U.S. patent application number 13/704951 was filed with the patent office on 2013-08-15 for energy aware sensor management for wearable medical systems optimization.
The applicant listed for this patent is Foad Dabiri, Saro Meguerdichian, Hyduke Noshadi, Miodrag Potkonjak, Majid Sarrafzadeh. Invention is credited to Foad Dabiri, Saro Meguerdichian, Hyduke Noshadi, Miodrag Potkonjak, Majid Sarrafzadeh.
Application Number | 20130211209 13/704951 |
Document ID | / |
Family ID | 45348904 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130211209 |
Kind Code |
A1 |
Sarrafzadeh; Majid ; et
al. |
August 15, 2013 |
ENERGY AWARE SENSOR MANAGEMENT FOR WEARABLE MEDICAL SYSTEMS
OPTIMIZATION
Abstract
In some aspects, the invention provides compositions and methods
for inhibiting viral infection. In some aspects, the invention
provides compositions and methods useful for identifying antiviral
compounds.
Inventors: |
Sarrafzadeh; Majid; (Anaheim
Hills, CA) ; Potkonjak; Miodrag; (Los Angeles,
CA) ; Dabiri; Foad; (San Francisco, CA) ;
Noshadi; Hyduke; (Sherman Oaks, CA) ; Meguerdichian;
Saro; (West Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sarrafzadeh; Majid
Potkonjak; Miodrag
Dabiri; Foad
Noshadi; Hyduke
Meguerdichian; Saro |
Anaheim Hills
Los Angeles
San Francisco
Sherman Oaks
West Hills |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Family ID: |
45348904 |
Appl. No.: |
13/704951 |
Filed: |
June 17, 2011 |
PCT Filed: |
June 17, 2011 |
PCT NO: |
PCT/US2011/040920 |
371 Date: |
December 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61356426 |
Jun 18, 2010 |
|
|
|
Current U.S.
Class: |
600/301 ;
600/300; 600/592; 600/595 |
Current CPC
Class: |
A61B 5/112 20130101;
A61B 5/7278 20130101; Y02A 50/30 20180101; A61B 5/1038 20130101;
C12Q 1/44 20130101; C12Q 1/18 20130101; A61K 38/00 20130101; A61B
5/7225 20130101; A61B 5/7285 20130101; A61P 31/14 20180101; A61P
31/12 20180101; A61B 5/0002 20130101; A61B 5/6807 20130101; A61P
31/16 20180101; Y02A 50/463 20180101; G01N 2333/918 20130101; A61P
43/00 20180101 |
Class at
Publication: |
600/301 ;
600/300; 600/595; 600/592 |
International
Class: |
A61B 5/11 20060101
A61B005/11; A61B 5/103 20060101 A61B005/103; A61B 5/00 20060101
A61B005/00 |
Claims
1. A method of inhibiting viral infection of a cell comprising
contacting the cell with a PLA2G16 inhibitor.
2. The method of claim 1, wherein the virus is a Picornavirus.
3. The method of claim 2, wherein the Picornavirus is an
enterovirus.
4. The method of claim 2, wherein the Picornavirus is a
coxsackievirus.
5. The method of claim 2, wherein the Picornavirus is a
hepatovirus.
6. The method of claim 2, wherein the Picornavirus is a
rhinovirus.
7. The method of claim 1, wherein the cell is a vertebrate
cell.
8. The method of claim 1, wherein the cell is a mammalian cell.
9. The method of claim 1, wherein the cell is a human cell.
10. The method of claim 1, wherein the inhibitor inhibits
expression of PLA2G16.
11. The method of claim 1, wherein the inhibitor inhibits enzymatic
activity of PLA2G16.
12. A method of treating a viral infection in a subject, the method
comprising administering a PLA2G16 inhibitor to a subject in need
of treatment for a viral infection.
13. The method of claim 12, wherein the viral infection is a
Picornavirus infection.
14. The method of claim 13, wherein the Picornavirus is an
enterovirus.
15. The method of claim 13, wherein the Picornavirus is a
coxsackievirus.
16. The method of claim 13, wherein the Picornavirus is a
hepatovirus.
17. The method of claim 13, wherein the Picornavirus is a
rhinovirus.
18. The method of claim 12, wherein the subject is a
vertebrate.
19. The method of claim 12, wherein the subject is a mammal.
20. The method of claim 12, wherein the subject is human.
21. The method of claim 12, wherein the inhibitor inhibits
expression of PLA2G16.
22. The method of claim 12, wherein the inhibitor inhibits
enzymatic activity of PLA2G16.
23. A method of identifying a candidate antiviral compound
comprising steps of: (a) providing a composition comprising a
PLA2G16 polypeptide and a test compound; (b) determining whether
the test compound inhibits the PLA2G16 polypeptide, wherein if the
compound inhibits the PLA2G16 polypeptide, the compound is
identified as a candidate antiviral compound.
24. The method of claim 23, wherein step (b) comprises determining
whether the test compound inhibits expression of the PLA2G16
polypeptide.
25. The method of claim 23, wherein step (b) comprises determining
whether the test compound inhibits an enzymatic activity of the
PLA2G16 polypeptide.
26. The method of claim 25, wherein the enzymatic activity is
phospholipase A2 activity.
27. The method of claim 23, wherein the composition of step (a) is
a cell-free composition comprising purified PLA2G16; and step (b)
comprises determining whether the test compound inhibits enzymatic
activity of PLA2G16.
28. The method of claim 23, wherein the composition of step (a)
comprises a cell that expresses a PLA2G16 polypeptide, and wherein
step (b) comprises determining whether the test compound inhibits
expression or enzymatic activity of PLA2G16.
29. The method of claim 23, wherein if the compounds inhibits the
PLA2G16 polypeptide, the compound is identified as a candidate
antiviral compound useful for inhibiting viral infection by a
Picornavirus.
30. The method of claim 23, further comprising assessing the
ability of the compound to inhibit viral infection of a cell or
subject.
31. The method of claim 23, further comprising the step of
contacting a cell with the compound and a virus, wherein the cell
would be susceptible to the virus in the absence of the
compound.
32. The method of claim 23, further comprising the step of
administering the compound to a subject, wherein the subject would
be susceptible to infection by the virus in the absence of the
compound.
33. The method of claim 23, further comprising the step of
contacting a cell that is infected by the virus with the
compound.
34. The method of claim 23, further comprising the step of
administering the compound to a subject, wherein the subject is
infected by a virus.
35. A method of validating a candidate antiviral compound
comprising steps of: (a) providing a candidate antiviral compound
identified according to the method of claim 23; and (b) determining
whether the compound inhibits infection of a cell or organism by a
virus, wherein if the compound inhibits infection of a cell or
organism by the virus, the compound is validated as an antiviral
compound.
36. The method of claim 35, wherein the virus is a
Picornavirus.
37. A composition comprising: (a) a PLA2G16 inhibitor; (b) a virus;
and (c) a population of cells.
38. The composition of claim 37, wherein the virus is present at a
multiplicity of infection (MOI) of at least 0.01.
39. The composition of claim 37, wherein the virus is a
Picornavirus.
40. The composition of claim 37, wherein the cells are in
culture.
41. The composition of claim 37, wherein the cells are vertebrate
cells.
42. The composition of claim 37, wherein the cells are mammalian
cells.
43. The composition of claim 37, wherein the cells are human
cells.
44. The composition of claim 37, wherein at least some of the cells
are infected by the virus.
45. The composition of claim 37, wherein the PLA2G16 inhibitor
binds to PLA2G16.
46. The composition of claim 37, wherein the PLA2G16 inhibitor
inhibits expression of PLA2G16.
47. The composition of claim 37, wherein the PLA2G16 inhibitor
inhibits an enzymatic activity of PLA2G16.
48. The composition of claim 37, wherein the PLA2G16 inhibitor is a
small molecule.
49. The composition of claim 37, wherein the PLA2G16 inhibitor is
present in an amount sufficient to detectably inhibit infection of
the cells by the virus.
50. A composition comprising a PLA2G16 inhibitor, wherein the
composition is useful for treating a viral infection in a
subject.
51. The composition of claim 50, wherein the PLA2G16 inhibitor
binds to PLA2G16.
52. The composition of claim 50, wherein the PLA2G16 inhibitor
inhibits expression of PLA2G16.
53. The composition of claim 50, wherein the PLA2G16 inhibitor
inhibits an enzymatic activity of PLA2G16.
54. The composition of claim 50, wherein the PLA2G16 inhibitor is a
small molecule.
55. The composition of claim 50, wherein the viral infection is a
picornavirus infection.
56. The composition of claim 50, wherein the subject is a
vertebrate.
57. The composition of claim 50, wherein the subject is a
mammal.
58. The composition of claim 50, wherein the subject is human.
59. A near-haploid mammalian cell that has a mutation in a gene
that encodes PLA2G16.
60. The near-haploid mammalian cell of claim 59, wherein the cell
expresses a mutant form of PLA2G16.
61. The near-haploid mammalian cell of claim 60, wherein the cell
expresses a mutant form of PLA2G16, wherein the mutant form has
reduced catalytic activity as compared with the non-mutant
form.
62. A method of identifying a non-human multicellular organism with
increased resistance to infection by a virus, the method comprising
determining whether the organism has reduced PLA2G16 expression or
activity, wherein if the organism has reduced PLA2G16 expression or
activity, the organism has increased resistance to infection by a
virus.
63. The method of claim 62, wherein the virus is a
Picornavirus.
64. The method of claim 62, wherein the organism is a commercially
important vertebrate animal.
65. A method comprising: (a) providing a multicellular organism
with reduced or absent functional PLA2G16; and (b) using the
organism in agriculture and/or animal husbandry.
66. The method of claim 65, wherein the organism is a commercially
important vertebrate animal.
67. The method of claim 65, wherein the organism is not genetically
modified.
68. The method of claim 65, wherein the organism has increased
resistance to infection by a Picornavirus relative to an organism
that does not have reduced or absent functional PLA2G16.
69. A farm animal having reduced or absent functional PLA2G16,
wherein the animal has increased resistance to infection by a
virus.
70. The farm animal of claim 69, wherein the virus is a
Picornavirus.
71. The farm animal of claim 69, wherein the farm animal is a cow,
pig, sheep, goat, horse, chicken, or turkey.
Description
RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Application No. 61/356,426, filed Jun. 18, 2010. The entire
contents of this application are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Viruses are major causes of disease and death throughout the
world. Although vaccines and public health measures have greatly
reduced the incidence of certain viral infections, such approaches
have been less successful in tackling many viruses of significant
medical and/or veterinary importance. Even if a generally
protective vaccine exists, it is challenging to achieve vaccination
of all individuals. Furthermore, obstacles to effective
immunization can arise due to factors such as immune senescence and
treatment with immunosuppressive medications. Pharmacological
therapies have been developed against some viruses, with human
immunodeficiency virus (HIV) being a notable example. However,
there are still relatively few viral diseases for which effective
drugs are available. There is a need for new antiviral compounds
and for new approaches to identifying such compounds.
SUMMARY OF THE INVENTION
[0003] The invention relates at least in part to identification of
a target for antiviral drug discovery. In one aspect, the invention
provides a method of inhibiting viral infection of a cell
comprising contacting the cell with a PLA2G16 inhibitor. In some
embodiments, the virus is a Picornavirus. In some embodiments, the
cell is a vertebrate cell. In some embodiments the vertebrate cell
is a mammalian cell, e.g., a human cell. In some embodiments, the
PLA2G16 inhibitor inhibits expression of PLA2G16. In some
embodiments, the PLA2G16 inhibitor inhibits enzymatic activity of
PLA2G16.
[0004] In another aspect, the invention provides a method of
treating a viral infection in a subject, the method comprising
administering a PLA2G16 inhibitor to a subject in need of treatment
for a viral infection. In some embodiments, the viral infection is
a Picornavirus infection. In some embodiments, the subject is a
vertebrate. In some embodiments, the subject is a mammal, e.g., a
human. In some embodiments, the PLA2G16 inhibitor inhibits
expression of PLA2G16. In some embodiments, the PLA2G16 inhibitor
inhibits enzymatic activity of PLA2G16.
[0005] In another aspect, the invention provides a method of
identifying a candidate antiviral compound comprising steps of: (a)
providing a composition comprising a PLA2G16 polypeptide and a test
compound; (b) determining whether the test compound inhibits the
PLA2G16 polypeptide, wherein if the compound inhibits the PLA2G16
polypeptide, the compound is identified as a candidate antiviral
compound. In some embodiments, step (b) comprises determining
whether the test compound inhibits expression of the PLA2G16
polypeptide. In some embodiments, step (b) comprises determining
whether the test compound inhibits an enzymatic activity of the
PLA2G16 polypeptide. In some embodiments, the enzymatic activity is
phospholipase A2 activity. In some embodiments, the composition of
step (a) is a cell-free composition comprising purified PLA2G16;
and step (b) comprises determining whether the test compound
inhibits enzymatic activity of PLA2G16. In some embodiments, the
composition of step (a) comprises a cell that expresses a PLA2G16
polypeptide, and wherein step (b) comprises determining whether the
test compound inhibits expression or enzymatic activity of PLA2G16.
In some embodiments, if the compounds inhibits the PLA2G16
polypeptide, the compound is identified as a candidate antiviral
compound useful for inhibiting viral infection by a Picornavirus.
In some embodiments, the method further comprises assessing the
ability of the compound to inhibit viral infection of a cell or
subject. In some embodiments, the method further comprises the step
of contacting a cell with the compound and a virus, wherein the
cell would be susceptible to the virus in the absence of the
compound. In some embodiments, the method further comprises the
step of administering the compound to a subject, wherein the
subject would be susceptible to infection by the virus in the
absence of the compound. In some embodiments, the method further
comprises the step of contacting a cell that is infected by the
virus with the compound. In some embodiments, the method further
comprises the step of administering the compound to a subject,
wherein the subject is infected by a virus.
[0006] In another aspect, the invention provides a method of
validating a candidate antiviral compound comprising steps of: (a)
providing a candidate antiviral compound identified according to a
method that comprises identifying or selecting a compound that
inhibits PLA2g16; and (b) determining whether the compound inhibits
infection of a cell or organism by a virus, wherein if the compound
inhibits infection of a cell or organism by the virus, the compound
is validated as an antiviral compound. In some embodiments, the
virus is a Picornavirus.
[0007] In another aspect, the invention provides a composition
comprising: (a) a PLA2G16 inhibitor; (b) a virus; and (c) a
population of cells. In some embodiments, the virus is present at a
multiplicity of infection (MOI) of at least 0.01. In some
embodiments, the virus is a Picornavirus. In some embodiments, the
cells are in culture. In some embodiments, the cells are vertebrate
cells. In some embodiments, the cells are mammalian cells, e.g.,
human cells. In some embodiments, the population of cells comprises
at least 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, or more cells. In some embodiments, the cells are human
cells. In some embodiments, at least some of the cells are infected
by the virus. In some embodiments, the PLA2G16 inhibitor binds to
PLA2G16. In some embodiments, the PLA2G16 inhibitor inhibits
expression of PLA2G16. In some embodiments, the PLA2G16 inhibitor
inhibits an enzymatic activity of PLA2G16. In some embodiments, the
PLA2G16 inhibitor is a small molecule. In some embodiments, the
PLA2G16 inhibitor is present in an amount sufficient to detectably
inhibit infection of the cells by the virus.
[0008] In another aspect, the invention provides a composition
comprising a PLA2G16 inhibitor, wherein the composition is useful
for treating a viral infection in a subject. In some embodiments,
the PLA2G16 inhibitor binds to PLA2G16. In some embodiments, the
PLA2G16 inhibitor inhibits expression of PLA2G16. In some
embodiments, the PLA2G16 inhibitor inhibits an enzymatic activity
of PLA2G16. In some embodiments, the PLA2G16 inhibitor is a small
molecule. In some embodiments, the viral infection is a
Picornavirus infection. In some embodiments, the subject is a
vertebrate. In some embodiments, the subject is a mammal, e.g., a
human.
[0009] In another aspect, the invention provides a mammalian cell
that has a mutation in a gene that encodes PLA2G16. In some
embodiments the cell is a near-haploid cell. In some embodiments,
the cell expresses a mutant form of PLA2G16. In some embodiments,
the cell expresses a mutant form of PLA2G16, wherein the mutant
form has reduced catalytic activity as compared with the non-mutant
form.
[0010] In another aspect, the invention provides a method of
identifying a non-human multicellular organism, e.g., a vertebrate
animal, that has increased resistance to viral infection, the
method comprising identifying a multicellular organism that has
reduced or absent functional PLA2G16. In some embodiments the
invention provides a method of identifying a non-human
multicellular organism with increased resistance to infection by a
virus, the method comprising determining whether the organism has
reduced PLA2G16 expression or activity, wherein if the organism has
reduced PLA2G16 expression or activity, the organism has increased
resistance to infection by a virus. In some embodiments, the method
further comprises providing or using an organism with reduced or
absent PLA2G16 in agriculture and/or animal husbandry. In some
embodiments, a virus-resistant animal is of a non-domesticated
species. Optionally the species is endangered. In some embodiments,
the organism is a commercially important vertebrate animal. In some
embodiments of the inventive methods, the organism is not
genetically modified.
[0011] In another aspect, the invention provides a farm animal
having reduced or absent functional PLA2G16, wherein the animal has
increased resistance to infection by a virus. In some embodiments
the animal is not genetically modified. In other embodiments the
animal is genetically modified.
[0012] In certain embodiments of any of the aspects of the
invention, the Picornavirus is an enterovirus (member of the
Enterovirus genus). In certain embodiments the enterovirus is a
human enterovirus, e.g., a virus classified within the Human
enterovirus A, Human enterovirus B, Human enterovirus C, Human
enterovirus D, Human rhinovirus A, Human rhinovirus B, or Human
rhinovirus C species. In some embodiments, the human enterovirus is
poliovirus 1, 2, or 3 or any of human enteroviruses 68-107, e.g.,
EV-71. In certain embodiments of any of the aspects of the
invention, the Picornavirus is a hepatovirus, e.g., human hepatitis
A virus. In certain embodiments of any of the aspects of the
invention, the Picornavirus is a coxsackievirus. In certain
embodiments the coxsackievirus is a human coxsackievirus, e.g., any
coxsackieviruses A1-A22, A24, or B1-B5. In certain embodiments of
any of the aspects of the invention, the Picornavirus is a
rhinovirus (member of Human rhinovirus A, Human rhinovirus B, or
Human rhinovirus C species), e.g., any of human rhinoviruses 1-100.
In certain embodiments of any of the aspects of the invention, the
Picornavirus is an echovirus. In certain embodiments of any of the
various aspects of the invention, the virus is a foot-and-mouth
disease virus, e.g., one of the seven foot-and-mouth disease virus
serotypes: O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1.
[0013] The practice of the present invention will typically employ,
unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant nucleic acid (e.g., DNA) technology,
immunology, and RNA interference (RNAi) which are within the skill
of the art. Non-limiting descriptions of certain of these
techniques are found in the following publications: Ausubel, F., et
al., (eds.), Current Protocols in Molecular Biology, Current
Protocols in Immunology, Current Protocols in Protein Science, and
Current Protocols in Cell Biology, all John Wiley & Sons, N.Y.,
edition as of December 2008; Sambrook, Russell, and Sambrook,
Molecular Cloning: A Laboratory Manual, 3.sup.rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and
Lane, D., Antibodies--A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I.,
"Culture of Animal Cells, A Manual of Basic Technique", 5th ed.,
John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting
information regarding viruses is found in, e.g., Knipe, D M and
Howley, P M (eds.) Fields Virology, Volumes I and II. 5.sup.th ed.
Lippincott Williams and Wilkins, 2007; Buchen-Osmond, C. (Ed),
(2006) Index to ICTVdB virus descriptions. In: ICTVdB--The
Universal Virus Database, version 4. ICTVdB Management, Mailman
School of Public Health, Columbia University, New York, N.Y., USA;
and "ICTVdB--The Universal Virus Database", version 4, April 2006.
http://www.ictvdb.org/Ictv/ICTVindex.htm) and ICTVdb Virus
Descriptions (http://www.ictvdb.org/ICTVdB/index.htm). (It is noted
that the online database is currently being rewritten.) The most
recent report of the International Committee on the Taxonomy of
Viruses (ICTV) of the International Union of Microbiological
Societies: "Virus Taxonomy: VIIIth Report of the International
Committee on Taxonomy of Viruses", 2005, C. M. Fauquet, M. A. Mayo,
J. Maniloff, U. Desselberger, and L. A. Ball (Eds), Elsevier
Academic Press, is considered the standard and definitive reference
for virus taxonomy (classification and nomenclature), as
supplemented by taxonomic proposals subsequently approved by the
ICTV (available as updates on the ICTV website as
http://talk.ictvonline.org/media/22/default.aspx/.
http://talk.ictvonline.org/files/ictv_official_taxonomy_updates_since_the-
.sub.--8th_report/default.aspx) (See also Carstens, E B and Ball,
L. Ratification vote on taxonomic proposals to the International
Committee on Taxonomy of Viruses. Archives of Virology, Volume 154,
Number 7, 2008, and Carstens, E. Ratification vote on taxonomic
proposals to the International Committee on Taxonomy of Viruses
(2009) Archives of Virology, Volume 155, Number 1, 2009). The Virus
Taxonomy: 2009 Release v4 (Mar. 20, 2010) (available on the ICTV
website at http://ictvonline.org/virusTaxonomy.asp) represents the
most recent taxonomy.
[0014] Non-limiting information regarding therapeutic agents and
human diseases is found in Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B.
(ed.) Basic and Clinical Pharmacology; McGraw-Hill/Appleton &
Lange; 10.sup.th ed. (2006) or 11th edition (July 2009). All
patents, patent applications, and other publications (e.g.,
scientific articles, books, websites, and databases) mentioned
herein are incorporated by reference in their entirety. In case of
a conflict between the specification and any of the incorporated
references, the specification (including any amendments thereof,
which may be based on an incorporated reference), shall control.
Standard art-accepted meanings of terms are used herein unless
indicated otherwise. Standard abbreviations for various terms are
used herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. A. Schematic outline of gene-trap vector integration
in an endogenous gene. B. Schematic overview of haploid genetic
screen for genes critical for poliovirus replication.
[0016] FIG. 2. Haploid genetic screen identifies PLA2G16 as
critical for poliovirus infection. Mutagenized haploid cells were
contacted with poliovirus and resistant colonies were allowed to
grow out. Gene trap insertion sites were determined using inverse
PCR and massively parallel sequencing. The plot shows the positions
on the human chromosome to which individual gene trap mutations
were mapped on the x-axis and the inverse of the distance of a
particular mutation to its neighbors on the y-axis. Mutations are
highly enriched in chromosome 19 in the known poliovirus receptor
(PVR) and on chromosome 11 in the phospholipase PLA2G16 that
contained 42 independent gene trap insertions.
[0017] FIG. 3. Western blot analysis for expression of PLA2G16 in
wild type haploid cells (WT; lane 1), cells containing a gene trap
insertion in PLA2G16 gene (PLA2G16.sup.GT:; lane 2) cells
containing a gene trap in PLA2G16 and expressing FLAG-tagged
PLA2G16 (lane 3); cells containing a gene trap in PLA2G16
expressing FLAG-tagged mutant PLA2G16 (lane 4); cells containing a
gene trap in PLA2G16 and expressing untagged PLA2G16 (lane 5);
cells containing a gene trap in PLA2G16 and expressing untagged
mutant PLA2G16 (lane 6).
[0018] FIG. 4. Haploid cells containing a PLA2G16 gene trap
insertion are resistant to poliovirus infection. Complementation of
PLA2G16 by retroviral overexpression restores sensitivity of these
cells to poliovirus. This requires the catalytic activity of
PLA2G16 because complementation with a catalytic site mutant
(C113A) does not restore sensitivity.
[0019] FIG. 5. Cells containing a PLA2G16 gene trap insertion are
resistant to coxsackievirus B1. Cells were plated in 24-well wells
and monolayers were virus was added at the indicated MOIs. Four
days after infection viable, adherent cells were stained using
crystal violet. Cells mutant for PLA2G16 were unaffected by high
concentrations of coxsackievirus B1. Complementation of PLA2G16 by
retroviral overexpression restores sensitivity of these cells to
coxsackievirus B1. This requires the catalytic activity of PLA2G16
because complementation with a catalytic site mutant (C113A) does
not restore sensitivity.
[0020] FIG. 6. (A) Sensitivity of wild type and gene trap mutant
cells to poliovirus. (B) Sensitivity of wild type gene trap mutant
cells to coxsackievirus B1. Poliovirus was added to cells at the
indicated MOIs (X-axis) and viability was measured three days later
using an MTT assay. HAP1: Wild type HAP1 cells (without gene trap)
[0021] PLA2G16: HAP1 cells containing gene trap insertion into
PLA2G16 gene (PLA2G16.sup.GT) [0022] PLA2G16+PM2G16WT: HAP1
PLA2G16.sup.GT cells infected with retrovirus encoding wild type
PLA2G16 [0023] PLA2G16+PM2G16MUT: HAP1 PLA2G16.sup.GT cells
infected with retrovirus encoding catalytically inactive mutant
PLA2G16 (with C113A mutation) [0024] PVR: HAP1 cells with gene trap
insertion into poliovirus receptor.
[0025] FIG. 7. Knock down of PLA2G16 in Hela cells results in
increased resistance to the human rhinoviruses HRV-2 and
HRV-14.
[0026] FIG. 8. Exemplary PLA2G16 sequences. Predicted transmembrane
domain is shown in bold in the human sequence.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
I. Definitions
[0027] The term "antibody" encompasses immunoglobulins and
derivatives thereof containing an immunoglobulin domain capable of
binding to an antigen. An antibody can originate from a mammalian
or avian species, e.g., human, rodent (e.g., mouse, rabbit), goat,
chicken, etc., or can be generated ex vivo using a technique such
as phage display. Antibodies include members of the various
immunoglobulin classes, e.g., IgG, IgM, IgA, IgD, IgE, or
subclasses thereof such as IgG1, IgG2, etc. In various embodiments
of the invention "antibody" refers to an antibody fragment or
molecule such as an Fab', F(ab')2, scFv (single-chain variable)
that retains an antigen binding site and encompasses recombinant
molecules comprising one or more variable domains (VH or VL). An
antibody can be monovalent, bivalent or multivalent in various
embodiments. The antibody may be a chimeric or "humanized"
antibody. An antibody may be polyclonal or monoclonal, though
monoclonal antibodies may be preferred. In some aspects, an
antibody is an intrabody, which may be expressed intracellularly.
In some embodiments a compound comprises a single-chain antibody
and a protein transduction domain (e.g., as a fusion
polypeptide).
[0028] An "effective amount" or "effective dose" of a compound or
other agent (or composition containing such compound or agent)
refers to the amount sufficient to achieve a desired biological
and/or pharmacological effect, e.g., when delivered to a cell or
organism according to a selected administration form, route, and/or
schedule. As will be appreciated by those of ordinary skill in this
art, the absolute amount of a particular compound, agent, or
composition that is effective may vary depending on such factors as
the desired biological or pharmacological endpoint, the agent to be
delivered, the target tissue, etc. Those of ordinary skill in the
art will further understand that an "effective amount" may be
contacted with cells or administered in a single dose, or the
desired effect may be achieved by use of multiple doses. An
effective amount of an antiviral compound may be an amount
sufficient to achieve one or more of the following: (i) reduce
virus replication (e.g., reduce production of progeny virus) in
cell culture and/or in vivo; (ii) reduce the severity of or prevent
one or more symptoms or signs of a viral infection; (iii)
significantly reduce the risk of recurrence of a viral infection
(e.g., reduce the risk of relapse); (iv) significantly reduce the
risk of a clinically significant infection in a subject who has
been exposed to an infectious agent, etc.
[0029] "Identity" or "percent identity" is a measure of the extent
to which the sequence of two or more nucleic acids or polypeptides
is the same. The percent identity between a sequence of interest A
and a second sequence B may be computed by aligning the sequences,
allowing the introduction of gaps to maximize identity, determining
the number of residues (nucleotides or amino acids) that are
opposite an identical residue, dividing by the minimum of TG.sub.A
and TG.sub.B (here TG.sub.A and TG.sub.B are the sum of the number
of residues and internal gap positions in sequences A and B in the
alignment), and multiplying by 100. When computing the number of
identical residues needed to achieve a particular percent identity,
fractions are to be rounded to the nearest whole number. Sequences
can be aligned with the use of a variety of computer programs known
in the art. For example, computer programs such as BLAST2, BLASTN,
BLASTP, Gapped BLAST, etc., generate alignments. The algorithm of
Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci.
USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc.
Natl. Acad Sci. USA 90:5873-5877, 1993 is incorporated into the
NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J.
Mol. Biol. 215:403-410, 1990). In some embodiments, to obtain
gapped alignments for comparison purposes, Gapped BLAST is utilized
as described in Altschul et al. (Altschul, et al. Nucleic Acids
Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs may be
used. See the Web site having URL www.ncbi.nlm.nih.gov. Other
suitable programs include CLUSTALW (Thompson J D, Higgins D G,
Gibson T J, Nuc Ac Res, 22:4673-4680, 1994) and GAP (GCG Version
9.1; which implements the Needleman & Wunsch, 1970 algorithm
(Needleman S B, Wunsch C D, J Mol Biol, 48:443-453, 1970.)
[0030] "Infection" refers to the (often detrimental) colonization
of a cell (sometimes referred to as a "host cell" or "host") or
multicellular organism (sometimes referred to as a "host"), by a
microorganism such as a virus. The process of infection encompasses
entry of the virus into one or more cell(s) (invasion) and, if the
infection proceeds, subsequent steps in the viral life cycle,
typically resulting in multiplication of the virus and, frequently
in the case of viruses of medical or veterinary importance,
detrimental effects of the virus on the host. A viral infection can
be any situation in which the presence of one or more virus
population(s) is damaging to a host cell or organism. The term
"infection" encompasses excessive replication of viruses that are
normally present in or on the body of a vertebrate, e.g., mammal,
or other organism, or the presence and, optionally, replication, of
viruses that are not normally present in or on the body of a
vertebrate, e.g., a mammal, or other organism.
[0031] "Inhibit" may be used interchangeably with terms such as
"suppress", "decrease", and the like, as appropriate in the
context. It will be understood that the extent of inhibition can
vary. For example, inhibition can refer to a reduction by at least
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
[0032] "Isolated" refers to a substance that is separated from at
least some other substances with which it is normally found in
nature, usually by a process involving the hand of man, or is
artificially produced, e.g., chemically synthesized, or present in
an artificial environment.
[0033] "Nucleic acid" is used interchangeably with "polynucleotide"
and encompasses naturally occurring polymers of nucleosides, such
as DNA and RNA, usually linked by phosphodiester bonds, and
non-naturally occurring polymers of nucleosides or nucleoside
analogs. In some embodiments a nucleic acid comprises standard
nucleotides (abbreviated A, G, C, T, U). In other embodiments a
nucleic acid comprises one or more non-standard nucleotides. In
some embodiments, one or more nucleotides are non-naturally
occurring nucleotides or nucleotide analogs. A nucleic acid can
comprise chemically or biologically modified bases (for example,
methylated bases), modified sugars (2'-fluororibose, arabinose, or
hexose), modified phosphate groups (for example, phosphorothioates
or 5'-N-phosphoramidite linkages), locked nucleic acids, or
morpholinos. In some embodiments, a nucleic acid comprises
nucleosides that are linked by phosphodiester bonds. In some
embodiments, at least some nucleosides are linked by a
non-phosphodiester bond. A nucleic acid can be single-stranded,
double-stranded, or partially double-stranded. An at least
partially double-stranded nucleic acid can have one or more
overhangs, e.g., 5' and/or 3' overhang(s). Nucleic acid
modifications (e.g., nucleoside and/or backbone modifications),
non-standard nucleotides, delivery vehicles and approaches, etc.,
known in the art as being useful in the context of RNA interference
(RNAi), aptamer, or antisense-based molecules for research or
therapeutic purposes are contemplated for use in various
embodiments of the instant invention. See, e.g., Crooke, S T (ed.)
Antisense drug technology: principles, strategies, and
applications, Boca Raton: CRC Press, 2008; Kurreck, J. (ed.)
Therapeutic oligonucleotides, RSC biomolecular sciences. Cambridge:
Royal Society of Chemistry, 2008. A nucleic acid may comprise a
detectable label, e.g., a fluorescent dye, radioactive atom, etc.
"Oligonucleotide" refers to a relatively short nucleic acid, e.g.,
typically between about 4 and about 60 nucleotides long.
[0034] A "polypeptide" refers to a polymer of amino acids linked by
peptide bonds. A protein is a molecule comprising one or more
polypeptides. A peptide is a relatively short polypeptide,
typically between about 2 and 60 amino acids in length. The terms
"protein", "polypeptide", and "peptide" may be used
interchangeably. Polypeptides of interest herein often contain
standard amino acids (the 20 L-amino acids that are most commonly
found in nature in proteins). However, other amino acids and/or
amino acid analogs known in the art can be used in certain
embodiments of the invention. One or more of the amino acids in a
polypeptide (e.g., at the N- or C-terminus or in a side chain) may
be modified, for example, by addition, e.g., covalent linkage, of a
moiety such as an alkyl group, carbohydrate group, a phosphate
group, a halogen, a linker for conjugation, etc. A polypeptide
sequence presented herein is presented in an N-terminal to
C-terminal direction unless otherwise indicated. "Polypeptide
domain" refers to a segment of amino acids within a longer
polypeptide. A polypeptide domain may exhibit one or more discrete
binding or functional properties, e.g., a catalytic activity. Often
a domain is recognizable by its conservation among polypeptides
found in multiple different species.
[0035] As used herein, the term "purified" refers to agents or
entities (e.g., compounds) that have been separated from most of
the components with which they are associated in nature or when
originally generated. In general, such purification involves action
of the hand of man. Purified agents or entities may be partially
purified, substantially purified, or pure. Such agents or entities
may be, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or more than 99% pure. In some
embodiments, a nucleic acid or polypeptide is purified such that it
constitutes at least 75%, 80%, 855%, 90%, 95%, 96%, 97%, 98%, 99%,
or more, of the total nucleic acid or polypeptide material,
respectively, present in a preparation. Purity can be based on,
e.g., dry weight, size of peaks on a chromatography tracing,
molecular abundance, intensity of bands on a gel, or intensity of
any signal that correlates with molecular abundance, or any
art-accepted quantification method. In some embodiments, water,
buffers, ions, and/or small molecules (e.g., precursors such as
nucleotides or amino acids), can optionally be present in a
purified preparation. A purified molecule may be prepared by
separating it from other substances (e.g., other cellular
materials), or by producing it in such a manner to achieve a
desired degree of purity. In some embodiments, a purified molecule
or composition refers to a molecule or composition that is prepared
using any art-accepted method of purification. In some embodiments
"partially purified" means that a molecule produced by a cell is no
longer present within the cell, e.g., the cell has been lysed and,
optionally, at least some of the cellular material (e.g., cell
wall, cell membrane(s), cell organelle(s)) has been removed.
[0036] "RNA interference" (RNAi) encompasses processes in which an
endogenous molecular complex known as an RNA-induced silencing
complex (RISC) silences gene expression in a sequence-specific
manner. The RISC contains a short RNA strand that directs or
"guides" sequence-specific degradation or translational repression
of mRNA to which it has complementarity. The complementarity
between the short RNA and mRNA need not be perfect (100%). For
example, the degree of complementarity and/or the characteristics
of the structure formed by hybridization of the mRNA and the short
RNA strand can be such that the strand can (i) guide cleavage of
the mRNA in the RNA-induced silencing complex (RISC) and/or (ii)
cause translational repression of the mRNA by RISC. It will be
appreciated that one or more mismatches between the guide strand
and the target mRNA can be tolerated, especially outside the seed
region (the nucleotides in positions 2-7 or 2-8) of the guide
strand. A short RNA that guides silencing often initially becomes
associated with RISC components (in a complex sometimes termed the
RISC loading complex) as part of a short double-stranded RNA
(dsRNA). RNAi is often used to knockdown a target gene. "Knockdown"
typically refers to a reduction in expression, which may occur,
e.g., at the level of transcription, mRNA stability, translation,
or protein stability. Reduction can be complete (e.g., the amount
of gene product is reduced to background levels) or less than
complete. For example, mRNA and/or protein level can be reduced by
50%, 60%, 70%, 75%, 80%, 85%, 90%, or more.
[0037] RNAi may be employed to inhibit expression in eukaryotic
cells, e.g., vertebrate cells, in a variety of ways as known in the
art. In some embodiments, a short double-stranded nucleic acid is
introduced into cells. In some embodiments, a nucleic acid that is
processed intracellularly (e.g., by one or more RNase III family
enzymes Dicer) to yield short dsRNA is introduced into or expressed
in cells. As used herein, the term "RNAi agent" encompasses nucleic
acids that can be used to achieve RNAi in eukaryotic cells.
Exemplary RNAi agents are short interfering RNA (siRNA) and short
hairpin RNA (shRNA). As known in the art, siRNAs typically comprise
two separate nucleic acid strands that are hybridized to each other
to form a duplex. They can be synthesized in vitro, e.g., using
standard nucleic acid synthesis techniques or by cleavage of a
longer dsRNA, e.g., by an RNase III or RNase III-like enzyme such
as Dicer. In certain embodiments an siRNA or shRNA comprises a
duplex portion about 15-29 nucleotides (nt) long, e.g., between
17-25 nt long, e.g., between 19-23 nt long, wherein either or both
strands optionally has a 3' overhang of 1-5 nucleotides long (e.g.,
2 nucleotides), which may be composed of deoxyribonucleotides. In
some embodiments, the strands are perfectly complementary within
the duplex portion, while in other embodiments, the duplex portion
could contain one or more mismatched nucleotide pairs or bulges. In
some embodiments, each strand of an siRNA is between 15-29
nucleotides in length, e.g., between 19-25 nt long, e.g., 21-23 nt
long. shRNA comprise a single nucleic acid strand that contains two
complementary portions separated by a predominantly
non-self-complementary region. The complementary portions hybridize
to form a duplex structure and the non-self-complementary region
forms a loop connecting the 3' end of one strand of the duplex and
the 5' end of the other strand. shRNAs can undergo intracellular
processing to generate siRNAs.
[0038] RNAi agents also include microRNA (miRNA) and miRNA
precursors. The terms "miRNA" and "miRNA precursor" are often used
in the art to refer to endogenously encoded RNAs. As used herein,
"miRNA" and "miRNA precursor" encompasses artificially designed
nucleic acids that function in an analogous manner to endogenous
miRNAs.
[0039] In certain embodiments an RNAi agent is a vector that
comprises a template for transcription of an siRNA (e.g., as two
separate strands that can hybridize to each other), shRNA, or
microRNA precursor. Such vectors can be used to introduce the
template into vertebrate cells, e.g., mammalian cells, and result
in transient or stable expression of the siRNA, shRNA, or miRNA
precursor.
[0040] A "small molecule" as used herein, is an organic molecule
that is less than about 2 kilodaltons (KDa) in mass. In some
embodiments, the small molecule is less than about 1.5 KDa, or less
than about 1 KDa. In some embodiments, the small molecule is less
than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200
Da, or 100 Da. Often, a small molecule has a mass of at least 50
Da. In some embodiments, a small molecule is non-polymeric. In some
embodiments, a small molecule is not an amino acid. In some
embodiments, a small molecule is not a nucleotide. In some
embodiments, a small molecule is not a saccharide. In some
embodiments, a small molecule contains multiple carbon-carbon bonds
and can comprise one or more heteroatoms and/or one or more
functional groups important for structural interaction with
proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl,
hydroxyl, or carboxyl group, and in some embodiments at least two
functional groups. Small molecules often comprise one or more
cyclic carbon or heterocyclic structures and/or aromatic or
polyaromatic structures, optionally substituted with one or more of
the above functional groups.
[0041] A "subject" can be any multicellular organism, e.g., a
multicellular organism that is susceptible to infection by a virus
or is or may be infected by a virus. Often at least some of the
cells of the subject express detectable amounts of PLA2G16. In some
embodiments a subject is an animal, e.g., a vertebrate, e.g., a
mammal or avian. Exemplary mammals include, e.g., humans, non-human
primates, rodents (e.g., mouse, rat, rabbit), ungulates (e.g.,
ovine, bovine, equine, caprine species), canines, and felines. In
some embodiments, the animal is a mammal of economic importance,
such as a cow, horse, pig, goat, or sheep. Often, a subject is an
individual to whom a compound is to be delivered, e.g., for
experimental, diagnostic, and/or therapeutic purposes or from whom
a sample is obtained or on whom a diagnostic procedure is performed
(e.g., a sample or procedure that will be used to determine whether
the subject has a viral infection or is at risk of a viral
infection).
[0042] "Treat", "treating" and similar terms refer to providing
medical and/or surgical management of a subject. Treatment can
include, but is not limited to, administering a compound or
composition (e.g., a pharmaceutical composition) to a subject.
Treatment is typically undertaken in an effort to alter the course
of a disease, disorder, or undesirable condition in a manner
beneficial to the subject. The effect of treatment can generally
include reversing, alleviating, reducing severity of delaying the
onset of, curing, inhibiting the progression of, and/or reducing
the likelihood of occurrence or reoccurence of the disease,
disorder, or condition to which such term applies, or one or more
symptoms or manifestations of such disease, disorder or condition.
A composition of this invention can be administered to a subject
who has developed an infection or is at increased risk of
developing an infection relative to a member of the general
population. A composition of this invention can be administered
prophylactically, i.e., before development of any symptom or
manifestation of a condition. Typically in this case the subject
will be at risk of developing the condition. For example, an
inventive composition can be administered prior to exposure of the
subject to an infectious agent or prior to the occurrence of a
pathogenic event. "Preventing" can refer to administering a
compound or composition (e.g., a pharmaceutical composition) to a
subject who has not developed a disease or condition, so as to
reduce the likelihood that the disease or condition will occur or
so as to reduce the severity of the disease or condition should it
occur. The subject may be identified as at risk of developing the
disease or condition (e.g., at increased risk relative to many most
other members of the population or as having a risk factor that
increases likelihood of developing the disease).
II. Overview
[0043] The present invention relates in part to the identification
of phospholipase A2, group XVI (PLA2G16) as a new molecular target
of use for identification and/or characterization of antiviral
compounds. PLA2G16 is a phospholipase that is widely or
ubiquitously expressed in mammalian tissues. It has now been
discovered that PLA2G16 polypeptide is a host cell factor that
plays an important role in infection of vertebrate cells, e.g.,
mammalian cells, by viruses of the Picornavirus family. The
invention encompasses the recognition that inhibiting PLA2G16
inhibits viral infection. As described in more detail in the
Examples, using a gene trap mutagenesis strategy in a near-haploid
mammalian cell line (the HAP1 cell line), it was shown that
insertions into the PLA2G16 gene (located on chromosome 11 in human
cells) rendered the cells resistant to infection by poliovirus and
Coxsackie virus B1. Restoring wild type PLA2G16 function by
expressing wild type PLA2G16 in the cells restored susceptibility
to infection, while expressing a catalytically inactive mutant
version of PLA2G16 did not. Furthermore, knockdown of endogenous
PLA2G16 expression in a rhinovirus-sensitive cell line (HeLa cells)
using short interfering RNA (siRNA) rendered these cells resistant
to rhinovirus infection. The discoveries described herein indicate
that PLA2G16 is required for infection of vertebrate cells by a
wide range of viruses.
[0044] The invention provides compositions and methods for
inhibiting viral infection. The invention further provides
compositions and methods useful for identifying candidate compounds
for inhibiting viral infection. In some aspects, the compositions
and methods relate to the use of the PLA2G16 gene and/or PLA2G16
polypeptide as targets for identification of antiviral compounds
(i.e., compounds that inhibit viral infection). Certain of the
inventive methods comprise identifying or providing a compound that
inhibits PLA2G16. In accordance with certain embodiments of the
invention, a compound that inhibits PLA2G16 is a candidate
antiviral compound. Certain of the inventive methods comprise (i)
identifying or providing a compound that inhibits PLA2G16; and (ii)
determining whether the compound inhibits viral infection of a cell
or multicellular organism, wherein if the compound inhibits viral
infection of a cell or multicellular organism, the compound is an
antiviral compound. In some embodiments, a compound that inhibits
PLA2G16 inhibits PLA2G16 expression. In some embodiments, a
compound that inhibits PLA2G16 inhibits a PLA2G16 molecular
function, e.g., the compound inhibits PLA2G16 catalytic
activity.
[0045] Inhibiting viral infection can comprise interventions that
inhibit one or more steps of the viral life cycle so that, for
example, there is reduced entry of virus into cells, reduced
production of viral gene product(s) (viral RNAs and/or proteins),
reduced production of progeny virus, reduced release of progeny
virus, and/or reduced spread of virus within a population of cells
(e.g., in cell culture or in a multicellular orgnanism) as compared
with an appropriate reference level, e.g., the level that would
exist in the absence of the intervention. Inhibition of viral
infection can be assessed based on any of a variety of suitable
indicators. In some embodiments, inhibition of an indicator of
viral infection is complete or substantially complete, e.g., an
indicator of viral infection such as production of a viral gene
product, production of progeny virus, infection of additional
cells, is reduced to background or undetectable level, e.g., a
level that would be expected in the absence of the virus. In some
embodiments, inhibition is not complete. In some embodiments,
inhibition of viral infection can refer to a reduction by about a
factor of at least 10, at least 10.sup.2, at least 10.sup.3, at
least 10.sup.4, or more, e.g., in production of progeny virus or of
a viral gene product.
[0046] In some aspects, the invention provides methods of
inhibiting viral infection of a cell. In some aspects, the methods
comprise inhibiting PLA2G16 in a cell, thereby inhibiting viral
infection of the cell. In some embodiments, the methods comprise
contacting a cell with a compound that inhibits PLA2G16, so that
viral infection of the cell is inhibited. In some embodiments, the
cell is an animal cell, e.g., a vertebrate cell. In some
embodiments, the vertebrate cell is a mammalian cell. In some
aspects, the invention provides methods of inhibiting viral
infection of a subject. In some embodiments, the subject is a
vertebrate. In some aspects, the methods comprise inhibiting
PLA2G16 in at least some cells of the organism, e.g., at least some
cells that are infected by a virus or are susceptible to infection
by a virus. In some embodiments, the methods comprise administering
a compound that inhibits PLA2G16 to the subject. In some
embodiments, the subject is an animal, e.g., a vertebrate. In some
embodiments, the vertebrate is a mammal.
[0047] In some aspects, the invention provides methods of
decreasing the susceptibility (or increasing the resistance) of a
cell or subject to a virus, the methods comprising inhibiting
PLA2G16 in a cell or in at least some cells of the subject. Thus
the invention provides methods of reducing the vulnerability or
propensity of a cell or subject to become infected and/or to
experience adverse effects due to a virus. "Resistance" to a virus
typically refers to the ability to defend against infection. For
purposes of description, the invention will mainly be described in
terms of inhibiting virus infection. However, it will be understood
that, unless otherwise indicated, the inventive methods of
inhibiting virus infection of a cell or subject could be described
as inhibiting susceptibility of the cell or subject to virus
infection or increasing resistance of the cell or subject to virus
infection.
[0048] In some aspects, the invention provides methods of selecting
a therapeutic agent for a subject, the method comprising (a)
determining whether the subject is infected by a virus for which
PLA2G16 is a host cell factor; and (b) selecting a compound that
inhibits PLA2G16 as a therapeutic agent for the subject if the
subject is infected by a virus for which PLA2S16 is a host cell
factor. In some embodiments, the method further comprises
administering a compound that inhibits PLA2G16 to the subject.
[0049] In some aspects, the invention provides methods of
determining whether a subject is a candidate for treatment with a
compound that inhibits PLA2G16. In some embodiments, the method
comprises determining whether the subject is infected by, or at
risk of infection by, a virus for which PLA2G16 is a host cell
factor, wherein if the subject is infected by a virus for which
PLA2G16 is a host cell factor, the subject is a candidate for
treatment with a compound that inhibits PLA2G16. In some
embodiments, the method comprises determining whether the subject
is infected by, or at risk of infection by, a picornavirus, wherein
if the subject is infected with a picornavirus, the subject is a
candidate for treatment with a compound that inhibits PLA2G16. In
some embodiments, the method further comprises administering a
compound that inhibits PLA2G16 to the subject.
[0050] In some aspects, the invention provides methods of treating
a subject in need of treatment for a viral infection. In some
embodiments, the methods comprise selecting a compound that
inhibits PLA2G16 as a therapeutic agent for the subject. In some
embodiments, the methods comprise administering a compound that
inhibits PLA2G16 to the subject. In some embodiments, the methods
of treatment comprise providing a subject in need of treatment for
a viral infection. In some embodiments, the methods of treatment
comprise diagnosing a subject as being infected with a virus. The
subject may have one or more symptoms or signs of a viral
infection, e.g., one or more symptoms or signs associated with a
pathological state resulting from infection by a virus. In some
embodiments, the method comprises administering a pharmaceutical
composition comprising the compound to the subject.
"Administration" can comprise direct administration or indirect
administration. "Indirect" administration comprises activities such
as providing, prescribing, directing another individual to
administer, or in any way making a compound available to a
subject.
III. Viruses and Viral Diseases
[0051] In some aspects, the invention relates to inhibiting
infection of a cell or subject by a virus, wherein PLA2G16 promotes
or plays a role in one or more steps of the life cycle of the
virus. In some embodiments, the virus is capable of infecting cells
of one or more animal species, e.g., one or more vertebrate
species, e.g., mammalian or avian species, wherein the cells
express PLA2G16. In various embodiments, the invention may be
applied to any virus whose capacity to infect a cell, e.g., an
animal cell, is reduced if PLA2G16 is inhibited. While the
invention is described herein mainly in reference to certain
viruses of interest, embodiments of the invention can be applied to
any virus wherein expression of a PLA2G16 polypeptide in the cell
promotes or plays a role in one or more steps of the viral life
cycle. In some embodiments, the virus is of medical importance,
e.g., it is recognized in the medical art as a causative agent of
one or more diseases that affect humans. In some embodiments, the
virus is of veterinary importance, e.g., it is recognized in the
veterinary art as a causative agent of one or more diseases that
affect non-human animals. See, e.g., Knipe & Howley, supra;
Buchen-Osmond, C. supra, and Virus Descriptions in "ICTVdB--The
Universal Virus Database", supra for discussion of various virus
families, including viruses of medical and/or veterinary
importance.
[0052] In some embodiments, the virus has a single-stranded RNA
(ssRNA) genome. In some embodiments, the ssRNA genome virus is
positive stranded. In some embodiments, the virus is a
non-enveloped virus and/or has an icosahedral virion or
nucleocapsid morphology. In some embodiments, the virus is a member
of the picornavirus-like superfamily (such viruses are also termed
"picorna-like viruses" herein). Viruses of the picornavirus-like
superfamily are positive-sense ssRNA viruses that are characterized
by a partially conserved set of genes that consists of an RNA
dependent RNA polymerase (RdRp), a chymotrypsin-like protease
(3CPro), a superfamily 3 helicase (S3H) and a genome-linked protein
(viral protein, genome linked, VPg). The picornavirus-like
superfamily encompasses the proposed order Picornavirales
(discussed below) as well as various virus genera and families
falling outside the proposed order, including, e.g., Caliciviridae
and Astroviridae. See, e.g., Koonin, E V, et al., Nature Reviews
Microbiology, 6:925-939, 2008.
[0053] In some embodiments, the virus is a member of the proposed
order Picornavirales. This order includes viruses that infect
eukaryotes and that share the following properties: (i) a
positive-sense RNA genome, usually with a 5'-bound VPg and
3'-polyadenylated, (ii) genome translation into autoproteolytically
processed polyprotein(s), (iii) capsid proteins organized in a
module containing three related jelly-roll domains which form small
icosahedral, non-enveloped particles with a pseudo-T=3 symmetry,
and (iv) a three-domain module containing a superfamily III
helicase, a (cysteine) proteinase with a chymotrypsin-like fold and
an RNA-dependent RNA polymerase. According to these criteria, the
order Picornavirales includes the families Picornaviridae,
Comoviridae, Dicistroviridae, Marnaviridae, Sequiviridae and the
genera Cheravirus, Iflavirus and Sadwavirus. Other taxa of
"picorna-like" viruses, e.g. Potyviridae, Caliciviridae,
Hypoviridae, do not conform to several of the above criteria and
are more remotely related. The family Caliciviridae is composed of
small (27-40 nm), nonenveloped, icosahedral viruses and include the
four genera Norovirus, Sapovirus, Vesivirus, and Lagovirus. The
major pathogens of medical importance are the noroviruses, which
are a major cause of acute gastroenteritis. Important veterinary
pathogens include vesirivurses such as feline calicivirus (FCV) and
rabbit hemorrhagic disease virus (RHDV). The family Astroviridae
includes human and animal astroviruses that have icosahedral
morphology and a characteristic starlike surface structure when
viewed by electron microscopy. They are important agents of
gastroenteritis and diarrhea in humans as well as various animals,
including mammals (e.g., pigs) and avians.
[0054] In some embodiments of particular interest, the invention
relates to inhibiting infection by viruses that are members of the
Picornaviridae family (also termed "picornaviruses" or
"Picornaviruses" herein). Picornaviruses (like other members of the
picornavirus-like superfamily) are nonenveloped viruses with a
single-stranded genome of positive polarity. They share a common
genomic organization with a long 5' untranslated region (UTR)
(e.g., at least about 500 nucleotides (nt) up to about 1200 nt
long) containing an internal ribosome entry site (IRES), a single
open reading frame (ORF) encoding a polyprotein that is
proteolytically processed, and a short 3' UTR followed by a polyA
tail (Knipe & Howley, supra). Major distinguishing features
among different picornaviruses include, among others, the secondary
structure of the 5' UTR and IRES.
[0055] The picornavirus family includes twelve genera: Aphthovirus,
Avihepatovirus, Cardiovirus, Enterovirus, Erbovirus, Hepatovirus,
Kobovirus, Parechovirus, Sapelovirus, Senecavirus, Teschovirus, and
Tremovirus (see, "ICTVdB--The Universal Virus Database", Virus
Taxonomy: 2009 Release v4, supra). A virus that is a members of one
of these genera may be referred to as anaphthovirus,
avihepatovirus, cardiovirus, enterovirus, erbovirus, hepatovirus,
kobovirus, parechovirus, rhinoviruses, sapelovirus, senecavirus,
teschovirus, or tremovirus, respectively These genera include
numerous viruses that infect vertebrates, and a number of them
contain members that are important causes of disease in humans
and/or in non-human animals. For example, aphthoviruses include
foot-and-mouth disease viruses, which infect cloven-footed animals
such as cattle, goats, pigs, and sheep. Cardioviruses include two
distinct clusters, the first of which includes
encephalomyocarditisvirus and the second of which includes
Theiler's murine encephalomyelitis virus and related viruses,
including some that infect humans.
[0056] Human enteroviruses are common causes of mild upper
respiratory symptoms and flu-like illnesses, among others. Less
commonly, they can result in more serious conditions such as viral
meningitis, myocarditis, or central nervous system conditions such
as encephalitis. The Enterovirus genus includes the following 10
species, as set forth by the ICTV in its 2009 release (available at
http://ictvonline.org/virusTaxonomy.asp?version=2009): Human
enterovirus A, Human enterovirus B, Human enterovirus C, Human
enterovirus D, Simian enterovirus A, Bovine enterovirus, Porcine
enterovirus B, Human rhinovirus A, Human rhinovirus B and Human
rhinovirus C. Many of these species encompass multiple serotypes,
which can in turn include multiple strains. The Human enterovirus
species collectively encompass polioviruses, coxsackievirus,
echoviruses, and numerous other enteroviruses that infect humans.
The Enterovirus genus also encompasses numerous nonhuman enteric
viruses. The poliovirus serotypes poliovirus (PV)-1, PV-2, and PV-3
are included within the Human enterovirus C species. Although
poliovirus has been largely eradicated through widespread use of
effective vaccines, other viruses within the Enterovirus genus are
frequent causes of acute and chronic human diseases.
[0057] Coxsackieviruses are divided into group A and group B
viruses based on early observations of their pathogenicity in mice.
Coxsackieviruses are associated with a range of diseases in human
including aseptic meningitis, hand-foot-mouth disease, herpangina,
myocarditis (sometimes leading to cardiomyopathy), and
pancreatitis, and may be an etiologic factor in type I diabetes
(See, e.g., articles in Curr Top Microbiol Immunol. Vol. 323,
2008). Coxsackieviruses are classified among the Human enterovirus
A, Human enterovirus B, and Human enterovirus C species. Exemplary
coxsackieviruses include serotypes CV-A2, CV-A3, CV-A4, CV-A5,
CV-A6, CV-A7, CV-A8, CV-A10, CV-A12, CV-A14, CV-A16, CV-B1, CV-B2,
CV-B3, CV-B4, CV-B5, CV-B6, CV-A9, CV-A1, CV-A11, CV-A13, CV-A17,
CV-A19, CV-A20, CV-A21, CV-A22, CV-A24.
[0058] Human enterovirus A, Human enterovirus B, Human enterovirus
C, and Human enterovirus D species include additional enteroviruses
such as serotypes EV-71, EV-76, EV-89, EV-90, EV-91, EV-92, EV-69,
EV-73, EV-74, EV-75, EV-77, EV-78, EV-79, EV-80, EV-81, EV-82,
EV-83, EV-84, EV-85, EV-86, EV-87, EV-88, EV-93, EV-97, EV-98,
EV-100, EV-101, EV-106, EV-107, EV-95, EV-96, EV-99, EV-102,
EV-104, EV-105, EV-109, EV-68, EV-70, and EV-94. For example,
enterovirus 71 (EV-71) is a pathogenic enterovirus serotype that
causes recurrent outbreaks in different parts of the world. It can
infect the central nervous system and may cause death and long-term
neurological sequelae in humans, especially infants and young
children (Lin, Y-W., et al., Journal of Virology, 83(13):
6477-6483, 2009, and references therein). EV-71 may also cause
diarrhea, rashes, and hand, foot and mouth disease.
[0059] Member of Human rhinovirus A and Human rhinovirus B species
("rhinoviruses") replicate in the nasopharynx and sometimes in the
lower respiratory tract. These viruses (of which more than 100
serotypes exist) are important etiologic agents of the common cold
in humans and can cause more severe disease as well, particularly
in susceptible individuals.
[0060] The Teschovirus genus includes porcine teschovirus, which
causes polioencephalitis in pigs. Hepatoviruses include human
hepatitis A virus (HAV), which causes hepatitis A, an acute liver
infection.
[0061] Additional picornaviruses continue to be discovered. See,
e.g., Kapoor, A., et al., A highly prevalent and genetically
diversified Picornaviridae genus in South Asian children. Proc Natl
Acad Sci USA. 105(51):20482-7, 2008, describing members of the
proposed cosavirus genus. More recently, a novel virus which has
been designated as klassevirus was discovered using high throughput
sequencing (see, e.g., Greninger, A L, et al., The complete genome
of klassevirus--a novel picornavirus in pediatric stool, Virol J.,
6:82-2009).
[0062] Those of skill in the art will appreciate that virus
taxonomy and classification continue to evolve and that viruses can
be reclassified, e.g., as additional viruses are discovered or
studied, e.g., as viral genes are sequenced, and/or as
relationships between viruses become evident. Thus certain viruses
may have been reclassified by the ICTV subsequent to publication of
certain references cited herein and/or may be reclassified in the
future. Furthermore, those of skill in the art will appreciate that
many publications and references relating to viruses do not adhere
to conventions established by the ICTV, may have preceded the
establishment of these conventions, and/or may employ formal and/or
informal vernacular nomenclature. Identifying characteristics of
viruses (and strains and variants thereof) are well established and
known in the art. Characterized reference samples of numerous
viruses are deposited in and typically available from various
internationally recognized biological resource centers or culture
collections such as the American Type Culture Collection (ATCC)
(Manassas, Va.; http://www.atcc.org/), National Collection of
Pathogenic Viruses (NCPV) of the Health Protection Agency Culture
Collections of the Health Protection Agency of the United Kingdom
(Porton Downm Salisbury UK;
http://www.hpacultures.org.uk/aboutus/ncpv.jsp) and/or
internationally recognized specialty groups, as are reagents of use
to identify and/or characterize viruses. Characterization and/or
classification can be based on properties such as nucleic acid
and/or polypeptide sequences, reactivity with immunological
reagents (e.g., antisera), etc. Genome sequences of numerous
enteroviruses, including those of numerous human enteroviruses, are
publicly available, e.g., on the website of the European
Bioinformatics Institute (http://www.ebi.ac.uk/), National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), and
in the scientific literature.
[0063] Picornavirus structure and life cycle have been extensively
studied (see, e.g., Knipe & Howley, supra). Briefly, the
picornavirus capsid is typically composed of four structural
proteins: VP1, VP2, VP3 and VP4. (Parechoviruses contain only VP1,
VP2, and VP0, the uncleaved precursor of VP2+VP4). The basic
building block of the picornavirus capsid, termed the protomer,
contains one copy of VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 form
a shell with VP4 on its inner surface. Differences in the amino
acid sequences of certain portions of VP 1, VP2, and VP3 give
different picornaviruses distinct morphologies and
antigenicities.
[0064] Replication of picornaviruses occurs in the cell cytoplasm.
Picornaviruses initiate infection by attaching to a receptor on the
host cell membrane, which is followed by uncoating and entry of the
viral genome into the cytoplasm. The poliovirus receptor (PVR,
CD155) and the major group rhinovirus (ICAM-1) were identified in
1989, and since that time receptors for a number of other
picornaviruses have been identified. Some picornaviruses typically
require co-receptors for infection. For example, many enteroviruses
bind to decay-accelerating factor (DAF; CD55) but infection
typically requires presence of an additional molecule, e.g., ICAM-1
or an integrin family member. The RNA genome is translated on entry
into the cytoplasm to a single polyprotein that is cleaved during
translation by virus-encoded proteases (mainly 2Apro and C3pro or
3CDpro) to produce all the viral proteins needed for viral
replication. Some of the uncleaved precursors also have functions
during viral replication. Among the viral proteins synthesized are
the viral RNA-dependent RNA polymerase and accessory proteins
required for genome replication and mRNA synthesis. The first step
of genome replication is copying of the positive-stranded RNA to
generate a negative-stranded intermediate, which is used as a
template for synthesis of additional positive strands.
Encapsidation begins once sufficient capsid proteins have
accumulated.
[0065] Many picornaviruses produce characteristic morphologic
changes termed "cytopathic effects" in infected cells. Cytopathic
effects can include chromatin condensation, nuclear blebbing,
proliferation of membranous vesicles, leakage of intracellular
contents, and shriveling of the entire cell. In the case of many
picornavirus species, virions are released from infected cells as a
consequence of cell lysis. Other picornaviruses (e.g., hepatitis A
virus) are released from cells in the absence of cell lysis. In
some embodiments of the invention, cytopathic effect(s) and/or
virion release is assessed to determine whether a cell or subject
is infected with a virus. In some embodiments, cytopathic effect(s)
and/or virion release is assessed to determine whether a compound
inhibits a virus infection.
IV. PLA2G16 Polypeptides
[0066] PLA2G16 is an .about.18 kilodalton protein that is highly
expressed in vertebrate adipose tissue (especially white adipose
tissue) and is also expressed at lower levels in a wide variety of
vertebrate tissues and cultured cell lines. PLA2G16 is also known
as adipose-specific phospholipase A2 (AdPLA), HRAS-like suppressor
3 (HRASLA3), and by several other names. One of skill in the art
will readily be able to obtain PLA2G16 genomic and mRNA sequences
and the PLA2G16 protein from publicly available databases. The
human gene encoding PLA2G16 has been assigned GeneID: 11145 in the
Gene database of the National Center for Biotechnology Information
(NCBI; www.ncbi.nlm.nih.gov). Genes encoding PLA2G16 from mouse and
rat have been assigned the following Gene IDs: Gene ID: 225845 (Mus
musculus); Gene ID: 24913 (Rattus norvegicus). One of skill in the
art will readily be able to obtain the sequences of PLA2G16 mRNA
and protein from these and other species. For example, accession
numbers for the human PLA2G16 mRNA and protein Reference Sequences
available at the NCBI are as follows: NM.sub.--001128203
(transcript variant 2) and NP.sub.--001121675 (protein).
NM.sub.--007069.3 (transcript variant 1) and NP.sub.--009000.2
(protein). Transcript variant 1 represents the longer transcript.
Variants 1 and 2 encode the same isoform but differ in the 5'
untranslated region (UTR).
[0067] PLA2G16 has phospholipase activity and significantly lower
but detectable lysophospholipase activity (Duncan, R E, et al., J
Biol Chem., 283(37):25428-36, 2008). PLA.sub.2 proteins are enzymes
that catalyze hydrolysis of the sn-2 bond of phospholipids
(Schaloske, R H and Dennis, E A, Biochim. Biophys. Acta 1761,
1246-1259, 2006). PLA2G16 was shown to generate free fatty acid and
lysophospholipid from phosphatidylcholine with a preference for
hydrolysis at the sn-2 position, suggesting that the protein is a
PLA.sub.2 (Duncan, supra). PLA2G16 was found in association with
intracellular membranes and has a C-terminal presumed membrane
spanning domain whose deletion caused a loss of activity.
Mutational analysis showed that certain highly conserved amino
acids, including His-23, Cys-113, Gln-129 and Asn-112, were
essential for catalysis, but that mutation of Asp-30 or His-80 to
alanine had no effect. Thus PLA2G16 appears to contain His and Cys
active catalytic residues rather than a His/Asp catalytic diad or a
Ser/His/Asp catalytic triad as found in some other PLA.sub.2s.
Calcium was found to activate PLA2G16 but is not essential for
activity. Since PLA2G16 does not fit clearly into any of the
previously identified 15 major groups of PLA.sub.2 it was proposed
to be the first member of a distinct group of calcium-dependent
phospholipase A.sub.2s (Group XVI) (Duncan, supra).
[0068] PLA2G16 is also known in the art as adipocyte phospholipase
A2 (AdPLA) (Jaworski, K., et al. Nat Med., 15(2):159-68, 2009). It
is the major PLA.sub.2 in adipose tissue and plays an important
role in regulating adipocyte lipolysis. PLA2G16 null mice were
viable and had normal weight at weaning but gained weight more
slowly than wild-type littermates despite having equivalent food
intakes (Jaworski, K., supra). By standard pathology analysis
PLA2G16 null mice showed no evidence of any gross, microscopic, or
functional abnormalities, aside from reduced adiposity. Blood cell
profile and immunological parameters in serum and adipose tissue
were not changed in these mice compared to wild-type mice. These
results suggest that methods of the present invention that comprise
inhibiting PLA2G16 in order to inhibit viral infection are likely
to be well tolerated in isolated cells and in subjects of interest,
e.g., humans and other vertebrates.
[0069] In some embodiments, a "PLA2G16 polypeptide" is a
polypeptide whose sequence comprises or consists of the sequence of
a PLA2G16 polypeptide of a multicellular organism (e.g., a
vertebrate, e.g., a mammal, such as a human, mouse, rat, bovine,
etc.). A naturally occurring PLA2G16 polypeptide or a polypeptide
identical in sequence to a naturally occurring PLA2G16 polypeptide
is referred to as a "native PLA2G16 polypeptide" or simply
"PLA2G16" herein. Exemplary native PLA2G16 polypeptides are
depicted in FIG. 8 and under the accession numbers mentioned above.
In some embodiments, a PLA2G16 polypeptide is a variant of PLA2G16
("PLA2G16 variant"). PLA2G16 variants include polypeptides that
differ by one or more amino acid substitutions, additions, or
deletions, relative to PLA2G16. An addition can be an insertion
within the polypeptide or an addition at the N- or C-terminus. In
some embodiments, the number of amino acids substituted, deleted,
or added can be for example, about 1 to 30, e.g., about 1 to 20,
e.g., about 1 to 10, e.g., about 1 to 5, e.g., 1, 2, 3, 4, or 5. In
some embodiments, a PLA2G16 variant comprises a polypeptide whose
sequence is homologous to the sequence of PLA2G16 over at least 50
amino acids, at least 100 amino acids, at least 150 amino acids, or
over the full length of PLA2G16 (but is not identical in sequence
to native PLA2G16). In some embodiments, a PLA2G16 variant
comprises a polypeptide at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or more identical to PLA2G16 (e.g., from human,
mouse, rat, dog, cow) over at least 50%, 60%, 70%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or 100% of PLA2G16. In some embodiments, a
PLA2G16 variant comprises a polypeptide at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99%, or more identical to at least amino acids
23-113 of human or mouse PLA2G16. In some embodiments, a PLA2G16
polypeptide comprises a polypeptide at least 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or more identical to at least amino acids
23-129 of human or mouse PLA2G16.
[0070] In some embodiments, a PLA2G16 polypeptide comprises or
consists of a PLA2G16 fragment. A PLA2G16 fragment is a polypeptide
that is shorter than PLA2G16 and is identical to PLA2G16 over the
length of the shorter polypeptide. In some embodiments, a PLA2G16
fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99% as long as native PLA2G16. In some embodiments, a
fragment consists of amino acids 23-113 or 23-129 of human or mouse
PLA2G16. In some embodiments, one or more amino acids at the
C-terminus are deleted. For example, in some embodiments at least
the membrane spanning domain at the C-terminus is deleted. For
example, in some embodiments, at least the C-terminal 30 amino
acids are deleted. In some embodiments, one or more amino acids at
the N-terminus are deleted.
[0071] In some embodiments, a PLA2G16 polypeptide comprises a
heterologous polypeptide portion. The heterologous portion often
has a sequence that is not present in or homologous to native
PLA2G16. A heterologous portion may be, e.g., between 5 and about
5,000 amino acids long, or longer. Often it is between 5 and about
1,000 amino acids long. In some embodiments, a heterologous portion
comprises a sequence that is found in a different polypeptide,
e.g., a functional domain. In some embodiments, a heterologous
portion comprises a sequence useful for purifying, expressing,
solubilizing, and/or detecting the polypeptide. In some
embodiments, a heterologous portion comprises a polypeptide "tag",
e.g., an affinity tag or epitope tag. For example, the tag can be
an affinity tag (e.g., HA, TAP, Myc, 6.times.His, Flag, GST),
fluorescent or luminescent protein (e.g., EGFP, ECFP, EYFP,
Cerulean, DsRed, mCherry), solubility-enhancing tag (e.g., a SUMO
tag, NUS A tag, SNUT tag, or a monomeric mutant of the Ocr protein
of bacteriophage T7). See, e.g., Esposito D and Chatterjee D K.
Curr Opin Biotechnol.; 17(4):353-8 (2006). In some embodiments, a
tag can serve multiple functions. A tag is often relatively small,
e.g., ranging from a few amino acids up to about 100 amino acids
long. In some embodiments a tag is more than 100 amino acids long,
e.g., up to about 500 amino acids long, or more. In some
embodiments, a PLA2G16 polypeptide has a tag located at the N- or
C-terminus, e.g., as an N- or C-terminal fusion. The polypeptide
could comprise multiple tags. In some embodiments, a 6.times.His
tag and a NUS tag are present, e.g., at the N-terminus. In some
embodiments, a tag is cleavable, so that it can be removed from the
polypeptide, e.g., by a protease. In some embodiments, this is
achieved by including a sequence encoding a protease cleavage site
between the sequence encoding the portion homologous to PLA2G16 and
the tag. Exemplary proteases include, e.g., thrombin, TEV protease,
Factor Xa, PreScission protease, etc. In some embodiments, a
"self-cleaving" tag is used. See, e.g., PCT/US05/05763. Sequences
encoding a tag can be located 5' or 3' with respect to a
polynucleotide encoding the polypeptide (or both). In some
embodiments a tag or other heterologous sequence is separated from
the rest of the polypeptide by a polypeptide linker. For example, a
linker can be a short polypeptide (e.g., 15-25 amino acids). Often
a linker is composed of small amino acid residues such as serine,
glycine, and/or alanine. A heterologous domain could comprise a
transmembrane domain, a secretion signal domain, etc.
[0072] In some embodiments, a PLA2G16 variant is a functional
variant, i.e., the variant at least in part retains at least one
biological activity of PLA2G16. In some embodiments, a functional
variant retains sufficient activity to be distinguishable from a
non-homologous protein or catalytically inactive PLA2G16
polypeptide (e.g., a PLA2G16 polypeptide having a C113A
substitution) when used in an assay of the present invention. In
some embodiments, the activity is phospholipase A2 activity, e.g.,
as measured by ability to catalyze hydrolysis of the sn-2 bond of
phospholipids. In some embodiments, the activity is
lysophospholipase activity. In some embodiments, a PLA2G16 variant
retains the ability of native PLA2G16 to serve as a host cell
factor for a virus. For example, the PLA2G16 variant has sufficient
activity so that expressing it in a vertebrate cell that is
resistant to viral infection because the cell's PLA2G16 gene is
disabled (e.g., by a gene trap insertion) renders the cell
sensitive to viral infection. In some embodiments, a functional
PLA2G16 variant retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the activity of
PLA2G16, e.g., about equal activity. In some embodiments, a
functional variant may have greater activity than PLA2G16.
[0073] One of skill in the art can readily generate functional
PLA2G16 variants or fragments. As discussed above, considerable
information is available regarding PLA2G16, including
identification of various residues important for activity and
various residues that may be altered without significantly
decreasing activity, as well as alignments with other PLA2
polypeptides (see, e.g., Duncan, et al, supra). In some
embodiments, a PLA2G16 variant comprises one or more conservative
amino acid substitutions relative to PLA2G16. Conservative
substitutions may be made on the basis of similarity in side chain
size, polarity, charge, solubility, hydrophobicity, hydrophilicity
and/or the amphipathic nature of the residues involved. As known in
the art, such substitutions are, in general, more likely to result
in a variant that retains activity as compared with
non-conservative substitutions. In one embodiment, amino acids are
classified as follows:
Special: C
[0074] Neutral and small: A, G, P, S, T Polar and relatively small:
N, D, Q, E Polar and relatively large: R, H, K Nonpolar and
relatively small: I, L, M, V Nonpolar and relatively large: F, W,
Y
Special: C
[0075] See, e.g., Zhang, J. J. Mol. Evol. 50:56-68, 2000). In some
embodiments, proline (P) is considered to be in its own group as a
second special amino acid. Within a particular group, certain
substitutions may be of particular interest, e.g., replacements of
leucine by isoleucine (or vice versa), serine by threonine (or vice
versa), or alanine by glycine (or vice versa). Of course
non-conservative substitutions are often compatible with retaining
function as well. In some embodiments, a substitution or deletion
does not alter or delete an amino acid important for activity,
e.g., amino acid His-23, Cys-113, Gln-129 or Asn-112. In some
embodiments, a deletion does not remove all or a substantial
portion of the C-terminal 36 amino acids. For example, in some
embodiments, a deletion does not remove the transmembrane domain.
In some embodiments, an alteration is at an amino acid that differs
among PLA2G16 of different species. In some embodiments, a
substitution alters an amino acid to that present at a
corresponding position in a different species. In some embodiments,
a functional PLA2G16 variant comprises a polypeptide at least 95%,
96%, 97%, 98%, 99% or 100% identical to PLA2G16, e.g., over at
least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100%
of the full length of PLA2G16. In some embodiments, a functional
PLA2G16 variant comprises a polypeptide at least 95%, 96%, 97%,
98%, 99% or 100% identical to PLA2G16 e.g., over at least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of the full
length of PLA2G16, and comprises a tag at the N- and/or C-terminus.
PLA2G16 variants could be tested in cell-free and/or cell-based
assays to assess their activity.
[0076] In some embodiments, a variant or fragment of PLA2G16 that
has substantially reduced activity as compared with the activity of
native PLA2G16 (e.g., less than 10% of the activity of native
PLA2G16) is useful as a PLA2G16 inhibitor or antiviral compound.
For example, such polypeptide could interfere with the function of
native PLA2G16 in viral infection, e.g., by competing with native
PLA2G16. In some embodiments, a variant or fragment of PLA2G16 that
has substantially reduced activity as compared with the activity of
native PLA2G16 is useful a control or as an immunogen or for
crystallization or binding studies.
[0077] A PLA2G16 polypeptide, e.g., a native PLA2G16 polypeptide or
a PLA2G16 variant can be produced using standard recombinant DNA
techniques. A nucleic acid encoding PLA2G16 can readily be
obtained, e.g., from cells that express PLA2G16 (e.g., by PCR or
other amplification methods or by cloning) or by synthesis based on
a known PLA2G16 cDNA or polypeptide sequence. One of skill in the
art would know that due to the degeneracy of the genetic code,
numerous different nucleic acid sequences would encode the desired
polypeptide. Optionally, a sequence is codon-optimized for
expression in a host cell of choice. A nucleic that encodes a
PLA2G16 variant can readily be generated, e.g., by modifying native
PLA2G16 using, e.g., site-directed mutagenesis, or by other
standard methods.
[0078] A nucleic acid encoding the desired polypeptide, operably
linked to appropriate expression control elements, usually in a
vector such as a plasmid or virus (e.g., as part of the viral
genome), is introduced into prokaryotic or eukaryotic cells. In
other embodiments, a PLA2G16 polypeptide is produced using in vitro
translation. Exemplary cells include, e.g., bacterial cells (e.g.,
E. coli), insect cells, mammalian cells, plant cells, fungal cells
(e.g., yeast). One of skill in the art will be aware of suitable
expression control elements (e.g., promoters). Promoters may be
constitutive or regulatable, e.g., inducible or repressible.
Exemplary promoters suitable for use in bacterial cells include,
e.g., Lac, Trp, Tac, araBAD (e.g., in a pBAD vectors), phage
promoters such as T7 or T3. Exemplary expression control sequences
useful for directing expression in mammalian cells include, e.g.,
the early and late promoters of SV40, adenovirus or cytomegalovirus
immediate early promoter, or viral promoter/enhancer sequences,
retroviral LTRs, promoters or promoter/enhancers from mammalian
genes, e.g., actin, EF-1 alpha, metallothionein, etc. The
polyhedrin promoter of the baculovirus system is of use to express
proteins in insect cells. One of skill in the art will be aware of
numerous expression vectors that contain appropriate expression
control element(s), selectable markers, cloning sites, etc., and
can be conveniently used to express a polypeptide of interest.
Optionally, such vectors include sequences encoding a tag, to allow
convenient production of a polypeptide comprising a tag. Suitable
methods for introducing vectors into bacteria, yeast, plant, or
animal cells (e.g., transformation, transfection, infection,
electroporation, etc.), and, if desired, selecting cells that have
taken up the vector and deriving stable cell lines. Transgenic
animals or plants that express the polypeptide could be produced
using methods known in the art.
[0079] To produce a PLA2G16 polypeptide, cells are maintained in
culture for a suitable time period, and the polypeptide is isolated
and optionally further purified. (Of course a PLA2G16 polypeptide
could also be isolated from cells or tissues obtained directly from
an organism that expresses it.) Standard protein
isolation/purification techniques can be used. In some embodiments,
affinity-based methods are used. For example, an antibody to
PLA2G16 can be employed. In the case of tagged PLA2G16
polypeptides, an appropriate isolation method can be selected
depending on the particular tag used.
V. Compositions and Methods for Inhibiting PLA2G16
[0080] The term "PLA2G16 inhibitor" refers to a compound that
inhibits PLA2G16 expression and/or inhibits one or more activities
of PLA2G16. For example, a compound is "PLA2G16 inhibitor" if one
or more PLA2G16 activities is reduced in the presence of the
compound as compared with its absence and/or if the level or amount
of PLA2G16 protein or gene product is reduced in the presence of
the compound as compared with its absence. In certain embodiments,
PLA2G16 inhibitors act directly on PLA2G16 in the sense that they
physically interact with PLA2G16. In other embodiments, inhibitors
act indirectly on PLA2G16. A PLA2G16 inhibitor can be, e.g., a
small molecule, nucleic acid, oligonucleotide, polypeptide,
peptide, lipid, phospholipid, etc. In some embodiments, a PLA2G16
inhibitor is an RNAi agent, antisense oligonucleotide, aptamer, or
antibody. In some embodiments, a PLA2G16 inhibitor is a small
molecule.
[0081] The invention provides a number of different methods of
inhibiting PLA2G16. As used herein, methods of inhibiting PLA2G16
encompass methods that result in a decreased amount of PLA2G16
polypeptide and methods that interfere with PLA2G16 molecular
function. In some embodiments, PLA2G16 is inhibited by inhibiting
or interfering with PLA2G16 expression, so that a decreased amount
of PLA2G16 polypeptide is produced. As used herein, "expression"
encompasses the cellular processes involved in producing a
polypeptide and include transcription, mRNA processing and
transport (in the case of eukaryotic cells), and mRNA translation.
A variety of methods useful for inhibiting or interfering with
expression can be applied in embodiments of the present invention.
In general, such methods result in decreased synthesis of PLA2G16
polypeptide and as a result, a reduction in the total level of
PLA2G16 molecular functional activity present.
[0082] In some embodiments, PLA2G16 expression is inhibited using
RNA interference (RNAi). Exemplary sequences for RNAi agents (e.g.,
siRNAs) that inhibit PLA2G16 expression are provided in the
Examples. Additional sequences can be selected using various
approaches known in the art including. If desired, such sequences
can be selected to minimize "off-target" effects. In some
embodiments, position-specific chemical modification is used to
reduce potential off-target effects. In some embodiments, at least
two different siRNAs targeted to the PLA2G16 gene are used (e.g.,
in combination). RNAi is use of herein for a variety of purposes.
For example, an RNAi agent can be used as a PLA2G16 inhibitor,
e.g., for therapeutic or research purposes. An RNAi agent that
inhibits PLA2G16 can be useful to confirm that the effect of a
second compound, e.g., a small molecule, is due to an effect on
PLA2G16 (rather than on another protein). For example, a small
molecule that is a putative specific inhibitor of PLA2G16 may be
expected not to have an effect in a cell in which PLA2G16
expression is inhibited by RNAi. In other aspects, RNAi is used to
inhibit expression of a PLA2 other than PLA2G16, which may be
expressed by a cell. Inhibiting other PLA2 enzymes may facilitate
identification of compounds that inhibit PLA2G16.
[0083] In some embodiments of the invention, PLA2G16 expression is
inhibited using an antisense approach in which one or more
oligonucleotides complementary to mRNA encoding PLA2G16 is
delivered to cells and hybridizes to the PLA2G16 mRNA resulting in,
e.g., degradation of the mRNA by RNase H or blockage of translation
by steric hindrance.
[0084] In some embodiments of the invention, a PLA2G16 inhibitor
inhibits at least one molecular function of PLA2G16. In some
embodiments, the molecular function is a catalytic activity, e.g.,
phospholipase activity and/or lysophospholipase activity. For
example, the activity may be phospholipase A2 activity, i.e.,
ability to catalyze hydrolysis of the sn-2 bond of phospholipids.
In some embodiments, a compound directly inhibits a molecular
function of PLA2G16. "Direct inhibition" refers to a physical
interaction (binding) with a target that inhibits a molecular
function of the target. For example, binding of a PLA2G16 inhibitor
to PLA2G16 can interfere with the enzyme's ability to catalyze a
reaction and/or prevent a substrate from entering the active site.
A variety of compounds can be used to directly inhibit PLA2G16
molecular function. Exemplary compounds that directly inhibit
PLA2G16 can be, e.g., small molecules, antibodies, or aptamers. In
some embodiments, a direct inhibitor is a substrate analog (e.g., a
phospholipid analog) or a transition state analog.
[0085] In some embodiments, an inhibitor is an irreversible
inhibitor. Most irreversible enzyme inhibitors react with the
enzyme and change it chemically, such as by modifying amino acid
residue(s) that are needed for enzymatic activity. For example, an
irreversible inhibitor can comprise one or more reactive functional
groups such as an aldehyde, haloalkane, alkene, fluorophosphonate
(e.g., alkyl fluorophosphonate), Michael acceptor, phenyl
sulfonate, methylketone, e.g., a halogenated methylketone or
diazomethylketone, fluorophosphonate, vinyl ester, vinyl sulfone,
or vinyl sulfonamide. In some embodiments, an irreversible PLA2G16
inhibitor comprises an electrophilic group that reacts with an
amino acid side chain of PLA2G16. For example, the electrophilic
group may react with an amino acid side chain containing a
nucleophile such as a hydroxyl or sulfhydryl group. For example,
the amino acid may be cysteine, serine, or threonine. In another
embodiment, an irreversible inhibitor reacts with a histidine.
Moieties sometimes referred to in the art as "cysteine traps" may
be used in various embodiments. In some embodiments a
cysteine-reactive moiety is a maleimide, isothiazolinone,
tetrazole, lactam, or carbamate. A reactive functional group can be
incorporated into a substrate analog or other molecule compatible
with binding to the enzyme, e.g., in or near the active site.
[0086] In other embodiments, a PLA2G16 inhibitor is a reversible
inhibitor. Reversible inhibitors bind non-covalently and may bind
to the enzyme, the enzyme-substrate complex, or both. Inhibition by
a reversible inhibitor may be classified as competitive inhibition,
uncompetitive inhibition, mixed inhibition, non-competitive
inhibition. See, e.g., Berg J. M, et al., Biochemistry, 6.sup.th
ed., W. H. Freeman and Company, 2007. In some embodiments, a
reversible inhibitor binds to the PLA2G16 active site and/or
competes with substrate(s) for access to the PLA2G16 active site.
In some embodiments a reversible inhibitor is a non-hydrolyzable
substrate analog.
[0087] In some embodiments, the PLA2G16 inhibitor is an analog of a
fatty acid, wherein the analog comprises an alkyl chain between 4
and about 30 carbons long, e.g., between 12 and 20 carbons long. In
some embodiments, the alkyl group is saturated. In some
embodiments, the alkyl group is unsaturated. In some embodiments,
the alkyl group is unbranched. In some embodiments, the alkyl group
has the structure of an alkyl group naturally found in a fatty acid
present in vertebrate cells. Exemplary fatty acids include, e.g.,
myristoleic acid, palmitoleic acid, sapienic acid, oleic acid,
linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic
acid, erucic acid, docosahexaenoic acid, lauric acid, myristic
acid, palmitic acid, stearic acid, and eicosanoic acid. In some
embodiments, the PLA2G16 inhibitor is an analog of arachidonic acid
or linoleic acid. In some embodiments, the analog is a methylated
fatty acid. In some embodiments, the arachidonic acid analog is an
eicosadienoic acid, such as 7,7-dimethyl-5,8-eicosadienoic
acid.
[0088] In some embodiments, the inhibitor comprises an analog of a
fatty acid, wherein the analog comprises a reactive functional
group. In some embodiments, the fatty acid analog comprises a
halogenated methyl ketone group instead of a carboxyl group. For
example, the halogen can be chlorine, fluorine, bromine, or iodine
in various embodiments. In some embodiments, the halogenated methyl
ketone group is fluoromethyl ketone, trifluoromethyl ketone, or
chloromethyl ketone. In some embodiments, the fatty acid is
arachidonic acid. In one embodiment, the inhibitor is a
trifluoromethyl ketone analog of arachidonic acid in which the COOH
group is replaced with COCF3, i.e., the compound arachidonyl
trifluoromethyl ketone (AACOCF3). AACOCF3 inhibits PLA2G16 and also
inhibits cPLA.sub.2 and sPLA.sub.2 (Duncan, supra). It is believed
that AACOCF3 binds in a hydrophobic pocket of cPLA.sub.2, and that
the carbonyl group of AACOCF3 forms a covalent bond with serine 228
in the active site (Street et al., 1993; Trimble et al., 1993).
Without wishing to be bound by theory, AACOCF3 may react with the
active site serine of PLA2G16. In another embodiment, the PLA2G16
inhibitor is methyl arachidonyl fluorophosphate (MAFP) or an analog
thereof (see, e.g., Martin, B R, et al., J. Pharm. Exp. Ther., 294
(3), 294:1209-1218, 2000). MAFP is believed to inhibit serine and
cysteine hydrolases by covalently binding to the enzyme. It
inhibits PLA2G16 as well as iPLA2 and cPLA2, but not sPLA2 (Duncan,
supra). Without wishing to be bound by theory, PLA2G16 may require
an active cysteine residue (i.e. Cys-113) that is inactivated by
MAFP.
[0089] A variety of other compounds that inhibit one or more Group
I-XV PLA2s have been identified. For example, U.S. Pat. Pub. No.
20080319065 discloses compounds that contain a 2-oxoamide with a
hydrocarbon tail and a four carbon tether and are reported to
inhibit PLA2 Group IVA c PLA2 and/or Group VIA iPLA2 and/or Group V
sPLA2. U.S. Pat. Pub. Nos. 20030144282 and 20100029645 disclose
inhibitors of various PLA2 enzymes. Other compounds that inhibit
one or more PLA2 enzymes include piperazines (see, e.g.,
WO2003048139); pyrimidone, pyridone, pyridinone, and pyrimidinone
compounds (see, e.g., WO2002030904; WO 2001060805; WO 2000027824;
WO 2003087088; WO 2003086400; WO 2003/042218; WO2003042206;
WO2002030911; WO2003041712), pyrrolidine derivatives (see, e.g.,
WO1998033797). Without wishing to be bound by any theory, at least
some of the compounds that inhibit one or more Group I-XV PLA2
enzymes may also inhibit PLA2G16. In some embodiments, the main
mechanism of action against such other Group I-XV PLA.sub.2 does
not involve specifically binding to a sequence motif that is
present in such other PLA2 but is absent in PLA2G16. For example,
in some embodiments the compound is not whose main mechanism of
action involves binding to a GXSXG consensus motif or a CCXXHDXC
motif.
[0090] In some embodiments, a PLA2G16 inhibitor comprises a
peptide, e.g., a peptide identified using a display technique, such
as phage display or ribosome display. In some embodiments, a
peptide comprises one or more non-standard amino acids. In some
embodiments, a peptide is cyclic. For example, the peptide can be
cyclized via a disulfide bond or covalent linkage, e.g., between
the N- and C-terminal amino acids, between the N- or C-terminal
amino acid an internal amino acid, or between two internal amino
acids.
[0091] In some embodiments, a PLA2G16 inhibitor comprises an
aptamer. In general, an aptamer is an often single-stranded
oligonucleotide (e.g., DNA or RNA, optionally containing one or
more non-standard nucleotides or modifications such as 2'-fluoro,
2'-amino, and/or 2'-methoxy nucleotides) that binds to a particular
molecule of interest. Aptamers are typically derived from an in
vitro evolution and selection process such as SELEX. Methods for
obtaining aptamers specific for a protein of interest are known in
the art. See, e.g., Brody E N, Gold L. J Biotechnol., 74(1):5-13,
2000.
[0092] In some embodiments, a PLA2G16 inhibitor comprises an
antibody or portion thereof. In some embodiments, the antibody is a
single-chain antibody, diabody, triabody, or minibody. Standard
methods of antibody production known in the art can be used to
produce an antibody, e.g., a monoclonal antibody, that binds to
PLA2G16. In some embodiments, an animal, e.g., a mouse or rabbit is
immunized with PLA2G16 or a portion thereof, antibody producing
cells are isolated, and a monoclonal antibody is identified using
hybridoma technology. In some embodiments, the mouse is a
transgenic mouse comprising at least some unrearranged human
immunoglobulin gene sequences and that preferably have a targeted
disruption of endogenous heavy and light chain murine sequences. In
some embodiments, an antibody is identified or produced using
recombinant nucleic acid technology (e.g., phage or yeast display).
See, e.g., Lonberg N. Fully human antibodies from transgenic mouse
and phage display platforms. Curr Opin Immunol. 20(4):450-9,
2008.
[0093] In some embodiments of the invention, a compound indirectly
inhibits PLA2G16. "Indirect inhibition" refers to inhibition of a
target (e.g., PLA2G16) by a mechanism that does not require
physical interaction between the compound and the target. For
example, the compound could inhibit expression or activity of a
polypeptide that is involved in localization or post-translational
modification of PLA2G16, wherein such localization or
post-translational modification is important for PLA2G16 molecular
function.
[0094] In some embodiments, a PLA2G16 inhibitor is not a compound
that is known or suggested in the art to have antiviral activity
and/or to be useful in treating a subject in need of treatment for
a viral infection. In some embodiments, a PLA2G16 inhibitor is a
compound that is known or suggested in the art to have antiviral
activity and/or to be useful in treating a subject in need of
treatment for a viral infection but, optionally, may be
administered or otherwise used in the present invention (i) to
inhibit infection by a different virus, e.g., a virus against which
the compound is not known or suggested to have antiviral activity;
(ii) in a different (e.g., more highly purified) form, in a
different amount or composition, or in combination with one or more
different substances; (iii) by a different route or to a subject of
a different species; and/or (iv) explicitly excluded from any one
or more of the inventive compositions and/or methods.
VI. Compositions and Methods for Identifying and/or Testing
Compounds
[0095] The invention provides methods of identifying compounds
useful for inhibiting viral infection and assay systems for
performing the inventive methods. In some aspects, the invention
provides a method of determining whether a test compound is a
candidate antiviral compound, the method comprising the step
determining whether the test compound inhibits PLA2G16 polypeptide,
wherein if the compound inhibits PLA2G16, the compound is a
candidate antiviral compound. In a related aspect, the invention
provides a method of identifying a candidate antiviral compound
comprising steps of: (a) providing a test compound; (b) determining
whether the test compound inhibits PLA2G16, wherein if the compound
inhibits PLA2G16, the compound is a candidate antiviral
compound.
[0096] In some embodiments, the method comprises determining
whether the test compound inhibits expression of PLA2G16, wherein
if the compound inhibits PLA2G16 expression the compound is a
candidate antiviral compound. In some embodiments, the method
comprises determining whether the compound inhibits a molecular
function of PLA2G16, wherein if the compound inhibits a molecular
function of PLA2G16 the compound is a candidate antiviral compound.
In some embodiments, the molecular function is an enzymatic
activity, e.g., phospholipase A2 activity or lysophospholipase
activity.
[0097] In some embodiments, a method is performed using a PLA2G16
polypeptide identical in sequence to PLA2G16 that is naturally
expressed by a multicellular organism, i.e., a native PLA2G16. In
some embodiments, a method is performed using a functional PLA2G16
variant. In some embodiments, the functional variant used in an
inventive assay retains at least 20%, 30%, 40%, 50%; 60%. 70%, 80%,
90%, 95%, 96%, 97%, 98%, 99%, or more of the phospholipase A2
activity of PLA2G16. In some embodiments, a functional variant
retains at least 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99%, or more of the lysophospholipase activity of
PLA2G16. In some embodiments, the functional variant retains at
least 50% or at least 75% or has about the same phospholipase A2
activity and/or lysophospholipase activity as native PLA2G16. A
PLA2G16 variant may have properties that make it convenient to use
in an inventive screening method, such as the presence of a tag
that facilitates production or purification of the protein. A
compound identified as an inhibitor using a PLA2G16 variant can be
further tested using native PLA2G16 to confirm its ability to
inhibit the native polypeptide.
[0098] A wide variety of test compounds can be used in the
inventive methods. For example, a test compound can be a small
molecule, polypeptide, peptide, nucleic acid, oligonucleotide,
lipid, carbohydrate, or hybrid molecule. Compounds can be obtained
from natural sources or produced synthetically. Compounds can be at
least partially pure or may be present in extracts or other types
of mixtures. Extracts or fractions thereof can be produced from,
e.g., plants, animals, microorganisms, marine organisms,
fermentation broths (e.g., soil, bacterial or fungal fermentation
broths), etc. In some embodiments, a compound collection
("library") is tested. The library may comprise, e.g., between 100
and 500,000 compounds, or more. Compounds are often arrayed in
multwell plates. They can be dissolved in a solvent (e.g., DMSO) or
provided in dry form, e.g., as a powder or solid. Collections of
synthetic, semi-synthetic, and/or naturally occurring compounds can
be tested. Compound libraries can comprise structurally related,
structurally diverse, or structurally unrelated compounds.
Compounds may be artificial (having a structure invented by man and
not found in nature) or naturally occurring. In some embodiments, a
library comprises at least some compounds that have been identified
as "hits" or "leads" in other drug discovery programs and/or
derivatives thereof. A compound library can comprise natural
products and/or compounds generated using non-directed or directed
synthetic organic chemistry. Often a compound library is a small
molecule library. Other libraries of interest include peptide or
peptoid libraries, cDNA libraries, and oligonucleotide
libraries.
[0099] A library can be focused (e.g., composed primarily of
compounds having the same core structure, derived from the same
precursor, or having at least one biochemical activity in common).
In some embodiments, compounds that have been identified as
inhibitors of one or more Group I-XV PLA2 enzymes are tested. In
some embodiments, the IC50 of a compound identified as a PLA2G16
inhibitor may be about 2, 5, 10, 20, 50, 100, 250, 500, or
1000-fold lower for PLA2G16 versus one or more other PLA2 enzymes
(e.g., one, more than one, or all other PLA2 enzymes present in
humans and known to date).
[0100] Compound libraries are available from a number of commercial
vendors such as Tocris BioScience, Nanosyn, BioFocus, and from
government entities. For example, the Molecular Libraries Small
Molecule Repository (MLSMR), a component of the U.S. National
Institutes of Health (NIH) Molecular Libraries Program is designed
to identify, acquire, maintain, and distribute a collection of
>300,000 chemically diverse compounds with known and unknown
biological activities for use, e.g., in high-throughput screening
(HTS) assays (see https://mli.nih.gov/mli/). The NIH Clinical
Collection (NCC) is a plated array of approximately 450 small
molecules that have a history of use in human clinical trials.
These compounds are highly drug-like with known safety profiles.
The NCC collection is arrayed in six 96-well plates. 50 .mu.l of
each compound is supplied, as an approximately 10 mM solution in
100% DMSO. In some embodiments, a collection of compounds
comprising "approved human drugs" is tested. An "approved human
drug" is a compound that has been approved for use in treating
humans by a government regulatory agency such as the US Food and
Drug Administration, European Medicines Evaluation Agency, or a
similar agency responsible for evaluating at least the safety of
therapeutic agents prior to allowing them to be marketed. The test
compound may be, e.g., an antineoplastic, antibacterial, antiviral,
antifungal, antiprotozoal, antiparasitic, antidepressant,
antipsychotic, anesthetic, antianginal, antihypertensive,
antiarrhythmic, antiinflammatory, analgesic, antithrombotic,
antiemetic, immunomodulator, antidiabetic, lipid- or
cholesterol-lowering (e.g., statin), anticonvulsant, anticoagulant,
antianxiety, hypnotic (sleep-inducing), hormonal, or anti-hormonal
drug, etc. In some embodiments, a compound is one that has
undergone at least some preclinical or clinical development or has
been determined or predicted to have "drug-like" properties. For
example, the test compound may have completed a Phase I trial or at
least a preclinical study in non-human animals and shown evidence
of safety and tolerability. In some embodiments, a test compound is
substantially non-toxic to cells of an organism to which the
compound may be administered or cells in which the compound may be
tested, at the concentration to be used or, in some embodiments, at
concentrations up to 10-fold, 100-fold, or 1,000-fold higher than
the concentration to be used. For example, there may be no
statistically significant effect on cell viability and/or
proliferation, or the reduction in viability or proliferation can
be no more than 1%, 5%, or 10% in various embodiments. Cytotoxicity
and/or effect on cell proliferation can be assessed using any of a
variety of assays (some of which are mentioned above). In some
embodiments, a test compound is not a compound that is found in a
cell culture medium known or used in the art, e.g., culture medium
suitable for culturing vertebrate, e.g., mammalian cells or, if the
test compound is a compound that is found in a cell culture medium
known or used in the art, the test compound is used at a different,
e.g., higher, concentration when used in a method of the present
invention.
[0101] In some embodiments, a test compound is a compound that is
recognized in the art as having antiviral activity against one or
more viruses, but that is not known to be useful to inhibit
infection by a virus of interest, e.g., a picornavirus. In some
embodiments, a test compound is not a compound that is recognized
in the art as having antiviral activity.
[0102] In some embodiments, one or more compounds or mixtures
thereof having known antiviral activity is tested, wherein the
molecular target of the compound or mixture and/or mechanism of
antiviral activity is unknown. Testing of such compounds or
mixtures according to the present invention to determine whether
they inhibit PLA2G16 may lead to discovering that PLA2G16 is the
molecular target. Such discovery may facilitate purification of an
active component from a mixture, development of more highly active
derivatives of the compound, and/or otherwise permit further
development of the compound or mixture as a therapeutic agent.
[0103] The step of determining whether a test compound inhibits
PLA2G16 expression can be carried out in a variety ways. Compounds
that inhibit PLA2G16 expression can be identified by contacting
cells with a test compound, maintaining the cells in culture for a
suitable period of time (e.g., sufficient time to allow degradation
of existing PLA2G16 mRNA and/or protein), and then measuring the
level of PLA2G16 mRNA or protein. Methods known in the art can be
used for measuring mRNA or protein. A variety of different
hybridization-based or amplification-based methods are available to
measure RNA. Examples include Northern blots, microarray (e.g.,
oligonucleotide or cDNA microarray), reverse transcription (RT)-PCR
(e.g., quantitative RT-PCR), or reverse transcription followed by
sequencing. The TaqMan.RTM. assay and the SYBR.RTM. Green PCR assay
are commonly used real-time PCR techniques. Other assays include
the Standardized (Sta) RT-PCR.TM. (Gene Express, Inc., Toledo,
Ohio) and QuantiGene.RTM. (Panomics, Inc., Fremont, Calif.). In
some embodiments the level of PLA2G16 mRNA is measured. In other
embodiments, a reporter-based system is used. In some embodiments,
a reporter-based system comprises a nucleic acid in which
expression control elements of the PLA2G16 gene are operably linked
to a sequence that encodes a reporter molecule ("reporter").
Reporters are often proteins but could be nucleic acids. Reporters
are often readily detectable molecules, such as proteins that
produce a fluorescent, luminescent, or colorimetric signal or are
capable of absorbing light of a particular wavelength. In some
embodiments, a reporter molecule comprises an enzyme that acts on a
substrate to produce a fluorescent, luminescent, or colorimetric
signal. Exemplary reporter molecules include, e.g., green, blue,
sapphire, yellow, red, orange, and cyan fluorescent proteins and
derivatives thereof; monomeric red fluorescent protein and
derivatives such as those known as "mFruits", e.g., mCherry,
mStrawberry, mTomato; luciferase; beta-galactosidase; horseradish
peroxidase; alkaline phosphatase; etc. In some embodiments, a
reporter is a secreted protein. In some embodiments, a reporter is
encoded by a sequence that is codon-optimized for expression in a
cell from an organism of interest. Methods for assessing the
efficacy of an RNAi agent to silence expression of a target gene
can involve use of a sequence in which the mRNA target of an shRNA
or siRNA (or a portion of the target) is cloned downstream of a
sequence that encodes a reporter, so that a bicistronic mRNA
transcript encoding both the target sequence and the reporter is
produced. Target gene knockdown results in the degradation (or
translational inhibition) of the mRNA transcript, which causes a
proportional decrease in the expression of the reporter
protein.
[0104] Compounds that inhibit PLAG16 molecular function can be
identified using a variety of different cell-free or cell-based
assays. A cell-free assay typically involves an isolated target
molecule. For example, the target molecule could be present in a
cell or tissue lysate or fraction thereof (e.g., a lysate made from
cells that express the target molecule) or could be an at least
partially purified or synthesized target molecule. A tissue lysate
could be made from any tissue containing cells that express
PLA2G16. In some embodiments, a tissue lysate is obtained from
adipose tissue, e.g., white adipose tissue. Various cells from
which a cell lysate could be prepared or from which a PLA2G16
polypeptide could be purified are mentioned below in the discussion
of cell-based assays. In some embodiments, an isolated polypeptide
is a polypeptide that has been synthesized using recombinant
nucleic acid techniques or in vitro translation. In order to
perform the assay, a test compound is contacted with the target
molecule, e.g., by preparing a composition comprising the test
compound and the target molecule. One or more parameters are
measured, e.g., binding, enzymatic activity, etc. The composition
can comprise other component(s) necessary or helpful for
identifying a compound of interest. In some embodiments, a
composition for use in a binding assay or activity assay comprises
cell membranes or cell membrane components. Such membranes or
components may be naturally occurring (e.g., components present in
animal cell membranes), articifical, or combination thereof in
various embodiments. For example, the composition can contain a
lipid membrane bilayer, lipid vesicles, etc. Optionally, a lipid
bilayer is immobilized on a surface. In some embodiments the lipids
comprise phospholipids.
[0105] A variety of cell-free assays may be performed to identify
compounds that inhibit a PLA2G16 polypeptide. In some embodiments,
an assay detects whether a test compound binds to a PLA2G16
polypeptide and/or quantifies one or more characteristics of such
binding. Numerous binding assay formats are known in the art. In
some embodiments, a label-free assay is used, while in other
embodiments either the PLA2G16 polypeptide or test compound is
detectably labeled. In some embodiments, a PLA2G16 polypeptide or a
compound to be tested for ability to bind to and/or inhibit
activity of a PLA2G16 polypeptide is attached to a solid support.
In some embodiments, a solid support is an article having a rigid
or semi-rigid surface. In some embodiments, at least one surface of
the support is substantially flat. In other embodiments, a support
is approximately spherical. A support can be composed of an
inorganic or organic material or combination thereof. In some
embodiments, a support is composed at least in part of a metal,
ceramic, glass, plastic, gel, or other matrix. Such articles may,
for example, take the form of plates (e.g., multiwell plates),
slides, particles (e.g., "beads", e.g., magnetic beads), pellets,
bars, rods, pins, disks, chips, filters, or other suitable forms.
In some embodiments, a support comprises a sensor, e.g., a sensor
capable of detecting changes in binding. For example, the sensor
could detect a change in weight or a signal such as fluorescence.
In some embodiments, the support comprises an electrode. In some
embodiments, compounds are arranged as a small molecule microarray.
Compounds could be present in multiple locations on a surface, in
individual wells or vessels, etc. See, e.g., Vegas A J, et al.,
Chem Soc Rev. 37(7):1385-94, 2008. In some embodiments, a PLA2G16
polypeptide or compound is noncovalently attachment or covalently
linked to the support. Noncovalent attachment could be, e.g., by
adsorption of the polypeptide or compound to the surface (which may
be coated with a substance to facilitate such adsorption), via an
affinity-based mechanism, or other means of immobilizing the
PLA2G16 polypeptide or test compound so that it remains physically
associated with the support. In some embodiments, an antibody is
used to attach a PLA2G16 polypeptide or test compound to a support.
In some embodiments, a PLA2G16 polypeptide or test compound is
attached to a support via a biotin-avidin interaction or other
strong binding interaction, wherein one of two binding partners is
attached directly or indirectly to the support and the other
binding partner is attached to the molecule to be immobilized.
[0106] In some embodiments, test compounds are immobilized in
multiple locations (e.g., in an array format. PLA2G16 polypeptide
is added and the composition is maintained for a suitable time
period to allow binding to occur. In some embodiments, unbound
material is removed by washing, and PLA2G16 polypeptide is detected
using an antibody or, if the polypeptide is detectably labeled, by
detecting a signal. In other embodiments, a washing step is not
necessary. For example, binding may be detected by measuring a
change in fluorescence polarization, fluorescence resonance energy
transfer, or electrochemiluminescence. In other embodiments,
PLA2G16 polypeptide is immobilized, test compounds are added, and
binding is measured using similar approaches.
[0107] In some embodiments, surface plasmon resonance (SPR) is used
to measure kinetics (on and/or off rates) and/or binding strength
(affinity) between a test compound and a PLA2G16 polypeptide. For
example, using SPR technology (e.g., systems such as those
available from Biacore, Life Sciences, GE Healthcare) the binding
and dissociation of a test compound to a protein immobilized on a
chip can be measured, and the measured values compared with those
obtained when a solution not containing the test compound is loaded
on the chip. A test compound capable of binding to the protein can
be selected on the basis of the binding and dissociation rate
and/or binding level. Other useful methods for detecting and/or
quantifying binding include use of a quartz crystal microbalance,
optical cantilever, microchannel resonator, dual polarisation
interferometer, coupled waveguide plasmon resonance,
immunoprecipitation or other antibody-based detection methods,
isothermal titration and differential scanning calorimetry,
capillary electrophoresis, resonance energy transfer,
electrochemiluninesce, and fluorescent correlation analysis.
[0108] In some embodiments, an aptamer, peptide, non-hydrolyzable
substrate analog, or small molecule that is known to bind to a
PLA2G16 polypeptide is labeled and used as a tool for screening
test compounds (e.g., small molecules) for ability to bind to
and/or inhibit activity of the polypeptide. The label can comprise,
e.g., a radioactive, fluorescent, luminescent, or other readily
detectable moiety. The ability of a test compound to compete with
the labeled aptamer, peptide, non-hydrolyzable substrate analog, or
small molecule can be detected and serves as an indicator of the
binding of the test compound to the PLA2G16 polypeptide. For
example, a scintillation proximity assay (SPA) can be used. In some
embodiments of an SPA for identifying compounds that bind to a
PLA2G16 polypeptide, the PLA2G16 polypeptide is attached to beads
containing a scintillant material. The beads are typically located
in wells or other vessels. In another embodiment, a PLA2G16
polypeptide is attached to scintillant material is embedded
directly into wells. A radiolabelled compound capable of binding to
the PLA2G16 polypeptide and a test compound are added to the well.
Binding of the radiolabelled compound to the PLA2G16 polypeptide
results in a signal. The signal is reduced in the presence of a
test compound that competes with the radiolabelled compound for
binding. See, e.g., J. Fraser Glickman, et al., Scintillation
Proximity Assays in High-Throughput Screening. Assay and Drug
Development Technologies. 6(3): 433-455, 2008, for review of SPA.
Similar assays can be performed using filters.
[0109] In some embodiments, a compound is selected that binds to
PLA2G16 polypeptide with a Kd equal to or less than approximately 1
mM, 500 .mu.M, 100 .mu.M, 50 .mu.M, 10 .mu.M, 5 .mu.M, or 1 .mu.M.
In some embodiments, a compound binds to a PLA2G16 polypeptide with
a Kd equal to or less than approximately 500 nM, 100 nM, 50 nM, or
10 nM. In some embodiments, a compound binds to a PLA2G16
polypeptide with a Kd between 0.1-10 nM. Compounds that bind to a
PLA2G16 polypeptide may be further tested, e.g., in cell-free or
cell-based assays, to determine the extent to which they inhibit
PLA2G16 activity (e.g., catalytic activity), e.g., as described
below.
[0110] A variety of different assays can be employed to identify
and/or characterize compounds that inhibit PLA2G16 activity. In
some embodiments, the ability of a compound to inhibit catalysis of
a chemical reaction by PLA2G16 polypeptide is assessed. In some
embodiments, the chemical reaction is hydrolysis of the sn-2 bond
of a phospholipid. A composition comprising a PLA2G16 polypeptide,
one or more PLA2G16 substrate(s), and a test compound is provided.
The PLA2G16 polypeptide, one or more PLA2G16 substrate(s), and test
compound are usually in a suitable liquid medium. In some
embodiments, the liquid medium is an aqueous medium that comprises
at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more
water (v/v). In some embodiments, the liquid medium may comprise an
organic solvent such as DMSO, e.g., in an amount that does not
significantly affect the activity of the PLA2G16 polypeptide as
compared with the activity in the absence of the organic solvent. A
"substrate" in this context is a molecule on which PLA2G16 acts,
i.e., a molecule that undergoes a chemical reaction that is
catalyzed by PLA2G16. Exemplary substrates are discussed below. The
concentration of the substrate and PLA2G16 polypeptide can vary. In
some embodiments, the substrate is present at between about 1 .mu.M
and 500 .mu.M, e.g., between about 10 .mu.M and about 50 .mu.M, 100
.mu.M, or 200 .mu.M. In some embodiments, the PLA2G16 polypeptide
is present at between 1 .mu.g/ml and about 100 .mu.g/ml. It will be
understood that the selection of concentrations and amounts can
depend at least in part on the particular assay and is within the
skill in the art. The composition is maintained for a suitable time
period under conditions that would otherwise (i.e., in the absence
of a compound that is a potential PLA2G16 inhibitor) be appropriate
for the PLA2G16 polypeptide to catalyze a reaction in which the
substrate(s) is/are converted to one or more product(s). The
reaction may be stopped after a desired time period, e.g., by
addition of (2:1) methanol:chloroform. The conditions and other
component(s) present in the composition can vary depending, e.g.,
on the particular assay. Suitable conditions for a PLA2G16
polypeptide to act on a substrate can include, e.g., a pH of
between about 6.5 and about 9.5, e.g., between about 7.0 and about
9.0, e.g., between about 7.5 and about 8.5, e.g., about 8.0. In
some embodiments, the temperature is between 10.degree. C. and
40.degree. C., e.g., between 20.degree. C. and 30.degree. C., e.g.,
about 25.degree. C. Other components may be present in the
composition. In some embodiments, the composition comprises a
buffer substance such as Tris-HCl or sodium borate, to help
regulate the pH. Other buffer substances include, e.g., HEPES,
MOPS, etc. In some embodiments, the composition comprises a
divalent cation, e.g., calcium (Ca2+). For example, in some
embodiments, the composition comprises up to about 5 mM calcium,
e.g., between about 0.5 mM and about 2.5 mM calcium. In exemplary
embodiments, the composition comprises about 1 mM calcium or about
2 mM calcium. In some embodiments, the composition does not
comprise a calcium chelator such as EDTA. In some embodiments, the
composition comprises a calcium chelator in an amount that does not
reduce the free calcium concentration below about 1.0 mM. In some
embodiments, the composition comprises a detergent, e.g.,
deoxycholate, e.g., at between 1-5 mM, e.g., about 2 mM or about 3
mM.
[0111] In some embodiments, the amount of product produced and/or
the rate of product formation is determined. The effect of the test
compound on the amount of product produced and/or the rate at which
the product is produced is assessed, e.g., by comparison with a
suitable reference value. If the amount of product or rate of
product production is decreased in the presence of the test
compound as compared with a suitable reference value, the test
compound inhibits the ability of the PLA2G16 polypeptide to
catalyze a reaction in which the substrate is converted to one or
more product(s), i.e., the test compound is an inhibitor of the
PLA2G16 polypeptide. In some embodiments, the rate of substrate
consumption or the amount of substrate consumed is determined.
Equivalently, the amount of substrate remaining can be determined.
If the amount of substrate consumed or the rate of substrate
consumption is decreased in the presence of the test compound as
compared with a suitable reference value, the test compound
inhibits the ability of the PLA2G16 polypeptide to catalyze a
reaction in which the substrate is converted to one or more
product(s), i.e., the test compound is an inhibitor of the PLA2G16
polypeptide. A reference value in any of these assays can be a
value measured under similar or identical conditions in the absence
of the test compound.
[0112] In some embodiments, a PLA2G16 substrate is useful to
measure phospholipase activity, e.g., phospholipase A2 activity.
For example, a PLA2G16 substrate can be a naturally occurring or
artificial phospholipid. As known in the art, most phospholipids
are composed of 1,2-diacylglycerol and a phosphate group, and an
organic molecule (often a nitrogenous base). A phosophodiester
bridge links the glycerol backbone to the base, which is sometimes
termed a "head group". Exemplary head groups are choline,
ethanolamine, inositol, and serine. For example, a substrate can be
a phosphatidylcholine or phosphatidylethanolamine. The hydrocarbon
chains of the acyl groups of a phospholipid molecule are often
different, e.g., they are derived from fatty acid molecules with
different hydrocarbon chains. In some embodiments, the hydrocarbon
chains are between 12 and 30 carbons in length. In some
embodiments, a PLA2G16 substrate has the structure of a naturally
occurring phospholipid, e.g., a phospholipid found in vertebrate
cells, e.g., mammalian cells. Exemplary PLA2G16 substrates include,
e.g., 1-palmitoyl-2-linoleoyl-PC, dilinoleoyl-PC,
1-palmitoyl-2-linoleoyl-PS, 1-palmitoyl-2-linoleoyl-PE,
phosphatidylinositol, 1-palmitoyl-2-arachidonyl-PC (abbreviations:
PC: phosphatidylcholine PE: phosphatidylethanolamine; PS:
phosphatidylserine). In some embodiments, the substrate comprises
choline as a head group. In some embodiments, a phospholipid analog
containing a thio ester bond instead of the sn-2 ester is used. In
some embodiments, a PLA2G16 substrate is useful for measuring
lysophospholipase activity. For example, the substrate can be a
lysophosphatidylcholine, e.g.,
1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocholine.
[0113] In some embodiments, a substrate comprises a moiety that
facilitates detection of a product of a biochemical reaction
catalyzed by a PLA2G16 polypeptide. For example, the substrate can
comprise one or more radioactive atoms, fluorescent labels, and/or
fluorescence quenchers. In some embodiments, the label comprises
14C, 3H, or 32P. In some embodiments, the substrate comprises a
moiety that emits a signal upon cleavage of the substrate. In some
embodiments, the substrate comprises a moiety that can be readily
detected upon release from the substrate. For example, the moiety
may react with another compound to produce a colorimetric,
fluorescent, or luminescent signal. Labels include, e.g., organic
materials (including "traditional" dye fluorophores, quenchers, and
polymers); inorganic materials such as metal chelates, metal and
semiconductor nanocrystals (e.g., "quantum dots", and fluorophores
of biological origin such as certain amino acids (e.g., tryptophan,
tyrosine); and compounds that exhibit luminescensce upon enzymatic
catalysis such as naturally occurring or synthetic luciferins
(e.g., firefly or Renilla luciferin, coelenterazine). Fluorescent
dyes include, e.g., acridine dyes; Alexa dyes; BODIPY, cyanine
dyes; fluorescein dyes, rhodamine dyes, and derivatives of any of
the foregoing. See, e.g., The Handbook--A Guide to Fluorescent
Probes and Labeling Technologies, 10th edition (Invitrogen Corp.),
which describes numerous fluorescent and otherwise detectable
molecules and methods for their use and modification. In another
embodiment, a phospholipid analogue containing a thio ester bond
instead of the sn-2 ester is used, and hydrolysis of the thioester
bond at the sn-2 position by PLA2 releases free thiol which can be
detected by DTNB (5,5'-dithiobis(2-nitrobenzoic acid)).
[0114] In some embodiments, a substrate is present in a vesicle or
micelle. For example, lipid-detergent micelles can be used. In some
embodiments, an ionic detergent such as deoxycholate is used. Other
detergents include, e.g., Triton X-100. In some embodiments, a
composition containing about 100 .mu.M 1-palmitoyl-2-linoleoyl-PC
with 2 mM deoxycholate and 2 mM CaCl.sub.2 is used. A test compound
can be incorporated into the vesicle or micelle.
[0115] A variety of assays can be used to measure PLA2 catalytic
activity. In some embodiments, an assays that has been used in the
art to measure activity of a Group I-XV PLA2 (e.g., a cytosolic or
secreted PLA2) is used or modified for use to measure catalytic
activity of PLA2G16. In some embodiments, a radiometric assay is
used, with a substrate of phospholipid (e.g., phosphatidylcholine
or phosphatidylethanolamine) containing a 14C- or 3H-labeled fatty
acid at the sn-2 position. The fatty acids released are separated
from the unreacted substrate and quantified by liquid scintillation
counting. In other embodiments, a fluorescence displacement assay,
is used. A fluorescent molecule can be detected using, e.g., a
spectrophotometer. An exemplary assay involves the displacement of
a fluorescent fatty acid probe from albumin or rat liver fatty
acid-binding protein by the decanoic acid released as a result of
the phospholipase A2-catalyzed hydrolysis of
didecanoyl-phosphatidylcholine A. R. Kinkaid & D. C. Wilton, A
continuous fluorescence displacement assay for phospholipase a2
using albumin and medium chain phospholipid substrates. Anal.
Biochem. 212: 65-70, 1993; D. C. Wilton, A continuous fluorescence
displacement assay for the measurement of phospholipase A2 and
other lipases that release long-chain fatty acids. Biochem. J. 266:
435-439, 1990). See also, Huang, Z., et al., Anal. Biochem. 222:
110-115, 1994, which describes an assay for cPLA2 activity based on
hydrolysis of fatty acid esters of 7-hydroxycoumarin by cPLA2,
producing the free fatty acid and highly fluorescent
7-hydroxycoumarin. Another assay is a fluorometric phospholipase
assay based on polymerized liposome substrates (Chu, W., et al.,
Fluorometric phospholipase assays based on polymerised liposome
substrates. Methods Mol. Biol. 109: 7-17, 1999). In another
embodiment, a phospholipid analogue containing a thio ester bond
instead of the sn-2 ester is used as a substrate to measure
phospholipase activity (Yu, L, et al. Carbonothioate phospholipids
as substrate for a spectrophotometric assay of phospholipase A2.
Anal. Biochem. 265: 35-41, 1998).
[0116] In another embodiment, a coupled spectrophotometric assay
using dilinoleoyl phosphatidylcholine (DL-PC) as PLA2 substrate and
lipoxygenase as the coupling enzyme is used. See, e.g., Jimenez,
M., et al. A continuous spectrophotometric assay for phospholipase
A(2) activity Anal Biochem., 319(1):131-7, 2003, and references
therein, and Duncan, supra. In this assay, lipoxygenase
(linoleate:oxygen oxidoreductase, EC 1.13.11.12) catalyzes the
addition of molecular oxygen to fatty acids containing at least one
(Z,Z)-pentadiene system to give the corresponding hydroperoxides.
Lipoxygenase oxidizes the linoleic acid released by the action of
phospholipase, the activity of which can then be followed
spectrophotometrically by recording the increase in absorbance at
234 nm due to the formation of the corresponding hydroperoxide from
the linoleic acid by the action of lipoxygenase. This method
provides a continuous record of phospholipid hydrolysis.
[0117] In some embodiments, a scintillation proximity assay (SPA)
is used. For example, a radiolabelled PLA2G16 substrate can be
attached to beads containing a scintillant material. The beads are
typically located in wells or other vessels. In another embodiment,
scintillant material is embedded directly into wells. A PLA2G16
polypeptide is added to the well in a suitable composition
(optionally containing calcium and/or a buffer). Hydrolysis of the
substrate releases the radioactive moiety, resulting in a decreased
signal. See, e.g., J. Fraser Glickman, supra for discussion of
SPA.
[0118] In some embodiments, an assay readout is based on resonance
energy transfer (RET), e.g., fluorescence resonance energy transfer
(FRET), luminescence resonance energy transfer (LRET), or
bioluminescence resonance energy transfer (BRET). A wide variety of
RET-based assays can be implemented. In general, such assays make
use of a distance-dependent interaction involving energy transfer
between two moieties (sometimes termed a donor and acceptor). If
both moieties are present as part of a PLA2G16 substrate and
positioned so that cleavage of the substrate releases one of the
moieties, a signal (e.g., an increase or decrease in a signal) can
be detected. FRET is a distance-dependent interaction between the
electronic excited states of two moieties in which excitation is
transferred from a donor moiety to an acceptor moiety without
emission of a photon, resulting in emission from the FRET acceptor.
LRET has similarities to FRET but uses a luminescent moiety, e.g.,
a lanthanide as the energy-transfer donor. BRET is analogous to
FRET but uses a luminescent or luminescence-generating biomolecule
such as luciferase, aequorin, or a derivative thereof as an energy
donor and a fluorescent moiety, e.g., a biomolecule such as green
fluorescent protein (GFP) as the acceptor, thus eliminating the
need for an excitation light source (reviewed in Pfleger, K. an
Eidne, K., Nature Methods, 3(3), 165-174, 2006).
[0119] Assays of the invention may detect acceptor emission, donor
quenching (decreased emission from the RET donor), and/or an
alteration in the fluorescence lifetime of the donor. Assays of the
invention can make use of increases in acceptor emission, decreases
in acceptor emission, donor quenching, reduction in donor
quenching, and/or increase or decrease in fluorescence lifetime of
the donor to detect cleavage of a PLA2G16 substrate. Nonfluorescent
acceptors, also referred to as quenchers are of use and include
dabcyl and QSY dyes. Such molecules are capable of absorbing the
energy of an excited fluorescent label when located in close
proximity and of dissipating that energy without the emission of
visible light. Numerous suitable donor/acceptor pairs are known in
the art. See, e.g, The Handbook--A Guide to Fluorescent Probes and
Labeling Technologies, 10th edition (Invitrogen Corp.).
[0120] In some embodiments of a FRET-based assay, a first acyl
chain of the PLA2G16 substrate has an attached fluorescence
quencher and the second acyl chain has an attached fluorophore.
Intramolecular FRET from the fluorophore to the quencher quenches
fluorescence until PLA2-mediated substrate cleavage, when at least
one fatty acid moiety becomes separated from the remainder of the
molecule, and the intermolecular distance exceeds that required for
efficient energy transfer. An increase in fluorescence signal
indicates substrate cleavage. The presence of an inhibitor will
cause a reduction in the fluorescence signal relative to that which
would be observed in the absence of the inhibitor. In some
embodiments the phospholipids sn-1-acyl chain contains an attached
fluorescence quencher (e.g., Dabcyl, also known as p-methyl red),
and the sn-2 acyl chain contains an appended BODIPY fluorophore.
Intramolecular FRET (fluorescence resonance energy transfer) to the
Dabcyl group quenches BODIPY fluorescence until PLA-mediated
substrate cleavage. See, e.g., Rose, T M & Prestwich, G D, ACS
Chemical Biology, 1(2): 83-89, 2006, for description of Dabcyl- and
BODIPY-containing phospholipids DBPA, DBPC, DBPE, and DBPG
(abbreviations: DB: Dabcyl-BODIPY; PG: phosphatidylglycerol).
[0121] Another assay format that can be used to measure PLA2
activity is a fluorescence based assay in which cationic conjugated
polyelectrolytes are supported on silica microspheres. (See, e.g.,
Chemburu S, et al. Conjugated polyelectrolyte supported bead based
assays for phospholipase A2 activity, Phys Chem B.,
112(46):14492-9, 2008, which describes such an assay for human
serum-derived PLA2. This assay can be modified for use to detect
compounds that inhibit activity of a PLA2G16 polypeptide.). The
polymer-coated beads are overcoated with an anionic phospholipid to
provide "lipobeads" that serve as a sensor for PLA2. The lipid
serves a dual role as a substrate for PLA2 and an agent to
attenuate quenching of the polymer fluorescence by the external
electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid
(AQS). Quenching of the polymer fluorescence by AQS increases as
the PLA2 digests the lipid. The lipid can also be used itself as a
quencher and substrate by employing a small amount of energy
transfer quencher substituted lipid in the anionic phospholipid
coating the beads. In this case the fluorescence of the polymer is
quenched when the lipid layer is intact; as the enzyme digests the
lipid, the fluorescence of the polymer is restored. The sensing of
PLA2 activity can be performed by monitoring fluorescence changes
in a multiwell plate reader and/or by flow cytometry.
[0122] A "cell-based assay" is an assay in which viable cells that
express or contain a PLA2G16 polypeptide are contacted with a test
compound and a parameter of interest such as PLA2G16 activity is
assessed. Typically, the cells are maintained in cell culture and
the test compound is added to the culture medium. In some
embodiments, the effect of the test compound on the ability of the
PLA2G16 polypeptide to act on a PLA2G16 substrate is assessed. For
example, a PLA2G16 substrate, e.g, a detectably labeled substrate,
can be added to the culture medium or synthesized by the cell from
a labeled precursor. Cleavage of the substrate may be detected by
detecting a free fatty acid or by detecting a downstream product
produced from a free fatty acid. For example, arachidonic acid is
modified by cyclooxygenases to form eicosanoids (e.g.,
prostaglandins, leukotrienes). In some embodiments, a cell that
substantially lacks other PLA2 enzymes that could act on the
PLA2G16 substrate can be used. In some embodiments, such cells are
identified by screening a variety of cell lines for expression of
known PLA2 enzymes. In other embodiments, a cell line is generated
by targeted deletion or insertion into the genes encoding one or
more PLA2 enzyme(s) or by causing the cell to express shRNA that
inhibit expression of such other PLA2 enzyme(s). In other
embodiments, the assay is performed in cells that have been
contacted with siRNA specific for such other PLA2 enzyme(s) to
knock down their expression.
[0123] A compound identified as an inhibitor of a PLA2G16
polypeptide can be tested in cell culture or in animal models ("in
vivo") to determine its ability to inhibit viral infection. In some
embodiments, host cells are contacted with a virus and a PLA2G16
inhibitor under conditions suitable for infection of the cells. The
ability of the test compound to inhibit viral infection is
assessed. If the compound detectably reduces viral infection, the
compound is identified as an antiviral compound. The virus can be,
e.g., any virus that utilizes PLA2G16 polypeptide, or a
PLA2G16-like polypeptide, in its life cycle.
[0124] A wide variety of cell types can be used in embodiments of
the inventive methods. Typically, the cell expresses or contains a
PLA2G16 polypeptide, either naturally or as a result of
modification by the hand of man, although cells that do not express
a PLA2G16 may be useful, e.g., for control purposes. A cell could
originate from any organism of interest, e.g., a vertebrate, e.g.,
a mammal. In some embodiments, a cell is a primate cell, e.g., a
monkey cell or a human cell. A cell could be a primary cell,
immortalized cell, cancer cell, etc. Often, a cell is a member of a
population of cells which is composed of cells that are
substantially genetically identical, e.g., a cell line. A cell line
can be descended from a single cell or from multiple cells isolated
from a single individual. A cell can originate from a tissue or
organ of interest or can have a property of interest. In some
embodiments, a cell is an epithelial cell, fibroblast, kidney cell,
rhabdosarcoma or rhabdomyosarcoma, lung, or bronchial cell,
pre-adipocyte, or adipocyte. In some embodiments a cell originates
from breast, bladder, bone, brain, bronchus, cervix, colon,
endometrium, esophagus, larynx, liver, lung, nerve, muscle, ovary,
pancreas, prostate, stomach, kidney, skin, testis, or thyroid
gland. Numerous cell lines are known in the art, many of which can
be obtained from repositories such as the American Type Culture
Collection, Coriell Cell Repositories, European Collection of Cell
Cultures, Japanese Collection of Research Bioresources, or from a
variety of commercial suppliers. In some embodiments, a
pre-adipocyte is a 3T3-L1 cell. In some embodiment, a cell is a COS
cell, e.g., a COS-1 or COS-7 cell. In some embodiments, a cell is a
HeLa cell. In some embodiments, a cell is a Vero, RD, CHO, HEK-293,
HMEC, MDCK, NIH-3T3, HEp-2, A549, or BEAS-2B cell. In some
embodiments, a cell is a tumor cell. In some embodiments a tumor
cell originates from a carcinoma. In some embodiments a tumor cell
originates from a sarcoma. In some embodiments a tumor cell
originates from a hematologic malignancy, e.g., a lymphoma or
leukemia or myeloma. In some embodiments a tumor cell originates
from a breast, bladder, bone, brain, cervical, colon, endometrial,
esophageal, head and neck, laryngeal, liver, lung (small cell or
non-small cell), ovarian, pancreatic, prostate, stomach, renal,
skin (e.g., basal cell, melanoma, squamous cell), testicular, or
thyroid cancer. The tumor cell may be a cell of an established
tumor cell line (e.g., one of the NCI-60 tumor cell lines) or
another tumor cell line known in the art or newly established.
[0125] In some embodiments, a cell is a hematopoietic cell. In some
embodiments, a cell is a KBM-7 cell or derivative thereof, such as
a HAP1 cell. In some embodiments, a cell is a KBM-7 cell or other
cell that has been partially reprogrammed by expressing at least
one "reprogramming factor" therein or exposing the cell to at least
one "reprogramming agent" (e.g., an agent that induces expression
of an endogenous reprogramming factor or substitutes for a
reprogramming factor). Reprogramming cells, e.g., near-haploid
mammalian cells may facilitate their use in identifying PLA2G16
inhibitors and/or antiviral compounds. Such reprogramming may
convert the KBM-7 cell (which normally grows in suspension) into an
adherent cell, such as a HAP1 cell. As known in the art, mouse and
human fibroblasts and various other normal somatic cell types can
be reprogrammed in vitro to a pluripotent state through
retroviral-mediated introduction of combinations of transcription
factors, e.g., the four transcription factors Oct4, Sox2, Klf4, and
c-Myc (with c-Myc being dispensable, although omitting c-Myc
reduced reprogramming efficiency), or the four transcription
factors Oct4, Nanog, Sox2, and Lin28 (see, e.g., Meissner, A., et
al., Nat Biotechnol., 25(10):1177-81 (2007); Yu, J., et al,
Science, 318(5858):1917-20 (2007); and Nakagawa, M., et al., Nat
Biotechnol., 26(1):101-6 (2008). Such transcription factors are
often referred to as "reprogramming factors").
[0126] In some embodiments, a cell naturally expresses PLA2G16. In
some embodiments a cell is modified so that it expresses a PLA2G16
polypeptide at a higher level than would be the case in the absence
of the modification. In some embodiments, a cell expresses PLA2G16
at a level at least 25%, 50%, 75%, 90%, 95%, or approximately 100%
as high as the expression level present in a HAP1 cell, HeLa cell,
or other cell capable of serving as a host cell for a virus of
interest. The expression level can be normalized, e.g., based on
expression of a "housekeeping" gene. Commonly used housekeeping
genes include, e.g., beta-actin, GAPDH, phosphoglycerate kinase,
etc. Standard methods of transiently or stably expressing
polypeptides in cells can be used.
[0127] In some embodiments, a cell is of a type that is known in
the art to be naturally susceptible to infection by a virus, e.g.,
a picornavirus. For example, the cell can be of a type that is
normally a target cell of the virus in vivo or a cell line that has
been used in the art as a host for a virus in culture. A compound
can be tested in cells of multiple different types. For example, a
compound can be initially identified as a PLA2G16 inhibitor or
antiviral compound in a cell that has convenient properties for
screening or performing tests for virus inhibition and then
subsequently tested in one or more cells that are natural targets
of a virus of interest.
[0128] In some embodiments, a cell used in a method described
herein is genetically modified or selected to have a property that
facilitates its use to test compounds. For example, the cell can be
genetically modified or selected to have reduced or absent
expression of one or more molecular pumps that may otherwise
transport a test compound out of the cell. In some embodiments, the
cell is modified to facilitate detection of viral infection. For
example, the cell could comprise a reporter gene in which a
promoter or other expression control element(s) active only in the
presence of viral protein(s) are operably linked to an open reading
frame that encodes a readily detectable polypeptide such as a
fluorescent protein or enzyme. In another embodiment, a cell
expresses a protein that comprises a cleavage site for a viral
protease, wherein cleavage of the protein is detectable. For
example, the protein may contain a FRET pair (e.g., polypeptides
that are a FRET donor and acceptor pair) separated by a domain
containing a protease cleavage site. Cleavage by the protease
results in separation of the members of the FRET pair, resulting in
a disruption of FRET, which can be detected and serve as an
indicator of viral infection. In another embodiment, a
cell-permeable substrate for a viral protease is introduced into
the cells. A candidate antiviral compound, e.g., a compound that
inhibits PLA2G16 activity, can be tested in such cells to confirm
that it inhibits viral infection.
[0129] Cells can be contacted with test compound(s) and/or virus
for various periods of time. In certain embodiments cells are
contacted with test compound(s) and/or virus for between 1 hour and
20 days, e.g., for between 12 and 48 hours, between 48 hours and 5
days, e.g., about 3 days, between 5 days and 10 days, or any
intervening range or particular value. In some embodiments, cells
are contacted with a virus for at least a time sufficient for
completion of one or more rounds of viral replication and
production of progeny virus. In some embodiments, cells are
contacted with a virus for at least a time sufficient for
production of plaques that are detectable under a light microscope.
Cells can be contacted with a test compound during all or part of a
culture period. If desired, the test compound can be removed prior
to assessing PLA2G16 activity or viral infection. In some
embodiments, cells are contacted with a virus prior to contacting
the cells with the test compound. In other embodiments, cells are
contacted with the test compound prior to contacting them with a
virus. The absolute number of virus and the multiplicity of
infection (MOI) can vary. "Multiplicity of infection" refers to the
ratio of infectious agents (e.g., viruses) to infection targets
(e.g., cells). In some embodiments an MOI of between 10.sup.-4 and
10.sup.2 is used. For example, an MOI of between 0.001 and 10,
e.g., between 0.01 and 1, can be used. In some embodiments, an
amount of virus suitable to produce a pathologic change in between
10% and 100% of cells is used. One of skill in the art will be able
to determine a suitable amount of virus to use so as to be able to
detect an effect on viral infection. A range of dilutions of a
virus stock can be tested to identify an appropriate amount. Cells
are maintained in culture for a suitable time period after
contacting them with the virus. Typically, the time period will be
sufficient for the virus to enter cells and for at least one event
indicative of viral infection to occur. Such event may be a
detectable effect of a viral gene product(s) on the cell and/or the
synthesis or partial synthesis of at least one viral gene product.
In general, the time period will be sufficient to detect a
difference between the effect of the virus on the cells in the
absence of a PLA2G16 inhibitor versus in the presence of a PLA2G16
inhibitor. A detectable effect of a virus on a cell could be an
alteration (e.g., a decrease) in synthesis of some or most cellular
RNA(s) or protein(s), induction of an antiviral response (e.g.,
induction of interferon target gene(s) such as the gene encoding
2'5'-oligoadenylate synthetase), a morphological effect such as
chromatin condensation, nuclear blebbing, proliferation of
membranous vesicles; leakage of intracellular contents;
cytotoxicity; cleavage of a substrate by a virus-specific enzyme
(e.g., a protease), etc. Cytotoxicity can be assessed e.g., by
detecting cell lysis (which may be evident as clear areas or
"plaques" in a cell monolayer) or using any of a variety of assays
for cell viability and/or proliferation such as a cell membrane
integrity assay, a cellular ATP-based viability assay, a
mitochondrial reductase activity assay, a BrdU, EdU, or
H3-Thymidine incorporation assay, a DNA content assay using a
nucleic acid dye, such as Hoechst Dye, DAPI, Actinomycin D,
7-aminoactinomycin D or propidium iodide, a cellular metabolism
assay such as AlamarBlue, MTT, XTT, and CellTitre Glo, etc. Plaque
assays are a well established means of assessing viral titer and
detecting the effect of compounds on viral infectivity. In some
embodiments, a plaque assay involves inoculating a standard viral
stock into multiple identical cell cultures, e.g., grown in wells
of a multiwell plate. A solidifying agent, e.g., agarose, may be
added to minimize spread of the virus through the culture medium.
The viral titer of the stock is usually predetermined and is
selected to yield a countable number of plaques in each well.
Different concentrations of the test compound are introduced into a
series of wells. The effect of the compound may be expressed as the
50% inhibitory concentration (IC.sub.50), defined as the lowest
concentration of compound that results in a 50% decrease in the
number of viral plaques compared with a control well that does not
contain the compound. If desired, an IC.sub.90 can be assessed in a
similar manner. A compound that significantly decreases an effect
of the virus is an inhibitor of infection by the virus. For
example, a compound that significantly decreases the number and/or
size of viral plaques caused by a given amount of virus is an
inhibitor of viral infection. Optionally, an IC50 or IC90 is
determined. In some embodiments, one or more compound(s) with a
desired C50 or IC90 is selected. In some embodiments, an IC50
and/or IC90 is no greater than 100 mg/ml, e.g., no greater than 10
mg/ml, e.g., no greater than 1.0 mg/ml, e.g., no greater than 100
.mu.g/ml, e.g., no greater than 10 .mu.g/ml, e.g., no greater than
5 .mu.g/ml or no greater than 1 .mu.g/ml. In some embodiments, an
IC50 and/or IC90 is less than or equal to 500 .mu.M. In some
embodiments, an IC50 and/or IC90 less than or equal to 100 .mu.M.
In some embodiments, an IC50 and/or IC90 less than or equal to 10
.mu.M. In some embodiments, an IC50 and/or IC90 is in the nanomolar
range, i.e., less than or equal to 1 .mu.M.
[0130] In some embodiments, a high throughput screen (HTS) is
performed. A high throughput screen can utilize cell-free or
cell-based assays. High throughput screens often involve testing
large numbers of compounds with high efficiency, e.g., in parallel.
For example, tens or hundreds of thousands of compounds can be
routinely screened in short periods of time, e.g, hours to days.
Often such screening is performed in multiwell plates containing,
e.g., e.g., 96, 384, 1536, 3456, or more wells (sometimes referred
to as microwell or microtiter plates or dishes) or other vessels in
which multiple physically separated cavities are present in a
substrate. High throughput screens can involve use of automation,
e.g., for liquid handling, imaging, data acquisition and
processing, etc. Without limiting the invention in any way, certain
general principles and techniques that may be applied in
embodiments of a HTS of the present invention are described in
Macarron R & Hertzberg R P. Design and implementation of
high-throughput screening assays. Methods Mol Biol., 565:1-32, 2009
and/or An W F & Tolliday N J., Introduction: cell-based assays
for high-throughput screening. Methods Mol Biol. 486:1-12, 2009,
and/or references in either of these. Exemplary methods are also
disclosed in High Throughput Screening: Methods and Protocols
(Methods in Molecular Biology) by William P. Janzen (2002) and
High-Throughput Screening in Drug Discovery (Methods and Principles
in Medicinal Chemistry) (2006) by Jorg H{umlaut over
(.upsilon.)}ser.
[0131] In some embodiments, a first screen is performed to identify
compounds that bind to and/or inhibit PLA2G16 polypeptide, and the
ability of such compounds to inhibit viral infection is then
assessed. In some embodiments, test compounds are first tested in a
cell-based assay to identify compound(s) that inhibit viral
infection and are then tested to determine whether they inhibit
PLA2G16.
[0132] The invention provides compositions comprising components
appropriate to perform any of the inventive methods, e.g., any of
the methods of identifying a candidate antiviral compound. In some
embodiments, an assay system comprises components suitable for
identifying a PLA2G16 inhibitor. In some embodiments, a composition
comprises components appropriate to perform any of the inventive
methods of validating a candidate antiviral compound. In some
embodiments, the composition comprises components appropriate to
confirm that a candidate antiviral compound inhibits viral
infection in cultured cells or in vivo. In one aspect, an inventive
composition comprises (i) isolated cells that express a PLA2G16
polypeptide; (ii) a virus capable of infecting the cells; and (iii)
a test compound. In some embodiments, the virus is a Picornavirus,
e.g., a pathogenic Picornavirus. The virus is typically present in
the composition in amounts suitable for detecting virus infection
by the cells. Such amounts are typically greater than might happen
by chance if cultured cells happen to be exposed to an environment
where there is an individual infected by the virus. In some
embodiments, the ratio of viral particles (e.g., infectious viral
particles) to cells is at least 1:10.sup.6, at least 1:10.sup.5,
e.g., at least 1:10.sup.4, at least 1:10.sup.3, at least
1:10.sup.2, at least 1:10, or at least 1:1. In some embodiments,
there are more viral particles (e.g., infectious viral particles)
than cells. The test compound can be, e.g, any of the compounds
discussed above. In some embodiments, the test compound is a
phospholipase A2 inhibitor, e.g, a PLA2G16 inhibitor. In some
embodiments, the test compound is a small molecule. In some
embodiments, the test compound has been determined to bind to
and/or inhibit PLA2G16 in at least one cell-free or cell-based
assay.
[0133] Compounds identified in cell-free and/or cell-based assays
can be tested in subjects (e.g., non-human vertebrates) to assess
their ability to inhibit viral infection in vivo. Animal models for
viral infection are known in the art. An animal can be, e.g., a
rodent, non-human primate, dog, cat, etc. In one embodiment, an
animal model is a murine model of coxsackievirus B3 (CVB3)-induced
myocarditis. See, e.g., Szalay G, Ongoing coxsackievirus
myocarditis is associated with increased formation and activity of
myocardial immunoproteasomes, Am J Pathol., 168(5):1542-52, 2006,
and references therein. In one embodiment, an animal model is a
mouse model for EV71 infection. See, e.g. Wang, Y. F., et al., A
mouse-adapted enterovirus 71 strain causes neurological disease in
mice after oral infection. J. Virol. 78:7916-7924, 2004, which
describe an animal model in which mice are orally inoculated with
EV71. Mice may be monitored daily for signs of disease and
survival. In another embodiment, an attenuated mengovirus is used
in a rodent model for rhinovirus infection. See, e.g., Rosenthal L
A, A rat model of picornavirus-induced airway infection and
inflammation. Virol J., 6:122, 2009. Tissues or body fluids can be
collected after infection to determine viral titers and/or to
evaluate other signs of viral infection. For example, viral RNA or
protein can be detected using standard methods such as RT-PCR (for
RNA) or immunological methods for proteins. See, e.g., Li, Z. H.,
et al., Ribavirin reduces mortality in enterovirus 71-infected mice
by decreasing viral replication. J. Infect. Dis. 197:854-857,
2008).
[0134] The invention further provides a non-human subject e.g., a
vertebrate, wherein the non-human subject has been inoculated with
or exposed to a virus to which it is normally susceptible, or that
is suffering from a viral infection, and wherein a PLA2G16
inhibitor has been administered to the subject. "Inoculation" with
a virus means that the virus has been introduced into the subject's
body. Exposure can involve inoculating a subject or placing the
virus and subject in reasonably close proximity so as to increase
the likelihood that the subject will encounter the virus.
Inoculation can be by any appropriate route. Inoculation or
exposure will typically involve sufficient amount of virus to
produce evident disease in at least 25% of a population of that
species in the absence of an antiviral compound. The PLA2G16
inhibitor can be, e.g, any of the compounds discussed above or
identified according to an inventive method. In some embodiments,
the test compound is a small molecule. In some embodiments, the
test compound has been determined to bind to and/or inhibit PLA2G16
in at least one cell-free or cell-based assay. The non-human
subject can be monitored, e.g., to assess the safety, tolerability,
and/or efficacy of the compound as an antiviral agent. Assessing
the effect of a PLA2G16 inhibitor in a subject infected with a
virus is an aspect of the invention.
[0135] In some embodiments, the invention provides a near-haploid
cell that has an insertion into the PLA2G16 locus or otherwise
lacks expression of PLA2G16. The near-haploid cell is of a species,
e.g., a mammal, whose somatic cells are normally diploid. In some
embodiments, the invention provides a near-haploid cell that
expresses a catalytically inactive mutant PLA2G16 polypeptide,
wherein optionally the near-haploid mutant cell line has an
insertion in the endogenous PLA2G16 gene. In some embodiments, the
invention provides a near-haploid cell that expresses a tagged
functional PLA2G16 polypeptide, wherein optionally the near-haploid
mutant cell line has an insertion in the endogenous PLA2G16 gene. A
near-haploid mammalian cell, as used herein, refers to a mammalian
cell in which no more than 5 chromosomes are present in two or more
copies. In some embodiments a near-haploid mammalian cell has no
more than 1, 2, 3, or 4 chromosomes present in two or more copies.
The "near-haploid" cell should be understood to include haploid
cells. Further provided are cell lines derived from cells that lack
expression of functional PLA2G16, e.g., cell lines composed of
cells that have an insertion into the PLA2G16 gene. In some
embodiments, a cell line expresses a catalytically inactive mutant
PLA2G16 polypeptide, which is tagged in some embodiments. In some
embodiments, a near-haploid cell line eventually gains chromosomes
during culture so that it is no longer near-haploid. In some
embodiments the cell line may become near diploid or diploid.
[0136] The invention further provides kits comprising one or more
components of any of the inventive compositions and/or components
suitable for performing any of the inventive methods. The
components can be packaged individually, e.g., in individual
containers, which may be provided within a larger container. A kit
can contain instructions for using the contents to perform any of
the methods, e.g., to identify or characterize an antiviral
compound.
[0137] In some embodiments, computational approaches are employed
to identify and/or characterize compounds that inhibit PLA2G16. For
example, a three-dimensional structure of a PLA2G16 polypeptide can
be determined or an approximate structure generated using, e.g.,
nuclear magnetic resonance, homology modeling, and/or X-ray
crystallography. Optionally the structure of the polypeptide with a
ligand (e.g., an inhibitor) bound thereto is determined. In some
embodiments, a computational approach is used in the initial
identification of candidate PLA2G16 inhibitors (sometimes termed
"virtual screening"). Structures of candidate compounds can be
screened for ability to bind to the PLA2G16 polypeptide, e.g., to a
region (e.g., a "pocket") accessible to the compound. The region
could be a known or potential active site or any region accessible
to the compound, e.g., a concave region on the surface or a cleft.
A variety of docking and pharmacophore-based algorithms have been
developed, and computer programs implementing such algorithms are
available. Commonly used programs include Gold, Dock, Glide, FlexX,
Fred, and LigandFit (including the most recent releases thereof).
See, e.g., Ghosh, S., et al., Current Opinion in Chemical Biology,
10(3): 194-2-2, 2006; McInnes C., Current Opinion in Chemical
Biology; 11(5): 494-502, 2007, and references in either of the
foregoing articles, which are incorporated herein by reference. In
some embodiments, a virtual screening algorithm involves two major
phases: searching (also called "docking") and scoring. During the
first phase, the program automatically generates a set of candidate
complexes of two molecules (test compound and target molecule) and
determines the energy of interaction of the candidate complexes.
The scoring phase assigns scores to the candidate complexes and
selects a structure that displays favorable interactions based at
least in part on the energy. To perform virtual screening, this
process is repeated with a large number of test compounds to
identify those that display the most favorable interactions with
the target. In some embodiments, low-energy binding modes of a
small molecule within an active site or possible active site are
identified. Variations can include the use of rigid or flexible
docking algorithms and/or including the potential binding of water
molecules.
[0138] Numerous small molecule structures are available and can be
used for virtual screening. For example, ZINC is a publicly
available database containing structures of millions of
commercially available compounds that can be used for virtual
screening (http://zinc.docking.org/; Shoichet, J. Chem. Inf.
Model., 45(1):177-82, 2005). A database containing about 250,000
small molecule structures is available on the National Cancer
Institute (U.S.) website (at http://129.43.27.140/ncidb2/). In some
embodiments, multiple small molecules are screened, e.g., up to
50,000; 100,000; 250,000; 500,000, or up to 1 million, 2 million, 5
million, 10 million, or more. Compounds can be scored and,
optionally, ranked by their potential to bind to the target.
Compounds identified in virtual screens can be tested in cell-free
or cell-based assays or in animal models to confirm their ability
to inhibit PLA2G16 activity and/or viral infection.
[0139] Computational approaches can be used to predict one or more
physico-chemical, pharmacokinetic and/or pharmacodynamic properties
of compounds identified in actual or virtual screens. For example,
absorption, distribution, metabolism, and excretion (ADME)
parameters can be predicted. Such information can be used, e.g., to
select hits for further testing or modification. For example, small
molecules having characteristics typical of "drug-like" molecules
can be selected and/or small molecules having one or more undesired
characteristics can be avoided. In one embodiment, compounds that
satisfy at least some of the Lipinski "rule of five" criteria are
selected.
[0140] In one aspect, the invention provides a computer-readable
medium on which are stored results of a screen to identify
compounds that inhibit PLA2G16. The results may be stored in a
database and can include any screening protocols, results obtained
from the screen or from additional screens, and/or protocols of or
results obtained from tests performed on compounds identified in
the screen (e.g., tests in animal models of viral infection).
[0141] Additional compounds that inhibit PLA2G16 can be identified
or designed based on initial compounds ("hits") identified in an
actual or virtual screen such as those described above. Such
additional compounds and methods of designing or synthesizing them
are an aspect of the invention. In some embodiments, structures of
hit compounds are examined to identify a pharmacophore, which can
be used to design additional compounds ("derivatives").
[0142] An additional compound may, for example, have one or more
improved (i.e, more desirable) pharmacokinetic and/or
pharmacodynamic properties as compared with an initial hit or may
simply have a different structure. For example, a compound may have
higher affinity for the molecular target of interest (e.g.,
PLA2G16), lower affinity for a non-target molecule, greater
solubility (e.g., increased aqueous solubility), increased
stability, increased bioavailability, and/or reduced side
effect(s), etc. Optimization can be accomplished through empirical
modification of the hit structure (e.g., synthesizing compounds
with related structures and testing them in cell-free or cell-based
assays or in non-human animals) and/or using computational
approaches. Such modification can make use of established
principles of medicinal chemistry to predictably alter one or more
properties.
[0143] In some embodiments, a PLA2G16 inhibitor is modified or
incorporates a moiety that enhances cell uptake, stability (e.g.,
in serum), increases half-life, reduces toxicity or immunogenicity,
or otherwise confers a desirable property on the compound. In some
embodiments, a PLA2G16 inhibitor comprises a protein transduction
domain (PTD). A PTD or cell penetrating peptide (CPP) is a peptide
or peptoid that can traverse the plasma membrane of many, if not
all, mammalian cells. A PTD can enhance uptake of a moiety to which
it is attached or in which it is present. Often such peptides are
rich in arginine. For example, the PTD of the Tat protein of human
immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) has been
widely studied and used to transport cargoes into mammalian cells.
See, e.g., Fonseca S B, et al., Adv Drug Deliv Rev., 61(11):953-64,
2009; Heitz F, et al., Br J Pharmacol., 157(2):195-206, 2009, and
references in either of the foregoing, which are incorporated
herein by reference. In some embodiments, a PTD is used to enhance
cell uptake of a small molecule, siRNA, aptamer, or polypeptide
that inhibits PLA2G16.
[0144] In some embodiments, a compound causes a decrease in PLA2G16
level or catalytic activity of at least 50% when used in a
cell-free or cell-based assay at a concentration equal to or less
than approximately 1 mM, 500 .mu.M, 100 .mu.M, 50 .mu.M, 10 .mu.M,
5 .mu.M, or 1 .mu.M. In some embodiments, a compound causes a
decrease in PLA2G16 activity of at least 50% (i.e., a decrease to
50% or less of the activity that would be expected in the absence
of the compound) when used in a cell-free or cell-based assay at
lower concentrations, e.g., equal to or less than approximately 500
nM, 100 nM, 50 nM, or 10 nM or less. In some embodiments, a
compound causes a decrease in PLA2G16 activity of at least 50% when
used at a concentration between 0.1-10 nM. Various methods suitable
for assessing PLA2G16 level or activity are mentioned above. In
some embodiments, a compound causes a decrease in production or
progeny virus of at least 50% (i.e., a decrease to 50% or less of
the number of progeny viruses that would be expected in the absence
of the compound) or a decrease in cytopathic effect of at least 50%
when used in a suitable cell culture system at a concentration
equal to or less than approximately 1 mM, 500 .mu.M, 100 .mu.M, 50
.mu.M, 10 .mu.M, 5 .mu.M, or 1 .mu.M. In some embodiments, a
compound causes a decrease in production or progeny virus or
cytopathic effect of at least 50% when used in a suitable cell
culture system at lower concentrations, e.g., equal to or less than
approximately 500 nM, 100 nM, 50 nM, or 10 nM or less. In some
embodiments, a compound causes a decrease in production or progeny
virus of at least 50% when used in a suitable cell culture system
when used at a concentration between 0.1-10 nM. Various methods
suitable for assessing virus production or cytopathic effect are
mentioned above. In other aspects, a compound causes a decrease of
at least 25%, or at least 75%, or at least 90%, in PLA2G16 level,
catalytic activity, and/or production of progeny virus or
cytopathic effect.
[0145] It is noted that, in general, the PLA2G16 inhibitors and
methods of use thereof do not depend on, and are not limited by,
the way in which an inhibitor was identified or generated or the
components used to identify or generate the PLA2G16 inhibitor. For
example, in certain embodiments of the invention a PLA216 inhibitor
identified using a human PLA2G16 polypeptide and/or using human
cells is used to treat humans. In certain embodiments of the
invention a PLA216 inhibitor identified using a human PLA2G16
polypeptide and/or using human cells is used to treat non-human
animals, e.g., non-human vertebrate animals. In some embodiments, a
PLA216 inhibitor identified using a PLA2G16 polypeptide of a
non-human animal and/or using cells derived from a non-human animal
is used to treat non-human animals of that species, different
non-human animal species, and/or humans. A PLA216 inhibitor that
inhibits infection by a virus that infects human cells could be
used to treat humans, non-human animals, or both, in various
embodiments of the invention. For example, in certain embodiments a
PLA216 inhibitor that inhibits infection by a virus that infects
human cells is used to inhibit infection by a virus that mainly or
only infects cells of a non-human animal.
VII. Pharmaceutical Compositions, Methods of Treatment, and Other
Applications
[0146] A compound identified, selected, or designed according to a
method described herein can have a variety of uses. In some
embodiments, a compound is useful for therapeutic purposes, e.g.,
as a therapeutic agent for a subject in need of treatment for a
viral infection.
[0147] In some embodiments, a subject is "suffering from" a viral
infection when excessive numbers of a viral population are present
in or on the organism's body and/or when the effects of the
presence of a virus population(s) is damaging the cells or other
tissue of an organism. A subject can be "in need of treatment for"
a viral infection if, for example, the subject is suffering from a
viral infection or is at increased risk of developing a viral
infection as compared with (i) most members of the general
population; and/or (ii) the level of risk that the subject
typically experiences.
[0148] The invention contemplates treatment of a wide variety of
viral infections in human and/or animal subjects, e.g., infection
due to any of the viruses discussed herein. In some embodiments,
the virus is a picornavirus, e.g., a cardiovirus, echovirus,
enterovirus (e.g., a coxsackievirus, rhinovirus, or echovirus), or
hepatovirus, or rhinovirus. In some embodiments, the virus clusters
phylogenetically within the enterovirus genus. In some embodiments,
the picornavirus is classified with a species selected from the
group consisting of: Human enterovirus A, Human enterovirus B,
Human enterovirus C, Human enterovirus D, Simian enterovirus A,
Bovine enterovirus, Porcine enterovirus B, Human rhinovirus A,
Human rhinovirus B and Human rhinovirus C. In some embodiments, the
picornavirus is classified with a species selected from the group
consisting of: Human enterovirus A, Human enterovirus B, Human
enterovirus C, Human enterovirus D, Human rhinovirus A, Human
rhinovirus B and Human rhinovirus C. In some embodiments, the virus
is of a serotype that has been deposited at the American Type
Culture Collection (ATCC) or National Collection of Pathogenic
Viruses (NCPV) of the Health Protection Agency of the UK and,
optionally, is available for distribution.
[0149] The invention provides methods of treating diseases and
medical conditions resulting from viral infection, e.g., by a
picornavirus. Exemplary diseases and conditions include, e.g.,
asthma exacerbation, bronchiolitis, colitis, common cold, COPD
exacerbation, encephalitis, encephalomyelitis, enterocolitis,
foot-and-mouth disease, hand-foot-and-mouth disease,
gastroenteritis, herpangina, hepatitis, meningitis,
meningoencephalitis, myocarditis, pancreatitis, polio, and
pneumonia. In some aspects, the invention contemplates ex vivo uses
of the PLA2G16 inhibitors. For example, organs, tissues, or cells
intended for use in transplantation (e.g., xenotransplantation or
transplantation into an individual of the same species) can be
contacted ex vivo with a PLA2G16 inhibitor, e.g., to reduce the
likelihood of transmitting a viral infection to the recipient. In
another embodiment, recipients of an organ, tissue, or cell
transplant can be treated with a PLA2G16 inhibitor, e.g., to reduce
the likelihood of contracting a viral infection from the
transplanted cells, tissues, or organ(s). Such treatment could
commence prior to, during, or after the transplant procedure.
[0150] In some embodiments, the virus is one for which an effective
vaccine does not exist, is not in commercial use, or is not widely
used. For example, coxsackievirus B3 is widespread in the human
population and causes serious diseases such as myocarditis or
pancreatitis. Coxsackievirus B4 can cause a broad range of diseases
such as aseptic meningitis, meningoencephalitis, myocarditis,
hepatitis, pancreatitis, gastroenteritis, necrotizing
enterocolitis, and pneumonia. However, despite the clinical
significance of these viruses, there is no commercially available
and clinically applicable prophylactic vaccine. Enterovirus 71 is
another virus of significant medical importance for which a vaccine
is not available.
[0151] In some embodiments, the virus is one for which an effective
vaccine is in commercial use and/or available. Without limitation,
the inventive methods may find use to treat subjects who are
unvaccinated or otherwise non-immune, to treat subjects infected
with a strain of virus against which a vaccine may not afford
sufficient immunity, etc. In some embodiments, the individual is
infected by a vaccine strain, e.g., an attenuated strain. In some
embodiments, the invention provides methods of treating human
subjects who may have been exposed to or infected by a poliovirus,
e.g., unvaccinated or otherwise non-immune individuals (e.g.,
immunocompromised individuals), in the setting of a polio outbreak,
individuals who travel to or from a region where polio has not been
eradicated, etc. In some embodiments, the invention contemplates
treating livestock in need of treatment for foot-and-mouth disease
virus, e.g., in the setting of a foot-and-mouth disease
outbreak.
[0152] In some embodiments, a PLA2G16 inhibitor, e.g., a PLA2G16
inhibitor identified according to the instant invention, can have
one or more therapeutic uses in addition to, or instead of, for
treating a viral infection. In some embodiments, a PLA2G16
inhibitor is useful as a therapeutic agent for a subject in need of
treatment for excess body fat, a disease associated with excess
body fat, or a metabolic disorder. Excess body fat can be a
condition of having more body fat than desired by the subject or
having an amount of body fat that is considered within sound
medical judgement to contribute to a disease or to confer an
increased risk of disease. In some embodiments, a compound is
useful to treat as atherosclerosis or vascular disease (e.g.,
cardiovascular or cerebrovascular disease). In some embodiments,
the compound is useful for treating obesity, e.g., in a subject
having a body mass index (BMI) greater than or equal to 30. In some
embodiments, a compound is useful to treat a metabolic disorder,
e.g., diabetes (e.g., type II diabetes, also called diabetes
mellitus), glucose intolerance, insulin resistance, metabolic
syndrome, leptin deficiency, or hypertriglyceridemia.
[0153] Inventive methods of treatment can include a step of
identifying a subject suffering from or at risk of a viral
infection, a step of identifying a virus suspected of causing an
infection, a step of selecting a therapeutic agent or combination
of agents based at least in part on the identity or suspected
identity of the virus and/or the location or characteristics of the
infection, and/or a step of prescribing, providing, or
administering a selected agent to the subject. In certain
embodiments of the invention, the method includes determining that
a subject has a significant likelihood (e.g., at least 5%) of
suffering from or being at risk of infection by a virus, e.g., a
picornavirus. A subject can be "at risk of an infection" in any of
a variety of circumstances. "At risk of" implies at increased risk
of, relative to the risk such subject would have in the absence of
one or more circumstances, conditions, or attributes of that
subject, and/or relative to the risk that an average, healthy
member of the population would have and/or relative to the risk
that the subject had at a previous time. The population is
typically a group of subjects of the same species. Examples of
conditions that place a subject "at risk" include, but are not
limited to, immunodeficiencies (e.g., genetic immunodeficiencies);
prior treatment with antibiotic agent(s) that may have reduced or
eliminated normal microbial flora; treatment with agent(s) that
suppress the immune system (e.g., cancer chemotherapy,
immunosuppressive agents); exposure to agents that damage the
immune system; chronic diseases such as diabetes, COPD, or cystic
fibrosis; coexisting or preceding bacterial or fungal infection;
surgery or other trauma; infancy or old age; occupations, events,
or living conditions that entail exposure to pathogenic viruses,
etc., or any other condition that within the judgement and skill of
the subject's health care provider place the subject at increased
risk. In some embodiments, subject can be at increased risk of
developing a viral infection if the subject has been recently
exposed to a pathogenic virus, e.g., the subject has had contact
with an individual known or believed to be suffering from a viral
infection (e.g., exposure within the preceding 1, 2, 3, or 4 weeks
or within the "incubation period" of the virus). In one embodiment,
an incubation period refers to the range of times following
exposure to a virus during which 10%-90% of individuals who develop
symptomatic infection would do so.
[0154] Any of a variety of methods may be employed to identify a
subject in need of treatment (e.g., a subject at risk of or
suffering from a viral infection) according to the present
invention. For example, such methods include clinical diagnosis
based at least in part on symptoms, medical history (if available),
physical examination, laboratory tests, imaging studies,
immunodiagnostic assays, nucleic acid based diagnostics, and/or
isolation and culture of potentially causative viruses from
samples, such as blood, urine, sputum, saliva, nasal secretions,
stool, synovial fluid, cerebrospinal fluid, bronchealveolar lavage,
pus, or any sample of body fluid, cells, or tissue. In some
embodiments, diagnosis can at least in part be based on serology
(e.g., detection of an antibody that specifically reacts with the
virus). In some embodiments, diagnosis can be based at least in
part on isolating the virus and/or a viral genome or gene product
from the subject. In some embodiment, the sample is tested for a
viral genome or gene product. For example, PCR or other nucleic
acid amplification methods can be used to amplify viral DNA or RNA,
which can be detected in a variety of ways such as
hybridization-based methods. Multiplexed PCR or other amplification
methods are useful. Signal amplification assays include branched
chain DNA assays and hybrid capture assays. Transcription based
amplification and nucleic acid sequence based amplification (NASBA)
may be used. Microarrays, e.g., oligonucleotide micorarrays, can be
used. A microarray can be a solid phase or suspension array (e.g.,
a microsphere-based approach such as the Luminex platform).
Immunological methods (e.g., ELISA or particle agglutination) can
be used to detect viral antigens, e.g., polypeptides. Labelled
compounds that specifically bind to a viral component can be used.
In some embodiments, a virus is grown in cell culture and
identified. Identification can be based on morphology, effect on
cultured cells, and/or detection of virus specific nucleic acids
and/or polypeptides. In some embodiments, a specific virus is not
identified, while in other embodiments a specific virus is
identified.
[0155] The compounds and compositions disclosed herein and/or
identified or validated using a method described herein may be
administered by any suitable means such as orally, intranasally,
subcutaneously, intramuscularly, intravenously, intra-arterially,
parenterally, intraperitoneally, intrathecally, intratracheally,
ocularly, sublingually, vaginally, rectally, dermally, or by
inhalation, e.g., as an aerosol. Depending upon the type of
condition (e.g., viral infection) to be treated, compounds of the
invention may, for example, be inhaled, ingested or administered by
systemic routes. Thus, a variety of administration modes, or
routes, are available. The particular mode selected will depend, of
course, upon the particular compound selected, the particular
condition being treated and the dosage required for therapeutic
efficacy. The methods of this invention, generally speaking, may be
practiced using any mode of administration that is medically or
veterinarily acceptable, meaning any mode that produces acceptable
levels of efficacy without causing clinically unacceptable (e.g.,
medically or veterinarily unacceptable) adverse effects. The term
"parenteral" includes intravenous, intramuscular, intraperitoneal,
subcutaneous, intraosseus, and intrasternal injection, or infusion
techniques. In some embodiments, a route of administration is
parenteral or oral. Optionally, a route or location of
administration is selected based at least in part on the particular
viral infection and/or location of infected tissue. For example, a
compound may be delivered to or near an infected tissue. In some
embodiments, inhaled medications are of use. Such administration
allows direct delivery to the lung, for example in subjects with a
respiratory infection, although it could also be used to achieve
systemic delivery. Several types of metered dose inhalers are
regularly used for administration by inhalation. These types of
devices include metered dose inhalers (MDI), breath-actuated MDI,
dry powder inhaler (DPI), spacer/holding chambers in combination
with MDI, and nebulizers. In other embodiments, intrathecal
administration may be of use, e.g., in a subject with a viral
infection of the central nervous system. Other appropriate routes
and devices for administering therapeutic agents will be apparent
to one of ordinary skill in the art.
[0156] Suitable preparations, e.g., substantially pure
preparations, of a PLA2G16 inhibitor may be combined with one or
more pharmaceutically acceptable carriers or excipients, etc., to
produce an appropriate pharmaceutical composition. The invention
provides a variety of pharmaceutically acceptable compositions for
administration to a subject comprising (i) a PLA2G16 inhibitor; and
(ii) a pharmaceutically acceptable carrier or excipient. The term
"pharmaceutically acceptable carrier or excipient" refers to a
carrier (which term encompasses carriers, media, diluents,
solvents, vehicles, etc.) or excipient which does not significantly
interfere with the biological activity or effectiveness of the
active ingredient(s) of a composition and which is not excessively
toxic to the host at the concentrations at which it is used or
administered. Other pharmaceutically acceptable ingredients can be
present in the composition as well. Suitable substances and their
use for the formulation of pharmaceutically active compounds is
well-known in the art (see, for example, "Remington's
Pharmaceutical Sciences", E. W. Martin, 19th Ed., 1995, Mack
Publishing Co.: Easton, Pa., and more recent editions or versions
thereof, such as Remington: The Science and Practice of Pharmacy.
21st Edition. Philadelphia, Pa. Lippincott Williams & Wilkins,
2005, for additional discussion of pharmaceutically acceptable
substances and methods of preparing pharmaceutical compositions of
various types which are incorporated herein by reference in their
entirety).
[0157] A pharmaceutical composition is typically formulated to be
compatible with its intended route of administration. For example,
preparations for parenteral administration include sterile aqueous
or non-aqueous solutions, suspensions, and emulsions. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media, e.g., sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; preservatives, e.g., antibacterial agents such
as benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. Such
parenteral preparations can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions and compounds for use in such
compositions may be manufactured under conditions that meet
standards or criteria prescribed by a regulatory agency. For
example, such compositions and compounds may be manufactured
according to Good Manufacturing Practices (GMP) and/or subjected to
quality control procedures appropriate for pharmaceutical agents to
be administered to humans.
[0158] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a subject to be treated. Suitable
excipients for oral dosage forms are, e.g., fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Optionally the oral formulations may also be formulated
in saline or buffers for neutralizing internal acid conditions or
may be administered without any carriers. Dragee cores are provided
with suitable coatings. For this purpose, concentrated sugar
solutions may be used, which may optionally contain gum arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for identification or to
characterize different combinations of active compound doses.
[0159] Pharmaceutical preparations which can be used orally include
push fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral
administration may also be used. Such microspheres have been well
defined in the art.
[0160] Formulations for oral delivery may incorporate agents to
improve stability in the gastrointestinal tract and/or to enhance
absorption.
[0161] For administration by inhalation, inventive compositions may
be delivered in the form of an aerosol spray from a pressured
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, a fluorocarbon, or a nebulizer.
Liquid or dry aerosol (e.g., dry powders, large porous particles,
etc.) can be used. The present invention also contemplates delivery
of compositions using a nasal spray or other forms of nasal
administration.
[0162] For topical applications, pharmaceutical compositions may be
formulated in a suitable ointment, lotion, gel, or cream containing
the active components suspended or dissolved in one or more
pharmaceutically acceptable carriers suitable for use in such
comporisition.
[0163] For local delivery to the eye, the pharmaceutically
acceptable compositions may be formulated as solutions or
micronized suspensions in isotonic, pH adjusted sterile saline,
e.g., for use in eye drops, or in an ointment.
[0164] Pharmaceutical compositions may be formulated for
transmucosal or transdermal delivery. For transmucosal or
transdermal administration, penetrants appropriate to the barrier
to be permeated may be used in the formulation. Such penetrants are
generally known in the art. Inventive pharmaceutical compositions
may be formulated as suppositories (e.g., with conventional
suppository bases such as cocoa butter and other glycerides) or as
retention enemas for rectal delivery.
[0165] In some embodiments, a pharmaceutical composition includes
one or more agents intended to protect the active agent(s) against
rapid elimination from the body, such as a controlled release
formulation, implants, microencapsulated delivery system, etc.
Compounds may be encapsulated or incorporated into particles, e.g.,
microparticles or nanoparticles. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, PLGA, collagen, polyorthoesters,
polyethers, and polylactic acid. Methods for preparation of such
formulations will be apparent to those skilled in the art. For
example, and without limitation, a number of particle-based
delivery systems are known in the art for delivery of siRNA. The
invention contemplates use of such compositions. Liposomes or other
lipid-based particles can also be used as pharmaceutically
acceptable carriers.
[0166] In some embodiments, the invention provides a
pharmaceutically acceptable derivative of a PLA2G16 inhibitor,
e.g., a PLA2G16 inhibitor described herein or identified or
validated according to an inventive method. According to the
present invention, a pharmaceutically acceptable derivative of a
particular compound includes, but is not limited to,
pharmaceutically acceptable salts, esters, salts of such esters, or
any other adduct or derivative which upon administration to a
subject in need thereof is capable of providing the compound,
directly or indirectly. Thus, pharmaceutically acceptable
derivatives can include salts, prodrugs, and/or active metabolites.
The term "pharmaceutically acceptable salt" refers to those salts
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of humans and/or lower animals
without undue toxicity, irritation, allergic response and the like,
and which are commensurate with a reasonable benefit/risk ratio. A
wide variety of appropriate pharmaceutically acceptable salts are
well known in the art. Pharmaceutically acceptable salts include,
but are not limited to, those derived from suitable inorganic and
organic acids and bases. A pharmaceutically acceptable derivative
of a PLA2G16 inhibitor may be formulated and, in general, used for
the same purpose(s).
[0167] Pharmaceutical compositions of the invention, when
administered to a subject, are preferably administered for a time
and in an amount sufficient to treat the disease or condition for
which they are administered, e.g., a viral infection. Therapeutic
efficacy and toxicity of active agents can be assessed by standard
pharmaceutical procedures in cell cultures or experimental animals.
The data obtained from cell culture assays and animal studies can
be used in formulating a range of dosages suitable for use in
humans or other subjects. Different doses for human administration
can be further tested in clinical trials in humans as known in the
art. The dose used may be the maximum tolerated dose or a lower
dose. A therapeutically effective dose of an active agent in a
pharmaceutical composition may be within a range of about 0.001 to
about 100 mg/kg body weight, about 0.01 to about 25 mg/kg body
weight, about 0.1 to about 20 mg/kg body weight, about 1 to about
10 mg/kg. Other exemplary doses include, for example, about 1
.mu.g/kg to about 500 mg/kg, about 100 .mu.g/kg to about 5 mg/kg).
In some embodiments a single dose is administered while in other
embodiments multiple doses are administered. Those of ordinary
skill in the art will appreciate that appropriate doses in any
particular circumstance depend upon the potency of the agent(s)
utilized, and may optionally be tailored to the particular
recipient. The specific dose level for a subject may depend upon a
variety of factors including the activity of the specific agent(s)
employed, severity of the disease or disorder, the age, body
weight, general health of the subject, etc.
[0168] It may be desirable to formulate pharmaceutical
compositions, particularly those for oral or parenteral
compositions, in unit dosage form for ease of administration and
uniformity of dosage. Unit dosage form, as that term is used
herein, refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active agent(s) calculated to produce the
desired therapeutic effect in association with an appropriate
pharmaceutically acceptable carrier. The invention provides a
pharmaceutically acceptable unit dosage form containing a
predetermined amount of a PLA2G16 inhibitor, such amount being
appropriate to treat a subject in need of treatment for a viral
infection.
[0169] It will be understood that a therapeutic regimen may include
administration of multiple unit dosage forms over a period of time.
In some embodiments, a subject is treated for between 1-7 days. In
some embodiments a subject is treated for between 7-14 days. In
some embodiments a subject is treated for between 14-28 days. In
other embodiments, a longer course of therapy is administered,
e.g., over between about 4 and about 10 weeks. In some embodiments
a subject is treated at least until at least one symptom or sign of
viral infection has started to decrease in severity or has
significantly decreased in severity or until a subject is no longer
at risk of viral infection. In some embodiments, treatment may be
continued indefinitely, e.g., in order to achieve prophylaxis. For
example, a subject at risk of recurrent viral infection or wanting
to avoid viral infection may be treated for any period during which
such risk exists or the subject desires to avoid viral infection. A
subject may receive one or more doses a day, or may receive doses
every other day or less frequently, within a treatment period.
[0170] In some embodiments, two or more different PLA2G16
inhibitors are administered. In some embodiments, a PLA2G16
inhibitor is administered in combination with a second compound
useful for treating a viral infection. The phrase "in combination,
as used herein, with regard to combination treatment means with
respect to administration of first and second compounds,
administration performed such that (i) a dose of the second
compound is administered before more than 90% of the most recently
administered dose of the first agent has been metabolized to an
inactive form or excreted from the body; or (ii) doses of the first
and second compound are administered within 48 hours of each other,
or (iii) the agents are administered during overlapping time
periods (e.g., by continuous or intermittent infusion); or (iv) any
combination of the foregoing. The compounds may, but need not be,
administered together as components of a single composition. In
some embodiments, they may be administered individually at
substantially the same time (by which is meant within less than 10
minutes of one another). In some embodiments they may be
administered individually within a short time of one another (by
which is meant less than 3 hours, sometimes less than 1 hour,
apart). The compounds may, but need not, be administered by the
same route of administration. When administered in combination with
a second compound, the effective amount of a first compound needed
to elicit a particular biological response may be less or more than
the effective amount of the first compound when administered in the
absence of the second compound (or vice versa), thereby allowing an
adjustment of the amount dose of the either or both agent(s)
relative to the amount that would be needed if one compound were
administered in the absence of the other. For example, when the
compounds of the invention are administered in combination (e.g., a
PLA2G16 inhibitor and a second antiviral compound), a
sub-therapeutic dosage of either of the compounds, or a
sub-therapeutic dosage of both, may be used in the treatment of a
subject in need of treatment for a viral infection. In some
embodiments, the two compounds are used in combination, the second
antiviral compound may in some embodiments be administered at a
sub-therapeutic amount to produce a desirable therapeutic result. A
"sub-therapeutic amount" as used herein refers to an amount which
is less than that amount which would be expected to produce a
therapeutic result in the subject if administered in the absence of
the other compound, e.g., less than a recommended amount. The
effects of multiple compounds may, but need not be, additive or
synergistic. One or more of the compounds may be administered
multiple times.
[0171] In some embodiments, an antiviral agent known in the art as
being useful for treating a subject infected with a particular
virus, e.g., a Picornavirus, is used as a second compound in
combination with a PLA2G16 inhibitor. In some embodiments, an
antibody that neutralizes or inhibits the virus is used. In some
embodiments, a compound that inhibits viral fusion is used. In some
embodiments a protease inhibitor or kinase inhibitor is used. In
some embodiments an RNAi agent is used, e.g., an siRNA, e.g.,
targeting a viral gene. In some embodiments a capsid binding agent
is used. In some embodiments, the second compound is, e.g.,
ruprintrivir, pleconaril, a pyridazinyl oxime ether, or arbidol.
See, e.g., Barnard D L., Current status of anti-picornavirus
therapies Curr Pharm Des.12(11):1379-90, 2006; DePalma, A M, et
al., Medicinal Research Reviews, 28(6): 823-884, 2008.
[0172] In some embodiments, a compound that is not sufficiently
active to be therapeutically useful is rendered therapeutically
useful when administered in combination with a PLA2G16 inhibitor.
In some embodiments, a lower dose of such compound can be used when
administered in combination with a PLA2G16 inhibitor.
[0173] In some embodiments, the invention provides a composition
comprising a PLA2G16 inhibitor and a second compound useful for
inhibiting a viral infection, e.g., an infection by a picornavirus.
In some embodiments, a unit dosage form comprising the two (or
more) agents is provided.
[0174] The present invention also provides pharmaceutical packs or
kits comprising one or more containers (e.g., vials, ampoules,
bottles) containing a pharmaceutically acceptable PLA2G16 inhibitor
and, optionally, one or more other pharmaceutically acceptable
ingredients. Optionally associated with such container(s) can be a
notice in the form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceutical products, which
notice reflects approval by the agency of manufacture, use or sale
for human administration. The notice may describe, e.g., doses,
routes and/or methods of administration, approved indications
(e.g., viral infections that the pharmaceutical composition has
been approved for use in treating), mechanism of action, or other
information of use to a medical practioner and/or patient.
Different ingredients may be supplied in solid (e.g. lyophilized)
or liquid form. Each ingredient will generally be suitable as
aliquoted in its respective container or provided in a concentrated
form. Kits may also include media for the reconstitution of
lyophilized ingredients. The individual containers of the kit are
preferably maintained in close confinement for commercial sale.
[0175] A virus to be inhibited according to the instant invention
may infect a cell type, organ or organ system of interest. For
example, in some embodiments the virus infects cells of the
gastrointestinal tract. In some embodiments the virus infects the
liver, e.g., hepatocytes. In some embodiments the virus infects the
respiratory system, e.g., cells of the upper and/or lower
respiratory tract. In some embodiments the virus infects muscle
cells, e.g., cardiac muscle cells. In some embodiments the virus
infects the nervous system (e.g., neurons). In some embodiments the
virus infects the central nervous system. In some embodiments the
virus infects skin cells (e.g., keratinocytes). In some embodiments
the virus infects mucosal cells. In some embodiments the virus
infects immune system cells, e.g., lymphocytes or macrophages. In
some embodiments, a virus infection is associated with damage to a
cell type, organ, or organ system of interest. Such damage could
arise due to infection of cells by the virus and/or due to
immune-mediated mechanisms.
[0176] In some embodiments, a compound is useful for research
purposes, e.g., to further study the role of PLA2G16 in normal
physiologic processes or pathologic processes. For example, a
compound can be used to further study the role of PLA2G16 in
metabolism and/or in viral infection.
[0177] In another aspect, the invention provides a method of
generating a non-human multicellular organism, e.g., a non-human
animal, e.g., a non-human vertebrate, that has increased resistance
to viral infection, e.g., by a picornavirus. In one aspect, the
non-human multicellular organism has reduced endogenous PLA2G16
activity as compared with a normal, non-transgenic organism of the
same species. In some embodiments, the organism is a transgenic,
non-human vertebrate that has a targeted insertion into, or
deletion of at least part of the PLA2G16 gene, so that the animal
has reduced expression of functional PLA2G16. In other embodiments,
the transgenic non-human animal expresses an RNAi agent, e.g., a
shRNA, that reduces PLA2G16 expression. In some embodiments, the
organism is not a rodent. In some embodiments the organism is not a
mouse. In some embodiments, the vertebrate is an animal of
commercial importance. For example, the organism may contribute at
least $10,000 to the gross national product of at least one country
and/or be an object of interstate or international commerce.
Exemplary animals of commercial importance are, e.g., cows, horses,
sheep, goats, pigs, chickens, turkeys, fish. In some embodiments,
an animal is a domesticated animal, e.g., a farm animal, e.g.,
livestock such as a cow, pig, sheep, goat, or horse. In some
embodiments, a virus-resistant animal is of a non-domesticated
species. Optionally the species is endangered. The method can be
used to identify individuals that are resistant to viral infection
and have improved likelihood of surviving in the wild or in
captivity. Animal resistance to viral infection may reduce the
spread of viruses that can infect both animal and human hosts.
Mutations or deletions can be engineered using, e.g., homologous
recombination, zinc finger nuclease-mediated recombination,
oligonucleotide-mediated gene modification, etc. The transgenic
organism can be generated using standard methods known in the art
for generating such organisms. For example, somatic cell nuclear
transfer (SCNT) can be used.
[0178] In another aspect, the invention provides a method
comprising identifying a non-human multicellular organism, e.g., a
non-human vertebrate, e.g., a non-human animal, with reduced or
absent functional PLA2G16. In some embodiments, the organism is not
a rodent. In some embodiments the animal is not a mouse. In some
embodiments, the organism has reduced expression of PLA2G16. In
some embodiments the organism expresses a functionally inactive
variant or fragment of PLA2G16. For example, the organism could
have a frameshift mutation or a deletion or alteration of at least
some residues needed for activity. The organism can be identified
using, e.g., genotyping (e.g., to identify animals that have
mutations or polymorphisms that result in decreased or altered
PLA2G16) and/or examining expression level in tissues and
identifying animals with low or absent PLA2G16 expression or
activity. In some embodiments, polymorphisms, e.g., single
nucleotide polymorphisms (SNPs) that are known in the art are
examined. For example, genome projects and other sequencing efforts
have identified numerous SNPs in animal genomes. SNPs, e.g., SNPs
located in or near the PLA2G16 gene can be assessed to identify
those that are associated with altered, e.g., reduced or absent,
functional PLA2G16. Animals carrying such SNPs can be identified.
In some embodiments, the reduced or absent PLA2G16 occurs in at
least some tissues and/or cells that are targets for infection by a
virus. In some embodiments, the reduced or absent PLA2G16 occurs in
most or all tissues. Organisms with a desirable trait (e.g.,
reduced or absent PLA2G16) can be selected. Standard breeding
techniques can be applied to produce animals with particularly low
PLA2G16 expression and/or activity. For example, standard methods
of livestock breeding could be used. Traditional breeding schemes
and/or marker-assisted selection may be employed. In some
embodiments, a mutation or polymorphism is a spontaneously arising
mutation, i.e., it is not generated by man. In some embodiments, a
mutation is generated by man, e.g., using radiation or chemical
mutagenesis. Thus the invention provides a method of producing a
non-genetically modified non-human organism, e.g., non-human
animal, with reduced or absent functional PLA2G16. In some
embodiments, the method comprising identifying or selecting an
organism with reduced or absent functional PLA2G16. In some
embodiments, the non-human organism, is produced using selective
breeding techniques. The invention further provides such organisms
and methods of use thereof.
[0179] In some embodiments, a method comprises providing or using
an organism with reduced or absent functional PLA2G16 in
agriculture and/or animal husbandry. The organism can be a
genetically modified organism or a non-genetically modified
organism. The organism may have reduced likelihood of infection
with a virus and/or may have reduced severity of infection. For
example, in some embodiments the animal has reduced likelihood of
infection and/or reduced severity of infection by a foot-and-mouth
disease virus. In some embodiments the animal has reduced
likelihood of infection and/or reduced severity of infection by a
bovine or porcine enterovirus. In some embodiments, the invention
provides a method comprising (a) providing an animal that has
reduced or absent functional PLA2G16; and (b) engaging in animal
husbandry using the animal. Animal husbandry encompasses the
breeding and raising of animals for meat or to harvest animal
products (such as milk, eggs, or wool) as well as the breeding and
care of species for work and/or companionship. Agriculture refers
to the production of food and/or goods through farming.
[0180] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The details of the description and the examples herein are
representative of certain embodiments, are exemplary, and are not
intended as limitations on the scope of the invention.
Modifications therein and other uses will occur to those skilled in
the art. These modifications are encompassed within the spirit of
the invention. It will be readily apparent to a person skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention.
[0181] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to include the plural referents.
Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention provides all
variations, combinations, and permutations in which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or more of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. It is contemplated that all embodiments described
herein are applicable to all different aspects of the invention
where appropriate. It is also contemplated that any of the
embodiments or aspects can be freely combined with one or more
other such embodiments or aspects whenever appropriate. Where
elements are presented as lists, e.g., in Markush group or similar
format, it is to be understood that each subgroup of the elements
is also disclosed, and any element(s) can be removed from the
group. It should be understood that, in general, where the
invention, or aspects of the invention, is/are referred to as
comprising particular elements, features, etc., certain embodiments
of the invention or aspects of the invention consist, or consist
essentially of, such elements, features, etc. For purposes of
simplicity those embodiments have not in every case been
specifically set forth in so many words herein. It should also be
understood that any embodiment or aspect of the invention can be
explicitly excluded from the claims, regardless of whether the
specific exclusion is recited in the specification. For example,
any one or more viral genera, viral species, viruses, assays,
compounds, diseases, subjects, or combinations thereof, can be
excluded.
[0182] Where the claims or description relate to a composition of
matter, e.g., a compound it is to be understood that methods of
making or using the composition of matter according to any of the
methods disclosed herein, and methods of using the composition of
matter for any of the purposes disclosed herein are aspects of the
invention, unless otherwise indicated or unless it would be evident
to one of ordinary skill in the art that a contradiction or
inconsistency would arise. Where the claims or description relate
to a method, e.g., a method of identifying a compound, it is to be
understood that methods of using the compound, or formulating a
composition comprising the compound, as described herein, are
aspects of the invention, unless otherwise indicated or unless it
would be evident to one of ordinary skill in the art that a
contradiction or inconsistency would arise.
[0183] Where ranges are given herein, the invention includes
embodiments in which the endpoints are included, embodiments in
which both endpoints are excluded, and embodiments in which one
endpoint is included and the other is excluded. It should be
assumed that both endpoints are included unless indicated
otherwise. Furthermore, it is to be understood that unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values that are
expressed as ranges can assume any specific value or subrange
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise. It is also understood that
where a series of numerical values is stated herein, the invention
includes embodiments that relate analogously to any intervening
value or range defined by any two values in the series, and that
the lowest value may be taken as a minimum and the greatest value
may be taken as a maximum. Numerical values, as used herein,
include values expressed as percentages. For any embodiment of the
invention in which a numerical value is prefaced by "about" or
"approximately", the invention includes an embodiment in which the
exact value is recited. For any embodiment of the invention in
which a numerical value is not prefaced by "about" or
"approximately", the invention includes an embodiment in which the
value is prefaced by "about" or "approximately". "Approximately" or
"about" generally includes numbers that fall within a range of 1%
or in some embodiments within a range of 5% of a number or in some
embodiments within a range of 10% of a number in either direction
(greater than or less than the number) unless otherwise stated or
otherwise evident from the context (except where such number would
impermissibly exceed 100% of a possible value). It should be
understood that, unless clearly indicated to the contrary, in any
methods claimed herein that include more than one act, the order of
the acts of the method is not necessarily limited to the order in
which the acts of the method are recited, but the invention
includes embodiments in which the order is so limited. It should
also be understood that unless otherwise indicated or evident from
the context, any product or composition described herein may be
considered "isolated".
EXAMPLES
Example 1
Characterization and Retroviral Infection of KBM7 Subclones
[0184] We first characterized a haploid genome setting in human
cells that we believed would be permissive for efficient forward
genetic approaches. A subclone of the CML cell line KBM7 has been
described to carry a near haploid chromosome set (Kotecki, M.,
Reddy, P. S., and Cochran, B. H. Isolation and characterization of
a near-haploid human cell line. Exp Cell Res 252, 273-280, 1999).
First we examined if this cell line (generously provided by Dr. B.
H. Cochran, Tufts University School of Medicine, Boston, Mass.)
could be easily propagated, was tolerant to viral infection and
could be efficiently subcloned. The term "KBM7 cell line" is used
herein to refer to this near-haploid cell line or to a subclone
thereof. Cells of the KBM7 cell line or a subclone thereof may be
referred to as "KBM7 cells". KBM7 cells had a high subcloning
efficiency (of around .about.80%), and several of the subclones
were examined further. The KBM7 subclones proliferated readily with
a generation time of approximately 24 hrs and could be maintained
at sparse and very high cell densities (e.g.,
.about.1.times.10.sup.7 cells/ml). Importantly, flow cytometric
analysis indicated that KBM7 subclones had a hypodiploid karyotype
as compared to diploid HCT116 colorectal carcinoma cells. One
subclone was examined further by 24-color FISH spectral karyotyping
and shown to be haploid for all chromosomes except chromosome 8 and
to contain a Philadelphia chromosome (t(9; 22)) characteristic of
BCR-ABL transformed chronic myelogenous leukemia cells. See also,
PCT Publication No. WO 2011/006145 and Carette J E, et al., Haploid
genetic screens in human cells identify host factors used by
pathogens, Science. 2009 Nov. 27; 326(5957):1231-5.
Example 2
Retroviral Infection of KBM7 Cells
[0185] We next showed that KBM-7 cells could be infected with
retroviruses. Virus was produced by transfection of a GFP
expressing retroviral vector with packaging vectors in 293T cells
(obtained from ATCC). The retroviral vector was pLIB-GFP (Clontech)
but it will be understood that many different retroviral vectors
could be used. Supernatant containing virus was used to infect KBM7
cells. To improve the infection efficiency of KBM7 cells with
retroviruses, different conditions were tested. Centrifugation of
the cells in a 24-well tissue culture dish for 45 minutes at 2,000
pm at room temperature resulted in a 2-fold increase in infection
efficiency compared to no centrifugation. Next the effect of
retronectin, polybrene and protamine sulphate addition was tested,
yielding efficiencies of 25%, 33% and 44%, respectively. Eight
microgram per milliliter culture medium of protamine sulphate is
the preferred addition. Concentration of virus by
ultracentrifugation for 1.5 h at 25,000 r.p.m. in a Beckman SW28
rotor dramatically improved infection rates compared to undiluted
virus and was preferred over concentration by Amicon filters. In
conclusion, KBM-7 cells are optimally infected when concentrated
virus is used for a spin-infection in the presence of protamine
sulphate. These subclones could be efficiently (.about.70-90%)
infected with GFP expressing retroviral or lentiviral viruses that
were VSV-G pseudotyped and maintained high levels of GFP expression
for several months.
Example 3
Construction of Gene Trap Vectors Containing Vectors Containing
Puromycin and GFP Selectable Markers
[0186] Retroviral gene trap vectors that contain an inactivated
LTR, a strong splice-acceptor site derived from the long fiber gene
of Adenovirus serotype 40 (Carette et al. 2005 The Journal of Gene
Medicine 7(8) 1053-1062), and either GFP or the puromycin
resistance gene (PURO) followed by a SV40 polyadenylation signal
were constructed as follows. The coding sequence of the PURO or GFP
was obtained by PCR amplification with primers containing
overhanging ClaI and NheI restriction sites as well as partial
splice acceptor sites:
(GFP:5'-GATCGCTAGCCGCATTTCTTTTTTCCAGATGGTGAGCAAGGGCGAGG-3' and
5'-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3' PURO:
5'-GATCGCTAGCCGCATTTCTTTTTTCCAGATGACCGAGTACAAGCCCAC-3' and
5'-GATCGGATCCTCAGGCACCGGGCTTGCGGGTC-3'). These PCR products were
inserted in pEGFPC1 (Clontech) replacing EGFP. Subsequently PCR was
performed to introduce the complete splice acceptor site and to
obtain either GFP or PURO followed by the poladenylation signal
using primers containing overhanging ClaI and BamHI sites as well
as the 5' end of the splice acceptor signal (GFP:
5'-GATCATCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAGATGG-3' and
5'-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3' PURO:
5'-GATCATCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAGATGAC-3' and
5'-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3'). These PCR products were
inserted in pRETRO-SUPER (Brummelkamp et al. 2002 Cancer Cell.
2(3):243-7) replacing the polIII promoter. The resulting plasmids
were named pGT-GFP and pGT-PURO. Gene trap constructs containing a
GFP or a puromycin reporter gene in all three reading frames were
generated.
[0187] The viral vectors contain an adenoviral splice acceptor site
immediately upstream of a promoterless reporter and polyadenylation
signal so that vector insertion into an intron of an active gene
inactivates the native locus, and transcription driven by the
gene's promoter results in a fusion transcript in which the
upstream exon(s) are spliced to the GFP or PURO gene. Since
transcription terminates at the inserted polyA site, the resulting
fusion transcript encodes a truncated and nonfunctional version of
the cellular protein and either GFP or PURO, as shown schematically
in FIG. 1B for a gene trap vector in which the gene encoding GFP
gene serves as a reporter gene.
Example 4
Generation of Mutant Cell Library
[0188] To generate a cell library with knock-out alleles in nearly
all genes, the near-haploid KBM7-cells were infected with the gene
traps generated as described in Example 3. Gene trap virus was made
by transfection of 293T cells in T175 dishes with either pGT-GFP or
pGT-PURO combined with retroviral packaging plasmids. The
virus-containing supernatant was concentrated using
ultracentrifugation for 1.5 h at 25,000 r.p.m. in a Beckman SW28
rotor. Batches of mutant KBM7 cells are typically made by infection
of one 24-well tissue culture dish containing 1.5 million cells per
well using the method described in Example 2. Cells infected with
the gene trap containing the puromycin resistance gene were
selected 2 days after infection using 500 ng puromycin per
milliliter. After selection by limiting dilution, cells were
expanded and frozen down for further screens. The GFP gene trap
infected cells were either used for screens unselected to negate
the gene trap introduced bias for actively expressed genes or were
selected using FACS sorting for GFP-expressing cells. In some cases
further stratification based on GFP expression was performed to
obtain batches of cells with different levels of GFP. To increase
the likelihood of identifying genes encoding gene products with a
relatively longer half-life, the screens were performed on or after
day 6 after gene trap infection, thereby allowing the gene products
to dilute during cell proliferation.
Example 5
Generation of a New Cell Type Useful for Haploid Genetics
[0189] We generated an additional cell type suitable for haploid
genetics using somatic cell reprogramming, a method that has
recently been described that allows reprogramming of the
differentiated cell state by, e.g., introduction of
pluripotency-inducing transcription factors such as OCT4, SOX2,
KLF4 and c-Myc (Zaehres, H., and Scholer, H. R. (2007). Induction
of pluripotency: from mouse to human. Cell 131, 834-835).
Introduction of these four transcription factors into KBM-7 cells
by retroviral infection (as described in Takahashi, K., et al.,
Cell, 131(5):861-72, 2007) resulted in the formation of adherent
cell clones. Some or most of these clones lost the hematopoietic
cell surface markers CD43 and CD45. The majority of these cells
were not pluripotent. A subclone was isolated and named "HAP1".
HAP1 cells could be cultured in medium containing 10% FCS and could
be expanded using trypsin. These cells were not hematopoietic and
the majority of these cells had a single copy of each chromosome
including chromosome 8
[0190] In contrast to influenza virus, KBM7 cells cannot be
productively infected with poliovirus (FIG. 1B, left panels). HAP1
cells however, are very susceptible to poliovirus infection and
undergo massive cell death within a few days (FIG. 1B, compare
upper right and lower right panels). Subsequently, fresh HAP1 cells
were infected with our gene trap retroviral construct and exposed
to poliovirus. Two resistant colonies were expanded and the
integrations were mapped. Both mutants contained independent
integrations in the known poliovirus entry receptor, PVR, thus
explaining their resistance. These results indicated that factors
essential for poliovirus infection can be found through haploid
genetic screens in reprogrammed, non-hematopoietic cell lines
derived from KBM7 cells, such as HAP1 cells.
Example 6
Identification of PLA2G16 as a Host Factor for Poliovirus
[0191] In order to identify new host factors for poliovirus, a
larger screen was undertaken using HAP1 cells (FIG. 1C). Retrovirus
was prepared and a mutant HAP1 cell library was generated as
described in Example 4. One hundred million mutagenized haploid
HAP1 cells were contacted with poliovirus and resistant colonies
were allowed to grow out. To identify gene trap insertion sites, an
inverse PCR protocol was adapted for use with massively parallel
sequencing techniques. In order to do so, genomic DNA was isolated
from 30 million cells that had been infected with a gene trap
vector. Four digestion reactions were performed per sample, two
using NlaIII and two using MseI. Subsequently the digested DNA was
column-purified (Qiagen) and 1 microgram DNA was ligated in a
volume of 300 microliter using T4 DNA ligase (NEB) at room
temperature overnight. After another round of column purification
the DNA was used as template for an inverse PCR with outward facing
primers. The oligonucleotides were designed to contain adaptor
sequences required for use with the "Illumina Genome Analyzer", a
massively parallel sequencing platform. Oligonucleotides used were:
5'-AATGATACGGCGACCACCGAGATCTGATGGTTCTCTAGCTTGCC-3'
5'-CAAGCAGAAGACGGCATACGACCCAGGTTAAGATCAAGGTC-3' for templates
digested with NlaIII. Oligonucleotides used were:
5'-AATGATACGGCGACCACCGAGATCTGATGGTTCTCTAGCTTGCC-3'
5'-CAAGCAGAAGACGGCATACGACGTTCTGTGTTGTCTCTGTCTG-3' for templates
digested with MseI. The four PCR reactions were pooled and used for
analysis on an Illumina Genome Analyzer according to manufacturer's
protocol and mapped against the human genome. Typically
.about.20,000 insertions sites mapping to different positions on
the human genome are obtained from this analysis. To facilitate
identification of genomic loci that are enriched for gene trap
insertions "insertion density" was plotted in a graph. Insertion
density was determined for every insertion by calculating
1/(average distance to three following insertions sites).
[0192] The plot in FIG. 2 shows the positions on the human
chromosome to which individual gene trap mutations were mapped on
the x-axis and the inverse of the distance of a particular mutation
to its neighbors on the y-axis. Mutations were found to be highly
enriched in chromosome 19 in the known poliovirus receptor (PVR)
and on chromosome 11 in a region that we identified as the gene
encoding the phospholipase PLA2G16. This gene contained 42
independent gene trap insertions.
Example 7
Confirmation that Gene Trap Insertion Ablates PLA2G16
Expression
[0193] Changing amino acid 113 from C to A (C113A mutation) renders
PLA2G16 catalytically inactive (Duncan, supra). Retroviral
constructs suitable for expressing wild type or mutant human
PLA2G16 in HAP1 cells with or without a FLAG tag were generated
using standard methods and introduced into HAP1 cells that
contained a gene trap insertion in the PLA2G16 locus
(PLA2G16.sup.GT) The pMX retroviral vector was used expressing
Flag-tagged human PLA2G16 and a IRES-Blasticidin selectable marker
gene or. For the non-tagged version of PLA2G16 human PLA2G16 cDNA
was cloned into the pBABEpuro retroviral vector). A Western blot
was performed using a polyclonal antibody to PLA2G16 to examine
PLA2G16 expression (FIG. 3). PLA2G16 was detected in wild type (WT)
HAP1 cells (i.e., HAP1 cells that had not been exposed to the gene
trap vector) (lane 1). As expected, PLA2G16.sup.GT cells lacked
detectable PLA2G16 (lane 2). As seen in lanes 3-6, PLA2G16 was
readily detected in PLA2G16.sup.GT cells that had received a
construct encoding PLA2G16 (wild type or C113A mutant). As expected
FLAG-tagged PLA2G16 was slightly larger in size than untagged
PLA2G16 (compare lanes 3 and 4 versus 5 and 6). This experiment
demonstrated that the gene trap had indeed effectively abrogated
PLA2G16 expression and that the constructs restored PLA2G16
expression when introduced into HAP 1 PLA2G16.sup.GT cells.
Example 8
Confirmation that Lack of PLA2G16 Renders Cells Resistant to
Poliovirus
[0194] To confirm that ablating PLA2G16 expression inhibits
infection by poliovirus, haploid PLA2G16.sup.GT cells were infected
with retrovirus encoding PLA2G16 or a catalytically inactive mutant
(containing a C113A alteration). PLA2G16.sup.GT grow robustly in
the absence of poliovirus (FIG. 4, left panel). As shown in FIG. 4
(second panel from left), PLA2G16.sup.GT cells (containing a
PLA2G16 gene trap insertion) also grow in the presence of
poliovirus. Complementation of PLA2G16 by retroviral overexpression
of wild type PLA2G16 in PLA2G16.sup.GT cells restores sensitivity
of these cells to poliovirus (second panel from right). This
requires the catalytic activity of PLA2G16 because complementation
with a catalytic site mutant (C113A) does not restore sensitivity
(right panel).
[0195] FIG. 6A shows the sensitivity of gene trap mutant cells to
poliovirus in graphical form. Cells were infected with indicated
MOIs and three days after infection viability was measured using an
MTT assay. Gene trap insertion into PLA2G16 renders cells sensitive
to infection by poliovirus, and sensitivity can be restored by
expressing wild type but not catalytically inactive mutant PLA2G16
in the cells. As expected, gene trap insertion into poliovirus
receptor renders haploid cells resistant to poliovirus infection.
Gene trap insertion into PLA2G16 has an essentially equivalent
effect to gene trap insertion into poliovirus receptor.
Example 9
PLA2G16 Insertion Renders Haploid Cells Resistant to
Coxsackievirus
[0196] FIG. 5 shows effect of coxsackievirus B1 on wild type
haploid cells and cells lacking functional PLA2G16. Cells were
plated in 24-well wells and monolayers were treated with
coxsackievirus B1 at the indicated MOIs. Four days after infection
viable, adherent cells were stained using crystal violet. Wild type
cells were highly sensitive to the virus at all MOIs tested (top
row). Cells mutant for PLA2G16 due to the gene trap insertion were
essentially unaffected by of coxsackievirus B1, even at high
concentrations of virus (second row from top). Complementation of
PLA2G16 by retroviral overexpression restores sensitivity of these
cells to coxsackievirus B1 (third row from top). This requires the
catalytic activity of PLA2G16 because complementation with a
catalytic site mutant (C113A) does not restore sensitivity (bottom
row). Thus, cells containing a PLA2G16 gene trap insertion are
resistant to coxsackievirus B1, and sensitivity can be restored by
expressing wild type but not catalytically inactive mutant
PLA2G16.
[0197] FIG. 6B shows the sensitivity of gene trap mutant cells to
coxsackievirus B1 in graphical form. Cells were contacted with
virus at the indicated MOIs and three days later viability was
measured using an MTT assay. Gene trap insertion into PLA2G16
renders cells sensitive to infection by coxsackievirus B1, and
sensitivity can be restored by expressing wild type but not
catalytically inactive mutant PLA2G16 in the cells. As expected,
gene trap insertion into the poliovirus receptor does not
significantly affect sensitivity to coxsackievirus B1.
Example 10
PLA2G16 Knockdown Increases Resistance to Rhinovirus
[0198] Ability of RNAi-mediated knockdown of PLA2G16 to inhibit
rhinovirus infection was studied in HeLa cells. PLA2G16 expression
was inhibited in HeLa cells using two different siRNAs targeted to
PLA2G16, and the ability of the cells to survive and proliferate
after exposure to human rhinoviruses HRV-2 and HRV-14 was examined.
The siRNAs were Ambion siRNA 223200, sequence
5'-CAAGAAACAAGCGACAAAtt-3' and siRNA 21977 sequence
5'-GUACCAGGUCAACAACAAAtt-3'. HeLa cells that had not been
transfected with siRNA or were transfected with a control siRNA
were highly susceptible to infection by human rhinoviruses HRV-2
and HRV-14 (FIG. 7, two left columns). Knock down of PLA2G16 in
Hela cells resulted in significantly increased resistance to both
HRV-2 and HRV-14 (FIG. 7, right two columns). Cells transfected
with siRNA targeted to PLA2G16 were able to survive and proliferate
well following exposure to HRV-2 and HRV-14.
* * * * *
References