U.S. patent application number 09/810644 was filed with the patent office on 2002-01-31 for production of adenine nucleotide translocator (ant), novel ant ligands and screening assays therefor.
Invention is credited to Anderson, Christen M., Clevenger, William, Davis, Robert E., Ghosh, Soumitra S., Miller, Scott W., Moos, Walter H., Pei, Yazhong, Szabo, Tomas R., Wiley, Sandra Eileen.
Application Number | 20020012992 09/810644 |
Document ID | / |
Family ID | 26881590 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012992 |
Kind Code |
A1 |
Anderson, Christen M. ; et
al. |
January 31, 2002 |
Production of adenine nucleotide translocator (ANT), novel ANT
ligands and screening assays therefor
Abstract
Compositions and methods are provided for producing adenine
nucleotide translocator (ANT) polypeptides and fusion proteins,
including the production and use of recombinant expression
constructs having a regulated promoter. ANT ligands and
compositions and methods for identifying ANT ligands, agents that
bind ANT and agents that interact with ANT are also disclosed.
Inventors: |
Anderson, Christen M.;
(Encinitas, CA) ; Davis, Robert E.; (San Diego,
CA) ; Clevenger, William; (Oceanside, CA) ;
Wiley, Sandra Eileen; (San Diego, CA) ; Miller, Scott
W.; (San Marcos, CA) ; Szabo, Tomas R.; (San
Diego, CA) ; Ghosh, Soumitra S.; (San Diego, CA)
; Moos, Walter H.; (Oakland, CA) ; Pei,
Yazhong; (San Diego, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Family ID: |
26881590 |
Appl. No.: |
09/810644 |
Filed: |
March 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09810644 |
Mar 14, 2001 |
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09393441 |
Sep 8, 1999 |
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09393441 |
Sep 8, 1999 |
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09185904 |
Nov 3, 1998 |
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Current U.S.
Class: |
435/325 ;
435/183; 435/252.3; 435/320.1; 536/23.1; 536/23.4 |
Current CPC
Class: |
C07K 14/47 20130101;
C12N 15/62 20130101; A61P 17/06 20180101; A61P 35/00 20180101; A61P
3/10 20180101; A61P 25/28 20180101; A61P 25/14 20180101; A61P 27/16
20180101; A61P 43/00 20180101; C07K 2319/23 20130101; A61P 25/08
20180101; A61P 25/16 20180101; C07K 2319/07 20130101; C07K 2319/21
20130101; C12N 2799/021 20130101; A61P 25/18 20180101; A61P 3/00
20180101; C07K 2319/00 20130101 |
Class at
Publication: |
435/325 ;
435/183; 435/320.1; 536/23.1; 536/23.4; 435/252.3 |
International
Class: |
C07H 021/02; C07H
021/04; C12N 009/00; C12N 001/20; C12N 015/00; C12N 015/09; C12N
015/63; C12N 015/70; C12N 015/74; C12N 005/00 |
Claims
We claim:
1. A recombinant expression construct comprising at least one
regulated promoter operably linked to a first nucleic acid encoding
an adenine nucleotide translocator polypeptide.
2. The expression construct of claim 1 further comprising at least
one additional nucleic acid sequence that regulates
transcription.
3. The expression construct of claim 2 wherein the additional
nucleic acid sequence that regulates transcription encodes a
repressor of said regulated promoter.
4. The expression construct of claim 1 wherein the adenine
nucleotide translocator polypeptide comprises a human adenine
nucleotide translocator polypeptide.
5. The expression construct of claim 4 wherein the human adenine
nucleotide translocator polypeptide is ANT1.
6. The expression construct of claim 4 wherein the human adenine
nucleotide translocator polypeptide is ANT2.
7. The expression construct of claim 4 wherein the human adenine
nucleotide translocator polypeptide is ANT3.
8. An expression construct according to claim 1 wherein the adenine
nucleotide translocator polypeptide is expressed as a fusion
protein with a polypeptide product of a second nucleic acid
sequence.
9. The expression construct of claim 8 wherein the polypeptide
product of said second nucleic acid sequence is an enzyme.
10. The expression construct of claim 8 wherein said fusion protein
localizes to membranes.
11. The expression construct of claim 10 wherein said membranes are
mitochondrial membranes.
12. An expression construct according to claim 1 wherein the
adenine nucleotide translocator polypeptide is expressed as a
fusion protein with at least one product of a second nucleic acid
sequence encoding a polypeptide cleavable by a protease, said
adenine nucleotide translocator polypeptide being separable from
the fusion protein by cleavage with the protease.
13. A host cell comprising a recombinant expression construct
according to claim 1.
14. A host cell according to claim 13 wherein the host cell is a
prokaryotic cell.
15. A host cell according to claim 13 wherein the host cell is a
eukaryotic cell.
16. The host cell of claim 15 wherein the eukaryotic cell is
selected from the group consisting of a yeast cell, an insect cell
and a mammalian cell.
17. The host cell of claim 16 wherein the insect cell is selected
from the group consisting of an Sf9 cell and a Trichoplusia ni
cell.
18. A host cell according to claim 13 that lacks at least one
isoform of an endogenous adenine nucleotide translocator.
19. A host cell according to claim 13 in which expression of at
least one gene encoding an endogenous adenine nucleotide
translocator isoform is substantially impaired.
20. A recombinant expression construct comprising at least one
promoter operably linked to a nucleic acid molecule comprising a
first nucleic acid sequence and a second nucleic acid sequence,
said first nucleic acid sequence encoding an animal adenine
nucleotide translocator polypeptide wherein the adenine nucleotide
translocator polypeptide is expressed as a fusion protein with a
polypeptide product of said second nucleic acid sequence.
21. The expression construct of claim 20 wherein the polypeptide
product of said second nucleic acid sequence is an enzyme.
22. The expression construct of claim 20 wherein said fusion
protein localizes to membranes.
23. The expression construct of claim 22 wherein said membranes are
mitochondrial membranes.
24. The expression construct of claim 20 further comprising at
least one additional nucleic acid sequence that regulates
transcription.
25. The expression construct of claim 24 wherein the additional
nucleic acid sequence that regulates transcription encodes a
repressor of said promoter.
26. The expression construct of claim 20 wherein the adenine
nucleotide translocator polypeptide comprises a human adenine
nucleotide translocator polypeptide.
27. The expression construct of claim 26 wherein the human adenine
nucleotide translocator polypeptide is ANT1.
28. The expression construct of claim 26 wherein the human adenine
nucleotide translocator polypeptide is ANT2.
29. The expression construct of claim 26 wherein the human adenine
nucleotide translocator polypeptide is ANT3.
30. An expression construct according to claim 20 wherein the
adenine nucleotide translocator polypeptide is expressed as a
fusion protein with at least one product of a second nucleic acid
sequence encoding a polypeptide cleavable by a protease, said
adenine nucleotide translocator polypeptide being separable from
the fusion protein by cleavage with the protease.
31. A host cell comprising a recombinant expression construct
according to claim 20.
32. A host cell according to claim 31 wherein the host cell is a
prokaryotic cell.
33. A host cell according to claim 31 wherein the host cell is a
eukaryotic cell.
34. The host cell of claim 33 wherein the eukaryotic cell is
selected from the group consisting of a yeast cell, an insect cell
and a mammalian cell.
35. The host cell of claim 34 wherein the insect cell is selected
from the group consisting of an Sf9 cell and a Trichoplusia ni
cell.
36. A host cell according to claim 20 that lacks at least one
isoform of an endogenous adenine nucleotide translocator.
37. A host cell according to claim 20 in which expression of at
least one gene encoding an endogenous adenine nucleotide
translocator isoform is substantially impaired.
38. A recombinant expression construct according to either claim 1
or claim 20 wherein the expression construct is a recombinant viral
expression construct.
39. A method of producing a recombinant adenine nucleotide
translocator polypeptide, comprising: culturing a host cell
comprising a recombinant expression construct comprising at least
one regulated promoter operably linked to a first nucleic acid
encoding an adenine nucleotide translocator polypeptide.
40. A method of producing a recombinant adenine nucleotide
translocator polypeptide, comprising: culturing a host cell
comprising a recombinant expression construct comprising at least
one promoter operably linked to a nucleic acid molecule comprising
a first nucleic acid sequence and a second nucleic acid sequence,
said first nucleic acid sequence encoding an animal adenine
nucleotide translocator polypeptide wherein the adenine nucleotide
translocator polypeptide is expressed as a fusion protein with a
polypeptide product of said second nucleic acid sequence.
41. A method of producing a recombinant adenine nucleotide
translocator polypeptide, comprising: culturing a host cell
infected with the recombinant viral expression construct of claim
38.
42. An ANT polypeptide produced by the method of any one of claims
39-41.
43. An isolated human adenine nucleotide translocator
polypeptide.
44. The isolated polypeptide of claim 43 wherein the human adenine
nucleotide translocator polypeptide is recombinant ANT1 or a
variant or fragment thereof.
45. The isolated polypeptide of claim 43 wherein the human adenine
nucleotide translocator polypeptide is recombinant ANT2 or a
variant or fragment thereof.
46. The isolated polypeptide of claim 43 wherein the human adenine
nucleotide translocator polypeptide is recombinant ANT3 or a
variant or fragment thereof.
47. An isolated human adenine nucleotide translocator fusion
protein comprising an adenine translocator polypeptide fused to at
least one additional polypeptide sequence.
48. The fusion protein of claim 47 wherein said one additional
polypeptide sequence is an enzyme sequence or a variant or fragment
thereof.
49. The fusion protein of claim 47 wherein said fusion protein
localizes to membranes.
50. The fusion protein of claim 49 wherein said membranes are
mitochondrial membranes.
51. An isolated human adenine nucleotide translocator fusion
protein comprising an adenine translocator polypeptide fused to at
least one additional polypeptide sequence cleavable by a protease,
said adenine nucleotide translocator polypeptide being separable
from the fusion protein by cleavage with the protease.
52. An isolated adenine nucleotide translocator fusion protein
comprising a first polypeptide that is an animal adenine
translocator polypeptide fused to at least one additional
polypeptide sequence.
53. The fusion protein of claim 52 wherein said one additional
polypeptide sequence is an enzyme sequence or a variant or fragment
thereof.
54. A fusion protein according to claim 52 that localizes to
membranes.
55. A fusion protein according to claim 54 wherein said membranes
are mitochondrial membranes.
56. An isolated recombinant animal adenine nucleotide translocator
fusion protein comprising an adenine translocator polypeptide fused
to at least one additional polypeptide sequence cleavable by a
protease, said adenine nucleotide translocator polypeptide being
separable from the fusion protein by cleavage with the
protease.
57. The fusion protein of either claim 47 or claim 52 wherein the
additional polypeptide sequence is a polypeptide having affinity
for a ligand.
58. A method for determining the presence of an ANT polypeptide in
a biological sample comprising: contacting a biological sample
suspected of containing an ANT polypeptide with an ANT ligand under
conditions and for a time sufficient to allow binding of the ANT
ligand to an ANT polypeptide; and detecting the binding of the ANT
ligand to an ANT polypeptide, and therefrom determining the
presence of an ANT polypeptide in said biological sample.
59. The method of claim 58 wherein the adenine nucleotide
translocator polypeptide comprises a human adenine nucleotide
translocator polypeptide.
60. The method of claim 59 wherein the human adenine nucleotide
translocator polypeptide is ANT1.
61. The method of claim 59 wherein the human adenine nucleotide
translocator polypeptide is ANT2.
62. The method of claim 59 wherein the human adenine nucleotide
translocator polypeptide is ANT3.
63. The method of claim 58 wherein the ANT ligand comprises
atractyloside substituted at 6' hydroxyl to form an atractyloside
derivative.
64. The method of claim 63 wherein the atractyloside is detectably
substituted at the 6' hydroxyl to form a detectable atractyloside
derivative.
65. The method of claim 64 wherein the detectable atractyloside
derivative comprises a radioloabeled substituent.
66. The method of claim 65 wherein the radiolabeled substituent is
selected from the group consisting of .sup.125I, .sup.131I,
.sup.3H, .sup.14C and .sup.35S.
67. The method of claim 64 wherein the detectable atractyloside
derivative comprises a fluorescent substituent.
68. The method of claim 67 wherein the ANT ligand further comprises
a Eu.sup.3+ atom complexed to the atractyloside derivative.
69. The method of claim 64 wherein the detectable atractyloside
derivative comprises covalently bound biotin.
70. The method of claim 63 wherein the atractyloside molecule is
substituted at 6' hydroxyl with an amine or an amine containing
functionality to form an amine modified atractyloside
derivative.
71. The method of any one of claims 63-70 wherein the atractyloside
molecule is a carboxyatractyloside molecule that is substituted at
6' hydroxyl to form an atractyloside derivative that is a
carboxyatractyloside derivative.
72. A method for isolating ANT from a biological sample,
comprising: contacting a biological sample suspected of containing
an ANT polypeptide with an ANT ligand under conditions and for a
time sufficient to allow binding of the ANT ligand to an ANT
polypeptide; and recovering the ANT polypeptide, and thereby
isolating ANT from a biological sample.
73. The method of claim 72 wherein the ANT ligand is covalently
bound to a solid phase.
74. The method of claim 72 wherein the ANT ligand is non-covalently
bound to a solid phase.
75. A method for identifying an agent that binds to an ANT
polypeptide, comprising: contacting a candidate agent with a host
cell expressing at least one recombinant ANT polypeptide under
conditions and for a time sufficient to permit binding of the agent
to said recombinant ANT polypeptide; and detecting binding of said
agent to the recombinant ANT.
76. The method of claim 75 wherein the host cell is a prokaryotic
cell.
77. The method of claim 76 wherein the prokaryotic cell is an E.
coli cell.
78. The method of claim 75 wherein the host cell is a eukaryotic
cell.
79. The method of claim 78 wherein the eukaryotic cell is selected
from the group consisting of a yeast cell, an insect cell and a
mammalian cell.
80. The method of claim 79 wherein the insect cell is selected from
the group consisting of an Sf9 cell and a Trichoplusia ni cell.
81. The method of any one of claims 75-80 wherein the host cell
lacks at least one isoform of an endogenous adenine nucleotide
translocator.
82. The method of any one of claims 75-80 wherein host cell
expression of at least one gene encoding an endogenous adenine
nucleotide translocator isoform is substantially impaired.
83. A method for identifying an agent that binds to an ANT
polypeptide, comprising: contacting a candidate agent with a
biological sample containing at least one recombinant ANT
polypeptide under conditions and for a time sufficient to permit
binding of the agent to said ANT polypeptide; and detecting binding
of said agent to the recombinant ANT polypeptide.
84. A method for identifying an agent that interacts with an ANT
polypeptide comprising: contacting a biological sample containing
recombinant ANT with a detectable ANT ligand in the presence of a
candidate agent; and comparing binding of the detectable ANT ligand
to recombinant ANT in the absence of said agent to binding of the
detectable ANT ligand to recombinant ANT in the presence of said
agent, and therefrom identifying an agent that interacts with an
ANT polypeptide.
85. An ANT ligand comprising atractyloside substituted at the 6'
hydroxyl to form an atractyloside derivative.
86. The ANT ligand of claim 85 wherein the atractyloside is
detectably substituted at the 6' hydroxyl to form a detectable
atractyloside derivative.
87. The ANT ligand of claim 86 wherein the detectable atractyloside
derivative comprises a radioloabeled substituent.
88. The ANT ligand of claim 87 wherein the radiolabeled substituent
is selected from the group consisting of .sup.125I, .sup.131I,
.sup.3H, .sup.14C and .sup.35S.
89. The ANT ligand of claim 86 wherein the detectable atractyloside
derivative comprises a fluorescent substituent.
90. The ANT ligand of claim 89 further comprising a Eu.sup.3+ atom
complexed to the atractyloside derivative.
91. The ANT ligand of claim 86 wherein the detectable atractyloside
derivative comprises covalently bound biotin.
92. The ANT ligand of claim 85 wherein the atractyloside molecule
is substituted at 6' hydroxyl with an amine or an amine containing
functionality to form an amine modified atractyloside
derivative.
93. The ANT ligand according to any one of claims 85-92 wherein the
atractyloside molecule is a carboxyatractyloside molecule that is
substituted at 6' hydroxyl to form an atractyloside derivative that
is a carboxyatractyloside derivative.
94. An ANT ligand having the following structure: 45and
stereoisomers and pharmaceutically acceptable salts thereof,
wherein R.sub.1 is hydroxyl, halogen, --OC(.dbd.O)R.sub.4 or
NHR.sub.4; R.sub.2 is hydrogen or --C(.dbd.O)R.sub.5; R.sub.3 is
--CH.sub.3 or .dbd.CH.sub.2; R.sub.4 is -X-aryl, -X-substituted
aryl, -X-arylalkyl, -X-substituted arylalkyl, X-heteroaryl, or
-X-heteroarylalkyl, wherein X is an optional amido or alkylamido
linker moiety; and R.sub.5 is alkyl.
95. The ANT ligand of claim 94 wheriein R.sub.1 is hydroxyl.
96. The ANT ligand of claim 94 wherein R.sub.1 is
--C(.dbd.O)R.sub.4.
97. The ANT ligand of claim 94 wherein R.sub.1 is --NHR.sub.4.
98. The ANT ligand of claim 94 wherein R.sub.2 is hydrogen.
99. The ANT ligand of claim 94 wherein R.sub.2 is
--C(.dbd.O)R.sub.5.
100. The ANT ligand of claim 94 wherein R.sub.3 is --CH.sub.3.
101. The ANT ligand of claim 94 wherein R.sub.3 is --CH.sub.2.
102. The ANT ligand of claim 94 wherien R.sub.4 is -X-aryl,
-X-substituted aryl, -X-arylalkyl or -X-substituted arylalkyl.
103. The ANT ligand of claim 95 wherein R.sub.5 is
--CH.sub.2CH(CH.sub.3).- sub.2.
104. An assay plate for high throughput screening of candidate
agents that bind to at least one ANT polypeptide, comprising: an
assay plate having a plurality of wells, each of said wells further
comprising at least one immobilized recombinant ANT polypeptide or
a variant or fragment thereof.
105. A method of targeting a polypeptide of interest to a
mitochondrial membrane, comprising: expressing a recombinant
expression construct encoding a fusion protein in a host cell, said
construct comprising at least one regulated promoter operably
linked to a nucleic acid molecule comprising a first nucleic acid
sequence and a second nucleic acid sequence, said first nucleic
acid sequence encoding an adenine nucleotide translocator
polypeptide that is expressed as a fusion protein with a
polypeptide product of said second nucleic acid sequence, wherein
said second nucleic acid sequence encodes the polypeptide of
interest.
106. A method of targeting a polypeptide of interest to a
mitochondrial membrane, comprising: expressing a recombinant
expression construct encoding a fusion protein in a host cell, said
construct comprising at least one promoter operably linked to a
nucleic acid molecule comprising a first nucleic acid sequence and
a second nucleic acid sequence, said first nucleic acid sequence
encoding an animal adenine nucleotide translocator polypeptide that
is expressed as a fusion protein with a polypeptide product of said
second nucleic acid sequence, wherein said second nucleic acid
sequence encodes the polypeptide of interest.
107. A pharmaceutical composition comprising an ANT ligand of claim
85.
108. A pharmaceutical composition comprising an ANT ligand of claim
94.
109. A pharmaceutical composition comprising an agent that binds to
an ANT polypeptide identified according to claim 75.
110. A pharmaceutical composition comprising an agent that binds to
an ANT polypeptide identified according to claim 83.
111. A pharmaceutical composition comprising an agent that
interacts with an ANT polypeptide identified according to claim
84.
112. A method of treatment comprising administering to a subject
the pharmaceutical composition of any one of claims 107-111.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/185,904, filed Nov. 3, 1998.
FIELD OF THE INVENTION
[0002] The invention relates to the adenine nucleotide translocator
(ANT) protein that is found in mitochondria of eukaryotic cells.
More particularly, the invention relates to the production of ANT
polypeptides and ANT fusion proteins using recombinant DNA
technology; to novel labeled ligands for ANT proteins; and to
assays useful for identifying and isolating ANT proteins and for
screening compounds that interact with ANT, including high
throughput screening.
BACKGROUND OF THE INVENTION
[0003] Mitochondria are the main energy source in cells of higher
organisms, and these organelles provide direct and indirect
biochemical regulation of a wide array of cellular respiratory,
oxidative and metabolic processes. These include electron transport
chain (ETC) activity, which drives oxidative phosphorylation to
produce metabolic energy in the form of adenosine triphosphate
(ATP), and which also underlies a central mitochondrial role in
intracellular calcium homeostasis.
[0004] Mitochondrial ultrastructural characterization reveals the
presence of an outer mitochondrial membrane that serves as an
interface between the organelle and the cytosol, a highly folded
inner mitochondrial membrane that appears to form attachments to
the outer membrane at multiple sites, and an intermembrane space
between the two mitochondrial membranes. The subcompartment within
the inner mitochondrial membrane is commonly referred to as the
mitochondrial matrix. (For a review, see, e.g., Ernster et al.,
1981 J. Cell Biol. 91:227s.) The cristae, originally postulated to
occur as infoldings of the inner mitochondrial membrane, have
recently been characterized using three-dimensional electron
tomography as also including tube-like conduits that may form
networks, and that can be connected to the inner membrane by open,
circular (30 nm diameter) junctions (Perkins et al., 1997, Journal
of Structural Biology 119:260). While the outer membrane is freely
permeable to ionic and non-ionic solutes having molecular weights
less than about ten kilodaltons, the inner mitochondrial membrane
exhibits selective and regulated permeability for many small
molecules, including certain cations, and is impermeable to large
(>.about.10 kDa) molecules.
[0005] Altered or defective mitochondrial activity, including but
not limited to failure at any step of the ETC, may result in
catastrophic mitochondrial collapse that has been termed
"permeability transition" (PT) or "mitochondrial permeability
transition" (MPT). According to generally accepted theories of
mitochondrial function, proper ETC respiratory activity requires
maintenance of an electrochemical potential (.DELTA..PSI.m) in the
inner mitochondrial membrane by a coupled chemiosmotic mechanism.
Altered or defective mitochondrial activity may dissipate this
membrane potential, thereby preventing ATP biosynthesis and halting
the production of a vital biochemical energy source. In addition,
mitochondrial proteins such as cytochrome c may leak out of the
mitochondria after permeability transition and may induce the
genetically programmed cell suicide sequence known as apoptosis or
programmed cell death (PCD).
[0006] MPT may result from direct or indirect effects of
mitochondrial genes, gene products or related downstream mediator
molecules and/or extramitochondrial genes, gene products or related
downstream mediators, or from other known or unknown causes. Loss
of mitochondrial potential therefore may be a critical event in the
progression of diseases associated with altered mitochondrial
function, including degenerative diseases.
[0007] Mitochondrial defects, which may include defects related to
the discrete mitochondrial genome that resides in mitochondrial DNA
and/or to the extramitochondrial genome, which includes nuclear
chromosomal DNA and other extramitochondrial DNA, may contribute
significantly to the pathogenesis of diseases associated with
altered mitochondrial function. For example, alterations in the
structural and/or functional properties of mitochondrial components
comprised of subunits encoded directly or indirectly by
mitochondrial and/or extramitochondrial DNA, including alterations
deriving from genetic and/or environmental factors or alterations
derived from cellular compensatory mechanisms, may play a role in
the pathogenesis of any disease associated with altered
mitochondrial function. A number of degenerative diseases are
thought to be caused by, or to be associated with, alterations in
mitochondrial function. These include Alzheimer's Disease (AD);
diabetes mellitus; Parkinson's Disease; Huntington's disease;
dystonia; Leber's hereditary optic neuropathy; schizophrenia;
mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS);
cancer; psoriasis; hyperproliferative disorders; mitochondrial
diabetes and deafness (MIDD) and myoclonic epilepsy ragged red
fiber syndrome. The extensive list of additional diseases
associated with altered mitochondrial function continues to expand
as aberrant mitochondrial or mitonuclear activities are implicated
in particular disease processes.
[0008] A hallmark pathology of AD and potentially other diseases
associated with altered mitochondrial function is the death of
selected cellular populations in particular affected tissues, which
results from apoptosis (also referred to as "programmed cell death"
or PCD) according to a growing body of evidence. Mitochondrial
dysfunction is thought to be critical in the cascade of events
leading to apoptosis in various cell types (Kroemer et al., FASEB
J. 9:1277-87, 1995), and may be a cause of apoptotic cell death in
neurons of the AD brain. Altered mitochondrial physiology may be
among the earliest events in PCD (Zamzami et al., J. Exp. Med.
182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995)
and elevated reactive oxygen species (ROS) levels that result from
such altered mitochondrial function may initiate the apoptotic
cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994).
[0009] Thus, in addition to their role in energy production in
growing cells, mitochondria (or, at least, mitochondrial
components) participate in apoptosis (Newmeyer et al., 1994, Cell
79:353-364; Liu et al., 1996, Cell 86:147-157). Apoptosis is
apparently also required for, inter alia, normal development of the
nervous system and proper functioning of the immune system.
Moreover, some disease states are thought to be associated with
either insufficient (e.g., cancer, autoimmune diseases) or
excessive (e.g., stroke damage, AD-associated neurodegeneration)
levels of apoptosis. For general reviews of apoptosis, and the role
of mitochondria therein, see Green and Reed (1998, Science
281:1309-1312), Green (1998, Cell 94:695-698) and Kromer (1997,
Nature Medicine 3:614-620). Hence, agents that effect apoptotic
events, including those associated with mitochondrial components,
might have a variety of palliative, prophylactic and therapeutic
uses.
[0010] The adenine nucleotide translocator (ANT), a nuclear encoded
polypeptide that is a major component of the inner mitochondrial
membrane, is responsible for mediating transport of ADP and ATP
across the mitochondrial inner membrane. For example, ANT is
believed to mediate stoichiometric ATP/proton exchange across the
inner mitochondrial membrane, and ANT inhibitors (such as
atractyloside or bongkrekic acid) induce MPT under certain
conditions. Three human ANT isoforms have been described that
differ in their tissue expression patterns and other mammalian ANT
homologues have been described. (See, e.g., Wallace et al., 1998 in
Mitochondria & Free Radicals in Neurodegenerative Diseases,
Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp.
283-307, and references cited therein.) ANT has also been
implicated as an important molecular component of the mitochondrial
permeability transition pore, a Ca.sup.2+-regulated inner membrane
channel that, as described above, plays an important modulating
role in apoptotic processes.
[0011] As inner mitochondrial membrane proteins are believed to
possess multiple hydrophobic membrane spanning domains, ANT
polypeptides may exhibit, inter alia, poor intracellular
solubility, toxic accumulations and/or the formation of inclusion
bodies and other deleterious effects on respiratory homeostasis
within a host cell due to ANT biological activity. Consequently,
those having ordinary skill in the art have heretofore been unable
to produce ANT reliably or in sufficient quantities for a variety
of uses, such as those provided herein. Because of the significance
of mitochondria to respiratory, metabolic and apoptotic processes,
and in view of the prominent role played by ANT in these and other
mitochondrial acitivities, there is clearly a need for compositions
and methods that permit the production of ANT proteins, including
ANT fusion proteins; for novel ANT ligands; for methods to identify
and isolate ANT proteins; and for methods of identifying and
isolating agents that interact with ANT.
[0012] The present invention fulfills these needs and provides
other related advantages. These and other aspects of the present
invention will become evident upon reference to the following
detailed description and attached drawings. In addition, various
references are set forth below which describe in more detail
certain procedures or compositions (e.g., plasmids, vectors, etc.),
and are therefore incorporated by reference in their
entireties.
SUMMARY OF THE INVENTION
[0013] In its various aspects and embodiments the invention is
directed to:
[0014] A recombinant expression construct comprising at least one
regulated promoter operably linked to a first nucleic acid encoding
an adenine nucleotide translocator polypeptide; further comprising
at least one additional nucleic acid sequence that regulates
transcription; wherein the additional nucleic acid sequence that
regulates transcription encodes a repressor of said regulated
promoter; wherein the adenine nucleotide translocator polypeptide
comprises a human adenine nucleotide translocator polypeptide;
wherein the human adenine nucleotide translocator polypeptide is
ANT1; wherein the human adenine nucleotide translocator polypeptide
is ANT2; wherein the human adenine nucleotide translocator
polypeptide is ANT3; wherein the adenine nucleotide translocator
polypeptide is expressed as a fusion protein with a polypeptide
product of a second nucleic acid sequence; wherein the polypeptide
product of said second nucleic acid sequence is an enzyme; wherein
said fusion protein localizes to membranes; wherein said membranes
are mitochondrial membranes; wherein the adenine nucleotide
translocator polypeptide is expressed as a fusion protein with at
least one product of a second nucleic acid sequence encoding a
polypeptide cleavable by a protease, said adenine nucleotide
translocator polypeptide being separable from the fusion protein by
cleavage with the protease; A host cell comprising a recombinant
expression construct as provided; wherein the host cell is a
prokaryotic cell; wherein the host cell is a eukaryotic cell;
wherein the eukaryotic cell is selected from the group consisting
of a yeast cell, an insect cell and a mammalian cell; wherein the
insect cell is an Sf9 cell or a Trichoplusia ni cell; at lacks at
least one isoform of an endogenous adenine nucleotide translocator;
in which expression of at least one gene encoding an endogenous
adenine nucleotide translocator isoform is substantially
impaired.
[0015] A recombinant expression construct comprising at least one
promoter operably linked to a nucleic acid molecule comprising a
first nucleic acid sequence and a second nucleic acid sequence,
said first nucleic acid sequence encoding an animal adenine
nucleotide translocator polypeptide wherein the adenine nucleotide
translocator polypeptide is expressed as a fusion protein with a
polypeptide product of said second nucleic acid sequence; wherein
the polypeptide product of said second nucleic acid sequence is an
enzyme; wherein said fusion protein localizes to membranes; wherein
said membranes are mitochondrial membranes; further comprising at
least one additional nucleic acid sequence that regulates
transcription; wherein the additional nucleic acid sequence that
regulates transcription encodes a repressor of said promoter;
wherein the adenine nucleotide translocator polypeptide comprises a
human adenine nucleotide translocator polypeptide; wherein the
human adenine nucleotide translocator polypeptide is ANT1; wherein
the human adenine nucleotide translocator polypeptide is ANT2;
wherein the human adenine nucleotide translocator polypeptide is
ANT3; wherein the adenine nucleotide translocator polypeptide is
expressed as a fusion protein with at least one product of a second
nucleic acid sequence encoding a polypeptide cleavable by a
protease, said adenine nucleotide translocator polypeptide being
separable from the fusion protein by cleavage with the protease; a
host cell comprising a recombinant expression construct as just
described; wherein the host cell is a prokaryotic cell; wherein the
host cell is a eukaryotic cell; wherein the eukaryotic cell is
selected from the group consisting of a yeast cell, an insect cell
and a mammalian cell; wherein the insect cell is an Sf9 cell or a
Trichoplusia ni cell; that lacks at least one isoform of an
endogenous adenine nucleotide translocator; in which expression of
at least one gene encoding an endogenous adenine nucleotide
translocator isoform is substantially impaired; wherein the
expression construct is a recombinant viral expression
construct;
[0016] A method of producing a recombinant adenine nucleotide
translocator polypeptide, comprising; culturing a host cell
comprising a recombinant expression construct comprising at least
one regulated promoter operably linked to a first nucleic acid
encoding an adenine nucleotide translocator polypeptide;
[0017] A method of producing a recombinant adenine nucleotide
translocator polypeptide, comprising culturing a host cell
comprising a recombinant expression construct comprising at least
one promoter operably linked to a nucleic acid molecule comprising
a first nucleic acid sequence and a second nucleic acid sequence,
said first nucleic acid sequence encoding an animal adenine
nucleotide translocator polypeptide wherein the adenine nucleotide
translocator polypeptide is expressed as a fusion protein with a
polypeptide product of said second nucleic acid sequence;
[0018] A method of producing a recombinant adenine nucleotide
translocator polypeptide, comprising culturing a host cell infected
with the recombinant viral expression construct as provided
above.
[0019] An ANT polypeptide produced by the methods just
described.
[0020] An isolated human adenine nucleotide translocator
polypeptide; wherein the human adenine nucleotide translocator
polypeptide is recombinant ANT1 or a variant or fragment thereof;
wherein the human adenine nucleotide translocator polypeptide is
recombinant ANT2 or a variant or fragment thereof, wherein the
human adenine nucleotide translocator polypeptide is recombinant
ANT3 or a variant or fragment thereof;
[0021] An isolated human adenine nucleotide translocator fusion
protein comprising an adenine translocator polypeptide fused to at
least one additional polypeptide sequence; wherein said one
additional polypeptide sequence is an enzyme sequence or a variant
or fragment thereof; wherein said fusion protein localizes to
membranes; wherein said membranes are mitochondrial membranes;
[0022] An isolated human adenine nucleotide translocator fusion
protein comprising an adenine translocator polypeptide fused to at
least one additional polypeptide sequence cleavable by a protease,
said adenine nucleotide translocator polypeptide being separable
from the fusion protein by cleavage with the protease.
[0023] An isolated adenine nucleotide translocator fusion protein
comprising a first polypeptide that is an animal adenine
translocator polypeptide fused to at least one additional
polypeptide sequence; wherein said one additional polypeptide
sequence is an enzyme sequence or a variant or fragment thereof;
that localizes to membranes; wherein said membranes are
mitochondrial membranes.
[0024] An isolated recombinant animal adenine nucleotide
translocator fusion protein comprising an adenine translocator
polypeptide fused to at least one additional polypeptide sequence
cleavable by a protease, said adenine nucleotide translocator
polypeptide being separable from the fusion protein by cleavage
with the protease; wherein the additional polypeptide sequence is a
polypeptide having affinity for a ligand.
[0025] A method for determining the presence of an ANT polypeptide
in a biological sample comprising contacting a biological sample
suspected of containing an ANT polypeptide with an ANT ligand under
conditions and for a time sufficient to allow binding of the ANT
ligand to an ANT polypeptide; and detecting the binding of the ANT
ligand to an ANT polypeptide, and therefrom determining the
presence of an ANT polypeptide in said biological sample; wherein
the adenine nucleotide translocator polypeptide comprises a human
adenine nucleotide translocator polypeptide; wherein the human
adenine nucleotide translocator polypeptide is ANT1; wherein the
human adenine nucleotide translocator polypeptide is ANT2; wherein
the human adenine nucleotide translocator polypeptide is ANT3;
wherein the ANT ligand comprises atractyloside substituted at 6'
hydroxyl to form an atractyloside derivative; wherein the
atractyloside is detectably substituted at the 6' hydroxyl to form
a detectable atractyloside derivative; wherein the detectable
atractyloside derivative comprises a radioloabeled substituent;
wherein the radiolabeled substituent is selected from the group
consisting of .sup.125I, .sup.131I, .sup.3H, .sup.14C and .sup.35S;
wherein the detectable atractyloside derivative comprises a
fluorescent substituent; wherein the ANT ligand further comprises a
Eu.sup.3+ atom complexed to the atractyloside derivative; wherein
the detectable atractyloside derivative comprises covalently bound
biotin; wherein the atractyloside molecule is substituted at 6'
hydroxyl with an amine or an amine containing functionality to form
an amine modified atractyloside derivative; wherein the
atractyloside molecule is a carboxyatractyloside molecule that is
substituted at 6' hydroxyl to form an atractyloside derivative that
is a carboxyatractyloside derivative.
[0026] A method for isolating ANT from a biological sample,
comprising contacting a biological sample suspected of containing
an ANT polypeptide with an ANT ligand under conditions and for a
time sufficient to allow binding of the ANT ligand to an ANT
polypeptide; and recovering the ANT polypeptide, and thereby
isolating ANT from a biological sample; wherein the ANT ligand is
covalently bound to a solid phase; wherein the ANT ligand is
non-covalently bound to a solid phase.
[0027] A method for identifying an agent that binds to an ANT
polypeptide, comprising contacting a candidate agent with a host
cell expressing at least one recombinant ANT polypeptide under
conditions and for a time sufficient to permit binding of the agent
to said recombinant ANT polypeptide; and detecting binding of said
agent to the recombinant ANT; wherein the host cell is a
prokaryotic cell; wherein the prokaryotic cell is an E. coli cell;
wherein the host cell is a eukaryotic cell; wherein the eukaryotic
cell is selected from the group consisting of a yeast cell, an
insect cell and a mammalian cell; wherein the insect cell is an Sf9
cell or a Trichoplusia ni cell; wherein the host cell lacks at
least one isoform of an endogenous adenine nucleotide translocator;
wherein host cell expression of at least one gene encoding an
endogenous adenine nucleotide translocator isoform is substantially
impaired.
[0028] A method for identifying an agent that binds to an ANT
polypeptide, comprising contacting a candidate agent with a
biological sample containing at least one recombinant ANT
polypeptide under conditions and for a time sufficient to permit
binding of the agent to said ANT polypeptide; and detecting binding
of said agent to the recombinant ANT polypeptide.
[0029] A method for identifying an agent that interacts with an ANT
polypeptide comprising contacting a biological sample containing
recombinant ANT with a detectable ANT ligand in the presence of a
candidate agent; and comparing binding of the detectable ANT ligand
to recombinant ANT in the absence of said agent to binding of the
detectable ANT ligand to recombinant ANT in the presence of said
agent, and therefrom identifying an agent that interacts with an
ANT polypeptide.
[0030] An ANT ligand comprising atractyloside substituted at the 6'
hydroxyl to form an atractyloside derivative; wherein the
atractyloside is detectably substituted at the 6' hydroxyl to form
a detectable atractyloside derivative; wherein the detectable
atractyloside derivative comprises a radioloabeled substituent;
wherein the radiolabeled substituent is selected from the group
consisting of .sup.125I, .sup.131I, .sup.3H, .sup.14C and .sup.35S;
wherein the detectable atractyloside derivative comprises a
fluorescent substituent; further comprising a Eu.sup.3+ atom
complexed to the atractyloside derivative; wherein the detectable
atractyloside derivative comprises covalently bound biotin; wherein
the atractyloside molecule is substituted at 6' hydroxyl with an
amine or an amine containing functionality to form an amine
modified atractyloside derivative; wherein the atractyloside
molecule is a carboxyatractyloside molecule that is substituted at
6' hydroxyl to form an atractyloside derivative that is a
carboxyatractyloside derivative.
[0031] An ANT ligand having the following structure (I): 1
[0032] including stereoisomers and pharmaceutically acceptable
salts thereof, wherein R.sub.1, R.sub.2 and R.sub.3 are as
identified below.
[0033] An assay plate for high throughput screening of candidate
agents that bind to at least one ANT polypeptide, comprising an
assay plate having a plurality of wells, each of said wells further
comprising at least one immobilized recombinant ANT polypeptide or
a variant or fragment thereof.
[0034] A method of targeting a polypeptide of interest to a
mitochondrial membrane, comprising expressing a recombinant
expression construct encoding a fusion protein in a host cell, said
construct comprising at least one regulated promoter operably
linked to a nucleic acid molecule comprising a first nucleic acid
sequence and a second nucleic acid sequence, said first nucleic
acid sequence encoding an adenine nucleotide translocator
polypeptide that is expressed as a fusion protein with a
polypeptide product of said second nucleic acid sequence, wherein
said second nucleic acid sequence encodes the polypeptide of
interest.
[0035] A method of targeting a polypeptide of interest to a
mitochondrial membrane, comprising expressing a recombinant
expression construct encoding a fusion protein in a host cell, said
construct comprising at least one promoter operably linked to a
nucleic acid molecule comprising a first nucleic acid sequence and
a second nucleic acid sequence, said first nucleic acid sequence
encoding an animal adenine nucleotide translocator polypeptide that
is expressed as a fusion protein with a polypeptide product of said
second nucleic acid sequence, wherein said second nucleic acid
sequence encodes the polypeptide of interest; a pharmaceutical
composition comprising an ANT ligand as just described.
[0036] A pharmaceutical composition comprising an agent that binds
to an ANT polypeptide identified as just described. A
pharmaceutical composition comprising an agent that binds to an ANT
polypeptide identified as described above. A pharmaceutical
composition comprising an agent that interacts with an ANT
polypeptide identified above. A method of treatment comprising
administering to a subject any one of the just described the
pharmaceutical compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows the nucleotide sequences of the coding regions
of human ANT1 ("ANT1m"), human ANT2 ("ANT2m") and human ANT3
("ANT3m").
[0038] FIG. 2 shows the polypeptide sequences of human ANT1
("ANT1p"), human ANT2 ("ANT2p") and human ANT3 ("ANT3p").
[0039] FIG. 3 shows induction of His-Tagged, XPRESS.TM.-epitope
containg huANT3 protein in E. coli as determined by Western
analysis.
[0040] FIG. 4 shows the localization of His-Tagged,
XPRESS.TM.-epitope containg huANT3 protein in E. coli as determined
by Western analysis.
[0041] FIG. 5 shows the expression of human ANT3 (huANT3) in E.
coli expression systems.
[0042] FIG. 6 shows the expression of huANT3 in
baculovirus-infected Sf9 cells.
[0043] FIG. 7 shows [.sup.32P]ATP binding to Sf9/huANT3
mitochondria.
[0044] FIG. 8 shows that ATP and atractyloside bind competitively
to Sf9/huANT3 mitochondria.
[0045] FIG. 9 shows high-affinity binding of atractyloside to
Sf9/huANT3 mitochondria.
[0046] FIG. 10 shows Northern blot analysis of huANT3 transcripts
detected in a yeast expression system. Lane contents: lane "M,"
molecular weight markers (positions of 1.4, 2.4, 4.4 and 7.5
kilobase markers indicated); lanes 1-3, 10 .mu.g of RNA from three
independent isolates of mock transformed AAC.sup.- yeast; lanes
4-6, 10 .mu.g of RNA from three independent isolates of AAC.sup.-
yeast transformed with pMK5C (pYPGE2-hANT3); lanes 7-9, 10 .mu.g of
RNA from three independent isolates of AAC.sup.- yeast transformed
with pMK5B (pYESTrp2-hANT3); lanes 10 and 11, 0.2 (lane 10) and 0.8
(lane 11) .mu.g of RNA prepared from samples of human spleen.
[0047] FIG. 11 shows binding of .sup.125I-compound 24 to bovine
mitochondria. Symbols: (.tangle-soliddn.), bovine mitochondria;
(.box-solid.), control (no mitochondria).
[0048] FIG. 12 shows binding of .sup.125I-compound 24 to
mitochondria comprising recombinant huANT3. Symbols:
(.tangle-soliddn.), mitochondria from T. ni cells expressing
huANT3; (.box-solid.), control (no mitochondria).
[0049] FIG. 13 shows competition of .sup.125I-compound 24 binding
to bovine mitochondria by unlabeled compound 24(.tangle-soliddn.),
ATR (.box-solid.) and ADP (.tangle-solidup.).
[0050] FIG. 14 shows competition of .sup.125I-compound 24 binding
to mitochondria from T. ni cells expressing huANT3 by unlabeled
compound 24 (.tangle-soliddn., dashed line), ATR (.box-solid.,
solid line) and ADP (.tangle-solidup.).
[0051] FIG. 15 shows competition of .sup.125I-compound 24 binding
by unlabeled ATR to mitochondria from T. ni cells expressing huANT3
(.tangle-solidup.) and control (nontransformed) T. ni cells
(.diamond-solid.).
[0052] FIG. 16 shows competition of .sup.125I-compound 24 binding
to beef heart mitochondria by (.box-solid.) BKA and () unlabeled
compound 24.
[0053] FIG. 17 shows competition of .sup.125I-compound 24 binding
to beef heart mitochondria by compound 23 (), compound 28
(.diamond-solid.) and ATR (.box-solid.).
[0054] FIG. 18 shows competition of .sup.125I-compound 24 binding
to beef heart mitochondria by compound 5 (.diamond-solid.) and ATR
(.box-solid.).
[0055] FIG. 19 shows competition of .sup.125I-compound 24 binding
to recombinant His-tagged huANT3 immobilized on Ni beads by BKA ()
and ATR (.box-solid.).
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention is directed generally toward adenine
nucleotide translocator (ANT) polypeptides, which as provided
herein may refer to any ANT isoform; to expression constructs
containing nucleic acids encoding ANT and to natural and synthetic
small molecules that interact with ANT, including ANT binding
ligands. The present invention relates in part to the unexpected
findings that bacterial, insect, yeast or mammalian expression
systems can be designed for reliable production of recombinant
human ANT polypeptides in significant quantities. In certain
aspects the invention provides compositions and methods for
producing recombinant ANT polypeptides that employ regulated
promoters, and in certain of these and other aspects the invention
provides compositions and methods for producing recombinant ANT
polypeptides that are ANT fusion proteins. In certain preferred
embodiments, the design of such expression systems includes the use
of a host cell that lacks endogenous ANT or in which endogenous ANT
gene expression is substantially impaired, as provided herein.
[0057] The present invention thus also pertains in part to methods
for producing and isolating recombinant ANT polypeptides, including
human ANT polypeptides and in preferred embodiments human ANT3
polypeptides, that may then be used in various binding assays and
screening assays and the like. In view of the surprising
observation that expression of recombinant human ANT polypeptides
can be achieved at levels enabling such uses of these ANT
polypeptide products, the present invention provides assays
(including high throughput assays) for identifying agents that bind
to recombinant human ANT. Accordingly, the present invention
further relates in part to novel human ANT ligands, the synthesis,
selection and characterization of which would heretofore have not
been possible given the need for expressed recombinant ANT
polypeptides to use in binding assays. The invention also pertains
to agents that interact with ANT, including agents that enhance or
impair any ANT functions known to the art, including but not
limited to those described herein, and to incorporation of such
agents into pharmaceutical compositions and their use in
therapeutic methods.
[0058] As dicussed above, the present invention relates in part to
the unexpected finding that recombinant adenine nucleotide
translocator (ANT) polypeptides, which includes full length ANT
proteins and polypeptides, fragments and variants thereof, and
further includes ANT fusion proteins as provided herein, can be
produced in useful amounts by using a recombinant expression vector
having a regulatory nucleic acid operably linked to a nucleic acid
encoding ANT. In particular, the invention provides compositions
and methods for producing recombinant ANT polypeptides through the
use of a regulated promoter; the invention also provides
recombinant ANT polypeptides that are ANT fusion proteins.
[0059] The invention also pertains to compositions and methods to
identify the presence of ANT polypeptides and to isolate
recombinant ANT, and in addition to screening assays for compounds
that interact with ANT. Accordingly, the invention provides certain
advantages with regard to regulation of mitochondrial function, and
in particular regulation of the mitochondrial permeability "pore".
By way of background, four of the five multisubunit protein
complexes (Complexes I, III, IV and V) that mediate ETC activity
are localized to the inner mitochondrial membrane, which is the
most protein rich of biological membranes in cells (75% by weight);
the remaining ETC complex (Complex II) is situated in the matrix.
ANT represents the most abundant of the inner mitochondrial
membrane proteins. In at least three distinct chemical reactions
known to take place within the ETC, positively-charged protons are
moved from the mitochondrial matrix, across the inner membrane, to
the intermembrane space. This disequilibrium of charged species
creates an electrochemical potential of approximately 220 mV
referred to as the "protonmotive force" (PMF), which is often
represented by the notation .DELTA..psi. or .DELTA..psi.m and
represents the sum of the electric potential and the pH
differential across the inner mitochondrial membrane (see, e.g.,
Ernster et al., 1981 J. Cell Biol. 91:227s and references cited
therein).
[0060] This membrane potential drives ANT-mediated stoichiometric
exchange of adenosine triphosphate (ATP) and adenosine diphosphate
(ADP) across the inner mitochondrial membrane, and provides the
energy contributed to the phosphate bond created when ADP is
phosphorylated to yield ATP by ETC Complex V, a process that is
"coupled" stoichiometrically with transport of a proton into the
matrix. Mitochondrial membrane potential, .DELTA..psi.m, is also
the driving force for the influx of cytosolic Ca.sup.2+ into the
mitochondrion. Under normal metabolic conditions, the inner
membrane is impermeable to proton movement from the intermembrane
space into the matrix, leaving ETC Complex V as the sole means
whereby protons can return to the matrix. When, however, the
integrity of the inner mitochondrial membrane is compromised, as
occurs during MPT that may accompany a disease associated with
altered mitochondrial function, protons are able to bypass the
conduit of Complex V without generating ATP, thereby "uncoupling"
respiration because electron transfer and associated proton pumping
yields no ATP. Thus, mitochondrial permeability transition involves
the opening of a mitochondrial membrane "pore", a process by which,
inter alia, the ETC and ATP synthesis are uncoupled, .DELTA..psi.m
collapses and mitochondrial membranes lose the ability to
selectively regulate permeability to solutes both small (e.g.,
ionic Ca.sup.2+, Na.sup.+, K.sup.+, H.sup.+) and large (e.g.,
proteins).
[0061] Without wishing to be bound by theory, it is unresolved
whether this pore is a physically discrete conduit that is formed
in mitochondrial membranes, for example by assembly or aggregation
of particular mitochondrial and/or cytosolic proteins and possibly
other molecular species, or whether the opening of the "pore" may
simply represent a general increase in the porosity of the
mitochondrial membrane.
[0062] MPT may also be induced by compounds that bind one or more
mitochondrial molecular components. Such compounds include, but are
not limited to, atractyloside and bongkrekic acid, which are known
to bind to ANT. Methods of determining appropriate amounts of such
compounds to induce MPT are known in the art (see, e.g., Beutner et
al., 1998 Biochim. Biophys. Acta 1368:7; Obatomi and Bach, 1996
Toxicol. Lett. 89:155; Green and Reed, 1998 Science 281:1309;
Kroemer et al., 1998 Annu. Rev. Physiol. 60:619; and references
cited therein). Thus certain mitochondrial molecular components,
such as ANT, may contribute to the MPT mechanism. As noted above,
it is believed that adenine nucleotide translocator (ANT) mediates
ATP/proton exchange across the inner mitochondrial membrane, and
that ANT inhibitors such as atractyloside or bongkrekic acid induce
MPT under certain conditions. Hence, it is desirable to obtain ANT
in sufficient quantities for structural and functional assays that
provide, for example, ANT ligands and other agents that interact
with ANT, which will be useful for therapeutic management of
mitochondrial pore activity. See also U.S. Ser. No. 09/161,172,
entitled "Compositions and Methods for Identifying Agents that
Alter Mitochondrial Permeability Transition Pores", which is hereby
incorporated by reference.
[0063] The compositions and methods of the present invention can be
adapted to any prokaryotic or eukaryotic ANT, including plant and
animal ANTs, which may further include, for example, yeast,
vertebrate, mammalian, rodent, primate and human ANTs, for which
amino acid sequences and/or encoding nucleic acids will be known to
those familiar with the art. Three human ANT isoforms have been
described that differ in their tissue expression patterns. (Stepien
et al., 1992 J. Biol. Chem. 267:14592; see also Wallace et al.,
1998 in Mitochondria & Free Radicals in Neurodegenerative
Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New
York, pp. 283-307, and references cited therein.) Nucleic acid
sequences for cDNAs encoding these three human ANT isoforms have
been reported (FIG. 1; See Neckelmann et al., Proc. Nat'l. Acad.
Sci. U.S.A. 84:7580-7584 (1987) for huANT1 cDNA [SEQ ID NO:1];
Battini et al., J. Biol. Chem. 262:4355-4359 (1987) for huANT2 cDNA
[SEQ ID NO:2], and Cozens et al., J. Mol. Biol. 206:261-280 (1989)
for huANT3 cDNA [SEQ ID NO:3]; see FIG. 2 for amino acid sequences
of huANT1 [SEQ ID NO:31] huANT2 [SEQ ID NO:32] and huANT3 [SEQ ID
NO:33].), and ANT gene sequences have been determined for a number
of species (See, e.g., Li et al., 1989 J. Biol. Chem. 264:13998 for
huANT1 genomic DNA; Liew et al. GenBank Acc. #N86710 for huANT2;
Shinohara et al., 1993 Biochim. Biophys. Acta 1152:192 for rat ANT
gene; for others see also, e.g., Ku et al., 1990 J. Biol. Chem.
265:16060; Adams et al., 1991 Science 252:1651; and WO 98/19714.).
ANT sequences among mammalian species are highly conserved; for
example, at the amino acid level murine ANT1 and ANT2 exhibit 98%
sequence identity with human ANT2. Full length amino acid sequences
of at least 29 ANT proteins have been reported to date from a
variety of animal and plant species, with most of these deduced
from nucleic acid sequences. (Fiore et al., 1998 Biochimie
80:137-150)
[0064] The present invention further relates to nucleic acids which
hybridize to ANT encoding polynucleotide sequences as provided
herein, as incorporated by reference or as will be readily apparent
to those familiar with the art, if there is at least 70%,
preferably at least 90%, and more preferably at least 95% identity
between the sequences. The present invention particularly relates
to nucleic acids which hybridize under stringent conditions to the
ANT encoding nucleic acids referred to herein. As used herein, the
term "stringent conditions" means hybridization will occur only if
there is at least 95% and preferably at least 97% identity between
the sequences. The nucleic acids which hybridize to ANT encoding
nucleic acids referred to herein, in preferred embodiments, encode
polypeptides which either retain substantially the same biological
function or activity as the ANT polypeptides encoded by the cDNAs
of FIG. 1 [SEQ ID NOS:1, 2 and 3], or the deposited expression
constructs.
[0065] As used herein, to "hybridize" under conditions of a
specified stringency is used to describe the stability of hybrids
formed between two single-stranded nucleic acid molecules.
Stringency of hybridization is typically expressed in conditions of
ionic strength and temperature at which such hybrids are annealed
and washed. Typically "high", "medium" and "low" stringency
encompass the following conditions or equivalent conditions
thereto: high stringency: 0.1.times.SSPE or SSC, 0.1% SDS,
65.degree. C.; medium stringency: 0.2.times.SSPE or SSC, 0.1% SDS,
50.degree. C.; and low stringency: 1.0.times.SSPE or SSC, 0.1% SDS,
50.degree. C.
[0066] The deposits referred to herein will be maintained under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Micro-organisms for purposes of Patent Procedure.
These deposits are provided merely as convenience to those of skill
in the art and are not an admission that a deposit is required
under 35 U.S.C. .sctn. 112. The sequences of the nucleic acids
contained in the deposited materials, as well as the amino acid
sequences of the polypeptides encoded thereby, are incorporated
herein by reference and are controlling in the event of any
conflict with any description of sequences herein. A licensee may
be required to make, use or sell the deposited materials, and no
such license is hereby granted.
NUCLEIC ACIDS
[0067] The nucleic acids of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. A coding sequence which encodes an
ANT polypeptide for use according to the invention may be identical
to the coding sequence known in the art for any given ANT, as
described above and, for example, as shown for human ANT1 [SEQ ID
NO:1], human ANT2 [SEQ ID NO:2] and human ANT3 [SEQ ID NO:3] in
FIG. 1, or to that of any deposited clone, or may be a different
coding sequence, which, as a result of the redundancy or degeneracy
of the genetic code, encodes the same ANT polypeptide as, for
example, the cDNAs of FIG. 1 or the deposited expression
constructs.
[0068] The nucleic acids which encode ANT polypeptides, for example
the human ANT polypeptides having the amino acid sequences of FIG.
2 [SEQ ID NOS:31-33] or any other ANT polypeptides for use
according to the invention, or for the ANT polypeptides encoded by
the deposited constructs may include, but are not limited to: only
the coding sequence for the ANT polypeptide; the coding sequence
for the ANT polypeptide and additional coding sequence; the coding
sequence for the ANT polypeptide (and optionally additional coding
sequence) and non-coding sequence, such as introns or non-coding
sequences 5' and/or 3' of the coding sequence for the ANT
polypeptide, which for example may further include but need not be
limited to one or more regulatory nucleic acid sequences that may
be a regulated or regulatable promoter, enhancer, other
transcription regulatory sequence, repressor binding sequence,
translation regulatory sequence or any other regulatory nucleic
acid sequence. Thus, the term "nucleic acid encoding an ANT
polypeptide" encompasses a nucleic acid which includes only coding
sequence for the polypeptide as well as a nucleic acid which
includes additional coding and/or non-coding sequence(s).
[0069] The present invention further relates to variants of the
herein described nucleic acids which encode for fragments, analogs
and derivatives of an ANT polypeptide, for example the human ANT1,
ANT2 and ANT3 polypeptides having the deduced amino acid sequences
of FIG. 2 [SEQ ID NOS:31-33] or any ANT polypeptide, including ANT
polypeptides encoded by the cDNAs of the deposited expression
constructs. The variants of the nucleic acids encoding ANTs may be
naturally occurring allelic variants of the nucleic acids or
non-naturally occurring variants. As is known in the art, an
allelic variant is an alternate form of a nucleic acid sequence
which may have at least one of a substitution, a deletion or an
addition of one or more nucleotides, any of which does not
substantially alter the function of the encoded ANT polypeptide.
Thus, for example, the present invention includes nucleic acids
encoding the same ANT polypeptides as shown in FIG. 2 [SEQ ID
NOS:31-33], or the same ANT polypeptides encoded by the cDNAs of
the deposited expression constructs, as well as variants of such
nucleic acids, which variants encode a fragment, derivative or
analog of any of the polypeptides of FIG. 2 (SEQ ID NO:2) or the
polypeptides encoded by the cDNAs of the deposited expression
constructs.
[0070] Variants and derivatives of ANT may be obtained by mutations
of nucleotide sequences encoding ANT polypeptides. Alterations of
the native amino acid sequence may be accomplished by any of a
number of conventional methods. Mutations can be introduced at
particular loci by synthesizing oligonucleotides containing a
mutant sequence, flanked by restriction sites enabling ligation to
fragments of the native sequence. Following ligation, the resulting
reconstructed sequence encodes an analog having the desired amino
acid insertion, substitution, or deletion.
[0071] Alternatively, oligonucleotide-directed site-specific
mutagenesis procedures can be employed to provide an altered gene
wherein predetermined codons can be altered by substitution,
deletion or insertion. Exemplary methods of making such alterations
are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al.
(Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);
Smith et al. (Genetic Engineering: Principles and Methods, Plenum
Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985);
Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Pat.
Nos. 4,518,584 and 4,737,462.
[0072] Equivalent DNA constructs that encode various additions or
substitutions of amino acid residues or sequences, or deletions of
terminal or internal residues or sequences not needed for
biological activity are also encompassed by the invention. For
example, sequences encoding Cys residues that are not essential for
biological activity can be altered to cause the Cys residues to be
deleted or replaced with other amino acids, preventing formation of
incorrect intramolecular disulfide bridges upon renaturation. Other
equivalents can be prepared by modification of adjacent dibasic
amino acid residues to enhance expression in yeast systems in which
KEX2 protease activity is present. EP 212,914 discloses the use of
site-specific mutagenesis to inactivate KEX2 protease processing
sites in a protein. KEX2 protease processing sites are inactivated
by deleting, adding or substituting residues to alter Arg-Arg,
Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these
adjacent basic residues. Lys-Lys pairings are considerably less
susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg
to Lys-Lys represents a conservative and preferred approach to
inactivating KEX2 sites.
POLYPEPTIDES AND FUSION PROTEINS
[0073] The present invention further relates to ANT polypeptides,
and in particular to methods for producing recombinant ANT
polypeptides by culturing host cells containing ANT expression
constructs, and to isolated recombinant human ANT polypeptides,
including, for example, the human ANT1, ANT2 and ANT3 polypeptides
which have the deduced amino acid sequence of FIG. 2 [SEQ ID
NOS:31-33] or which have the amino acid sequence encoded by the
deposited recombinant expression constructs, as well as fragments,
analogs and derivatives of such polypeptides The polypeptides and
nucleic acids of the present invention are preferably provided in
an isolated form, and in certain preferred embodiments are purified
to homogeneity.
[0074] The terms "fragment," "derivative" and "analog" when
referring to ANT polypeptides or fusion proteins, or to ANT
polypeptides or fusion proteins encoded by the deposited
recombinant expression constructs, refers to any ANT polypeptide or
fusion protein that retains essentially the same biological
function or activity as such polypeptide. Thus, an analog includes
a proprotein which can be activated by cleavage of the proprotein
portion to produce an active ANT polypeptide. The polypeptide of
the present invention may be a recombinant polypeptide or a
synthetic polypeptide, and is preferably a recombinant
polypeptide.
[0075] A fragment, derivative or analog of an ANT polypeptide or
fusion protein, including ANT polypeptides or fusion proteins
encoded by the cDNAs of the deposited constructs, may be (i) one in
which one or more of the amino acid residues are substituted with a
conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the ANT polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which additional amino acids are fused to the ANT
polypeptide, including amino acids that are employed for
purification of the ANT polypeptide or a proprotein sequence. Such
fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0076] The polypeptides of the present invention include ANT
polypeptides and fusion proteins having amino acid sequences that
are identical or similar to sequences known in the art. For example
by way of illustration and not limitation, the human ANT ("huANT")
polypeptides of FIG. 2 [SEQ ID NOS:31-33] are contemplated for use
according to the instant invention, as are polypeptides having at
least 70% similarity (preferably a 70% identity) to the
polypeptides of FIG. 2 [SEQ ID NOS:31-33] and more preferably 90%
similarity (more preferably a 90% identity) to the polypeptides of
FIG. 2 [SEQ ID NOS: 31-33] and still more preferably a 95%
similarity (still more preferably a 95% identity) to the
polypeptides of FIG. 2 [SEQ ID NOS:31-33] and to portions of such
polypeptides, wherein such portions of an ANT polypeptide generally
contain at least 30 amino acids and more preferably at least 50
amino acids.
[0077] As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and conserved amino
acid substitutes thereto of the polypeptide to the sequence of a
second polypeptide. Fragments or portions of the polypeptides of
the present invention may be employed for producing the
corresponding full-length polypeptide by peptide synthesis;
therefore, the fragments may be employed as intermediates for
producing the full-length polypeptides. Fragments or portions of
the nucleic acids of the present invention may be used to
synthesize full-length nucleic acids of the present invention.
[0078] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally occurring nucleic
acid or polypeptide present in a living animal is not isolated, but
the same nucleic acid or polypeptide, separated from some or all of
the co-existing materials in the natural system, is isolated. Such
nucleic acids could be part of a vector and/or such nucleic acids
or polypeptides could be part of a composition, and still be
isolated in that such vector or composition is not part of its
natural environment.
[0079] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region "leader and trailer" as well as
intervening sequences (introns) between individual coding segments
(exons).
[0080] As described herein, the invention provides ANT fusion
proteins encoded by nucleic acids that have the ANT coding sequence
fused in frame to an additional coding sequence to provide for
expression of an ANT polypeptide sequence fused to an additional
functional or non-functional polypeptide sequence that permits, for
example by way of illustration and not limitation, detection,
isolation and/or purification of the ANT fusion protein. Such ANT
fusion proteins may permit detection, isolation and/or purification
of the ANT fusion protein by protein-protein affinity, metal
affinity or charge affinity-based polypeptide purification, or by
specific protease cleavage of a fusion protein containing a fusion
sequence that is cleavable by a protease such that the ANT
polypeptide is separable from the fusion protein.
[0081] Thus ANT fusion proteins may comprise polypeptide sequences
added to ANT to facilitate detection and isolation of ANT. Such
peptides include, for example, poly-His or the antigenic
identification peptides described in U.S. Pat. No. 5,011,912 and in
Hopp et al., (1988 Bio/Technology 6:1204), or the XPRESS.TM.
epitope tag (Invitrogen, Carlsbad, Calif.). The affinity sequence
may be a hexa-histidine tag as supplied, for example, by a pBAD/His
(Invitrogen) or a pQE-9 vector to provide for purification of the
mature polypeptide fused to the marker in the case of a bacterial
host, or, for example, the affinity sequence may be a hemagglutinin
(HA) tag when a mammalian host, e.g., COS-7 cells, is used. The HA
tag corresponds to an antibody defined epitope derived from the
influenza hemagglutinin protein (Wilson et al., Cell 37:767
(1984)).
[0082] ANT fusion proteins may further comprise immunoglobulin
constant region polypeptides added to ANT to facilitate detection,
isolation and/or localization of ANT. The immunoglobulin constant
region polypeptide preferably is fused to the C-terminus of an ANT
polypeptide. General preparation of fusion proteins comprising
heterologous polypeptides fused to various portions of
antibody-derived polypeptides (including the Fc domain) has been
described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991) and
Byrn et al. (Nature 344:677, 1990). A gene fusion encoding the
ANT:Fc fusion protein is inserted into an appropriate expression
vector. In certain embodiments of the invention, ANT:Fc fusion
proteins may be allowed to assemble much like antibody molecules,
whereupon interchain disulfide bonds form between Fc polypeptides,
yielding dimeric ANT fusion proteins.
[0083] ANT fusion proteins having specific binding affinities for
pre-selected antigens by virtue of fusion polypeptides comprising
immunoglobulin V-region domains encoded by DNA sequences linked
in-frame to sequences encoding ANT are also within the scope of the
invention, including variants and fragments thereof as provided
herein. General strategies for the construction of fusion proteins
having immunoglobulin V-region fusion polypeptides are disclosed,
for example, in EP 0318554; U.S. Pat. No. 5,132,405; U.S. Pat. No.
5,091,513; and U.S. Pat. No. 5,476,786.
[0084] The nucleic acid of the present invention may also encode a
fusion protein comprising an ANT polypeptide fused to other
polypeptides having desirable affinity properties, for example an
enzyme such as glutathione-S-transferase. As another example, ANT
fusion proteins may also comprise an ANT polypeptide fused to a
Staphylococcus aureus protein A polypeptide; protein A encoding
nucleic acids and their use in constructing fusion proteins having
affinity for immunoglobulin constant regions are disclosed
generally, for example, in U.S. Pat. No. 5,100,788. Other useful
affinity polypetides for construction of ANT fusion proteins may
include streptavidin fusion proteins, as disclosed, for example, in
WO 89/03422; U.S. Pat. No. 5,489,528; U.S. Pat. No. 5,672,691; WO
93/24631; U.S. Pat. No. 5,168,049; U.S. Pat. No. 5,272,254 and
elsewhere, and avidin fusion proteins (see, e.g., EP 511,747). As
provided herein and in the cited references, ANT polypeptide
seqences may be fused to fusion polypeptide sequences that may be
full length fusion polypeptides and that may alternatively be
variants or fragments thereof.
[0085] The present invention also provides a method of targeting a
polypeptide of interest to a membrane, and in particular
embodiments to a cellular membrane, and in further embodiments to a
mitochondrial membrane. This aspect of the invention is based on
the unexpected observation that certain recombinant expression
constructs as provided herein, which constructs include a nucleic
acid encoding a first polypeptide that is an ANT polypeptide, and
that is expressed as a fusion protein with a second polypeptide
sequence, provide recombinant ANT fusion proteins capable of
preferentially localizing to cell membranes. In certain embodiments
the cell membrane is a prokaryotic cell membrane such as a
bacterial cell membrane, for example an E. coli membrane. In other
embodiments the cell membrane is a eukaryotic cell membrane such as
a yeast or a mammalian cell membrane, for example a membrane of any
eukaryotic cell contemplated herein.
[0086] A cell membrane as used herein may be any cellular membrane,
and typically refers to membranes that are in contact with
cytosolic components, including intracellular membrane bounded
compartments such as mitochondrial inner and outer membranes as
described above, and also intracellular vesicles, ER-Golgi
constituents, other organelles and the like, as well as the plasma
membrane. In preferred embodiments, an ANT fusion protein may be
targeted to a mitochondrial membrane. In other preferred
embodiments, for example, recombinant expression constructs
according to the invention may encode ANT fusion proteins that
contain polypeptide sequences that direct the fusion protein to be
retained in the cytosol, to reside in the lumen of the endoplasmic
reticulum (ER), to be secreted from a cell via the classical
ER-Golgi secretory pathway, to be incorporated into the plasma
membrane, to associate with a specific cytoplasmic component
including the cytoplasmic domain of a transmembrane cell surface
receptor or to be directed to a particular subcellular location by
any of a variety of known intracellular protein sorting mechanisms
with which those skilled in the art will be familiar. Accordingly,
these and related embodiments are encompassed by the instant
compositions and methods directed to targeting a polypeptide of
interest to a predefined intracellular, membrane or extracellular
localization.
VECTORS
[0087] The present invention also relates to vectors and to
constructs that include nucleic acids of the present invention, and
in particular to "recombinant expression constructs" that include
any nucleic acids encoding ANT polypeptides according to the
invention as provided above; to host cells which are genetically
engineered with vectors and/or constructs of the invention and to
the production of ANT polypeptides and fusion proteins of the
invention, or fragments or variants thereof, by recombinant
techniques. ANT proteins can be expressed in mammalian cells,
yeast, bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989).
[0088] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences. Optionally, the heterologous sequence can
encode a fission protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0089] Useful expression constructs for bacterial use are
constructed by inserting into an expression vector a structural DNA
sequence encoding a desired protein together with suitable
translation initiation and termination signals in operable reading
phase with a functional promoter. The construct may comprise one or
more phenotypic selectable marker and an origin of replication to
ensure maintenance of the vector construct and, if desirable, to
provide amplification within the host. Suitable prokaryotic hosts
for transformation include E. coli, Bacillus subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, although others may also be
employed as a matter of choice. Any other plasmid or vector may be
used as long as they are replicable and viable in the host.
[0090] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0091] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter, if it is a regulated promoter as provided
herein, is induced by appropriate means (e.g., temperature shift or
chemical induction) and cells are cultured for an additional
period. Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification. Microbial cells employed in
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents; such methods are well know to those
skilled in the art.
[0092] Thus, for example, the nucleic acids of the invention as
provided herein may be included in any one of a variety of
expression vector constructs as a recombinant expression construct
for expressing an ANT polypeptide. Such vectors and constructs
include chromosomal, nonchromosomal and synthetic DNA sequences,
e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of
plasmids and phage DNA, viral DNA, such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used for preparation of a recombinant expression construct as long
as it is replicable and viable in the host.
[0093] The appropriate DNA sequence(s) may be inserted into the
vector by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Standard techniques for cloning, DNA
isolation, amplification and purification, for enzymatic reactions
involving DNA ligase, DNA polymerase, restriction endonucleases and
the like, and various separation techniques are those known and
commonly employed by those skilled in the art. A number of standard
techniques are described, for example, in Ausubel et al. (1993
Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc.
& John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al.
(1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory,
Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold
Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.
[0094] The DNA sequence in the expression vector is operatively
linked to at least one appropriate expression control sequences
(e.g., a promoter or a regulated promoter) to direct mRNA
synthesis. Representative examples of such expression control
sequences include LTR or SV40 promoter, the E. coli lac or trp, the
phage lambda P.sub.L promoter and other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses. Promoter regions can be selected from any desired gene
using CAT (chloramphenicol transferase) vectors or other vectors
with selectable markers. Two appropriate vectors are pKK232-8 and
pCM7. Particular named bacterial promoters include lacI, lacZ, T3,
T7, gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters
include CMV immediate early, HSV thyridine kinase, early and late
SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection
of the appropriate vector and promoter is well within the level of
ordinary skill in the art, and preparation of certain particularly
preferred recombinant expression constructs comprising at least one
promoter or regulated promoter operably linked to a nucleic acid
encoding an ANT polypeptide is described herein.
[0095] In certain preferred embodiments the expression control
sequence is a "regulated promoter", which may be a promoter as
provided herein and may also be a repressor binding site, an
activator binding site or any other regulatory sequence that
controls expression of a nucleic acid sequence as provided herein.
In certain particularly preferred embodiments the regulated
promoter is a tightly regulated promoter that is specifically
inducible and that permits little or no transcription of nucleic
acid sequences under its control in the absence of an induction
signal, as is known to those familiar with the art and described,
for example, in Guzman et al. (1995 J. Bacteriol. 177:4121), Carra
et al. (1993 EMBO J. 12:35), Mayer (1995 Gene 163:41), Haldimann et
al. (1998 J. Bacteriol. 180:1277), Lutz et al. (1997 Nuc. Ac. Res.
25:1203), Allgood et al. (1997 Curr. Opin. Biotechnol. 8:474) and
Makrides (1996 Microbiol. Rev. 60:512), all of which are hereby
incorporated by reference. In other preferred embodiments of the
invention a regulated promoter is present that is inducible but
that may not be tightly regulated. In certain other preferred
embodiments a promoter is present in the recombinant expression
construct of the invention that is not a regulated promoter; such a
promoter may include, for example, a constitutive promoter such as
an insect polyhedrin promoter as described in the Examples or a
yeast phosphoglycerate kinase promoter (see, e.g., Giraud et al.,
1998 J. Mol. Biol. 281:409). The expression construct also contains
a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0096] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act on a promoter to increase its transcription. Examples including
the SV40 enhancer on the late side of the replication origin bp 100
to 270, a cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus
enhancers.
[0097] As noted above, in certain embodiments the vector may be a
viral vector such as a retroviral vector. For example, retroviruses
from which the retroviral plasmid vectors may be derived include,
but are not limited to, Moloney Murine Leukemia Virus, spleen
necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey
Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus,
human immunodeficiency virus, adenovirus, Myeloproliferative
Sarcoma Virus, and mammary tumor virus.
[0098] The viral vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller, et al.,
Biotechniques 7:980-990 (1989), or any other promoter (e.g.,
cellular promoters such as eukaryotic cellular promoters including,
but not limited to, the histone, pol III, and .beta.-actin
promoters). Other viral promoters which may be employed include,
but are not limited to, adenovirus promoters, thymidine kinase (TK)
promoters, and B19 parvovirus promoters. The selection of a
suitable promoter will be apparent to those skilled in the art from
the teachings contained herein, and may be from among either
regulated promoters or promoters as described above.
[0099] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, .psi.-2, .psi.-AM, PA12, T19-14X,
VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAm12, and DAN cell
lines as described in Miller, Human Gene Therapy, 1:5-14 (1990),
which is incorporated herein by reference in its entirety. The
vector may transduce the packaging cells through any means known in
the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0100] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the ANT polypeptides or fusion proteins. Such retroviral
vector particles then may be employed, to transduce eukaryotic
cells, either in vitro or in vivo. The transduced eukaryotic cells
will express the nucleic acid sequence(s) encoding the ANT
polypeptide or fusion protein. Eukaryotic cells which may be
transduced include, but are not limited to, embryonic stem cells,
embryonic carcinoma cells, as well as hematopoietic stem cells,
hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial
cells, and bronchial epithelial cells.
[0101] As another example of an embodiment of the invention in
which a viral vector is used to prepare the recombinant ANT
expression construct, in one preferred embodiment, host cells
transduced by a recombinant Viral construct directing the
expression of ANT polypeptides or fusion proteins may produce viral
particles containing expressed ANT polypeptides or fusion proteins
that are derived from portions of a host cell membrane incorporated
by the viral particles during viral budding. In another preferred
embodiment, ANT encoding nucleic acid sequences are cloned into a
baculovirus shuttle vector, which is then recombined with a
baculovirus to generate a recombinant baculovirus expression
construct that is used to infect, for example, Sf9 or Trichoplusia
ni (PharMingen, Inc., San Diego, Calif.) host cells, as described
in Baculovirus Expression Protocols, Methods in Molecular Biology
Vol. 39, Christopher D. Richardson, Editor, Human Press, Totowa,
N.J., 1995; Piwnica-Worms, "Expression of Proteins in Insect Cells
Using Baculoviral Vectors," Section II in Chapter 16 in: Short
Protocols in Molecular Biology, 2.sup.nd nd Ed., Ausubel et al.,
eds., John Wiley & Sons, New York, N.Y., 1992, pages 16-32 to
16-48.
HOST CELLS
[0102] In another aspect, the present invention relates to host
cells containing the above described recombinant ANT expression
constructs. Host cells are genetically engineered (transduced,
transformed or transfected) with the vectors and/or expression
constructs of this invention which may be, for example, a cloning
vector, a shuttle vector or an expression construct. The vector or
construct may be, for example, in the form of a plasmid, a viral
particle, a phage, etc. The engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying
particular genes such as genes encoding ANT polypeptides or ANT
fusion proteins. The culture conditions for particular host cells
selected for expression, such as temperature, pH and the like, will
be readily apparent to the ordinarily skilled artisan.
[0103] The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or the host cell can be a prokaryotic cell, such as a bacterial
cell. Representative examples of appropriate host cells according
to the present invention include, but need not be limited to,
bacterial cells, such as E. coli, Streptomyces, Salmonella
typhimurium; fungal cells, such as yeast; insect cells, such as
Drosophila S2, Trichoplusia ni (PharMingen, San Diego, Calif.) and
Spodoptera Sf9; animal cells, such as CHO, COS or 293 cells;
adenoviruses; plant cells, or any suitable cell already adapted to
in vitro propagation or so established de novo. The selection of an
appropriate host is deemed to be within the scope of those skilled
in the art from the teachings herein.
[0104] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences, for example as described herein regarding
the preparation of ANT expression constructs. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements. Introduction
of the construct into the host cell can be effected by a variety of
methods with which those skilled in the art will be familiar,
including but not limited to, for example, calcium phosphate
transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis et al., 1986 Basic Methods in Molecular
Biology).
[0105] As will be aprreciated by those of ordinary skill in the
art, in certain situations it may be desirable to prepare the
compositions of the invention and to practice the methods of the
invention under conditions where endogenous ANT expression by a
host cell is compromised, in order to provide advantages associated
with the expression of a desired ANT encoding construct. For
example, detection of particular ANT encoding nucleic acid
sequences or ANT polypeptides that are highly similar to those
encoded by the host cell genome may be facilitated by inhibiting
host cell ANT gene expression. As another example, where functional
activity of an exogenously introduced recombinant ANT polypeptide
is to be determined in a host cell or in a biological sample
derived therefrom, it may also be advantageous to inhibit
endogenous host cell ANT gene expression.
[0106] Thus, in certain preferred embodiments of the invention,
host cells may lack at least one isoform of an endogenous ANT, and
in certain preferred embodiments the host cells may lack all
endogenous ANT isoforms. For example, in the yeast system described
by Giraud et al. (1998 J. Mol. Biol. 281:409) a S. cerevisiae
triple null mutant is described that lacks all three yeast ANT
isoforms and is unable to grow under anaerobic conditions. In other
preferred embodiments, expression in host cells of at least one
gene encoding an endogenous ANT isoform is substantially impaired
Substantial impairment of endogenous ANT isoform expression may be
achieved by any of a variety of methods that are well known in the
art for blocking specific gene expression, including site-specific
or site-directed mutagenesis as described above, antisense
inhibition of gene expression, ribozyme mediated inhibition of gene
expression and generation of mitochondrial DNA depleted
(.rho..sup.0) cells.
[0107] Identification of oligonucleotides and ribozymes for use as
antisense agents and DNA encoding genes for targeted delivery for
genetic therapy involve methods well known in the art. For example,
the desirable properties, lengths and other characteristics of such
oligonucleotides are well known. Antisense oligonucleotides are
typically designed to resist degradation by endogenous nucleolytic
enzymes by using such linkages as: phosphorothioate,
methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate,
phosphoramidate, phosphate esters, and other such linkages (see,
e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542 (1987); Miller
et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec et al.,
Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. Acids
Res. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);
Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu.
Rev. Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci.
14:97-100 (1989); Stein In: Oligodeoxynucleotides. Antisense
Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London,
pp. 97-117 (1989); Jager et al., Biochemistry 27:7237-7246
(1988)).
[0108] Antisense nucleotides are oligonucleotides that bind in a
sequence-specific manner to nucleic acids, such as mRNA or DNA.
When bound to mRNA that has complementary sequences, antisense
prevents translation of the mRNA (see, e.g., U.S. Pat. No.
5,168,053 to Altman et al.; U.S. Pat. No. 5,190,931 to Inouye, U.S.
Pat. No. 5,135,917 to Burch; U.S. Pat. No. 5,087,617 to Smith and
Clusel et al. (1993) Nucl. Acids Res. 21:3405-3411, which describes
dumbbell antisense oligonucleotides). Triplex molecules refer to
single DNA strands that bind duplex DNA forming a colinear triplex
molecule, thereby preventing transcription (see, e.g., U.S. Pat.
No. 5,176,996 to Hogan et al., which describes methods for making
synthetic oligonucleotides that bind to target sites on duplex
DNA).
[0109] According to this embodiment of the invention, particularly
useful antisense nucleotides and triplex molecules are molecules
that are complementary to or bind the sense strand of DNA or mRNA
that encodes an ANT polypeptide or a protein mediating any other
process related to expression of endogenous ANT genes, such that
inhibition of translation of mRNA encoding the ANT polypeptide is
effected.
[0110] A ribozyme is an RNA molecule that specifically cleaves RNA
substrates, such as mRNA, resulting in specific inhibition or
interference with cellular gene expression. There are at least five
known classes of ribozymes involved in the cleavage and/or ligation
of RNA chains. Ribozymes can be targeted to any RNA transcript and
can catalytically cleave such transcripts (see, e.g., U.S. Pat. No.
5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053,
5,180,818, 5,116,742 and 5,093,246 to Cech et al.). According to
certain embodiments of the invention, any such ANT mRNA-specific
ribozyme, or a nucleic acid encoding such a ribozyme, may be
delivered to a host cell to effect inhibition of ANT gene
expression. Ribozymes, and the like may therefore be delivered to
the host cells by DNA encoding the ribozyme linked to a eukaryotic
promoter, such as a eukaryotic viral promoter, such that upon
introduction into the nucleus, the ribozyme will be directly
transcribed.
[0111] As used herein, expression of a gene encoding an endogenous
adenine nucleotide translocator isoform is substantially impaired
by any of the above methods for inhibiting when cells are
substantially but not necessarily completely depleted of functional
DNA or functional mRNA encoding the endogenous ANT isoform, or of
the relevant ANT polypeptide. ANT isoform expression is
substantially impaired when cells are preferably at least 50%
depleted of DNA or mRNA encoding the endogenous ANT (as measured
using high stringency hybridization as described above) or depleted
of ANT polypeptide (as measured by Western immunoblot as described
herein, see also, e.g., Giraud et al. 1998 J. Mol. Biol. 281:409);
and more preferably at least 75% depleted of endogenous ANT DNA,
mRNA or polypeptide. Most preferably, ANT isoform expression is
substantially impaired when cells are depleted of >90% of their
endogenous ANT DNA, mRNA, or polypeptide.
[0112] Alternatively, expression of a gene encoding an endogenous
adenine nucleotide translocator isoform may be substantially
impaired through the use of mitochondrial DNA depleted .rho..sup.0
cells, which are incapable of mitochondrial replication and so may
not continue to express functional ANT polypeptides. Methods for
producing .rho..sup.0 cells are known and can be found, for example
in PCT/US95/04063, which is hereby incorporated by reference.
PROTEIN PRODUCTION
[0113] The expressed recombinant ANT polypeptides or fusion
proteins may be useful in intact host cells; in intact organelles
such as mitochondria, cell membranes, intracellular vesicles other
cellular organelles; or in disrupted cell preparations including
but not limited to cell homogenates or lysates, submitochondrial
particles, uni- and multilamellar membrane vesicles or other
preparations. Alternatively, expressed recombinant ANT polypeptides
or fusion proteins can be recovered and purified from recombinant
cell cultures by methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0114] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
SAMPLES
[0115] A "biological sample containing mitochondria" may comprise
any tissue or cell preparation in which intact mitochondria capable
of maintaining a membrane potential when supplied with one or more
oxidizable substrates such as glucose, malate or galactose are or
are thought to be present. Mitochondrial membrane potential may be
determined according to methods with which those skilled in the art
will be readily familiar, including but not limited to detection
and/or measurement of detectable compounds such as fluorescent
indicators, optical probes and/or sensitive pH and ion-selective
electrodes (See, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s
and references cited therein; see also Haugland, 1996 Handbook of
Fluorescent Probes and Research Chemicals--Sixth Ed., Molecular
Probes, Eugene, Oreg., pp. 266-274 and 589-594.). By "capable of
maintaining a potential" it is meant that such mitochondria have a
membrane potential that is sufficient to permit the accumulation of
a detectable compound (e.g., DASPMI
[2-,4-dimethylaminostyryl-N-methylpyridinium], TMRM
[tetramethylrhodamine methyl ester], etc.) used in the particular
instance. A biological sample containing mitochondria may, for
example, be derived from a normal (i.e., healthy) individual or
from an individual having a disease associated with altered
mitochondrial function. Biological samples containing mitochondria
may be provided by obtaining a blood sample, biopsy specimen,
tissue explant, organ culture or any other tissue or cell
preparation from a subject or a biological source. The subject or
biological source may be a human or non-human animal, a primary
cell culture or culture adapted cell line including but not limited
to genetically engineered cell lines that may contain chromosomally
integrated or episomal recombinant nucleic acid sequences,
immortalized or immortalizable cell lines, somatic cell hybrid or
cytoplasmic hybrid "cybrid" cell lines, differentiated or
differentiatable cell lines, transformed cell lines and the
like.
[0116] A "biological sample" may comprise any tissue or cell
preparation as just described for a biological sample containing
mitochondria, but does not require the presence of intact
mitochondria. Thus a "biological sample" may comprise any tissue or
cell preparation and a "biological sample containing at least one
recombinant ANT polypeptide" comprises any tissue or cell
preparation in which an expressed recombinant ANT polypeptide or
fusion protein as provided herein is thought to be present. A
biological sample may, for example, be derived from a recombinant
cell line or from a transgenic animal. Biological samples
containing recombinant ANT may be provided by obtaining a blood
sample, biopsy specimen, tissue explant, organ culture or any other
tissue or cell preparation from a subject or a biological source.
The subject or biological source may be a human or non-human
animal, a primary cell culture or culture adapted cell line
including but not limited to genetically engineered cell lines that
may contain chromosomally integrated or episomal recombinant
nucleic acid sequences, immortalized or immortalizable cell lines,
somatic cell hybrid or cytoplasmic hybrid "cybrid" cell lines,
differentiated or differentiatable cell lines, transformed cell
lines and the like.
PROTEINS
[0117] As described herein, isolation of a mitochondrial pore
component or a mitochondrial molecular species with which an agent
identified according to the methods of the invention interacts
refers to physical separation of such a complex from its biological
source, and may be accomplished by any of a number of well known
techniques including but not limited to those described herein, and
in the cited references. Without wishing to be bound by theory, a
compound that "binds a mitochondrial component" can be any discrete
molecule, agent compound, composition of matter or the like that
may, but need not, directly bind to a mitochondrial molecular
component, and may in the alternative bind indirectly to a
mitochondrial molecular component by interacting with one or more
additional components that bind to a mitochondrial molecular
component. These or other mechanisms by which a compound may bind
to and/or associate with a mitochondrial molecular component are
within the scope of the claimed methods, so long as isolating a
mitochondrial pore component also results in isolation of the
mitochondrial molecular species that directly or indirectly binds
to the identified agent. Thus, for example, as provided herein, any
ANT polypeptide including recombinant ANT polypeptides and fusion
proteins may be a mitochondrial molecular component and/or a
mitochondrial pore component, and any ANT ligand or agent that
binds to an ANT polypeptide may be a compound that binds a
mitochondrial component and/or an agent that affects mitochondrial
pore activity.
[0118] As described herein, the mitochondrial permeability
transition "pore" may not be a discrete assembly or multisubunit
complex, and the term thus refers instead to any mitochondrial
molecular component (including, e.g., a mitochondrial membrane per
se) that regulates the inner membrane selective permeability where
such regulated function is impaired during MPT. As used herein,
mitochondria are comprised of "mitochondrial molecular components",
which may be any protein, polypeptide, peptide, amino acid, or
derivative thereof, any lipid, fatty acid or the like, or
derivative thereof, any carbohydrate, saccharide or the like or
derivative thereof, any nucleic acid, nucleotide, nucleoside,
purine, pyrimidine or related molecule, or derivative thereof, or
the like; or any other biological molecule that is a constituent of
a mitochondrion. "Mitochondrial molecular components" includes but
is not limited to "mitochondrial pore components". A "mitochondrial
pore component" is any mitochondrial molecular component that
regulates the selective permeability characteristic of
mitochondrial membranes as described above, including those
responsible for establishing .DELTA..PSI.m and those that are
functionally altered during MPT.
[0119] Isolation and, optionally, identification and/or
characterization of the mitochondrial pore component or components
with which an agent that affects mitochondrial pore activity
interacts may also be desirable and are within the scope of the
invention. Once an agent is shown to alter MPT according to the
methods provided herein and in U.S. Ser. No. 09/161,172, those
having ordinary skill in the art will be familiar with a variety of
approaches that may be routinely employed to isolate the molecular
species specifically recognized by such an agent and involved in
regulation of MPT, where to "isolate" as used herein refers to
separation of such molecular species from the natural biological
environment. Thus, for example, once an ANT ligand is prepared
according to the methods provided herein, such approaches may be
routinely employed to isolate the ANT polypeptide. Techniques for
isolating a mitochondrial pore component such as an ANT polypeptide
or fusion protein may include any biological and/or biochemical
methods useful for separating the complex from its biological
source, and subsequent characterization may be performed according
to standard biochemical and molecular biology procedures. Those
familiar with the art will be able to select an appropriate method
depending on the biological starting material and other factors.
Such methods may include, but need not be limited to, radiolabeling
or otherwise detectably labeling cellular and mitochondrial
components in a biological sample, cell fractionation, density
sedimentation, differential extraction, salt precipitation,
ultrafiltration, gel filtration, ion-exchange chromatography,
partition chromatography, hydrophobic chromatography,
electrophoresis, affinity techniques or any other suitable
separation method that can be adapted for use with the agent with
which the mitochondrial pore component interacts. Antibodies to
partially purified components may be developed according to methods
known in the art and may be used to detect and/or to isolate such
components.
[0120] Affinity techniques may be particularly useful in the
context of the present invention, and may include any method that
exploits a specific binding interaction between a mitochondrial
pore component and an agent identified according to the invention
that interacts with the pore component. For example, because ANT
ligands as provided herein and other agents that influence MPT can
be immobilized on solid phase matrices, an affinity binding
technique for isolation of the pore component may be particularly
useful. Alternatively, affinity labeling methods for biological
molecules, in which a known MPT-active agent or a novel ANT ligand
as provided herein may be modified with a reactive moiety, are well
known and can be readily adapted to the interaction between the
agent and a pore component, for purposes of introducing into the
pore component a detectable and/or recoverable labeling moiety.
(See, e.g., Pierce Catalog and Handbook, 1994 Pierce Chemical
Company, Rockford, Ill.; Scopes, R. K., Protein Purification:
Principles and Practice, 1987, Springer-Verlag, New York; and
Hermanson, G. T. et al., Immobilized Affinity Ligand Techniques,
1992, Academic Press, Inc., California; for details regarding
techniques for isolating and characterizing biological molecules,
including affinity techniques.
[0121] Characterization of mitochondrial pore component molecular
species, isolated by MPT-active agent affinity techniques described
above or by other biochemical methods, may be accomplished using
physicochemical properties of the pore component such as
spectrometric absorbance, molecular size and/or charge, solubility,
peptide mapping, sequence analysis and the like. (See, e.g.,
Scopes, supra.) Additional separation steps for biomolecules may be
optionally employed to further separate and identify molecular
species that co-purify with mitochondrial pore components. These
are well known in the art and may include any separation
methodology for the isolation of proteins, lipids, nucleic acids or
carbohydrates, typically based on physicochemical properties of the
newly identified components of the complex. Examples of such
methods include RP-HPLC, ion exchange chromatography, hydrophobic
interaction chromatography, hydroxyapatite chromatography, native
and/or denaturing one- and two-dimensional electrophoresis,
ultrafiltration, capillary electrophoresis, substrate affinity
chromatography, immunoaffinity chromatography, partition
chromatography or any other useful separation method. Preferably
extracts of cultured cells, and in particularly preferred
embodiments extracts of biological tissues or organs may be sources
of mitochondrial molecular components, including ANT polypeptides.
Preferred sources may include blood, brain, fibroblasts, myoblasts,
liver cells or other cell types.
ANT LIGANDS
[0122] As noted above, the binding of the adenine nucleotide
translocator (ANT) is responsible for mediating transport of ADP
and ATP across the mitochondrial inner membrane. ANT has also been
implicated as the critical component of the mitochondrial
permeability transition pore, a Ca.sup.2+ regulated inner membrane
channel that plays an important modulating role in apoptotic
processes. Additionally, ANT activity appears to be related to
changes in ANT polypeptide conformation within the mitochondrial
membrane, as evidenced by studies using agents that are capable of
binding to ANT. (Block et al., 1986 Meths. Enzymol. 125:658)
Accordingly, it is another aspect of the present invention to
provide compositions and methods for producing and identifying
agents that bind to ANT, which agents are also referred to herein
as ANT ligands.
[0123] Binding interactions between ANT and a variety of small
molecules are known to those familiar with the art. For example,
these interactions include binding to ANT by atractyloside,
carboxyatractyloside, palmitoyl-CoA, bongkrekic acid, thyroxin,
eosin Y and erythrosin B. (See, e.g., Stubbs, 1979 Pharm. Ther.
7:329; Klingenberg et al., 1978 Biochim. Biophys. Acta 503:193;
Sterling, 1986 Endocrinol. 119:292; Majima et al., 1998 Biochem.
37:424; Block et al. 1986 Meths. Enzymol. 125:658; for erythrosin B
and additional ANT inhibitors, see Beavis et al. 1993 J. Biol.
Chem. 268:997; Powers et al. 1994 J. Biol. Chem. 269:10614.)
[0124] The ANT ligands of the present invention represent novel
atractyloside derivatives. Atractyloside (ATR) and its known
derivatives, including carboxyatractyloside (CATR), naphthoyl-ATR,
MANT-ATR and other ATR derivatives (see, e.g., Boulay et al.,
Analytical Biochemistry 128:323-330,1983; Roux et al., Analytical
Biochemistry 234:31-37,1996; Lauquin et al., FEBS Letters
67:306-311,1976; and Gottikh et al., Tetrahedron 26:4419-4433,
1970; for other known ATR derivatives see, e.g., Block et al., 1986
Meths. Enzymol. 125:658) have proven invaluable in the elucidation
of the structure and the mechanism of action of the adenine
nucleotide translocator. According to the ANT ligands of the
invention, the binding mode of ATR to ANT allows for modifications
of the ATR 6'-hydroxyl functionality without significantly altering
ATR binding affinity for ANT. Thus, ANT ligands as provided herein
may be ATR derivatives modified by chemical substitution at the 6'
hydroxyl position. In particular, the novel ANT ligands as provided
herein further include long linker moieties at the 6' position,
which linkers may include a 6'-amine linker, thereby permitting
additional chemical modification to the ANT ligand as will be
appreciated by those skilled in the art and as illustrated in the
non-limiting Examples. Also, as shown in Examples 6-11, such
linkers as provided herein may have carbon chain backbones of 2-20
carbon atoms, and in preferred embodiments 2-6 carbon atoms.
[0125] The invention therefore provides ANT ligands that may be
intermediates for conjugation to a variety of additional chemical
moieties to yield further ATR derivatives that are ANT ligands
within the scope of the invention. These include ANT ligands to
which .sup.125I may be covalently attached under mild reaction
conditions; the invention also includes ANT ligands to which
reactive amine groups may be covalently linked. ANT ligands which
are such amine-containing ATR derivatives may then be reacted with
a variety of fluorophores and haptens bearing, for example,
reactive isothiocyanate, N-hydroxysuccinimide ester, anhydride and
other useful functionalities to yield stable ATR derivatives
including, for example, derivatives that have thiourea, amide or
other linkages.
[0126] Thus, ANT ligands as provided herein also include ATR
derivatives that are detectable by virtue of substituents
introduced at the 6' position. Accordingly, detectable ATR
derivatives as herein provided include ATR derivatives having a 6'
hydroxyl substitution that includes a radiolabeled substituent, for
example .sup.125I, .sup.131I, .sup.3H, .sup.14C or .sup.35S. Other
ANT ligands that are detectable ATR derivatives may comprise
fluorescent substituents, including those appropriately tagged with
reporter molecules such as fluorophores and haptens having utility
in high throughput screening assays for identifying agents that
bind to ANT. More specifically, in preferred embodiments, an ANT
ligand according to the present invention that includes fluorescent
substituents has an extinction coefficient .gtoreq.10,000 M.sup.-1
(see Table 1); further, this property provides an advantage for
using such ANT ligands according to the methods provided herein,
and in particular for use in high throughput screening assays.
Additionally, the ANT ligands of the invention exhibit high
affinities for ANT, and in preferred embodiments have binding
constants in the nanomolar range.
[0127] In certain embodiments of the invention, ANT ligands may be
ATR derivatives such as ATR-lanthanide chelating agents, which have
utility in time-resolved fluorescence detection, for example
detection of these compounds complexed to a lanthanide ion such as
Eu.sup.3+, Tb.sup.3+, Sm.sup.3+ and Dy.sup.3+. In addition, ANT
ligands may comprise ATR conjugated to readily detectable
substituents such as highly fluorescent moieties, for example by
way of illustration and not limitation, cyanine and coumarin
derivatives. These and other highly fluorescent substituents permit
the synthesis, according to the methods of the invention, of ANT
ligands that are detectable with extremely high sensitivities.
Those familiar with the art are aware of additional fluorescent
substituents that may be used, for example, those disclosed in
Haugland, 1996 Handbook of Fluorescent Probes and Research
Chemicals--Sixth Ed., Molecular Probes, Eugene, Oreg. In other
embodiments, the invention provides detectable ANT ligands produced
by coupling of biotin-NHS ester with the ATR derivatives as
disclosed herein; these and other ANT ligands similarly generated
according to the instant methods can be detected with commercially
available enzyme-avidin conjugates using, for example,
colorimetric, fluorescent or chemiluminescent techniques.
[0128] In one embodiment, the ANT ligands of this invention have
the following structure (I): 2
[0129] including stereoisomers and pharmaceutically acceptable
salts thereof, wherein
[0130] R.sub.1 is hydroxyl, halogen, --OC(.dbd.O)R.sub.4 or
--NHR.sub.4;
[0131] R.sub.2 is hydrogen or --C(.dbd.O)R.sub.5;
[0132] R.sub.3 is --CH.sub.3 or .dbd.CH.sub.2;
[0133] R.sub.4 is -X-aryl, -X-substituted aryl, -X-arylalkyl,
-X-substituted arylalkyl, X-heteroaryl, or -X-heteroarylalkyl,
wherein X is an optional amido or alkylamido linker moiety; and
[0134] R.sub.5 is alkyl.
[0135] As used herein, the above terms have the meanings set forth
below.
[0136] "Amido" means --NHC(.dbd.O)-- or --C(.dbd.O)NH--.
[0137] "Alkylamido" means -(alkyl)-NHC(.dbd.O)-- or
-(alkyl)-C(.dbd.O)NH--, such as --CH.sub.2NHC(.dbd.O)--,
--CH.sub.2CH.sub.2NHC(.dbd.O)--, --CH.sub.2C(.dbd.O)NH--,
--CH.sub.2CH.sub.2C(.dbd.O)NH--, and the like.
[0138] "Alkyl" means a straight chain or branched, noncyclic or
cyclic, unsaturated or saturated aliphatic hydrocarbon containing
from 1 to 8 carbon atoms. Representative saturated straight chain
alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,
and the like; while saturated branched alkyls include isopropyl,
sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Representative saturated cyclic alkyls include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like; while
unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl,
and the like. Unsaturated alkyls contain at least one double or
triple bond between adjacent carbon atoms (referred to as an
"alkenyl" or "alkynyl", respectively). Representative straight
chain and branched alkenyls include ethylenyl, propylenyl,
1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like; while representative straight chain and branched alkynyls
include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,
2-pentynyl, 3-methyl-1 butynyl, and the like.
[0139] "Aryl" means an aromatic carbocyclic moiety such as phenyl
or naphthyl (i.e., 1- or 2-naphthyl).
[0140] "Arylalkyl" means an alkyl having at least one alkyl
hydrogen atoms replaced with an aryl moiety, such as benzyl,
--(CH.sub.2).sub.2phenyl, --(CH.sub.2).sub.3phenyl, and the
like.
[0141] "Heteroaryl" means an aromatic heterocycle ring of 5- to 10
members and having at least one heteroatom selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and bicyclic ring systems. Representative heteroaryls
are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,
quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl,
benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl.
[0142] "Heteroarylalkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heteroaryl moiety, such as
--CH.sub.2pyridinyl, --CH.sub.2pyrimidinyl, and the like.
[0143] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 to 4
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Heterocycles include
heteroaryls as defined above. Thus, in addition to the heteroaryls
listed above, heterocycles also include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl,
valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0144] "Heterocyclealkyl" means an alkyl having at least one alkyl
hydrogen atom replaced with a heterocycle, such as
--CH.sub.2morpholinyl, and the like.
[0145] The term "substituted" as used herein means any of the above
groups (i.e., alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
heterocycle and heterocyclealkyl) wherein at least one hydrogen
atom is replaced with a substituent. In the case of a keto
substituent ("C(.dbd.O)") two hydrogen atoms are replaced.
Substituents include halogen, hydroxy, alkyl, haloalkyl, aryl,
substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,
substitued heterocycle, heterocyclealkyl or substituted
heterocyclealkyl.
[0146] "Halogen" means fluoro, chloro, bromo and iodo.
[0147] "Haloalkyl" means an alkyl having at least one hydrogen atom
replaced with halogen, such as trifluoromethyl and the like.
[0148] "Alkoxy" means an alkyl moiety attached through an oxygen
bridge (i.e., --O-alkyl) such as methoxy, ethoxy, and the like.
[0149] In one embodiment, R.sub.2 is
--C(.dbd.O)CH.sub.2CH(CH.sub.3).sub.2- , R.sub.3 is .dbd.CH.sub.2,
and the ANT ligand is an atractyloside derivative having the
following structure (II): 3
[0150] wherein R.sub.1 is as defined above.
[0151] In another embodiment, R.sub.2 is
--C(.dbd.O)CH.sub.2CH(CH.sub.3).s- ub.2, R.sub.3 is --CH.sub.3, and
the ANT ligand is a dihydro-atractyloside derivative having the
following structure (III): 4
[0152] wherein R.sub.1 is as defined above.
[0153] In still a further embodiment, R.sub.2 is --OH, R.sub.3 is
.dbd.CH.sub.2, and the ANT ligand is an apoatractyloside derivative
having the following structure (IV): 5
[0154] wherein R.sub.1 is as defined above.
[0155] In more specific embodiments of structures (II), (III) and
(IV), R.sub.1 is --OC(.dbd.O)(aryl), --OC(.dbd.O)(substituted
aryl), --OC(.dbd.O)(arylalkyl), --OC(.dbd.O)(substituted
arylalkyl), --NH(CH.sub.2).sub.2NHC(.dbd.O)(arylalkyl),
--NH(CH.sub.2).sub.2NHC(.dbd.- O)(substituted arylalkyl).
Representative R.sub.1 moieties in this regard include
--OC(.dbd.O)(phenyl), --OC(.dbd.O)(1-naphthyl),
--OC(.dbd.O)(substituted phenyl), --OC(.dbd.O)(substituted
1-naphthyl), --OC(.dbd.O)(CH.sub.2).sub.1-3(phenyl),
--OC(.dbd.O)(CH.sub.2).sub.1-3(su- bstituted phenyl),
--NH(CH.sub.2).sub.2NHC(.dbd.O)(CH.sub.2).sub.1-3(pheny- l),
--NH(CH.sub.2).sub.2NHC(.dbd.O)(CH.sub.2).sub.1-3(substituted
phenyl). In this context, representative substituted phenyl
moieties include (but are not limited to) 4-hydroxyphenyl,
3-iodo-4-hydroxyphenyl, 3,5-iodo-4-hydroxyphenyl,
4-(4-hydoxyphenyl)phenyl, 4-(3-iodo-4-hyroxyphenyl)phenyl,
3-methyl-4-hyroxyphenyl, and 3-methyl-4-hydroxy-5-iodophenyl.
[0156] The ANT ligands of structure (I) may readily be made by one
skilled in the art of organic chemisty and, more particularly, by
the techniques disclosed in Examples 6-11.
[0157] The compounds of the present invention may generally be
utilized as the free base. Alternatively, the compounds of this
invention may be used in the form of acid addition salts. Acid
addition salts of the free amino compounds of the present invention
may be prepared by methods well known in the art, and may be formed
from organic and inorganic acids. Suitable organic acids include
maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic,
acetic, oxalic, propionic, tartaric, salicylic, citric, gluconic,
lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic,
glutamic, and benzenesulfonic acids. Suitable inorganic acids
include hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric
acids. Thus, the term "pharmaceutically acceptable salt" of
structure (I) is intended to encompass any and all acceptable salt
forms.
[0158] With regard to stereoisomers, the compounds of structure (I)
may have chiral centers and may occur as recemates, reacemic
mixtures and as individual enantiomers or diastereomers. All such
isomeric forms are included within the present invention, including
mixtures thereof. Furthermore, some of the crystalline forms of the
compounds of structure (I) may exist as polymorphs, which are
included in the present invention. In addition, some of the
compounds of structure (I) may also form solvates with water or
other organic solvents. Such solvates are similarly included within
the scope of this invention.
[0159] Activities of ANT ligands are typically calculated from the
IC.sub.50 as the concentration of a compound necessary to displace
50% of the detectable (i.e., detectably labeled, for example,
radiolabeled) ligand from ANT molecules, which may be present as
isolated or purified polypeptides or as components of preparations
containing isolated mitochondria or submitochondrial particles
(SMP) using established ligand binding assays or modifications
thereof. For example, ANT ligands may be tested for their ability
to compete with radiolabeled ATR, or with a radiolabeled ATR
derivative such as compound 24 as provided herein, for binding to
isolated ANT polypeptides or to ANT present in isolated
mitochondria or SMP.
[0160] As another example, the relative affinities for ANT of
various ANT ligands as provided herein may be determined by a
fluorescence assay that exploits the flourescent properties of
compound 22 (Example 11), a naphthoyl-ATR derivative that is an ANT
ligand having a fluorescence excitation peak at 312 nm and an
emission peak at 400 nm. When compound 22 is bound to ANT, the
fluorescence is quenched. When, however, compound 22 is displaced
from ANT by a known concentration of ATR or an ATR derivative that
is an ANT ligand, fluorescence dequenching that results from
displacement of the fluorophore can be measured in real time.
[0161] Briefly, a mitochondrial preparation (see, e.g., Example 13)
is washed and resuspended in a suitable buffer in the presence of
compound 22 (e.g., 10 mM Tris-120 mM KCl containing 3.6 nmoles of
compound 22 per mg mitochondrial protein, 10 min at room
temperature), washed to remove unbound fluorophore and placed in a
fluorometer equipped with a light source and filter set appropriate
for the fluorophore. Fluorescence intensity is monitored as a
function of time, and a candidate ANT ligand is then added to
determine its ability to compete with compound 22 for binding to
ANT, as evidenced by a change in detectable relative fluorescence
intensity units. After the fluorescence signal has stabilized, any
additional compound 22 that remains bound to ANT is displaced by
adding an excess (e.g., .mu.M quantities) of ATR as a competitive
inhibitor, to determine maximal signal intensity and therefrom
calculate the proportion of compound 22 displaced by the candidate
ANT ligand. Those having familiarity with the art will appreciate
that variations and modifications may be made to ANT-binding assays
such as those illustrated above and described in the Examples for
determing IC.sub.50 values of candidate ANT ligands, and which are
not intended to be limiting.
[0162] Activity of each ANT ligand is reported as a "K.sub.i" value
calculated by the following equation: 1 K i = IC 50 1 + L / K D
[0163] where L=radioligand and K.sub.D=affinity of radioligand for
receptor (Cheng and Prusoff, Biochem. Pharmacol. 22:3099, 1973).
ANT ligands of this invention have a K.sub.i of 100 .mu.M or less.
In a preferred embodiment of this invention, the ANT ligands have a
K.sub.i of less than 10 .mu.M, and more preferably less than 1
.mu.M. To this end, ANT ligands of this invention having a K.sub.i
of less than 100 .mu.M include compound 5 (Example 7), compound 6
(Example 8), and compounds 22, 23, 24, 26, 29, 33, 35, 37, and 38
(Example 11). Preferred ANT ligands having a K.sub.i of less than
10 .mu.M include compounds 6, 22 23, 24, 29, 33, 35, and 38, and
more preferred ANT ligands having a K.sub.i of less than 1 .mu.M
include compounds 6, 24, 33, and 38, as well as ATR.
ASSAYS
[0164] It is another aspect of the invention to provide
compositions and methods for the determination of the presence of
ANT polypeptides and for the identification of agents that bind to,
or that interact with, ANT polypeptides. Such compositions and
methods will be useful for diagnostic and prognostic purposes, for
example in the determination of the existence of altered
mitochondrial function which, as described above, may accompany
both normal and disease states. These compositions and methods will
also be useful for the identification of agents that alter or
regulate mitochondrial function based on ANT roles in mitochondrial
activities, for example by way of illustration and not limitation,
maintenance of mitochondrial membrane potential, ATP biosynthesis,
induction of apoptosis, MPT and other mitochondrial function. In
certain preferred embodiments these compositions and methods are
useful as high throughput screening assays.
[0165] In certain aspects the invention provides a method for
determining the presence of an ANT polypeptide in a biological
sample, comprising contacting a sample suspected of containing an
ANT polypeptide with an ANT ligand under conditions and for a time
sufficieint to allow binding of the ANT ligand to an ANT
polypeptide, and detecting such binding. "ANT ligands" according to
these aspects of the invention may include any novel ANT ligands as
provided herein. The use of human ANT1, ANT2 and ANT3 according to
these methods represent particularly preferred embodiments. Other
preferred embodiments include the use of any ANT polypeptide or ANT
fusion protein as provided herein. Accordingly, the instant method
for determining the presence of ANT polypeptide in a sample will be
useful for monitoring expression of ANT encoding constructs
provided herein. In some preferred embodiments an ANT fusion
protein is used that is a GST fusion protein, and in other
preferred embodiments the ANT fusion protein is a His-tagged fusion
protein. As provided herein, the biological sample may be a cell, a
mitochondrion, submitochondrial particles, a cell membrane
(including any cellular membrane as described herein), a cell
extract, cell conditioned medium, a tissue homogenate or an
isolated ANT.
[0166] In other aspects, the invention provides a method for
identifying an agent that binds to an ANT polypeptide, comprising
contacting a candidate agent with a host cell expressing at least
one recombinant ANT polypeptide under conditions and for a time
sufficient to permit binding of the agent to the ANT polypeptide
and detecting such binding. In various preferred embodiments the
host cell may be a prokaryotic cell or a eukaryotic cell. In
certain other preferred embodiments the host cell may lack at least
one isoform of an endogenous ANT, for example, due to a mutation in
one or more endogenous ANT encoding genes. In certain other
embodiments host cell expression of at least one gene encoding an
endogenous ANT isoform is substantially impaired, for example,
through the use of ANT nucleic acid-specific ribozyme or antisense
constructs as provided herein, or through the use of .rho..sup.0
cells, as also provided herein. According to other embodiments of
this aspect of the invention, it may be preferred to use intact
cells or, alternatively, to use permeabilized cells. Those having
ordinary skill in the art are familiar with methods for
permeabilizing cells, for example by way of illustration and not
limitation, through the use of surfactants, detergents,
phospholipids, phospholipid binding proteins, enzymes, viral
membrane fusion proteins and the like; through the use of
osmotically active agents; by using chemical crosslinking agents;
by physicochemical methods including electroporation and the like,
or by other permeabilizing methodologies.
[0167] In other aspects, the invention provides a method for
identifying an agent that binds to an ANT polypeptide comprising
contacting a candidate agent with a biological sample containing at
least one recombinant ANT polypeptide under conditions and for a
time sufficient to permit binding of the agent to the ANT
polypeptide, and detecting such binding. The use of human ANT1,
ANT2 and ANT3 according to these methods represent particularly
preferred embodiments. Other preferred embodiments include the use
of any ANT polypeptide or ANT fusion protein as provided herein. In
some preferred embodiments an ANT fusion protein is used that is a
GST fusion protein, and in other preferred embodiments the ANT
fusion protein is a His-tagged fusion protein. As provided herein,
the biological sample may be a cell, a mitochondrion,
submitochondrial particles, a cell membrane (including any cellular
membrane as described herein), a cell extract, cell conditioned
medium, a recombinant viral particle, a tissue homogenate or an
isolated ANT. Detection of binding may be by any of a variety of
methods and will depend on the nature of the candidate agent being
screened. For example, certain candidate agents are inherently
detectable as a consequence of their physicochemical properties,
such as will be apparent to those skilled in the art and including
spectrophotometric, colorimetric, fluorimetric, solubility,
hydrophobic, hydrophilic, electrostatic charge, molecular mass or
other physicochemical properties. As another example, certain
candidate agents may be radioactively labeled with a readily
detectable radionuclide, as is well known in the art. Certain
candidate agents may also be directly or indirectly detectable by
ANT protein affinity methodologies, for example by their ability to
interfere with binding of an ANT-specific antibody to an ANT; or by
their being removable from an assay solution using a protein
affinity reagent that binds to a fusion polypeptide present as a
portion of an ANT fusion protein. A candidate agent bound to an ANT
polypeptide may be detected by any method known for the detection,
identification or characterization of relevant molecules, including
spectrophotometric, mass spectrometric, chromatographic,
electrophoretic, calorimetric or any other suitable analytical
technique.
[0168] In another aspect the invention provides a method for
identifying an agent that interacts with an ANT polypeptide
comprising contacting a biological sample containing recombinant
ANT with a detectable ANT ligand (or a known detectable molecule
capable of binding to ANT) in the presence of a candidate agent,
and comparing binding of the detectable ANT ligand (or known
detectable ANT binding molecule) to recombinant ANT in the absence
of the agent to binding of the detectable ANT ligand (or known
detectable ANT binding molecule) to recombinant ANT in the presence
of the agent, and therefrom identifying an agent that interacts
with an ANT polypeptide. It will be appreciated that in certain
preferred embodiments this aspect provides competitive binding
assays wherein novel ANT ligands as provided hereinabove are
useful. However, this aspect of the invention need not be so
limited and may be modified to employ known detectable ANT binding
molecules, in which case it should be pointed out that the
selection of biological sample and/or of recombinant ANT as
provided by the present invention offer unexpected advantages
heretofore unknown in the art. Examples of known detectable
ANT-binding molecules include suitably labeled ATP, ADP, ATR, CATR,
palmitoyl-CoA, bongkrekic acid, thyroxin, eosin Y and erythrosin B
or other ANT-binding molecules known in the art. (See, e.g., Block
et al., 1986 Meths. Enzymol. 125:658.) The use of human ANT1, ANT2
and ANT3 according to these methods represent particularly
preferred embodiments. Other preferred embodiments include the use
of any ANT polypeptide or ANT fusion protein as provided herein. In
some preferred embodiments an ANT fusion protein is used that is a
GST fusion protein, and in other preferred embodiments the ANT
fusion protein is a His-tagged fusion protein. As provided herein,
the biological sample may be a cell, a mitochondrion,
submitochondrial particles, a cell membrane (including any cellular
membrane as described herein), a cell extract, cell conditioned
medium, a recombinant viral particle, a tissue homogenate or an
isolated ANT.
[0169] The ANT ligands compounds are preferably part of a
pharmaceutical composition when used in the methods of the present
invention. The pharmaceutical composition will include at least one
of a pharmaceutically acceptable carrier, diluent or excipient, in
addition to one or more ANT ligands and, optionally, other
components.
[0170] "Pharmaceutically acceptable carriers" for therapeutic use
are well known in the pharmaceutical art, and are described, for
example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co.
(A. R. Gennaro edit. 1985). For example, sterile saline and
phosphate-buffered saline at physiological pH may be used.
Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. For example, sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. Id. at 1449. In addition, antioxidants and
suspending agents may be used. Id.
[0171] "Pharmaceutically acceptable salt" refers to salts of the
compounds of the present invention derived from the combination of
such compounds and an organic or inorganic acid (acid addition
salts) or an organic or inorganic base (base addition salts). The
compounds of the present invention may be used in either the free
base or salt forms, with both forms being considered as being
within the scope of the present invention.
[0172] The pharmaceutical compositions that contain one or more ANT
substrates/ligands compounds may be in any form which allows for
the composition to be administered to a patient. For example, the
composition may be in the form of a solid, liquid or gas (aerosol).
Typical routes of administration include, without limitation, oral,
topical, parenteral (e.g., sublingually or buccally), sublingual,
rectal, vaginal, and intranasal. The term parenteral as used herein
includes subcutaneous injections, intravenous, intramuscular,
intrasternal, intracavernous, intrameatal, intraurethral injection
or infusion techniques. The pharmaceutical composition is
formulated so as to allow the active ingredients contained therein
to be bioavailable upon administration of the composition to a
patient. Compositions that will be administered to a patient take
the form of one or more dosage units, where for example, a tablet
may be a single dosage unit, and a container of one or more
compounds of the invention in aerosol form may hold a plurality of
dosage units.
[0173] For oral administration, an excipient and/or binder may be
present. Examples are sucrose, kaolin, glycerin, starch dextrins,
sodium alginate, carboxymethylcellulose and ethyl cellulose.
Coloring and/or flavoring agents may be present. A coating shell
may be employed.
[0174] The composition may be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension The liquid may be
for oral administration or for delivery by injection, as two
examples. When intended for oral administration, preferred
composition contain, in addition to one or more ANT
substrates/ligands compounds, one or more of a sweetening agent,
preservatives, dye/colorant and flavor enhancer. In a composition
intended to be administered by injection, one or more of a
surfactant, preservative, wetting agent, dispersing agent,
suspending agent, buffer, stabilizer and isotonic agent may be
included.
[0175] A liquid pharmaceutical composition as used herein, whether
in the form of a solution, suspension or other like form, may
include one or more of the following adjuvants: sterile diluents
such as water for injection, saline solution, preferably
physiological saline, Ringer's solution, isotonic sodium chloride,
fixed oils such as synthetic mono or digylcerides which may serve
as the solvent or suspending medium, polyethylene glycols,
glycerin, propylene glycol or other solvents; antibacterial agents
such as benzyl alcohol or methyl paraben; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic. Physiological saline is a preferred
adjuvant. An injectable pharmaceutical composition is preferably
sterile.
[0176] A liquid composition intended for either parenteral or oral
administration should contain an amount of ANT substrates/ligands
compound such that a suitable dosage will be obtained. Typically,
this amount is at least 0.01 wt % of an ANT substrates/ligands
compound in the composition. When intended for oral administration,
this amount may be varied to be between 0.1 and about 70% of the
weight of the composition. Preferred oral compositions contain
between about 4% and about 50% of ANT substrates/ligands
compound(s). Preferred compositions and preparations are prepared
so that a parenteral dosage unit contains between 0.01 to 1% by
weight of active compound.
[0177] The pharmaceutical composition may be intended for topical
administration, in which case the carrier may suitably comprise a
solution, emulsion, ointment or gel base. The base, for example,
may comprise one or more of the following: petrolatum, lanolin,
polyethylene glycols, beeswax, mineral oil, diluents such as water
and alcohol, and emulsifiers and stabilizers. Thickening agents may
be present in a pharmaceutical composition for topical
administration. If intended for transdermal administration, the
composition may include a transdermal patch or iontophoresis
device. Topical formulations may contain a concentration of the ANT
substrates/ligands compound of from about 0.1 to about 10% w/v
(weight per unit volume).
[0178] The composition may be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient.
Such bases include, without limitation, lanolin, cocoa butter and
polyethylene glycol.
[0179] In the methods of the invention, the ANT substrates/ligands
compound(s) may be administered through use of insert(s), bead(s),
timed-release formulation(s), patch(es) or fast-release
formulation(s).
[0180] It will be evident to those of ordinary skill in the art
that the optimal dosage of the ANT substrates/ligands compound(s)
may depend on the weight and physical condition of the patient; on
the severity and longevity of the physical condition being treated;
on the particular form of the active ingredient, the manner of
administration and the composition employed. It is to be understood
that use of an ANT substrates/ligands compound in a chemotherapy
can involve such a compound being bound to an agent, for example, a
monoclonal or polyclonal antibody, a protein or a liposome, which
assist the delivery of said compound.
EXAMPLES
[0181] The following Examples are offered by way of illustration
and not by way of limitation.
Example 1
Cloning and Expression of His-Tagged Human Ant Proteins in
Bacteria
[0182] A. PCR Amplification of ANT cDNAs
[0183] Total cellular RNA prepared from whole human brain was
obtained from a commercial source (Clontech, Palo Alto, Calif.).
The RNA was purified by treatment with RNase-free DNase I (Roche
Molecular Biochemicals, formerly Boehringer Mannheim Biochemicals,
Indianapolis, Ind.) using 1 ul of DNase I (10 u/ul) in a buffer
containing 40 mM Trsi-HCl, pH 7.0, 6 mM magnesium chloride and 2 mM
calcium chloride for 30 minutes at 37.degree. C. This treatment was
followed by two phenol/chloroform extractions, one chloroform
extraction and an ethanol precipitation in the presence of sodium
acetate. The RNA pellet was collected by centrifugation, washed
with 70% ethanol, air dried, and resuspended in RNase-free sterile
water. The RNA was reverse transcribed to generate cDNA using RNase
H-deficient Reverse Transcriptase (SUPERSCRIPT.TM.; Life
Technologies, Rockville, Md.).
[0184] ANT cDNAs were amplified by polymerase chain reactions (PCR)
in a thermal cycler using the following primers, AMPLITAQ.TM. DNA
Polymerase (Perkin-Elmer), and reagents and buffers supplied in a
GENEAMP.TM. PCR Reagent Kit (Perkin-Elmer), according to the
manufacturer's instructions. In the following representations of
the PCR primers, underlined nucleotides indicate sequences
complementary to the 5'-ends and 3'-ends of the ANT cDNAs and
double-underlined nucleotides indicate recognition sequences for
the restriction enzymes XhoI (recognition sequence: 5'-CTCGAG) and
Asp718 (recognition sequence: 5'-GGTACC).
[0185] For human ANT1 (huANT1; SEQ ID NO:1), the following primers
were used:
[0186] Forward (sense):
5'-TTATATCTCGAGTATGGGTGATCACGCTTGGAGCTTCCTAAAG SEQ ID NO:4
[0187] and Reverse (antisense):
5'-TATATAGGTACCTTAGACATATTTTTTGATCTCATCATACAAC SEQ ID NO:5.
[0188] For human ANT2 (huANT2; SEQ ID NO:2), the following primers
were used:
[0189] Forward (sense):
5'-TTATATCTCGAGTATGACAGATGCCGCTGTGTCCTTCGCCAAG SEQ ID NO:6
[0190] and Reverse (antisense):
5'-TATATAGGTACCTTATGTGTACTTCTTGATTTCATCATACAAG SEQ ID NO:7.
[0191] For human ANT3 (huANT3; SEQ ID NO:3), the following primers
were used:
[0192] Forward (sense):
5'-TTATATCTCGAGTATGACGGAACAGGCCATCTCCTTCGCCAAA SEQ ID NO:8
[0193] and Reverse (antisense):
5'-TATATAGGTACCTTAGATCACCTTCTTGAGCTCGTCGTACAGG SEQ ID NO:9.
[0194] B. Generation of ANT Expression Constructs
[0195] PCR products were digested with the restriction
endonucleases XhoI and Asp718 (both enzymes from Roche Molecular
Biochemicals) according to the manufacturer's recommendations using
manufacturer-supplied reaction buffers. Restricted DNAs were
purified by horizontal agarose gel electrophoresis and band
extraction using the UltraClean GelSpin kit (Mo Bio Laboratories,
Inc., Solana Beach, Calif.).
[0196] The expression vector pBAD/His ("B" derivative; Invitrogen,
Carlsbad, Calif.) was used. This vector contains the following
elements operably linked in a 5' to 3' orientation: the inducible,
but tightly regulatable, araBAD promoter; optimized E. coli
translation initiation signals; an amino terminal
polyhistidine(6xHis)-encoding sequence (also referred to as a
"His-Tag"); an XPRESS.TM. epitope-encoding sequence; an
enterokinase cleavage site which can be used to remove the
preceding N-terminal amino acids following protein purification, if
so desired; a multiple cloning site; and an in-frame termination
codon.
[0197] Plasmid pBAD/His DNA was prepared by digestion with the
restriction endonucleases XhoI and Asp718 according to the
manufacturer's instructions and subjected to horizontal agarose gel
electrophoresis and band extraction using the UltraClean GelSpin
kit (Mo Bio Laboratories). Restricted ANT cDNAs were ligated into
the linearized plasmid with restricted expression vector DNA using
T4 DNA ligase (New England Biolabs, Beverly, Mass.) using the
manufacturer's reaction buffer and following the manufacturer's
instructions. Competent recA1 hsdR endA1E. coli cells (strain
TOP10F'; Invitrogen, Catalog #C3030-03) were transformed with
ligation mixtures containing the prokaryotic vector construct
according to the manufacturer's instructions. Single colonies were
selected and grown in 3-5 ml of LB broth (Sambrook, J., Fritsch, E.
F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)
containing 50 .mu.g/ml ampicillin (Roche Molecular Biochemicals).
Plasmid DNA was isolated from the bacterial cultures using the
WIZARD.TM. Plus Series 9600 Miniprep Reagents System (Promega,
Madison, Wis.).
[0198] The recombinant huANT nucleotide sequences present in the
expression constructs were determined and their authenticity
confirmed relative to the published ANT sequences (FIG. 1; See
Neckelmann et al., Proc. Nat'l. Acad. Sci. U.S.A. 84:7580-7584
(1987) for huANT1; Battini et al., J. Biol. Chem. 262:4355-4359
(1987) for huANT2, and Cozens et al., J. Mol. Biol. 206:261-280
(1989) for huANT3.) by DNA sequencing using the PRISM.TM. Ready BIG
DYE.TM. Terminator Cycle Sequencing Kit (The Perkin-Elmer Corp.,
Norwalk, Conn.) and the following sequencing primers
5'-TATGCCATAGCATTTTTATCC (SEQ ID NO:10) and 5'-CGCCAAAACAGCCAAGCT
(SEQ ID NO:11). For each human ANT sequence, both primers are
located inside the vector sequence adjacent to the DNA insertion.
Sequence data was analyzed using the SEQUENCE NAVIGATOR.TM.
analysis software package (Perkin-Elmer). This huANT3 expression
construct was named pMK3A-huANT3.
[0199] The expression plasmids encoding His-tagged human ANT1, ANT2
and ANT3 are referred to herein as follows: For human ANT1, "pMK1
(His-tagged huANT1)" or "pMK1"; for human ANT2, "pMK2 (His-tagged
huANT2)" or "pMK2"; for human ANT3 "pM3A (His-tagged hu ANT3" or
"pMK3A"; for human ANT3 from which extraneous linker N-terminal
amino acids are deleted as detailed below, "pMK3B (His-tagged hu
ANT3, shortened epiotpe linker)" or "pMK3B". Plasmids pMK1, pMK2
and pMK3A have been deposited at the American Type Culture
Collection (ATCC; Manassas, Va.) on Nov. 3, 1998, and given the
accession numbers ATCC 98969, ATCC 98970 and ATCC 98971,
respectively.
[0200] The expression constructs comprising nucleotide sequences
encoding human ANT1 (pMK1-huANT1) and human ANT2 (pMK2-huANT2) were
restriction mapped to confirm their structures. The nucleotide
sequences of plasmids pMK1-huANT1 and pMK2-huANT2 are determined
using the methods and primers (SEQ ID NOS:10 and 11) described
above.
[0201] Treatment of the recombinant huANT3 protein expressed from
pMK3A-huANT3 with enterokinase liberates the His-Tag/XPRESS.TM.
epitope polypeptide from the huANT3 protein; however, the resultant
huANT3 protein comprises several extraneous N-terminal amino acids
(i.e., Pro-Ser-Ser-Ser-Met, where "Met" indicates the amino acid
encoded by the translation initiation codon of huANT3). Although
the extraneous amino acids probably have little or no effect on the
recombinant huANT3 protein, a derivative expression construct in
which the nucleotide sequence encoding the extraneous amino acids
are deleted was prepared in the following manner.
[0202] The QUIK-CHANGE.TM. Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, Calif.) essentially according to the
manufacturer's instructions. Briefly, a reaction mixture comprising
purified pMK3A-huANT3 DNA, the mutagenic oligonucleotide primers
5'-GGAGATGGCCTGTTCCGTCATCTTATCGTCATCGTC- GTACAGATC (SEQ ID NO:12;
the underlined sequence is the reverse complement of the 5' end of
the huANT3 reading frame), and 5'-GATCTGTACGACGATGACGATA-
AGATGACGGAACAGGCCATCTCC (SEQ ID NO:13; the underlined sequence
corresponds to the 5' end of the huANT3 reading frame), Pfu DNA
polymerase and dNTPs in manufacturer-supplied reaction buffer was
prepared. The mutagenic oligonucleotide primers were present in
excess and cycles of DNA synthesis was carried out in a thermal
cycler according to the manufacturer's protocol. The reaction
products were treated with the restriction enzyme DpnI, which
cleaves methylated and hemi-methylated DNAs but leaves unmethylated
DNA (i.e., annealed products of the reaction) intact, and used to
transform EPICUREAN COLI.TM. XL-1-Blue E. coli cells (Stratagene).
Plasmid DNA was prepared from twelve randomly selected
transformants and the nucleotide sequence of the region containing
the multiple cloning site cassette was determined according to the
methods described above. Of the twelve plasmids, only one retained
the original sequence found in pMK-huANT3, and three contained
undesired point mutations. One of the eight "correct" plasmids was
chosen and named pMK3B-huANT3.
[0203] C. Expression of His-Tagged huANT3
[0204] Cultures of E. coli cells containing pMK3A-huANT3 were grown
in LB media containing 50 ug/ml ampicillin to mid-log phase
(OD.sub.600.about.0.5) and induced for 3-4 hours with increasing
doses of arabinose (i.e., 0.00002%, 0.0002%, 0.002%, 0.02%, and
0.2%). One ml of each culture was centrifuged at 5,000.times.g for
10 minutes at 4.degree. C. to pellet the cells. Cell pellets were
resuspended, and the cells were lysed, by adding 100 ul of
Phosphate Buffered Saline (PBS; pH 7.4) containing 1% cholate, 1%
n-dodecyl maltoside, and 0.1% 2-mercaptoethanol (in the preceding
text, and throughout the specification, unless specified otherwise,
all chemicals are from Sigma, St. Louis, Mo.) Total protein content
in the lysates was determined using the BCA (bicinchoninic acid;
Smith et al., 1985, Anal. Biochem. 150:76-85) Protein Assay kit
(Pierce Chemical Co., Rockford, Ill.). Ten .mu.g of total protein
were loaded per lane onto an SDS polyacrylamide gel,
electrophoresed and transferred to a nitrocellulose membrane
(HYBOND.TM. ECL Nitrocellulose Membrane, Amersham Pharmacia
Biotech, formerely Amersham Life Sciences, Piscataway, N.J.). Human
ANT3 fusion proteins were detected in a western blot using
ANTI-XPRESS.TM. Antibody (Invitrogen) and horseradish
peroxidase-conjugated anti-mouse secondary antibody (Amersham
Pharmacia Biotech) according to the manufacturers'
instructions.
[0205] The results are shown in FIG. 3. From left to right in the
figure, the following samples are shown: lanes "M", molecular
weight markers; lane "0", untransformed E. coli cells; lane "o/n",
E. coli comprising pMK3A-huANT3 grown overnight without induction;
lane "1"-"5", E. coli comprising pMK3A-huANT3 grown induced with
increasing doses of arabinose (0.00002%, 0.0002%, 0.002%, 0.02% and
0.2%, respectively). As expected, untransformed (lane 0) and
uninduced (lane o/n) E. coli showed no XPRESS.TM.-huANT3 material.
However, expression of recombinant ANT3 fusion protein with a
molecular weight of 36.6 kD was observed in lanes 3 and 4 (0.002%
and 0.02% arabinose, respectively). No XPRESS.TM.-huANT3 material
was detected in lanes 1 and 2 (0.00002% and 0.0002% arabinose,
respectively) indicating that the degree of induction was
insufficient under these conditions.
[0206] Cells that were grown in the presence of the highest
concentration of arabinose (0.2%, lane 5) began to lyse and died
before the time of harvest; consequently, no recombinant protein
was detected. This indicated that very high expression of
recombinant huANT in E. coli caused cell death, as is sometimes the
case during overexpression of heterologous proteins in
bacteria.
[0207] D. Recombinant huANT3 Localizes to the Bacterial
Membrane
[0208] In order to locate the expressed human ANT 3 within E. coli
cells, cells were grown in culture and induced with arabinose as
described above, and then fractionated into different compartments
(e.g., membranes, inclusion bodies and cytosol). Bacteria were
pelleted by centrifugation at 5,000.times.g for 10 minutes at
4.degree. C. The cell pellets were resuspended in 1/10 volume of
cell buffer A (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 ug/ml
lysozyme, and 0.1% Triton X-100) and incubated for 15 minutes at
30.degree. C. in an orbital shaker. The cell mixture was sonicated
for 2 minutes and membranes were pelleted by centrifugation at
12,000.times.g for 15 minutes at 4.degree. C. The supernatant,
representing the cytosol, was removed for analysis (FIG. 4, lane
4), as was a portion of the pellet containing membranes and
inclusion bodies (FIG. 4, lane 3). The remaining portion of the
pellet was washed twice with cell buffer B (10 mM Tris-HCl, pH 7.0,
0.1 mM EDTA, and 1 mM DTT) and centrifuged at 12,000.times.g for 15
minutes at 4.degree. C. The pellet was resuspended in cell buffer C
(20 mM Tris-HCl, pH 8.0, 100 mM sodium chloride, and 6 M
guanidinium hyrochloride) and incubated for 1 hour at room
temperature. The solution was then centrifuged at 12,000.times.g
for 15 minutes at 4.degree. C. The supernatant (containing
solubilized inclusion bodies; lane 1, FIG. 4) and the pellet
(containing insoluble inclusion bodies; lane 2, FIG. 4) were
analyzed by Western blotting as described above.
[0209] The results are shown in FIG. 4. Recombinant huANT 3
(molecular weight 36.6 kD) was detected in lanes 2, 3, and 4, as
well as the positive control lane (+) (total cell lysate previously
tested for presence of ANT3 protein by Western immunoblot analysis,
as described above). The greatest amount of recombinant huATN3 was
detected in lane 3, which represents the membrane fraction. This
indicates that the majority of the huANT3 fusion protein integrated
into the E. coli cellular membrane. Smaller protein signals were
visible in lanes 2 and 4, representing the insoluble inclusion body
fraction which might have contained some membranes with integrated
ANT 3, and the cytosolic fraction where protein synthesis takes
place. No protein was detectable in the soluble inclusion body
fraction in lane 1, indicating that controlled expression of ANT3
in the bacteria did not result in the formation of inclusion
bodies, which is an undesirable consequence of over-expression of
some heterologous proteins in bacteria.
[0210] E. Purification of ANT Proteins
[0211] ANT proteins, and ANT fusion proteins, produced by the
expression systems described herein have been purified using a
variety of methods. The purification of ANT proteins, particularly
human ANT proteins, is described in this Example.
[0212] Regardless of which of following protein purification
methods is used, or others that can be derived from the present
disclosure, it is important to add sufficient amounts of DNase and
RNase to eliminate the viscosity associated with some bacterial
lysates (typically 10 .mu.g/mL of each enzyme; both from Roche
Biochemicals) when the bacterial cells are lysed (or immediately
thereafter). An alternative or additional means by which viscosity
has been minimized and ANT solubility has been optimized is
vigorous sonication, as opposed to standard sonication, of the
lysates. The term "vigorous sonication" refers to, for example,
sonication with a Branson Sonifier (Model 450) 2.times. (30 seconds
each time) at 50% duty cycle and 80% output using a tapered,
flat-tipped probe (as opposed to sonication with a cup and horn
apparatus). Although either type of sonication will suffice, better
yields have typically been observed when vigorous sonication has
been used.
[0213] Furthermore, in various ANT purification methods that have
been used, it was often desirable to make the lysate at least 1%
Triton-X, in order to solubilize the maximum possible amount of ANT
protein, after which insoluble material is removed by a high-speed
(i.e., about 100,000 g) spin. Typically, protease inhibitors such
as, for example, pepstatin, leupeptin, phenylmethylsulfonyl
fluoride (PMSF) and/or aprotinin (all from Sigma) have been present
at effective levels (typically 10 .mu.g/mL) during the preparation.
Depending on the particular ANT protein or ANT fusion protein being
isolated, all four protease inhibitors or any effective combination
thereof are used. For example, in preparations of GST-huANT3 fusion
proteins, best results were seen when all four protease inhibitors
were used, although acceptable results have been obtained when only
leupeptin and pepstatin were used.
[0214] One method incorporates novel methods with several
techniques previously used only for purifying ANT proteins from
non-human mammals, i.e., bovine cardiac tissue and rats (Aquila et
al., 1982, Hoppe-Seyler's Z. Physiol. Chem. 363:345-349; and
Sterling, 1986, Endocrinology 119:292-295). In brief, bacterial
cells expressing a GST-ANT3 fusion protein were lysed by lysozyme
treatment, and .sup.14C-palmityl-CoA (Sigma) was added at a
concentration of 50 nmol per gram of E. coli. Because it associates
with ANT proteins, .sup.14C-palmityl-CoA acts as a radiolabeled
tracer that can be used to follow the ANT protein in subsequent
purification steps. The lysates were then sonicated and made 6%
Triton X-100 (Sigma) and incubated at 4.degree. C. for 1 hr to
solubilize material. A high-speed spin was used to remove insoluble
material, and the resulting solute was applied either (1) for small
scale preparations, to hydroxyapatite beads (Bio-Rad Laboratories,
Hercules, Calif.), or (2) in the case of larger preparations (i.e.,
.gtoreq.1 liter of bacterial culture), to a hydroxyapatite column
(Bio-Rad) essentially according to the manufacturer's instructions.
Unlike other intramembrane mitochondrial proteins, ANT has a low
affinity for hydroxyapatite (Klingenberg et al., 1978, Biochim. et
Biophys. Acta 503:193-210). The hydroxyapatite column was eluted
with Column Buffer A (10 mM MOPS, pH 7.2, 100 Mm NaCl, 9.5% Triton
X100) and washed with Column Buffer B (10 mM MOPS, pH 7.2, 100 mM
NaCl, 400 mM sodium phosphate). Non-recombinant ANT proteins from
non-human species are eluted in the void volume with Column Buffer
A, and the GST-huANT3 fusion protein was expected to be present in
the void volume as well; Column Buffer B was used to wash the
column in the event that GST-huANT3 fusion protein behaves
differently. Samples were collected in such a manner as to have a
final concentration of 30 of mM octyl glucoside (Calbiochem), a
nonionic detergent that helps solubulize ANT proteins with minimal
effect on activity (Sterling, 1986, Endrocrinol. 119: 292-295). The
bead-extracted supernatant or column eluent was collected, and
Triton X-100 was removed therefrom using the EXTRACTI-GEL.TM.
affinity matrix (Pierce) essentially according to the
manufacturer's instructions (see also Berman et al., 1985,
Biochemistry 24:7140-7147).
[0215] Varying amounts of GST-huANT3 prepared in the above manner
were subject to PAGE and the gel was stained using a colloidal blue
protein stain (Novex, San Diego, Calif.). The stained gel displayed
a single band having a molecular weight corresponding to that
predicted for the fusion protein. Based on the intensity of bands
from samples of varying volumes, and the known volume of the
preparation and minimal sensitivity of the stain, the yield from
100 mL of bacterial culture was estimated to be about 50 ug. In one
of the lanes of the gel, approximately 500 ng of protein was
loaded, and no contaminating bands were detected; this indicates
that the GST-huANT3 protein was from at least about 90% pure to at
least about 95% pure.
[0216] GST-huANT3 fusion proteins (see preceding Examples) have
been purified by this method, and other ANT fusion proteins,
including His-tagged huANT3 and other His-tagged ANT proteins, are
purified in like fashion. Purified huANT fusion proteins are used
to produce purified human ANT proteins as follows.
[0217] GST-huANT fusion proteins are further purified via
glutathione-agarose beads (Sigma) essentially according to the
manufacturer's instructions. In brief, a solution comprising
GST-huANT fusion proteins is contacted with glutathione-agarose
beads, and the beads are washed to release undesirable
contaminants. Next, the [bead:GST-huANT] complexes are treated with
an appropriate enzyme, i.e., one that separates the huANT
polypeptide from the remainder of the fusion protein. In the case
of the GST-huANT3 fusion protein described herein (i.e., that
encoded by pMK3C), thrombin (Sigma) cleaves the fusion protein in
such a manner so as to produce two polypeptides: a first
polypeptide corresponding to the GST moiety, and a second
polypeptide which corresponds to human ANT3 with an additional six
amino acids (i.e., Gly-Ser-Pro-Gly-Ile-Leu) present at its
N-terminus.
[0218] His-tagged huANT fusion proteins are further purified via
Nickel-coated resins (such as, e.g., PROBOND.TM. Ni.sup.2+ charged
agarose resin; Invitrogen) essentially according to the
manufacturer's instructions. In brief, a solution comprising
His-tagged huANT fusion proteins is contacted with the
Nickel-coated resin, and the resin is washed to release undesirable
contaminants. Next, the [resin:His-tagged huANT] complexes are
treated with an appropriate enzyme, i.e., one that separates the
huANT polypeptide from the remainder of the fusion protein. In the
case of the His-tagged huANT3 fusion proteins described herein,
enterokinase (Sigma, or EKMAX.TM. from Invitrogen may be used)
cleaves the fusion protein in such a manner so as to produce two
polypeptides: a first polypeptide comprising the His-tag and
XPRESS.TM. epitope moieties, and a second polypeptide which
corresponds to human ANT3. If the expression construct used is
pMK3A, the resultant purified human ANT3 protein has an additional
four amino acids (i.e., Pro-Ser-Ser-Ser) at its N-terminus. If
pMK3B is the expression construct present in the cells from which
His-tagged huANT3 is isolated, the resultant purified human ANT3
protein has the sequence of native huANT3, i.e., SEQ ID NO:3.
[0219] In both of the preceding purification steps, an ANT fusion
protein bound to a solid support is treated with an enzyme (i.e.,
thrombin or enterokinase) that liberates an ANT protein from the
remainder of the fusion protein, which remains bound to the solid
support. ANT protein is released into the liquid phase which is
then collected to generate a solution comprising the ANT protein
and some amount of the liberating enzyme. The amount of liberating
enzyme needed is minimal because the treatment is catalytic in
nature; nevertheless, some enzyme remains in the preparation. If
desired, enzyme molecules may be removed from the preparation using
any of a variety of means known in the art. For example, an enzyme
may be removed from a solution by contacting the solution with a
resin conjugated to a ligand having a high affinity for the enzyme.
In the case of enterokinases, one such resin is the EK-AWAY.TM.
resin (Invitrogen) which comprises the soybean trypsin inhibitor
having a high affinity for enterokinases. Methods of treating GST
fusion proteins with thrombin and purifying the desired recombinant
protein have been described previously (see, for example, Smith and
Corcoran, Unit 16.7 in Chapter 16 in Short Protocols in Molecular
Biology 2.sup.nd Ed., Ausubel et al., eds, John Wiley & Sons,
New York, N.Y., 1992, pages 16-28 to 16-31. In general, however,
any suitable means for separating the liberating enzyme from any
given ANT protein may be used.
[0220] F. Growth Inhibition
[0221] As noted in the above discussion of the results presented in
FIG. 3, very high expression of recombinant huANT3 in E. coli
caused cell death. Such a result is sometimes observed during
over-expression of heterologous proteins in bacteria. Although not
wishing to be bound by any particular theory, because the
recombinant huANT3 protein localized to the bacterial membrane, and
because ANT3 functions as an ATP/ADP exchanger in the inner
mitochondrial membrane and under appropriate conditions may exhibit
pore properties suggestive of a role in membrane permeability, one
possible explanation for the observed cell death would be an
inappropriate enhancement of the permeability of the bacterial
membrane. If this in fact the case, inhibitors of mitochondrial ANT
might prevent the death of E. coli overexpressing huANT3. As noted
above, under certain conditions atractyloside or bongkrekic acid
may exhibit inhibition of ANT activity, such that either of these
inhibitors, other known ANT-active agents and potentially other ANT
ligands as provided herein may be employed in the instant Example
described using bongkrekic acid.
[0222] In order to test this hypothesis, the following experiments
are carried out. E. coli harboring pMK3A-huANT3 are grown with no
arabinose or with 0.2% or more arabinose, the latter concentration
having been previously shown to induce toxic levels of huANT3, and
various concentrations (0, 5, 20, 50 and 200 .mu.M) of bongkrekic
acid (Biomol Research Laboratories, Inc., Plymouth Meeting, Mass.),
an inhibitor of ANT (Henderson and Lardy, 1970, J. Biol. Chem.
245:1319-1326) that binds to ANT (see, e.g., Vignais et al., 1976,
Biochim. Biophys. 440:688-696). The ability of bongkrekic acid to
prevent the lysis of E. coli overexpressing huANT3, or any other
ANT protein for that matter, indicates that the toxic effect of
such overexpression is due to an activity associated with normally
functioning ANT.
[0223] ANT proteins produced by this expression system, and others
described herein, are also purified using known methods for
purifying ANT proteins from humans and other mammals. See for
example, Klingenberg et al., 1978 Biochim. Biophys. Acta
503:193-210; Aquila et al., 1982 Hoppe-Seyler's Z. Physiol. Chem.
363:345-349; and Sterling, 1986 Endocrinol. 119:292-295.
[0224] The bacterial toxicity of extreme overexpression of ANT in
this system can be used to screen and identify novel inhibitors of
ANT, as such compounds will be expected to also prevent lysis of E.
coli overexpressing ANT proteins. In order to achieve a greater
degree of specificity for the ANT protein produced from an
expression vector, the yeast expression system for ANT proteins
(see Example 4, infra) is used in a mutant yeast strain that is
resistant to bongkrekic acid (Lauquin et al., 1975 FEBS Letters
35:198-200).
Example 2
Expression of GST-huANT3 Fusion Proteins
[0225] A. Generation of GST-huANT3 Expression Constructs
[0226] Human ANT3 cDNA was amplified from pMK3A-huANT3 by PCR as in
Example 1 but using the following primers. In the following
representations of PCR primers, underlined nucleotides indicate
sequences complementary to the 5'-ends and 3'-ends of the ANT cDNAs
and double-underlined nucleotides indicate recognition sequences
for the restriction enzymes XhoI (recognition sequence: 5'-CTCGAG)
or EcoRI (recognition sequence: 5'-GAATTC).
[0227] The primers used for PCR amplification were:
[0228] Forward (sense):
5'-CCCGGGGAATTCTGATGACGGAACAGGCCATCTCC SEQ ID NO:14
[0229] and Reverse (antisense):
5'-CCCGGGCTCGAGTTAGAGTCACCTTCTTGAGCTC SEQ ID NO:15
[0230] The expression vector pGEX-4T-2 (Amersham Pharmacia Biotech)
was used to generate huANT3 fusion proteins comprising an enzymatic
polypeptide and an ANT polypeptide. This vector comprises a
lacI.sup.q (repressor) gene a tac promoter operably linked to a
glutathione S-transferase (GST) gene from Schistosoma japonicum.
(Smith et al., 1988, Gene 67:31-40), the coding sequence of which
has been modified to comprise a thrombin cleavage site-encoding
nucleotide sequence immediately 5' from a multiple cloning site GST
fusion proteins can be detected by Western blots with anti-GST or
by using a colorimetric assay; the latter assay utilizes
glutathione and 1-chloro-2-4-dinitrobenzene (CDNB) as substrates
for GST and yields a yellow product detectable at 340 nm (Habig et
al., 1974, J. Biol. Chem. 249:7130-7139). GST fusion proteins
produced from expression constructs derived from this expression
vector can be purified by, e.g., glutathione affinity
chromatography, and the desired polypeptide released from the
fusion product by thrombin. Thus, this expression vector provides
for the rapid purification of fusion proteins, and release of
proteins with relatively few extraneous N-terminal amino acids,
although the resulting recombinantly produced protein contains two
additional amino acids at the amino terminus (Gly-Ser). The tac
promoter may be induced by the addition to cultured cells of, e.g.,
1-5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG; Fluka,
Milwaukee, Wis.) and provides for high-level expression.
[0231] Plasmid pGEX-4T-2 was prepared by digestion with the
restriction endonucleases EcoRI and Asp718 according to the
manufacturer's instructions and subjected to horizontal agarose gel
electrophoresis and band extraction using the UltraClean GelSpin
kit (Mo Bio Laboratories). Restricted ANT cDNAs were ligated with
the restricted expression vector DNA as described in the preceding
Example. Single colonies were selected for grown in 3-5 ml of LB
broth containing 50 ug/ml ampicillin (Roche Molecular
Biochemicals), and plasmid DNA was isolated from the bacterial
cultures using the WIZARD.TM. Plus Series 9600 Miniprep Reagents
System (Promega). To confirm their authenticity, the recombinant
huANT nucleotide sequences present in the pGEX deriavtive plasmid
were determined as described in the preceding Example using the
previously described oligonucleotide primers and 5' and 3' PGEX
Sequencing Primers (Amersham Pharmacia Biotech).
[0232] The resultant GST-huANT3 expression construct was named
pMK3C-GST-huANT3 (also referred to herein as pMK3C). Plasmid pMK3C
has been deposited at the American Type Culture Collection (ATCC;
Manassas, Va.) on Nov. 3, 1998, and given the accession number ATCC
98973. Thrombin treated recombinant huANT3 protein produced from
the pM3C-GST-huANT3 expression construct includes several
extraneous N-terminal amino acids, i.e.,
Gly-Ser-Pro-Gly-Ile-Leu-Met, where "Met" indicates the amino acid
encoded by the translation initiation codon of huANT3. There is,
however, no evidence that the extraneous six amino terminal amino
acids have any effect on the resultant recombinant huANT3
protein.
[0233] In order to confirm expression of the GST-huANT3 fusion
protein, the following experiments were carried out. Eight
independently isolated pMK3C-GST-huANT3 transformants and one
control (vector-transformed) isolate were grown overnight in
LB-ampicillin and then diluted 1:20 in 2 ml of fresh media. After 3
hours of growth at 37.degree. C., IPTG was added to a final
concentration of 0.1 mM. Cell growth was continued for 2 hours,
after which 1.5 of cells were tranferred to microfuge tubes,
pelleted, resuspended in 300 uL of cold PBS containing 1% Triton
X-100, and sonicated twice for 8 seconds. The sonicates were spun
for 5 min. at 4.degree. C., the supernatant was transferred to
fresh microfuge tubes and 50 uL of glutathione-agarose beads
(Sigma) were added to produce a 50% slurry. After a 5 min.
incubation at ambient temperature, the beads were spun and washed
with 1 ml of PBS three times. The washed pellet was resuspended in
SDS spl buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5%
beta-mercaptoethanol and sufficient bromophenol blue to provide
visible coloration), and 30 uL of each preparation (equivalent to
15 uL of culture) was subjected to SDS-PAGE. The gel was stained
using a Colloidal Coomassie (G-250) Staining Kit (Novex, San Diego,
Calif.). A band of the predicted molecular weight of the GST-huANT3
fusion protein was readily apparent, with the same intensity, in
each of the 8 preparations from pMK3C-GST-huANT3 transformants;
this band was absent in the control preparation.
[0234] B. Western Blot Analysis of Expression of huANT3 Fusion
Proteins
[0235] E. coli transformed with either (1) pMK3A-huANT3 (the
pBAD/His-huANT3 expression construct) or (2) pMK3C-GST-huANT3 (the
pGEX/GST-huANT3 expression construct) were lysed by the addition of
lysozyme (100 .mu.g/.mu.l; Sigma) for 20 min at room temperature,
followed by one freeze/thaw cycle. The negative control for the
former transformant was a parallel culture of the transformed cells
that had not undergone arabinose induction. The control for the
latter transformant was a parallel culture of E. coli that had been
transformed with the pGEX-4T-2 vector only.
[0236] Total protein concentrations of each lysate were determined
using the BCA Protein Assay kit (Pierce Chemical Co.), and
equivalent amounts of total protein from each lysate preparation
were mixed with equivalent volumes of 2.times.Laemmli
electrophoresis buffer and subjected to SDS-PAGE. The proteins were
electrophoretically transferred to nitrocellulose, which was then
contacted with antibodies against the appropriate epitope included
in each vector (i.e., ANTI-XPRESS.TM. from Invitrogen for
pMK3A-huANT3 and polyclonal goat anti-GST from Amersham Pharmacia
Biotech, formerly Nycomed Amersham plc and Pharmacia & UpJohn
Inc. for pMK3C-GST-huANT3).
[0237] In a separate experiment, the bacterial lysate from the
pMK3C-GST-huANT3 transformants was incubated with
agarose-glutathione beads (Sigma) according to the manufacturer's
instructions (see the preceding section and Smith et al.,
Expression and Purification of Glutathione S-Transferase Fusion
Proteins, Unit 16.7 of Chapter 16 in: Short Protocols in Molecular
Biology, 2nd Ed., Asubel et al., eds., John Wiley & Sons, New
York, N.Y., 1992, pages 16-28 to 16-31). The beads were suspended
in Laemmli sample buffer and subjected to SDS-PAGE and Western
analysis as described above. Although the yield of GST-huANT3 was
low, perhaps because the fusion protein is inserted into the
bacterial membrane, a sufficient amount of material was recovered
for the experiment.
[0238] The results (FIG. 5) show that a specific band of the
predicted molecular weight (His-Tag+enterokinase site+antigenic
site+huANT3=38 kDa) was observed in the arabinose induced E. coli
that were transformed with the pBAD/his-huANT3 vector, but was
absent in the non-induced control culture. Similarly, a band
corresponding to GST-huANT3 was observed in the
pMK3C-GST-huANT3-transformed E. coli, while only the unaltered GST
band was observed in control E. coli transformed with the
expression vector. Purification of the GST-huANT3 fusion protein
using agarose-GSH beads produced a band of equivalent size to that
observed in the crude lysate of pMK3C-GST-huANT-transformed
bacteria.
Example 3
Expression of ANT3 in Insect Cells
[0239] A. Generation of Baculovirus Expression Constructs
[0240] DNA comprising nucleotide sequences encoding huANT3 was
amplified by PCR from a whole human brain cDNA library (Clontech)
using the following primers. In the following representations of
PCR primers, underlined nucleotides indicate sequences
complementary to the 5'-ends and 3'-ends of the ANT cDNAs and
double-underlined nucleotides indicate recognition sequences for
the restriction enzymes BamHI (recognition sequence: 5'-GGATCC) or
EcoRI (recognition sequence: 5'-GAATTC).
[0241] The PCR primers used were:
[0242] Forward (sense):
5'-TTATAGGATCCATGACGGAACAGGCCATCTCCTTCGCCAAA SEQ ID NO:16
[0243] and Reverse (antisense):
5'-TTAAAGAATTCTTAGATCACCTTCTTGAGCTCGTCGTACAG SEQ ID NO:17.
[0244] PCR products were digested with the restriction
endonucleases BamHI (New England Biolabs) and EcoRI (New England
Biolabs) according to the manufacturer's recommendations.
Subsequent purification was carried out by horizontal agarose gel
electrophoresis and band extraction using the UltraClean GelSpin
kit (Mo Bio Laboratories, Inc.).
[0245] The Baculovirus transfer vector pBlueBacHis2 (B version,
Invitrogen) comprises, in 5' to 3' orientation, a constitutive
polyhedrin promotor operably linked to nucleotide sequences
encoding (1) a translation initiation sequence, (2) an N-terminal
polyhistidine sequence, (3) an XPRESS.TM. epitope tag for detection
and purification of the recombinant protein and (4) an enterokinas
cleavage site, followed by a multiple cloning site wherein cDNAs
can be inserted.
[0246] The transfer vector pBlueBacHis2 was prepared by digestion
with the restriction endonucleases BamHI and EcoRI according to the
manufacturer's recommendation, and the restricted DNA was subject
to horizontal agarose gel electrophoresis and band extraction using
the UltraClean GelSpin kit (Mo Bio Laboratories, Inc.). The
restricted PCR products were ligated with the restricted expression
vector DNA as in the preceding Examples.
[0247] Competent E. coli TOP10F' cells (Invitrogen) were
transformed with the ligation recation following the manufacturer's
instructions. Single colonies were selected for growth in 3-5 ml of
LB broth containing 50 ug/ml ampicillin. Plasmid DNA was isolated
from the bacterial cultures using the WIZARD.TM. Plus Series 9600
Miniprep Reagents System (Promega).
[0248] The recombinant ANT gene sequences were determined and their
authenticities confirmed (SEQ ID NOS:1, 2 and 3 correspond to human
ANTs 1, 2 and 3, respectively) by DNA sequencing using the Prism
Ready Dye Terminator Cycle Sequencing Kit (Perkin-Elmer, Catalog
#402080) and the following primers: Polyhedrin Forward Sequencing
Priming Site, 5'-AAATGATAACCATCTCGC (SEQ ID NO:18); Baculovirus
Reverse Sequencing Priming Site, 5'-ACTTCAAGGAGAATTTCC (SEQ ID
NO:19); primers internal to the ANT 3 coding sequence (sense
strand), 5'-ACTTCGCCTTCACGGATA (SEQ ID NO:20); and
5'-TACGGCCAAGGGCATTCT (SEQ ID NO:21); primers internal to the ANT 3
coding sequence (antisense strand), 5'-TGAAGCGGAAGTTCCTAT (SEQ ID
NO:22); and 5'-ATGCCGGTTCCCGTACGA (SEQ ID NO:23). Sequence data
were analysed using the SEQUENCE NAVIGATOR.TM. analysis software
package (Perkin-Elmer). An isolated plasmid having the correct
sequence was named pMK4A-huANT3.
[0249] Although pMK4A-huANT3 contains authentic huANT3-encoding
sequences, the ANT3 reading frame is not synchronous with the
reading frame of the His-Tag/XPRESS.TM. epitope of the expression
vector. Accordingly, pMK4A-huANT3 is not expected to produce
recombinant ANT protein, although cells harboring it may be used as
controls.
[0250] In order to generate an in-frame derivative of pMK4A-huANT3,
the plasmid was mutagenized using the QUIK-CHANGE.TM. Site-Directed
Mutagenesis Kit (Stratagene) as in Example 1, except that the
mutagenic oligonucleotide primers used were
5'-GGCCTGTTCCGTCATCTTATCGTCATCGTCG (SEQ ID NO:24; the underlined
sequence is the reverse complement of the 5' end of the huANT3
reading frame), and 5'-CGACGATGACGATAAGATGACGGAACAGGCC (SEQ ID
NO:25; the underlined sequence corresponds to the 5' end of the
huANT3 reading frame). Several transformants were isolated, and
plasmid DNA purified therefrom. The nucleotide sequences of the
plasmid DNAs were determined and one having the "correct" sequence
was identified and named pMK4B-huANT3.
[0251] The baculovirus expression plasmids encoding human ANT3 are
referred to as "pMK4A (baculovirus shuttle, out-of-frame hu ANT3)
or "pMK4A"; and "pMK4B (baculovirus shuttle, in-frame hu ANT3)" or
"pMK4B". Plasmid pM4B has been deposited at the American Type
Culture Collection (ATCC; Manassas, Va.) on Nov. 3, 1998, and given
the accession number ATCC 98972.
[0252] In order to insert sequences encoding the huANT3 protein
(and assoicated regulatory sequences) into the baculovirus genome,
insect cells (MAXBAC.TM. Spodoptera frugiperda Sf9 cells,
Invitrogen, Carlsbad, Calif.; or Trichoplusia ni cells, PharMingen,
San Diego, Calif.) were co-transfected with the baculoviral
transfer construct pMK4B-huANT3 and linear baculoviral (Autographa
californica nuclear polyhedrosis virus, AcMNPV) DNA engineered to
contain a promoterless 3' fragment of the lacZ gene
(BAC-N-BLUE.TM., Invitrogen) using the BAC-N-BLUE.TM. Transfection
Kit (Invitrogen) following the manufacturer's instructions.
Recombinant baculovirus plaques express functional
beta-galactosidase and were identified as blue plaques in the
presence of X-gal (5-bromo4-chloro-3-indoyl-beta-D-glactosidase).
These recombinant viruses are expression constructs that express
human ANT3 polypeptide in insect cells, as shown by the following
experiments.
[0253] B. Western Blot Analysis of Baculovirus Expression
Systems
[0254] High titer viral stock was produced, and recombinant protein
was expressed in infected Sf9 (Invitrogen, Carlsbad, Calif.) or T.
ni (PharMingen, San Diego, Calif.) cells according to the
manufacturer's instructions (see also Piwnica-Worms, Expression of
Proteins in Insect Cells Using Baculovirus Vectors, Section II of
Chapter 16 in: Short Protocols in Molecular Biology, 2nd Ed.,
Asubel et al., eds., John Wiley & Sons, New York, N.Y., 1992,
pages 16-32 to 16-48; Kitts, Chapter 7 in: Baculovirus Expression
Protocols, Methods in Molecular Biology, Vol. 39, C. R. Richardson,
Ed., Humana Press, Totawa, N.J., 1995, pages 129-142).
[0255] Transfected Sf9 cells were pelleted by centrifugation and
lysed by adding 100 .mu.l of MSB buffer (210 mM mannitol (Sigma),
70 mM sucrose (Fluka), 50 mM Tris-HCl, pH 7.4, 10 mM EDTA) and
performing three freeze-thaw cycles. A total cellular fraction, a
cytosolic fraction, a submitochondrial partical fraction, a
mitochondrial fraction and a plasma membrane fraction were prepared
as follows. The cell lysate was centrifuged at 600 g for 10 minutes
at 4.degree. C. to prepare a plasma membrane pellet. The
supernatant was removed and set aside. The plasma membrane pellet
was washed with 100 ul of MSB, centrifuged at 600 g for 10 minutes
at 4.degree. C., and used for the analysis. The supernatant was
removed, combined with the first supernatant and mixed. Half of the
supernatant was used to prepare a mitochondrial fraction and a
cytosolic fraction by centrifugation at 14,000 g for 15 minutes at
4.degree. C.; the pellet represents the mitochondrial fraction and
the supernatant represents the cytosol. The other half of the
supernatant was centrifuged at 14,000 g for 15 minutes at 4.degree.
C. to produce a mitochondria-containing pellet that was resuspended
in MSB, incubated with 0.25 mg/ml digitonin (Roche Molecular
Biochemicals, formerly Boehringer Mannheim, Indianapolis, Ind.) for
2 min and sonicated for 3 min at 50% duty cycle in a cup-horn
sonicator to produce submitochondrial particles (SMPs). (See
Example 13 for details regarding mitochondrial preparation from
transfected T. ni cells.)
[0256] The protein content for each fraction was determined using
the BCA Protein Assay kit (Pierce Chemical Co.), and 8 ug of total
protein were loaded per lane onto an SDS polyacrylamide gel,
electrophoresed and transferred to a HYBOND.TM. ECL Nitrocellulose
Membrane (Amersham Life Science). Fusion proteins were detected in
a western blot using ANTI-XPRESS.TM. Antibody (Invitrogen, Catalog
#R910-25) and horseradish peroxidase-conjugated anti-mouse
secondary antibody (Amersham Life Science) following the
manufacturers' instructions.
[0257] The results of the Western analysis are shown in FIG. 6.
Recombinant GST-huANT3 fusion protein (molecular weight 36.6 kD)
was detected in total cells, mitochondria, submitochondrial
particles and the plasma membrane. The signal was most intense in
mitochondria and submitochondrial particles, whereas no band was
detectable in the cytosolic fraction. These data suggest that the
human recombinant huANT3 fusion protein integrated into the
mitochondrial membranes much more efficiently than into the plasma
membranes. Furthermore, all of the recombinant protein integrated
into membranes since no signal was detected in the cytosolic
fraction. The final lane of the autoradiogram shows His-tagged
huANT3 isolated from cell lysates using magnetic agarose beads
coupled to Ni according to the manufacturers instructions (Qiagen;
Hilden, Germany).
[0258] Thus, as in E. coli, huANT3 is expressed in the
baculovirus/Sf9 system. Furthermore, recombinantly produced 6xHis-
and epitope-tagged huANT3 fusion protein is appropriately localized
to the mitochondria in Sf9 cells despite the presence of over 35
extraneous N-terminal amino acids, and can be isolated from
cellular fractions by means that take advantage of the His-Tag
moiety's affinity for metals such as, e.g., nickel.
Example 4
Expression of ANT3 in Yeast
[0259] A. Expression Constructs and Host Cells
[0260] Human ANT3 cDNA was amplified by PCR as in Example 1 but
using the following primers. In the following representations of
PCR primers, underlined nucleotides indicate sequences
complementary to the 5'-ends and 3'-ends of the ANT cDNAs and
double-underlined nucleotides indicate recognition sequences for
the restriction enzymes XhoI (recognition sequence: 5'-CTCGAG) or
Asp718 (recognition sequence: 5'-GGTACC).
[0261] The primers used for PCR amplification were:
[0262] Forward (sense; SEQ ID NO:28):
5'-TTAATGGGTACCATGACGGAACAGGCCATCTCCTTCGCCAAA, and
[0263] Reverse (antisense; SEQ ID NO:29):
5'-TTATACTCGAGTTAGATCACCTTCTTGAGCTCGTCGTACAGG.
[0264] PCR products, and expression vector DNAs, were digested with
the restriction endonucleases XhoI and Asp718 (both enzymes from
Roche Molecular Biochemicals) according to the manufacturer's
recommendations using manufacturer-supplied reaction buffers. The
expression vector pYES2 (Invitrogen) was used. This vector contains
a multiple cloning site located immediately downstream from an
inducible GAL1 promoter, as well as the 2u origin of replication
and the S. cerevisiae URA3 gene for high-copy maintenance and
selection in ura3 yeast cells, respectively.
[0265] The restricted DNAs were purified by horizontal agarose gel
electrophoresis and band extraction using the UltraClean GelSpin
kit (Mo Bio Laboratories), ligated to each other, and used to
transform E. coli cells, as in the preceding Examples. Plasmid DNA
was isolated from several transformants, and the nucleotide
sequence of the insert DNA was determined and confirmed to be that
of huANT3. One confirmed plasmid was chosen to be used for further
study and was designated pMK5A (huANT3).
[0266] A second yeast huANT3 expression vector, pMK5B, was
constructed as follows. Plasmids pMK5A and pYESTrp2 (Invitrogen)
were digested with restriction enzymes BglI and PvuII (both from
New England Biolabs) and gel purified, ligated and used to
transform E. coli as above. The expression vector pYES2Trp is
similar to pYES2 but comprises a TRP1 selectable marker. Plasmid
DNA was isolated from several transformants and restriction mapped
to confirm the structure of the expected expression construct. One
confirmed plasmid was chosen to be used for further study and was
designated pMK5B (huANT3).
[0267] A third yeast huANT3 expression vector, pMK5C, was
constructed using the expression vector pYPGE2, which comprises a
TRP1 selectable marker and the strong PGK promoter upstream from a
multiple cloning site (Brunelli and Pall, 1993 Yeast 9:1299-1308).
Plasmid pYPGE2 DNA was digested with XhoI and Asp718, gel-purified
and ligated with the XhoI- and Asp718-restricted huANT3 PCR product
of Example 1. The ligation mixture was used to transform E. coli,
and plasmid DNA was isolated from several transformants and
restriction mapped to confirm the structure of the expected
expression construct. One confirmed plasmid was chosen to be used
for further study and was designated pMK5C (huANT3).
[0268] In order to generate yeast expression systems, the S.
cerevisiae strain INVSc1 (MAT.alpha., his3.DELTA.1, leu2, trp1-289,
ura3-52) was transformed with purified pMK5A, pMK5B and pMK5C DNAs
using the S.c. EASYCOMP.TM. Transformation Kit (Invitrogen). A
second S. cerevisiae strain, J.DELTA.1.DELTA.3 (MAT.alpha., ade2-1,
leu2-3, leu2-112, his3-11, his3-15, trp1-1, ura3-1, can1-100,
AAC1::LEU2, AAC2::HIS3, AAC3::URA3) was also transformed with the
expression constructs. The AAC genes encode the three isoforms of
the mitochondrial ADP/ATP translocator in S. cerevisiae and are
interrupted in strain J.DELTA.1.DELTA.3 (Giraud et al., J. Mol.
Biol. 281:409-418 (1998)). It is thus expected that transformants
of J.DELTA.1.DELTA.3, which are incapable of expressing endogenous
ANT (AAC) proteins, will only express the human ANT protein encoded
by the expression construct with which they have been
transformed.
[0269] B. Northern blot analyses of yeast expression systems
[0270] In order to examine levels of huANT3 mRNA production in
strain J.DELTA.1.DELTA.3, Northern analyses of cells transformed
with pMK5B and pMK5C were performed according to methods known in
the art. In brief, transformed cells and control (untransformed)
cells grown to mid-log phase, harvested and lysed. RNA was
extracted from the lysates, electrophoresed and transferred to a
nitrocellulose filter (see Treco, Preparation of Yeast RNA, Unit
13.12 of Chapter 13 in Short Protocols in Molecular Biology, 2nd
Ed., Asubel et al., eds., John Wiley & Sons, New York, N.Y.
(1992), 13:44-46 and Seldon, Analysis of RNA by Northern
Hybridization, Unit 4.9 of Chapter 4, Id., 4:23-25). The XhoI- and
Asp718-restricted huANT3 PCR product of Example 1 was radiolabelled
and used as a probe, and an RNA preparation from human spleen
tissue was used as a positive control.
[0271] The results (FIG. 10) demonstrate the appropriately-sized
ANT3-specific RNA is produced in human spleen and in yeast cells
transformed with either expression vector, but not in untransformed
yeast cells. The pYPGE2-derived expression construct pMK5C, which
directs ANT3 expression from the PGK promoter, clearly results in
more ANT3 RNA than the pYES2Trp-derived construct pMK5B, in which
ANT3 expression is driven by the GAL1 promoter. In either case,
however, significant levels of huANT3-specific RNA were produced in
a yeast background that lacks any endogenous adenosine nucleotide
translocator proteins.
[0272] C. Western Analyses of Yeast Expression Systems
[0273] 1. Production of antibody to huANT3
[0274] As the huANT3 produced from the yeast expression constructs
lacks an epitope tag, it was necessary to produce an antibody to
huANT3 in order to evaluate recombinant production of the protein.
A monspecific (antipeptide) antibody specific to huANT3 was
prepared as follows.
[0275] A synthetic polypeptide corresponding to a portion of huANT3
located near the carboxy terminus and predicted to have high
antigenicity according to the Jameson-Wolf Index (Wolf et al.,
Comput. Appl. Biosci. 4:187-191 (1988)) was synthesized using known
means by Alpha Diagnostic International (San Antonio, Tex.) and
determined to be at least about 70% pure, preferably at least about
90% pure, by HPLC and MS analyses. The sequence of the synthetic
polypeptide (SEQ ID NO:30) is:
Cys-Trp-Arg-Lys-Ile-Phe-Arg-Asp-Glu-Gly-Gly-Lys-Ala-Phe-Phe
[0276] The synthetic polypeptide was conjugated to a carrier
molecule, keyhole limpet hemocyanin (KLH), using MSB
(m-maleimidobenzoyl-N-hydroxys- uccinimide ester; Pierce Chemical
Co., Rockford, Ill.), and the conjugated material was used to
immunize several rabbits, according to known means (Collawn and
Paterson, Units 11.14 and 11.15 in Chapter 11 in: Short Protocols
in Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley
& Sons, New York, N.Y. (1992) 11:37-41. The rabbits were or are
bled at 0 (preimmune, 2 mL), 7, 9, 11, 13 (15 mL for each bleed) or
15 weeks (50 mL) post-inoculation. Sodium azide (0.1%) was or is
added to the bleeds as preservative.
[0277] 2. Western analyses
[0278] Western analyses of yeast expression systems are performed
essentially as described in the preceding Examples, except that
different methods are used to prepare protein preparations from
yeast cells as opposed to bacterial or insect cells. Such methods
of isolating proteins from yeast are known in the art (see, for
example, Dunn and Wobbe, Preparation of Protein Extracts from
Yeast, Unit 13.13 of Chapter 13 in Short Protocols in Molecular
Biology, 2nd Ed., Asubel et al., eds. John Wiley & Sons, New
York, N.Y. (1992), 13:46-50). The intracellular distribution of
huANT3 in, e.g., membrane or mitochondrial fractions, is determined
as in the preceding Examples.
Example 5
Expression of ANT4 in Mammalian Cells
[0279] The preceding Examples describe a variety of means by which
ANT and ANT fusion proteins can be recombinantly produced in
various systems. Although such ANT proteins can be used in a
variety of assays (see infra), it may be desirable to isolate large
amounts of the native ANT protein from mammalian cells. In
particular, as described in this Example, it may be desirable to
produce recombinant viral particles in which ANT proteins are
displayed in the viral envelope. Such ANT-displaying viral
particles are expected to be very stable and useful in a variety of
assays including, for example, those in which compounds binding to
ANT proteins are screened and identified.
[0280] Another useful outcome of mammalian expression systems is
the generation and isolation of human mitochondria in which a
particular ANT isoform is over-represented in order to determine
the specific biological role(s) of such isoforms. For example, ANT3
is apparently ubiquitously expressed in human tissues, whereas ANT1
is primarily expressed in heart and skeletal muscle (Stepien et
al., 1992, J. Biol. Chem. 267:14592-14597). Directed overexpression
of huANT1 in cultured heart or muscle cells is expected to result
in mitochondria that contain mostly the ANT1 isoform. Such "ANT
isoform-enriched" mitochondria can be isolated and tested for
various mitochondrial functions.
[0281] Constructs for expressing ANT proteins in mammalian cells
are prepared in a stepwise process. First, expression cassettes
that comprise a promoter (and associated regulatory sequences)
operably linked to nucleotide sequences encoding an ANT protein are
constructed in bacterial plasmid-based systems; these expression
cassette-comprising constructs are evaluated and optimized for
their ANT-producing ability in mammalian cells that are transiently
transfected therewith. Second, the ANT expression cassettes are
transferred to viral systems that produce recombinant proteins
during lytic growth of the virus (e.g., SV40, BPV, EBV, adenovirus;
see below) or from a virus that can stably integrate into and
transduce a mammalian cellular genome (e.g., a retroviral
expression construct).
[0282] A. Transient expression
[0283] With regards to the first step, commercially available
"shuttle" (i.e., capable of replicaton in both E. coli and
mammalian cells) vectors that comprise promoters that function in
mammalian cells and can be operably linked to an ANT-encoding
sequence include, but are not limited to, SV40 late promoter
expression vectors (e.g., pSVL, Pharmacia),
glucocorticoid-inducible promoter expression vectors (e.g., pMSG,
Pharmacia), Rous sarcoma enhancer-promoter expression vectors
(e.g., pRc/RSV, Invitrogen) and CMV early promoter expression
vectors, including deriavtives thereof having selectable markers to
agents such as Neomycin, Hygromycin or ZEOCIN.TM. (e.g., pRc/CMV2,
pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo and
pcDNA3.1/Hygro, Invitrogen) In general, preferred shuttle vectors
for ANT genes are those having selectable markers (for ease of
isolation and maintenance of transformed cells) and inducible, and
thus regulatable, promoters (as overexpression of ANT genes may
have toxic effects).
[0284] Methods for transfecting mamallian cells are known in the
art (see, Kingston et al., "Transfection of DNA into Eukaryotic
Cells," Section I of Chapter 9 in: Short Protocols in Molecular
Biology, 2nd Ed., Asubel et al., eds., John Wiley & Sons, New
York, N.Y., 1992, pages 9-3 to 9-16). A control plasmid, such as
pCH110 (Pharmacia), may be cotransfected with the ANT expression
construct being examined so that levels of ANT can be normalized to
a gene product expressed from the control plasmid.
[0285] Western analyses of mammalian expression systems are
performed essentially as described in the preceding Examples,
except that different methods are used to prepare protein
preparations from mamallian cells as opposed to bacterial, insect
or yeast cells. Such methods of isolating proteins from yeast are
known in the art (see, for example, Kingston and Sheen, Unit 9.6A
and Brasier, Unit 9.6B of Chapter 9 in: Short Protocols in
Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley &
Sons, New York, N.Y., 1992, pages 9-17 to 9-23). Preferred
expression cassettes, consisting essentially of a promoter and
associated regulatory sequences operably linked to an ANT gene of
interest, are identified by the ability of cells transiently
transformed with a vector comprising a given ANT expression
cassette to express high levels of ANT protein when induced to do
so; these expression cassettes are incorporated into viral
expression vectors.
[0286] B. Viral expression
[0287] Nucleic acids, preferably DNA, comprising preferred
expression cassettes are isolated from the transient expression
constructs in which they were prepared, characterized and optimized
(see preceding section). A preferred method of isolating such
expression cassettes is by amplification by PCR, although other
methods (e.g., digestion with appropriate restriction enzymes) can
be used. Preferred expression cassettes are introduced into viral
expression vectors, preferably retroviral expression vectors, in
the following manner.
[0288] A DNA molecule comprising a preferred expression cassette is
introduced into a retroviral transfer vector by ligation (see
preceding Examples). Two types of retroviral transfer vectors are
known in the art: replication-incompetent and
replication-competent. Replication-incompetent vectors lack viral
genes necessary to produce infectious particles but retain
cis-acting viral sequences necessary for viral transmission. Such
cis-acting sequences include the .PSI. packaging sequence, signals
for reverse transcription and integration, and viral promoter,
enhancer, polyadenylation and other regulatory sequences.
Replication-competent vectors retain all these elements as well as
genes encoding virion structural proteins (typically, those encoded
by genes designated gag, pol and env) and can thus form infectious
particles in a variety of cell lines. In contrast, these functions
are supplied in trans to replication-incompetent vectors in a
packaging cell line, i.e, a cell line that produces mRNAs encoding
gag, pol and env genes but lacking the .PSI. packaging sequence.
See, generally, Cepko, Unit 9.10 of Chapter 9 in: Short Protocols
in Molecular Biology, 2nd Ed., Asubel et al., eds., John Wiley
& Sons, New York, N.Y., 1992, pages 9-30 to 9-35.
[0289] A retroviral construct comprising an ANT expression cassette
produces RNA molecules comprising the cassette sequences and the
.PSI. packaging sequence. These RNA molecules correspond to viral
genomes that are encapsidated by viral structural proteins in an
appropriate cell line (by "appropriate" it is meant that, for
example, a packaging cell line must be used for constructs based on
replication-incompetent retroviral vectors). Infectious viral
particles are then produced, and released into the culture
supernatant, by budding from the cellular membrane. The infectious
particles, which comprise a viral RNA genome that includes the ANT
expression cassette, are prepared and concentrated according to
known methods. It may be desirable to monitor undesirable helper
virus, i.e., viral particles which do not comprise an ANT
expression cassette. See, generally, Cepko, Units 9.11, 9.12 and
9.13 of Chapter 9 in: Short Protocols in Molecular Biology, 2nd
Ed., Asubel et al., eds., John Wiley & Sons, New York, N.Y.,
1992, pages 9-36 to 9-45.
[0290] Viral particles comprising an ANT expression cassette are
used to infect in vitro (e.g., cultured cells) or in vivo (e.g.,
cells of a rodent, or of an avian species, which are part of a
whole animal). Tissue explants or cultured embryos may also be
infected according to methods known in the art. See, generally,
Cepko, Unit 9.14 of Chapter 9 in: Short Protocols in Molecular
Biology, 2nd Ed., Asubel et al., eds., John Wiley & Sons, New
York, N.Y., 1992, pages 9-45 to 9-48. Regardless of the type of
cell used, production of ANT protein is directed by the recombinant
viral genome.
[0291] In a preferred embodiment, recombinantly produced ANT
proteins are inserted into the cell membrane of cultured cells.
Because the retroviral expression construct produces viral
particles by budding of the cell membrane, the resultant viral
particles delivered to the culture supernatant have ANT protein
incorporated into their capsules, preferably on the surface of the
particles. Such ANT-displaying viral particles are expected to
provide a stable format for ANT proteins and to thus be useful in
assays using ANT proteins, either directly or as a source material
from which ANT can be further purified. If it is desired to
minimize the amount of ANT protein inserted into mitochondrial
membranes, .rho..sup.0 cells, which have been treated in such a
manner as to be nearly or completely devoid of mitochondria, are
used as host cells.
[0292] C. ANT Antisense Constructs
[0293] Antisense versions of the preceding transient and viral ANT
expression constructs are prepared by exchanging the antisense
(non-encoding) strand for a sense (ANT protein encoding) strand in
a construct. Such ANT antisense constructs are useful as research
reagents, i.e., to reduce levels of expression of one or more
isoforms in a cell transformed or infected with such a construct in
order to determine the effects of such treatment on cellular
physiology. ANT antisense constructs are also useful as gene
therapy agents that interfere with the translation of one or more
isoforms of ANT.
Example 6
Synthesis and Properties of Representative ATR Derivatives
[0294] A number of atractyloside (ATR) derivatives were prepared
for use as ligands for adenine nucleotide translocators (ANTS) in
the context of high-throughput screening assays. These compounds
bind with high affinity (i.e., in the nM range) to ANT and are thus
useful for screening libraries of chemical compounds for molecules
having high specificity for ANT (regardless of isoform) The
structure of ATR is set forth below as compound (1). Compounds (3)
and (4) represent novel fluorescent derivatives of ATR, while
compound (2) is an ATR derivative which permits introduction of the
.sup.125I under mild conditions. 6
Purification
[0295] Compounds 2, 3 and 4 were purified by silica gel
chromatography using CH.sub.2Cl.sub.2/MeOH/AcOH (75:25:1) as the
eluting solution. Detection was achieved by staining with a 0.5%
solution of vanillin in H.sub.3PO.sub.4/H.sub.2O (1/1). Further
purification was accomplished by reversed-phase HPLC using a
Microsorb C8 column (250.times.10 mm). The column was eluted at a
flow rate of 2.0 mL/min with a linear gradient of methanol/acetic
acid/1 M ammonium acetate 98:1:1 ("Solvent B") and H.sub.2O/acetic
acid/1 M ammonium acetate aqueous solution 98:1:1 ("Solvent A").
The effluent was monitored for absorbance at 254 nm.
Compound-containing fractions were pooled, evaporated, and
repeatedly co-evaporated with added methanol (3.times.5 mL).
Synthesis of Compound 2
[0296] Atractyloside 1 (0.10 mmol) was dried by repeated
evaporation of added pyridine (3.times.5 mL) and the resulting
gummy residue dissolved in pyridine (5 mL). To the resulting
solution, 0.20 mmol of toluenesulfonyl chloride was added. The
reaction mixture was stirred at ambient temperature for 1.5 h.
Then, another portion of toluenesulfonyl chloride (0.20 mmol) was
added and the reaction left stirring an additional 1.5 h. 1 mL of
methanol was added to the reaction mixture which was then stirred
for 0.5 h, after which solvents were removed by evaporation.
Residual pyridine was removed by evaporation of additional methanol
(5.times.10 mL). Silica gel chromatography followed by
reversed-phase HPLC using a linear gradient of 50-80% of solvent B
in solvent A for 30 min. resulted in the compound 2 eluting at 68%
solvent B. Yield: 4.3 mg, 4.9%. ESI-MS (M-H) found:879,
calc.:879.
Synthesis of Compounds 3 and 4
[0297] 7-Diethylamino-2-oxo-2H-chromene-3-carboxylic acid or 0.20
mmol of 1-pyrenebutyric acid and 0.60 mmol of
1,1'-carbonyldiimidazole in 1 mL of dimethylformamide were allowed
to react for 15 min. To the activated carboxylic acid was added a
solution of atractyloside 1 in H.sub.2O (4 mL) and the resulting
reaction mixture was stirred at ambient temperature for 16 h.
Evaporation left a gummy residue which was purified by silica gel
chromatography followed by reversed-phase HPLC. Using a linear
gradient of 10-80% of solvent B in solvent A for 50 min (for
compound 3) or 50-100% of solvent B in solvent A for 50 min (for
compound 4) resulted in compound 3 eluting at 75% B and compound 4
eluting at 82% B. Yields: compound 3, 3.1 mg, 8.0%; compound 4, 1.3
mg, 3.6%. ESI-MS (M-H) compound 3 found:968, calc.: 968; compound 4
found:995, calc.:995.
Properties of Representative ATR Derivatives
[0298] As summarized in Table 1 below, compounds 3 and 4 were found
to be more advantageous in terms of fluorescence characteristics
and sensitivity compared to the existing ATR derivatives
Naphthoyl-ATR and MANT-ATR as reported by Boulay et al., Analytical
Biochemistry 128:323-330,1983; Roux et al., Analytical Biochemistry
234:31-37,1996; and Lauquin et al., FEBS Letters
67:306-311,1976.
1TABLE 1 Excitation Emission Extinction Coefficient (M.sup.-1) ATR
Derivative (nm) (nm) (Predicted) Naphthoyl-ATR 300 405 6,200
MANT-ATR 350 460 5,800 Compound 4 341 391 17,420 Compound 3 417 470
46,400
Example 7
Synthesis of Representative ATR Derivative
[0299] The further representative ATR derivative, compound 5, was
prepared by the procedure set forth below. 7
Synthesis of Compound 5
[0300] Dipotassium atractylate (0.10 mmol) was dissolved in 50% aq.
ethanol (5 mL) and palladium on charcoal (10%, 17 mg) was added to
the reaction mixture. After flushing the system with hydrogen, the
reaction mixture was stirred under an atmosphere of hydrogen gas
for 3 h. Removal of catalyst by filtration through Celite, washing
with 50% aq. ethanol (10 mL), and evaporation of solvents afforded
a white solid. Yield after thorough drying under high vacuum; 78.3
mg (97.3%). ESI-MS (M-2H+K) found:765, calc. :765. .sup.1H-NMR
analysis confirmed the absence of alkenic protons: DMSO-d.sub.6)
.delta.0.88(d, 3H), 0.89(d, 3H), 1.02(d, 3H).
Example 8
Synthesis of Representative Iodinated ATR Derivative
[0301] Compound 2 of Example 6 may be used as intermediate for
conjugation of variety of chemical moieties to yield further ATR
derivatives. In this ex ample, compound 2 is employed to introduce
.sup.125I under mild conditions to yield the following compound 6.
8
Synthesis of Compound 6
[0302] Five .mu.l of 0.2 M sodium phosphate (pH 5) was combined
with 21 ul of Na.sup.125I (9.25 mCi) in its shipping container
(specific activity, 2100 Ci/mmol; Amersham, Piscataway, N.J.). Ten
ul (200 ug, 212 nmol) of compound 2 of Example 1 was added to the
mixture. The pH was checked with litmus paper to confirm that it
did not rise above pH 5. The mixture was allowed to stand at
ambient temperature overnight (17.5 hours) to yield radiolabelled
compound 6. (Non-radioactive iodinated ATR derivative, for use as a
"cold" competitor in binding studies, may be prepared in the same
manner using unlabeled iodine). The iodinated derivative was
purified over a C18 analytical column (4.times.6.times.250 mm)
(Phenomenex, Torrance, Calif.) using a 25%-55% acetonitrile
gradient in running buffer (1% triethylammonium acetate, pH 4.5). A
flow rate of 1 ml/min was used to run the gradient over 30 min. The
desired product eluted at 25 min. ESI-MS: 835 (M-H), 707
(m-2H-1).
Example 9
Synthesis of Representative ATR Derivatives
[0303] Activation of carboxylic acids with carbonyl diimidazole and
their reaction with ATR has been the method of choice for synthesis
of various 6'-O-acyl derivatives. The relatively low reactivity of
the 6'-hydroxyl of ATR and the presence of an allylic secondary
hydroxyl in the aglycon as well as the sulfated glucose moiety, are
all factors that have a negative impact on the efficiencies of
these acylation reactions. Hence, yields are generally poor and the
approach requires a large excess of acylating reagents.
[0304] Two strategies for introduction of an amine functionality in
the ATR system are described below that permit synthesis of a
broader range of ATR derivatives. In the first strategy, as
depicted by Scheme 1, displacement of the primary tosylate from
compound 2 (Example 1) with azide followed by reduction yields the
corresponding 6'-amine (compound 7). Alternatively, the amine group
can be introduced as part of a spacer, which permits introduction
of more sterically demanding functional moieties. Thus, reacting
the 6'-O-succinoyl derivative (compound 8; see Brandolin et al.,
1974 FEBS Lett. 46:149.) with a monoprotected diamine followed by
deprotection affords compound 9 as illustrated by Reaction Scheme
2. 9 10
[0305] The amine-containing ATR derivatives 7 and 9 may then be
reacted with a variety of fluorophors and haptens bearing reactive
isothiocyanate, N-hydroxysuccinimide ester and anhydride
functionalities to yield stable ATR-derivatives having thiourea and
amide linkages. Representative ATR derivatives that were prepared
include ATR-lanthanide chelating agents (compounds 10, 11, 12, 13,
20 and 21) that have utility for time-resolved fluorescence
detection of these compounds complexed to Eu.sup.3+. In addition,
ATR was conjugated to cyanine (compounds 14 and 15) and fluorescein
analogues (compounds 16 and 17) that are detectable by fluorescence
with extremely high sensitivities. Coupling of biotin-NHS ester
with the ATR derivatives of compounds 7 and 9 provided ATR-biotin
conjugates (compounds 18 and 19) that can be detected with
commercially available enzyme-avidin conjugates using calorimetric,
fluorescent or chemiluminescent techniques.
[0306] More specifically, a solution of compound 2 in DMF was
treated with azide ion for 8 hours at 80.degree. C. to give the
6'-azido-ATR, that was purified by silica gel chromatography using
a CH.sub.2Cl.sub.2/CH.sub.3OH solvent system supplemented with 1%
acetic acid. Staudinger-reduction using 1.5 equivalents of
triphenylphosphine in a THF/H.sub.2O mixture for 4 hours at RT
afforded the amine of compound 7, that was isolated after silica
gel chromatographic purification.
[0307] To accommodate more sterically demanding functional
moieties, 6'-O-succinoyl-ATR may be condensed with commercially
available monoprotected diamines (Calbiochem-Novabiochem Corp, San
Diego, Calif.) to produce ATR-mono-protected amine derivatives.
Thus, EDC-mediated coupling of 6'-O-succinoyl-ATR in DMF with 1.1
equivalents of mono-protected FMOC diamines yield the amide that
was deprotected using piperidine or
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrile to furnish
the ATR derivative of compound 9. The amines were purified by silca
gel chromatography as described above.
[0308] The ATR-amine derivatives of compounds 7 and 9 were coupled
to a variety of fluorophors, chelates and haptens that contained
amine-reactive functionalities, such as isothiocyanates, anhydrides
and NHS esters in aqueous DMF to generate the ATR derivatives of
compounds 10 through 19: These compounds were purified by a
combination of silica gel chromatography and preparative reverse
phase chromatography on a C-8 column using CH.sub.3OH/H.sub.2O
gradient containing 0.1-1% acetic acid. 11
Example 10
Synthesis of Representative ATR Derivatives and Intermediates
Therefor
General Procedure for Coupling Atractyloside or
Dihydroatractyloside to Organic Acids
[0309] 12
[0310] Carboxylic acid (200 .mu.mol) and 1,1'-carbonyldiimidazole
(700 .mu.mol) were dissolved in DMF (2 mL) and stirred for 15 min.
To the activated acid, a solution of atractyloside (ATR) (100
.mu.mol, 85 mg) or dihydroatractyloside (100 .mu.mol) in
DMF:H.sub.2O (1:2, 6 mL) was added (in 1 mL portions over ca 30
sec). The reaction mixture was stirred at room temperature for 60
min, after which solvents were removed by rotary evaporation on a
water bath in which the temperature was kept below 40.degree. C.
The residue was stripped of traces of DMF by repeated evaporation
of added EtOH:H.sub.2O (1:1, 3.times.20 mL). The residue was then
taken up in MeOH: H.sub.2O (1:1, 10 mL), sonicated if necessary,
and filtered through a 0.45 .mu.m filter. Evaporation,
re-dissolution in .about.1.3 mL buffer, and purification by reverse
phase HPLC furnished the desired 6'-O-acyl-ATR in yields ranging
from 5-15%.
Reverse Phase HPLC Conditions for Purification of Atractyloside
Derivatives
[0311] Purification by reverse phase HPLC (RP-HPLC) was performed
in a 10.times.250 mm C-8 column, using a gradient of MeOH:AcOH:1 M
NH.sub.4OAc (buffer B, 98:1:1) in H.sub.2O:AcOH:1 M NH.sub.4OAc
(buffer A, 98:1:1). Typically, a gradient of B in A from 50-80%
over 30 min was employed. For more lipophilic derivatives, the
gradient was from 60-90% or 70-100% B over the same time
period.
Synthesis of Dihydroatractyloside
[0312] 13
[0313] Atractyloside, dipotassium salt (254 mg, 0.3 mmole) in 15 ml
of EtOH/H.sub.2O (1:1) was hydrogenated under atmospheric pressure
for 3 hours using 51 mg of activated palladium/carbon (10%) as
catalyst. The catalyst was removed by filtration through celite and
the celite bed was washed with 15 ml of EtOH/H.sub.2O. The
filtrates were concentrated by rotary evaporation, followed by
drying under high vacuum overnight to provide the product as white
solid (236 mg), that was pure by NMR.
6'-Tosyl-Atractyloside
[0314] 14
[0315] Atractyloside.3H.sub.2O (255 mg. 0.3 mmole) was dried by
co-evaporation with dry pyridine (3.times.5 ml) and then was kept
under high vacuum for 16 hrs. The dried atractyloside was dissolved
in 15 ml of dry pyridine and 114 mg of tosyl chloride (0.6 mmole)
was added. The reaction was stirred for 2 hrs at 23.degree. C. and
then an additional 114 mg of tosyl chloride was added and the
reaction was allowed to continue for an additional 1.5 hrs.
Methanol (1 ml) was added to the reaction mixture to scavenge
excess tosyl chloride and the mixture was stirred for several
minutes. The mixture was evaporated to dryness, and residual
pyridine was removed by evaporation of added methanol (2.times.30
ml). The crude product was partially purified by silica gel flash
chromatography (CH.sub.2Cl.sub.2/MeOH, 3:1 with 1% AcOH). The
product containing fractions were dissolved in 3 ml of
H.sub.2O:MeOH:1M NH.sub.4OAc:HOAc (49:49:1:1) and purified by
RP-HPLC using a 50%-80% gradient of MeOH:AcOH:1 M NH.sub.4OAc
(buffer B, 98:1:1) in H.sub.2O:AcOH:1 M NH.sub.4OAc (buffer A,
98:1:1). Product containing fractions were pooled, evaporated, and
subjected to additional co-evaporation of 3.times.10 ml of MeOH.
Tosyl-atractyloside was obtained as a glassy material weighing 60
mg.
General Procedure for Lactoperoxidase-Catalyzed Iodination of
4-Hydroxyphenyl Derivatives
[0316] 15
[0317] To a solution of 4'-hydroxy-biphenyl-4-carboxylic acid
atractylosid-6'-yl ester (1.0 mg, 1.0 mmol) in H.sub.2O (120 .mu.L)
were added aqueous solutions of lactoperoxidase (50 .mu.L, 200
IU/mL), NaI (10 .mu.L, 100 mM) and H.sub.2O.sub.2 (20 .mu.L, 100
mM). The reaction was left at room temperature for 1 h after which
it was frozen at -20.degree. C. The next day (.about.20 hours
later) the reaction mixture was thawed and subjected to RP-HPLC.
Three major peaks were eluted and electrospray ionization-mass
spectrometic (ESI-MS) analysis confirmed their identity as
unreacted starting material, and monoiodinated, and diiodinated
atractyloside derivatives.
[0318] These conditions can be modified to drive the reaction
completely to the mono and di-iodinated forms with additional
aliquots of NaI and/or enzyme.
General for Synthesis of Iodophenols
[0319] 16
[0320] The iodination procedure described in Acta Chem, Scand. 12,
188 (1958) was used for mono-iodination of
3-(4-hydroxyphenyl)propionic acid, 4'-hydroxy-4-biphenylcarboxylic
acid and 4-hydroxybenzoic acid. This method is also applicable for
the mono-iodination of 3-(4-methoxyphenyl)-propionic acid and
di-iodination of 3-(3-iodo-4-hydroxyphenyl)propionic acid.
[0321] Thus a solution of KI (1.99 gm, 12 mmole) and iodine (1.22
gm, 4.8 mmole) in 20 ml of H.sub.2O was added in a dropwise fashion
to a solution of 3-(4-hydroxyphenyl)propionic acid (0.83 gm, 5
mmole) in 100 ml of concentrated aqueous ammonia solution over 20
min. The reaction mixture was stirred for an additional 40 min and
then subjected to vacuum to remove the ammonia. The mixture was
dried further by rotary evaporation to afford an oily residue. The
crude material was partitioned between 2M HCl (50 ml) and ether
(2.times.50 ml) and the ether layers were combined and concentrated
to give a yellowish solid residue. Flash silica gel chromatography
using 95:5 CH.sub.2Cl.sub.2/MeOH as eluant, concentration of
product containing fractions and recrystallization in 1:1 benzene
hexane afforded 790 mg of 3-iodo4-hydroxyphenylpropionic acid.
[0322] 3-(3,5-Diiodo-4-hydroxyphenyl)propionic acid was prepared in
similar fashion using 5.2 equivalents of KI and 2.1 equivalents of
I.sub.2. Following crystallization from toluene, the di-iodo
derivative was obtained in 77% yield.
5-iodo-6-hydroxy-2-naphthoic acid
[0323] 17
[0324] Mono-iodination of 6-hydroxy-2-naphthoic acid and
4-hydroxybenzoic acid was carried out following the procedure of
Edgar and Falling (J. Org. Chem. 55, 5287, 1990). Thus, 0.75 gm of
(4.34 mmol) was dissolved in 19 ml of MeOH and 1.04 gm of NaI (and
0.27 gm of NaOH was added. The solution was cooled to
0.quadrature.C. and aqueous NaOCl (4% solution, 12.9 ml) was added
dropwise over 75 min. The resulting mixture was stirred for 1 hr at
0.degree. C. and then treated with 7 ml of 10% aqueous sodium
metabisulfite. The mixture was adjusted to pH 7 using 5% HCl and
extracted with 40 ml of ether. The organic layer was washed with
brine and dried over MgSO.sub.4. The solution was concentrated to
an off-white solid, that was recrystallized from toluene/CH.sub.3OH
to provide 0.42 gm of 5-iodo-6-hydroxy-2-naphthoic acid.
Reaction of 6'-Tostylatractyloside with 1.2-ethylenediamine
[0325] 18
[0326] 6'-Tosyl-Atractyloside (25 mg) was dissolved in 2 ml of
1,2-ethylenediamine and the mixture was stirred at 23.degree. C.
overnight. The 1,2-ethylenediamine was removed in vacuo, the
residue was dissolved in MeOH/H.sub.2O (2:1) and 10.6 mg of the
product was isolated in by RP-HPLC using the conditions described
above. Proton nmr and mass spectra indicate the loss of the
isovaleryl group.
Reaction of N-(6-Deoxy-apo-atractylosyl)-ethanediamine with Bolton
Hunter Reagent
[0327] N-(6-Deoxy-apo-atractylosyl)-ethanediamine (10.6 mg) in 2.75
ml of DMF/DMSO (8:3) was reacted with 60 mg of
4-hydroxyphenylpropionyl-N-hydro- xysuccinimidyl ester (Bolton
Hunter reagent) at 23.degree. C. for 16 hrs. The solution was
diluted with water and purified by RP-HPLC to afford 9.1 mg of the
desired compound. 19
[0328] Lactoperoxidase catalyzed iodination of the compound leads
to a quantitative conversion to the di-iodo-apo-atractyloside
derivative.
Reaction of Atractyloside with Succinic Anhydride
[0329] 20
[0330] Atractyloside.3H.sub.2O (255 mg. 0.3 mmole) was dried by
co-evaporation with dry pyridine (3.times.5 ml) and then was kept
under high vacuum for 16 hrs. The dried atractyloside was dissolved
in 6 ml of dry pyridine and 60 mg of succinic anhydride (0.6 mmole)
was added. The mixture was kept at 80.degree. C. for 30 min,
another 60 mg (0.6 mmole) of succinic anhydride was added and the
reaction mixture was stirred for an additional 3 hrs. The pyridine
was removed in vacuo, and the residue was triturated with 10 ml of
MeOH. The 6'-O-succinyl-ATR derivative was collected by filtration
as a white solid, washed with MeOH and dried overnight over
P.sub.2O.sub.5.
Reaction of 6'-O-succinylatractyloside with Tyramine and
Iodination
[0331] 21
[0332] 6'-O-Succinylatractyloside (85 mg) was dissolved in 2 ml of
DMF and 28 mg of tyramine and 100 mg of PyBOP was added. The
mixture was stirred at 23.degree. C. for 16 hrs. The crude mixture
was subjected to RP-HPLC and the desired amide was isolated in 19.7
mg. Lactoperoxidase catalyzed iodination of the product using the
standard conditions described above provides the mono and the
di-iodinated products.
Synthesis of 4-(4-Hydroxy-3-Methyl)-Butyric Acid
[0333] 22
[0334] To 4 gm (30 mmol) of AlCl3 in 50 ml of 1,2-dichloroethane at
0.degree. C. was added 2.7 gm (27 mmol) of succinic anhydride and
the mixture was stirred for 20 min. 2-Methylanisole (3.1 ml, 25
mmol) was added, and the reaction mixture was warmed to 23.degree.
C. and stirred for 12 hrs. The mixture was poured into 300 ml of
ice-cold water and the precipitate was filtered off The precipitate
was washed with 2.times.300 ml of water to afford a white solid.
The solid material was dried under vacuum to afford 3.51 gm of
product that was used in the next reaction.
[0335] 3-(4-Methoxy-3-methylbenzoyl)propionic acid (4.4 gm, 20
mmol) and 4.49 gm of KOH pellets (80 mmol) were dissolved in 30 ml
of ethylene glycol and 3.88 ml of hydrazine hydrate (80 mmol) was
added to the stirred solution in four portions. The resulting
reaction mixture was heated at 155.degree. C. for 24 hrs in an oil
bath. After cooling, the reaction mixture was taken up in 100 ml of
benzene and washed with 10% aqueous citric acid. The organic layer
was washed with another portion of citric acid, dried over
anhydrous sodium sulfate, and concentrated in vacuo to afford an
oily residue that crystallized upon standing. The material was
triturated with hot hexane and the solvent was evaporated off to
afford 3.55 gm of a crystalline solid that was homogeneous by
silica gel tic using hexane/ethyl acetate (8:2) as eluting
solvent.
[0336] 4-(4-Methoxy-3-methylphenyl)butyric acid (3.12 gm) was
heated in 120 ml of a 1:1 mixture of 48% aqueous HBr/acetic acid at
155.degree. C. for 24 hrs. The reaction mixture was cooled to room
temperature and was extracted with 200 ml of benzene/ether (1:1).
The organic layer was dried over anhydrous magnesium sulfate and
concentrated to afford a light brown solid residue. The reaction
products were separated by silica gel flash chromatography using
hexane/ethyl acetate (3:1) as eluting solvent to provide 0.86 mg of
4-(4-hydroxy-3-methyl)-butyric acid as a light yellow solid.
Representative Synthesis of Atractyloside Derivatives
[0337] To a solution of 3-(4-hydroxyphenyl)propionic acid (HPP)
(0.498 g, 3.0 mmole) in 10 ml of anhydrous DMF was added
carbonyldiimidazole (0.486 g, 3.0 mmole). The mixture was stirred
at room temperature for 30 minutes and added in portions (2
ml/hour) to a solution of atractyloside (ATR) (0.086 g, 0.1 mmole)
in 1 ml of anhydrous DMF. The reaction mixture was stirred at room
temperature overnight and quenched with 1 ml of water. The solvent
was evaporated under vacuum and the residue was dissolved in ethyl
acetate (75 ml) and water (50 ml). The aqueous layer is separated,
extracted with ethyl acetate (3.times.75 ml) and concentrated under
vacuum. The residue was dissolved in 1.5 ml of methanol/water(1/1),
filtered through a 0.2 mm filter and purified using HPLC with a
preparative C-8 column (microsorb, 10.times.250 mm) using a linear
gradient elution of 30%-60% solvent B with a flow rate of 2.0
ml/min (solvent A: H2O/HOAc/NH4Oac (1.0 M, aq.): {1000/1/1};
solvent B: CH3OH/HOAc/NH.sub.4OAc (1.0 M, aq.): {1000/1/1}). The
title compound (compound 36 of Example 11 below) was obtained as a
white film (6.2 mg).
Example 11
Further Representative ATR Derivatives
[0338] Following the procedures set forth in Example 10, the
following ATR derivatives were prepared.
Atractyloside Derivatives
[0339]
2 23 Cpd R MS NMR 22 24 879.3 (M-H).sup.-799.3
(M-SO.sub.3--H).sup.- .sup.1H NMR (CD.sub.3OD) .delta.0.63(s, 3H),
0.95(d, 3H), 0.96(d, 3H), 4.94(s, 1H), 5.09(s, 1H), 7.53(m, 2H),
7.60(m, 1H), 7.93(d, 1H), 8.07(d, 1H), 8.26(d, 1H), 8.88(d,1H) 23
25 1083 (M-H).sup.-1003 (M-SO.sub.3--H).sup.- .sup.1H NMR
(CD.sub.3OD) .delta.0.65(s, 3H), 0.95(d, 3H), 0.96(d, 3H), 5.02(s,
1H), 5.13(s, 1H), 6.61(ddd, 2H), 6.69(d, 2H), 6.86(dd, 2H), 7.34(d,
1H), 8.33(dd, 1H), 8.67(s, 1H) 24 26 999 (M-H).sup.- .sup.1H NMR
(CD.sub.3OD) .delta.0.94(s, 3H), 0.95(d, 3H), 0.96(d, 3H), 5.05(s,
1H), 5.15(s, 1H), 6.75(d, 1H), 7.04(dd, 1H), 7.52(d, 1H) 25 27 921
(M-H).sup.-841 (M-SO.sub.3--H).sup.- .sup.1H NMR (CD.sub.3OD)
.delta.0.75(s, 3H), 0.95(d, 3H), 0.96(d, 3H), 4.97(s, 1H), 5.08(s,
1H), 6.89(d, 2H), 7.52(d, 2H), 7.64(d, 2H), 8.07(d, 2H) 26 28 1047
(M-H).sup.-967 (M-SO.sub.3--H).sup.- .sup.1H NMR (CD.sub.3OD)
.delta.0.73(s, 3H), 0.95(d, 3H), 0.96(d, 3H), 4.98(s, 1H), 5.09(s,
1H), 6.93(d, 1H), 7.52(dd, 1H), 7.61(d, 2H), 7.98(d, 1H), 8.08(d,
2H) 27 29 -- .sup.1H NMR (CD.sub.3OD) .delta.0.81(s, 3H), 0.95(d,
3H), 0.96(d, 3H), 5.04(s, 1H), 5.15(s, 1H), 6.80(d, 2H), 7.90(d,
2H), 7.64(d, 2H) 28 30 1097.1 (M-H).sup.-1017.0
(M-SO.sub.3--H).sup.- .sup.1H NMR (CD.sub.3OD) .delta.0.83(s, 3H),
0.95(d, 3H), 0.96(d, 3H), 5.03(s, 1H), 5.15(s, 1H), 6.84(d, 1H),
7.88(dd, 1H), 8.33(d, 1H) 29 31 1043.2 (M-2H + Na).sup.-1021.2
(M-H).sup.- .sup.1H NMR (CD.sub.3OD) .delta.0.66(s, 3H), 0.95(d,
3H), 0.96(d, 3H), 4.93(s, 1H), 5.05(s, 1H), 7.22(d, 1H), 7.87(d,
1H), 8.04(dd, 1H), 8.08(d, 1H), 8.50(d, 1H) 30 32
944(M-H).sup.-864(M-SO.sub.3--H).sup.- .sup.1H NMR (CD.sub.3OD)
.delta.0.97(s, 3H), 0.98(d, 6H), 5.06(s, 1H), 5.17(s, 1H), 5.70(d,
2H), 7.03(d, 2H). 31 33 1070(M-H).sup.-990(M-SO.s- ub.3--H).sup.-
-- 32 34 1116(M-SO.sub.3-H).sup.- -- 33 35 1125(M-H).sup.-1147(M-2H
+ Na).sup.- .sup.1H NMR(CD.sub.3OD) .delta. 0.95(s, 3H), 0.96(s,
6H), 5.05(s, 1H), 5.15(s, 1H), 7.58(s, 2H) 34 36 923.2 (M-2H +
Na).sup.-901.3 (M-H).sup.- -- 35 37 1049.3 (M-2H + Na).sup.- -- 36
38 873 (M-H).sup.-895 (M-2H + Na).sup.- .sup.1H NMR (CD.sub.3OD)
.delta.0.94(s, 3H), 0.96(d, 6H), 5.05(s, 1H), 5.16(s, 1H),
Dihydroactractyloside Derivatives
[0340]
3 39 Cpd R MS NMR 37 40 897 (M-2H + Na).sup.-875 (M-H).sup.-
.sup.1H NMR (CD.sub.3OD) .delta. 0.93(s, 3H), 0.95(d, 3H), 0.96(d,
3H), 1.08(d, 3H), 6.69(d, 2H), 7.02(d, 2H), resonances from alkenic
protons absent 38 41 1105 (M-2H + Na).sup.-1083 (M-H).sup.- .sup.1H
NMR (CD.sub.3OD) .delta. 0.93(s, 3H), 0.95(d, 3H), 0.96(d, 3H),
1.08(d, 3H), 6.74(d, 1H), 7.05(dd, 1H), 7.53(d, 1H), resonances
from alkenic protons absent
Apoactractyloside Derivatives
[0341]
4 42 Cpd R MS NMR 39 43 831(M-H).sup.- .sup.1H NMR (CD.sub.3OD)
.delta. 1.00(s, 3H), 5.07(s, 1H), 5.17(s, 1H), 6.69(d, 2H), 7.03(d,
2H) 40 44 1083(M-H).sup.-1105(M- 2H + Na).sup.- --
Example 12
Binding Assays Using Recombinant huANT3
[0342] A. GST-huANT3 Recombinantly Produced in E. coli:
[0343] Following arabinose induction, transformed or sham
transformed (vector only) E. coli were collected by centrifugation
at 2000 g for 10 min. The bacterial pellets were resuspended in
MSB, to which lysozyme (100 .mu.g/ml) was added. After 20 min at
room temperature, the lysates were subjected to one freeze-thaw
cycle followed by sonication as described above. The resultant
membrane preparation was used for binding assays.
[0344] To estimate maximal binding and the extent of overexpression
of the huANT3, 25 .mu.g of membrane protein was incubated with
varying amounts of [.sup.32P]ATP (1-500 .mu.M) in binding buffer
(120 mM KCl, 10 mM Tris, 1 mM EDTA, pH 7.4) for 2 hr at room
temperature. The membranes with bound ATP were sedimented by
centrifugation at 5000 g for 5 min, and washed once with binding
buffer. Membrane pellets were then mixed with 5 ml scintillation
cocktail and counted. The results are presented in Table 2.
5TABLE 2 Saturation Binding of [.sup.32P]ATP to E. coli Membranes
cpm bound by: [ATP], uM Sham Transfomed Cells huANT3-Producing
Cells 0.1 109 191 0.5 95 49 1.0 147 325 5.0 123 N.D. 10 214 263 50
549 2,727 100 718 5,772 500 2,140 9,715
[0345] N.D., not determined
[0346] The data presented in Table 2 indicate that the affinity of
the ATP binding was .about.6 .mu.M. ATP binding was completed
abolished by the addition of atractyloside (10 .mu.M) to the assay.
These results support the contention that the measured ATP binding
was predominantly to recombinantly produced ANT3 protein.
[0347] Agarose-glutathione beads were incubated with solubilized
(using Dnase, Rnase and 0.1% Triton X-100; see Example 1, section
D) bacterial lysate (see Example 2), and substituted for the E.
coli membranes in binding assays. Best results (i.e., more specific
binding) were seen when the beads were preincubated with bovine
serum albumin (BSA, 0.1%) Specific ATP binding (1,070 cpm) was also
observed in this experiment (compare to nonspecific binding of 279
cpm in the presence of 10 mM non-radiolabeled ATP).
[0348] B. huANT3 from a Sf9/Baculovirus Expression System
[0349] Sf9 cells expressing huANT3 were grown in spinner flasks.
The cells were harvested by centrifugation at 2,000 g for 5 min.
The cell pellet was resuspended in MSB and subjected to 3
freeze-thaw cycles. Cell membranes and debris were removed by
centrifugation at 600 g for 5 min; mitochondria were collected by
centrifuging the supernatant at 20,000 g for 15 min. The
mitochondrial pellets were suspended in MSB, and used for binding
assays as described above. Homologous competition of [.sup.32P]ATP
binding was performed using 25 .mu.g mitochondrial protein per
assay.
[0350] As illustrated in FIG. 7, ATP bound to the mitochondria with
Kd=13 .mu.M, a value consistent with ATP binding to ANT.
Furthermore, the ATP binding was displaced by low concentrations of
atractyloside (FIG. 8). Homologous competition binding assays using
[.sup.125I]atractyloside revealed specific binding with Kd=12 nM
(FIG. 9). These findings are consistent with the presence of
functional huANT3 in the mitochondrial preparations.
[0351] His-tagged huANT3 protein was purified from
baculovirus-infected Sf9 solubilized cell lysates using Ni-agarose
magnetic beads; Sf9 cells that had not been infected were used as
negative controls. The beads were incubated with [.sup.32P]ATP (1
or 100 .mu.M) for 2 hr. The beads were washed and then counted to
determine the amount of bound ATP. As shown in Table 3, the
[.sup.32P]ATP binding was significantly higher in material
recovered from the infected cells than in the controls. Binding
saturation had essentially been achieved with 1 .mu.M ATP.
6TABLE 3 Binding of [.sup.32P]ATP to Purified His-Tagged huANT3 cpm
bound by: [ATP], uM Control (Uninfected) Cells huANT3-Producing
Cells 1.0 43 149 100 30 160
Example 13
Competitive Binding Assays
[0352] Atractyloside analogs (Example 6; Table 1; see also Examples
7-9) were used in pseudo-homologous competition binding assays
using Sf9/huANT3 mitochondria. Mitochondria (see Example 12, 25
.mu.g/tube) were incubated with 0.5 nM [.sup.125I]atractyloside and
varying concentrations of non-radiolabeled atractyloside or
fluorescent atractyloside derivatives as described above (FIGS.
7-9).
[0353] The results (Table 4) show that three of the atractyloside
derivatives (MANT-, Pyrene- and Coumarin-atractyloside) had
relative binding affinities similar to that of authentic
atractyloside (IC.sub.50<500 mM relative to atractyloside). Each
of these derivatives is fluorescent, and may therefore be useful as
detectable ligands for binding assays.
7TABLE 4 Competitive Binding Assays Using [.sup.125I]Atractyloside
cpm [.sup.125I]Atractyloside bound in the presence of: [ATR
Derivative], nM ATR COU-ATR PYR-ATR MANT-ATR 0.0 227 437 437 437
1.0 224 391 350 -- 5.0 146 -- -- -- 10 42 371 349 229 50 26 -- --
-- 100 -- 277 362 195 1,000 36 174 238 -- 5,000 45 -- -- -- 10,000
0 100 0 --
[0354] Abbreviations and symbols: ATR, atractlyoside. COU-ATR,
Coumarin-atractyloside, (Table 1, compound 3). PYR-ATR,
Pyrene-atractyloside, (Table 1, compound 4). MANT-ATR,
MANT-atractyloside, (Table 1, Roux et al. 1996 Anal. Bioch. 234:31)
- - - , not determined.
[0355] Atractyloside analogs (Example 6; Table 1; see also Examples
7-9) were also used in pseudo-homologous competition binding assays
using T. ni/huANT3 mitochondria or bovine mitochondria.
Mitochondria from noninfected T. ni cells, or T. ni cells infected
with a baculovirus expressing huANT3 (see Example 3) were prepared
as follows: T. ni cells were prepared by a subcontractor
(PharMingen, San Diego, Calif.) as portions of about 250 mg of
cells per tube. Each portion was suspended in 1 ml of MSB with
protease inhibitors (leupeptin, final concentration 10 ug/ml;
pepstatin, final concentration 10 ug/ml; aprotinin, final
concentration, 2 ug/ml; phenylmethylsulfonyl fluoride, [PMSF],
final concentration, 100 .mu.M; all from Sigma Chemical Co., St.
Louis, Mo.). The resuspended cell suspensions were frozen and
thawed twice, then homogenized using a rotating teflon-coated probe
and a close-fitting glass container (10 passes). The cellular
homogenate was centrifuged (3,700 rpm, approximately 1,500.times.g)
at 4.degree. C. for 5 minutes; this supernatant from the first spin
was saved. The pellet was washed with about 500 .mu.l of MSB with
protease inhibitors, centrifuged (3,800 rpm, approximately
1,600.times.g) at 4.degree. C. for 5 minutes, and supernatant from
this spin was combined with the supernatant from the first spin.
The combined supernatant was centrifuged (14,000 rpm, approximately
20,800.times.g) at 4.degree. C. for 15 minutes, and the pellet was
resuspended in 300 .mu.l of a 1:1 solution of (a) 20 mM MOPS and
(b) MSB, wherein both (a) and (b) contain the previously described
protease inhibitors. The resultant suspension was frozen and thawed
three times.
[0356] Bovine mitochondria were prepared as follows: Essentially
all of the fat and cholesterol in clogged arteries was removed from
two bovine hearts which were then cut into 1-inch cubes. The cubes
were ground in a meat grinder using the fine setting. Three hundred
(300) gm portions of the ground heart were weighed out and, to each
was added 400 ml of Isolation Buffer (IB; 250 mM sucrose, 1 mM
sodium succinate, 0.2 mM K.sup.+ EDTA, 10 mM Tris-base, pH 7.8).
(All buffers were filter sterilized, and column buffers were
degassed, and, unless otherwise noted, all steps were carried out
at 0 to 4.degree. C. on ice or in pre-cooled rotors and
centrifuges.) The preparations were mixed in a blender two times
for 15 seconds on high setting and, in between and after blends,
the pH was adjusted to 7.8 with 2M Tris-base. The homogenate was
centrifuged for 20 minutes at 1,200.times.g, and the supernatant
was poured through two layers of cheese cloth and adjusted to pH
7.8 with 2M Tris-base. The supernatant was then centrifuged for 30
minutes at 11,000.times.g. The supernatant was decanted, and the
buff-colored outer pellet was dislodged with about 10 ml of IB and
discarded. The brown inner pellet (heavy mitochondria) was
resuspended in IB (about 10 ml per pellet). The pellets were
homogenized in a glass-teflon homogenizer (2 passes at high drill
speed). Samples were combined and centrifuged for 30 minutes at
11,000.times.g. The supernatant was decanted, and the pellets were
resuspended in 60 ml of IB per 900 gm of ground heart. This
centrifugation step was repeated and the pellets were finally
resuspended in IB (60 ml per 900 gm of ground heart). One kilogram
of beef heart typically yields about one (1) gram of
mitochondria.
[0357] The mitochondrial preparations were divided into aliquots
(typically, 50 .mu.l for T. ni mitochondria or 20 .mu.l for bovine
mitochondria) and then either used directly in assays or flash
frozen and stored at -80.degree. C. The total protein content in
the mitochondrial preparations was determined using the enhanced
protocol (30 minutes at 60.degree. C.; see
http://www.ruf.rice.edu/.about.bioslabs/methods/protei- n/BCA.html)
of the bicinchoninic acid (BCA) assay (available in kit form from
Pierce, Rockford, Ill.).
[0358] In the "Tube Assay," mitochondria (from about 1 to 10 .mu.g
of total protein) were resuspended in 100 .mu.l of Tris-KCl buffer
with 01% BSA, pH 7.4. .sup.125I-labeled compound 24 (Example 11)
was added to a final concentration of 0.5 nM. When used,
competitors were added at these concentration ranges: unlabeled
atractyloside or compound 24, final concentration from 5 nM to 10
.mu.M; unlabeled ADP (a lower affinity competitor) was added at a
final concentration of 500 nM to 1 mM.
[0359] The reaction mixes were incubated on ice for 60 minutes and
then pelleted by centrifugation (approximately 16,000.times.g) for
11 minutes at 4.degree. C. Unbound .sup.125I-compound 24 was
removed by aspiration. The pellets were contact-washed with
Tris-KCl buffer, pH 7.4, and recentrifuged. The resultant pellets
were aspirated and the radioactivity (dpm) in each was determined
by gamma counting.
[0360] Representative results are shown in FIGS. 11-18. The data
presented in FIGS. 11 and 12 show that mitochondria (5 .mu.g of
protein/tube) from both beef heart (FIG. 11) and T. ni cells
expressing huANT3 (FIG. 12) specifically bind .sup.125I-compound 24
in a manner that is inhibited by increasing concentrations of
unlabeled compound 24, but, as expected, little or no binding is
seen when mitochondria are excluded from the reaction mixes.
[0361] FIGS. 13 and 14 show competitive inhibition of
.sup.125I-compound 24 binding to mitochondria (1 .mu.g of
protein/tube) from beef heart (FIG. 13) and T. ni cells expressing
huANT3 (FIG. 14) by compound 24 that is not detectably labeled,
unlabeled atractyloside (ATR), and unlabeled adenosine diphosphate
(ADP). In both instances, ATR and compound 24 yield comparable
competition curves, although ATR appears to have a slightly higher
affinity than compound 24. However, both ATR and compound 24 bind
with much higher (about 1,000 fold) affinity than the low affinity
ANT ligand ADP.
[0362] FIG. 15 shows the competitive inhibition, by unlabeled ATR,
of binding of .sup.125I-compound 24 to mitochondria (1 .mu.g of
protein/tube) from T. ni cells expressing huANT3 and control T. ni
cells (i.e., non-infected T. ni cells). As shown in FIG. 15, there
was only slight inhibition of .sup.125I-compound 24 binding to
control (nontransformed) mitochondria by higher concentrations of
ATR. In contrast, binding of .sup.125I-compound 24 to mitochondria
from T. ni cells expressing huANT3 was increasingly inhibited by
higher concentrations of ATR.
[0363] FIG. 16 shows competitive inhibition, by unlabeled compound
24 and by bongkrekic acid (BKA), of .sup.125I-compound 24 binding
to mitochondria (1 .mu.g of protein/tube) from beef heart. BKA
effectively displaced labeled compound 24, albeit with a slightly
lower affinity than unlabeled compound 24. FIG. 17 shows
competitive inhibition of .sup.125I-compound 24 binding to beef
heart mitochondria by either of the ATR derivatives, compound 23
(see Example 11) and compound 28 (see Example 11). As shown in FIG.
17, ATR exhibited an IC.sub.50 of approximately 44 nM, compound 23
an IC.sub.50 of approximately 105 nM, and compound 28 an IC.sub.50
of approximately 695 nM.
[0364] In FIG. 18, data are presented depicting competitive
inhibition of .sup.125I-compound 24 binding to beef heart
mitochondria by the ATR derivative compound 5 (see Example 7). As
shown in FIG. 18, the IC.sub.50 for compound 5 was approximately
3.3 .mu.M.
[0365] Competitive binding assays were also performed using
recombinant His-tagged huANT3 (see Example 3) immobilized on Ni
beads (FIG. 19) instead of mitochondria. To prepare the
bead-immobilzed huANT3, mitochondria from T. ni cells infected with
a baculovirus expressing huANT3 (see Example 3) were solubilized
with 0.5% O-glucopyranoside in the presence of 0.5 nM
.sup.125I-compound 24, Ni-agarose beads (Qiagen, Hilden, Germany),
and various concentrations of ATR or BKA as unlabeled competitors.
After 1 hour at 4.degree. C., the beads were washed and
radioactivity that remained associated with the beads was counted.
(Background binding of [.sup.125I] compound 24 to Ni-agarose beads
(Qiagen) in the absence of His-tagged huANT3 was approximately
700-800 cpm and was not subtracted from the radioactivity shown in
FIG. 19.) The results (FIG. 19) show that both ATR and BKA
effectively compete with compound 24 in a manner similar to that
observed in assays using intact mitochondria (cf. FIGS. 14 and
15).
Example 14
High Throughput Screening Assay for Compounds Targeted to ANT
Proteins and Polypeptides
[0366] The recombinantly produced ANT proteins, ANT fusion proteins
and detectably labeled ANT ligands described herein are
incorporated into automated assay systems. Such automated systems
are useful for high throughput screening (HTS) of candidate
ANT-binding compounds or chemical libraries comprising such
compounds. Such compounds may be further characterized and
developed as drug candidates and drugs useful for preventing,
treating or curing diseases or disorders resulting from the
overexpression or dysfunction of one or more ANT proteins or from
the overexpression or dysfunction of a factor that positively
regulates or stimulates ANT proteins.
[0367] A preferred element of many automated assay systems is the
incorporation of a target molecule (in the present instance, an ANT
protein) into a 96-well plate. This format is readily adaptable for
use in a variety of automated label detection systems. For HTS
assays, robotic label detection systems are preferred.
[0368] As one example of an HTS comprising the elements describes
herein, the GST-huANT3 fusion protein of Example 2 is contacted
with REACTI-BIND.TM. glutathione-coated 96-well plates (Pierce).
Glutathione coated strip-well plates are preferably used for assays
comprising radiolabeled ANT ligands (e.g., iodinated atractyloside
derivates; see Example 7), whereas black opaque glutathione coated
96-well plates are preferred for assays comprising fluorescent ANT
ligands (such as are described in, e.g., Examples 6-9); both types
of glutathione coated plates are commercially available
(Pierce).
[0369] In a typical assay, 1 to 50 ug of GST-huANT3 protein (i.e.,
total solubilized protein prepared as in Example 2) is added per
gluthathione-coated well to each well of a 96-well plate. Iodinated
atractyloside derivate (.sup.125I-ATR) is added to the wells (0.5
nmol/well). In a control experiment, unlabeled atractyloside (ATR;
Sigma) is used as a `mock` drug at a concentration of from about 1
to about 10,000 nM. That is, unlabeled ATR is used to displace a
labeled atractyloside derivative (e.g., .sup.125I-ATR). Unlabeled
ATR thus acts as a positive control for an HTS in which various
compounds are screened for their ability to displace a labeled ANT
ligand.
[0370] As an example of the automated label detection systems used
in the HTS assays of the Example, when the detectably labeled ANT
ligand of the assay is 125I-ATR, an automatic gamma counter is
used. Alternatively, .sup.125I-ATR can be used in scintillation
proximity assays (SPA). For example, a GST-huANT fusion protein is
contacted with ScintiStrip 96-well plates coated with glutathione
(EG&G Wallac). The polystyrene of these plates contains a
scintillating agent that emits beta radiation when excited by a
gamma emitter in close proximity thereto. The beta radiation is
then detected by any appropriate automatic beta counter. When
fluorescent ANT ligands are used in the HTS assay, an automatic
fluorescence counter is used and may be, for example, a
FLUOROCOUNT.TM. Counter (Packard Instrument Company, Meriden,
Conn.).
Sequence CWU 1
1
37 1 894 DNA Homo sapien 1 atgggtgatc acgcttggag cttcctaaag
gacttcctgg ccggggcggt cgccgctgcc 60 gtctccaaga ccgcggtcgc
ccccatcgag agggtcaaac tgctgctgca ggtccagcat 120 gccagcaaac
agatcagtgc tgagaagcag tacaaaggga tcattgattg tgtggtgaga 180
atccctaagg agcagggctt cctctccttc tggaggggta acctggccaa cgtgatccgt
240 tacttcccca cccaagctct caacttcgcc ttcaaggaca agtacaagca
gctcttctta 300 gggggtgtgg atcggcataa gcagttctgg cgctactttg
ctggtaacct ggcgtccggt 360 ggggccgctg gggccacctc cctttgcttt
gtctacccgc tggactttgc taggaccagg 420 ttggctgctg atgtgggcag
gcgcgcccag cgtgagttcc atggtctggg cgactgtatc 480 atcaagatct
tcaagtctga tggcctgagg gggctctacc agggtttcaa cgtctctgtc 540
caaggcatca ttatctatag agctgcctac ttcggagtct atgatactgc caaggggatg
600 ctgcctgacc ccaagaacgt gcacattttt gtgagctgga tgattgccca
gagtgtgacg 660 gcagtcgcag ggctgctgtc ctaccccttt gacactgttc
gtcgtagaat gatgatgcag 720 tccggccgga aaggggccga tattatgtac
acggggacag ttgactgctg gaggaagatt 780 gcaaaagacg aaggagccaa
ggccttcttc aaaggtgcct ggtccaatgt gctgagaggc 840 atgggcggtg
cttttgtatt ggtgttgtat gatgagatca aaaaatatgt ctaa 894 2 897 DNA Homo
sapien 2 atgacagatg ccgcattgtc cttcgccaag gacttcctgg caggtggagt
ggccgcagcc 60 atctccaaga cggcggtagc gcccatcgag cgggtcaagc
tgctgctgca ggtgcagcat 120 gccagcaagc agatcactgc agataagcaa
tacaaaggca ttatagactg cgtggtccgt 180 attcccaagg agcaggaagt
tctgtccttc tggcgcggta acctggccaa tgtcatcaga 240 tacttcccca
cccaggctct taacttcgcc ttcaaagata aatacaagca gatcttcctg 300
ggtggtgtgg acaagagaac ccagttttgg cgctactttg cagggaatct ggcatcgggt
360 ggtgccgcag gggccacatc cctgtgtttt gtgtaccctc ttgattttgc
ccgtacccgt 420 ctagcagctg atgtgggtaa agctggagct gaaagggaat
tccgaggcct cggtgactgc 480 ctggttaaga tctacaaatc tgatgggatt
aagggcctgt accaaggctt taacgtgtct 540 gtgcagggta ttatcatcta
ccgagccgcc tacttcggta tctatgacac tgcaaaggga 600 atgcttccgg
atcccaagaa cactcacatc gtcatcagct ggatgatcgc acagactgtc 660
actgctgttg ccgggttgac ttcctatcca tttgacaccg ttcgccgccg catgatgatg
720 cagtcagggc gcaaaggaac tgacatcatg tacacaggca cgcttgactg
ctggcggaag 780 attgctcgtg atgaaggagg caaagctttt ttcaagggtg
catggtccaa tgttctcaga 840 ggcatgggtg gtgcttttgt gcttgtcttg
tatgatgaaa tcaagaagta cacataa 897 3 897 DNA Homo sapien 3
atgacggaac aggccatctc cttcgccaaa gacttcttgg ccggaggcat cgccgccgcc
60 atctccaaga cggccgtggc tccgatcgag cgggtcaagc tgctgctgca
ggtccagcac 120 gccagcaagc agatcgccgc cgacaagcag tacaagggca
tcgtggactg cattgtccgc 180 atccccaagg agcagggcgt gctgtccttc
tggaggggca accttgccaa cgtcattcgc 240 tacttcccca ctcaagccct
caacttcgcc ttcaaggata agtacaagca gatcttcctg 300 gggggcgtgg
acaagcacac gcagttctgg aggtactttg cgggcaacct ggcctccggc 360
ggtgcggccg gcgcgacctc cctctgcttc gtgtacccgc tggattttgc cagaacccgc
420 ctggcagcgg acgtgggaaa gtcaggcaca gagcgcgagt tccgaggcct
gggagactgc 480 ctggtgaaga tcaccaagtc cgacggcatc cggggcctgt
accagggctt cagtgtctcc 540 gtgcagggca tcatcatcta ccgggcggcc
tacttcggcg tgtacgatac ggccaagggc 600 atgctccccg accccaagaa
cacgcacatc gtggtgagct ggatgatcgc gcagaccgtg 660 acggccgtgg
ccggcgtggt gtcctacccc ttcgacacgg tgcggcggcg catgatgatg 720
cagtccgggc gcaaaggagc tgacatcatg tacacgggca ccgtcgactg ttggaggaag
780 atcttcagag atgagggggg caaggccttc ttcaagggtg cgtggtccaa
cgtcctgcgg 840 ggcatggggg gcgccttcgt gctggtcctg tacgacgagc
tcaagaaggt gatctaa 897 4 43 DNA Artificial Sequence PCR Primer 4
ttatatctcg agtatgggtg atcacgcttg gagcttccta aag 43 5 43 DNA
Artificial Sequence PCR Primer 5 tatataggta ccttagacat attttttgat
ctcatcatac aac 43 6 43 DNA Artificial Sequence PCR Primer 6
ttatatctcg agtatgacag atgccgctgt gtccttcgcc aag 43 7 43 DNA
Artificial Sequence PCR Primer 7 tatataggta ccttatgtgt acttcttgat
ttcatcatac aag 43 8 43 DNA Artificial Sequence PCR Primer 8
ttatatctcg agtatgacgg aacaggccat ctccttcgcc aaa 43 9 44 DNA
Artificial Sequence PCR Primer 9 tatataggta ccttagagtc accttcttga
gctcgtcgta cagg 44 10 21 DNA Artificial Sequence Sequence primer 10
tatgccatag catttttatc c 21 11 18 DNA Artificial Sequence Sequence
primer 11 cgccaaaaca gccaagct 18 12 45 DNA Artificial Sequence
Mutagenic oligonucleotide primer 12 ggagatggcc tgttccgtca
tcttatcgtc atcgtcgtac agatc 45 13 45 DNA Artificial Sequence
Mutagenic oligonucleotide primer 13 gatctgtacg acgatgacga
taagatgacg gaacaggcca tctcc 45 14 35 DNA Artificial Sequence PCR
primer 14 cccggggaat tctgatgacg gaacaggcca tctcc 35 15 34 DNA
Artificial Sequence PCR primer 15 cccgggctcg agttagagtc accttcttga
gctc 34 16 41 DNA Artificial Sequence PCR primer 16 ttataggatc
catgacggaa caggccatct ccttcgccaa a 41 17 41 DNA Artificial Sequence
PCR primer 17 ttaaagaatt cttagatcac cttcttgagc tcgtcgtaca g 41 18
18 DNA Artificial Sequence Sequencing primer 18 aaatgataac catctcgc
18 19 18 DNA Artificial Sequence Sequencing primer 19 acttcaagga
gaatttcc 18 20 18 DNA Artificial Sequence Sequencing primer 20
acttcgcctt cacggata 18 21 18 DNA Artificial Sequence Sequencing
primer 21 tacggccaag ggcattct 18 22 18 DNA Artificial Sequence
Sequencing primer 22 tgaagcggaa gttcctat 18 23 18 DNA Artificial
Sequence Sequencing primer 23 atgccggttc ccgtacga 18 24 31 DNA
Artificial Sequence Mutagenic oligonucleotide primer 24 ggcctgttcc
gtcatcttat cgtcatcgtc g 31 25 31 DNA Artificial Sequence Mutagenic
oligonucleotide primer 25 cgacgatgac gataagatga cggaacaggc c 31 26
41 DNA Artificial Sequence PCR primer 26 ttaaagaatt catgacggaa
caggccatct ccttcgccaa a 41 27 41 DNA Artificial Sequence PCR primer
27 ttataggatc cttagatcac cttcttgagc tcgtcgtaca g 41 28 42 DNA
Artificial Sequence PCR primer 28 ttaatgggta ccatgacgga acaggccatc
tccttcgcca aa 42 29 42 DNA Artificial Sequence PCR primer 29
ttatactcga gttagatcac cttcttgagc tcgtcgtaca gg 42 30 15 PRT
Artificial Sequence Synthetic polypeptide 30 Cys Trp Arg Lys Ile
Phe Arg Asp Glu Gly Gly Lys Ala Phe Phe 1 5 10 15 31 297 PRT Homo
sapien 31 Met Gly Asp His Ala Trp Ser Phe Leu Lys Asp Phe Leu Ala
Gly Ala 1 5 10 15 Val Ala Ala Ala Val Ser Lys Thr Ala Val Ala Pro
Ile Glu Arg Val 20 25 30 Lys Leu Leu Leu Gln Val Gln His Ala Ser
Lys Gln Ile Ser Ala Glu 35 40 45 Lys Gln Tyr Lys Gly Ile Ile Asp
Cys Val Val Arg Ile Pro Lys Glu 50 55 60 Gln Gly Phe Leu Ser Phe
Trp Arg Gly Asn Leu Ala Asn Val Ile Arg 65 70 75 80 Tyr Phe Pro Thr
Gln Ala Leu Asn Phe Ala Phe Lys Asp Lys Tyr Lys 85 90 95 Gln Leu
Phe Leu Gly Gly Val Asp Arg His Lys Gln Phe Trp Arg Tyr 100 105 110
Phe Ala Gly Asn Leu Ala Ser Gly Gly Ala Ala Gly Ala Thr Ser Leu 115
120 125 Cys Phe Val Tyr Pro Leu Asp Phe Ala Arg Thr Arg Leu Ala Ala
Asp 130 135 140 Val Gly Arg Arg Ala Gln Arg Glu Phe His Gly Leu Gly
Asp Cys Ile 145 150 155 160 Ile Lys Ile Phe Lys Ser Asp Gly Leu Arg
Gly Leu Tyr Gln Gly Phe 165 170 175 Asn Val Ser Val Gln Gly Ile Ile
Ile Tyr Arg Ala Ala Tyr Phe Gly 180 185 190 Val Tyr Asp Thr Ala Lys
Gly Met Leu Pro Asp Pro Lys Asn Val His 195 200 205 Ile Phe Val Ser
Trp Met Ile Ala Gln Ser Val Thr Ala Val Ala Gly 210 215 220 Leu Leu
Ser Tyr Pro Phe Asp Thr Val Arg Arg Arg Met Met Met Gln 225 230 235
240 Ser Gly Arg Lys Gly Ala Asp Ile Met Tyr Thr Gly Thr Val Asp Cys
245 250 255 Trp Arg Lys Ile Ala Lys Asp Glu Gly Ala Lys Ala Phe Phe
Lys Gly 260 265 270 Ala Trp Ser Asn Val Leu Arg Gly Met Gly Gly Ala
Phe Val Leu Val 275 280 285 Leu Tyr Asp Glu Ile Lys Lys Tyr Val 290
295 32 298 PRT Homo sapien 32 Met Thr Asp Ala Ala Leu Ser Phe Ala
Lys Asp Phe Leu Ala Gly Gly 1 5 10 15 Val Ala Ala Ala Ile Ser Lys
Thr Ala Val Ala Pro Ile Glu Arg Val 20 25 30 Lys Leu Leu Leu Gln
Val Gln His Ala Ser Lys Gln Ile Thr Ala Asp 35 40 45 Lys Gln Tyr
Lys Gly Ile Ile Asp Cys Val Val Arg Ile Pro Lys Glu 50 55 60 Gln
Glu Val Leu Ser Phe Trp Arg Gly Asn Leu Ala Asn Val Ile Arg 65 70
75 80 Tyr Phe Pro Thr Gln Ala Leu Asn Phe Ala Phe Lys Asp Lys Tyr
Lys 85 90 95 Gln Ile Phe Leu Gly Gly Val Asp Lys Arg Thr Gln Phe
Trp Arg Tyr 100 105 110 Phe Ala Gly Asn Leu Ala Ser Gly Gly Ala Ala
Gly Ala Thr Ser Leu 115 120 125 Cys Phe Val Tyr Pro Leu Asp Phe Ala
Arg Thr Arg Leu Ala Ala Asp 130 135 140 Val Gly Lys Ala Gly Ala Glu
Arg Glu Phe Arg Gly Leu Gly Asp Cys 145 150 155 160 Leu Val Lys Ile
Tyr Lys Ser Asp Gly Ile Lys Gly Leu Tyr Gln Gly 165 170 175 Phe Asn
Val Ser Val Gln Gly Ile Ile Ile Tyr Arg Ala Ala Tyr Phe 180 185 190
Gly Ile Tyr Asp Thr Ala Lys Gly Met Leu Pro Asp Pro Lys Asn Thr 195
200 205 His Ile Val Ile Ser Trp Met Ile Ala Gln Thr Val Thr Ala Val
Ala 210 215 220 Gly Leu Thr Ser Tyr Pro Phe Asp Thr Val Arg Arg Arg
Met Met Met 225 230 235 240 Gln Ser Gly Arg Lys Gly Thr Asp Ile Met
Tyr Thr Gly Thr Leu Asp 245 250 255 Cys Trp Arg Lys Ile Ala Arg Asp
Glu Gly Gly Lys Ala Phe Phe Lys 260 265 270 Gly Ala Trp Ser Asn Val
Leu Arg Gly Met Gly Gly Ala Phe Val Leu 275 280 285 Val Leu Tyr Asp
Glu Ile Lys Lys Tyr Thr 290 295 33 298 PRT Homo sapien 33 Met Thr
Glu Gln Ala Ile Ser Phe Ala Lys Asp Phe Leu Ala Gly Gly 1 5 10 15
Ile Ala Ala Ala Ile Ser Lys Thr Ala Val Ala Pro Ile Glu Arg Val 20
25 30 Lys Leu Leu Leu Gln Val Gln His Ala Ser Lys Gln Ile Ala Ala
Asp 35 40 45 Lys Gln Tyr Lys Gly Ile Val Asp Cys Ile Val Arg Ile
Pro Lys Glu 50 55 60 Gln Gly Val Leu Ser Phe Trp Arg Gly Asn Leu
Ala Asn Val Ile Arg 65 70 75 80 Tyr Phe Pro Thr Gln Ala Leu Asn Phe
Ala Phe Lys Asp Lys Tyr Lys 85 90 95 Gln Ile Phe Leu Gly Gly Val
Asp Lys His Thr Gln Phe Trp Arg Tyr 100 105 110 Phe Ala Gly Asn Leu
Ala Ser Gly Gly Ala Ala Gly Ala Thr Ser Leu 115 120 125 Cys Phe Val
Tyr Pro Leu Asp Phe Ala Arg Thr Arg Leu Ala Ala Asp 130 135 140 Val
Gly Lys Ser Gly Thr Glu Arg Glu Phe Arg Gly Leu Gly Asp Cys 145 150
155 160 Leu Val Lys Ile Thr Lys Ser Asp Gly Ile Arg Gly Leu Tyr Gln
Gly 165 170 175 Phe Ser Val Ser Val Gln Gly Ile Ile Ile Tyr Arg Ala
Ala Tyr Phe 180 185 190 Gly Val Tyr Asp Thr Ala Lys Gly Met Leu Pro
Asp Pro Lys Asn Thr 195 200 205 His Ile Val Val Ser Trp Met Ile Ala
Gln Thr Val Thr Ala Val Ala 210 215 220 Gly Val Val Ser Tyr Pro Phe
Asp Thr Val Arg Arg Arg Met Met Met 225 230 235 240 Gln Ser Gly Arg
Lys Gly Ala Asp Ile Met Tyr Thr Gly Thr Val Asp 245 250 255 Cys Trp
Arg Lys Ile Phe Arg Asp Glu Gly Gly Lys Ala Phe Phe Lys 260 265 270
Gly Ala Trp Ser Asn Val Leu Arg Gly Met Gly Gly Ala Phe Val Leu 275
280 285 Val Leu Tyr Asp Glu Leu Lys Lys Val Ile 290 295 34 41 DNA
Artificial Sequence Primer for PCR amplification of human ANT3 for
expression construct 34 ttaatggtac catgacggaa caggccatct ccttcgccaa
a 41 35 42 DNA Artificial Sequence Primer for PCR amplification of
human ANT3 for expression construct 35 ttatactcga gttagatcac
cttcttgagc tcgtcgtaca gg 42 36 30 DNA Artificial Sequence Primer
for PCR amplification of EYFP 36 gggcccctcg agatggtgag caagggcgag
30 37 33 DNA Artificial Sequence Primer for PCR amplification of
EYFP 37 gggccctcta gactacttgt acagctcgtc cat 33
* * * * *
References