U.S. patent application number 15/536314 was filed with the patent office on 2017-12-21 for anti-arrhythmicity agents.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Jau-Nian CHEN, Yi Chiao FAN, Jie HUANG, Ohyun KWON, Kui LU, Johann SCHREDELSEKER, Hirohito SHIMIZU.
Application Number | 20170362173 15/536314 |
Document ID | / |
Family ID | 56127483 |
Filed Date | 2017-12-21 |
United States Patent
Application |
20170362173 |
Kind Code |
A1 |
CHEN; Jau-Nian ; et
al. |
December 21, 2017 |
ANTI-ARRHYTHMICITY AGENTS
Abstract
Agents, compositions, and methods for regulating cardiac
rhythmicity are disclosed.
Inventors: |
CHEN; Jau-Nian; (Los
Angeles, CA) ; KWON; Ohyun; (Los Angeles, CA)
; HUANG; Jie; (Los Angeles, CA) ; SHIMIZU;
Hirohito; (Los Angeles, CA) ; LU; Kui; (Los
Angeles, CA) ; SCHREDELSEKER; Johann; (Los Angeles,
CA) ; FAN; Yi Chiao; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
56127483 |
Appl. No.: |
15/536314 |
Filed: |
December 15, 2015 |
PCT Filed: |
December 15, 2015 |
PCT NO: |
PCT/US15/65876 |
371 Date: |
June 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62092185 |
Dec 15, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/11 20130101;
C07D 207/48 20130101; C12N 15/113 20130101; C12N 2320/31 20130101;
A61K 38/1709 20130101; A61K 31/40 20130101; A61K 31/7088 20130101;
A61K 48/005 20130101 |
International
Class: |
C07D 207/48 20060101
C07D207/48; A61K 38/17 20060101 A61K038/17; C12N 15/113 20100101
C12N015/113; A61K 31/40 20060101 A61K031/40; A61K 48/00 20060101
A61K048/00; A61K 31/7088 20060101 A61K031/7088 |
Goverment Interests
FEDERAL GOVERNMENT GRANT INFORMATION
[0002] This invention was made with Government support under Grant
Nos. HL09698, GM071779, and GM081282 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
1.-76. (canceled)
77. A compound of structure of Formula Ic: ##STR00032## wherein:
R.sub.1 is an alkane, phenyl, heteroaryl, or substituted phenyl
group; R.sub.2 is a phenyl, heteroaryl, substituted phenyl, or
hydrocarbyl group with or without a heteroatom; and R.sub.3 is an
alkoxy, amino, amino ether, N-Boc-protected
2-aminoethoxyethoxyethylamino group, or a C1-C10 straight or
branched, acyclic or cyclic alkyloxy group, aryloxy, or amino group
with or without heteroatom; and wherein the compound is effective
to potentiate mitochondrial Ca.sup.2+ uptake so as to modulate
cardiac rhythmicity in a subject.
78. The compound of claim 77, wherein R.sub.1 is para-tolyl,
R.sub.2 is phenyl, and R.sub.3 is ethoxy.
79. The compound of claim 77, wherein R.sub.1 is orth-fluoro
substituted phenyl, a meta-fluoro substituted phenyl, or
para-fluoro substituted phenyl.
80. The compound of claim 77, wherein R.sub.3 is ethoxy,
menthyloxy, or N-Box-protected 2-aminoethoxyethoxyethalamino
group.
81. The compound of claim 77, wherein R.sub.2 is para-fluoro
substituted phenyl or a meta-fluoro substituted phenyl.
82. The compound of claim 77, wherein R.sub.3 is ethyl, C3-C6 short
alkyl, menthyloxy group, C1-C10 straight or branched, acyclic or
cyclic alkyl group, or aryl group.
83. The compound of claim 77, which is in an optically active
form.
84. The compound of claim 77, wherein the compound is in an
enantiomerically pure form of Formula Ia or Formula Ib:
##STR00033##
85. The compound of claim 77, wherein the compound is a compound
having the structure of ##STR00034## ##STR00035##
86. A method of regulating cardiac rhythmicity in a subject,
comprising potentiating mitochondrial Ca.sup.2+ uptake by i)
inducing VDAC2 or VDAC1 overexpression in the subject to restore
rhythmic contraction, ii) inducing overexpression of VDAC2 or VDAC1
and/or MCU or MICU1 complex, iii) administering to the subject in
need thereof an agent effective to activate VDAC2 or VDAC1 and/or
MCU or MICU1 complex, or iv) administering to the subject in need
thereof an agent effective to induce Ca2+ transporting activity of
VDAC2 or VDAC1.
87. The method of claim 86, wherein inducing VDAC2 or VDAC1
overexpression in the subject is via gene therapy.
88. The method of claim 86, wherein the agent is a VDAC2 or VDAC1
gene product.
89. The method of claim 88, wherein the VDAC2 or VDAC1 gene product
is a VDAC2 or VDAC1 protein, or a VDAC2 or VDAC1 RNA.
90. The method of claim 86, wherein the agent is the compound of
Formula Ic.
91. The method of claim 90, wherein the compound of Formula Ic is
in an optically active form.
92. The method of claim 90, wherein the agent is efsevin or a
compound having the structure of ##STR00036## ##STR00037##
93. The method of claim 92, wherein the agent is an enantiomer of
the agent.
94. A method of forming the compound of Formula Ic: ##STR00038##
where R.sub.1 is substituted phenyl or substituted or unsubstituted
heteroaryl; and R.sub.2 is substituted phenyl or substituted or
unsubstituted heteroaryl; and R.sub.3 is R.sub.3 is an alkoxy,
amino, amino ether, N-Boc-protected 2-aminoethoxyethoxyethylamino
group, or a C1-C10 straight or branched, acyclic or cyclic alkyloxy
group, aryloxy, or amino group with or without heteroatom
comprising reacting ##STR00039## according to a reaction of Scheme
III ##STR00040## to form the compound of Formula Ic.
95. The method of claim 94, wherein the reaction of Scheme III is
carried out under asymmetric synthesis conditions.
96. The method of claim 94, wherein the compound of Formula Ic is
formed in an optically active form.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/092,185 filed Dec. 15, 2014, the contents of
which are incorporated into the present application by
reference.
BACKGROUND OF THE INVENTION
[0003] A. Field of the Invention
[0004] The present invention relates to agents, compositions, and
methods for regulating cardiac rhythmicity.
[0005] B. Description of Related Art Cardiac diseases are the
leading cause of death in Western countries. Many of these
conditions, including hypertrophy, heart failure and arrhythmias,
have a root in aberrant Ca2+ homeostasis. Identifying clinically
relevant targets and pharmacological agents that can effectively
modulate cardiac Ca2+ homeostasis may lead to the development of
new therapeutic strategy for cardiac diseases.
[0006] The present invention addresses such needs for targets and
agents for regulating cardiac rhythmicity.
SUMMARY OF THE INVENTION
[0007] In one embodiment, there are methods of regulating cardiac
rhythmicity in a subject, comprising potentiating mitochondrial
Ca.sup.2+ uptake by inducing VDAC2 or VDAC1 overexpression in the
subject to restore rhythmic contraction, inducing overexpression of
or activating VDAC (VDAC2 or VDAC1) and/or MCU (MCU or MICU1)
complex, or administering to the subject in need thereof an agent
effective to induce Ca2+ transporting activity of VDAC2 or
VDAC1.
[0008] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, there are methods
of regulating cardiac rhythmicity in a subject in need thereof
comprising administering to the subject a composition that
comprises an agent or compound that increases the activity of VDAC2
or VDAC1. In some embodiments, the agent or compound binds to VDAC2
or VDAC1. In some embodiments, the agent or compound increases Ca2+
transporting activity of VDAC2.
[0009] The agent or compound disclosed in the embodiments may be
any agent or compound of the formulas described herein or compounds
described herein. In some embodiments, one or more specific
compounds described herein may be excluded.
[0010] In some embodiments, optionally in combination with any or
all of the various to embodiments disclosed herein, the compound is
in a composition. In some embodiments, the composition comprises a
pharmaceutically acceptable carrier.
[0011] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the subject
suffers from a disorder related to cardiac arrhythmicity or cardiac
disorder with a root in aberrant Ca2+ handling. Such disorder
includes, for example, cardiac fibrillation, arrhythmia, atrial
fibrillation, sick sinus syndrome, catecholaminergic polymorphic
ventricular tachycardia (CPVT), or cardiomyopathy.
[0012] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, inducing VDAC2 or
VDAC1 overexpression in the subject is via gene therapy.
[0013] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the agent is a
VDAC2 or VDAC1 gene product.
[0014] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the agent is a
VDAC2 or VDAC1 protein, or a VDAC2 or VDAC1 RNA.
[0015] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the agent is the
compound of Formula I or a derivative thereof, wherein compound of
Formula I comprises
##STR00001##
wherein: R.sub.1 is tosyl, or mesyl group; and wherein R.sub.2 is a
hydrocarbyl group with or without a heteroatom or wherein the
carboxylic ester of R.sub.2 is attached to a mono-N-Boc protected
2,2'-(ethylenedioxy)bis(ethylamine), and wherein the compound is
effective to potentiate mitochondrial Ca.sup.2+ uptake so as to
modulate cardiac rhythmicity in a subject, provided that when
R.sub.1 is tosyl and R.sub.2 is ethyl, the compound is in an
optionally active (e.g., substantially enantiomerically pure) form
Formula Ia or Formula Ib:
##STR00002##
[0016] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, R.sub.2 is methyl,
ethyl, C3-C6 short alkyl, or menthyl group.
[0017] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, R.sub.2 is a
C1-C10 straight or branched, acyclic or cyclic alkyl group, or aryl
group.
[0018] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the compound is in
an optionally active (e.g., substantially enantiomerically pure)
form.
[0019] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the agent is
efsevin.
[0020] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the efsevin is an
efsevin enantiomer.
[0021] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the compound of
Formula I is in a composition.
[0022] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the composition
comprises a pharmaceutically acceptable carrier.
[0023] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the agent is the
compound of Formula II, wherein compound of Formula II
comprises:
##STR00003##
or wherein the carboxylic ester is attached to a mono-N-Boc
protected 2,2'-(ethylenedioxy)bis(ethylamine).
[0024] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the compound is in
an optionally active (e.g., substantially enantiomerically pure)
form.
[0025] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the compound of
Formula II is in a composition.
[0026] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the composition
comprises a pharmaceutically acceptable carrier.
[0027] In another aspect, embodiments provide an anti-arrhythmicity
compound of structure of Formula I or derivative thereof:
##STR00004##
wherein: R.sub.1 is tosyl, or mesyl group; and wherein R.sub.2 is a
hydrocarbyl group with or without a heteroatom or wherein the
carboxylic ester of R.sub.2 is attached to a mono-N-Boc protected
2,2'-(ethylenedioxy)bis(ethylamine), and wherein the compound is
effective to potentiate mitochondrial Ca.sup.2+ uptake so as to
modulate cardiac rhythmicity in a subject, provided that when
R.sub.1 is tosyl and R.sub.2 is ethyl, the compound is in an
optionally active (e.g., substantially enantiomerically pure) form
Formula Ia or Formula Ib:
##STR00005##
[0028] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, R.sub.2 is methyl,
ethyl, C3-C6 short alkyl, or menthyl group.
[0029] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, R.sub.2 is a
C1-C10 straight or branched, acyclic or cyclic alkyl group, or aryl
group.
[0030] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the compound is in
an optionally active (e.g., substantially enantiomerically pure)
form.
[0031] In a further aspect, embodiments involve methods of forming
the compound of Formula I:
##STR00006##
where R.sub.1 is tosyl, or mesyl group; and R.sub.2 is a
hydrocarbyl group with or without a heteroatom or wherein the
carboxylic ester of R.sub.2 is attached to a mono-N-Boc protected
2,2'-(ethylenedioxy)bis(ethylamine), comprising reacting
##STR00007##
according to a reaction of Scheme I
##STR00008##
to form the compound of Formula I.
[0032] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the reaction of
Scheme I is carried out under asymmetric synthesis conditions.
[0033] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the method further
comprises performing chiral resolution of compound of Formula I to
yield R- or S-enantiomers of the compound in an optionally active
(e.g., substantially enantiomerically pure) form.
[0034] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the chiral
resolution is performed on an HPLC chiral stationary phase.
[0035] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the chiral
resolution is achieved by reacting the compound with a chiral
agent.
[0036] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the chiral agent
is menthol.
[0037] In a further aspect, there is provided a composition
comprising the compound of Formula I or a derivative thereof:
##STR00009##
wherein: R.sub.1 is tosyl, or mesyl group; and wherein R.sub.2 is a
hydrocarbyl group with or without a heteroatom or wherein the
carboxylic ester of R.sub.2 is attached to a mono-N-Boc protected
2,2'-(ethylenedioxy)bis(ethylamine), and wherein the compound is
effective to potentiate mitochondrial Ca.sup.2+ uptake so as to
modulate cardiac rhythmicity in a subject, provided that when
R.sub.1 is tosyl and R.sub.2 is ethyl, the compound is in an
optionally active (e.g., substantially enantiomerically pure) form
Formula Ia or Formula Ib:
##STR00010##
[0038] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, R.sub.2 is methyl, ethyl, C3-C6 short alkyl, or menthyl
group.
[0039] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, R.sub.2 is a C1-C10 straight or branched, acyclic or cyclic
alkyl group, or aryl group.
[0040] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0041] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the composition is in a formulation suitable for
administration to a subject.
[0042] In a further aspect of the present invention, it is provided
a method of forming a composition, comprising providing a compound
of Formula I or a derivative thereof in an effective amount, and
forming the composition, wherein the compound of Formula I
comprises:
##STR00011##
wherein: R.sub.1 is tosyl, or mesyl group; and wherein R.sub.2 is a
hydrocarbyl group with or without a heteroatom or wherein the
carboxylic ester of R.sub.2 is attached to a mono-N-Boc protected
2,2'-(ethylenedioxy)bis(ethylamine), and wherein the compound is
effective to potentiate mitochondrial Ca.sup.2+ uptake so as to
modulate cardiac rhythmicity in a subject, provided that when
R.sub.1 is tosyl and R.sub.2 is ethyl, the compound is in an
optionally active (e.g., substantially enantiomerically pure) pure
form Formula Ia or Formula Ib:
##STR00012##
[0043] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, R.sub.2 is methyl, ethyl, C3-C6 short alkyl, or menthyl
group.
[0044] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, R.sub.2 is a C1-C10 straight or branched, acyclic or cyclic
alkyl group, or aryl group.
[0045] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0046] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the composition is in a formulation suitable for
administration to a subject.
[0047] In another aspect, embodiments provide an anti-arrhythmicity
compound of structure of Formula II:
##STR00013##
or wherein the carboxylic ester is attached to a mono-N-Boc
protected 2,2'-(ethylenedioxy)bis(ethylamine).
[0048] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the compound is in
an optionally active (e.g., substantially enantiomerically pure)
form.
[0049] In a further aspect, there is provided a composition
comprising the compound of Formula II:
##STR00014##
or wherein the carboxylic ester is attached to a mono-N-Boc
protected 2,2'-(ethylenedioxy)bis(ethylamine).
[0050] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0051] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the composition is in a formulation suitable for
administration to a subject.
[0052] In a further aspect of the present invention, it is provided
a method of forming a composition, comprising providing a compound
of Formula II in an effective amount, and forming the composition,
wherein the compound of Formula II comprises:
##STR00015##
or wherein the carboxylic ester is attached to a mono-N-Boc
protected 2,2'-(ethylenedioxy)bis(ethylamine).
[0053] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0054] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the composition is in a formulation suitable for
administration to a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIGS. 1A-G show test results demonstrating that efsevin
restores rhythmic cardiac contractions in zebrafish tremblor
embryos. (A,B) Fractional shortening (FS) deduced from line scans
across the atria of Tg(myl7: GFP) embryonic hearts at 48 hpf.
Rhythmically alternating systoles and diastoles were recorded from
vehicle- or efsevin-treated wild type and efsevin-treated tre
embryos, while only sporadic unsynchronized contractions were
recorded from vehicle-treated tre embryos. While cardiac
contraction was not observed in Ire, efsevin-treated wild type and
tre hearts have similar levels of FS to those observed in control
hearts. Ventricular FS of wild type v.s. wild type+efsevin vs.
tre+efsevin: 39.+-.0.6%, n=8 vs. 39.+-.1%, n=10 vs. 35.+-.3%, n=6;
and Atrial FS: 37.+-.1%, n=11 vs. 35.+-.2%, n=11 vs. 33.+-.2%,
n=15. (C) While efsevin restored a heart rate of 46.+-.2 beats per
minute (bpm) in tre embryos, same treatment does not affect the
heart rate in wild type embryos (126.+-.2 bpm in vehicle-treated
embryos vs. 123.+-.3 bpm in efsevin-treated wild-type embryos).
***, p<0.001 by one-way ANOVA. (D) Dose-dependence curve for
efsevin. The Ire embryos were treated with various concentrations
of efsevin from 24 hpf and cardiac contractions were analyzed at 48
hpf. (E-G) Representative time traces of local field potentials for
wild type (E), tre (F) and efsevin-treated tre (G) embryos clearly
display periods of regular, irregular, and restored periodic
electrical activity.
[0056] FIGS. 2A-F show test results demonstrating efsevin reduces
arrhythmogenic events in ES cell-derived cardiomyocytes (A-D).
Representative graph of rhythmic Ca.sup.2+ transients detected in
mESC-CMs of line-scan analysis of Ca.sup.2+ transients in mESC-CMs
after 10 days of differentiation (A). After treatment with 10 mM
Ca.sup.2+ for 10 minutes, the EB showed an irregular pattern of
Ca.sup.2+ transients (B). Efsevin treatment restores regular
Ca.sup.2+ transients under Ca.sup.2+ overload conditions in
mESC-CMs (C). (D) Plotted intervals between peaks of Ca.sup.2+
signals detected in mESC-CMs prior to treatment (control), in 10 mM
Ca.sup.2+.sub.ext (Ca.sup.2+) and in 10 mM Ca.sup.2+.sub.ext+10
.mu.M efsevin (Ca.sup.2++efsevin). (E,F) Plotted intervals of
contractions detected in EBs prior to treatment (control), in 10 mM
Ca.sup.2+.sub.ext (Ca.sup.2+) and in 10 mM Ca.sup.2+.sub.ext 10
.mu.M efsevin (Ca.sup.2++efsevin) for mouse ESC-CMs (E) and 5 mM
Ca.sup.2+.sub.ext (Ca.sup.2+) and in 5 mM Ca.sup.2+.sub.ext+5 .mu.M
efsevin (Ca.sup.2++efsevin) for human ESC-CMs (F). ***, p<0.001
by F-test.
[0057] FIGS. 3A-E show test results demonstrating VDAC2 is a
protein target of efsevin. (A) Structures of efsevin and two
derivatives, OK-C125 and OK-C19. (B) Efsevin and OK-C125 restored
rhythmic contractions in the majority of tremblor embryos, whereas
OK-C19 failed to rescue the tremblor phenotype. (C) Structures of
linker-attached compounds (indicated by superscript L). (D)
Compounds efsevin.sup.L and OK-C125.sup.L retained their ability to
restore rhythmic contractions in NCX1hMO injected embryos, while
the inactive derivative OK-C19.sup.L was still unable to induce
rhythmic contraction. (E) Mass Spectrometry identification of VDAC
2, a 32 kD band pulled down with affinity agarose beads covalently
linked with efsevin (efsevin.sup.LB) or OK-C125 (OK-C125.sup.LB)
that was sensitive to competition with a 100 fold excess free
efsevin.sup.L. The 32 kD band was not detected in proteins eluted
from beads capped with ethanolamine alone (beads.sup.C) or beads
linked to OK-C19 (OK-C19.sup.LB). Peptides identified by mass
spectrometry (underlined) account for 30% of the total sequence
(SEQ ID NO: 1).
[0058] FIGS. 4A-F show test results demonstrating VDAC2 restores
rhythmic cardiac contractions in tre. In situ hybridization
analysis showed that VDAC2 is expressed in embryonic hearts at 36
hpf and 48 hpf. (A) Injection of 25 pg in-vitro synthesized VDAC2
mRNA restored cardiac contractions in 52.9.+-.12.1% (n=78) of
one-day-old tre embryos, compared to 21.8.+-.5.1% in uninjected
siblings (n=111). (B) Schematic diagram of myl7:VDAC2 construct.
(C) While only .about.20% of myl7:VDAC2; NCX1hMO embryos have
coordinated contractions (n=116), 52.3.+-.2.4% of these embryos
established persistent, rhythmic contractions after TBF induction
of VDAC2 (n=154). (D) On average, 71.2.+-.8.8% efsevin treated
embryos have coordinated cardiac contractions (n=131). Morpholino
antisense oligonucleotide knockdown of VDAC2 (MO.sup.VDAc2)
attenuates the ability of efsevin to suppress cardiac fibrillation
in tre embryos (45.3.+-.7.4% embryos with coordinated contractions,
n=94). (E) Efsevin treatment restores coordinated cardiac
contractions in 76.2.+-.8.7% NCX1MO embryos, only 54.1.+-.3.6%
VDAC2.sup.zfn/zfn; NCX1MO embryos have coordinated contractions
(n=250). (F) Diagram of Zinc finger target sites. VDAC2.sup.zfn/zfn
carries a 34 bp deletion in exon 3 which results in a premature
stop codon (asterisk).
[0059] FIGS. 5A-5D show test results demonstrating efsevin enhances
mitochondrial Ca.sup.2+ uptake. HeLa cells were transfected with a
flag-tagged zebrafish VDAC2 (VDAC2.sup.flag), immunostained against
the flag epitope and counterstained for mitochondria with
MitoTracker Orange and for nuclei with DAPI. (A) Representative
traces of mitochondrial matrix [Ca.sup.2+] ([Ca.sup.2+].sub.m)
detected by Rhod2. Arrows denote the addition of Ca.sup.2+.
Mitochondrial Ca.sup.2+ uptake was assessed when VDAC2 was
overexpressed (left), cells were treated with 1 .mu.M efsevin
(middle) and combination of both at suboptimal doses (right).
Control-traces with ruthenium red (RuRed) show mitochondrial
specificity of the signal. (B) Representative traces of cytosolic
[Ca.sup.2+] ([Ca.sup.2+].sub.c) changes upon the application of 7.5
.mu.M IP.sub.3 in the presence (+) or absence (-) of RuRed.
Mitochondrial Ca.sup.2+ uptake was assessed by the difference of
the - and + RuRed conditions normalized to the total release (n=4;
mean.+-.SE). (C) MEFs overexpressing zebrafish VDAC2 (polycistronic
with mCherry) were stimulated with 1 .mu.M ATP in a nominally
Ca.sup.2+ free buffer. Changes in [Ca.sup.2+].sub.c and
[Ca.sup.2+].sub.m were imaged using fura2 and mitochondria-targeted
inverse pericam, respectively. Black and gray traces show the
[Ca.sup.2+].sub.c (in nM) and [Ca.sup.2+].sub.m (F.sub.0/F
mtpericam) time courses in the absence (left) or present (right) of
efsevin. (D) Bar charts: Cell population averages for the peak
[Ca.sup.2+].sub.c (left), the corresponding [Ca.sup.2+].sub.m
(middle), and the coupling time (time interval between the maximal
[Ca.sup.2+].sub.c and [Ca.sup.2+].sub.m responses) in the presence
(black, n=24) or absence (gray, n=28) of efsevin.
[0060] FIGS. 6A-C show effects of efsevin on isolated
cardiomyocytes. (A) Electrically paced Ca.sup.2+ transients at 0.5
Hz (top). Normalized quantification of Ca.sup.2+ transient
parameters reveals no difference for transient amplitude
(efsevin-treated at 98.6.+-.4.5% of vehicle-treated) and time to
peak (95.+-.3.9%), but a significant decrease for the rate of decay
(82.8.+-.4% of vehicle- for efsevin-treated) (lower panel). (B)
Representation of typical Ca.sup.2+ sparks of vehicle- and efsevin
treated cardiomyocytes (top). No differences were observed for
spark frequency (101.1.+-.7.7% for efsevin-compared to
vehicle-treated), maximum spark amplitude (101.6.+-.2.5%) and
Ca.sup.2+ release flux (98.7.+-.2.8%). In contrast, the decay phase
of the single spark was significantly faster in efsevin treated
cells (82.5.+-.2.1% of vehicle-treated). Consequently, total
duration of the spark was reduced to 85.7.+-.2% and the total width
was reduced to 89.5.+-.1.4% of vehicle-treated cells. *, p<0.05;
***, p<0.001. (C) Quantitative analysis of spontaneous Ca.sup.2+
waves spanning more than half of the entire cell. Addition of 1
.mu.M efsevin reduced Ca.sup.2+ waves to approximately half.
Increasing the concentration of efsevin to 10 .mu.M further reduced
the number of spontaneous Ca.sup.2+ waves and 25 .mu.M efsevin
almost entirely blocked the formation of Ca.sup.2+ waves.
[0061] FIGS. 7A-7E show test results demonstrating that
mitochondria regulate cardiac rhythmicity through a VDAC2-dependent
mechanism. MCU and MICU1 are expressed in the developing zebrafish
hearts. (A) Overexpression of MCU is sufficient to restore
coordinated cardiac contractions in tre embryos (47.1.+-.1.6%
embryos, n=112 as opposed to 18.3.+-.5.3% of uninjected siblings,
n=64) while this effect is significantly attenuated when
co-injected with morpholino antisense oligonucleotide targeted to
VDAC2 (27.1.+-.1.9% embryos, n=135). (B) Suboptimal overexpression
of MCU (MCU.sup.S) and VDAC2 (VDAC2.sup.S) in combination is able
to suppress cardiac fibrillation in tre embryos (42.9.+-.2.6%
embryos, n=129). (C) The ability of VDAC2 to restore rhythmic
contractions in tre embryos (48.5.+-.3.5% embryos, n=111) is
significantly attenuated when MCU is knocked down by antisense
oligonucleotide (MO.sup.MCU) (25.6.+-.2.4% embryos, n=115). (D)
Overexpression of MICU1 is sufficient to restore rhythmic cardiac
contractions in tre embryos (49.3.+-.3.4% embryos, n=127 compared
to 16.8.+-.1.4% of uninjected siblings, n=150). This effect is
abrogated by VDAC2 knockdown (MO.sup.VDAc2, 25.3.+-.5.5% embryos,
n=97). (E) Suboptimal overexpression of MICU1 (MICU1.sup.S) and
VDAC2 (VDAC2.sup.S) in combination is able to restore rhythmic
cardiac contractions in tre embryos (48.6.+-.6.0%, n=106). Error
bars represent s.d.; *p<0.05; ***p<0.001.
[0062] FIG. 8 shows that local Ca.sup.2+ delivery between IP3
receptors and VDAC2. V1/V3DKO MEFs were stimulated with 100 .mu.M
ATP (left) or 2 .mu.M thapsigargan (Tg) (right). Changes in
[Ca.sup.2+].sub.c and [Ca.sup.2+].sub.m were imaged using fura 2
and mitochondria targeted inverse pericam, respectively.
Representative traces obtained in 3 cells are shown.
[0063] FIGS. 9A-G show that mitochondria regulate cardiac
rhythmicity through a VDAC dependent mechanism.
[0064] A) Injection of 25 pg in-vitro synthesized VDAC1 and VDAC2
mRNA restored cardiac contractions in 53.0.+-.10.2% (n=126) and
52.9.+-.12.1% (n=78) of one-day-old tre embryos, respectively,
compared to 21.8.+-.5.1% in uninjected siblings (n=111).
[0065] B) While only .about.20% of myl7:VDAC2; NCX1hMO embryos have
coordinated contractions (n=116), 52.3.+-.2.4% of these embryos
established persistent, rhythmic contractions after TBF induction
of VDAC2 (n=154).
[0066] C) On average, 71.2.+-.8.8% efsevin treated embryos have
coordinated cardiac contractions (n=131). Morpholino antisense
oligonucleotide knockdown of VDAC2 (MO.sup.VDAC2) or VDAC1
(MO.sup.VDAC1) attenuates the ability of efsevin to suppress
cardiac fibrillation in tre embryos (45.3.+-.7.4% and 46.9.+-.10.7%
embryos with coordinated contractions, n=94 and 114, respectively).
Knocking down VDAC1/2 simultaneously further suppresses efsevin's
effect (30.3.+-.6.3%, n=75).
[0067] D) Efsevin treatment restores coordinated cardiac
contractions in 76.2.+-.8.7% NCX1MO embryos, only 54.1.+-.3.6%
VDAC2.sup.zfn/zfn; NCX1MO embryos and 35.7.+-.7.1%
VDAC2.sup.zfn/zfn; VDAC1MO; NCX1MO embryos have coordinated
contractions (n=250).
[0068] E) Overexpression of MCU is sufficient to restore
coordinated cardiac contractions in tre embryos (47.1.+-.1.6%
embryos, n=112 as opposed to 18.3.+-.5.3% of uninjected siblings,
n=64) while this effect is significantly attenuated when
co-injected with morpholino antisense oligonucleotide targeted to
VDAC2 (27.1.+-.1.9% embryos, n=135).
[0069] F) Suboptimal overexpression of MCU (MCU.sup.S) and VDAC2
(VDAC2.sup.S) in combination is able to suppress cardiac
fibrillation in tre embryos (42.9.+-.2.6% embryos, n=129).
[0070] G) The ability of VDAC2 to restore rhythmic contractions in
tre embryos (48.5.+-.3.5% embryos, n=111) is significantly
attenuated when MCU is knocked down by antisense oligonucleotide
(MO.sup.MCU) (25.6.+-.2.4% embryos, n=115). Error bars represent
s.d.; *p<0.05; ***p<0.001.
[0071] FIG. 10 shows resolution of (R)- and (S)-efsevin from a
mixture of (R)- and (S)-efsevin through HPLC separation.
[0072] FIG. 11 shows purification of (R)-efsevin through HPLC
separation.
[0073] FIG. 12 shows purification of (S)-efsevin through HPLC
separation.
[0074] FIG. 13 shows resolution of (R)- and (S)-efsevin from a
racemic mixture of efsevin through HPLC separation.
[0075] FIG. 14 shows .sup.1H NMR (top) and .sup.13C NMR (bottom) of
(R)-Efsevin menthol ester.
[0076] FIG. 15 shows .sup.1H NMR (top) and .sup.13C NMR (bottom) of
(S)-Efsevin menthol ester.
[0077] FIGS. 16A and B show active compounds found through forward
chemical genetics.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0078] The term "effective amount", as used herein, is an amount of
an agent that is sufficient to produce a statistically significant,
measurable change of a condition in cardiac rhythmicity as compared
with the condition in cardiac rhythmicity without using the agent.
Such effective amounts can be gauged in clinical trials as well as
animal studies. Such a statistically significant, measurable, and
positive change of a condition in cardiac rhythmicity using the
agent disclosed herein as compared with the condition in the
cardiac rhythmicity to without using the agent is referred to as
being an "improved condition".
[0079] As used herein, the term "agent" refers to an agent that
capable of potentiating mitochondrial Ca.sup.2+ uptake to effect
VDAC2 expression in a subject. In some embodiments, the term agent
can refer to as "anti-arrhythmicity" drug or compound.
[0080] As used herein, the term "subject" as used herein is any
vertebrate. Subjects include individuals in need of drug (e.g. an
agent disclosed herein such as efsevin) treatment (patients) and
individuals not in need of drug treatment (e.g. normal healthy
volunteers). Humans are preferred subjects and patients.
[0081] "Treat" or "treatment" refers to any treatment of a disorder
or disease, such as preventing the disorder or disease from
occurring in a subject which may be predisposed to the disorder or
disease, but has not yet been diagnosed as having the disorder or
disease; inhibiting the disorder or disease, e.g., arresting the
development of the disorder or disease, relieving the disorder or
disease, causing regression of the disorder or disease, relieving a
condition caused by the disease or disorder or reducing the
symptoms of the disease or disorder.
[0082] As used herein, the term "disorder" generally refers to a
condition related to cardiac cardiac arrhythmicity or cardiac
disorder with a root in aberrant Ca2+ handling. Such disorder
includes, for example, cardiac fibrillation, arrhythmia, atrial
fibrillation, sick sinus syndrome, catechol aminergic polymorphic
ventricular tachycardia, or cardiomyopathy.
[0083] As used herein, the term "derivative" refers to a form of
the agent or compound disclosed herein, which derivative is capable
of generating an active species or moiety in vitro or in vivo
having anti-arrhythmicity activities as is the agent or compound
disclosed herein. Non-limiting examples of such "derivative"
includes prodrug or metabolite.
[0084] As used herein, the term "optically active" refers to the
compound Formula I disclosed herein that is not a 50/50 R/S racemic
mixture of the compound of Formula I.
[0085] As used herein, the term "substantially enantiomerically
pure" refers to the purity of S- or R-enantiomer of the compound of
Formula I of about 60%-about 100%, of about 70%-about 100%, of
about 80%-about 100%, of about 90%-about 100%, of about 95%-about
100%, e.g., of about 75%, of about 80%, of about 85%, or of about
99%.
[0086] As used herein, the term "enantiomer" refers to a compound
disclosed herein that is optically active or substantially
enantiomerically pure.
[0087] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0088] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of elements that do not materially affect the basic
and novel or functional characteristic(s) of that embodiment of the
invention.
[0089] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0090] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" include one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
B. Mitochondrial Ca.sup.2+ Uptake Regulates Cardiac Rhythmicity
[0091] In an aspect of the present invention, it is provided a
method of regulating cardiac rhythmicity in a subject, comprising
potentiating mitochondrial Ca.sup.2+ uptake by inducing VDAC2 or
VDAC1 overexpression in the subject to restore rhythmic
contraction, inducing overexpression of or activating VDAC (VDAC2
or VDAC1) and/or MCU (MCU or MICU1) complex, or administering to
the subject in need thereof an agent effective to induce Ca2+
transporting activity of VDAC2 or VDAC1.
[0092] In some embodiments, optionally in combination with any or
all of the various embodiments disclosed herein, the subject
suffers from a disorder related to cardiac arrhythmicity or cardiac
disorder with a root in aberrant Ca2+ handling. Such disorder
includes, for example, cardiac fibrillation, arrhythmia, atrial
fibrillation, sick sinus syndrome, catechol aminergic polymorphic
ventricular tachycardia, or cardiomyopathy.
[0093] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, inducing VDAC2 or VDAC1 overexpression in the subject is
via gene therapy.
[0094] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the agent is a VDAC2 or VDAC1 gene product.
[0095] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the agent is a VDAC2 or VDAC1 protein, or a VDAC2 or VDAC1
RNA.
[0096] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the agent is the compound of Formula I, which is described
in detail below. For concise description of the present invention,
the description of the compound of Formula I is not repeated here
but is fully incorporated hereto by reference.
[0097] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the agent is efsevin.
[0098] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the efsevin is an efsevin enantiomer.
[0099] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the compound of Formula I is in a composition.
[0100] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the composition comprises a pharmaceutically acceptable
carrier.
C. Gene Therapy
[0101] In some embodiments, potentiating mitochondrial Ca2+ uptake
can be achieved by overexpression of VDAC2 or VDAC1 by delivery to
a subject in need thereof a VDAC2- or VDAC1-encoding gene sequence
using a viral or non-viral vector. Vectors for transduction of a
VDAC2- or VDAC1-encoding sequence are well known in the art. While
overexpression using a strong non-specific promoter, such as a CMV
promoter, can be used, it can be helpful to include a tissue- or
cell-type-specific promoter on the expression construct. Further,
treatment can include the administration of viral vectors that
drive the overexpression of VDAC2 or VDAC1 proteins in infected
host cells. Viral vectors are well known to those skilled in the
art.
[0102] These vectors are readily adapted for use in the methods of
the present invention. By the appropriate manipulation using
recombinant DNA/molecular biology techniques to insert an
operatively linked VDAC2 or VDAC1 encoding nucleic acid segment
into the selected expression/delivery vector, many equivalent
vectors for the practice of the methods described herein can be
generated. It will be appreciated by those of skill in the art that
cloned genes readily can be manipulated to alter the amino acid
sequence of a protein.
[0103] The cloned gene for VDAC2 or VDAC1 can be manipulated by a
variety of well-known techniques for in vitro mutagenesis, among
others, to produce variants of the naturally occurring human
protein, herein referred to as muteins or variants or mutants of
VDAC2 or VDAC1, which may be used in accordance with the methods
and compositions described herein. The variation in primary
structure of muteins of VDAC2 or VDAC1 protein useful in the
invention, for instance, may include deletions, additions and
substitutions. The substitutions may be conservative or
non-conservative. The differences between the natural protein and
the mutein generally conserve desired properties, mitigate or
eliminate undesired properties and add desired or new properties.
The VDAC2 or VDAC1 protein can also be a fusion polypeptide, fused,
for example, to a polypeptide that targets the product to a desired
location, or, for example, a tag that facilitates its purification,
if so desired. Fusion to a polypeptide sequence that increases the
stability of the VDAC2 protein is also contemplated. For example,
fusion to a serum protein, e.g., serum albumin, can increase the
circulating half-life of a VDAC2 or VDAC1 protein. Tags and fusion
partners can be designed to be cleavable, if so desired. Another
modification specifically contemplated is attachment, e.g.,
covalent attachment, to a polymer. In one aspect, polymers such as
polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG) can
increase the in vivo half-life of proteins to which they are
conjugated. Methods of PEGylation of polypeptide agents are well
known to those skilled in the art, as are considerations of, for
example, how large a PEG polymer to use. In another aspect,
biodegradable or absorbable polymers can provide extended, often
localized, release of polypeptide agents. Such synthetic
bioabsorbable, biocompatible polymers, which may release proteins
over several weeks or months can include, for example,
poly-a-hydroxy acids (e.g. polylactides, polyglycolides and their
copolymers), polyanhydrides, polyorthoesters, segmented block
copolymers of polyethylene glycol and polybutylene terephtalate
(Polyactive.TM.), tyrosine derivative polymers or
poly(ester-amides). Suitable bioabsorbable polymers to be used in
manufacturing of drug delivery materials and implants are discussed
e.g. in U.S. Pat. Nos. 4,968,317; 5,618,563, among others, and in
"Biomedical Polymers" edited by S. W. Shalaby, Carl Hanser Verlag,
Munich, Vienna, N.Y., 1994 and in many references cited in the
above publications. The particular bioabsorbable polymer that
should be selected will depend upon the particular patient that is
being treated.
D. Anti-Arrhythmicity Compound
[0104] In another aspect of the present invention, it is provided
an anti-arrhythmicity compound of structure of Formula I or a
derivative thereof:
##STR00016##
wherein: R.sub.1 is tosyl, or mesyl group; and wherein R.sub.2 is a
hydrocarbyl group with or without a heteroatom, and wherein the
compound is effective to potentiate mitochondrial Ca.sup.2+ uptake
so as to modulate cardiac rhythmicity in a subject, provided that
when R.sub.1 is tosyl and R.sub.2 is ethyl, the compound is in an
optionally active (e.g., substantially enantiomerically pure) form
Formula Ia or Formula Ib:
##STR00017##
[0105] In some embodiments of the invention compound, optionally in
combination with any or all of the various embodiments disclosed
herein, R.sub.2 is methyl, ethyl, C3-C6 short alkyl, or menthyl
group.
[0106] In some embodiments of the invention compound, optionally in
combination with any or all of the various embodiments disclosed
herein, R.sub.2 is a C1-C10 straight or branched, acyclic or cyclic
alkyl group, or aryl group.
[0107] In some embodiments of the invention compound, optionally in
combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0108] Non-limiting examples of derivatives of Formula I include
Formula Ic:
##STR00018##
wherein: R1 is an alkane, phenyl, heteroaryl, or substituted phenyl
group; R2 is a phenyl, heteroaryl, substituted phenyl, or
hydrocarbyl group with or without a heteroatom, and R3 is an
alkoxy, amino, or amino ether. Non-limiting examples of a
substituted phenyl group is a phenyl group wherein one or more
hydrogens are replaced with an alkane, amino, amino ether, or
heteroatom.
[0109] In some embodiments, R3 is an alkoxy, amino, amino ether,
N-Boc-protected 2-aminoethoxyethoxyethylamino group, or a C1-C10
straight or branched, acyclic or cyclic alkyloxy group, aryloxy, or
amino group with or without heteroatom
[0110] In some embodiments, the R.sub.1 is para-toyl, R.sub.2 is
phenyl, and R.sub.3 is ethoxy.
[0111] In some embodiments, R3 is ethoxy, menthyloxy, or
N-Box-protected 2-aminoethoxyethoxyethalamino group.
[0112] In some embodiments, there is a method of forming the
compound of Formula Ic:
##STR00019##
where R.sub.1 is substituted phenyl or substituted or unsubstituted
heteroaryl; and R.sub.2 is substituted phenyl or substituted or
unsubstituted heteroaryl; and R3 is R.sub.3 is an alkoxy, amino,
amino ether, N-Boc-protected 2-aminoethoxyethoxyethylamino group,
or a C1-C10 straight or branched, acyclic or cyclic alkyloxy group,
aryloxy, or amino group with or without heteroatom comprising
reacting
##STR00020##
according to a reaction of Scheme I
##STR00021##
to form the compound of Formula Ic.
[0113] In some embodiments, the compound is any one of, (FIG.
16A-B)
E. Method of Synthesis
[0114] In a further aspect of the present invention, it is provided
a method of forming the compound of Formula I:
##STR00022##
where R.sub.1 is tosyl, or mesyl group; and R.sub.2 is a
hydrocarbyl group with or without a heteroatom, comprising
reacting
##STR00023##
according to a reaction of Scheme I
##STR00024##
to form the compound of Formula I.
[0115] Description of the compound of Formula I is fully provided
above. For concise description of the present invention, the
description of the compound of Formula I is not repeated here but
is fully incorporated hereto by reference.
[0116] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the reaction of Scheme I is carried out under asymmetric
synthesis conditions.
[0117] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the method further comprises performing chiral resolution
of compound of Formula I to yield R- or S-enantiomers of the
compound in an optionally active (e.g., substantially
enantiomerically pure) form.
[0118] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the chiral resolution is performed on an HPLC chiral
stationary phase.
[0119] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the chiral resolution is achieved by reacting the compound
with a chiral agent.
[0120] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the chiral agent is menthol.
F. Compositions
[0121] In a further aspect of the present invention, it is provided
a composition comprising an anti-arrhythmicity compound of Formula
I, which is described above. For concise description of the present
invention, the description of the compound of Formula I is not
repeated here but is fully incorporated hereto by reference.
[0122] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0123] In some embodiments of the invention composition, optionally
in combination with any or all of the various embodiments disclosed
herein, the composition is in a formulation suitable for
administration to a subject.
[0124] In a further aspect of the present invention, it is provided
a method of forming a composition, comprising providing a compound
of Formula I in an effective amount, and forming the
composition.
[0125] The compound of Formula I is fully described above. For
concise description of the present invention, the description of
the compound of Formula I is not repeated here but is fully
incorporated hereto by reference.
[0126] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the compound is in an optionally active (e.g.,
substantially enantiomerically pure) form.
[0127] In some embodiments of the invention method, optionally in
combination with any or all of the various embodiments disclosed
herein, the composition is in a formulation suitable for
administration to a subject to treat or ameliorate a disorder
related to cardiac arrhythmicity, e.g., cardiac fibrillation.
G. Formulations
[0128] The compound of invention or composition of invention can be
formulated into any desirable formulation. Such formulations can
include a pharmaceutically acceptable carrier, which can be, e.g.,
salient or can comprise a polymeric material.
[0129] In some embodiments, the carrier disclosed herein can be a
polymeric material. Exemplary polymeric material that can be used
here include but are not limited to a biocompatible or
bioabsorbable polymer that is one or more of poly(.sub.DL-lactide),
poly(.sub.L-lactide), poly(.sub.L-lactide),
poly(.sub.L-lactide-co-.sub.D,L-lactide), polymandelide,
polyglycolide, poly(lactide-co-glycolide),
poly(.sub.D,L-lactide-co-glycolide),
poly(.sub.L-lactide-co-glycolide), poly(ester amide), poly(ortho
esters), poly(glycolic acid-co-trimethylene carbonate),
poly(.sub.D,L-lactide-co-trimethylene carbonate), poly(trimethylene
carbonate), poly(lactide-co-caprolactone),
poly(glycolide-co-caprolactone), poly(tyrosine ester),
polyanhydride, derivatives thereof. In some embodiments, the
polymeric material comprises a combination of these polymers.
[0130] In some embodiments, the polymeric material comprises
poly(.sub.D,L-lactide-co-glycolide). In some embodiments, the
polymeric material comprises poly(.sub.D,L-lactide). In some
embodiments, the polymeric material comprises poly(.sub.L-lactide).
[0065] Additional exemplary polymers include but are not limited to
poly(.sub.D-lactide) (PDLA), polymandelide (PM), polyglycolide
(PGA), poly(.sub.L-lactide-co-D,L-lactide) (PLDLA),
poly(.sub.D,L-lactide) (PDLLA), poly(.sub.D,L-lactide-co-glycolide)
(PLGA) and poly(.sub.L-lactide-co-glycolide) (PLLGA). With respect
to PLLGA, the stent scaffolding can be made from PLLGA with a mole
% of GA between 5-15 mol %. The PLLGA can have a mole % of (LA:GA)
of 85:15 (or a range of 82:18 to 88:12), 95:5 (or a range of 93:7
to 97:3), or commercially available PLLGA products identified as
being 85:15 or 95:5 PLLGA. The examples provided above are not the
only polymers that may be used. Many other examples can be
provided, such as those found in Polymeric Biomaterials, second
edition, edited by Severian Dumitriu; chapter 4.
[0131] In some embodiments, polymers that are more flexible or that
have a lower modulus that those mentioned above may also be used.
Exemplary lower modulus bioabsorbable polymers include,
polycaprolactone (PCL), poly(trimethylene carbonate) (PTMC),
polydioxanone (PDO), poly(3-hydrobutyrate) (PHB),
poly(4-hydroxybutyrate) (P4HB), poly(hydroxyalkanoate) (PHA), and
poly(butylene succinate), and blends and copolymers thereof.
[0132] In exemplary embodiments, higher modulus polymers such as
PLLA or PLLGA may be blended with lower modulus polymers or
copolymers with PLLA or PLGA. The blended lower modulus polymers
result in a blend that has a higher fracture toughness than the
high modulus polymer. Exemplary low modulus copolymers include
poly(.sub.L-lactide)-b-polycaprolactone (PLLA-b-PCL) or
poly(.sub.L-lactide)-co-polycaprolactone (PLLA-co-PCL). The
composition of a blend can include 1-5 wt % of low modulus
polymer.
[0133] More exemplary polymers include but are not limited to at
least partially alkylated polyethyleneimine (PEI); at least
partially alkylated poly(lysine); at least partially alkylated
polyornithine; at least partially alkylated poly(amido amine), at
least partially alkylated homo- and co-polymers of vinylamine; at
least partially alkylated acrylate containing aminogroups,
copolymers of vinylamine containing aminogroups with hydrophobic
monomers, copolymers of acrylate containing aminogroups with
hydrophobic monomers, and amino containing natural and modified
polysaccharides, polyacrylates, polymethacryates, polyureas,
polyurethanes, polyolefins, polyvinylhalides,
polyvinylidenehalides, polyvinylethers, polyvinylaromatics,
polyvinylesters, polyacrylonitriles, alkyd resins, polysiloxanes
and epoxy resins, and mixtures thereof. Additional examples of
biocompatible biodegradable polymers include, without limitation,
polycaprolactone, poly(.sub.L-lactide), poly(.sub.D,L-lactide),
poly(.sub.D,L-lactide-co-PEG) block copolymers,
poly(.sub.D,L-lactide-co-trimethylene carbonate),
poly(lactide-co-glycolide), polydioxanone (PDS), polyorthoester,
polyanhydride, poly(glycolic acid-co-trimethylene carbonate),
polyphosphoester, polyphosphoester urethane, poly(amino acids),
polycyanoacrylates, poly(trimethylene carbonate),
poly(iminocarbonate), polycarbonates, polyurethanes, polyalkylene
oxalates, polyphosphazenes, PHA-PEG, and combinations thereof. The
PHA may include poly(a-hydroxyacids), poly(.beta.-hydroxyacid) such
as poly(3-hydroxybutyrate) (PHB),
poly(3-hydroxybutyrate-co-valerate) (PHBV),
poly(3-hydroxyproprionate) (PHP), poly(3-hydroxyhexanoate) (PHH),
or poly(4-hydroxyacid) such as poly poly(4-hydroxybutyrate),
poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate),
poly(hydroxyvalerate), poly(tyrosine carbonates), poly(tyrosine
arylates), poly(ester amide), polyhydroxyalkanoates (PHA),
poly(3-hydroxyalkanoates) such as poly(3-hydroxypropanoate),
poly(3-hydroxybutyrate), poly(3-hydroxyvalerate),
poly(3-hydroxyhexanoate), poly(3-hydroxyheptanoate) and
poly(3-hydroxyoctanoate), poly(4-hydroxyalkanaote) such as
poly(4-hydroxybutyrate), poly(4-hydroxyvalerate),
poly(4-hydroxyhexanote), poly(4-hydroxyheptanoate),
poly(4-hydroxyoctanoate) and copolymers including any of the
3-hydroxyalkanoate or 4-hydroxyalkanoate monomers described herein
or blends thereof, poly(.sub.D,L-lactide), poly(.sub.L-lactide),
polyglycolide, poly(.sub.D,L-lactide-co-glycolide),
poly(.sub.L-lactide-co-glycolide), polycaprolactone,
poly(lactide-co-caprolactone), poly(glycolide-co-caprolactone),
poly(dioxanone), poly(ortho esters), poly(anhydrides),
poly(tyrosine carbonates) and derivatives thereof, poly(tyrosine
ester) and derivatives thereof, poly(imino carbonates),
poly(glycolic acid-co-trimethylene carbonate), polyphosphoester,
polyphosphoester urethane, poly(amino acids), polycyanoacrylates,
poly(trimethylene carbonate), poly(iminocarbonate),
polyphosphazenes, silicones, polyesters, polyolefins,
polyisobutylene and ethylene-alphaolefin copolymers, acrylic
polymers and copolymers, vinyl halide polymers and copolymers, such
as polyvinyl chloride, polyvinyl ethers, such as polyvinyl methyl
ether, polyvinylidene halides, such as polyvinylidene chloride,
polyacrylonitrile, polyvinyl ketones, polyvinyl aromatics, such as
polystyrene, polyvinyl esters, such as polyvinyl acetate,
copolymers of vinyl monomers with each other and olefins, such as
ethylene-methyl methacrylate copolymers, acrylonitrile-styrene
copolymers, ABS resins, and ethylene-vinyl acetate copolymers,
polyamides, such as Nylon 66 and polycaprolactam, alkyd resins,
polycarbonates, polyoxymethylenes, polyimides, polyethers,
poly(glyceryl sebacate), poly(propylene fumarate), poly(n-butyl
methacrylate), poly(sec-butyl methacrylate), poly(isobutyl
methacrylate), poly(tert-butyl methacrylate), poly(n-propyl
methacrylate), poly(isopropyl methacrylate), poly(ethyl
methacrylate), poly(methyl methacrylate), epoxy resins,
polyurethanes, rayon, rayon-triacetate, cellulose acetate,
cellulose butyrate, cellulose acetate butyrate, cellophane,
cellulose nitrate, cellulose propionate, cellulose ethers,
carboxymethyl cellulose, polyethers such as poly(ethylene glycol)
(PEG), copoly(ether-esters) (e.g. poly(ethylene oxide-co-lactic
acid) (PEO/PLA)), polyalkylene oxides such as poly(ethylene oxide),
poly(propylene oxide), poly(ether ester), polyalkylene oxalates,
phosphoryl choline containing polymer, choline, poly(aspirin),
polymers and co-polymers of hydroxyl bearing monomers such as
2-hydroxyethyl methacrylate (HEMA), hydroxypropyl methacrylate
(HPMA), hydroxypropylmethacrylamide, PEG acrylate (PEGA), PEG
methacrylate, methacrylate polymers containing
2-methacryloyloxyethyl-phosphorylcholine (MPC) and n-vinyl
pyrrolidone (VP), carboxylic acid bearing monomers such as
methacrylic acid (MA), acrylic acid (AA), alkoxymethacrylate,
alkoxyacrylate, and 3-trimethylsilylpropyl methacrylate (TMSPMA),
poly(styrene-isoprene-styrene)-PEG (SIS-PEG), polystyrene-PEG,
polyisobutylene-PEG, polycaprolactone-PEG (PCL-PEG), PLA-PEG,
poly(methyl methacrylate), MED610, poly(methyl methacrylate)-PEG
(PMMA-PEG), polydimethylsiloxane-co-PEG (PDMS-PEG), poly(vinylidene
fluoride)-PEG (PVDF-PEG), PLURONIC.TM. surfactants (polypropylene
oxide-co-polyethylene glycol), poly(tetramethylene glycol), hydroxy
functional poly(vinyl pyrrolidone), biomolecules such as collagen,
chitosan, alginate, fibrin, fibrinogen, cellulose, starch, dextran,
dextrin, hyaluronic acid, fragments and derivatives of hyaluronic
acid, heparin, fragments and derivatives of heparin, glycosamino
glycan (GAG), GAG derivatives, polysaccharide, elastin, elastin
protein mimetics, or combinations thereof.
[0134] In some embodiments, polyethylene is used to construct at
least a portion of the device. For example, polyethylene can be
used in an orthopedic implant on a surface that is designed to
contact another implant, as such in a joint or hip replacement.
Polyethylene is very durable when it comes into contact with other
materials. When a metal implant moves on a polyethylene surface, as
it does in most joint replacements, the contact is very smooth and
the amount of wear is minimal. Patients who are younger or more
active may benefit from polyethylene with even more resistance to
wear. This can be accomplished through a process called
crosslinking, which creates stronger bonds between the elements
that make up the polyethylene. The appropriate amount of
crosslinking depends on the type of implant. For example, the
surface of a hip implant may require a different degree of
crosslinking than the surface of a knee implant.
[0135] Additional examples of polymeric materials can be found, for
example, in U.S. Pat. No. 6,127,448 to Domb, US Pat. Pub. No.
2004/0148016 by Klein and Brazil, US Pat. Pub. No. 2009/0169714 by
Burghard et al, U.S. Pat. No. 6,406,792 to Briquet et al, US Pat.
Pub. No. 2008/0003256 by Martens et al, each of which is hereby
incorporated by reference herein in its entirety.
H. Dosage and Administration
[0136] The dosage can be determined by one of skill in the art and
can also be adjusted by the individual physician in the event of
any complication. Typically, the dosage ranges from 0.0005 mg/kg
body weight to 1 g/kg body weight. In some embodiments, the dosage
range is from 0.001 mg/kg body weight to 0.5 g/kg body weight, from
0.0005 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg
body weight to 0.05 g/kg body weight.
[0137] As another alternative, dosages are selected for localized
delivery and are not necessarily selected for body weight or to
achieve a certain serum level, but to achieve a localized effect,
e.g., as for a localized injection, implantation or other localized
administration to the eye.
[0138] Administration of the doses recited above can be repeated
for a limited period of time. In some embodiments, the doses are
given once a day, or multiple times a day, for example, but not
limited to, three times a day. In a preferred embodiment, the doses
recited above are administered daily for several weeks or months.
The duration of treatment depends upon the subject's clinical
progress and responsiveness to therapy. Continuous, relatively low
maintenance doses are contemplated after an initial higher
therapeutic dose.
[0139] Agents useful in the methods and compositions described
herein can be administered topically, intravenously (by bolus or
continuous infusion), orally, by inhalation, intraperitoneally,
intramuscularly, subcutaneously, intracavity, and can be delivered
by peristaltic means, if desired, or by other means known by those
skilled in the art. It is preferred that the agents for the methods
described herein are administered topically to the eye. For the
treatment of tumors, the agent can be administered systemically, or
alternatively, can be administered directly to the tumor e.g., by
intratumor injection or by injection into the tumor's primary blood
supply.
[0140] Therapeutic compositions containing at least one agent
disclosed herein can be conventionally administered in a unit dose.
The term "unit dose" when used in reference to a therapeutic
composition refers to physically discrete units suitable as unitary
dosage for the subject, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect in association with the required physiologically
acceptable diluent, i.e., carrier, or vehicle.
[0141] The compositions are administered in a manner compatible
with the dosage formulation, and in a therapeutically effective
amount. The quantity to be administered and timing depends on the
subject to be treated, capacity of the subject's system to utilize
the active ingredient, and degree of therapeutic effect desired. An
agent can be targeted by means of a targeting moiety, such as e.g.,
an antibody or targeted liposome technology.
[0142] Precise amounts of active ingredient required to be
administered depend on the judgment of the practitioner and are
particular to each individual. However, suitable dosage ranges for
systemic application are disclosed herein and depend on the route
of administration. Suitable regimes for administration are also
variable, but are typified by an initial administration followed by
repeated doses at one or more intervals by a subsequent injection
or other administration. Alternatively, continuous intravenous
infusion sufficient to maintain concentrations in the blood in the
ranges specified for in vivo therapies are contemplated.
[0143] An agent may be adapted for catheter-based delivery systems
including coated balloons, slow-release drug-eluting stents or
other drug-eluting formats, microencapsulated PEG liposomes, or
nanobeads for delivery using direct mechanical intervention with or
without adjunctive techniques such as ultrasound, together with an
active agent as described herein, dissolved or dispersed therein as
an active ingredient. In a preferred embodiment, the therapeutic
composition is not immunogenic when administered to a mammal or
human patient for therapeutic purposes. As used herein, the terms
"pharmaceutically acceptable", "physiologically tolerable" and
grammatical variations thereof, as they refer to compositions,
carriers, diluents and reagents, are used interchangeably and
represent that the materials are capable of administration to or
upon a mammal without the production of undesirable physiological
effects such as nausea, dizziness, gastric upset and the like. A
pharmaceutically acceptable carrier will not promote the raising of
an immune response to an agent with which it is admixed, unless so
desired. The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
understood in the art and need not be limited based on formulation.
Typically such compositions are prepared as injectable either as
liquid solutions or suspensions; however, solid forms suitable for
solution, or suspensions, in liquid prior to use can also be
prepared. The preparation can also be emulsified or presented as a
liposome composition. The active ingredient can be mixed with
excipients which are pharmaceutically acceptable and compatible
with the active ingredient and in amounts suitable for use in the
therapeutic methods described herein. Suitable excipients include,
for example, water, saline, dextrose, glycerol, ethanol or the like
and combinations thereof. In addition, if desired, the composition
can contain minor amounts of auxiliary substances such as wetting
or emulsifying agents, pH buffering agents and the like which
enhance the effectiveness of the active ingredient. The therapeutic
composition of the present invention can include pharmaceutically
acceptable salts of the components therein. Pharmaceutically
acceptable salts include the acid addition salts (formed with the
free amino groups of the polypeptide) that are formed with
inorganic acids such as, for example, hydrochloric or phosphoric
acids, or such organic acids as acetic, tartaric, mandelic and the
like. Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine and the like. Physiologically tolerable
carriers are well known in the art. Exemplary liquid carriers are
sterile aqueous solutions that contain no materials in addition to
the active ingredients and water, or contain a buffer such as
sodium phosphate at a physiological pH value, physiological saline
or both, such as phosphate-buffered saline. Still further, aqueous
carriers can contain more than one buffer salt, as well as salts
such as sodium and potassium chlorides, dextrose, polyethylene
glycol and other solutes. Liquid compositions can also contain
liquid phases in addition to and to the exclusion of water.
Exemplary of such additional liquid phases are glycerin, vegetable
oils such as cottonseed oil, and water-oil emulsions. The amount of
an active agent used in the methods described herein that will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques.
[0144] It is understood that the foregoing detailed description and
the following examples are illustrative only and are not to be
taken as limitations upon the scope of the invention. Various
changes and modifications to the disclosed embodiments, which will
be apparent to those of skill in the art, may be made without
departing from the spirit and scope of the present invention.
Further, all patents, patent applications, and publications
identified are expressly incorporated herein by reference for the
purpose of describing and disclosing, for example, the
methodologies described in such publications that might be used in
connection with the present invention. These publications are
provided solely for their disclosure prior to the filing date of
the present application. Nothing in this regard should be construed
as an admission that the inventors are not entitled to antedate
such disclosure by virtue of prior invention or for any other
reason. All statements as to the date or representation as to the
contents of these documents are based on the information available
to the applicants and do not constitute any admission as to the
correctness of the dates or contents of these documents.
EXAMPLES
[0145] The following examples illustrate rather than limit the
embodiments of the present invention.
Example 1
Studies on Mitochondrial Ca.sup.2+ Uptake by the Voltage-Dependent
Anion Channel 2 Regulates Cardiac Rhythmicity
[0146] A. Summary
[0147] Tightly regulated Ca.sup.2+ homeostasis is a prerequisite
for proper cardiac function. To dissect the regulatory network of
cardiac Ca.sup.2+ handling, we performed a chemical suppressor
screen on zebrafish tremblor embryos, which suffer from Ca.sup.2+
extrusion defects. Efsevin was identified based on its potent
activity to restore coordinated contractions in tremblor. We show
that efsevin binds to VDAC2, potentiates mitochondrial Ca.sup.2+
uptake and accelerates the transfer of Ca.sup.2+ from intracellular
stores into mitochondria. In cardiomyocytes, efsevin restricts the
temporal and spatial boundaries of Ca.sup.2+ sparks and thereby
inhibits Ca.sup.2+ overload-induced erratic Ca.sup.2+ waves and
irregular contractions. We further show that overexpression of
VDAC2 recapitulates the suppressive effect of efsevin on tremblor
embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect
and that VDAC2 functions synergistically with MCU to suppress
cardiac fibrillation in tremblor. Together, these findings
demonstrate a critical modulatory role for VDAC2-dependent
mitochondrial Ca.sup.2+ uptake in the regulation of cardiac
rhythmicity.
[0148] B. Introduction
[0149] During development, well-orchestrated cellular processes
guide cells from diverse lineages to integrate into the primitive
heart tube and establish rhythmic and coordinated contractions.
While many genes and pathways important for cardiac morphogenesis
have been identified, molecular mechanisms governing embryonic
cardiac rhythmicity are poorly understood. The findings that Ca2+
waves traveling across the heart soon after the formation of the
primitive heart tube (Chi et al., 2008, PLoS Biol 6, e109) and that
loss of function of key Ca2+ regulatory proteins, such as the
L-type Ca2+ channel, Na/K-ATPase and sodium-calcium exchanger 1
(NCX1), severely impairs normal cardiac function (Rottbauer et al.,
2001, Dev Cell 1, 265-275; Shu et al., 2003, Development 130,
6165-6173; Ebert et al., 2005, Proc Natl Acad Sci USA 102,
17705-17710; Langenbacher et al., 2005, Proc Natl Acad Sci USA 102,
17699-17704), indicate an essential role for Ca2+ handling in the
regulation of embryonic cardiac function.
[0150] Ca.sup.2+ homoeostasis in cardiac muscle cells is tightly
regulated at the temporal and spatial level by a subcellular
network involving multiple proteins, pathways, and organelles. The
release and reuptake of Ca.sup.2+ by the sarcoplasmic reticulum
(SR), the largest Ca.sup.2+ store in cardiomyocytes, constitutes
the primary mechanism governing the contraction and relaxation of
the heart. Ca.sup.2+ influx after activation of the L-type
Ca.sup.2+ channel in the plasma membrane induces the release of
Ca.sup.2+ from the SR via ryanodine receptor (RyR) channels, which
leads to an increase of the intracellular Ca.sup.2+ concentration
and cardiac contraction. During diastolic relaxation, Ca.sup.2+ is
transferred back into the SR by the SR Ca.sup.2+ pump or extruded
from the cell through NCX1. Defects in cardiac Ca.sup.2+ handling
and Ca.sup.2+ overload, for example during cardiac
ischemia/reperfusion or in long QT syndrome, are well known causes
of contractile dysfunction and many types of arrhythmias including
early and delayed after depolarizations and Torsade des pointes
(Bers, 2002, Nature 415, 198-205; Choi et al., 2002, J Physiol 543,
615-631; Yano et al., 2008, Circ J 72, A22-30; Greiser et al.,
2011, Cardiovasc Res 89, 722-733).
[0151] Ca.sup.2+ crosstalk between mitochondria and ER/SR has been
noted in many cell types and the voltage-dependent anion channel
(VDAC) and the mitochondrial Ca.sup.2+ uniporter (MCU) serve as
primary routes for Ca.sup.2+ entry through the outer and inner
mitochondrial membranes, respectively (Rapizzi et al., 2002, J Cell
Biol 159, 613-624; Bathori et al., J Biol Chem 2006, 281,
17347-17358; Shoshan-Barmatz et al., 2010, Mol Aspects Med 31,
227-285; Baughman et al., 2011, Nature 476, 341-345; De Stefani et
al., 2011, Nature 476, 336-340). In the heart, mitochondria are
tethered to the SR and are located in close proximity to Ca.sup.2+
release sites (Garcia-Perez et al., 2008, J Biol Chem 283,
32771-32780; Boncompagni et al., 2009, Mol Biol Cell 20, 1058-1067;
Hayashi et al., 2009, J Cell Sci 122, 1005-1013). This subcellular
architecture exposes the mitochondria near the Ca.sup.2+ release
sites to a high local Ca.sup.2+ concentration that is sufficient to
overcome the low Ca.sup.2+ affinity of MCU and facilitates
Ca.sup.2+ crosstalk between SR and mitochondria (Garcia-Perez et
al., 2008; Dorn et al., 2010, Circ Res 107, 689-699; Kohlhaas et
al., 2013, Cardiovasc Res 98, 259-268). Increase of the
mitochondrial Ca.sup.2+ concentration enhances energy production
during higher workload and dysregulation of SR-mitochondrial
Ca.sup.2+ signaling results in energetic deficits and oxidative
stress in the heart and may trigger programmed cell death (Brandes
et al., 1997, Circ Res 80, 82-87; Maack et al., 2006, Circ Res 99,
172-182; Kohlhaas et al., 2013). However, whether SR-mitochondrial
Ca.sup.2+ crosstalk also contributes significantly to cardiac
Ca.sup.2+ signaling during excitation-contraction coupling requires
further investigation.
[0152] In zebrafish, the tremblor (tre) locus encodes a
cardiac-specific isoform of the Na+/Ca2+ exchanger 1, NCX1h (also
known as slc8ala) (Ebert et al., 2005; Langenbacher et al., 2005).
The tre mutant hearts lack rhythmic Ca2+ transients and display
chaotic Ca2+ signals in the myocardium leading to unsynchronized
contractions resembling cardiac fibrillation (Langenbacher et al.,
2005). In this study, we used tre as an animal model for aberrant
Ca2+ handling-induced cardiac dysfunction and took a chemical
genetic approach to dissect the Ca2+ regulatory network important
for maintaining cardiac rhythmicity. A synthetic compound named
efsevin was identified from a suppressor screen due to its potent
ability to restore coordinated contractions in tre. Using
biochemical and genetic approaches we show that efsevin interacts
with VDAC2 and potentiates its mitochondrial Ca2+ transporting
activity and spatially and temporally modulates cytosolic Ca2+
signals in cardiomyocytes. The important role of mitochondrial Ca2+
uptake in regulating cardiac rhythmicity is further supported by
the suppressive effect of VDAC2 and MCU overexpression on cardiac
fibrillation in tre.
C. Materials and Methods
Zebrafish Husbandry and Transgenic Lines.
[0153] Zebrafish of the mutant line tremblor (tre.sup.tc318) were
maintained and bred as described previously (Langenbacher et al.,
2005). Transgenic lines, myl7:gCaMP4.1.sup.L42124 and
myl7:VDAC2.sup.L42309 were created using the Tol2kit (Esengil et
al., 2007, Nat Chem Biol 3, 154-155; Kwan et al., 2007, Dev Dyn
236, 3088-3099; Shindo et al., 2010, PLoS One 5, e8897). The
VDAC2.sup.LA2256 was created using the zinc finger array OZ523 and
OZ524 generated by the zebrafish Zinc Finger Consortium (Foley et
al., 2009a, Nature protocols 4, 1855-1867; Foley et al., 2009b,
PLoS One 4, e4348).
Molecular Biology.
[0154] Full length VDAC2 cDNA was purchased from Open Biosystems
and cloned into pCS2+ or pCS2+3XFLAG. Full length cDNA fragments of
zebrafish MCU (Accession number: JX424822) and MICU1 (JX42823) were
amplified from 2 dpf embryos and cloned into pCS2+. For mRNA
synthesis, plasmids were linearized and mRNA was synthesized using
the SP6 mMESSAGE mMachine kit according to the manufacturers manual
(Ambion).
Zebrafish Injections.
[0155] VDAC2 mRNA and morpholino antisense oligos
(5'-GGGAACGGCCATTTTATCTGTTAAA-3') (Genetools) (SEQ ID NO: 9) were
injected into one-cell stage embryos collected from crosses of
tre.sup.tc318 heterozygotes. Cardiac performance was analyzed by
visual inspection on 1 dpf. The tre mutant embryos were identified
either by observing the fibrillation phenotype at 2-3 dpf or by
genotyping as previously described (Langenbacher et al., 2005).
Chemical Screen.
[0156] Chemicals from a synthetic library (Castellano et al., 2007,
J Am Chem Soc 129, 5843-5845; Choi et al., 2011, Development 138,
1173-1181; Cruz et al., 2011, Proc Natl Acad Sci USA 108,
6769-6774) and from Biomol International LP were screened for their
ability to partially or completely restore persistent heartbeat in
tre embryos. 12 embryos collected from crosses of tre.sup.tc318
heterozygotes were raised in the presence of individual compounds
at a concentration of 10 .mu.M from 4 hpf (Choi et al., 2011).
Cardiac function was analyzed by visual inspection at 1 and 2 dpf.
The hearts of tre.sup.tc318 embryos manifest a chaotic movement
resembling cardiac fibrillation with intermittent contractions in
rare occasion (Ebert et al., 2005; Langenbacher et al., 2005).
Compounds that elicit persistent coordinated cardiac contractions
were validated on large number of tre mutant embryos and NCX1h
morphants (>500 embryos).
Zebrafish Cardiac Imaging.
[0157] Movies of GFP-labelled myl7:GFP hearts were taken at 30
frames per second. Line-scan analysis was performed along a line
through the atria or the ventricles of these hearts (Nguyen et al.,
2009, Drug Discovery Today: Disease Models 5). Fraction of
shortening was deduced from the ratio of diastolic and systolic
width and heart rate was determined by beats per minute. Cardiac
parameters were analyzed in tremblor.sup.tc318 and VDAC2.sup.LA2256
at 2 dpf.
Zebrafish Optical Mapping.
[0158] 36 hpf myl7:gCaMP4.1 embryos were imaged at a frame rate of
30 ms/frame. Electromechanical isolation was achieved by tnnt2MO
(Milan et al., 2006, Development 133, 1125-1132). The fluorescence
intensity of each pixel in a 2D map was normalize to generate heat
maps and isochronal lines at 33 ms intervals were obtained by
identifying the maximal spatial gradient for a given time point
(Chi et al., 2008).
Mouse and Human Embryonic Stem Cells.
[0159] The mouse E14Tg2a ESC and human H9 ESC line were cultured
and differentiated as previously described (Blin et al., 2010, J
Clin Invest 120, 1125-1139; Arshi et al., 2013, Sci Technol Adv
Mater 2013, 025003). At day 10 of differentiation, beating mouse
EBs were exposed to external solution containing 10 mM CaCl.sub.2
for 10 minutes before DMSO or efsevin (10 .mu.M) treatment. Human
EBs were differentiated for 15 days and treated with 5 mM
CaCl.sub.2 for 10 minutes before DMSO or efsevin (5 .mu.M)
treatment. Images of beating EBs were acquired at a rate of 30
frames/sec and analyzed by motion-detection software. For calcium
recording, the EBs were loaded with 10 .mu.M fluo-4 AM in culture
media for 30 minutes at 37.degree. C. Line-scan analysis was
performed and fluorescent signals were acquired by a Zeiss LSM510
confocal microscope.
Microelectrode Array Measurements.
[0160] Two-day-old wild type, tre, and efsevin-treated tre embryos
were placed on uncoated, microelectrode arrays (MEAs) containing
120 integrated TiN electrodes (30 .mu.m diameter, 200 .mu.m
interelectrode spacing). Local field potentials (LFPs) at each
electrode were collected for three trials per embryo type over a
period of three minutes at a sampling rate of 1 kHz using the
MEA2100-HS120 system (Multichannel Systems, Reutiligen, Germany).
Raw data was low-pass filtered at a cutoff frequency of 10 Hz using
a third-order Butterworth filter. Data analysis was carried out
using the MC DataTool (Multichannel Systems) and Matlab
(MathWorks).
Ca.sup.2+ Imaging
[0161] Murine ventricular cardiomyocytes were isolated as
previously described (Reuter et al., 2004, J Physiol 554, 779-789).
Cells were loaded with 5 .mu.M fluo-4 AM in external solution
containing: 138.2 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl, 15 mM glucose,
20 mM HEPES for 1 hr and imaged in external solution supplemented
with 2, 5 or 10 mM CaCl.sub.2. For the recording of Ca.sup.2+
sparks and transients, the external solution contained 2 mM
CaCl.sub.2. For Ca.sup.2+ transients, cells were field stimulated
at 0.5 Hz with a 5 ms pulse at a voltage of 20% above contraction
threshold. For all measurements, efsevin was added 2 hours prior to
the actual experiment. Images were recorded on a Zeiss LSM 5 Pascal
confocal microscope. Data analysis was carried out using the Zeiss
LSM Image Browser and ImageJ with the SparkMaster plugin (Picht et
al., 2007, Am J Physiol Cell Physiol 293, C1073-1081). Cells were
visually inspected prior to and after each recording. Only those
recordings from healthy looking cells with distinct borders,
uniform striations and no membrane blebs or granularity were
included in the analysis.
Biochemistry.
[0162] For pull down assays mono-N-Boc protected
2,2'-(ethylenedioxy)bis(ethylamine) was attached to the carboxylic
ester of efsevin and its derivatives through the amide bond. After
removal of the Boc group using TFA, the primary amine was coupled
to the carboxylic acid of Affi-Gel 10 Gel (Biorad). Two-day-old
zebrafish embryos were deyolked by centrifugation before being
lysed with Rubinfeld's lysis buffer (Rubinfeld et al., 1993,
Science 262, 1731-1734). The lysate was precleaned by incubation
with Affi-Gel 10 Gel to eliminate non-specific binding. Precleaned
lysate was incubated with affinity beads overnight. Proteins were
eluted from the affinity beads and separated on SDS-PAGE. Protein
bands of interest were excised. Gel plugs were dehydrated in
acetonitrile (ACN) and dried completely in a Speedvac. Samples were
reduced and alkylated with 10 mM dithiotreitol and 10 mM TCEP
solution in 50 mM NH.sub.4HCO.sub.3 (30 min at 56.degree. C.) and
100 mM iodoacetamide (45 min in dark), respectively. Gel plugs were
washed with 50 mM NH.sub.4HCO.sub.3, dehydrated with ACN, and dried
down in a Speedvac. Gel pieces were then swollen in digestion
buffer containing 50 mM NH.sub.4HCO.sub.3, and 20.0 ng/.mu.L of
chymotrypsin (25.degree. C., overnight). Peptides were extracted
with 0.1% TFA in 50% ACN solution, dried down and resuspended in LC
buffer A (0.1% formic acid, 2% ACN).
Mass Spectrometry Analyses and Database Searching.
[0163] Extracted peptides were analyzed by nano-flow LC/MS/MS on a
Thermo Orbitrap with dedicated Eksigent nanopump using a reversed
phase column (New Objective). The flow rate was 200 nL/min for
separation: mobile phase A contained 0.1% formic acid, 2% ACN in
water, and mobile phase B contained 0.1% formic acid, 20% water in
ACN. The gradient used for analyses was linear from 5% B to 50% B
over 60 min, then to 95% B over 15 min, and finally keeping
constant 95% B for 10 min. Spectra were acquired in data-dependent
mode with dynamic exclusion where the instrument selects the top
six most abundant ions in the parent spectra for fragmentation.
Data were searched against the Danio rerio IPI database v3.45 using
the SEQUEST algorithm in the BioWorks software program version
3.3.1 SP1. All spectra used for identification had deltaCN>0.1
and met the following Xcorr criteria: >2 (+1), >3 (+2), >4
(+3), and >5 (+4). Searches required full cleavage with the
enzyme, <4 missed cleavages and were performed with the
differential modifications of carbamidomethylation on cysteine and
methionine oxidation.
In Situ Hybridization.
[0164] In situ hybridization was performed as previously described
(Chen et al., 1996, Development 122, 3809-3816). DIG-labeled RNA
probe was synthesized using the DIG RNA labeling kit (Roche).
Immunostaining.
[0165] HeLa cells were transfected with a C-terminally flag-tagged
zebrafish VDAC1 or VDAC2 in plasmid pCS2+ using Lipofectamine.TM.
2000 (Invitrogen). After staining with Mit.RTM. Tracker.RTM. Orange
(Invitrogen) cells were fixed in 3.7% formaldehyde and
permeabilized with acetone. Immunostaining was performed using
primary antibody ANTI-FLAG.RTM. M2 (Sigma Aldrich) at 1:100 and
secondary antibody Anti-Mouse IgG1-FITC (Southern Biotechnology
Associates) at 1:200. Cells were mounted and counterstained using
Vectashield.RTM. Hard Set.TM. with DAPI (Vector Laboratories).
Mitochondria Ca.sup.2+ Uptake Assay in HeLa Cells.
[0166] HeLa cells were transfected with zebrafish VDAC2 using
Lipofectamine.TM. 2000 (Invitrogen). 36 hrs after transfection,
cells were loaded with 5 .mu.M Rhod2-AM (Invitrogen), a Ca.sup.2+
indicator preferentially localized in mitochondria, for 1 hour at
15.degree. C. followed by a 30 min de-esterification period at
37.degree. C. Subsequently, cells were permeabilized with 100 .mu.M
digitonin for 1 min at room temperature. Fluorescence changes in
Rhod2 (ex: 544 nm, em: 590 nm) immediately after the addition of
Ca.sup.2+ (final free Ca.sup.2+ concentration is calculated to be
approximately 10 .mu.M using WEBMAXC at
http://web.stanford.edu/.about.cpatton/webmaxcS.htm) were monitored
in internal buffer (5 mM K-EGTA, 20 mM HEPES, 100 mM K-aspartate,
40 mM KCl, 1 mM MgCl.sub.2, 2 mM maleic acid, 2 mM glutamic acid, 5
mM pyruvic acid, 0.5 mM KH.sub.2PO.sub.4, 5 mM MgATP, pH adjusted
to 7.2 with Trizma base) using a FLUOSTAR plate reader (BMG
Labtech).
Mitochondria Ca.sup.2+ Uptake Assay in VDAC1/VDAC3 Double Knockout
(V1/V3 DKO) MEFs.
[0167] V1/V3 DKO MEFs were cultured as previously described (Roy et
al., 2009a, EMBO Rep 10, 1341-1347). Efsevin-treated (15 .mu.M for
30 min) or mock-treated MEFs were used for measurements of
[Ca.sup.2+].sub.c in suspensions of permeabilized cells or imaging
of [Ca.sup.2+].sub.m simultaneously with [Ca.sup.2+].sub.c in
intact single cells. Permeabilization of the plasma membrane was
performed by digitonin (40 .mu.Mimi). Changes in [Ca.sup.2+] in the
cytoplasmic buffer upon IP.sub.3 (7.5 .mu.M) addition in the
presence or absence of ruthenium red (3 .mu.M) was measured by
fura2 in a fluorometer (Csordas et al., 2006, J Cell Biol 174,
915-921; Roy et al., 2009b, Mol Cell 33, 377-388). To avoid
endoplasmic reticulum Ca.sup.2+ uptake 2 .mu.M thapsigargin was
added before IP.sub.3. For imaging of [Ca.sup.2+].sub.m and
[Ca.sup.2+].sub.c, MEFs were co-transfected with plasmids encoding
polycistronic zebrafish VDAC2 with mCherry and
mitochondria-targeted inverse pericam for 40 hours. Cells were
sorted to enrich the transfected cells and attached to glass
coverslips. In the final 10 min, of the efsevin or mock-treatment,
the cells were also loaded with fura2AM (2.5.mu..quadrature.) and
subsequently transferred to the microscope stage. Stimulation with
1 .mu.M ATP was carried out in a norminally Ca.sup.2+ free buffer.
Changes in [Ca.sup.2+].sub.c and [Ca.sup.2+].sub.m were imaged
using fura2 (ratio of ex:340 nm to 380 nm) and
mitochondria-targeted inverse pericam (ex: 495 nm), respectively
(Csordas et al., 2010, Mol Cell 39, 121-132).
Statistics.
[0168] All values are expressed as mean.+-.SEM. Significance values
are calculated by unpaired student's t-test unless noted
otherwise.
Test data not shown include the following information: [0169] 1) a
heart of a wild-type zebrafish embryo at 2 dpf. Robust rhythmic
contractions can be observed in atrium and ventricle. [0170] 2) a
heart of a tremblor embryo at 2 dpf. Embryos of the mutant line
tremblor display only local, unsynchronized contractions,
comparable to cardiac fibrillation. [0171] 3) a heart of a tremblor
embryo at 2 dpf treated with efsevin. Treatment of tremblor embryos
with efsevin restores rhythmic contractions with comparable atrial
fractional shortening compared to wild-type embryos and
approximately 40% of wild-type heart rate. [0172] 4) a heart of a
wild-type zebrafish embryo at 2 dpf treated with efsevin. Treatment
of wild-type embryos with efsevin did not affect cardiac
performance, indicated by robust, rhythmic contractions comparable
to untreated wild-type embryos. [0173] 5) heat map of Ca.sup.2+
transients recorded in one day old wild type heart. [0174] 6) heat
map of Ca.sup.2+ transients recorded in one day old tremblor heart.
[0175] 7) heat map of Ca.sup.2+ transients recorded in one day old
efsevin treated tremblor heart. [0176] 8) a heart of a wild-type
zebrafish embryo at 1 dpf. Robust rhythmic contractions can be
observed in atrium and ventricle. [0177] 9) a heart of a wild-type
zebrafish embryo injected with zebrafish VDAC2 mRNA at 1 dpf.
Robust rhythmic contractions can be observed in atrium and
ventricle. [0178] 10) a heart of a tremblor embryo at 1 dpf.
Tremblor embryos display only local, unsynchronized contractions,
comparable to cardiac fibrillation. [0179] 11) a heart of a
tremblor embryo injected with zebrafish VDAC2 mRNA at 1 dpf.
Overexpression of zebrafish VDAC2 mRNA restores rhythmic
contractions in tremblor embryos. [0180] 12) a heart of a 2 dpf
Tg-VDAC2 embryo injected with a morpholino targeting NCX1h.
Morpholino knock-down of NCX1h results in a fibrillating heart.
[0181] 13) a heart of a 2 dpf NCX1h morphant in the Tg-VDAC2
genetic background. TBF treatment induces VDAC2 expression and
restores coordinated cardiac contractions. [0182] 14) a heart of a
2 dpf wild type zebrafish embryo injected with a morpholino
targeting VDAC2. Morpholino knockdown of VDAC2 did not have obvious
effects on cardiac performance. [0183] 15) a heart of a 2 dpf
tremblor mutant embryo injected with a morpholino targeting VDAC2.
[0184] 16) a heart of a 2 dpf tremblor mutant embryo injected with
a morpholino targeting VDAC2. Efsevin treatment cannot restore
coordinated cardiac contractions in the absence of VDAC2.
D. Results and Discussion
Identification of a Chemical Suppressor of Tre Cardiac
Dysfunction
[0185] Homozygous tre mutant embryos suffer from Ca2+ extrusion
defects and manifest chaotic cardiac contractions resembling
fibrillation (Ebert et al., 2005; Langenbacher et al., 2005). To
dissect the regulatory network of Ca2+ handling in cardiomyocytes
and to identify mechanisms controlling embryonic cardiac
rhythmicity, we screened the BioMol library and a collection of
synthetic compounds for chemicals that are capable of restoring
heartbeat either completely or partially in tre embryos. A
dihydropyrrole carboxylic ester compound named efsevin was
identified based on its ability to restore persistent and rhythmic
cardiac contractions in tre mutant embryos in a dose-dependent
manner (FIG. 1D). To validate the effect of efsevin, we assessed
cardiac performance of wild type, tre and efsevin-treated tre
embryos (Nguyen et al., 2009). Line scans across the atria of
Tg(myl7:GFP) embryonic hearts at 48 hpf showed rhythmically
alternating systoles and diastoles from vehicle- or efsevin-treated
wild type and efsevin-treated tre embryos, while only sporadic
unsynchronized contractions were seen from vehicle-treated tre
embryos. Fractional shortening of efsevin treated tre mutant hearts
was comparable to that of their wild type siblings and heart rate
was restored to approximately 40% of that observed in controls
(FIG. 1A-C). Periodic local field potentials accompanying each
heartbeat were detected in wild type and efsevin-treated tre
embryos using a microelectrode array (FIG. 1E-G). Furthermore,
while only sporadic Ca.sup.2+ signals were detected in tre hearts,
in vivo Ca.sup.2+ imaging revealed steady Ca.sup.2+ waves
propagating through efsevin-treated tre hearts, demonstrating that
cardiomyocytes are functionally coupled and that efsevin treatment
restores regular Ca.sup.2+ transients in tre hearts.
Efsevin Suppresses Ca.sup.2+ Overload-Induced Irregular
Contraction
[0186] We next examined whether efsevin could suppress aberrant
Ca2+ homeostasis-induced arrhythmic responses in mammalian
cardiomyocytes. Mouse embryonic stem cell-derived cardiomyocytes
(mESC-CMs) establish a regular contraction pattern with rhythmic
Ca2+ transients (FIG. 2A, D, E). Mimicking Ca2+ overload by
increasing extracellular Ca2+ levels was sufficient to disrupt
normal Ca2+ cycling and induce irregular contractions in mESC-CMs
(FIG. 2B, D, E). Remarkably, efsevin treatment restored rhythmic
Ca2+ transients and cardiac contractions in these cells (FIG.
2C-E). Similar effect was observed in human embryonic stem
cell-derived cardiomyocytes (hESC-CMs) (FIG. 2F). Together, these
findings suggest that efsevin targets a conserved Ca2+ regulatory
mechanism critical for maintaining rhythmic cardiac contraction in
fish, mice and humans.
VDAC2 Mediates the Suppressive Effect of Efsevin on Tre
[0187] To identify the protein target of efsevin, we generated a
N-Boc-protected 2-aminoethoxyethoxyethylamine linker-attached
efsevine (efsevine.sup.L) (FIGS. 3A and C). This modified compound
retained the activity of efsevin to restore cardiac contractions in
ncxlh deficient embryos (FIGS. 3B and D) and was used to create
efsevin-conjugated agarose beads (efsevin.sup.LB). A 32 kD protein
species was detected from zebrafish lysate due to its binding
ability to efsevin.sup.LB and OK-C125.sup.LB, an active efsevin
derivative conjugated to beads, but not to beads capped with
ethanolamine alone or beads conjugated with an inactive efsevin
analog (OK-C19.sup.LB) (FIG. 3A-D). Furthermore, preincubation of
zebrafish lysate with excess efsevin prevented the 32 kD protein
from binding to efsevin.sup.LB or OK-C125.sup.LB. Mass spectrometry
analysis revealed that this 32 kD band represents a zebrafish
homologue of the mitochondrial voltage-dependent anion channel 2
(VDAC2) (FIG. 3E) by identification of VDAC2 peptide LTFDTTFSPNTGK
by b- and y-series ions analysis (SEQ ID NO: 8).
[0188] VDAC2 is expressed in the developing zebrafish heart, as
confirmed by in situ hybridization analysis of embryonic hearts at
36 hpf and 48 hpf, making it a good candidate for mediating
efsevin's effect on cardiac Ca.sup.2+ handling. To examine this
possibility, we injected in vitro synthesized VDAC2 RNA into tre
embryos and found that the majority of these embryos had
coordinated cardiac contractions similar to those subjected to
efsevin treatment (FIG. 4A). In addition, we generated myl7: VDAC2
transgenic fish in which VDAC2 expression can be induced in the
heart by tebufenozide (TBF) (FIG. 4B). In situ hybridization
analysis showed that TBF treatment induces VDAC2 expression in the
heart. Knocking down NCX1h in myl7:VDAC2 embryos results in chaotic
cardiac movement similar to tre. Like efsevin treatment, induction
of VDAC2 expression by TBF treatment restored coordinated and
rhythmic contractions in myl7:VDAC2; NCX1h MO hearts (FIG. 4C).
Conversely, knocking down VDAC2 in tre hearts attenuated the
suppressive effect of efsevin (FIG. 4D). Furthermore, we generated
VDAC2 null embryos by the Zinc Finger Nuclease gene targeting
approach (FIG. 4F). In situ hybridization analysis showed loss of
VDAC2 transcripts in VDAC2.sup.zfn/zfn embryos (SEQ ID NO: 2-7).
Similar to that observed in morpholino knockdown embryos,
homozygous VDAC2.sup.LA2256 embryos do not exhibit noticeable
morphological defects, but the suppressive effect of efsevin was
attenuated in homozygous VDAC2.sup.LA2256; NCX1MO embryos (FIG.
4E). These findings demonstrate that VDAC2 is a major mediator for
efsevin's effect on ncxlh deficient hearts.
VDAC2-Dependent Effect of Efsevin on Mitochondrial Ca.sup.2+
Uptake
[0189] VDAC is an abundant channel located on the outer
mitochondrial membrane serving as a primary passageway for
metabolites and ions (Rapizzi et al., 2002; Bathori et al., 2006;
Shoshan-Barmatz et al., 2010). At its close state, VDAC favours
Ca.sup.2+ flux (Tan et al., 2007, Biochim Biophys Acta 1768,
2510-2515). To examine whether efsevin would modulate mitochondrial
Ca.sup.2+ uptake via VDAC2, we transfected HeLa cells with VDAC2.
HeLa cells transfected with a flag-tagged zebrafish VDAC2
(VDAC2.sup.flag) were immunostained against the flag epitope and
counterstained for mitochondria with MitoTracker Orange and for
nuclei with DAPI to confirm transfection. We noted increased
mitochondrial Ca.sup.2+ uptake in permeabilized VDAC2 transfected
and efsevin-treated cells after the addition of Ca.sup.2+ and the
combined treatment further enhanced mitochondrial Ca.sup.2+ levels
(FIG. 5A).
[0190] Mitochondria are located in close proximity to Ca.sup.2+
release sites of the ER/SR and an extensive crosstalk between the
two organelles exists (Garcia-Perez et al., 2008; Hayashi et al.,
2009; Brown et al., 2010, Cardiovasc Res 88, 241-249; Dorn et al.,
2010; Kohlhaas et al., 2013). We examined whether Ca.sup.2+
released from intracellular stores could be locally transported
into mitochondria through VDAC2 in VDAC1/VDAC3 double knockout
(V1/V3DKO) MEFs where VDAC2 is the only VDAC isoform being
expressed (Roy et al., 2009a). While treatments with ATP, an
IP3-linked agonist, and Thapsigargin, a SERCA inhibitor, stimulated
similar global cytoplasmic [Ca.sup.2+] elevation in intact cells,
only ATP induced a rapid mitochondrial matrix [Ca.sup.2+] rise
(FIG. 8). This finding is consistent with observations obtained in
other cell types (Rizzuto et al., 1994, J Cell Biol 126, 1183-1194;
Hajnoczky et al., 1995, Cell 82, 415-424) and suggests that
Ca.sup.2+ was locally transferred from IP3 receptors to
mitochondria through VDAC2 at the close ER-mitochondrial
associations. We next investigated whether this process could be
modulated by efsevin. In permeabilized V1/V3DKO MEFs, treatment
with efsevin increased the amount of Ca.sup.2+ transferred into
mitochondria during IP.sub.3-induced Ca.sup.2+ release (FIG. 5B).
Also, in intact V1/V3 DKO MEFs, efsevin accelerated the transfer of
Ca.sup.2+ released from intracellular stores into mitochondria
during stimulation with ATP (FIGS. 5C and D).
Efsevin Modulates Ca.sup.2+ Sparks and Suppresses Erratic Ca.sup.2+
Waves in Cardiomyocytes
[0191] We next examined the effect of efsevin on cytosolic
Ca.sup.2+ signals in isolated adult murine cardiomyocytes. We found
that efsevin treatment induced faster inactivation kinetics without
affecting the amplitude or time to peak of paced Ca.sup.2+
transients (FIG. 6A). Similarly, efsevin treatment did not
significantly alter the frequency, amplitude or Ca.sup.2+ release
flux of spontaneous Ca.sup.2+ sparks, local Ca.sup.2+ release
events, but accelerated the decay phase resulting in sparks with a
shorter duration and a narrower width (FIG. 6B). These results
indicate that by activating mitochondrial Ca.sup.2+ uptake, efsevin
accelerates Ca.sup.2+ removal from the cytosol in cardiomyocytes
and thereby restricts local cytosolic Ca.sup.2+ sparks to a
narrower domain for a shorter period of time without affecting SR
Ca.sup.2+ load or RyR Ca.sup.2+ release. Under conditions of
Ca.sup.2+ overload, single Ca.sup.2+ sparks can trigger opening of
neighbouring Ca.sup.2+ release units and thus induce the formation
of erratic Ca.sup.2+ waves. Efsevin treatment significantly reduced
the number of propagating Ca2+ waves in a dosage-dependent manner
(FIG. 6C), demonstrating a potent suppressive effect of efsevin on
the propagation of Ca2+ overload-induced Ca2+ waves and suggesting
that efsevin could serve as a pharmacological tool to manipulate
local Ca2+ signals.
Mitochondrial Ca2+ Uptake Modulates Embryonic Cardiac
Rhythmicity
[0192] We believe that efsevin treatment/VDAC2 overexpression
suppresses aberrant Ca.sup.2+ handling-associated arrhythmic
cardiac contractions by buffering excess Ca.sup.2+ into
mitochondria. Therefore we predict that activating other
mitochondrial Ca.sup.2+ uptake molecules would likewise restore
coordinated contractions in tre. To test this model, we cloned
zebrafish MCU and MICU1, an inner mitochondrial membrane Ca.sup.2+
transporter and its regulator (Perocchi et al., 2010, Nature 467,
291-296; Baughman et al., 2011; De Stefani et al., 2011;
Mallilankaraman et al., 2012, Cell 151, 630-644; Csordas et al.,
2013, Cell Metab 17, 976-987). In situ hybridization showed that
MCU and MICU1 were expressed in the developing zebrafish heart and
their expression levels were comparable between the wild type and
tre hearts and embryos with and without efsevin treatment.
Overexpression of MCU restored coordinated contractions in tre,
akin to what was observed with VDAC2 (FIG. 7A). In addition, tre
embryos injected with suboptimal concentrations of MCU or VDAC2 had
a fibrillating heart, but embryos receiving both VDAC2 and MCU at
the suboptimal concentration manifested coordinated contractions
(FIG. 7B), demonstrating a synergistic effect of these proteins.
Furthermore, overexpression of MCU failed to suppress the tre
phenotype in the absence of VDAC2 activity and VDAC2 could not
restore coordinated contractions in tre without functional MCU
(FIG. 7A,C). Similar results were observed by manipulating MICU1
activity (FIGS. 7D and E). Together, these findings indicate that
mitochondrial Ca.sup.2+ uptake mechanisms on outer and inner
mitochondrial membranes act cooperatively to regulate cardiac
rhythmicity.
Mitochondria Regulate Cardiac Rhythmicity Through a VDAC Dependent
Mechanism
[0193] Affinity agarose beads covalently linked with efsevin
(efsevin.sup.LB) pulled down 2 protein species from zebrafish
embryonic lysate, whereof one, the 32 kD upper band, was sensitive
to competition with a 100 fold excess free efsevin.sup.L. The 32 kD
band was not detected in proteins eluted from beads capped with
ethanolamine alone (beads.sup.C) or beads linked to an inactive
derivative of efsevin, OK-C19.sup.LB, but was detected in samples
eluted from beads attached with a biologically active derivative,
OK-C125.sup.LB. Also for the OK-C125.sup.LB pull-down the 32 kD
band was again sensitive to competition with free efsevin. In situ
hybridization analysis showed that VDAC1, VDAC2 and MCU are
expressed in embryonic hearts at 36 hpf and 48 hpf.
[0194] FIG. 9 A)-G) show that mitochondria regulate cardiac
rhythmicity through a VDAC dependent mechanism.
[0195] A) Injection of 25 pg in-vitro synthesized VDAC1 and VDAC2
mRNA restored cardiac contractions in 53.0.+-.10.2% (n=126) and
52.9.+-.12.1% (n=78) of one-day-old tre embryos, respectively,
compared to 21.8.+-.5.1% in uninjected siblings (n=111).
[0196] B) While only .about.20% of myl7:VDAC2; NCX1hMO embryos have
coordinated contractions (n=116), 52.3.+-.2.4% of these embryos
established persistent, rhythmic contractions after TBF induction
of VDAC2 (n=154).
[0197] C) On average, 71.2.+-.8.8% efsevin treated embryos have
coordinated cardiac contractions (n=131). Morpholino antisense
oligonucleotide knockdown of VDAC2 (MO.sup.VDAC2) or VDAC1
(MO.sup.VDAC1) attenuates the ability of efsevin to suppress
cardiac fibrillation in tre embryos (45.3.+-.7.4% and 46.9.+-.10.7%
embryos with coordinated contractions, n=94 and 114, respectively).
Knocking down VDAC1/2 simultaneously further suppresses efsevin's
effect (30.3.+-.6.3%, n=75).
[0198] D) Efsevin treatment restores coordinated cardiac
contractions in 76.2.+-.8.7% NCX1MO embryos, only 54.1.+-.3.6%
VDAC2.sup.zfn/zfn; NCX1MO embryos and 35.7.+-.7.1%
VDAC2.sup.zfn/zfn; VDAC1MO; NCX1MO embryos have coordinated
contractions (n=250).
[0199] E) Overexpression of MCU is sufficient to restore
coordinated cardiac contractions in tre embryos (47.1.+-.1.6%
embryos, n=112 as opposed to 18.3.+-.5.3% of uninjected siblings,
n=64) while this effect is significantly attenuated when
co-injected with morpholino antisense oligonucleotide targeted to
VDAC2 (27.1.+-.1.9% embryos, n=135).
[0200] F) Suboptimal overexpression of MCU (MCU.sup.S) and VDAC2
(VDAC2.sup.S) in combination is able to suppress cardiac
fibrillation in tre embryos (42.9.+-.2.6% embryos, n=129).
[0201] G) The ability of VDAC2 to restore rhythmic contractions in
tre embryos (48.5.+-.3.5% embryos, n=111) is significantly
attenuated when MCU is knocked down by antisense oligonucleotide
(MO.sup.MCU) (25.6.+-.2.4% embryos, n=115). Error bars represent
s.d.; *p<0.05; ***p<0.001.
E. Conclusion
[0202] In summary, we conducted a chemical suppressor screen in
zebrafish to dissect the regulatory network critical for
maintaining rhythmic cardiac contractions and to identify
mechanisms underlying aberrant Ca.sup.2+ handling-induced cardiac
dysfunction. We show that activation of VDAC2 through
overexpression or efsevin treatment potently restores rhythmic
contractions in NCX1h deficient zebrafish hearts and effectively
suppresses Ca.sup.2+ overload-induced arrhythmogenic Ca.sup.2+
events and irregular contractions in mouse and human
cardiomyocytes. We provide evidence that potentiating VDAC2
activity enhances mitochondrial Ca.sup.2+ uptake, accelerates
Ca.sup.2+ transfer from intracellular stores into mitochondria and
spatially and temporally restricts single Ca.sup.2+ sparks in
cardiomyocytes. The crucial role of mitochondria in the regulation
of cardiac rhythmicity is further supported by the findings that
VDAC2 functions in concert with MCU; these genes have a strong
synergistic effect on suppressing cardiac fibrillation and loss of
function of either gene abrogates the rescue effect of the other in
Ire.
[0203] The regulatory roles of mitochondrial Ca.sup.2+ in cardiac
metabolism, cell survival and fate have been studied extensively
(Brown et al., 2010; Dorn et al., 2010; Doenst et al., 2013, Circ
Res 113, 709-724; Kasahara et al., 2013, Science 342, 734-737;
Kohlhaas et al., 2013; Luo et al., 2013, Circ Res 113, 690-708).
Our study provides genetic and physiologic evidence supporting an
additional role for mitochondria in regulating cardiac rhythmicity
and reveals VDAC2 as a modulator of Ca.sup.2+ handling in
cardiomyocytes. Our findings, together with recent reports of the
physical interaction between VDAC2 and RyR2 (Min et al., 2012,
Biochem J 447, 371-379) and the close proximity of outer and inner
mitochondrial membranes at the contact sites between the
mitochondria and the SR (Garcia-Perez et al., 2011, Am J Physiol
Heart Circ Physiol 301, H1907-1915), suggest an intriguing model.
We propose that mitochondria facilitate an efficient clearance
mechanism in the Ca.sup.2+ microdomain, which modulates Ca.sup.2+
handling without affecting global Ca.sup.2+ signals in
cardiomyocytes. In this model, VDAC facilitates mitochondrial
Ca.sup.2+ uptake via MCU complex and thereby controls the duration
and the diffusion of cytosolic Ca.sup.2+ near the Ca.sup.2+ release
sites to ensure rhythmic cardiac contractions. This model is
consistent with our observation that efsevin treatment induces
faster inactivation kinetics of cytosolic Ca.sup.2+ transients
without affecting the amplitude or the time to peak in
cardiomyocytes and the reports that blocking mitochondrial
Ca.sup.2+ uptake has little impact on cytosolic Ca.sup.2+
transients (Maack et al., 2006; Kohlhaas et al., 2010, Circulation
121, 1606-1613). Further support for this model comes from the
observation of the Ca.sup.2+ peaks on the OMM (Drago et al., 2012,
Proc Natl Acad Sci USA 109, 12986-12991) and the finding that
downregulating VDAC2 extends Ca.sup.2+ sparks (Subedi et al., 2011,
Cell Calcium 49, 136-143; Min et al., 2012) and that blocking
mitochondrial Ca.sup.2+ uptake by Ru360 leads to an increased
number of spontaneous propagating Ca.sup.2+ waves (Seguchi et al.,
2005, Cell Calcium 38, 1-9). Future studies on the kinetics of
VDAC2-dependent mitochondrial Ca.sup.2+ uptake and exploring
potential regulatory molecules for VDAC2 activity will provide
insights into how the crosstalk between SR and mitochondria
contributes to Ca.sup.2+ handling and cardiac rhythmicity.
[0204] Aberrant Ca.sup.2+ handling is associated with many cardiac
dysfunctions including arrhythmia. Establishing animal models to
study molecular mechanisms and develop new therapeutic strategies
are therefore major preclinical needs. Our chemical suppressor
screen identified a potent effect of efsevin and its biological
target VDAC2 on manipulating cardiac Ca.sup.2+ handling and
restoring regular cardiac contractions in fish and mouse and human
cardiomyocytes. This success indicates that fundamental mechanisms
regulating cardiac function are conserved among vertebrates despite
the existence of species-specific features and suggests a new
paradigm of using zebrafish cardiac disease models for the
dissection of critical genetic pathways and the discovery of new
therapeutic approaches. Future studies examining the effects of
efsevin on other arrhythmia models would further elucidate the
potential for efsevin as a pharmacological tool to treat cardiac
arrhythmia associated with aberrant Ca.sup.2+ handling.
Examples 2-4
Synthesis of Efsevin: Synthesis of (R)-Efsevin and (S)-Efsevin
& Identification of (R)-Efsevin as the Active Antipode for the
Previously Reported Defibrillator Activity of Efsevin
Example 2
(Resolution of (R)- and (S)-Efsevin Through HPLC Separation on
Chiral Stationary Phase)
##STR00025##
[0206] Racemic-efsevin (50 mg, 0.13 mmol) was dissolved in DCM (0.2
mL) and injected into a Shimadzu CBM Lite system using a REGIS (R,
R)-DACH DNB 5/100 preparatory column (25 cm.times.30 mm) with
DCM/hexanes (70:30) as eluent at a flow rate of 10.0 mL/min.
(R)-efsevin eluted at 75.78 min and (S)-efsevin at 102.72 min
(FIGS. 10-13). Fractions were collected and concentrated in vacuo.
The protocol was repeated six more times to give (R)-efsevin (158
mg) which was recrystallized with hexanes/EtOAc to give (R)-efsevin
crystals (112 mg, >99% ee).
[0207] Enantiomeric excess were determined by a REGIS (R, R)-DACH
DNB 5/100 analytical column (25 cm.times.4.6 mm) with DCM/hexanes
(60:40) as eluent at a flow rate of 2.0 mL/min.
Example 3
(Resolution of (R)-Efsevin and (S)-Efsevin Through Derivatization
Using Menthol)
##STR00026##
[0209] Racemic-efsevin (500 mg, 1.35 mmol) was dissolved in 1:1
H.sub.2O/THF (34.0 mL) at room temperature. Lithium hydroxide
monohydrate (141.2 mg, 3.37 mmol) was added to the reaction
mixture. The reaction was allowed to stir overnight at room
temperature. The reaction was monitored by TLC. Upon completion,
mixture was cooled to 0.degree. C. using an ice bath and acidified
to pH 1 with aq. 1N HCl. The mixture was extracted with DCM (30
mL.times.3). The combined organic layer was dried with
Na.sub.2SO.sub.4 and concentrated in vacuo. The resulting efsevin
carboxylic acid was used in the next step without further
purification.
[0210] Racemic-efsevin carboxylic acid (1.35 mmol) and (-)-menthol
(317.0 mg, 2.0 mmol) were dissolved in DCM (1.0 mL) and cooled to
0.degree. C. using an ice bath. DCC (279.0 mg, 1.35 mmol) and DMAP
(1.6 mg, 0.014 mmol) were dissolved in DCM (1.0 mL) and added to
the reaction mixture over 1 h using a syringe pump. After addition,
the reaction mixture was allowed to warm to room temperature by
removing the cooling bath. Upon completion, the mixture was
filtered through a short pad of celite and concentrated in vacuo.
The crude product was purified using FCC on silica gel (20% EtOAc
in hexanes) to yield two diastereoisomeric efsevin menthol esters
(385.0 mg, 58%). Selective crystallization in 9:1 hexanes/EtOAc
yielded (R)-efsevin menthol ester (180 mg) and (S)-efsevin menthol
ester [200 mg with trace (R)-ester]. Both (R)- and (S)-efsevin
menthol esters can be hydrolyzed and esterified to give
enantiomerically pure (R)- and (S)-efsevin. See (a) Jonas, R.;
Wurziger, H. Tetrahedron 1987, 43, 4539-4547. (b) I to, Y.; Miyake,
T.; Hatano, S.; Shima, R.; Ohara, T.; Suginome, M. J. Org. Chem.
1998, 120, 11880-11893. (d) Yang, D.; Ye, X.-Y.; Xu, M. J. Org.
Chem. 2000, 65, 2208-2217. (e) Holy, R.; Kova{hacek over (c)}, M.;
Tich , M.; Zavada, J.; Bud{hacek over (e)}{hacek over (s)}ink , M.;
Cisa{hacek over (r)}ova, I. Tetrahedron: Asymmetry 2005, 16,
2031-2038.
##STR00027##
(R)-Efsevin menthol ester
[0211] .sup.1H NMR (500 MHz, CDCl3) (FIG. 14 top) .delta. 7.34 (d,
J=8.3 Hz, 2H), 7.22-7.16 (m, 5H), 7.09 (d, J=8.0 Hz, 2H), 6.86 (q,
J=2.0 Hz, 1H), 5.74 (dt, J=5.9, 1.9 Hz, 1H), 4.55 (td, J=10.5, 3.9
Hz, 1H), 4.51 (dt, J=16.9, 2.4 Hz, 2H), 2.35 (s, 3H), 1.87-1.82 (m,
1H), 1.63-1.59 (m, 1H), 1.54-1.51 (m, 2H), 1.41-1.35 (m, 1H), 1.05
(tt, J=5.4, 3.1 Hz, 1H), 0.93-0.84 (m, 5H), 0.82-0.71 (m, 2H), 0.54
(d, J=7.0 Hz, 3H), 0.40 (d, J=7.0 Hz, 3H)
[0212] .sup.13C NMR (125 MHz, CDCl3) (FIG. 14 bottom) .delta.
161.6, 143.1, 139.2, 136.2, 136.1, 135.9, 129.4, 128.2, 128.01,
127.96, 127.0, 74.8, 68.8, 54.6, 46.8, 40.8, 34.1, 31.4, 25.0,
22.7, 22.0, 21.5, 21.0, 15.4.
Example 4
(Catalytic Asymmetric Synthesis of (S)-Efsevin)
##STR00028##
[0214] N-Tosyl benzaldimine (5.67 g, 21.9 mmol) was dissolved in
benzene (175.0 mL) at room temperature. Exo-phenyl Kwonphos (756.0
mg, 2.19 mmol) was added to the reaction mixture. Ethyl allenoate
(2.94 g, 26.3 mmol) was then added into the reaction mixture
dropwise. The reaction was allowed to stir at room temperature and
monitored by TLC. Upon completion, the mixture was concentrated in
vacuo. The resulting mixture was purified using FCC on silica gel
(20% EtOAc in hexanes) to yield a 23:77 mixture of (R)- and
(S)-efsevin (6.78 g, 90%). Selective crystallization in 4:1
hexanes/EtOAc gave racemic-efsevin (3.12 g), leaving (S)-efsevin
(3.66 g) in the mother liquor. The mother liquor was recrystallized
in 4:1 hexanes/EtOAc to give (S)-efsevin (2.1 g, >99% ee)
[0215] A. Synthesis of (S)-Efsevin Menthol Ester
##STR00029##
[0216] (S)-Efsevin (50 mg, 0.135 mmol) was dissolved in 1:1
H.sub.2O/THF (3.4 mL) at room temperature. Lithium hydroxide
monohydrate (14.1 mg, 0.34 mmol) was added into the reaction
mixture. The reaction was allowed to stir overnight at room
temperature. The reaction was monitored by TLC. Upon completion,
mixture was cooled to 0.degree. C. using an ice bath and acidified
to pH 1 with aq. 1N HCl. The mixture was extracted with DCM (10
mL.times.3). The combined organic layer was dried with
Na.sub.2SO.sub.4 and concentrated in vacuo. The resulting
carboxylic acid was used in the next step without further
purification. (S)-Efsevin carboxylic acid (0.135 mmol) and
(-)-menthol (31.7 mg, 0.2 mmol) were dissolved in DCM (1.0 mL) and
cooled to 0.degree. C. using an ice bath. DCC (27.9 mg, 0.135 mmol)
and DMAP (0.2 mg, 0.0014 mmol) were dissolved in DCM (1.0 mL) and
added to the reaction mixture over 1 h using a syringe pump. After
addition, the reaction mixture was allowed to warm to room
temperature by removing the cooling bath. Upon completion, the
mixture was filtered through a short pad of celite and concentrated
in vacuo. The crude product was purified using FCC on silica gel
(20% EtOAc in hexanes) to yield (S)-efsevin menthol ester (50.0 mg,
77%).
##STR00030##
(S)-Efsevin Menthol Ester
[0217] .sup.1H NMR (500 MHz, CDCl3) (FIG. 15 top) .delta. 7.39 (d,
J=8.3 Hz, 2H), 7.22-7.17 (m, 5H), 7.12 (d, J=8.0 Hz, 2H), 6.74 (q,
J=2.0 Hz, 1H), 5.72 (dt, J=5.8, 2.0 Hz, 1H), 4.54-4.49 (m, 2H),
4.35 (ddd, J=16.9, 5.9, 2.0 Hz, 1H), 2.36 (s, 3H), 1.69-1.63 (m,
1H), 1.61-1.58 (m, 3H), 1.35-1.20 (m, 3H), 0.99-0.90 (m, 1H), 0.83
(d, J=7.1 Hz, 3H), 0.80-0.71 (m, 4H), 0.61 (d, J=7.0 Hz, 3H), 0.51
(q, J=11.8 Hz, 1H)
[0218] .sup.13C NMR (125 MHz, CDCl3) (FIG. 15 bottom) .delta.
161.5, 143.2, 139.4, 136.5, 135.7, 134.9, 129.4, 128.2, 127.93,
127.86, 127.1, 75.0, 69.1, 54.9, 46.9, 40.2, 34.1, 31.2, 26.5,
23.6, 21.9, 21.5, 20.6, 16.5.
Examples 5-7
Synthesis of Screening Library & Zebrafish Screening
[0219] A. Introduction
[0220] We generation a large number of diverse small molecules to
screen zebrafish tremblor mutants, defective in a cardiac-specific
sodium calcium exchanger gene, NCX1h, to study cardiac arrhythmia
induced by abnormal calcium ion handling. To achieve this, our
group build up a diverse library by constructing diverse scaffolds
such as dihydropyrroles (Zhu, et al., 2005, Tetrahedron., 61,
6276-6282), tetrahydropyridines (Zhu, et al., 2003, J. Am. Chem.
Soc., 125, 4716-4717), cyclohexenes (Tran, et al., 2007, J. Am.
Chem. Soc., 129, 12632-12633), bicyclic succinimides, coumarins
(Henry, et al., 2007, Org. Lett., 9, 3069-3072), dioxanyidenes
(Zhu, et al., 2005, Org. Lett., 7, 1387-1390.), dihydropyrones
(Creech, et al., 2008, Org. Lett., 10, 429-432), and pyrenes (Zhu,
et al., 2005, Org. Lett., 7, 2977-2980) using phosphine-catalyzed
reactions, phosphine-catalyzed reactions combined with Michael
addition, or the sequence of phosphine-catalyzed reactions/Tebbe
reactions/Diels-Alder reactions (see non-limiting examples in FIG.
18A-E). The library included a large number of efsevin analogs,
including: analogs with ethyl ester motif by using different imines
as one of the annulation coupling partners; and analogs with meta-,
para-methyl substitutes, halogen substitutes, and unsubstituted
benzaldehyde to react with orth-, para-methyl substitutes and
halogen substituted benzenesulfonamide for imine synthesis.
[0221] B. General Information
[0222] Benzene and dichloromethane were distilled fresh from
CaH.sub.2. THF was distilled fresh from sodium. All other reagents
were used as received from commercial sources. Reactions were
monitored using thin layer chromatography (TLC) performed on
0.25-mm E. Merck silica gel plates (60F-254) and visualized under
UV light or through permanganate staining. Flash column
chromatography was performed using E. Merck silica gel 60 (230-400
mesh) and compressed air. NMR spectra were obtained on Bruker
ARX-400, or Bruker AXR-300 instruments (as indicated), calibrated
using residual undeuterated chloroform as an internal reference
(7.26 and 77.0 ppm for .sup.1H and .sup.13C NMR spectra,
respectively). .sup.1H NMR spectral data are reported as follows:
chemical shift (.delta., ppm), multiplicity, coupling constant
(Hz), and integration. .sup.13C spectral data are reported in terms
of the chemical shift. The following abbreviations are used to
indicate multiplicities: s=singlet; d=doublet; t=triplet;
q=quartet; m=multiplet; br=broad. Affi-gel-10 was purchased from
BioRad. EDTA, EGTA, Glycine, NaF, phenylmethylsulfonylfluoride
(PMSF) and Trizma base were purchased from Sigma Chemicals. Silver
Stain Kit and pre-casted tris-glycine gel were obtained from
Invitrogen. Protein inhibitor cocktail was purchased from Roche.
Nonidet P-40 was from Fluka. Resin filtration procedures were
carried out using a 70.mu. PE frit cartridges from Applied
Separations (cat. #2449).
Example 5
Synthesis of N-Sulfonyimines and Allenoates
[0223] N-sulfonylimines for the screening library were synthesized
from corresponding aldehydes and sulfonamides through the use of
TiCl.sub.4, according to the procedure in McKay, et al. (McKay, et
al., 1981, J. Chem. Soc., Perkin Trans. 1, 2435). The rest of the
imine were synthesized through the condensation of the
corresponding aldehydes with the sulfonamides catalyzed by
BF.sub.3.OEt.sub.2 with azeotropic water removal (Dean-Stark),
according to the procedure in Jennings, et al. (Jennings, et al.,
1991, Tetrahedron, 47, 5561). Ethyl buta-2,3-dienoate was synthesis
according to the procedure in Lang, et al. (Lang, et al., 1984,
Organic Syntheses., 62, 202).
Example 6
Synthesis of Dihydropyrroles
##STR00031##
[0225] Dihydropyrroles for the screening library were synthesized
according to the following general procedure. PPh.sub.3 (1 mmol)
and imine (1 mmol) were dissolved in dry benzene. Ethyl
buta-2,3-dienoate (1.2 mmol) was added dropwised. The mixture was
heated to 40.degree. C. and stirred overnight (Scheme II). Solvent
was removed under vacuum and the residue was purified by flash
chromatography. (Ethyl acetate: Hexane=1:6-1:4).
Example 7
Zebrafish Screening
[0226] We screened our above mention library by using tremblor
mutants Zebrafish embryos. The heart cells of tremblor mutant
embryos do not establish rhythmic synchronized contraction but
rather contract independently and create a chaotic contraction
pattern. Compounds were screened for their ability to suppress
cardiac fibrillation and/or restore rhythmic synchronized
contraction. Briefly, we treated the embryo by soaking in 20 .mu.M
solution or injecting compounds after 24 hours fertilization and
observed the phenotype after 48 hours. Further, we determined the
structure-activity relationship (SAR) between the active compound's
structures and the ability to suppress cardiac fibrillation and/or
restore rhythmic synchronized contraction.
[0227] C. Methods
Soaking
[0228] Zebrafish tremblor mutant eggs were collected after
fertilization, and arrayed in 24-well plates (20 twenty
embryos/well) in 500 .mu.L buffer. After 24 hour fertilization in
29.degree. C., 20 .mu.M testing compound was added and using E3
buffer as background control. After 48 hour fertilization in
29.degree. C., phenotypic changes were observed using a high
magnification dissecting microscope.
Injection with Morpholine
[0229] Zebrafish eggs were collected at 1 cell stages. 1 nL of
compound in Daneu's buffer and 1 nL of morpholino (1 mM) to NCX1h
was injected into the embryo. After 24 hours the embryo was arrayed
in 24-well plates (20 twenty embryos/well) in 500 .mu.L buffer.
After 48 hour fertilization in 29.degree. C., phenotypic changes
were observed using a high magnification dissecting microscope.
Injection into Tremblor Mutants
[0230] Zebrafish tremblor mutant eggs were collected at 1 cell
stages. 1 nL of compound in Daneu's buffer were injected into the
embryo. After 24 hours the embryo was arrayed in 24-well plates (20
twenty embryos/well) in 500 .mu.L buffer. After 48 hour
fertilization in 29.degree. C., phenotypic changes were observed
using a high magnification dissecting microscope.
[0231] D. Results
Activity Analysis
[0232] We identified multiple active compounds from the library
with similar or greater activity than efseven (see non-limiting
examples in FIGS. 0A and 0B). Table 1 shows some of the most active
compounds found in the screens.
TABLE-US-00001 TABLE 1 Soaking: Injection with Injection into
Number of tremblor Morpholino: tremblor Mutants: Mutants/ Number of
Fish/ Number of fish/ Chemical % Rescued % Rescued % Rescued
Formula Id 18/80% Formula Ie 8/75% Formula If 19/78% Formula Ig
17/88% Formula Ih 14/50% 10/80% Formula Ii 8/75% 15/87% Formula Ij
8/88% 28/79% Formula Ik 8/75% 26/85% Formula Il 8/63% 12/83%
Formula Im 8/88% Formula In 8/88% Formula Io 8/75% Formula Ip 8/38%
27/74% Formula Iq 6/67% 21/81% Formula Ir 21/76% Formula Is 6/67%
23/87% Formula It 6/0% 30/73% Formula Iu 8/88% 43/74%
[0233] Tables 2, 3, and 4 show additional screening results (see
FIG. 18A-D for structures). Table 2 shows screening results using
tremblor mutant soaked in 20 .mu.M test compounds.
TABLE-US-00002 TABLE 2 Number of Rescue Chemical fish percent F7 89
72% F7-I-A02 12 33% F7-I-A03 21 38% F7-I-A04 27 31% F7-I-A05 10 40%
F7-I-A06 17 88% F7-I-A07 0 Dead F7-I-A08 14 57% F7-I-A09 19 42%
F7-I-A10 14 50% F7-I-A11 17 50% F7-I-A12 14 60% F7-I-B01 20 11%
F7-I-B02 15 40% F7-I-B03 23 0% F7-I-B04 8 75% F7-I-B05 15 29%
F7-I-B06 13 0% F7-I-B07 18 80% F7-I-B08 13 43% F7-I-B09 16 67% F7
ketone 51 26% F7-I-B10 15 63% F7-I-B11 19 30% F7-I-B12 19 78%
F7-I-C01 15 33% F7-I-C02 14 86%* F7-I-C03 17 29% F7-I-C04 26 62%
F7-I-C05 26 23% F7-I-C06 15 11% F7-I-C07 16 25% F7-I-C08 9 11%
F7-I-C09 0 Dead F7-I-C10 0 Dead F7-I-C11 0 Dead F7-I-C12 15 100%**
F7-I-D01 1 Dead F7-I-D02 9 0.0% F7-I-D03 0 Dead F7-I-D04 19 15.8%
F7 acid 47 21% F7 methyl ester 46 44% F7 isopropyl 42 41% ester
[0234] Table 3 shows screening results using morpholino
mutants.
TABLE-US-00003 TABLE 3 Number of Rescue Chemical fish percent
F7-I-D05 8 0% F7-I-D06 8 38% F7-I-D07 8 38% F7-I-D08 15 73%
F7-I-D09 8 13% F7-I-D10 8 0% F7-I-D11 8 0% F7-I-D12 8 50% F7-I-E01
16 63% F7-I-E02 8 13% F7-I-E03 8 0% F7-I-E04 8 0% F7-I-E05 16 75%
F7-I-E06 8 13% F7-I-E07 16 50% F7-I-E08 14 71% F7-I-E09 8 50%
F7-I-E10 8 25% F7-I-E11 8 0% F7-I-E12 8 25% F7-I-F01 16 81%
F7-I-F02 14 79% F7-I-F03 8 0% F7-I-F04 8 0% F7-I-F05 14 50%
F7-I-F06 8 100% F7-I-F07 8 0% F7-I-F08 8 75% F7-I-F09 8 75%
F7-I-F10 8 88% F7-I-F11 8 88% F7-I-F12 8 63% F7-I-G01 8 100%
F7-I-G02 8 75% F7-I-G03 8 100% F7-I-G04 8 75% F7-I-G05 8 100%
F7-I-G06 8 75% F7-I-G07 8 88% F7-I-G08 8 38% F7-I-G09 6 67%
F7-I-G10 6 67% F7-I-G11 7 86% F7-I-G12 8 38% F7-I-H01 8 63%
F7-I-H02 8 88% F7-I-H03 8 88% F7-I-H04 8 75% F7-I-H05 8 88%
F7-I-H06 8 63% F7-I-H07 8 63% F7-I-H08 7 100% F7-I-H09 10 10%
F7-I-H10 8 25% F7-I-H11 8 88% F7-I-H12 8 100% F7-II-A01 8 38%
F7-II-A02 8 0% F7-II-A03 8 88% F7-II-A04 8 100% F7-II-A05 8 13%
F7-II-A06 7 86% F7-II-A07 6 17% F7-II-A08 7 86% F7-II-A09 6 17%
F7-II-A10 5 80% F7-II-A11 6 0% F7-II-A12 8 0% F7-II-B01 6 67%
F7-II-B02 8 38%
[0235] Table 4 shows screening results using injection into
tremblor mutants.
TABLE-US-00004 TABLE 4 Screen 1 Screen 2 Screen 3 Total Average
Number Rescue Number Rescue Number Rescue number rescue Chemical of
fish percent of fish percent of fish percent of fish ratio F7-I-D08
13 23% 13 23% F7-I-E01 8 0% 8 0% F7-I-E05 8 50% 8 50% F7-I-E07 8
50% 8 50% F7-I-E08 12 42% 12 42% F7-I-F01 7 14% 7 14% F7-I-F02 12
58% 12 58% F7-I-F05 10 80% 10 80% F7-I-F06 10 40% 10 40% F7-I-F08
10 20% 10 20% F7-I-F09 15 87% 15 87% F7-I-F10 8 50% 8 50% F7-I-F11
10 80% 18 78% 28 79% F7-I-F12 5 60% 5 60% F7-I-G01 11 73% 12 50% 23
61% F7-I-G02 7 57% 7 57% F7-I-G03 10 20% 10 20% F7-I-G04 9 78% 17
88% 26 85% F7-I-G05 11 73% 14 50% 25 60% F7-I-G06 11 55% 11 55%
F7-I-G07 10 40% 10 40% F7-I-G09 23 78% 23 78% F7-I-G10 28 66% 28
66% F7-I-G11 31 62% 31 62% F7-I-G12 42 61% 42 61% F7-I-H01 12 83%
12 83% F7-I-H02 5 100% 19 74% 24 79% F7-I-H03 7 86% 9 78% 16 81%
F7-I-H04 6 100% 6 67% 12 83% F7-I-H05 7 86% 16 69% 20 75% 43 74%
F7-I-H06 9 44% 9 44% F7-I-H07 10 90% 21 52% 31 65% F7-I-H08 9 44% 9
44% F7-I-H09 8 88% 15 46% 23 61% F7-I-H10 5 60% 5 60% F7-I-H11 9
56% 9 56% F7-I-H12 13 54% 13 54% F7-II-A01 27 74% 27 74% F7-II-A02
18 62% 18 62% F7-II-A03 17 60% 17 60% F7-II-A04 22 48% 22 48%
F7-II-A05 7 29% 7 29% F7-II-A06 36 64% 36 64% F7-II-A07 15 33% 15
33% F7-II-A08 20 26% 20 26% F7-II-A09 16 22% 16 22% F7-II-A10 33
40% 33 40% F7-II-A11 30 73% 30 73% F7-II-A12 14 5% 14 5% F7-II-B01
7 91% 14 79% 21 81% F7-II-B02 13 7% 13 7% F7-II-B10 33 58% 33 58%
F7-II-B11 35 37% 35 37% F7-II-B12 21 76% 21 76% F7-II-C01 25 60% 25
60% F7-II-C02 17 65% 17 65% F7-II-C03 22 55% 22 55%
E. SAR Analysis
[0236] Further, SAR analysis showed that para-halogen, especially
para-fluoro substituted benzaldehyde has good structure-function
correlation. For benznensulfonamide the orth-fluoro and para-fluro
substituted groups also showed good structure-function
correlation.
[0237] Those skilled in the art will know, or be able to ascertain,
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
91283PRTArtificial SequenceSynthetic Peptide 1Met Ala Val Pro Pro
Ala Tyr Ala Asp Leu Gly Lys Ser Ala Lys Asp 1 5 10 15 Ile Phe Asn
Lys Gly Tyr Gly Phe Gly Met Val Lys Leu Asp Val Lys 20 25 30 Thr
Lys Ser Ala Ser Gly Val Glu Phe Lys Thr Ser Gly Ser Ser Asn 35 40
45 Thr Asp Thr Ser Lys Val Val Gly Ser Leu Glu Thr Lys Tyr Lys Arg
50 55 60 Ser Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp
Asn Thr 65 70 75 80 Leu Gly Thr Glu Ile Asn Ile Glu Asp Gln Ile Ala
Lys Gly Leu Lys 85 90 95 Leu Thr Phe Asp Thr Thr Phe Ser Pro Asn
Thr Gly Lys Lys Ser Gly 100 105 110 Lys Val Lys Thr Ala Tyr Lys Arg
Glu Phe Val Asn Leu Gly Cys Asp 115 120 125 Val Asp Phe Asp Phe Ala
Gly Pro Thr Ile His Gly Ala Ala Val Val 130 135 140 Gly Tyr Glu Gly
Trp Leu Ala Gly Tyr Gln Met Ser Phe Asp Thr Ala 145 150 155 160 Lys
Ser Lys Met Thr Gln Asn Asn Phe Ala Val Gly Tyr Lys Thr Gly 165 170
175 Asp Phe Gln Leu His Thr Asn Val Asn Asp Gly Ser Glu Phe Gly Gly
180 185 190 Ser Ile Tyr Gln Lys Val Ser Asp Lys Leu Glu Thr Ala Val
Asn Leu 195 200 205 Ala Trp Thr Ala Gly Ser Asn Ser Thr Arg Phe Gly
Ile Ala Ala Lys 210 215 220 Tyr Gln Leu Asp Lys Ser Ala Ser Ile Ser
Ala Lys Val Asn Asn Thr 225 230 235 240 Ser Leu Val Gly Val Gly Tyr
Thr Gln Ser Leu Arg Pro Gly Ile Lys 245 250 255 Leu Thr Leu Ser Ala
Leu Val Asp Gly Lys Ser Ile Asn Ser Gly Gly 260 265 270 His Lys Leu
Gly Leu Gly Leu Glu Leu Glu Ala 275 280 235DNAArtificial
SequenceSynthetic Primer 2aacactgaca ccagcaaagt ggttgggagc ctgga
35335DNAArtificial SequenceSynthetic Primer 3tccaggctcc caaccacttt
gctggtgtca gtgtt 35496DNAArtificial SequenceSynthetic Primer
4aggaattcaa gacctctggt tcctccaaca ctgacaccag caaagtggtt gggagcctgg
60aaaccaaata caagaggtct gaatatggcc tgacct 96531PRTArtificial
SequenceSynthetic Peptide 5Glu Phe Lys Thr Ser Gly Ser Ser Asn Thr
Asp Thr Ser Lys Val Val 1 5 10 15 Gly Ser Leu Glu Thr Lys Tyr Lys
Arg Ser Glu Tyr Gly Leu Thr 20 25 30 662DNAArtificial
SequenceSynthetic Primer 6aggaattcaa gaccttggga gcctggaaac
caaatacaag aggtctgaat atggcctgac 60ct 62719PRTArtificial
SequenceSynthetic Peptide 7Glu Phe Lys Thr Leu Gly Ala Trp Lys Pro
Asn Thr Arg Gly Leu Asn 1 5 10 15 Met Ala Lys 813PRTArtificial
SequenceSynthetic Peptide 8Leu Thr Phe Asp Thr Thr Phe Ser Pro Asn
Thr Gly Lys 1 5 10 925DNAArtificial SequenceSynthetic Primer
9gggaacggcc attttatctg ttaaa 25
* * * * *
References