±s.d.).

(5) FIGS. 3A-F: Transgenic Overexpression of SRSF1 Induces Enhanced Camk2d Splicing and Signs of Heart Failure

(6) A, Generation of transgenic mice with myocardium-specific SRSF1 expression under control of the myocardium-specific alpha-MHC Promoter. B, Increased cardiac SRSF1/Srsf1 protein of Tg-SRSF1 mice was detected in immunoblot (n=4 hearts/group). C, Enhanced cardiac splicing of Camk2d protein isoforms B and C in Tg-SRSF1 mice (n=4 hearts/group). D, Myocardial necrosis in Tg-SRSF1 hearts was determined with von Kossa stain (n=5, ±s.d.). E, Histological assessment of a Tg-SRSF1 heart shows cardiac hypertrophy with dilation relative to the non-transgenic B6 control. Cardiac sections were stained with hematoxylin-eosin (HE) and are representative of 4 mice/group. F, Cardiac dysfunction of Tg-SRSF1 mice relative to non-transgenic B6 controls was determined by echocardiography (n=5; ±s.d.).

(7) FIGS. 4A-D: Inhibition of GRK2 In Vivo Retards Activating Srsf1 Phosphorylation, and Signs of Heart Failure in a Chronic Pressure Overload Model of Cardiac Dysfunction

(8) A, Generation of transgenic mice with myocardium-specific expression of dominant-negative GRK2-K220R and immunoblot detection of GRK2-K220R in Tg-GRK2-K220R hearts (n=3 (B6 controls) and n=5 (Tg-GRK2-K220R hearts). B, Immunoblot detection of cardiac pSrsf1 and Camk2d isoforms B/C in hearts from 4 month-old B6 controls (B6), Tg-GRK2-K220R and Tg-GRKInh mice with 8 weeks of chronic pressure overload imposed by abdominal aortic constriction (AAC); n=4 mice/group. C, Myocardial necrosis was determined by von Kossa stain (n=6/group; ±s.d.).D, Cardiac dysfunction in B6 mice with 8 weeks of AAC was retarded by GRK2 inhibition with GRK2-K220R or GRKInh (n=6/group; ±s.d.).

(9) FIGS. 5A-G: The GRK2 Inhibitor RKIP does not Inhibit Activating Srsf1 Phosphorylation and Induces Signs of Heart Failure In Vivo

(10) A, Human RKIP does not inhibit the phosphorylation of SRSF1 by GRK2 in vitro. In contrast, human RKIP inhibited the phosphorylation of phosducin by GRK2 (IC50=950 nM, n=6-7). B, C, Immunoblot detection of cardiac RKIP in Tg-RKIP2 and Tg-RKIP3 line with myocardium-specific expression of human RKIP (B; n=3 mice/group), and reduced left ventricular ejection fraction of Tg-RKIP2 and Tg-RKIP3 line (n=5/group; ±s.d.). D, Histological assessment of an 8 month-old Tg-RKIP2 heart relative to B6 and Tg-GRK2-K220R hearts (left) and heart-weight to body-weight determination (n=6, ±s.d). Histological sections are representative of 4 hearts/group. E, F, Cardiac fibrosis was determined with picrosirius red staining (E) and necrosis was determined by von Kossa stain (F) in Tg-RKIP2 hearts, B6 and Tg-GRK2-K220R hearts (n=6; ±s.d.). G, Immunoblot detection of cardiac Srsf1 (total Srsf1) and activated phosphorylated p-Srsf1 in heart lysates from the different groups of mice (n=3/group).

(11) FIGS. 6A-G: Tg-RKIP Mouse Lines in the FVB Background Also Develop Signs of Heart Failure

(12) A, Immunoblot detection of human/mouse RKIP in Tg-RKIP mouse lines generated in the FVB background (n=8/group, ±s.d.). B, Immunohistological detection of the RKIP protein in a Tg-RKIP1 heart relative to a non-transgenic FVB control. Immunohistology is representative of 4 hearts/group. C, D, Left ventricular ejection fraction (C; n=5) and heart-weight to body-weight ratio of Tg-RKIP1 and Tg-RKIP2 mice relative to non-transgenic FVB controls (n=6; ±s.d.). E, Histological analysis of Tg-RKIP hearts relative to a non-transgenic FVB control. The histological sections are representative of four mice/group. F, G, Left ventricular ejection fraction of Tg-RKIP1 and TgRKIP2 mice relative to non-transgenic FVB controls, and RNAi-mediated down-regulation of human RKIP by lentiviral transduction of miRKIP (F; n=5; ±s.d.). Down-regulation of human RKIP was confirmed by real-time qRT-PCR (G; n=5; ±s.d.)

(13) FIGS. 7A-F: Transgenic RKIP Expression Induces a GRK2 Inhibition-Related Gene Expression Signature

(14) A, Immunoblot detection of serine-153 phosphorylation of RKIP (pS153) in Tg-RKIP and B6 control hearts (n=5 mice/group). B, Removal of RKIP by affinity purification (AP) with immobilized RKIP-specific antibodies depletes the GRK2 protein from Tg-RKIP heart lysates. A control affinity purification with unrelated antibodies did neither deplete RKIP nor GRK2. Upper panels show representative experiments and lower panels show quantitative data evaluation (±s.d., n=4; *, p=0.0286; Mann Whitney Test). C, Isoproterenol-stimulated cAMP response in neonatal cardiomyocytes from Tg-RKIP and Tg-GRK2-K220R mice (n=6; ±s.d.). D, Significantly increased expression of the cAMP inducible gene, Ttc14, in Tg-RKIP and Tg-GRK2-K220R hearts relative to the B6 control (n=3; ±s.d.). E, F, Whole genome gene expression profiling revealed concordant regulation of 45% of significantly altered probe sets between Tg-RKIP and Tg-GRK2-K220R hearts. Panel F shows concordantly regulated probe sets in Tg-RKIP and Tg-GRK2-K220R hearts (two gene chips/group; **, p<0.01 and ***, p<0.001, t-test relative to B6 control).

(15) FIGS. 8A-8F: The Cardio-Protective GRK2 Inhibitor Paroxetine Retards the Heart Failure Phenotype of Tg-RKIP Mice

(16) A-C, Treatment with paroxetine (5 mg/kg body weight in drinking water per day) for 8 weeks decreased the cardiac content of Srsf1 and activating Srsf1 phosphorylation (A), and the splicing of Camk2d isoforms B/C (B) in 5 month-old Tg-RKIP hearts as determined in immunoblot (B, n=5 hearts/group) and real-time qRT-PCR (C, n=6; ±s.d). C, Myocardial necrosis with calcium overload was determined on cardiac sections by von Kossa stain (n=6 hearts/group; ±s.d.). D, The left ventricular ejection fraction was determined by echocardiography (n=6 mice/group; ±s.d.). D-F, the development of myocardial necrosis and signs of heart failure were retarded.

(17) FIG. 9: Development of Small Molecule Compounds, which Inhibit GRK2-Mediated Srsf1 Phosphorylation.

(18) The GRK2-mediated phosphorylation of SRSF1 was determined with purified proteins in the presence of increasing concentrations of paroxetine or different small molecule compounds. SRSF1 phosphorylation is presented as % of control, which is the SRSF1 phosphorylation in the absence of inhibitor. The IC50 value of 0.45 μM of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) is more than 5-fold lower than that of paroxetine (±s.d., n=4).

(19) FIGS. 10A-B: Small Molecule Compounds Inhibit the GRK2-Mediated SRSF1 Phosphorylation in Intact Tg-SRSF1 Cardiomyocytes and Human HEK Cells.

(20) A, The GRK2-mediated phosphorylation of SRSF1/Srsf1 was determined by immunoblot in lysates from isolated neonatal cardiomyocytes from Tg-SRSF1 mice after incubation for 60 h with 10 microM of paroxetine, 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) or 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”) relative to vehicle-treated control (“C”) cardiomyocytes. B, Immunoblot detection of GRK2-mediated phosphorylation of SRSF1 (pSRSF1) was performed with lysates from HEK (human embryonic kidney) cells incubated for 60 h with 10 microM of 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide (“Compound-24”) or (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]-methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) relative to vehicle-treated controls (“C”).

(21) FIGS. 11A-B: Cardio-Protective Effects of Small Molecule Compounds In Vivo.

(22) A, The GRK2-mediated phosphorylation of Srsf1 was determined by immunoblot in cardiac lysates prepared from hearts of non-transgenic B6 mice after treatment (i.p.) for 5 h with 10 mg/kg of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”), 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrole-3-carboxamide (“Compound-2”), or (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]-methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) relative to vehicle-treated B6 controls (“con”). The right panel shows quantitative immunoblot evaluation. B, Left panel: The AAC-induced cardiac p-Srsf1 content was determined in cardiac lysates from B6 mice with AAC after treatment for 7 days with 5 mg/kg/d of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) or (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]-methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) relative to vehicle-treated B6 controls with AAC (“AAC-con”). The middle panel shows quantitative immunoblot evaluation. Right and lower panels: Cardiac function parameters were determined by echocardiography in B6 mice with AAC after treatment for 7 days with “Compound-1” or “Compound-5” relative to vehicle-treated B6 controls with AAC ((±s.d.; n=5 (A) and n=3 (B); Dunnett's Multiple Comparison Test vs. con (A) or AAC-con (B)).

(23) FIGS. 12A-D: Oral Treatment with Compound-1 and Compound-4 Counteracts Heart Failure and Ageing Induced by Cardiovascular Disease.

(24) A, The left ventricular ejection fraction was determined in 6 month-old male B6 mice with chronic pressure overload imposed by 3 months of AAC and oral treatment with Compound-1 (AAC+compd-1) and Compound-4 (AAC+compd-4) relative to untreated AAC controls. Treatment was started one month after AAC induction and was continued for 2 months. Only mice with heart failure (ejection fraction <34% after one month of AAC) were included in the study (±s.d., n=5/group, ***p=0.0006, **p=0.0051 vs. untreated AAC; Dunnett's multiple comparison test). B, Histological assessment of hearts with 3 months of AAC without (controls, upper panels) or with 2 months of treatment with Compound-1 (lower panels) reveals that Compound-1 retards the chronic pressure overload-induced cardiac hypertrophy (n=4 hearts/group). C,D, Treatment with Compound-1 also retards cardiovascular disease-induced ageing, e.g. induced by chronic pressure overload, i.e. the grey coloring of the hair was substantially retarded after 2 months of treatment with Compound-1 (left (D) and right (D) panels, n=5 different mice).

(25) FIGS. 13A-J: Identification of Tested Compounds According to the Present Invention Molecule and Exemplary Chemical Synthesis Route

(26) A-H, Compounds-1 to -5 and -22 to -24 were analyzed by electrospray mass spectrometry and HPLC analysis. I, J, exemplary chemical synthesis routes of Compound-1 and Compound-4.

EXAMPLE 1: IDENTIFICATION OF SRSF1 AS NOVEL NON-RECEPTOR GRK2 SUBSTRATE

(27) In view of the pathophysiological importance of GRK2 and its yet unknown targets novel GRK2-interacting proteins were searched for. GRK2-specific antibodies were covalently coupled to an affinity matrix. A protein lysate from biopsy specimens from heart failure patients was applied, bound proteins were eluted, and enriched GRK2 and co-enriched proteins were separated by SDS-PAGE. Stained protein bands were cut out and Nano-LC-ESI-MS/MS analysis identified SRSF1 (Serine/arginine-rich splicing factor 1) ASF/SF2) as a previously unrecognized GRK2-interacting protein (FIG. 1A, right panel). As a control, immunoblot detection confirmed the protein enrichment of GRK2 and the co-enrichment of SRSF1 (FIG. 1A, left panels). It was found that SRSF1 is a kinase substrate of GRK2. In an in vitro kinase assay, GRK2 phosphorylated the recombinant SRSF1 protein (FIG. 1B). Nano-LC-ESI-MS/MS analysis identified phospho-containing peptides with serine199/201 as GRK2 phosphorylation site(s) (FIG. 1B). The phosphorylation of SRSF1 on serine 199/201 by GRK2 is of relevance because these residues and phosphorylation of these residues are essentially involved in the splicing function of SRSF1 (Zuo P and Manley J L, EMBO J 12, 4727-4737 (1993); Xiao S H and Manley J L, Genes Dev 11, 334-344 (1997)). As a control for GRK2-specificity of the in vitro phosphorylation assay, the cardio-protective ATP-site-directed GRK2 inhibitor, paroxetine (Thal D M, et al., ACS Chem Biol 7, 1830-1839 (2012); Schumacher S M, et al., Sci. Transl. Med. 7, 277ra31 (2015)) was applied. Paroxetine inhibited the GRK2-mediated phosphorylation of SRSF1 with a half maximum inhibitory concentration (IC50 value) of 2.38 μM (FIG. 1C). This value is comparable to the paroxetine-mediated inhibition of other GRK2 substrates (Thal D M, et al., ACS Chem Biol 7, 1830-1839 (2012); Schumacher S M, et al., Sci. Transl. Med. 7, 277ra31 (2015)). In addition, GRKInh, another cardio-protective GRK2 inhibitor (6,7), also inhibited the in vitro phosphorylation of SRSF1 by GRK2 (FIG. 1D). The activating phosphorylation of SRSF1 by GRK2 was also confirmed in immune-blot with the phospho-specific SR antibody 1H4 (FIG. 1E), which detects phosphorylated RS repeats in the carboxyl terminal domain of SRSF1 (Neugebauer K M, et al., Genes Dev. 11, 1148-1159 (1997)).

EXAMPLE 2: GRK2 INDUCES ACTIVATING SRSF1 PHOSPHORYLATION IN VIVO

(28) It was investigated whether GRK2 phosphorylates Srsf1 in vivo and Tg-GRK2 mice were generated with myocardium-specific human GRK2 (ADRBK1) expression under control of the myocardium-specific alpha-MHC promoter (FIG. 2A). The hearts of Tg-GRK2 mice had an increased GRK2 protein level (2-fold over the non-transgenic B6 control, FIGS. 2B and 2C). In agreement with the pathophysiological role of an increased cardiac GRK2 protein level (Hullmann J, et al., Pharmacol. Res. 110, 52-64 (2016)), Tg-GRK2 mice developed cardiac dysfunction with increased age as documented by a significantly reduced cardiac ejection fraction in 8 month-old Tg-GRK2 mice (FIG. 2D). The cardiac content of activated phospo-Srsf1 was determined to analyze whether the increased GRK2 protein level in transgenic Tg-GRK2 mice resulted in increased Srsf1 phosphorylation and activation. Immunoblot detection revealed that Tg-GRK2 hearts had an increased level of activating Srsf1 phosphorylation (FIG. 2E). The splicing factor activity of activated Srsf1 is required for Camk2d (calcium calmodulin-dependent kinase II isoform 6) isoform B and C splicing (Xu X, et al., Cell 120, 59-72 (2005)). In agreement with the enhanced activation of Srsf1, Tg-GRK2 hearts had an increase in heart-specific Camk2d isoforms B and C as detected in immunoblot (FIG. 2G). An increase in Camk2d isoforms B and C is sufficient to promote cardiac dysfunction and heart failure (Zhang T, et al., Circ. Res. 92, 912-919 (2003); Zhang T, et al., J. Biol. Chem. 277, 1261-1267 (2002)).

(29) Moreover, Camk2d promotes cardiac necrosis (Zhang T, et al., Nat. Med. 22, 175-182 (2016)). Tg-GRK2 hearts showed an increase in necrotic areas with calcium overload as determined with von Kossa stain (FIG. 2H). The causal relationship between GRK2-mediated Srsf1 activation and the cardiac phenotype of Tg-GRK2 hearts was also demonstrated by down-regulation of Srsf1 by lentiviral transduction of an miRNA targeting Srsf1 by RNAi (FIG. 2I). The down-regulation of Srsf1 retarded the induction of Camk2d isoforms B and C, the development of signs of heart failure and cardiac necrosis with calcium overload in Tg-GRK2 hearts (FIG. 2J-2L).

EXAMPLE 3: TRANSGENIC OVEREXPRESSION OF SRSF1 INDUCES ENHANCED CAMK2D SPLICING AND SIGNS OF HEART FAILURE

(30) Tg-SRSF1 mice with increased cardiac SRSF1 protein due to myocardium-specific SRSF1 expression were generated under control of the myocardium-specific alpha-MHC Promoter (FIG. 3A). Tg-SRSF1 mice had an increased cardiac SRSF1 protein (FIG. 3B) and showed enhanced splicing of Camk2d isoforms B and C (FIG. 3C). Concomitantly, Tg-SRSF1 mice developed myocardial necrosis with calcium overload, cardiac hypertrophy with dilation (indicative of cardiomyocyte loss) and cardiac dysfunction (FIG. 3D-F).

EXAMPLE 4: INHIBITION OF GRK2 IN VIVO RETARDS ACTIVATING SRSF1 PHOSPHORYLATION, AND SIGNS OF HEART FAILURE IN A CHRONIC PRESSURE OVERLOAD MODEL OF CARDIAC DYSFUNCTION

(31) Srsf1 and GRK2 are up-regulated in experimental models of pressure overload-induced cardiac dysfunction (Hullmann J, et al., Pharmacol. Res. 110, 52-64 (2016); Kim T, et al., Mol Cells 37, 81-87 (2014)). The pressure overload-triggered induction of activating Srsf1 phosphorylation was retarded by GRK2 inhibition with transgenic expression of the dominant-negative GRK2-K220R mutant (FIG. 4B). The transgenic Tg-GRK2-K220R mice with myocardium-specific expression of GRK2-K220R were generated (FIG. 4A). The cardio-protective GRK2 inhibitor, GRKInh (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016); Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)), which inhibits SRSF1 phosphorylation in vitro (cf. FIG. 1), also retarded the pressure overload-induced activating Srsf1 phosphorylation by GRK2. Concomitantly, the two different modes of GRK2 inhibition retarded Camk2d isoform B/C splicing, myocardial necrosis with calcium overload and signs of heart failure (FIG. 4C,D). Thus, cardio-protective GRK2 inhibition blunts pressure overload-induced Srsf1 activation, cardiomyocyte death and signs of heart failure.

EXAMPLE 5: THE GRK2 INHIBITOR RKIP DOES NOT INHIBIT ACTIVATING SRSF1 PHOSPHORYLATION AND INDUCES SIGNS OF HEART FAILURE IN VIVO

(32) The role of another GRK2 inhibitor, i.e. the raf kinase inhibitor protein, RKIP (Lorenz K, et al., Nature 426, 574-579 (2003)) was analyzed, which is a dual-specific GRK2 and Raf kinase inhibitor. RKIP shows up-regulation in cardiac biopsy specimens of patients with heart failure (Schmid E, et al., Nat. Med. 21, 1298-1306 (2015)). Human RKIP did not inhibit the phosphorylation of SRSF1 by GRK2 in vitro (FIG. 5A). As a control, human RKIP is an efficient GRK2 inhibitor and inhibited the phosphorylation of phosducin (FIG. 5A), which is another non-receptor substrate of GRK2 (Ruiz-Gomez A, et al., J. Biol. Chem. 275, 29724-29730 (2000)). The IC50 value for human RKIP-mediated inhibition of GRK2-induced phosducin phosphorylation was 950 nM (FIG. 5A), which is comparable to the reported IC50 value of 460 nM for RKIP-mediated inhibition of GPCR phosphorylation (Lorenz K, et al., Nature 426, 574-579 (2003)). Tg-RKIP mice with myocar-dium-specific expression of human RKIP developed signs of heart failure in a dose-dependent manner (FIG. 5B,C). Concomitantly, cardiac hypertrophy with dilation, cardiac fibrosis and necrosis were evident (FIG. 5D-F). In agreement with the in vitro experiments, which document-ted the inability of human RKIP to inhibit activating SRSF1 phosphorylation by GRK2 (cf. FIG. 5A), the cardiac content of activating Srsf1 phosphorylation was higher in Tg-RKIP hearts than that in non-transgenic B6 controls (FIG. 5G).

EXAMPLE 6: TG-RKIP MOUSE LINES IN THE FVB BACKGROUND ALSO DEVELOP SIGNS OF HEART FAILURE

(33) The heart failure phenotype induced by transgenic human RKIP expression was similarly detected in a dose-dependent manner in human RKIP-expressing transgenic mouse lines generated in the FVB background (FIG. 6A-D). Histology analysis revealed that Tg-RKIP mice in the FVB background developed a strong cardiac hypertrophy with dilation and cardiomyocyte loss (FIG. 6E). The cardiac dysfunction of Tg-RKIP mice was partially reversed by down-regulation of transgenic RKIP (PEBP1) by lentiviral transduction of an miRNA targeting RKIP (PEBP1) by RNAi (FIG. 6F,G). Thus, the heart failure phenotype in Tg-RKIP mice was attributed to transgenic RKIP expression.

EXAMPLE 7: TRANSGENIC RKIP INTERACTS WITH GRK2 AND INDUCES A GRK2 INHIBITION-RELATED GENE EXPRESSION SIGNATURE

(34) The GRK2-inhibitory activity of transgenic RKIP (human RKIP˜PEBP1) was controlled in vivo. The phosphorylation of RKIP on serine-153 switches RKIP from Raf1 inhibition to GRK2 inhibition (Lorenz K, et al., Nature 426, 574-579 (2003)). Substantial serine-153 phosphorylation of RKIP was documented by immunoblot detection in Tg-RKIP hearts (FIG. 7A). The serine-153 phosphorylated RKIP was sufficient to bind and neutralize 90.4% (±3.6%, n=4) of the GRK2 protein in Tg-RKIP hearts (FIG. 7B). We determined the GRK2 inhibition-mediated resensitization of the isoproterenol-stimulated cAMP response in neonatal cardiomyocytes as readout for GRK2 inhibition by RKIP and GRK2-K220R, respectively (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016); Kong K C, et al., Biochemistry 47, 9279-9288 (2008)). Neonatal cardiomyocytes from Tg-RKIP and Tg-GRK2-K220R mice showed comparable signs of GRK2 inhibition as documented by the significantly enhanced β-adrenoceptor-mediated cAMP response (FIG. 7C). Resensitized cAMP signaling was confirmed in vivo by the significantly increased expression of the cAMP inducible gene, Ttc14, in Tg-RKIP and Tg-GRK2-K220R hearts (FIG. 7D). Whole genome microarray gene expression profiling further revealed a GRK2 inhibition-related gene expression signature in Tg-RKIP hearts (FIGS. 7E,F). Notably, there was concordant regulation of 45% of significantly altered probe sets between Tg-RKIP and Tg-GRK2-K220R hearts (FIGS. 7E,F).

EXAMPLE 8: THE CARDIO-PROTECTIVE GRK2 INHIBITOR PAROXETINE RETARDS THE HEART FAILURE PHENOTYPE IN TG-RKIP MICE

(35) It was analyzed, whether the incapability of RKIP to inhibit the activating Srsf1 phosphorylation by GRK2 contributed to the heart failure phenotype in Tg-RKIP mice. Tg-RKIP mice were treated with the GRK2 inhibitor, paroxetine, which is cardio-protective in an experimental model of myocardial infarction (Schumacher S M, et al., Sci. Transl. Med. 7, 277ra31 (2015)). Paroxetine also inhibits SRSF1 phosphorylation in vitro (cf. FIG. 1C). Treatment with paroxetine for 8 weeks decreased the cardiac content of activating Srsf1 phosphorylation and the splicing of Camk2d isoforms B/C in Tg-RKIP hearts (FIG. 8A-C). Concomitantly, the development of myocardial necrosis and signs of heart failure were retarded (FIG. 8D-F). Taken together, data with transgenic mice and different GRK2 inhibitors show that cardio-protective GRK2 inhibition relies on inhibition of the activating Srsf1 phosphorylation by GRK2.

EXAMPLE 9: DEVELOPMENT OF SMALL MOLECULE COMPOUNDS, WHICH INHIBIT GRK2-MEDIATED SRSF1 PHOSPHORYLATION

(36) Small molecule compounds were developed, which inhibit GRK2-mediated SRSF1 phosphorylation (FIG. 9). 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) inhibits the GRK2-mediated SRSF1 phosphorylation with an IC50 value of 0.45 microM. The IC50 value of 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) is more than 5-fold lower than that of paroxetine (FIG. 9), which is the only available small molecule GRK2 inhibitor with documented cardio-protective activity under experimental conditions in vivo (Schumacher S M, et al., Sci. Transl. Med. 7, 277ra31 (2015)). The four compounds 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrol-3-carboxamide, (3R)—N-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide, 4-(1,3-benzodioxol-5-yl)pyrimidine, and (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]-methyl]pyrrolidine-3,4-dicarboxamide (“Compounds 2-5”) inhibited the SRSF1 phosphorylation with IC50 values ranging between 1.87 microM (4-(1,3-benzodioxol-5-yl)pyrimidine, “Compound-4”) and 12.87 microM ((3R)—N-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)thiomorpholine-3-carboxamide, “Compound-3”) (FIG. 9). An unrelated control compound, Ibuprofen ((±)-2-(4-Isobutylphenyl)propanoic acid (“Compound-6”, Sigma-Aldrich, St. Louis, USA) had no inhibitory effect up to 1 mM (FIG. 9).

EXAMPLE 10: SMALL MOLECULE COMPOUNDS INHIBIT THE GRK2-MEDIATED SRSF1/SRSF1 PHOSPHORYLATION IN INTACT CARDIOMYOCYTES AND HUMAN KIDNEY CELLS

(37) 1-(1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”) and 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”) also decreased the heart failure-promoting SRSF1/Srsf1 phosphorylation in isolated Tg-SRSF1 cardiomyocytes (FIG. 10A). GRK2 inhibition promotes survival of human kidney cells (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)). 4-(1,3-benzodioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-chlorophenyl)-2-methyl-pyrrole-3-carboxamide (“Compound-24”) and (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]-methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) inhibited the cell-damaging SRSF1 phosphorylation in human kidney cells (FIG. 10B).

EXAMPLE 11: CARDIO-PROTECTIVE EFFECTS OF SMALL MOLECULE COMPOUNDS IN VIVO

(38) Short-term treatment for 5 h with small molecule compounds (1-((1,3-benzodioxol-5-yl)-4-(cyclopropane-carbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (“Compound-1”), 4-(1,3-benzo-dioxol-5-yl)pyrimidine (“Compound-4”), 1-(1,3-benzodioxol-5-ylmethyl)-5-(4-fluorophenyl)-2-methyl-pyrrol-3-carboxamide (“Compound-2”) and (4R)-N3-(1,3-benzodioxol-5-ylmethyl)-N4-[[3-(trifluoromethyl)phenyl]methyl]pyrrolidine-3,4-dicarboxamide (“Compound-5”) decreased the heart failure promoting GRK2-mediated Srsf1 phosphorylation under basal conditions in vivo, in the hearts of non-transgenic B6 mice (FIG. 11A). Treatment with “Compound-1” or “Compound-5” also retarded the chronic pressure overload-induced cardiac p-Srsf1 content and improved the cardiac performance of B6 mice after chronic pressure overload imposed by AAC (FIG. 11B).

EXAMPLE 12: ORAL TREATMENT WITH COMPOUND-1 AND COMPOUND-4 COUNTERACTS HEART FAILURE AND AGEING INDUCED BY CARDIOVASCULAR DISEASE

(39) We investigated whether the new small molecule GRK2 inhibitors, Compound-1 (1-(1,3-benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one)) and Compound-4 (4-(1,3-benzodioxol-5-yl)pyrimidine) could retard symptoms of heart failure in a chronic pressure overload model of heart failure imposed by abdominal aortic constriction (AAC). Compound-1 (3 mg/kg/d) and Compound-4 (5 mg/kg/d) were applied orally in the AAC-induced model of heart failure. Oral treatment with Compound-1 and Compound-4 was initiated in B6 mice with symptoms of heart failure (left ventricular ejection fraction <34%) induced by four weeks of AAC. Two months of treatment with Compound-1 and Compound-4, respectively, counteracted the AAC-induced cardiac dysfunction as documented by a significantly improved left ventricular ejection fraction of 41.4±4.8% and 38.2±3.7% after treatment with Compound-1 and Compound-4, respectively, compared to the ejection fraction of the untreated AAC controls of 28.6%±3.5% (FIG. 12A). In addition to the improved cardiac function, histological analysis showed a decrease in the AAC-induced cardiac hypertrophy after oral treatment with Compound-1 (FIG. 12B). Concomitantly, as a consequence of the improved cardiac function, cardiovascular disease-induced ageing of the mice was visibly retarded, i.e. the grey coloring of the black hair was substantially retarded after 8 weeks of treatment with Compound-1 compared to untreated B6 control animals (FIG. 12C, D).

EXAMPLE 13: MATERIAL AND METHODS

(40) Generation of Transgenic Mice

(41) The study used the following transgenic mouse lines, which were generated by our group: Tg-RKIP (PEBP1) mice in the B6 (C57BL/6J) and FVB background, Tg-GRK2 (ADRBK1) mice in the B6 background, Tg-SRSF1 mice in the B6 background, Tg-GRK2-K220R and Tg-GRKInh mice in the B6 background. All the transgenes were expressed under control of the myocardium-specific alpha-MHC promoter (the MyHC plasmid was kindly provided by James Gulick, Gulick J, et al., J. Biol. Chem. 266, 9180-9185 (1991)). Transgenic mice were generated according to standard procedures. Briefly, the DNAs encoding the indicated proteins/peptides were inserted into the alpha-MHC (MyHC) plasmid, plasmid sequences were removed by Not I digestion and the purified DNA (2 ng/microL) was injected into fertilized oocytes of superovulated B6 (C57BL/6J) and FVB (FVB/N) mice. Oviduct transfer of injected embryos into pseudopregnant CD-1 mice was performed according to standard procedures. Genomic DNA of the FO generation was isolated from ear punch biopsies taken at an age of 3-4 weeks and analyzed by PCR for integration of the transgene (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)).

(42) Chronic Pressure Overload-Induced Model of Experimental Heart Failure and Transthoracic Echocardiography

(43) Chronic pressure overload imposed by abdominal aortic constriction (AAC) was used as an experimental model to induce cardiac hypertrophy and signs of heart failure. Aortic constriction of the abdominal aorta was performed in tribromoethanol-anesthetized 8-12 week-old transgenic mice or non-transgenic B6 controls as described (AbdAlla S, et al., Cardiovasc. Hematological Agents Med. Chem. 9, 190-206 (2011)). The abdominal aorta was constricted above the suprarenal artery by tying a 7-0 silk suture ligature against a blunt 26-gauge needle. Age-matched controls underwent identical surgical procedure except for ligation of the aorta (sham-operated mice).

(44) Cardiac function parameters were determined by transthoracic echocardiography, which was performed on anesthetized mice with a Vivid 7 echocardiograph equipment (GE Healthcare, Glattbrugg, Schweiz) and a 12 MHz linear array transducer. The left ventricular ejection fraction was calculated in the M-mode of the parasternal long-axis view using the formula of Teichholz. M-mode imaging was performed according to the recommendations of the American Society of Cardiology (Sahn D J, et al., Circulation 58, 1072-1083 (1978)) adapted to mice. Recordings were interpreted offline using EchoPac Pc 3.0 software (GE Healthcare, Glattbrugg, Schweiz).

(45) Animal experiments were performed in accordance with the NIH guidelines, and reviewed and approved by the local committee on animal care and use (University of Zurich).

(46) Nano-LC-ESI-MS/MS

(47) To enrich proteins interacting with human heart GRK2 protein, a protein lysate was prepared from small myocardial biopsy specimens from patients with signs of heart failure undergoing mitral valve replacement. Informed consent was obtained from all participants. The study was conducted in conformity with the principles of the declaration of Helsinki, with approval of the protocol by the ethical committee of Ain Shams University. Patient characterization of study participants was published previously (AbdAlla S, et al., Cardiovasc. Hematological Agents Med. Chem. 9, 190-206 (2011)). The enrichment of GRK2 and co-enrichment of GRK2-interacting proteins was performed similarly as described (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)). Briefly, myocardial proteins were solubilized for 30 min at 4° C. with solubilization buffer (1% sodium deoxycholate, 0.05% SDS, 0.05% Tween 20 in PBS, pH 7.4 supplemented with protease inhibitors), insoluble material was removed by centrifugation, the supernatant was diluted 1:5 in PBS supplemented with protease inhibitors and subjected to affinity chromatography with anti-GRK2 antibodies (6 mg of affinity-purified IgG coupled to 1 ml of Affigel 10, Bio-Rad Gmbh, München, Germany; polyclonal anti-GRK2 antibodies were raised in rabbit against full-length recombinant GRK2 protein expressed in Sf9 insect cells). After overnight incubation at 4° C., unbound proteins were removed by washing with PBS (20 column volumes), and bound proteins were eluted with 0.25 M NH.sub.4OH, 10% dioxane, pH 11. The pH of the eluate was immediately adjusted to pH 7.4, eluted proteins were concentrated by acetone precipitation, dissolved in 8 M urea, and subjected to 8% urea-containing SDS-PAGE under reducing conditions. After Coomassie Brilliant Blue staining, enriched protein bands were cut and subjected to nano-LC-ESI-MS/MS. The SRSF1 protein was identified in the gel slice encompassing the 30-40 kDa protein range. Protein identification was performed by nano-LC-ES-MS/MS (Proteome Factory AG, Berlin). The MS system consisted of an Agilent 1100 nano-LC system (Agilent, Boeblingen, Germany), a PicoTip emitter (New Objective, Woburn, Mass.) and an Esquire 3000 plus ion trap MS (Bruker, Bremen, Germany). The cut protein band was in-gel digested by trypsin (Promega, Mannheim, Germany) and applied to non-LC-MS/MS. After trapping and desalting the peptides on an enrichment column (Zorbax SB C18, 0.3×5 mm, Agilent Boeblingen, Germany) using 1% acetonitrile, 0.5% formic acid solution for 5 min, peptides were separated on a Zorbax 300 SB C18, 75 microm×150 mm column (Agilent Boeblingen, Germany) using an acetonitrile, 0.1% formic acid gradient from 5% to 40% acetonitrile within 40 min. MS spectra were automatically taken by Esquire 3000 plus according to the manufacturer's instrument settings for nan-LC-MS/MS analyses. Proteins were identified using MS/MS ion search of the Mascot search engine (Matrix Science, London, England) and nr protein database (National Center for Biotechnology Information, Bethesda, Md.). Ion charge in search parameters for ions form ESI-MS/MS data acquisition were set to “1+, 2+ or 3+” according to the common charge state distribution for the instrument and the method (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)).

(48) For identification of residues in SRSF1, which were phosphorylated by GRK2, the nano-LC-ES-MS/MS analysis was performed with purified recombinant SRSF1 after the in vitro phosphorylation assay. The procedure was performed as detailed above but the MS system consisted of an Agilent 1100 nanoLC system (AGILENT®, Waldbronn, Germany), a Nanomate 100 electrospray system (ADVION®, Ithaca, USA) and a Finnigan LTQ-FT mass spectrometer (THERMO FISHER®, Bremen, Germany). Settings of the Mascot search engine were adjusted to identify variable modifications, i.e. deamidated (NQ), Oxidation (M), Phospho (ST) and Phospho (Y).

(49) Expression and Purification of Recombinant Proteins.

(50) Recombinant human GRK2 protein and GRK2-S670A protein was expressed in and purified from Spodoptera frugiperda (Sf9) cells by the baculoviral expression system. The cDNAs encoding hexahistidine-tagged human GRK2 (ADRBK1) GRK2-S670A were subcloned into the pFastBac1 expression plasmid (INVITROGEN™, THERMO FISHER SCIENTIFIC®, Waltham, Mass., USA) using the Sal I/Hind III restriction sites, recombinant baculovirus was generated by the Bac-To-Bac Baculovirus expression system (THERMO FISHER SCIENTIFIC®, Waltham, Mass., USA). Sf9 cells were infected with recombinant baculoviruses at an MOI of 2-3, 48 h after infection cells were harvested by centrifugation, lysed with lysis buffer (300 mM NaCl, 50 mM HEPES, pH 7.5 supplemented with 1% NP40, 1 mM PMSF and protease inhibitor cocktail) and applied to Ni-NTA chromatography. After overnight incubation at 4° C., unbound proteins were removed by washing with lysis buffer (20× column volumes) followed by 1× column volume of 30 mM imidazole-containing lysis buffer. Bound GRK2 was eluted by 300 mM imidazole in lysis buffer, desalted by PD10 column chromatograpy, supplemented with 20% glycerol and stored at −80° C. for further use.

(51) The cDNAs encoding human His6-SRSF1 and phosducin-His6, and the carboxyl terminal domain of GRK2 (SEQ ID NO: 10, His-6 tagged) respectively, were subcloned into the PET-3d expression plasmid (NOVAGEN®, EMD MILLIPORE®, Merck KGaA, Darmstadt, Germany) under control of the T7 promoter, which allows protein expression by T7 RNA polymerase induction with IPTG in BL21(DE3) pLysS bacteria. Protein-expressing bacteria were collected by centrifugation, frozen in liquid nitrogen and thawed on ice in lysis buffer (8 M urea, 300 mM NaCl, 50 mM HEPES, 10 mM imidazole, pH 7.5), which was supplemented with 2-mercaptoethanol (0.7 ml/L) freshly before use (10 ml of lysis buffer for a bacterial pellet from 200 ml of culture medium). The bacterial lysate was incubated for 1 h at room temperature, sonicated and centrifuged for 15 min at 4000×g at 4° C. The supernatant was applied to the Ni-NTA column matrix prewashed with 20 ml of lysis buffer. After overnight incubation at 4° C., the flow-through was discarded, the Ni-NTA affinity matrix was subjected to 3 different washing steps (30 min of incubation at 4° C. with 20 ml of each washing buffer for 1 ml of 5% Ni-NTA matrix) with wash buffer-1 (4 M urea, 300 mM NaCl, 50 mM HEPES, 20 mM imidazole, pH 7.5, supplemented freshly with 0.7 ml/I 2-mercaptoethanol), wash buffer-2 (2 M urea, 300 mM NaCl, 50 mM Hepes, 20 mM imidazole, pH 7.5, supplemented freshly with 0.7 ml/L 2-mercaptoethanol) and wash buffer-3 (300 mM NaCl, 50 mM HEPES, 20 mM imidazole, pH 7.5, supplemented freshly with 0.7 ml/I 2-mercaptoethanol). Finally, proteins were eluted with elution buffer (300 mM NaCl, 50 mM HEPES, 500 mM imidazole, pH 7.5, supplemented freshly with 0.7 ml/L 2-mercaptoethanol). Buffer of eluted proteins was exchanged to 150 mM NaCl, 50 mM HEPES (pH 7.5) by PD10 column chromatography.

(52) In Vitro Phosphorylation Assay

(53) Recombinant proteins were used for an in vitro phosphorylation assay with GRK2 and GRK2-S670A. GRK2 and GRK2-5670A were expressed in and purified from Spodoptera frugiperda (Sf9) cells by the baculoviral expression system. Substrate phosphorylation was performed in reaction buffer (50 microL of 20 mM Tris, 2 mM EDTA, 5 mM MgCl2 pH 7.5; or 20 mM Hepes, 2 mM MgCl2, 0.025% DDM (n-Dodecyl beta-D-maltoside), pH 7.4) supplemented with 50 microM or 5 microM of ATP, respectively, and [gamma-32P]-ATP, 1×10 6 DPM, specific activity 3000 Ci/mmol)) with 300-500 nM of substrate (SRSF1, phosducin) in the presence of increasing concentrations of GRK2 inhibitor as indicated. The reaction was started by the addition of GRK2-5670A or GRK2 (50 nM or 130 nM). After an incubation for 30 min at 30° C., the reaction was stopped by the addition of 5×SDS-Laemmli buffer. Proteins were separated by SDS-PAGE and subjected to autoradiography. For the analysis of small molecule inhibitors, the phosphorylation assay was performed in reaction buffer (20 mM Tris, 2 mM EDTA, 5 mM MgCl2, 0.05% BSA, pH 7.5; or 20 mM Hepes, 2 mM MgCl2, 0.025% DDM (n-Dodecyl beta-D-maltoside), pH 7.4) supplemented with 5 microM ATP, [gamma-32P]-ATP (1×10 6 DPM, specific activity 3000 C.sub.1/mmol)) and 300-500 nM of substrate (SRSF1, phosducin) (=reaction mixture). The reaction mixture was added to GRK2-S670A or GRK2 (50 nM or 60 nM in reaction buffer, without or with increasing concentrations of the small molecule compound) to give a final reaction volume of 50 microL. After an incubation for 30-60 min at 30° C., the phosphorylation was stopped by the addition of 5 volumes of ice-cold reaction buffer. The reaction mixtures were immediately applied to glass fiber filters (GF/C, WHATMAN™, GE Healthcare Life Sciences, Glattbrugg, Switzerland). After three washing steps with 5 ml of reaction buffer, filter-bound radioactivity was determined in a β-counter.

(54) Immunoblot Detection of Proteins

(55) For immunoblot detection of proteins, cardiac tissue was pulverized in liquid nitrogen and extracted with RIPA buffer supplemented with protease/phosphatase inhibitor cocktail, as previously described (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)) with minor modifications. Particulate material was removed by centrifugation at 20,000×g for 15 min at 4° C. Solubilized proteins were precipitated and delipidated by acetone/methanol (12:2; final concentration 83%) for 90 min at 4° C. The precipitate was collected by centrifugation (5000×g, 10 min, 4° C.), which was followed by three washing steps with 0.2 ml of cold acetone. The pellet was dissolved in SDS-sample buffer containing 2% SDS, 0.1 M DTT, and 6 M urea for 90 min at room temperature. After the addition of iodoacetamide (10 mM), samples were stored for further use at −70° C. Detection of proteins was performed with affinity-purified antibodies or F(ab)2 fragments of the respective antibodies after separation of proteins by SDS-PAGE (10% gel for proteins <100 kDa; 7.5% gel for proteins >100 kDa) and subsequent electrophoretic protein transfer to PVDF membranes by semidry blotting (TRANS-BLOT® SD Semi-Dry Transfer Cell, Bio-Rad GmbH, Munchen, Germany). For the electrophoretic transfer of the Fasn protein, a tank transfer cell (Mini TRANS-BLOT® cell, BIO-RAD® GmbH, Munchen, Germany) was used. Bound antibody was visualised with F(ab)2 fragments of enzyme-coupled secondary antibodies (Dianova GmbH, Hamburg, Germany), or by enzyme-coupled protein A (Calbiochem, EMD MILLIPORE®, Merck KGaA, Darmstadt, Germany) as applicable, and was followed by enhanced chemiluminescent detection (ECL Prime, AMERSHAM®, GE Healthcare Life Sciences, Glattbrugg, Switzerland).

(56) Antibodies

(57) The following antibodies were used for immunoblot detection of proteins: anti-Gnb antibodies were raised in rabbit against purified Gnb (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016)); anti-GRK2 antibodies were raised in rabbit against recombinant GRK2 expressed in Sf9 cells (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016)); anti-pRKIP antibodies were raised in rabbit against a short amino acid sequence from human RKIP containing phosphorylated serine-153 (sc-32623, Santa Cruz Biotechnology Inc., Dallas, Tex., USA); anti SRSF1 antibody is a mouse monoclonal antibody epitope mapping near the N-terminus of the SF2/ASF protein (sc-33652, Santa Cruz Biotechnology Inc. USA); SR (1H4) antibody is a mouse monoclonal antibody raised against full length SR of Xenopus origin (sc-13509 from Santa Cruz Biotechnology Inc. USA); anti-CAMK2D polyclonal antibodies were raised in rabbit against full-length human protein (purified polyclonal antibody, no. H00000817-D01P; Abnova, Taipei, Taiwan) and monoclonal anti-CAMK2D antibody, which was produced in mouse against CAMK2D (amino acids 301-410) partial recombinant protein with GST-tag (WH0000817M2; Sigma-Aldrich, St. Louis, Mo., USA) were used for detection of CAMK2D/Camk2d isoforms B/C.

(58) Compound Synthesis.

(59) Chemicals were synthesized by EMC microcollections GmbH, Tubingen, Germany and CHIROBLOCK® GmbH, Wolfen, Germany. The synthesis of compounds was performed in a small scale by solid phase chemical synthesis methods, which were adapted from established protocols (For “Compound-1”: Poncet J, et al., J. Chem. Soc. Perkin Trans I., 611-616 (1990); for “Compound-2”, “Compound-22”, “Compound-23”, and “Compound-24”: Trautwein A W, et al., Bioorg. Med. Chem. Lett. 8, 2381-2384 (1998); for Compound-3: Sakai K, et al., Chem. Pharm. Bull. 29(6) 1554-1560 (1981); for “Compound-4”: Coombs T C, et al., Bioorg. Med. Chem. Lett. 23, 3654-3661 (2013); and for “Compound-5”: Baber J C, et al., Bioorg. Med. Chem. 20, 3565-3574 (2012)). In addition, Compound-1 and Compound-4 were synthesized in a larger scale as detailed below.

Synthesis of Compound-1

(60) Synthesis of Compound-1 (1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one) was performed by a 6-step chemical reaction process (CHIROBLOCK® GmbH, Wolfen, Germany). Step-1 encompassed the synthesis of methyl 2-(1,3-benzodioxol-5-ylamino)-2-phenyl-acetate. A mixture of methyl 2-oxo-2-phenyl-acetate (96 g, 584 mmol, 4.0 equivalents), 1,3-benzodioxol-5-amine (20 g, 146 mmol, 1.0 equivalents), and Na.sub.2SO.sub.4 in cyclohexane (800 ml) was refluxed under N.sub.2 for 21 h. 5% Pd/C (7.8 g) was added, and the obtained suspension was hydrogenated at 20 bar and 20° C. for 48 h. The resulting heteroge-neous mixture was diluted with EtOAc (ca. 800 ml) and filtered through Celite. The filtrate was concentrated in vacuo (40° C., 100 mbar) to yield a brown oil (135 g) that was purified by flash chromatography (silica gel, ethyl acetate—petroleum ether 12:88 to 30:70) to yield target 3, which was an off-white solid (18.46 g; purity 95%, yield 44%).

(61) Step-2 was the synthesis of S-tert-butyl ethanethioate. A solution of pyridine (87.0 g, 1.1 mol, 1.1 equivalents) in chloroform (800 ml) was cooled in an ice bath and treated with acetyl chloride (86.4 g, 1.1. mol, 1.1 equivalents), with the reaction temperature not exceeding 11° C. To the resulting orange suspension, 2-methylpropane-2-thiol (90.2 g, 1.0 mol, 1.0 equivalents) was dropwise added over 40 min., and the mixture was stirred for 48 h and subsequently quenched with water (500 ml). The phases were separated and the aqueous phase was extracted with chloroform (400 ml). The combined organic extracts were washed with 400 ml each of water, 10% H2SO.sub.4, sat. NaHCO.sub.3, and water being subsequently dried over Na.sub.2SO.sub.4. The obtained chloroformic solution was subjected to fractional distillation, which afforded target S-tert-butyl ethanethioate as a clear liquid (55.8 g, purity 95%, yield 45%).

(62) In Step-3 the synthesis of S-(2-pyridyl) cyclopropanecarbothioate was performed. Cyclopropanecarbonyl chloride (23.5 g, 225 mmol, 1.0 equiv.) was dropwise added to solution of pyridine-2-thiol (25.0 g, 225 mmol, 1.0 equiv.) in THF (250 ml) at 20° C. The mixture was stirred for 10 min, filtered, and the filter cake was washed with 1:4 Et.sub.2O/petrol ether (250 ml). The thus obtained solid was dissolved in water (250 ml) and treated with NaHCO.sub.3 (19 g, 225 mmol, 1.0 equiv.), and the aqueous solution was extracted with 2*250 ml EtOAc. The combined organic fractions were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford S-(2-pyridyl) cyclopropanecarbothioate as a yellow oil (37 g, purity 95%; yield 92%).

(63) Step-4 was the synthesis of S-tert-butyl 3-cyclopropyl-3-oxo-propanethioate. A 2-L 3-neck round-bottom flask was charged with HMDS (83.3 g, 516 mmol, 2.5 equiv) and freshly distilled THF (800 ml). The obtained mixture was cooled in an acetone/dry ice bath, and 1.6 M nBuLi in hexanes (323 ml, 516 mmol, 2.5 equiv.) was dropwise added while keeping the temperature below −50° C. Subsequently, the obtained mixture was sequentially treated with solutions of S-(2-pyridyl) cyclopropanecarbothioate (37.0 g, 206 mmol, 1.0 equiv.) and S-tert-butyl ethanethioate (23.4 g, 214 mmol, 1.04 equiv.). The obtained solution was stirred for 1 h at −30° C., and the reaction was quenched (under TLC process control) by 1 N H.sub.2SO.sub.4 (800 ml). The resulting suspension was extracted with EtOAc (3*900 ml), and the organic fractions combined, washed with brine (2 L), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, ethyl acetate—petroleum ether 25:75) to yield target S-tert-butyl 3-cyclopropyl-3-oxo-propanethioate as a brown oil (29.5 g, purity 83%, yield: 59%).

(64) In Step-5, the synthesis of Methyl 2-[1,3-benzodioxol-5-yl-(3-cyclopropyl-3-oxo-propanoyl)amino]-2-phenyl-acetate was performed. A 1-L round-bottom flask was charged with Methyl 2-(1,3-benzodioxol-5-ylamino)-2-phenyl-acetate (18.5 g, 61 mmol, 1.0 equiv.), S-tert-butyl 3-cyclopropyl-3-oxo-propanethioate (15.9 g, 66 mmol, 1.073 equiv.), CF.sub.3COOAg 814.6 g, 66 mmol, 1.073 equiv.), and distilled THF (400 ml), and the obtained mixture was stirred at 20° C. for 36 h (the process was controlled by TLC). The dark-brown reaction mixture was concentrated in vacuo and purified by flash chromatography (silica gel, ethyl acetate—petroleum ether 25:75 to 50:50) to yield target Methyl 2-[3-benzodioxol-5-yl-(3-cyclopropyl-3-oxo-propanoyl)amino]-2-phenyl-acetate as a brown oil (21.0 g, purity: 90%, yield: 78%).

(65) The final Step-6 yielded the final target 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbo-nyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one (Compound-1). A 500 ml round-bottom flask was charged with Methyl 2-[3-benzodioxol-5-yl-(3-cyclopropyl-3-oxo-propanoyl)amino]-2-phenyl-acetate (20.0 g; 45.5 mmol, 1.0 equiv.), CsF (6.9 g, 45.5. mmol, 1.0 equiv.), and DMF (140 ml), and the obtained mixture was stirred at 60° C. for 20 h (the process was controlled by TLC). The dark-brown reaction mixture was concentrated in vacuo and the residue was treated with 1N H.sub.2SO.sub.4 (400 ml). The obtained mixture was extracted with EtOAc (500 ml), and the organic phase was washed with brine (2*300 ml), dried over Na.sub.2SO.sub.4, and concentrated in vacuo to afford crude 1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one as a brown solid (19 g). The above solid was washed on filter with EtOAc until becoming colorless, affording target Compound-1 (1-(1,3-Benzodioxol-5-yl)-4-(cyclopropanecarbonyl)-3-hydroxy-2-phenyl-2H-pyrrol-5-one) as an off-white solid (5.0 g, purity: 98%, yield: 30%).

(66) Synthesis of Compound-4 Compound-4 (4-(1,3-Benzodioxol-5-yl)pyrimidine) was synthesized by the following procedure (ChiroBlock GmbH, Wolfen, Germany). A 250 ml round-bottom flask was loaded with 1-(1,3-Benzodioxol-5-yl)ethanone (10.0 g, 60.9 mmol, 1.0 equivalent), (EtO).sub.3CH (27 g, 183 mmol, 3.0 equivalents), ZnCl.sub.2 (0.83 g, 6.1 mmol, 0.1 equivalent), NH.sub.4CH.sub.3COO (0.4 g, 122 mmol, 2.0 equivalents) and toluene (120 ml), and the obtained mixture was stirred at reflux for 48 g and subsequently at 20° C. for 48 h (the process was controlled by TLC). The reaction mixture was quenched with saturated NaHCO.sub.3 (400 ml) and extracted with chloroform (400 ml). The organic phase was dried over Na.sub.2SO.sub.4 and concentrated in vacuo, and the resulting crude product was purified by flash chromatography (silica gel, MeOH—CHCl.sub.3 (0:100 to 5:95) to yield target Com-pound-4 (4-(1,3-Benzodioxol-5-yl)pyrimidine) as an off-white solid (3.0 g, purity 97%; yield 25%).

(67) Isolation of Neonatal Cardiomyocytes and Cell Experiments

(68) Neonatal mouse and rat cardiomyocytes were isolated as described (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013); Lorenz K, et al., Nature 426, 574-579 (2003)). Briefly, the hearts were dissected out from 2-3 day-old mice, atria and aorta were removed, and the hearts were transferred into sterile buffer A (137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4, 5.55 mM dextrose, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 20 mM HEPES, 100 U/ml penicillin, 100 microg/ml streptomycin, pH 7.4). The hearts were cut into small pieces and incubated (on a magnetic stirrer) for 15 min with buffer A supplemented with 150 mg/L trypsin (Becton&Dickinson, Franklin Lakes, N.J., USA). The supernatant was discarded, and the procedure was repeated once. Thereafter the heart tissue was digested by sequential 5 min incubations with trypsin-supplemented buffer A at room temperature until the heart tissue was completely digested. Cells in the supernatants were collected by centrifugation (10 min, 700×g, at room temperature), suspended in MEM with 5% FCS, and filtered through a nylon mesh (40 microm). Fibroblasts were removed by pre-plating for 1 h at 37° C. Non-adherent cardiomyocytes were collected and cultivated in MEM (supplemented with 5% FCS and 25 mg/I BrdU). Cellular cAMP levels of isolated cardiomyocytes were determined with cAMP Enzyme Immunoassay kit (CA200, Sigma Aldrich, St. Louis, Mo., USA) after beta-adrenoceptor stimulation with 100 nM isoproterenol similarly as described (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016)). Human embryonic kidney cells were cultivated as described (Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)).

(69) Histology Techniques.

(70) For histology analyses, paraffin sections of mouse heart specimens were used. Immunohistological detection of RKIP was performed with affinity-purified, polyclonal antibodies raised in rabbit against recombinant RKIP. After antigen retrieval, sections were incubated with primary antibodies (dilution 1:200) in blocking buffer [PBS, pH 7.4, supplemented with 5% (w/v) bovine serum albumin, 0.05% Tween-20] for 1 h at 37° C. Unbound antibody was removed by three washing steps with PBS supplemented with 0.05% Tween-20. After incubation with peroxidase-conjugated secondary antibody (Dianova, Dianova GmbH, Hamburg, Germany; dilution 1:500), followed by washing steps, an enzyme substrate reaction was performed (DAB Enhanced Liquid Substrate System, Sigma-Aldrich, St. Louis, Mo., USA). Immunohistology sections were imaged with a Leica DM16000 microscope equipped with a DFC420 camera. Myocardial necrosis was determined with von Kossa stain (Calcium stain kit, modified Von Kossa No. KT028 Diagnostic Biosystems Pleasanton, Calif., USA)

(71) Lentiviral-Mediated Down Regulation of Srsf1 by RNAi In Vivo

(72) For the down regulation of Srsf1 expression in vivo, Tg-GRK2 mice were transduced by i.p. administration of a replication-incompetent lentivirus (1×10.sup.8 copies/mouse in PBS), which down-regulates Srsf1 by polymerase II (Pol II)-dependent expression of a pre-miRNA targeting the Srsf1 RNA by RNAi. The lentiviral expression plasmid was generated by inserting the indicated double-stranded oligonucleotides that encoded an engineered pre-miRNA sequence targeting the murine Srsf1 gene by RNAi interference into the pLenti6/V5-DEST™ GATEWAY™ Vector (INVITROGEN™, THERMO FISHER SCIENTIFIC®, Waltham, Mass., USA): miSrsf top strand 5′-TGC TGT TTA AGT CCT GCC AGC TTC CAG TTT TGG CCA CTG ACT GAC TGG AAG CTC AGG ACT TAA A-3′ (SEQ ID NO: 1); and miSrsf1 bottom strand 5′-CCT GTT TAA GTC CTG AGC TTC CAG TCA GTC AGT GGC CAA AAC TGG AAG CTG GCA GGA CTT AAA C-3′ (SEQ ID NO: 2). A pseudotyped lentivirus was produced by cotransfection of 293FT cells with the lentiviral plasmid and a mixture of packaging plasmids pLP1, pLP2 and pLP/VSVG (INVITROGEN™, THERMO FISHER SCIENTIFIC®, Waltham, Mass., USA). Down regulation of Srsf1 protein expression was confirmed by immunblot detection after the transduction of mice with miSrsf1-lentivirus.

(73) Whole Genome Microarray Gene Expression Analysis

(74) Whole genome microarray gene expression analysis of cardiac tissue from Tg-RKIP and Tg-GRK2-K220R mice was performed using Affymetrix GeneChip Mouse genome MG430 2.0 Arrays essentially as described (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016)). GO analyses of microarray data were performed with GCOS and/or RMA-processed data using GeneSpring GX software (Agilent, Santa Clara, Calif., USA). The data were compared between two groups using the unpaired two-tailed Student's t-test. Probe sets, which were significantly up-regulated (fold change 2 relative to the respective control group and P0.01) were used for GO classification. Real-time qRT-PCR of Camk2d isoform splicing was performed with a LightCycler 480 (Roche Molecular Diagnostics, Pleasanton, Calif., USA) as described (Abd Alla, J, et al., J. Biol. Chem. 291, 2583-2600 (2016); Fu X, et al., J. Biol. Chem. 288, 7738-7755 (2013)). The following primers were used: Camk2d-forward 5′-ACG AGA AAT TTT TCA GCA GCC-3′ (SEQ ID NO: 3); Camk2d-reverse-A 5′-AC AGT AGT TTG GGG CTC CAG C-3′ (SEQ ID NO: 4); Camk2d-reverse-B 5′-T CAT CTG AAC ACT CGA ACT GG-3′ (SEQ ID NO: 5); Camk2d-reverse-C 5′-CTC AGT TGA CTC CTT TAC CCC-3′ (SEQ ID NO: 6).

(75) Statistical Analyses

(76) The results are presented as the means±s.d. unless otherwise specified. The P values were calculated with Student's t-test. Analysis of variance was performed for comparisons between more than two groups followed by a post-test, and statistical significance was set at a P value of <0.05 unless otherwise stated.