초록 ▼

The present invention relates to generating hiPSC-derived cardiomyocytes and embryoid bodies that recapitulate the disease phenotype of Long QT Syndrome and their use in developing pharmacological treatments thereof. The present invention also includes the use of a compound which inhibits the ubiqui

The present invention relates to generating hiPSC-derived cardiomyocytes and embryoid bodies that recapitulate the disease phenotype of Long QT Syndrome and their use in developing pharmacological treatments thereof. The present invention also includes the use of a compound which inhibits the ubiquitin-proteasome pathway for the preparation of a medicament for the prophylaxis or treatment of a disease associated with prolonged ventricular repolarization (cardiac arrhythmia) caused by one or more mutations in the amino acid sequence of the hERG potassium channel.

대표청구항 ▼

1. A method of testing compounds for activity in ameliorating long QT syndrome 2 (LQTS2) phenotype, comprising the steps; (a) contact LQTS2-specific cardiomyocytes or embryoid bodies (EBs) with the test compound, and(b) quantitate the level of human ether-a-go-go related gene (hERG) protein in the e

1. A method of testing compounds for activity in ameliorating long QT syndrome 2 (LQTS2) phenotype, comprising the steps; (a) contact LQTS2-specific cardiomyocytes or embryoid bodies (EBs) with the test compound, and(b) quantitate the level of human ether-a-go-go related gene (hERG) protein in the endoplasmic reticulum (ER) and in the sarcolemma, and compare the relative level of hERG in the ER and sarcolemma with untreated cardiomyocytes or EBs, wherein an increase in sarcolemma level and a decrease in ER level of hERG in the treated cells indicates re-trafficking of hERG and the compound has LQTS2-ameliorating activity, and/or(c) quantitate the level of glycosylated (mature) hERG protein and compare with untreated cardiomyocytes or EBs, wherein increased glycosylation indicates the compound has LQTS2-ameliorating activity, and/or(d) measure the local field potential duration (FPD), correct for the beating rate of contracting areas (cFPD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in cFPD indicates the compound has LQTS2-ameliorating activity, and/or(e) measure the action potential duration (APD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in APD, indicates the compound has LQTS2-ameliorating activity. 2. The method according to claim 1, further comprising the step; culture LQTS2-specific cardiomyocytes or EBs until spontaneously contracting or induced to contract. 3. The method according to claim 1, wherein the phenotype is cardiac rhythm disturbance, comprising the steps; (a) culture LQTS2-specific cardiomyocytes or embryoid bodies (EBs) until spontaneously contracting or induced to contract,(b) contact the cardiomyocytes or EBs with the test compound, and (i) an agent that triggers arrhythmia, and/or(ii) an agent that increases cardiomyocyte or EB beating rate,(c) measure the local field potential duration (FPD), correct for the beating rate of contracting areas (cFPD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in cFPD indicates the compound has rhythm normalizing activity, and/or(d) measure the action potential duration (APD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in APD indicates the compound has repolarization normalizing activity. 4. The method according to claim 1, comprising the steps; (a) culture LQTS2-specific cardiomyocytes or embryoid bodies (EBs) until spontaneously contracting or induced to contract,(b) contact the cardiomyocytes or EBs with the test compound, and (i) an agent that modulates a single allele of hERG gene in cardiomyocytes or EBs, and/or(ii) an agent that modulates any one or more of calpain, calpastatin, and ubiquitin expression, and/or (iii) an agent that modulates any one or more of HSP70, HSP90 and CAV3 expression,(c) measure the local field potential duration (FPD), correct for the beating rate of contracting areas (cFPD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in cFPD indicates the compound has LQTS2-ameliorating activity, and/or(d) measure the action potential duration (APD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in APD indicates the compound has LQTS2-ameliorating activity, and/or(e) measure the hERG channel kinetic and compare with the untreated cardiomyocytes or EBs, wherein a normalizing channel kinetic indicates the compound has LQTS2-ameliorating activity. 5. The method according to claim 4, wherein the hERG gene modulator is hERG gene-specific siRNA. 6. The method according to claim 4, wherein the HSP70 modulator is any one or more selected from the group comprising 2-Phenylethynesulfonamide, Pifithrin-μ, 2,5′-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, 3-(phenylthio)pyridines, MKT-077, rapamycin and VER155008. 7. The method according to claim 4, wherein the ubiquitin modulator is any one or more selected from the group comprising rapamycin, N—[N—(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine, PYR-41, MLN4924, SMER3, BAY11-7082 and Nutlin-3. 8. The method according to claim 4, wherein the calpain modulator is any one or more selected from the group comprising 4PBA, N—[N—(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine, MG-132, 3-[6-[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino]-3-methylpyridin-2-yl]benzoic acid, AK275, MDL28170, PD150606, SJA6017, ABT-705253 and SNJ-1945. 9. The method according to claim 4, wherein the CAV3 modulator is SB203580. 10. The method according to claim 4, wherein the HSP90 modulator is any one or more selected from the group comprising 4-hydroxytamoxifen, tomoxifen, activator of Hsp90 ATPase homolog1 (AHA1). 11. The method according to claim 1, wherein the phenotype is cardiac rhythm instability, comprising the steps; (a) culture LQTS2-specific cardiomyocytes or embryoid bodies (EBs) until spontaneously contracting with normal rhythm,(b) contact the cardiomyocytes or EBs with the test compound, and (i) an agent that increases cardiomyocyte or EB beating rate, and/or(ii) an agent that triggers abnormal cardiac rhythm,(c) measure the local field potential duration (FPD), correct for the beating rate of contracting areas (cFPD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in cFPD indicates the compound has rhythm normalizing activity, and/or(d) measure the action potential duration (APD), and compare with untreated cardiomyocytes or EBs, wherein a time-dependent reduction in APD indicates the compound has repolarization normalizing activity, and/or(e) measure the presence of early after-depolarizations (EADs) and compare to untreated cardiomyocytes or EBs, wherein the suppression of EADs indicates the compound has arrhythmia suppressing activity. 12. The method according to claim 11, wherein the agent that increases cardiomyocyte or EB beating rate is any one or more selected from the group comprising isoprenaline, dobutamine, epinephrine, norepinephrine and xamoterol. 13. The method according to claim 11, wherein the agent that triggers abnormal cardiac rhythm is any one or more selected from the group comprising E-4031, terfenadine, roxithromycin, fluconazole, cisapride and astemizole. 14-17. (canceled) 18. A method of prophylaxis or treatment of a disease associated with prolonged ventricular repolarization (cardiac arrhythmia), caused by one or more mutations in the amino acid sequence of the hERG potassium channel, comprising administering to a subject in need of such prophylaxis or treatment an efficacious amount of a compound which redirects (re-trafficks) hERG towards the sarcolemma. 19. The method according to claim 18, wherein the disease is LQTS2. 20. The method according to claim 18, wherein the compound is an inhibitor of the ubiquitin-proteasome pathway. 21. The method according to claim 20, wherein the compound is any one or more selected from the group comprising N—[N—(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine and 3-[6-[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino]-3-methylpyridin-2-yl]benzoic acid.