U.S. patent application number 15/569851 was filed with the patent office on 2018-05-03 for compositions and methods for assessing toxicity using dynamic bh3 profiling.
This patent application is currently assigned to Dana-Farber Cancer Institute, Inc.. The applicant listed for this patent is Dana-Farber Cancer Institute, Inc.. Invention is credited to Patrick Bhola, Anthony Letai, Jeremy Ryan.
Application Number | 20180120297 15/569851 |
Document ID | / |
Family ID | 57198812 |
Filed Date | 2018-05-03 |
United States Patent
Application |
20180120297 |
Kind Code |
A1 |
Letai; Anthony ; et
al. |
May 3, 2018 |
COMPOSITIONS AND METHODS FOR ASSESSING TOXICITY USING DYNAMIC BH3
PROFILING
Abstract
The present invention provides methods of assessing toxicity
using Dynamic BH3 profiling.
Inventors: |
Letai; Anthony; (Medfield,
MA) ; Bhola; Patrick; (Cambridge, MA) ; Ryan;
Jeremy; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Dana-Farber Cancer Institute,
Inc.
Boston
MA
|
Family ID: |
57198812 |
Appl. No.: |
15/569851 |
Filed: |
April 27, 2016 |
PCT Filed: |
April 27, 2016 |
PCT NO: |
PCT/US16/29510 |
371 Date: |
October 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62153475 |
Apr 27, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 2500/10 20130101; G01N 33/5044 20130101; G01N 2800/52
20130101; G01N 2333/47 20130101; G01N 33/5079 20130101; G01N
33/5014 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A method of screening a plurality of agents for determining
toxicity, said method comprising: a) providing a plurality of
agents; b) providing a sample of cells, wherein the cells are
non-cancerous; c) providing a test aliquot of the sample of cells
for each of the plurality of agents; d) separately contacting each
test aliquot with its respective agent; e) providing a control
aliquot of the sample of cells, wherein the control aliquot is not
contacted with any of the plurality of agents; f) permeabilizing
the cells in each aliquot; g) contacting the permeabilized cells
with a pro-apoptotic BH3 domain peptide; h) determining a value for
percent BH3 domain peptide-induced mitochondrial outer membrane
permeabilization (MOMP) in the cells in each of the test aliquots;
i) determining a value for percent BH3 domain peptide-induced MOMP
in the cells in the control aliquot; and j) determining a value for
delta percent priming for each of the test aliquots, wherein delta
percent priming is the difference between the value for percent
MOMP determined in (h) and the value for percent MOMP determined in
(i), wherein a delta percent priming of greater than 20 percent
indicates that the agent with which the cells in the test aliquot
were contacted is toxic to the cells.
2. The method of claim 1, further comprising conducting in vivo or
in vitro toxicity testing with the agent if the delta percent
priming is 20 percent or less, 18 percent or less, 16 percent or
less, 14 percent or less, 12 percent or less, 10 percent or less, 8
percent or less, 6 percent or less, 4 percent or less, 2 percent or
less, or 1 percent or less.
3. The method of claim 1 or 2, wherein the non-cancerous cells are
healthy cells.
4. The method of any one of claims 1-3, further comprising: k)
providing a sample of cells, wherein the cells are cancerous; l)
providing a test aliquot of the sample of cancerous cells for each
of the plurality of agents; m) separately contacting each test
aliquot with its respective agent; n) providing a control aliquot
of the sample of cancerous cells, wherein the control aliquot is
not contacted with any of the plurality of agents; o)
permeabilizing the cancerous cells in each aliquot; p) contacting
the permeabilized cancerous cells with a pro-apoptotic BH3 domain
peptide; q) determining a value for percent BH3 domain
peptide-induced MOMP in the cancerous cells in each of the test
aliquots; r) determining a value for percent BH3 domain
peptide-induced MOMP in the cancerous cells in the control aliquot;
and s) determining a value for delta percent priming for each of
the test aliquots, wherein delta percent priming is the difference
between the value for percent MOMP determined in (q) and the value
for percent MOMP determined in (r), wherein a delta percent priming
of greater than 20 percent indicates that the agent with which the
cancerous cells in the test aliquot were contacted is toxic to the
cancerous cells.
5. The method of claim 4, further comprising conducting in vivo or
in vitro toxicity testing with the agent if the delta percent
priming in the cancerous cells is greater than 20 percent, greater
than 30 percent, greater than 40 percent, greater than 50 percent,
greater than 60 percent, greater than 70 percent, greater than 80
percent, greater than 90 percent, greater than 95 percent, or 100
percent.
6. The method of claim 4, further comprising conducting in vivo or
in vitro toxicity testing with the agent if the delta percent
priming in the cancerous cells is greater than the delta percent
priming in the non-cancerous cells.
7. The method of claim 4, further comprising conducting in vivo or
in vitro toxicity testing with the agent if the delta percent
priming in the cancerous cells is at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 100%, at least 150%, or at
least 200%, greater than the delta percent priming in the
non-cancerous cells.
8. The method of claim 4, further comprising conducting in vivo or
in vitro toxicity testing with the agent if the delta percent
priming in the cancerous cells is at least 2-fold, at least 3-fold,
at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold,
at least 8-fold, at least 9-fold, or at least 10-fold greater than
the delta percent priming in the non-cancerous cells.
9. The method of any one of claims 4-8, wherein the cancerous cells
are primary human tumor cells.
10. The method of any one of claims 4-8, wherein the cancerous
cells are obtained from a patient derived xenograft (PDX).
11. The method of any one of claims 1-10, wherein the plurality of
agents includes 20 or more agents.
12. The method of any one of claims 1-10, wherein the plurality of
agents includes 100 or more agents.
13. The method of any one of claims 1-12, wherein the each test
aliquot is contacted with its respective agent for 24 hours or
less.
14. The method of any one of claims 1-13, wherein the permeabilized
cells are contacted with the pro-apoptotic BH3 domain peptide for 3
hours or less.
15. The method of any one of claim 1-14, wherein the cells in each
of the test aliquots are permeabilized with digitonin or
saponin.
16. The method of any one of claims 1-15, wherein the percent BH3
domain peptide-induced MOMP is determined by one or more of: i)
contacting the cells with a potentiometric dye and measuring the
emission of said potentiometric dye; ii) measuring the release of a
molecule from the mitochondrial intermembrane space; and iii)
measuring the retention of a molecule from the mitochondrial
intermembrane space.
17. The method of claim 16, wherein the potentiometric dye is
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl
ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE).
18. The method of claim 14 or 15, wherein the molecule from the
mitochondrial intermembrane space is cytochrome c, SMAC/Diablo,
Omi, adenylate kinase-2, or apoptosis-inducing factor.
19. The method of any one of claims 1-18, wherein the BH3 domain
peptide is derived from the BH3 domain of a BID, a BIM, a BAD, a
NOXA, a PUMA, a BMF, or an HRK polypeptide.
20. The method of any one of claims 1-19, wherein the BH3 domain
peptide is selected from the group consisting of SEQ ID NO:
1-16.
21. The method of any one of claims 1-20, wherein an agent from the
plurality of agents comprises one or more compounds.
22. The method of any one of claims 1-21, wherein an agent from the
plurality of agents is an anticancer agent.
23. The method of any one of claims 1-21, wherein an agent from the
plurality of agents is a chemotherapeutic agent.
24. The method of any one of claims 1-23, wherein one or more of
the plurality of agents are selected from small organic molecules,
small inorganic molecules, peptides, proteins, protein analogs,
enzymes, nucleic acids, nucleic acid analogs, antibodies, antigens,
hormones, lipids, polysaccharides, growth factors, viruses, cells,
bioactive agents, pharmaceutical agents, and combinations and
prodrugs thereof.
25. The method of any one of claims 1-24, wherein one or more of
the plurality of agents are selected from gases, fine particles,
radiation, electromagnetic radiation, and aerosols.
26. A method of predicting toxicity of an agent to a tissue, said
method comprising: a) providing an agent; b) providing a plurality
of cell samples comprising non-cancerous cells, wherein each cell
sample is obtained from a tissue; c) providing a test aliquot of
each of the plurality of cell samples; d) separately contacting
each test aliquot with the agent; e) providing a control aliquot of
each of the plurality of cell samples, wherein the control aliquot
is not contacted with the agent; f) permeabilizing the cells in
each test aliquot and control aliquot; g) contacting the
permeabilized cells with a pro-apoptotic BH3 domain peptide; h)
determining a value for percent BH3 domain peptide-induced MOMP in
the cells in each test aliquot; i) determining a value for percent
BH3 domain peptide-induced MOMP in the cells in each control
aliquot; and j) determining a value for delta percent priming for
each test aliquot, wherein delta percent priming is the difference
between the value for percent MOMP determined in (h) and the value
for percent MOMP determined in (i), wherein a delta percent priming
of greater than 20 percent indicates that the agent is toxic to the
tissue from which the cells were obtained.
27. The method of claim 26, further comprising conducting in vivo
or in vitro toxicity testing with the agent if the delta percent
priming is 20 percent or less, 18 percent or less, 16 percent or
less, 14 percent or less, 12 percent or less, 10 percent or less, 8
percent or less, 6 percent or less, 4 percent or less, 2 percent or
less, or 1 percent or less.
28. The method of claim 26 or 27, wherein the non-cancerous cells
are healthy cells.
29. The method of any one of claims 26-28, wherein the cells are
mammalian cells.
30. The method of any one of claims 26-29, wherein the cells are
derived from samples obtained by biopsy or autopsy of an
animal.
31. The method of any one of claims 26-30, wherein the cells in
each test aliquot are contacted with the agent for 24 hours or
less.
32. The method of any one of claims 26-31, wherein the
permeabilized cells are contacted with the pro-apoptotic BH3 domain
peptide for 3 hours or less.
33. The method of any one of claims 26-32, wherein the cells are
permeabilized with digitonin or saponin.
34. The method of any one of claims 26-33, wherein the percent BH3
domain peptide-induced MOMP is determined by one or more of: i)
contacting the cells with a potentiometric dye and measuring the
emission of said potentiometric dye; ii) measuring the release of a
molecule from the mitochondrial intermembrane space; and iii)
measuring the retention of a molecule from the mitochondrial
intermembrane space.
35. The method of claim 34, wherein the potentiometric dye is
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl
ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE).
36. The method of claim 34 or 35, wherein the molecule from the
mitochondrial intermembrane space is cytochrome c, SMAC/Diablo,
Omi, adenylate kinase-2, or apoptosis-inducing factor.
37. The method of any one of claims 26-36, wherein the BH3 domain
peptide is derived from the BH3 domain of a BID, a BIM, a BAD, a
NOXA, a PUMA, a BMF, or an HRK polypeptide.
38. The method of any one of claims 26-37, wherein the BH3 domain
peptide is selected from the group consisting of SEQ ID NO:
1-16.
39. The method of any one of claims 26-38, wherein the agent
comprises one or more compounds.
40. The method of any one of claims 26-39, wherein the agent is an
anticancer agent.
41. The method of any one of claims 26-39, wherein the agent is a
chemotherapeutic agent.
42. The method of any one of claims 26-41, wherein the agent is a
small organic molecule, small inorganic molecule, peptide, protein,
protein analog, enzyme, nucleic acid, nucleic acid analog,
antibody, antigen, hormone, lipid, polysaccharide, growth factor,
virus, cell, bioactive agent, pharmaceutical agent, or some
combination or prodrug thereof.
43. The method of any one of claims 26-42, wherein the agent is
gas, fine particles, radiation, electromagnetic radiation, or
aerosol.
44. A method of predicting toxicity of an agent, said method
comprising: a) contacting an non-human animal with a sublethal dose
of an agent; b) sacrificing the animal; c) providing one or more
cell samples, wherein each cell sample is obtained from a tissue
from the animal; d) providing a test aliquot of each of the one or
more cell samples; e) separately contacting each test aliquot with
the agent; f) providing a control aliquot of each of the one or
more cell samples, wherein the control aliquot is not contacted
with the agent; g) permeabilizing the cells in each aliquot; h)
contacting the permeabilized cells with a pro-apoptotic BH3 domain
peptide; i) determining a value for percent BH3 domain
peptide-induced MOMP in the cells in the test aliquot; j)
determining a value for percent BH3 domain peptide-induced MOMP in
the cells in the control aliquot; and k) determining a value for
delta percent priming for the test aliquot, wherein delta percent
priming is the difference between the value for percent MOMP
determined in (i) and the value for percent MOMP determined in (j),
wherein a delta percent priming of greater than 20 percent
indicates that the agent is toxic to the tissue of the animal from
which the cells were obtained.
45. The method of claim 44, wherein the animal is a mammal.
46. The method of claim 45, wherein the mammal is a non-human
primate or a rodent.
47. The method of any one of claims 44-46, wherein the animal is
contacted with the agent by injection, inhalation, topical
administration, or oral administration.
48. The method of any one of claims 44-47, wherein the animal is
contacted with the agent for 24 hours or less.
49. The method of any one of claims 44-48, wherein the
permeabilized cells are contacted with the pro-apoptotic BH3 domain
peptide for 3 hours or less.
50. The method of any one of claims 44-49, wherein the cells are
permeabilized with digitonin or saponin.
51. The method of any one of claims 44-50, wherein the percent BH3
domain peptide-induced MOMP is determined by one or more of: i)
contacting the cells with a potentiometric dye and measuring the
emission of said potentiometric dye; ii) measuring the release of a
molecule from the mitochondrial intermembrane space; and iii)
measuring the retention of a molecule from the mitochondrial
intermembrane space.
52. The method of claim 51, wherein the potentiometric dye is
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl
ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE).
53. The method of claim 51 or 52, wherein the molecule from the
mitochondrial intermembrane space is cytochrome c, SMAC/Diablo,
Omi, adenylate kinase-2, or apoptosis-inducing factor.
54. The method of any one of claims 44-53, wherein the BH3 domain
peptide is derived from the BH3 domain of a BID, a BIM, a BAD, a
NOXA, a PUMA, a BMF, or an HRK polypeptide.
55. The method of any one of claims 44-54, wherein the BH3 domain
peptide is selected from the group consisting of SEQ ID NO:
1-16.
56. The method of any one of claims 44-55, wherein the agent
comprises one or more compounds.
57. The method of any one of claims 44-56, wherein the agent is an
anticancer agent.
58. The method of any one of claims 44-57, wherein the agent is a
chemotherapeutic agent.
59. The method of any one of claims 44-58, wherein the agent is a
small organic molecule, small inorganic molecule, peptide, protein,
protein analog, enzyme, nucleic acid, nucleic acid analog,
antibody, antigen, hormone, lipid, polysaccharide, growth factor,
virus, cell, bioactive agent, pharmaceutical agent, or some
combination or prodrug thereof.
60. The method of any one of claims 44-59, wherein the agent is
gas, fine particles, radiation, electromagnetic radiation, or
aerosol.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. provisional application number 62/153,475, filed
Apr. 27, 2015, the contents of which are incorporated herein by
reference in their entirety.
BACKGROUND
[0002] Dynamic BH3 Profiling is a technique used to measure the
sensitivity of cells to a test compound or therapeutic. Dynamic BH3
profiling is applied to a cancer cell sample, and drugs that move
the cancer cells in that sample closer to the threshold of
programmed cell death can be readily identified. The drugs so
identified are those likely to provide clinical anti-cancer benefit
to the subject from which the cancer cell sample was derived,
thereby providing personalized therapy for individual cancer
patients.
SUMMARY
[0003] The present disclosure relates to the discovery that the
principles of dynamic BH3 profiling can be utilized to assess the
toxicity of an agent in non-cancerous cells. In various aspects,
the disclosure provides methods for determining how an agent alters
the apoptotic sensitivity of non-cancerous cells.
[0004] Accordingly, in some aspects, the disclosure provides a
method of screening a plurality of agents for determining toxicity,
said method comprising: a) providing a plurality of agents; b)
providing a sample of cells, wherein the cells are non-cancerous;
c) providing a test aliquot of the sample of cells for each of the
plurality of agents; d) separately contacting each test aliquot
with its respective agent; e) providing a control aliquot of the
sample of cells, wherein the control aliquot is not contacted with
any of the plurality of agents; f) permeabilizing the cells in each
aliquot; g) contacting the permeabilized cells with a pro-apoptotic
BH3 domain peptide; h) determining a value for percent BH3 domain
peptide-induced mitochondrial outer membrane permeabilization
(MOMP) in the cells in each of the test aliquots; i) determining a
value for percent BH3 domain peptide-induced MOMP in the cells in
the control aliquot; and j) determining a value for delta percent
priming for each of the test aliquots, wherein delta percent
priming is the difference between the value for percent MOMP
determined in (h) and the value for percent MOMP determined in (i),
wherein a delta percent priming of greater than 20 percent
indicates that the agent with which the cells in the test aliquot
were contacted is toxic to the cells.
[0005] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming is 20 percent or less, 18 percent or less, 16
percent or less, 14 percent or less, 12 percent or less, 10 percent
or less, 8 percent or less, 6 percent or less, 4 percent or less, 2
percent or less, or 1 percent or less.
[0006] In some embodiments, the non-cancerous cells are healthy
cells.
[0007] In some embodiments, the method further comprises k)
providing a sample of cells, wherein the cells are cancerous; 1)
providing a test aliquot of the sample of cancerous cells for each
of the plurality of agents; m) separately contacting each test
aliquot with its respective agent; n) providing a control aliquot
of the sample of cancerous cells, wherein the control aliquot is
not contacted with any of the plurality of agents; o)
permeabilizing the cancerous cells in each aliquot; p)contacting
the permeabilized cancerous cells with a pro-apoptotic BH3 domain
peptide; q) determining a value for percent BH3 domain
peptide-induced MOMP in the cancerous cells in each of the test
aliquots; r) determining a value for percent BH3 domain
peptide-induced MOMP in the cancerous cells in the control aliquot;
and s) determining a value for delta percent priming for each of
the test aliquots, wherein delta percent priming is the difference
between the value for percent MOMP determined in (q) and the value
for percent MOMP determined in (r), wherein a delta percent priming
of greater than 20 percent indicates that the agent with which the
cancerous cells in the test aliquot were contacted is toxic to the
cancerous cells. In some embodiments, the method further comprises
conducting in vivo or in vitro toxicity testing with the agent if
the delta percent priming in the cancerous cells is greater than 20
percent, greater than 30 percent, greater than 40 percent, greater
than 50 percent, greater than 60 percent, greater than 70 percent,
greater than 80 percent, greater than 90 percent, greater than 95
percent, or 100 percent.
[0008] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming in the cancerous cells is greater than the delta
percent priming in the non-cancerous cells.
[0009] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming in the cancerous cells is at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 100%, at least
150%, or at least 200%, greater than the delta percent priming in
the non-cancerous cells.
[0010] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming in the cancerous cells is at least 2-fold, at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, at least 8-fold, at least 9-fold, or at least 10-fold
greater than the delta percent priming in the non-cancerous
cells.
[0011] In some embodiments, the cancerous cells are primary human
tumor cells. In some embodiments, the cancerous cells are obtained
from a patient derived xenograft (PDX).
[0012] In some embodiments, the plurality of agents includes 20 or
more agents. In some embodiments, the plurality of agents includes
100 or more agents.
[0013] In some embodiments, each test aliquot is contacted with its
respective agent for 24 hours or less. In some embodiments, the
permeabilized cells are contacted with the pro-apoptotic BH3 domain
peptide for 3 hours or less.
[0014] In some embodiments, the cells in each of the test aliquots
are permeabilized with digitonin or saponin.
[0015] In some embodiments, the percent BH3 domain peptide-induced
MOMP is determined by one or more of: i) contacting the cells with
a potentiometric dye and measuring the emission of said
potentiometric dye; ii) measuring the release of a molecule from
the mitochondrial intermembrane space; and iii) measuring the
retention of a molecule from the mitochondrial intermembrane space.
In some embodiments, the potentiometric dye is
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl
ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE). In some
embodiments, the molecule from the mitochondrial intermembrane
space is cytochrome c, SMAC/Diablo, Omi, adenylate kinase-2, or
apoptosis-inducing factor.
[0016] In some embodiments, the BH3 domain peptide is derived from
the BH3 domain of a BID, a BIM, a BAD, a NOXA, a PUMA, a BMF, or an
HRK polypeptide. In some embodiments, the BH3 domain peptide is
selected from the group consisting of SEQ ID NO: 1-16.
[0017] In some embodiments, an agent from the plurality of agents
comprises one or more compounds. In some embodiments, an agent from
the plurality of agents is an anticancer agent. In some
embodiments, an agent from the plurality of agents is a
chemotherapeutic agent. In some embodiments, one or more of the
plurality of agents are selected from small organic molecules,
small inorganic molecules, peptides, proteins, protein analogs,
enzymes, nucleic acids, nucleic acid analogs, antibodies, antigens,
hormones, lipids, polysaccharides, growth factors, viruses, cells,
bioactive agents, pharmaceutical agents, and combinations and
prodrugs thereof. In some embodiments, one or more of the plurality
of agents are selected from gases, fine particles, radiation,
electromagnetic radiation, and aerosols.
[0018] In another aspect, the disclosure provides a method of
predicting toxicity of an agent to a tissue, said method
comprising: a) providing an agent; b) providing a plurality of cell
samples comprising non-cancerous cells, wherein each cell sample is
obtained from a tissue; c) providing a test aliquot of each of the
plurality of cell samples; d) separately contacting each test
aliquot with the agent; e) providing a control aliquot of each of
the plurality of cell samples, wherein the control aliquot is not
contacted with the agent; f) permeabilizing the cells in each test
aliquot and control aliquot; g) contacting the permeabilized cells
with a pro-apoptotic BH3 domain peptide; h) determining a value for
percent BH3 domain peptide-induced MOMP in the cells in each test
aliquot; i) determining a value for percent BH3 domain
peptide-induced MOMP in the cells in each control aliquot; and j)
determining a value for delta percent priming for each test
aliquot, wherein delta percent priming is the difference between
the value for percent MOMP determined in (h) and the value for
percent MOMP determined in (i), wherein a delta percent priming of
greater than 20 percent indicates that the agent is toxic to the
tissue from which the cells were obtained.
[0019] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming is 20 percent or less, 18 percent or less, 16
percent or less, 14 percent or less, 12 percent or less, 10 percent
or less, 8 percent or less, 6 percent or less, 4 percent or less, 2
percent or less, or 1 percent or less.
[0020] In some embodiments, the non-cancerous cells are healthy
cells. In some embodiments, the cells are mammalian cells. In some
embodiments, the cells are derived from samples obtained by biopsy
or autopsy of an animal.
[0021] In some embodiments, the cells in each test aliquot are
contacted with the agent for 24 hours or less. In some embodiments,
the permeabilized cells are contacted with the pro-apoptotic BH3
domain peptide for 3 hours or less.
[0022] In some embodiments, the cells are permeabilized with
digitonin or saponin.
[0023] In some embodiments, the percent BH3 domain peptide-induced
MOMP is determined by one or more of: i) contacting the cells with
a potentiometric dye and measuring the emission of said
potentiometric dye; ii) measuring the release of a molecule from
the mitochondrial intermembrane space; and iii) measuring the
retention of a molecule from the mitochondrial intermembrane space.
In some embodiments, the potentiometric dye is
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl
ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE). In some
embodiments, the molecule from the mitochondrial intermembrane
space is cytochrome c, SMAC/Diablo, Omi, adenylate kinase-2, or
apoptosis-inducing factor.
[0024] In some embodiments, the BH3 domain peptide is derived from
the BH3 domain of a BID, a BIM, a BAD, a NOXA, a PUMA, a BMF, or an
HRK polypeptide. In some embodiments, the BH3 domain peptide is
selected from the group consisting of SEQ ID NO: 1-16.
[0025] In some embodiments, the agent comprises one or more
compounds. In some embodiments, the agent is an anticancer agent.
In some embodiments, the agent is a chemotherapeutic agent. In some
embodiments, the agent is a small organic molecule, small inorganic
molecule, peptide, protein, protein analog, enzyme, nucleic acid,
nucleic acid analog, antibody, antigen, hormone, lipid,
polysaccharide, growth factor, virus, cell, bioactive agent,
pharmaceutical agent, or some combination or prodrug thereof. In
some embodiments, the agent is gas, fine particles, radiation,
electromagnetic radiation, or aerosol.
[0026] In another aspect, the disclosure provides a method of
predicting toxicity of an agent, said method comprising: a)
contacting an non-human animal with a sublethal dose of an agent;
b)sacrificing the animal; c) providing one or more cell samples,
wherein each cell sample is obtained from a tissue from the animal;
d) providing a test aliquot of each of the one or more cell
samples; e) separately contacting each test aliquot with the agent;
f) providing a control aliquot of each of the one or more cell
samples, wherein the control aliquot is not contacted with the
agent; g) permeabilizing the cells in each aliquot; h) contacting
the permeabilized cells with a pro-apoptotic BH3 domain peptide; i)
determining a value for percent BH3 domain peptide-induced MOMP in
the cells in the test aliquot; j) determining a value for percent
BH3 domain peptide-induced MOMP in the cells in the control
aliquot; and k) determining a value for delta percent priming for
the test aliquot, wherein delta percent priming is the difference
between the value for percent MOMP determined in (i) and the value
for percent MOMP determined in (j), wherein a delta percent priming
of greater than 20 percent indicates that the agent is toxic to the
tissue of the animal from which the cells were obtained.
[0027] In some embodiments, the animal is a mammal. In some
embodiments, the mammal is a non-human primate or a rodent.
[0028] In some embodiments, the animal is contacted with the agent
by injection, inhalation, topical administration, or oral
administration. In some embodiments, the animal is contacted with
the agent for 24 hours or less.
[0029] In some embodiments, the permeabilized cells are contacted
with the pro-apoptotic BH3 domain peptide for 3 hours or less.
[0030] In some embodiments, the cells are permeabilized with
digitonin or saponin.
[0031] In some embodiments, the percent BH3 domain peptide-induced
MOMP is determined by one or more of: i) contacting the cells with
a potentiometric dye and measuring the emission of said
potentiometric dye; ii) measuring the release of a molecule from
the mitochondrial intermembrane space; and iii) measuring the
retention of a molecule from the mitochondrial intermembrane space.
In some embodiments, the potentiometric dye is
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl
ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE). In some
embodiments, the molecule from the mitochondrial intermembrane
space is cytochrome c, SMAC/Diablo, Omi, adenylate kinase-2, or
apoptosis-inducing factor.
[0032] In some embodiments, the BH3 domain peptide is derived from
the BH3 domain of a BID, a BIM, a BAD, a NOXA, a PUMA, a BMF, or an
HRK polypeptide. In some embodiments, the BH3 domain peptide is
selected from the group consisting of SEQ ID NO: 1-16.
[0033] In some embodiments, the agent comprises one or more
compounds. In some embodiments, the agent is an anticancer agent.
In some embodiments, the agent is a chemotherapeutic agent. In some
embodiments, the agent is a small organic molecule, small inorganic
molecule, peptide, protein, protein analog, enzyme, nucleic acid,
nucleic acid analog, antibody, antigen, hormone, lipid,
polysaccharide, growth factor, virus, cell, bioactive agent,
pharmaceutical agent, or some combination or prodrug thereof. In
some embodiments, the agent is gas, fine particles, radiation,
electromagnetic radiation, or aerosol.
[0034] In some embodiments of any of the provided methods, the
cells are cultured on an adhesive solid surface in a culture medium
having serum in the presence and absence of an agent. In some
embodiments of any of the provided methods, the method further
comprises washing the culture media from the cells prior to
contacting the cells with the BH3 profiling buffer and the
pro-apoptotic BH3 domain peptide.
[0035] In some embodiments, the BH3 profiling buffer is added at a
concentration of 2.times., 3.times. or 4.times.. In some
embodiments, the BH3 profiling buffer is added at a concentration
of 2.times. and the amount of BH3 domain peptide induced MOMP is
measured by microscopy. In some embodiments, the BH3 profiling
buffer is added at a concentration of 3.times. or 4.times. and the
amount of BH3 domain peptide induced MOMP is measured by
Fluorescence-activated cell sorting (FACS).
[0036] In some embodiments, the adhesive solid surface is coated
with one or more pro-adhesive compounds. In some embodiments, the
one or more pro-adhesive compounds is an extracellular matrix (ECM)
protein. In some embodiments, the ECM protein is selected from the
group consisting of collagen 1, laminin, collagen 4 and
fibronectin. In some embodiments, the one or more pro-adhesive
compounds is an antibody. In some embodiments, the one or more
pro-adhesive compounds is streptavidin.
[0037] In some embodiments, the BH3 profiling buffer is Derived
from Trehalose Experimental Buffer (DTEB) or Mitochondria
Experimental Buffer (MEB). In some embodiments, the cells are
permeabilized after, prior to, or simultaneously when contacting
with the BH3 domain peptide. In some embodiments, the BH3 profiling
buffer is supplemented with a permeabilizing agent. In some
embodiments, the permeabilizing agent is digitonin or saponin.
[0038] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0039] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1A-1E depict exemplary BH3 profiling on human foreskin
fibroblasts (HFF). To demonstrate BH3 profiling on healthy HFF
cells, loss of cytochrome c from mitochondria was measured in
response to the synthetic BH3 peptides. Cytochrome c
immunofluorescence (FIGS. 1A-1D) was assessed at varied
concentrations of the synthetic Bim BH3 peptide along a
dose-response curve (FIG. 1E).
[0041] FIGS. 2A-2B depict exemplary drug-induced changes in BH3
profiles in healthy human foreskin fibroblasts. After treating
healthy HFFs with a library of compounds or with DMSO (an inactive
chemical used as an experimental control) for 20 hours, increases
in cell death sensitivity were determined by BH3 profiling.
Specifically, a compound that increases apoptotic sensitivity
(delta percent priming) is a compound that accelerates loss of
cytochrome c. FIG. 2A depicts delta a histograms of the increases
in cell death sensitivity (delta percent priming), with FIG. 2B
depicting a zoomed view of the y-axis (percentage of compounds in
the library) in 2A.
[0042] FIG. 3 shows an exemplary comparison of drug induced changes
in BH3 profiles between mouse mammary tumor cells (y-axis) and
healthy human foreskin fibroblasts. In comparing the effects of
.about.2400 compounds on cell death sensitivity in the healthy HFF
cells or in the tumor cells, several compounds appear to sensitize
tumors, but not healthy cells for death (shaded area). Conversely,
several compounds appear to sensitize both the healthy HFF cells
and the mouse mammary tumor cells for apoptosis (circled area).
[0043] FIGS. 4A-4E depict exemplary BH3 profiling of adult mouse
hepatocytes. The loss of cytochrome c from mitochondria was
measured in response to the synthetic BH3 peptides. Cytochrome c
immunofluorescence was assessed using the synthetic Bim BH3 peptide
at 0.1 .mu.M (FIG. 4A), 1.56 .mu.M (FIG. 4B), 25 .mu.M (FIG. 4C),
and 100 .mu.M (FIG. 4D). A corresponding dose-response curve is
further depicted (FIG. 4E).
[0044] FIGS. 5A-5E depicts examples of compounds that increase cell
death sensitivity in adult mouse hepatocytes. The loss of
cytochrome c from mitochondria was measured in response to DMSO
control (FIG. 5A), Navitoclax (FIG. 5B), Doxorubicin (FIG. 5C),
and
[0045] Tanespimycin (FIG. 5D). Increase in cell death sensitivity
is indicated by loss of cytochrome c, which is quantified as an
increase in apoptotic sensitivity, or delta priming (e.g., delta
percent priming), and is depicted in FIG. 5E.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present disclosure relates to the discovery that the
principles of dynamic BH3 profiling can be utilized to assess the
toxicity of an agent in non-cancerous cells. In various aspects,
the disclosure provides methods for quickly and effectively
determining how an agent alters the apoptotic sensitivity of
non-cancerous cells. For example, many current means of assessing
drug toxicity during drug development is limited because
pre-clinical toxicity largely relies on testing in mammals, which
can be expensive, time consuming, and unreliable. Dynamic BH3
profiling methods provided herein provide inexpensive and robust
alternatives to traditional pre-clinical toxicity testing. For
example, dynamic BH3 profiling may be carried out on normal cells,
e.g., a panel of normal cells, to perform toxicity screens of drugs
on normal primary tissues. As another example, many current means
of assessing whether a particular environment (e.g., a room, a
building, a street, a city) is toxic depend on chromatographic
analyses that require prior knowledge of the potential toxin.
Dynamic BH3 profiling methods provided herein permit profiling of
normal cells to rapidly detect the presence of toxic agents (e.g.,
radiation, gas, biological agents, etc.) in an environment.
[0047] In some aspects, the disclosure provides a method of
screening a plurality of agents for determining toxicity,
including: a) providing a plurality of agents; b) providing a
sample of cells, wherein the cells are non-cancerous; c) providing
a test aliquot of the sample of cells for each of the plurality of
agents; d) separately contacting each test aliquot with its
respective agent; e) providing a control aliquot of the sample of
cells, wherein the control aliquot is not contacted with any of the
plurality of agents; f) permeabilizing the cells in each aliquot;
g) contacting the permeabilized cells with a pro-apoptotic BH3
domain peptide; h) determining a value for percent BH3 domain
peptide-induced MOMP in the cells in each of the test aliquots; i)
determining a value for percent BH3 domain peptide-induced MOMP in
the cells in the control aliquot; and j) determining a value for
delta percent priming for each of the test aliquots, wherein delta
percent priming is the difference between the value for percent
MOMP determined in (h) and the value for percent MOMP determined in
(i), wherein a delta percent priming of greater than 20 percent
indicates that the agent with which the cells in the test aliquot
were contacted is toxic to the cells. In some aspects, the
disclosure provides a method of predicting toxicity of an agent to
a tissue, said method comprising: a) providing an agent; b)
providing a plurality of cell samples comprising non-cancerous
cells, wherein each cell sample is obtained from a tissue; c)
providing a test aliquot of each of the plurality of cell samples;
d) separately contacting each test aliquot with the agent; e)
providing a control aliquot of each of the plurality of cell
samples, wherein the control aliquot is not contacted with the
agent (e.g., the control aliquot contains cells not contacted with
the agent or contains cells from an animal not contacted with the
agent); f) permeabilizing the cells in each test aliquot and
control aliquot; g) contacting the permeabilized cells with a
pro-apoptotic BH3 domain peptide; h) determining a value for
percent BH3 domain peptide-induced MOMP in the cells in each test
aliquot; i) determining a value for percent BH3 domain
peptide-induced MOMP in the cells in each control aliquot; and j)
determining a value for delta percent priming for each test
aliquot, wherein delta percent priming is the difference between
the value for percent MOMP determined in (h) and the value for
percent MOMP determined in (i), wherein a delta percent priming of
greater than 20 percent indicates that the agent is toxic to the
tissue from which the cells were obtained.
[0048] In some embodiments, the non-cancerous cells are normal
healthy cells (e.g., cells that do not have any patent or latent
pathological condition). In some embodiments, the non-cancerous
cells are infected with an infectious agent, e.g., a virus or an
intracellular bacterium. In some embodiments, the non-cancerous
cells are from an organ that is otherwise not functioning normally.
In some embodiments, the non-cancerous cells are subject to stress,
e.g., vascular stress such as hypoxia, stroke, myocardial
infarction, among others. In some embodiments, the cells are
mammalian cells. In some embodiments, the cells are derived from
samples obtained by biopsy or autopsy of an animal. In some
embodiments, the cells are non-malignant human or mouse primary
cells.
[0049] In some embodiments, provided methods further include k)
providing a sample of cells, wherein the cells are cancerous; l)
providing a test aliquot of the sample of cancerous cells for each
of the plurality of agents; m) separately contacting each test
aliquot with its respective agent; n) providing a control aliquot
of the sample of cancerous cells, wherein the control aliquot is
not contacted with any of the plurality of agents; o)
permeabilizing the cancerous cells in each aliquot; p) contacting
the permeabilized cancerous cells with a pro-apoptotic BH3 domain
peptide; q) determining a value for percent BH3 domain
peptide-induced MOMP in the cancerous cells in each of the test
aliquots; r) determining a value for percent BH3 domain
peptide-induced MOMP in the cancerous cells in the control aliquot;
and s) determining a value for delta percent priming for each of
the test aliquots, wherein delta percent priming is the difference
between the value for percent MOMP determined in (q) and the value
for percent MOMP determined in (r), wherein a delta percent priming
of greater than 20 percent indicates that the agent with which the
cancerous cells in the test aliquot were contacted is toxic to the
cancerous cells.
[0050] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming in the cancerous cells is at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 100%, at least
150%, or at least 200%, greater than the delta percent priming in
the non-cancerous cells.
[0051] In some embodiments, the method further comprises conducting
in vivo or in vitro toxicity testing with the agent if the delta
percent priming in the cancerous cells is at least 2-fold, at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, at least 8-fold, at least 9-fold, or at least 10-fold
greater than the delta percent priming in the non-cancerous
cells.
[0052] In some embodiments, the cancerous cells comprise an
immortalized cancer cell line. In some embodiments, the cells are
immortalized mouse or human cancer cell lines. Established cancer
cell lines are well-known in the art and include for example
pancreatic cancer cell lines (e.g., YAPC, Panc02.03 and SU86.86,
etc.), breast cancer cell lines (e.g., AU565, BT-20, CAL-120, HMEL
and KPL-1, etc.), kidney cancer lines (e.g., 769-P, ACNH, HEK TE,
SLR 20 and UMRC2, etc.), bone cancer cell lines (e.g., CAL-78, HOS,
MG-63 and SK-ES-1, etc.) and lymphoid cancer cell lines (e.g.,
AML-193, BDCM, CML-T1 and JM1, etc.). The skilled artisan
recognizes other cancer cell lines, for example those disclosed in
Barretina et al. (The Cancer Cell Line Encyclopedia enables
predictive modelling of anticancer drug sensitivity. Nature. 2012
Mar 28;483(7391):603-7. doi: 10.1038/nature11003).
[0053] In some embodiments, the cells are derived from a subject.
For example, cancer cells may be isolated from a subject by a
surgical technique (e.g., biopsy). Thus, in some embodiments, the
cells are primary tumor cells. In some embodiments, the cells
comprise a patient-derived xenograft (PDX). As used herein, the
term "patient-derived xenograft" (PDX) refers to tissue generated
by the implantation of cancerous primary tumor into an
immunodeficient mouse.
[0054] In some embodiments, contacting the cells includes culturing
the cells in the presence or absence of an agent. In some
embodiments, the cells are cultured in a culture medium under
suitable culture conditions in the presence or absence of an agent.
A "culture medium" (also referred to herein as a "cell culture
medium" or "medium") is a medium for culturing cells containing
nutrients that maintain cell viability and support proliferation.
The disclosure contemplates various parameters and conditions for
culturing the cell. In some embodiments, the culture medium
contains serum. The cell culture medium may contain any of the
following nutrients in appropriate amounts and combination:
salt(s), buffer(s), amino acids, glucose or other sugar(s),
antibiotics, serum or serum replacement, and other components such
as peptide growth factors, etc. Cell culture media are known in the
art and may be classified as natural or artificial media. Examples
of cell culture media include but are not limited to Minimum
Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM)
and Roswell Park Memorial Institute Medium (RPMI). Selection of an
appropriate medium for culturing the cell is within the capability
of the skilled artisan. Suitable conditions include growing the
cell under standard cell culture conditions in a cell culture
incubator (e.g., at 37.degree. C. in a humidified atmosphere of
>80% relative humidity air and 5 to 10% CO.sub.2).
[0055] In some embodiments, the cells are contacted with an agent,
e.g., cultured in the presence of an agent, for at least 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours,
16 hours, 20 hours, 1 day, at least 2 days, at least 3 days, or at
least 4 days. In some embodiments, the cells are contacted with an
agent, e.g., cultured in the presence of an agent, for 48 hours or
less, 36 hours or less, 24 hours or less, or 12 hours or less.
[0056] In some aspects, the disclosure provides a method of
predicting toxicity of an agent, said method comprising: a)
contacting a non-human animal with a sublethal dose of an agent; b)
sacrificing the animal; c) providing one or more cell samples,
wherein each cell sample is obtained from a tissue from the animal;
d) providing a test aliquot of each of the one or more cell
samples; e) separately contacting each test aliquot with the agent;
f) providing a control aliquot of each of the one or more cell
samples, wherein the control aliquot is not contacted with the
agent; g) permeabilizing the cells in each aliquot; h) contacting
the permeabilized cells with a pro-apoptotic BH3 domain peptide; i)
determining a value for percent BH3 domain peptide-induced MOMP in
the cells in the test aliquot; j) determining a value for percent
BH3 domain peptide-induced MOMP in the cells in the control
aliquot; and k) determining a value for delta percent priming for
the test aliquot, wherein delta percent priming is the difference
between the value for percent MOMP determined in (i) and the value
for percent MOMP determined in (j), wherein a delta percent priming
of greater than 20 percent indicates that the agent is toxic to the
tissue of the animal from which the cells were obtained.
[0057] As used herein, "a subject" is preferably a mammal. The
mammal is, for example, a human, non-human primate, mouse, rat,
dog, cat, horse, or cow. In some embodiments, the mammal is a
non-human primate or a rodent. In some embodiments, the subject is
a genetically-modified animal. For example, a mouse can be
genetically engineered to develop a particular cancer. In some
embodiments, the subject has not been previously diagnosed as
having cancer. Alternatively, in some embodiments, the subject has
been previously diagnosed as having cancer, and possibly has
already undergone treatment for cancer.
[0058] As described herein, aspects of the disclosure can relate to
contacting a subject (e.g., a non-human animal) with a sublethal
dose of an agent. The non-human animal can be contacted with the
agent by methods known in the art. In some embodiments, the agent
is administered to the animal, e.g., by injection, inhalation,
topical administration, or oral administration. For example, the
agent can be administered by topical administration (e.g.,
epicutaneous or inhalational administration), enteral
administration (e.g., oral, gastric, or rectal administration), or
parenteral administration (e.g., intravenous, intra-arterial,
intraosseous, intra-muscular, intracerebral,
intracerebroventricular, intrathecal, or subcutaneous
administration).
[0059] Cell sensitivity to the agent is determined by contacting
the cells or cellular component (e.g., mitochondria) with a BH3
profiling buffer and a BH3 domain peptide from the pro-apoptotic
BCL-2 family or small molecules with direct mitochondrial activity.
This includes, but is not limited to ABT-199, ABT-263, ABT-737,
WEHI-539, A-1210477, and ABT-199. The ability of BH3 peptides to
induce MOMP is measured in the cells (or cellular component, e.g.,
mitochondria) exposed to the agent and the control cells (or
cellular component, e.g., mitochondria) not exposed to the agent.
An increase in BH3 peptide-induced MOMP in the cells cultured in
the presence of the agent compared to the amount of BH3 domain
peptide induced MOMP in the cells cultured in the absence of the
agent indicates that the cells are responsive (e.g., cell death
will be induced) to the agent. No change in MOMP in the cells
cultured in the presence of the agent compared to the amount of BH3
domain peptide induced MOMP in the cells cultured in the absence of
the agent indicates that the drug has no effect on inducing cell
death. A decrease in MOMP in the cells cultured in the presence of
the agent compared to the amount of BH3 domain peptide induced MOMP
in the cells cultured in the absence of the agent indicates that
the agent has a desensitizing or protective effect on the
cells.
[0060] The difference in the level of BH3 domain peptide-induced
MOMP of cells that have been exposed to the agent as compared to
the level of BH3 domain peptide-induced MOMP of cells that have not
been exposed to the agent is statistically significant. By
statistically significant, it is meant that the alteration is
greater than what might be expected to happen by chance alone.
Significant differences may be identified by using an appropriate
statistical test. Tests for statistical significance are well known
in the art and are exemplified in Applied Statistics for Engineers
and Scientists by Petruccelli, Chen and Nandram 1999 Reprint
Ed.
[0061] As used herein, the term "BH3 Profiling Buffer" refers to an
aqueous solution comprising a sugar, a pH buffer, salts, chelators
and a carbon source for the electron transport chain that is useful
for performing measurements of MOMP.
[0062] Pro-apoptotic BCL-2 BH3 proteins and peptides include: Bcl-2
interacting mediator of cell death (BIM); a mutant thereof (BIM
AV); BH3 interacting domain death agonist (BID); Bcl-2-associated
death promoter (BAD); NOXA; p53 up-regulated modulator of apoptosis
(PUMA); Bcl-2-modifying factor (BMF) and harakiri (HRK) (See, Table
1).
[0063] In some embodiments, the method comprises permeabilizing the
cell after, prior to, or simultaneously when contacting with the
BH3 domain peptide. Generally, permeabilization refers to the
process of treating a cell with a reagent such that the cell
membrane becomes permeable without permeabilizing the mitochondrial
outer membrane. Reagents used for permeabilization include organic
solvents (e.g., acetone and methanol) and detergents (e.g.,
digitonin, saponin, Triton X-100 and Tween-20). Without wishing to
be bound by any particular theory, the cell is permeabilized to
permit the BH3 peptides access to the mitochondria. Cells are
permeabilized by methods known in the art. For example, the cell
are permeabilized by contacting the cell with digitonin, saponin,
methanol, Triton X-100 or other art-recognized
cell-permeabilization agents. In some embodiments, the BH3
profiling buffer comprises the permeabilizing reagent, such as
digitonin or saponin.
[0064] In some embodiments, permeabilized cells are contacted with
the pro-apoptotic BH3 domain peptide for 3 hours or less, 2 hours
or less, or 1 hour or less.
[0065] The skilled artisan recognizes several methods for
contacting the cells with the agent, BH3 profiling buffer and/or
BH3 domain peptide. For example, automated liquid handling systems
are generally utilized for high throughput drug screening.
Automated liquid handling systems utilize arrays of liquid
dispensing vessels, controlled by a robotic arm, to distribute
fixed volumes of liquid to the wells of an assay plate. Generally,
the arrays comprise 96, 384 or 1536 liquid dispensing tips.
Non-limiting examples of automated liquid handling systems include
digital dispensers (e.g., HP D300 Digital Dispenser) and pinning
machines (e.g., MULTI-BLOT.TM. Replicator System, CyBio, Perkin
Elmer Janus). Non-automated methods are also contemplated by the
disclosure, and include but are not limited to a manual digital
repeat multichannel pipette.
[0066] After the cells are treated with the BH3 domain peptide, the
MOMP is measured. Outer membrane permeabilization can be measured
in several ways. For example, outer membrane permeabilization can
be measured by determining the loss of mitochondrial membrane
potential. Loss of mitochondrial membrane potential is measured by,
for example, treating the cells with a potentiometric or
radiometric dye. Examples of potentiometric dyes include, but are
not limited to, the fluorescent JC-1 probe
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyani-
ne iodide) or dihydrorhodamine 123, or tetramethylrhodamine methyl
ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE). These and
other potentiometric dyes are well-known in the art.
[0067] Alternatively, outer membrane permeabilization is determined
by measuring the release of a molecule from the mitochondrial
intermembrane space or by measuring the retention of a molecule
from the mitochondrial intermembrane space. Examples of a molecule
from the mitochondrial intermembrane space include cytochrome c,
SMAC/Diablo, Omi, adenylate kinase-2, or apoptotic-inducing factor
(AIF). The release or retention of a molecule from the
mitochondrial intermembrane space can be measured by methods
well-known in the art. For example, the release or retention of a
molecule can be measured by using an antibody to the molecule,
i.e., an antibody to cytochrome c, SMAC/Diablo, Omi, adenylate
kinase-2 or apoptotic-inducing factor (AIF). Detection can be for
example, by ELISA, FACS, immunoblot, immunofluorescence,
immunohistochemistry, plate fluorimetry, fluorescent imaging or
automated image analysis. Analysis of the cells can be manually
accomplished using a microscope or automated for example by using
software such as Cellprofiler to locate nuclei.
[0068] Optionally, the cells are fixed prior to measuring outer
membrane permeabilization. Cells are fixed by methods known in the
art, such as by using an aldehyde (e.g., formaldehyde), or
methanol.
[0069] Mitochondrial outer membrane permeabilization can be
measured at the single cell level or multi-cell level.
Additionally, some of the methods disclosed herein allow for
subpopulations of cells to be assayed. For example, a
tumor-specific or tumor-associated marker (e.g., EpCam) can be used
to identify tumor cells. This allows for the ability to
discriminate between normal and tumor cells. MOMP is then measured
as described herein in the tumor cells.
[0070] Advantageously, measurements of MOMP (e.g., measurements of
mitochondrial membrane potential, measurements of the release of a
molecule from the mitochondrial intermembrane space, measurements
of the retention of a molecule from the mitochondrial intermembrane
space) can be normalized to facilitate comparative assessments. For
example, some aspects of the methods provided herein relate to
screening a plurality of agents for determining toxicity by
determining BH3 domain peptide-induced MOMP in a cell for each of
the plurality of agents. As described herein, BH3 domain
peptide-induced MOMP can be measured by methods known in the art.
Normalization of measured BH3 peptide-induced MOMP can be performed
by methods known in the art, such as described in Ryan, J., Letai,
A. (2013) Methods 61(2): 156-164 and Friedman, A., Letai, A.,
Fisher, D., Flaherty, K. (2015) Nature Reviews Cancer 15: 747-756,
the contents of each of which are incorporated by reference their
entireties.
[0071] The following is an exemplary, non-limiting, method of
normalizing BH3 domain peptide-induced MOMP measurements: A test
aliquot of a sample of cells is contacted with an agent. The test
aliquot is contacted with a pro-apoptotic BH3 domain peptide. The
BH3 domain peptide-induced MOMP in these "treated" cells is
measured as described herein and by methods known in the art (e.g.,
by measuring mitochondrial membrane polarization, by measuring the
release of a molecule from the mitochondrial intermembrane space,
or by measuring the retention of a molecule from the mitochondrial
intermembrane space). A separate aliquot of the sample of cells, a
control aliquot, is not contacted with the agent. The control
aliquot is contacted with a pro-apoptotic BH3 domain peptide. The
BH3 domain peptide-induced MOMP in these "untreated" cells is
measured as described herein and by methods known in the art. In
some embodiments, the measured MOMP in "treated" and "untreated"
cells can be compared by measuring a maximum range for MOMP
measurements in a given sample of cells. The maximum range for MOMP
measurements can be determined by measuring an "upper limit" value
for MOMP and a "lower limit" value for MOMP. For example, an "upper
limit" value for MOMP measurements can be obtained by contacting a
separate aliquot of the sample of cells with a molecule that is
known to be toxic to a cell and measuring a value for MOMP. In some
embodiments, the molecule that is known to be toxic to a cell is an
uncoupling agent such as carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) or carbonyl
cyanide m-chlorophenyl hydrazine (CCCP). A "lower limit" value for
MOMP measurements can be obtained by measuring a value for MOMP in
a separate aliquot of cells that have not been contacted with an
agent, a pro-apoptotic BH3 domain peptide, or a molecule that is
known to be toxic to a cell. The value for percent BH3 domain
peptide-induced MOMP can be calculated for the test aliquot and the
control aliquot using the equation:
% M O M P = [ 1 - ( ( " Sample " - " UpperLimit " ) ( " LowerLimit
" - " UpperLimit " ) ) ] .times. 100 , ##EQU00001##
where "% MOMP" represents the value for percent BH3 domain
peptide-induced MOMP; "Sample" represents either the value for BH3
domain peptide-induced MOMP measured in the test aliquot or the
value for BH3 domain peptide-induced MOMP measured in the control
aliquot; "UpperLimit" represents the measured "upper limit" value
for MOMP; and "LowerLimit" represents the measured "lower limit"
value for MOMP.
[0072] In some aspects, methods provided herein relate to
determining the toxicity of an agent in a sample of cells. In some
embodiments, the toxicity of the agent can be determined by
determining a value for delta percent priming. As used herein,
"delta percent priming" refers to the extent to which an agent
shifts a cell closer to the threshold of apoptosis. In some
embodiments, normalized values of MOMP (e.g., percent BH3 domain
peptide-induced MOMP, or "% MOMP") are used to determine a value
for delta percent priming. In such embodiments, the value for delta
percent priming can be calculated using the equation:
Delta Percent Priming =(% MOMP.sub.TEST)-(% MOMP.sub.CONTROL),
where "% MOMP .sub.TEST" represents the value for percent BH3
domain peptide-induced MOMP determined in a test aliquot (cells
contacted with the agent); and "% MOMP.sub.CONTROL" represents the
value for percent BH3 domain peptide-induced MOMP determined in a
control aliquot (cells not contacted with the agent).
[0073] Determining a value for delta percent priming can provide an
assessment of the toxicity attributable to a particular agent in a
given sample of cells. As described herein, the greater the value
for delta percent priming of a cell contacted with an agent, the
greater the toxicity of the agent to the cell. Thus, in some
embodiments, the toxicity of a first agent in a sample of cells can
be compared to the toxicity of a second agent in the sample of
cells by comparing the value for delta percent priming determined
for each agent. Such methods can be useful to screen for agents
that are more toxic to a given sample of cells or less toxic to a
given sample of cells, depending upon the outcome desired by a
practitioner.
[0074] In yet other aspects, methods provided herein relate to
determining the toxicity of an agent in each of a plurality of cell
samples. Thus, in some embodiments, the toxicity of the agent in a
first cell sample can be compared to the toxicity of the agent in a
second cell sample by comparing the value for delta percent priming
determined for the agent in each of the cell samples. Such methods
can be useful to assess the toxicity of a given agent across
different cell types (e.g., cells obtained from different tissue
types).
[0075] In some aspects, the disclosure provides methods for
determining the toxicity of an agent to a sample of cells, wherein
the cells are non-cancerous. In some embodiments, a delta percent
priming of greater than 20% indicates that the agent is toxic to
the non-cancerous cells. In some embodiments, a delta percent
priming of greater than 0% indicates that the agent is toxic to the
non-cancerous cells. In some embodiments, methods provided herein
further comprise conducting in vivo or in vitro toxicity testing
with the agent if the delta percent priming in the non-cancerous
cells is 20% or less. For example, in vivo or in vitro toxicity
testing is conducted with an agent if the delta percent priming in
the non-cancerous cells is 20% or less, 18% or less, 16% or less,
14% or less, 12% or less, 10% or less, 8% or less, 6% or less, 5%
or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or
less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4%
or less, 0.3% or less, 0.2% or less, 0.1% or less, or 0%.
[0076] As described herein, aspects of the disclosure provide
methods relating to determining toxicity of an agent in a sample of
non-cancerous cells. In some embodiments, methods provided herein
can further comprise steps relating to determining toxicity of the
agent in a sample of cancerous cells. In some embodiments, the
toxicity of the agent in the cancerous cells can be assessed using
methods provided herein, for example, by determining a value for
delta percent priming. In some embodiments, a delta percent priming
of greater than 20% indicates that the agent is toxic to the
cancerous cells. In some embodiments, a delta percent priming of
greater than 0% indicates that the agent is toxic to the cancerous
cells. In some embodiments, methods provided herein further
comprise conducting in vivo or in vitro toxicity testing with the
agent if the delta percent priming in the cancerous cells is
greater than 20%. For example, in vivo or in vitro toxicity testing
is conducted with an agent if the delta percent priming in the
cancerous cells is greater than 20%, greater than 25%, greater than
30%, greater than 35%, greater than 40%, greater than 45%, greater
than 50%, greater than 55%, greater than 60%, greater than 65%,
greater than 70%, greater than 75%, greater than 80%, greater than
85%, greater than 90%, greater than 95%, greater than 98%, greater
than 99%, or 100%. In some embodiments, methods provided herein
further comprise conducting in vivo or in vitro toxicity testing
with the agent if the delta percent priming in the non-cancerous
cells is lower than the delta percent priming in the cancerous
cells.
[0077] It should be appreciated that in vivo or in vitro toxicity
testing can comprise methods provided herein and methods known in
the art related to determining the toxicity of an agent. For
example, certain aspects of the disclosure relate to the screening
of a plurality of agents for determining toxicity in a sample of
non-cancerous cells. In some embodiments, if an agent is not
determined to be toxic to the non-cancerous cells, the agent can be
selected for further toxicity testing (e.g., in vivo or in vitro
toxicity testing). In such embodiments, it can be useful to conduct
further testing comprising determining the toxicity of the agent in
a different sample of cells (e.g., cancerous cells, cells obtained
from a different type of tissue, cells obtained from a different
subject). In some embodiments, it can be useful to conduct further
testing comprising determining the toxicity of the agent in a
subject (e.g., by administering the agent to the subject, by
contacting the agent with a sample of cells obtained from the
subject).
[0078] A person of skill in the art would appreciate that aspects
of the present disclosure can be useful in the optimization of an
agent for therapeutic purposes. For example, in some embodiments,
methods of determining the toxicity of an agent to both cancerous
cells and non-cancerous cells are provided herein. It should be
appreciated that, in some embodiments, it is advantageous for an
agent (e.g., a therapeutic agent, a cancer therapeutic) to have
minimal toxicity in non-cancerous (e.g., healthy) cells and maximal
toxicity in cancerous cells. As described herein, such an agent
would manifest as having a minimal value for delta percent priming
in non-cancerous cells and a maximal value for delta percent
priming in cancerous cells.
[0079] In some aspects, the disclosure relates to drug discovery.
In some embodiments, methods described by the disclosure are useful
for screening large libraries of candidate agents to identify new
drugs that do not move cells closer to programmed cell death, e.g.,
agents that are not toxic to the cells. In some embodiments, the
library of candidate agents includes 20 or more, 30 or more, 40 or
more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more,
100 or more, 150 or more, 200 or more, 250 or more, or 300 or more
agents.
[0080] In some embodiments, an agent includes a compound. In some
embodiments, an agent includes more than one compound. Exemplary
candidate agents include, but are not limited to small organic
molecules, small inorganic molecules, peptides, proteins, protein
analogs, enzymes, nucleic acids, nucleic acid analogs, antibodies,
antigens, hormones, lipids, polysaccharides, growth factors,
viruses, cells, bioactive agents, pharmaceutical agents, and
combinations and prodrugs thereof. In some embodiments, an agent is
an anticancer agent, such as, a chemotherapeutic agent. Further
exemplary candidate agents include, but are not limited to, gases,
fine particles, radiation, electromagnetic radiation, and
aerosols.
[0081] In some embodiments, a candidate agent is a chemotherapeutic
agent. Non-limiting examples of chemotherapeutic agents include
small molecules, peptides or proteins (e.g., peptide antibiotics
and antibodies) and RNA-interference (RNAi) molecules. Examples of
small molecule chemotherapeutic agents include alkylating agents
(cyclophosphamide, chlormethine, temozolomide), anthracyclines
(daunorubicin, doxorubicin, mitoxantrone), taxanes (paclitaxel,
docetaxel), histone deacetylase inhibitors (vorinostat,
romidepsin), topoisomerase I/II inhibitors (irinotecan, topotecan,
etoposide), kinase inhibitors (gefitinib, imatinib, bortezomib),
nucleotide analogs and precursor analogs (azacitidine,
fluorouracil, methotrexate), platinum-based agents (cisplatin,
carboplatin), retinoids (alitretinoin, bexarotene) and vinca
alkaloids (vinblastine, vindesine, vinorelbine). Examples of
peptides and proteins include bleomycin, dactinomycin, antitumor
antibodies (anti-HER2/neu, alemtuzumab, trastuzumab, brentuximab).
The skilled artisan recognizes chemotherapeutic RNAi molecules as
RNAi molecules that target expression of genes related to cancer.
For example, RNAi molecules directed against HoxA1 can inhibit
mammary tumor cell formation, as disclosed by Brock et al. Sci
Transl Med 6: 217ra2 (2014). In some embodiments, chemotherapeutic
agents include, but are not limited to, kinase inhibitors,
apoptosis inducers, angiogenesis inhibitors, and monoclonal
antibodies.
[0082] In some embodiments, methods provided herein can be useful
for determining the toxicity of an environment or environmental
condition. For example, in some embodiments, the environment
comprises an agent that may or may not be toxic to a cell. In some
embodiments, agents in an environment or environmental condition
can include, without limitation, gases, fine particles, radiation,
electromagnetic radiation, and aerosols. Environmental agents are
known in the art, for example, as provided by the National
Institute of Environmental Health Sciences
(www.niehs.nih.gov/health/topics/agents/). In such embodiments, it
should be appreciated that cells can be contacted with the agent
using various methods known in the art and described herein. For
example, a cell can be exposed to an environment that is suspected
of being radioactive or known to be radioactive. In such an
embodiment, the agent being tested for toxicity could comprise
radiation (e.g., alpha particles, beta particles, positron
particles, gamma rays, X-rays, and neutrons).
[0083] In some aspects, the disclosure relates to personalized
medicine. In some embodiments, methods described herein are useful
for the customization of chemotherapeutic regimens. For example,
methods provided herein can relate to determining the toxicity of
an agent in both non-cancerous cells and cancerous cells. In some
embodiments, the non-cancerous cells and the cancerous cells are
obtained from a subject. By determining the toxicity of the agent
in non-cancerous cells (e.g., normal cells, healthy cells) from the
subject and determining the toxicity of the agent in cancerous
cells from the subject, the agent can be identified as being more
or less appropriate for treating the subject. As described herein,
the agent becomes more appropriate for treating the subject as the
ratio of delta percent priming in cancerous cells to delta percent
priming in non-cancerous cells increases. For example, in
determining an appropriate agent for treating a subject who is
known to have or is suspected of having cancer, it is preferable to
use an agent that has minimal toxicity (e.g., minimal value for
delta percent priming) in non-cancerous cells of the patient and
maximal toxicity (e.g., maximal value for delta percent priming) in
cancerous cells of the patient. Such an agent would selectively
target the cells intended for treatment while minimizing negative
peripheral effects.
[0084] In some aspects, the disclosure provides methods of
determining the toxicity of an agent, wherein the toxicity of the
agent is determined by comparing BH3 domain peptide-induced MOMP in
test cells contacted with the agent to BH3 domain peptide-induced
MOMP to control cells not contacted with the agent. In some
embodiments, the control cells have not been contacted with any
agent. In some embodiments, the control cells have been contacted
with buffer or solvent, e.g., buffer or solvent used as a vehicle
for the agent. For example, the test cells can be contacted with an
agent that has been dissolved in a suitable buffer or solvent
(e.g., DMSO), and the control can be contacted with a suitable
buffer or solvent (e.g., DMSO) lacking the agent.
[0085] In some embodiments, methods described herein can be
suitable for a high-throughput format. For example, aspects of the
disclosure relate to methods of screening of a plurality of agents
to determine toxicity. In such methods, it can be desirable to
utilize a multi-well plate. In some embodiments, the plate
comprises an agent. In some embodiments, the plate comprises a
plurality of agents. For example, the plate may be used to test a
panel of agents, each well comprising an agent. In some
embodiments, one or more wells of the plate do not comprise an
agent. For example, as described herein, control aliquots are not
contacted with an agent. In some embodiments, one or more wells
designated for control aliquots can comprise buffer or solvent
(e.g., DMSO). In some embodiments, the multi-well plate comprises a
BH3 domain peptide. In some embodiments, the BH3 domain peptide is
derived from the BH3 domain of a BID, a BIM, a BAD, a NOXA, a PUMA
a BMF, or a HRK polypeptide. In some embodiments, the BH3 domain
peptide is selected from the group consisting of SEQ ID NO: 1-15.
In some embodiments, the plate comprises more than one BH3 domain
peptide. For example, in some embodiments, a plate comprises at
least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9 or at least 10 BH3 domain peptides. In
some embodiments, one or more wells of the plate do not comprise a
BH3 domain peptide. For example, as described herein, it may be
desirable to establish a lower limit for MOMP measurements. In such
embodiments, aliquots are not contacted with a BH3 domain peptide.
In some embodiments, one or more wells designated for lower limit
measurements can comprise buffer or solvent (e.g., DMSO).
[0086] It will be appreciated that cells do not need to be removed
from a culture plate to perform dynamic BH3 profiling described
herein. The presence of cell culture media and serum has been
demonstrated not to interfere with the ability of BH3 peptides to
induce mitochondrial outer membrane permeabilization. Furthermore,
wash steps where there is little residual volume above the cells
have been demonstrated to be unnecessary. Instead, the cells can be
successively treated in the plate well with the drug and the BH3
domain peptide.
[0087] In some embodiments of any of the provided methods, the
cells are cultured on an adhesive solid surface in a culture medium
having serum in the presence and absence of an agent. In some
embodiments of any of the provided methods, the method further
comprises washing the culture media from the cells prior to
contacting the cells with the BH3 profiling buffer and the
pro-apoptotic BH3 domain peptide.
[0088] In some embodiments of any of the provided methods, the
adhesive solid surface is coated with one or more pro-adhesive
compounds. In some embodiments of any of the provided methods, the
one or more pro-adhesive compounds is an extracellular matrix (ECM)
protein. In some embodiments of any of the provided methods, the
ECM protein is selected from the group consisting of collagen 1,
laminin, collagen 4 and fibronectin. In some embodiments, the one
or more pro-adhesive compounds is an antibody. In some embodiments,
the one or more pro-adhesive compounds is streptavidin.
[0089] In some embodiments of any of the provided methods, the BH3
profiling buffer is Derived from Trehalose Experimental Buffer
(DTEB) or Mitochondria Experimental Buffer (MEB). In some
embodiments of any of the provided methods, the cells are
permeabilized after, prior to, or simultaneously when contacting
with the BH3 domain peptide. In some embodiments, the BH3 profiling
buffer is supplemented with a permeabilizing agent. In some
embodiments, the permeabilizing agent is digitonin or saponin
[0090] As used here, the term "BH3 Profiling Buffer" refers to an
aqueous solution comprising a sugar, a pH buffer, salts, chelators
and a carbon source for the electron transport chain that is useful
for performing measurements of MOMP. In some embodiments of any of
the provided methods, the BH3 Profiling Buffer is a Derived from
Trehalose Experimental Buffer (DTEB). In some embodiments, the BH3
Profiling Buffer is a Mitochondria Experimental Buffer (MEB). DTEB
is comprised of 135 mM trehalose, 10 mM Hepes, 50 mM KCl,20 .mu.M
EGTA, 20 .mu.M EDTA, 0.1% BSA and 5 mM Succinate. MEB is comprised
of 150 mM mannitol, 10 mM Hepes, 50 mM KCl, 20 .mu.M EGTA, 20 .mu.M
EDTA, 0.1% BSA and 5 mM Succinate. Sucrose and other sugars may be
used in the place of mannitol. Some increases in KCl are tolerated,
however can be detrimental to BH3 profiling. Concentrated buffers
(2.times.-5.times.) involve proportionally increasing the
concentration of the above reagents
[0091] In some embodiments of any of the provided methods, the BH3
profiling buffer is added at a concentration of 2.times.,
3.times.or 4.times.. In some embodiments of any of the provided
methods, the BH3 profiling buffer is added at a concentration of
2.times. and the amount of BH3 domain peptide induced MOMP is
measured by microscopy. In some embodiments of any of the provided
methods, the BH3 profiling buffer is added at a concentration of
3.times. or 4.times. and the amount of BH3 domain peptide induced
MOMP is measured by Fluorescence-activated cell sorting (FACS).
[0092] The skilled artisan recognizes several methods for adding
the agent, BH3 profiling buffer and/or BH3 domain peptide to the
cultured cells. For example, automated liquid handling systems are
generally utilized for high throughput drug screening. Automated
liquid handling systems utilize arrays of liquid dispensing
vessels, controlled by a robotic arm, to distribute fixed volumes
of liquid to the wells of an assay plate. Generally, the arrays
comprise 96, 384 or 1536 liquid dispensing tips. Non-limiting
examples of automated liquid handling systems include digital
dispensers (e.g., HP D300 Digital Dispenser) and pinning machines
(e.g., MULTI-BLOT.TM. Replicator System, CyBio, Perkin Elmer
Janus). Non-automated methods are also contemplated by the
disclosure, and include but are not limited to a manual digital
repeat multichannel pipette.
Pro-Apoptotic BCL-2 BH3 Domain Peptides
[0093] Pro-Apoptotic BCL-2 BH3 domain peptides have been described
previously in WO 2014/047,342, the contents of which are
incorporated by reference herein. In particular, a Pro-apoptotic
BCL-2 BH3 domain peptide is less than 195 amino acids in length,
e.g., less than or equal to 150, 100, 75, 50, 35, 25 or 15 amino
acid in length. Non-limiting examples of pro-apoptotic BCL-2 BH3
peptides include: Bcl-2 interacting mediator of cell death (BIM); a
mutant thereof (BIM AV); BH3 interacting domain death agonist
(BID); Bcl-2-associated death promoter (BAD); NOXA; p53
up-regulated modulator of apoptosis (PUMA); Bcl-2-modifying factor
(BMF) and harakiri (HRK).
[0094] In some embodiments, a pro-apoptotic BCL-2 BH3 domain
peptide includes the sequence of SEQ ID NO: 1-15 shown in Table 1.
PUMA2A (SEQ ID NO: 16) is a negative control peptide.
TABLE-US-00001 TABLE 1 Pro-Apoptotic Peptide Sequence SEQ ID NO:
BIM Ac-MRPEIWIAQELRRIGDEFNA-NH2 1 BIM AC
AC-MRPEIWIAQELRRIGDEFNV-NH2 2 BID EDIIRNIARHLAQ VGD SMDR 3 BIM AV
MRPEIWIAQELRRIGDEFNA 4 BID mut EDIIRNIARHAAQVGASMDR 5 BAD
LWAAQRYGRELRRMSDEFEGSFKGL 6 BIK MEGSDALALRLACIGDEMDV 7 NOXA A
AELPPEFAAQLRKIGDKVYC 8 NOXA B PADLKDECAQLRRIGDKVNL 9 HRK S S
AAQLTAARLKALGDELHQ 10 PUMA EQWAREIGAQLRRMADDLNA 11 BMF
HQAEVQIARKLQLIADQFHR 12 huBAD NLWAAQRYGRELRRMSDEFVDSFKK 13 BAD mut
LWAAQRYGREARRMSDEFEGSFKGL 14 MS1 RPEIWMTQGLRRLGDEINAYYAR 15 PUMA2A
EQWAREIGAQARRMAADLNA 16
[0095] In some embodiments, a BH3 domain peptide include a peptide
which includes (in whole or in part) the sequence
NH.sub.2-XXXXXXXXXXLXXXXDXXXX-COOH (SEQ ID NO: 17). As used herein
X may be any amino acid. Alternatively, the BH3 domain peptides
include at least 5, 6, 7, 8, 9, 15 or more amino acids of SEQ ID
NO: 17.
[0096] The BH3 domain peptides can be modified using standard
modifications. Modifications may occur at the amino (N-), carboxy
(C-) terminus, internally or a combination of any of the preceding.
In one aspect described herein, there may be more than one type of
modification on the polypeptide. Modifications include but are not
limited to: acetylation, amidation, biotinylation, cinnamoylation,
farnesylation, formylation, myristoylation, palmitoylation,
phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation,
sulfurylation and cyclisation (via disulfide bridges or amide
cyclisation), and modification by Cys3 or Cys5. The modified BH3
domain peptides retain the biological activity of BH3 domain
peptides. By retaining the biological activity, it is meant that
cell death is induced by the BH3 polypeptide, although not
necessarily at the same level of potency as that of the
naturally-occurring BH3 domain polypeptide. The terms induced and
stimulated are used interchangeably throughout the
specification.
[0097] Optionally, the BH3 domain peptide is attached to a
transduction domain. A transduction domain directs a peptide in
which it is present to a desired cellular destination. Thus, the
transduction domain can direct the peptide across the plasma
membrane, e.g., from outside the cell, through the plasma membrane,
and into the cytoplasm. Alternatively, or in addition, the
transduction domain can direct the peptide to a desired location
within the cell, e.g., the nucleus, the ribosome, the ER,
mitochondria, a lysosome, or peroxisome. In some embodiments, the
transduction domain is derived from a known membrane-translocating
sequence. Alternatively, transduction domain is a compound that is
known to facilitate membrane uptake such as polyethylene glycol,
cholesterol moieties, octanoic acid and decanoic acid. The
transduction domain may be linked either to the N-terminal or the
C-terminal end of BH3 domain peptide.
[0098] The BH3 domain peptides and/or the transduction domain
peptides can be polymers of L-amino acids, D-amino acids, or a
combination of both. Alternatively, the BH3 domain peptides and/or
the transduction domain peptides are cyclic peptides. Cyclic
peptides are prepared by methods known in the art. For example,
macrocyclization is often accomplished by forming an amide bond
between the peptide N- and C-termini, between a side chain and the
N- or C-terminus [e.g., with K3Fe(CN)6 at pH 8.5] (Samson et al.,
Endocrinology, 137: 5182-5185 (1996)), or between two amino acid
side chains. See, e.g., DeGrado, Adv Protein Chem, 39: 51-124
(1988).
[0099] BH3 domain peptides and/or the transduction domain peptides
are prepared using modern cloning techniques, or may be synthesized
by solid state methods or by site-directed mutagenesis. In some
embodiments, native BH3 domain peptides and/or transduction domain
peptides can be isolated from cells or tissue sources by an
appropriate purification scheme using standard protein purification
techniques. In another embodiment, BH3 domain polypeptides and/or
transduction domain peptides are produced by recombinant DNA
techniques. Alternative to recombinant expression, BH3 domain
peptides and/or transduction domain peptides can be synthesized
chemically using standard peptide synthesis techniques.
[0100] In various embodiments, the BH3 peptide maintains its
secondary structure, e.g., .alpha.-helical structure. Methods of
helix stabilization are known in the art.
[0101] An "isolated" or "purified" BH3 domain peptide is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the BH3 domain
peptide is derived, or substantially free from chemical precursors
or other chemicals when chemically synthesized.
Kits
[0102] Some aspects of the invention include kits for performing
BH3 Profiling. In some embodiments, the kit comprises a multi-well
plate having an agent and a BH3 domain peptide. In some
embodiments, the kit comprises a multi-well plate having an agent.
In some embodiments, the agent is a chemotherapeutic agent. In some
embodiments, the kit comprises a multi-well plate having at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9 or at least 10 agents. In some embodiments, the
kit comprises a multi-well plate, wherein each well of the plate
comprises a different agent. In some embodiments, the kit comprises
a multi-well plate having a BH3 domain peptide. In some
embodiments, the BH3 domain peptide is derived from the BH3 domain
of a BID, a BIM, a BAD, a NOXA, a PUMA a BMF, or a HRK polypeptide.
In some embodiments, the BH3 domain peptide is selected from the
group consisting of SEQ ID NO: 1-15.
[0103] In some aspects, the kit further comprises a vial containing
a BH3 profiling buffer. In some embodiments, the vial is a glass or
plastic vial. In some embodiments, the vial comprises volumetric
markings on its surface. In some embodiments, the BH3 profiling
buffer is supplemented with a permeabilizing agent. In some
embodiments, the permeabilizing agent is digitonin or saponin.
[0104] In some embodiments, the kit further comprises a
potentiometric dye. In some embodiments, the potentiometric dye is
5,5',6,6'-
tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide
(JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl ester
(TMRM) or tetramethylrhodamine ethyl ester (TMRE). Alternatively,
in some embodiments, the kit further comprises an antibody for
cytochrome c, SMAC/Diablo, Omi, adenylate kinase-2 or
apoptosis-inducing factor.
[0105] In some aspects, the kit further comprises instructions for
using the kit to predict the sensitivity of cells to an agent.
Instructions are generally provided as a printed pamphlet or paper
sheet but may also include any oral or electronic instructions
provided in any manner such that a user will clearly recognize that
the instructions are to be associated with the kit, for example,
audiovisual (e.g., videotape, DVD, etc.), Internet, and/or
web-based communications, etc.
[0106] The invention will be further illustrated in the following
non-limiting examples.
EXAMPLES
Example 1
[0107] Predicting Toxicity in Normal Cells with Dynamic BH3
Profiling
[0108] There is a great need to predict toxicity of agents to
normal tissues. This is most apparent in the case of drug
development. Pre-clinical toxicity largely relies on testing in
mammals which is expensive and can be unreliable. Testing using
dynamic BH3 profiling on normal tissues derived from animals,
including human tissues, could alleviate concerns regarding expense
and reliability. The concept includes measurement of the
provocation of death signaling at short time points after exposure
of normal cells to the agent of interest. Using an array of normal
cells, measurement of death signaling may be interpreted as being a
flag for potential toxicity.
[0109] To demonstrate BH3 profiling on healthy human foreskin
fibroblasts (HIT) cells, loss of cytochrome c from mitochondria was
measured in response to the synthetic BH3 peptides. To do this,
cytochrome c immunofluorescence (FIGS. 1A-D) was assessed at
increasing concentrations of the synthetic Bim BH3 peptide. An
exemplary dose-response curve of cytochrome c loss for increasing
concentrations of the synthetic Bim BH3 peptide is depicted in FIG.
1E (standard deviations depicted as error bars). Note that at low
concentrations of the Bim peptide there is little or no loss of
cytochrome c, whereas at high peptide concentrations a complete
loss of cytochrome c is observed.
[0110] Next, drug-induced changes in BH3 profiles in healthy human
foreskin fibroblasts was examined. After treating healthy HFFs with
a library of .about.2400 different drug compounds or with DMSO (an
inactive chemical used as an experimental control) for 20 hours,
increases in cell death sensitivity were determined by BH3
profiling. Specifically, a compound that increases apoptotic
sensitivity (delta priming) is a compound that accelerates loss of
cytochrome c (FIG. 2). FIG. 2A depicts an exemplary histogram of
the increases in cell death sensitivity (delta priming) represented
by values of delta percent priming, with FIG. 2B representing a
zoomed view of the y-axis of the histogram (percentage of compounds
in the library). Note that the drug treated healthy HFF cells show
an increase in cell death sensitivity compared to the healthy cells
treated with DMSO (the inactive chemical) with approximately 2.41%
of compounds tested. This indicates that molecules that increase
cell death sensitivity in healthy cells relative to a control
compound that has no effect can be reliably detected.
[0111] The experiments exemplified in FIG. 2 were repeated using
mouse mammary tumor cells. A comparison of drug induced changes in
BH3 profiles between mouse mammary tumor cells (y-axis) and healthy
human foreskin fibroblasts (x-axis) is depicted in FIG. 3. When
comparing the effects of .about.2400 compounds on increased cell
death sensitivity, it was found that there are several compounds
that sensitize tumors, but not healthy cells for death (shaded
area). Conversely, several compounds appear to sensitize both the
healthy HFF cells and the mouse mammary tumor cells for apoptosis.
These latter compounds are those that likely have generally toxic
effects to both tumor and healthy cells, and have little
therapeutic value. However, the former compounds (shaded area) are
those that would preferentially target apoptosis in tumor cells
over healthy cells as delta priming increases along the y-axis.
[0112] BH3 profiling on healthy adult mouse hepatocytes was
examined by measuring the loss of cytochrome c from mitochondria in
response to the synthetic BH3 peptides. To do this,
immunofluorescence for cytochrome c was assessed at increasing
concentrations of Bim BH3 peptide (0.1 .mu.M (FIG. 4A), 1.56 .mu.M
(FIG. 4B), 25 .mu.M (FIG. 4C), and 100 .mu.M (FIG. 4D)). A
dose-response curve of cytochrome c loss is depicted in FIG. 4E.
Note that at low concentrations of the Bim peptide, there is little
or no loss of cytochrome c, whereas at high peptide concentrations
a complete loss of cytochrome c is observed.
[0113] Next, various drug compounds were tested to assess the
extent of drug-induced increased cell death sensitivity in adult
mouse hepatocytes. Freshly isolated adult mouse hepatocytes were
treated with different drugs for 20 hours, and the loss of
cytochrome c induced by a single concentration of the synthetic Bim
BH3 peptide was subsequently assessed by immunofluorescence (FIGS.
5A-D). Increase in cell death sensitivity is indicated by loss of
cytochrome c, which is quantified as an increase in apoptotic
sensitivity (delta priming, FIG. 5E). Navitoclax, doxorubicin, and
tanespimycin all increase apoptotic sensitivity.
OTHER EMBODIMENTS
[0114] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
17120PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified by
AcMOD_RES(20)..(20)AMIDATION 1Met Arg Pro Glu Ile Trp Ile Ala Gln
Glu Leu Arg Arg Ile Gly Asp 1 5 10 15 Glu Phe Asn Ala 20
220PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified by
ACMOD_RES(20)..(20)AMIDATION 2Met Arg Pro Glu Ile Trp Ile Ala Gln
Glu Leu Arg Arg Ile Gly Asp 1 5 10 15 Glu Phe Asn Val 20
320PRTArtificial SequenceSynthetic Polypeptide 3Glu Asp Ile Ile Arg
Asn Ile Ala Arg His Leu Ala Gln Val Gly Asp 1 5 10 15 Ser Met Asp
Arg 20 420PRTArtificial SequenceSynthetic Polypeptide 4Met Arg Pro
Glu Ile Trp Ile Ala Gln Glu Leu Arg Arg Ile Gly Asp 1 5 10 15 Glu
Phe Asn Ala 20 520PRTArtificial SequenceSynthetic Polypeptide 5Glu
Asp Ile Ile Arg Asn Ile Ala Arg His Ala Ala Gln Val Gly Ala 1 5 10
15 Ser Met Asp Arg 20 625PRTArtificial SequenceSynthetic
Polypeptide 6Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg
Met Ser Asp 1 5 10 15 Glu Phe Glu Gly Ser Phe Lys Gly Leu 20 25
720PRTArtificial SequenceSynthetic Polypeptide 7Met Glu Gly Ser Asp
Ala Leu Ala Leu Arg Leu Ala Cys Ile Gly Asp 1 5 10 15 Glu Met Asp
Val 20 820PRTArtificial SequenceSynthetic Polypeptide 8Ala Glu Leu
Pro Pro Glu Phe Ala Ala Gln Leu Arg Lys Ile Gly Asp 1 5 10 15 Lys
Val Tyr Cys 20 920PRTArtificial SequenceSynthetic Polypeptide 9Pro
Ala Asp Leu Lys Asp Glu Cys Ala Gln Leu Arg Arg Ile Gly Asp 1 5 10
15 Lys Val Asn Leu 20 1018PRTArtificial SequenceSynthetic
Polypeptide 10Ala Ala Gln Leu Thr Ala Ala Arg Leu Lys Ala Leu Gly
Asp Glu Leu 1 5 10 15 His Gln 1120PRTArtificial SequenceSynthetic
Polypeptide 11Glu Gln Trp Ala Arg Glu Ile Gly Ala Gln Leu Arg Arg
Met Ala Asp 1 5 10 15 Asp Leu Asn Ala 20 1220PRTArtificial
SequenceSynthetic Polypeptide 12His Gln Ala Glu Val Gln Ile Ala Arg
Lys Leu Gln Leu Ile Ala Asp 1 5 10 15 Gln Phe His Arg 20
1325PRTArtificial SequenceSynthetic Polypeptide 13Asn Leu Trp Ala
Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser 1 5 10 15 Asp Glu
Phe Val Asp Ser Phe Lys Lys 20 25 1425PRTArtificial
SequenceSynthetic Polypeptide 14Leu Trp Ala Ala Gln Arg Tyr Gly Arg
Glu Ala Arg Arg Met Ser Asp 1 5 10 15 Glu Phe Glu Gly Ser Phe Lys
Gly Leu 20 25 1523PRTArtificial SequenceSynthetic Polypeptide 15Arg
Pro Glu Ile Trp Met Thr Gln Gly Leu Arg Arg Leu Gly Asp Glu 1 5 10
15 Ile Asn Ala Tyr Tyr Ala Arg 20 1620PRTArtificial
SequenceSynthetic Polypeptide 16Glu Gln Trp Ala Arg Glu Ile Gly Ala
Gln Ala Arg Arg Met Ala Ala 1 5 10 15 Asp Leu Asn Ala 20
1720PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(1)..(10)Xaa can be any amino
acidmisc_feature(12)..(15)Xaa can be any amino
acidmisc_feature(17)..(20)Xaa can be any amino acid 17Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Asp 1 5 10 15 Xaa
Xaa Xaa Xaa 20
* * * * *