U.S. patent application number 11/318940 was filed with the patent office on 2006-06-22 for methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated rna decay.
Invention is credited to Holger Beckmann, Kevin Czaplinski, Marc Learned, Stuart Peltz.
Application Number | 20060134681 11/318940 |
Document ID | / |
Family ID | 23832841 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134681 |
Kind Code |
A1 |
Beckmann; Holger ; et
al. |
June 22, 2006 |
Methods of assaying for compounds that inhibit premature
translation termination and nonsense-mediated RNA decay
Abstract
The present application provides methods of assaying for
compounds that inhibit premature translation termination and
nonsense mediated RNA decay in cells.
Inventors: |
Beckmann; Holger; (El
Cerrito, CA) ; Learned; Marc; (El Granada, CA)
; Peltz; Stuart; (Piscataway, NJ) ; Czaplinski;
Kevin; (Summerset, NJ) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Family ID: |
23832841 |
Appl. No.: |
11/318940 |
Filed: |
December 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10226803 |
Aug 21, 2002 |
7026122 |
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11318940 |
Dec 22, 2005 |
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09461508 |
Dec 14, 1999 |
6458538 |
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10226803 |
Aug 21, 2002 |
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Current U.S.
Class: |
435/6.11 ;
435/15; 435/6.12; 435/7.1 |
Current CPC
Class: |
G01N 33/5008 20130101;
G01N 2333/70503 20130101; G01N 2500/00 20130101; G01N 2333/90
20130101; C12Q 1/6897 20130101 |
Class at
Publication: |
435/006 ;
435/007.1; 435/015 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53; C12Q 1/48 20060101
C12Q001/48 |
Claims
1. A method of in vitro screening for compounds that modulate
premature translation termination and nonsense-mediated mRNA decay,
the method comprising the steps of: (i) incubating a translation
assay, the assay comprising an in vitro translation cellular
extract; a nucleic acid encoding a polypeptide, wherein the coding
sequence for the polypeptide comprises a premature stop codon; and
a candidate modulator compound; and (ii) detecting the polypeptide
translated from the nucleic acid.
2. The method of claim 1, wherein the nucleic acid encodes an
enzyme.
3. The method of claim 1, wherein the nucleic acid encodes an
immunoglobin.
4. The method of claim 1, wherein the nucleic acid encodes
luciferase, green fluorescent protein, red fluorescent protein,
phosphatase, peroxidase, kinase, chloramphenicol transferase, or
.beta.-galactosidase.
5. (canceled)
6. The method of claim 1, wherein the cellular extract is from
yeast, plants, mammals, or amphibians.
7. The method of claim 1, wherein the cellular extract is a
eukaryotic reticulocyte lysate.
8. (canceled)
9. The method of claim 1, wherein the cellular extract is a
mammalian tissue culture cell extract.
10. (canceled)
11. The method of claim 1, wherein the polypeptide is detected by
ELISA, light emission, colorimetric measurements, enzymatic
activity, or radioactivity.
12. The method of claim 1, wherein the assay is performed in a well
of a microtiter dish.
13-15. (canceled)
16. The method of in vivo screening for compounds that modulate
premature translation termination and nonsense-mediated mRNA decay,
the method comprising the steps of: (i) expressing in a cell a
nucleic acid encoding a polypeptide, wherein the coding sequence
for the polypeptide comprises a premature stop codon; (ii)
contacting the cell with a candidate modulator compound; and (iii)
detecting either the polypeptide translated from the nucleic acid
or RNA transcribed from the nucleic acid.
17. The method of claim 16, wherein the nucleic acid comprises a
promoter operably linked to a heterologous nucleic acid encoding
the polypeptide.
18. The method of claim 17, wherein the heterologous nucleic acid
encoding the polypeptide comprises an intron and at least two exons
comprising coding sequence.
19. (canceled)
20. The method of claim 18, wherein the heterologous nucleic acid
encodes a chimeric polypeptide.
21. (canceled)
22. The method of claim 17, wherein the nucleic acid encodes an
enzyme.
23. The method of claim 17, wherein the nucleic acid encodes
luciferase, green fluorescent protein, red fluorescent protein,
phosphatase, peroxidase, kinase, chloramphenicol transferase, or
.beta.-galactosidase.
24. The method of claim 17, wherein the nucleic acid encodes an
immunoglobin.
25. The method of claim 16, wherein the nucleic acid is an
endogenous gene.
26. The method of claim 25, wherein the endogenous gene is
.alpha.-globin, .beta.-globin, factor VIII, factor IX, vWF, p53,
dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1, hexosaminidase A,
neurofibromin 1, or neurofibromin 2.
27. The method of claim 25, wherein the endoegenous gene encodes an
immunoglobin.
28. The method of claim 16, wherein the polypeptide is detected by
ELISA, light emission, colorimetric measurements, enzymatic
activity, drug resistance, FACS, or radioactivity.
29. The method of claim 16, wherein the assay is performed in a
well of a microtiter dish.
30-34. (canceled)
35. The method of claim 16, wherein the cell is a human cell or a
mouse cell.
36. The method of claim 16, wherein the cell is stably transfected
with the nucleic acid.
37. A kit for screening for compounds that modulate translation
termination and nonsense-mediated mRNA decay, the kit comprising a
nucleic acid encoding a polypeptide, wherein the polypeptide coding
sequence comprises a premature stop codon; instructions for
practicing a method of screening for compounds that inhibit
translation termination at premature stop codons and nonsense
mediated RNA decay; and a control compound that inhibits
nonsense-mediated RNA decay.
38-43. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] Not applicable.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] Chain termination mutations are those in which a base
substitution or frameshift mutation changes a sense codon into one
of three stop codons (UAA, UAG, or UGA). Studies of yeast, human
genetic disorders, and immunoglobulin family gene expression have
identified an RNA surveillance mechanism that minimizes the
translation and regulates the RNA stability of nonsense RNAs
containing such chain termination mutations. This surveillance
mechanism is called "nonsense-mediated mRNA decay" ("NMD," see,
e.g., Hentze & Kulozik, Cell 96:307-310 (1999); Culbertson,
Trends in Genetics 15:74-80 (1999); Li & Wilkinson, Immunity
8:135-141 (1998); and Ruiz-Echevarria et al., TIBS 21-433-438
(1996)). NMD is a post transcriptional mechanism that is
operational in both normal cells (i.e., B and T cells) and cells
with genetic mutations (i.e., cells with mutations in
.beta.-globin, CFTR, and dystrophin).
[0004] The NMD machinery discriminates between normal and premature
stop codons, and then commits many RNAs with premature stop codons
to degradation. In some cases, when the premature stop codon is
located near the end of an ORF or in the last exon, the RNA is not
subject to NMD and results in production of a truncated
polypeptide.
[0005] A number of human diseases are caused by nonsense mutations,
e.g., p53 associated cancers, retinoblastoma, Duchenne muscular
dystrophy, cystic fibrosis, von Willebrand's disease, thalassemias,
neurofibromatosis, Tay-Sachs disease, and hemophilia. In cultured
cells having premature stop codons in the CFTR gene, synthesis of
full length CFTR was observed when the cells were treated with
aminoglycosides (see, e.g., Bedwell et al., Nat. Med. 3:1280-1284
(1997); Howard et al., Nat. Med. 2:467-469 (1996)). Furthermore, in
a mouse model for Duchenne muscular dystrophy, gentamycin sulfate
was found to suppress translational termination at premature stop
codons in the dystrophin gene. These antibiotics mediated
misreading and insertion of alternative amino acids at the site of
the premature stop codon (see, e.g., Barton-Davis et al., J. Clin
Invest. 104:375-381 (1999)). Dystrophin produced in this manner
provided protection against contraction-induced damage in the mdx
mice.
[0006] Compounds that suppress premature translation termination
would be a useful treatment for numerous diseases caused by
nonsense mutations. Accordingly, high throughput assays for drug
discovery related to NMD and inhibition of premature translation
termination is desirable. This invention provides these assays, as
well as other features which will become apparent upon review.
SUMMARY OF THE INVENTION
[0007] The present application therefore provides high throughput
methods of assaying for compounds that inhibit premature
translation termination and nonsense mediated RNA decay in
cells.
[0008] In one aspect, the present invention provides a method of in
vitro screening for compounds that modulate premature translation
termination and nonsense-mediated mRNA decay, the method comprising
the steps of: (i) incubating a translation assay, the assay
comprising an in vitro translation cellular extract; a nucleic acid
encoding a polypeptide, wherein the coding sequence for the
polypeptide comprises a premature stop codon; and a candidate
modulator compound; and (ii) detecting the polypeptide translated
from the nucleic acid.
[0009] In one embodiment, the cellular extract is from yeast,
plants, mammals, or amphibians. In another embodiment, the cellular
extract is a eukaryotic reticulocyte lysate, e.g., a rabbit
reticulocyte lysate. In another embodiment, the cellular extract is
a mammalian tissue culture cell extract, e.g., a HeLa cell S100
extract. In one embodiment, the nucleic acid is an in vitro
transcribed RNA.
[0010] In another aspect, the present invention provides a method
of in vivo screening for compounds that modulate premature
translation termination and nonsense-mediated mRNA decay, the
method comprising the steps of: (i) expressing in a cell a nucleic
acid encoding a polypeptide, wherein the coding sequence for the
polypeptide comprises a premature stop codon; (ii) contacting the
cell with a candidate modulator compound; and (iii) detecting
either the polypeptide translated from the nucleic acid or RNA
transcribed from the nucleic acid.
[0011] In one embodiment, the nucleic acid comprises a promoter
operably linked to a heterologous nucleic acid encoding the
polypeptide. In another embodiment, the heterologous nucleic acid
encoding the polypeptide comprises an intron and at least two exons
comprising coding sequence. In another embodiment, the premature
stop codon is located in a last exon. In another embodiment, the
heterologous nucleic acid encodes a chimeric polypeptide. In
another embodiment, the nucleic acid is an endogenous gene, e.g.,
an immunoglobulin, .alpha.-globin, .beta.-globin, factor VIII,
factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1,
hexosaminidase A, neurofibromin 1, or neurofibromin 2. In another
embodiment the cell is adhered to a solid substrate, e.g., a bead,
a membrane, and a microtiter plate. In another embodiment, the cell
is a human cell or a mouse cell. In another embodiment, the cell is
stably transfected with the nucleic acid.
[0012] In one embodiment, the nucleic acid encodes an enzyme. In
another embodiment, the nucleic acid encodes an immunoglobulin. In
another embodiment, the nucleic acid encodes luciferase, green
fluorescent protein, red fluorescent protein, phosphatase,
peroxidase, kinase, chloramphenicol transferase, or
.beta.-galactosidase. In another embodiment, the polypeptide is
detected by ELISA, light emission, colorimetric measurements,
enzymatic activity, or radioactivity.
[0013] In one embodiment, the assay is performed in a well of a
microtiter dish, e.g., a microtiter dish having 96 or 384 wells. In
another embodiment, the steps of the method are repeated in
parallel in a microtiter plate format, wherein between at least
about 100 and at least about 6,000 different compounds are tested.
In another embodiment, the assay is performed in a high throughput
integrated system comprising an automatic pipetting station, a
robotic armature, and a robotic controller.
[0014] In another aspect, the present invention provides a kit for
screening for compounds that modulate translation termination and
nonsense-mediated mRNA decay, the kit comprising a nucleic acid
encoding a polypeptide, wherein the polypeptide coding sequence
comprises a premature stop codon; instructions for practicing a
method of screening for compounds that inhibit translation
termination at premature stop codons and nonsense mediated RNA
decay; and a control compound that inhibits nonsense-mediated RNA
decay.
[0015] In one embodiment, the control compound is G418 or
gentamycin sulfate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows an overview of constructs for assays with
transiently or stably transiently infected cells. Positions of stop
codons, genes, and promoters are indicated.
[0017] FIG. 2 A-C shows that G418-mediated suppression is not cell
line specific. Cells, transiently infected with the indicated
plasmids for 24 hours, were exposed to the indicated concentration
of G418 (panel A, pMLT49; panel B, pMLT54; panel C, pMLT55).
Luciferase activity was measured after a 12 hour exposure.
[0018] FIG. 3 A-B shows G418-mediated suppression in various clones
of stably transfected 293 cells with plasmid pMLT55 (panel A,
relative luciferase activity; panel B, fold activation). The
indicated independent clones were exposed to the indicated
concentrations of G418. Luciferase activity was measured after a 12
hour exposure.
[0019] FIG. 4 shows G418-mediated suppression in a stably
transfected 293 cell line (clone #31) in high throughput format (96
well plate). The indicated cell numbers were plated per well of a
96 well plate. Cells were exposed to the indicated concentrations
of G418. Luciferase activity was measured after a 12 hour exposure
on a robotic 96 well plate reader. Gentamycin was also used to
inhibit NMD and premature translation termination.
[0020] FIG. 5 shows an overview of constructs for use in a
biochemical suppression assay using RRL (rabbit reticulocyte
lysate). Positions of stop codons, genes, IRES sequence, and
promoter regions are indicated.
[0021] FIG. 6 A-D shows properties of the biochemical suppression
assay using RRL. In vitro translation reactions were performed in
the presence of the indicated amounts of RRL and luciferase RNA
(panels A and B, wild type; panels C and D, stop codon). Low levels
of luciferase activity is RNA (panels A and C) and RRL (panels B
and D) dependent. WT and stop codon-containing luciferase RNA are
schematically shown. Luciferase activity was measured after 90
minutes of incubation. Reactions were performed in a 96 well
format.
[0022] FIG. 7 A-B shows that suppression by gentamycin is time
dependent. In vitro translation assays were performed in the
presence of RRL, luciferase RNA (panel A, WT; panel B, stop codon)
and the indicated concentrations of gentamycin. Reactions were
incubated 45 or 90 minutes before luciferase activity was
determined. WT and stop codon containing RNA are schematically
shown. The reactions were performed in either a 96 or 384 well
format.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0023] The present invention provides high throughput assay methods
for screening compounds that inhibit NMD and premature translation
termination caused by nonsense mutations in RNA. The assays can be
performed in vitro, using cellular translation extracts or combined
transcription-translation extracts and heterologous or endogenous
reporter nucleic acids, either DNA or RNA. The assays can also be
performed in vivo, using cells with heterologous (transiently or
stably transfected) or endogenous reporter nucleic acids.
Inhibitory compounds are detected by measuring whether or not
full-length reporter RNA or full-length reporter protein produced
in the assay.
[0024] Inhibitory compounds identified using the assays of the
invention are used to treat diseases related to nonsense mutations.
The compounds of the invention allow read-through of the nonsense
codons, leading to production of full-length polypeptide. Diseases
that are related to nonsense mutations include, e.g., blood
diseases, e.g., thalassemia (.alpha.-globin and .beta.-globin
genes), hemophilia A and B (factor VIII and factor IX genes), and
von Willebrand's disease (vWF gene); p53 related cancers, e.g.
lung, breast, colon, pancreatic, esophageal, non-Hodgkin's
lymphoma, and ovarian cancer (p53 gene); colorectal cancers (APC,
MSH1, and MSH2 genes); cystic fibrosis (CFTR gene); Duchenne
muscular dystrophy; (dystrophin gene) Tay-Sachs disease
(hexosaminidase A gene); Wilms tumor (Wt1 gene); retinoblastoma (Rb
gene), neurofibromatosis (NF1 and NF2 genes); kidney stones, and
collagen disorders.
[0025] As described above, in one embodiment, the assay is an in
vitro assay. The in vitro assay uses a translation extract or a
transcription-translation extract and a reporter nucleic acid
molecule encoding a reporter polypeptide with a premature stop
codon to test compounds that are potential inhibitors of
nonsense-mediated mRNA decay and premature translation termination.
Inhibitory compounds are identified by detecting production of a
full length polypeptide translated from the nucleic acid
molecule.
[0026] The reporter nucleic acid encodes any polypeptide, where the
full length polypeptide can be labeled or detected by any of the
methods described herein. In one embodiment, the polypeptide is an
enzyme such as luciferase, phosphatase, e.g., alkaline phosphatase,
peroxidase, e.g., horseradish peroxidase, kinase, chloramphenicol
transferase, or .beta.-galactosidase. In another embodiment, the
polypeptide is green fluorescent protein or red fluorescent
protein. In another embodiment, the polypeptide is any polypeptide
detectable with an antibody, or by radioactive labeling, etc. The
stop codon can be at any position in the polypeptide that is
N-terminal with respect to the correct stop codon for the
full-length polypeptide.
[0027] The in vitro reaction can be a translation reaction, or a
combined transcription-translation reaction. Cellular extracts for
translation and transcription-translation are commercially
available or can be prepared using standard methods known to those
of skill in the art (see, e.g., Sambrook et al., Molecular Cloning,
A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994); and Jackson &
Hunt, Methods in Enzymology 96:50-75 (1983)). For a combined
transcription-translation reaction, the nucleic acid is typically a
DNA molecule that is transcribed into RNA, which is then
translated. For a translation reaction, the nucleic acid is
typically an RNA, either an in vitro transcribed RNA or an
endogenous RNA. Methods of making in vitro transcribed RNA are well
know in the art, e.g., by using bacterial RNA polymerases such as
SP6 and T7 (see, e.g., Contreras et al., Nuc. Acids Res. 10:6353
(1982)). Suitable cellular extracts for the translation or
transcription-translation reaction include those from yeast,
fission yeast, plants, amphibians (e.g., Xenopus), plants (e.g.,
wheat germ) and mammals (see, e.g., Krieg & Melton, Nature
308:203 (1984); Dignam et al., Methods Enzymol. 182:194-203
(1990)). In one embodiment, the extract is a reticulocyte lysate,
preferably from rabbits. In another embodiment, the extract is from
cultured cells, preferably mouse, rat, or human cells, e.g., an
S100 extract from HeLa cells (Dignam et al., Nuc. Acids Res. 11:
1475-1489 (1983)).
[0028] In another embodiment, the assay is an in vivo, cell based
assay. The in vivo assay uses a cell, typically a cultured cell,
and a reporter nucleic acid molecule encoding a reporter
polypeptide with a premature stop codon to test compounds that are
potential inhibitors of nonsense-mediated mRNA decay and premature
translation termination. Inhibitory compounds are identified by
detecting production of a full length reporter polypeptide
translated from the nucleic acid molecule, or by detecting full
length reporter RNA transcribed from the reporter nucleic acid.
[0029] The reporter nucleic acid encodes any polypeptide, where the
full length polypeptide can be labeled or detected by any of the
methods described herein. The reporter nucleic acid can be an
endogenous gene or a heterologous gene that is stably or
transiently expressed in the cell. In addition, a heterologous
reporter nucleic acid can encode a chimeric polypeptide, e.g., a
chimera of different polypeptides and/or different polypeptides
from different species, for example, a chimera comprising an exon
and an intron from human .beta.-globin and a sequence encoding fly
luciferase. In one embodiment, the reporter nucleic acid is a
heterologous nucleic acid encoding an enzyme such as luciferase,
phosphatase, e.g., alkaline phosphatase, peroxidase, e.g.,
horseradish peroxidase, kinase, chloramphenicol transferase, or
.beta.-galactosidase. In another embodiment, the heterologous
nucleic acid encodes green fluorescent protein, red fluorescent
protein, or an immunoglobulin or a fragment thereof. In another
embodiment, the reporter nucleic acid is an endogenous gene, e.g.,
an immunoglobulin gene, .alpha.-globin, .beta.-globin, factor VIII,
factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1,
hexosaminidase A, neurofibromin 1, or neurofibromin 2. In another
embodiment, the polypeptide is any polypeptide detectable with an
antibody, or by radioactive labeling, etc.
[0030] The premature stop codon can be at any position in the
polypeptide that is N-terminal with respect to the correct stop
codon for the full-length polypeptide. The reporter polypeptide can
include introns, with the premature stop codon positioned in any
one of the exons. The premature stop codon can be naturally
occurring, or can be produced by in vitro mutagenesis techniques
such as PCR, linker insertion, oligonucleotide mediated
mutagenesis, and random chemical mutagenesis, both in vivo and in
vitro.
[0031] Any suitable eukaryotic cultured cell can be used in the
methods of the invention, e.g., insect cells, yeast cells, and
manmmalian cells, preferably human cells. The cells can be
transformed cell lines or primary cells, and can either be adherent
or in suspension. In one embodiment the cell is a hybridoma or a
pre-B cell or a cancer cell. In another embodiment, the cell is a
293 cell, a HeLa cell, a HepG2 cell, a K562 cell, or a 3T3 cell.
The cell can either be in solution or can be anchored to a solid
substrate such as a bead or a plate.
[0032] Preferably, the in vitro or in vivo assay is performed in a
high throughput format, using microtiter plates and liquid robotic
handling, or cell sorting using fluorescent antibodies or ligands
(FACS). The in vitro and in vivo assays can be incubated with the
test compound for any suitable length of time, i.e., from about 15,
30, or 45 minutes up to an hour, one hour, one and a half hours,
two hours, four hours, six hours, twelve hours or more up to a day
or more for an in vitro reaction, and from one hour, two hours,
four hours, six hours, twelve hours, or more, up to a day, two
days, three days or more for an in vivo reaction. The time period
of incubation of the test compound and the reporter reaction can be
determined by one of skill in the art using standard
methodology.
Definitions
[0033] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0034] By "detecting either the polypeptide translated from the
nucleic acid or RNA transcribed from the nucleic acid" is meant
assays for a compound that increases or decreases a parameter that
is indirectly or directly under the influence of premature
translation termination and/or NMD, e.g., functional, physical and
chemical effects. Such effects can be measured by any means known
to those skilled in the art, e.g., changes in spectroscopic
characteristics (e.g., fluorescence, absorbance, refractive index),
enzyme activity, protein expression, voltage-sensitive dyes,
radioisotope efflux, inducible markers, levels of RNA expression,
proper splicing of an mRNA, ligand binding assays, changes in
intracellular second messengers such as cAMP, cGMP, and inositol
triphosphate (IP3), changes in intracellular calcium levels, and
the like.
[0035] "Inhibitors" "suppressors" and "modulators" of are used
interchangeably to refer to inhibiting, suppressing, or modulating
premature translation termination and NMD with compounds identified
using in vitro and in vivo assays. Inhibitors are compounds that,
e.g., bind to, partially or totally block, decrease, prevent,
delay, inactivate, desensitize, or down regulate premature
translation termination and NMD. Modulators include naturally
occurring and synthetic ligands, antagonists, agonists, small
chemical molecules and the like. Such assays for inhibitors
include, e.g., expressing a test nucleic acid in an in vitro assay
or in a cell, applying putative modulator compounds, and then
determining the functional effects on RNA or protein levels of the
test nucleic acid. Samples or assays that are treated with a
potential inhibitor or modulator are compared to control samples
without the inhibitor or modulator to examine the extent of
inhibition. Control samples (untreated with inhibitors) are
assigned a relative activity value of 100%. Inhibition of premature
translation termination and NMD is achieved when the activity value
relative to the control is at least 110%, optionally 150%, 200%,
500%, 100%, 200%, 5000%, 10,000% or higher, i.e., 11/2 fold, 2
fold, 3 fold, 4 fold, 5 fold, 10 fold or more increase in activity.
Inhibition can be measured by detecting either full length
polypeptide or full length RNA. Optionally, an inhibitory control
reaction is included, using a known inhibitor such as an
aminoglycoside antibiotic, e.g., G418.
[0036] "Antibody" or "immunoglobulin" are used interchangeably and
refer to a polypeptide comprising a framework region from an
immunoglobulin gene or fragments thereof that specifically binds
and recognizes an antigen. The recognized immunoglobulin genes
include the kappa, lambda, alpha, gamma, delta, epsilon, and mu
constant region genes, as well as the various immunoglobulin
diversity/joining/variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0037] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms variable light chain (V.sub.L) and variable heavy chain
(V.sub.H) refer to these light and heavy chains respectively.
[0038] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H--C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990)).
[0039] For preparation of monoclonal or polyclonal antibodies, any
technique known in the art can be used (see, e.g., Kohler &
Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology
Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies
and Cancer Therapy (1985)). Techniques for the production of single
chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to
produce antibodies to polypeptides of this invention. Also,
transgenic mice, or other organisms such as other mammals, may be
used to express humanized antibodies. Alternatively, phage display
technology can be used to identify antibodies and heteromeric Fab
fragments that specifically bind to selected antigens (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990); Marks et al.,
Biotechnology 10:779-783 (1992)).
[0040] A "chimeric antibody" or "chimeric immunoglobulin" is an
antibody molecule in which (a) the constant region, or a portion
thereof, is altered, replaced or exchanged so that the antigen
binding site (variable region) is linked to a constant region of a
different or altered class, effector function and/or species, or an
entirely different molecule which confers new properties to the
chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor,
drug, etc.; or (b) the variable region, or a portion thereof, is
altered, replaced or exchanged with a variable region having a
different or altered antigen specificity.
[0041] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0042] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0043] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0044] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0045] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0046] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a finctionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0047] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0048] The following eight groups each contain amino acids that are
conservative substitutions for one another: [0049] 1) Alanine (A),
Glycine (G); [0050] 2) Aspartic acid (D), Glutamic acid (E); [0051]
3) Asparagine (N), Glutamine (Q); [0052] 4) Arginine (R), Lysine
(K); [0053] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V); [0054] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0055] 7) Serine (S), Threonine (T); and [0056] 8) Cysteine (C),
Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0057] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and proteins which can be made detectable, e.g., by
incorporating a radiolabel into the peptide or used to detect
antibodies specifically reactive with the peptide.
[0058] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0059] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0060] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element or a initiator element.
A promoter also optionally includes nearby activator or repressor
binding site(s) (e.g., SP1 or NF kappa B sites), or distal enhancer
or repressor elements, which can be located as much as several
thousand base pairs from the start site of transcription. A
"constitutive" promoter is a promoter that is active under most
environmental and developmental conditions. An "inducible" promoter
or a "tissue specific" promoter is a non-constitutive promoter that
is active under, e.g., environmental, temporal, tissue specific, or
developmental regulation. The term "operably linked" refers to a
functional linkage between a nucleic acid expression control
sequence (such as a promoter, or array of transcription factor
binding sites) and a second nucleic acid sequence, wherein the
expression control sequence directs transcription of the nucleic
acid corresponding to the second sequence.
[0061] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed
operably linked to a promoter.
Cloning and Expression of Reporter Genes
[0062] A. General Recombinant DNA Methods
[0063] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)). Standard
mutagenesis methods can be used to introduce nonsense mutations
into the reporter genes or endogenous genes of choice.
[0064] For nucleic acids, sizes are given in either kilobases (kb)
or base pairs (bp). These are estimates derived from agarose or
acrylamide gel electrophoresis, from sequenced nucleic acids, or
from published DNA sequences. For proteins, sizes are given in
kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are
estimated from gel electrophoresis, from sequenced proteins, from
derived amino acid sequences, or from published protein
sequences.
[0065] Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage & Caruthers,
Tetrahedron Letts. 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Devanter et al., Nucleic Acids
Res. 12:6159-6168 (1984). Purification of oligonucleotides is by
either native acrylamide gel electrophoresis or by anion-exchange
HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0066] The sequence of the cloned genes and synthetic
oligonucleotides can be verified after cloning using, e.g., the
chain termination method for sequencing double-stranded templates
of Wallace et al., Gene 16:21-26 (1981).
[0067]
[0068] B. Cloning Methods for the Isolation of Nucleotide
Sequences
[0069] In general, the nucleic acid sequences encoding heterologous
reporter genes of the invention are isolated from cDNA and genomic
DNA libraries by hybridization with probes, or isolated using
amplification techniques with oligonucleotide primers. For example,
reporter sequences are typically isolated from mammalian nucleic
acid (genomic or cDNA) libraries by hybridizing with a nucleic acid
probe, the sequence of which can be derived from Genebank or other
database or publication, or by using a cloned ortholog. A suitable
tissue from which RNA and cDNA is isolated is determined by one of
skill in the art. Methods for making and screening genomic and cDNA
libraries are well known (see, e.g., Gubler & Hoffman, Gene
25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).
Recombinant phage are analyzed by plaque hybridization as described
in Benton & Davis, Science 196:180-182 (1977). Colony
hybridization is carried out as generally described in Grunstein et
al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
[0070] Amplification techniques such as PCR and LCR using primers
can also be used to amplify and isolate reporter sequences from DNA
or RNA (see, e.g., Dieffenfach & Dveksler, PCR Primer: A
Laboratory Manual (1995); U.S. Pat. Nos. 4,683,195 and 4,683,202;
PCR Protocols: A Guide to Methods and Applications (Innis et al.,
eds, 1990)). These primers can be used, e.g., to amplify either the
full length sequence or a probe of one to several hundred
nucleotides, which is then used to screen a mammalian library for
full-length clones. In addition, degenerate primers encoding the
following amino acid sequences can be used to amplify a sequence of
a particular reporter gene. As described above, such primers can be
used to isolate a full length sequence, or a probe which can then
be used to isolated a full length sequence, e.g., from a library.
Nucleic acids encoding a reporter gene can also be isolated from
expression libraries using antibodies as probes.
[0071] Synthetic oligonucleotides can be used to construct
recombinant reporter genes. This method is performed using a series
of overlapping oligonucleotides usually 40-120 bp in length,
representing both the sense and nonsense strands of the gene. These
DNA fragments are then annealed, ligated and cloned.
[0072] Optionally, nucleic acids encoding chimeric proteins
comprising an reporter gene, e.g., a combination of luciferase and
.beta.-globin can be made according to standard techniques. The
chimeric genes can also be combinations of genes or exons from
different species, e.g., bacteria (e.g., E. coli), yeast,
invertebrates such as flies and nematodes, and mammalian genes
(e.g., rat, mouse, or human genes). Often the chimeric gene has an
exon and an intron from one gene, and a second exon from another
gene. Other heterologous proteins of choice for the production of
chimeric genes include, e.g., red or green fluorescent protein, a
phosphatase, a peroxidase, a kinase, chloramphenicol transferase,
luciferase, .beta.-galactosidase, a glutamate receptor, and the
rhodopsin presequence.
[0073] C. Expression in Prokaryotes and Eukaryotes
[0074] To obtain expression of a cloned gene or nucleic acid one
typically subclones the sequence into an expression vector that
contains a promoter to direct transcription, optionally a
transcription or translation terminator, and optionally for a
nucleic acid encoding a protein, a ribosome binding site for
translational initiation. Suitable bacterial promoters are well
known in the art and described, e.g., in Sambrook et al. and
Ausubel et al. Bacterial expression systems are available in, e.g.,
E. coli, Bacillus sp., and Salmonella (Palva et al., Gene
22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits
for such expression systems are commercially available. Eukaryotic
expression systems for mammalian cells, yeast, and insect cells are
well known in the art and are also commercially available. RNA
expression systems such as those using SP6, T3 or T7 RNA polymerase
are also well known to those in the art and are commercially
available.
[0075] The promoter used to direct expression of a heterologous
nucleic acid depends on the particular application. The promoter is
optionally positioned about the same distance from the heterologous
transcription start site as it is from the transcription start site
in its natural setting. As is known in the art, however, some
variation in this distance can be accommodated without loss of
promoter function.
[0076] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of the
nucleic acid in host cells. A typical expression cassette thus
contains a promoter operably linked to the nucleic acid sequence
and signals required for efficient polyadenylation of the
transcript, ribosome binding sites, and translation termination.
Additional elements of the cassette may include enhancers and, if
genomic DNA is used as the structural gene, introns with functional
splice donor and acceptor sites.
[0077] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0078] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as GST and LacZ. Epitope
tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc.
[0079] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors
derived from Epstein-Barr virus. Other exemplary eukaryotic vectors
include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5,
baculovirus pDSVE, and any other vector allowing expression of
proteins under the direction of the SV40 early promoter, SV40 later
promoter, metallothionein promoter, murine mammary tumor virus
promoter, Rous sarcoma virus promoter, polyhedrin promoter, or
other promoters shown effective for expression in eukaryotic
cells.
[0080] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are optionally
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0081] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express the nucleic
acid. Transformation of eukaryotic and prokaryotic cells are
performed according to standard techniques (see, e.g., Morrison, J.
Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in
Enzymology 101:347-362 (Wu et al., eds, 1983).
[0082] Any of the well known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, liposomes, microinjection, plasma vectors,
viral vectors and any of the other well known methods for
introducing cloned genomic DNA, cDNA, synthetic DNA or other
foreign genetic material into a host cell (see, e.g., Sambrook et
al., supra). It is only necessary that the particular genetic
engineering procedure used be capable of successfully introducing
at least one gene into the host cell.
[0083] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of the reporter gene, using standard techniques
identified below.
[0084] In addition to heterologous reporter genes, endogenous genes
can also be used as reporters. Cells comprising suitable endogenous
genes such as CFTR, dystrophin, immunoglobulins, .beta.-globin,
p53, retinoblastoma, neurofibromin, etc. are cultured under
conditions favoring expression of the endogenous reporter gene,
using standard techniques described below.
Cell Culture and Selection
[0085] The culture of cells used in the assays of the present
invention, including cell lines and cultured cells from tissue or
blood samples is well known in the art. Freshney (Culture of Animal
Cells, a Manual of Basic Technique (3.sup.rd ed. 1994)) and the
references cited therein provides a general guide to the culture of
cells. Additional information on cell culture is found in Ausubel
and Sambrook, supra. Cell culture media are described in The
Handbook of Microbiological Media (Atlas & Parks, eds., 1993).
Additional information is found in commercial literature such as
the Life Science Research Cell Culture Catalogue (1998) from
Sigma-Aldrich, Inc (St Louis, Mo.) and, e.g., the Plant Culture
Catalogue and Supplement (1997) also from Sigma-Aldrich, Inc (St
Louis, Mo.). Cells can be grown in bulk flasks and added to the
substrate (e.g., microtiter plate) or can be grown directly on the
substrate (e.g., in the wells of the microtiter plate, depending on
the intended application and available equipment.
[0086] Selection of cells is based upon the intended application.
Where inhibition of premature translation termination of a gene in
a particular cell is a target of the assay, the particular cell, or
a related cell culture form of the cell is typically the target. In
some embodiments, adherent cells which will adhere during culture
to the substrate of the container in which the cells are placed are
preferred. Many examples of adherent cell types are known,
including epithelial and endothelial cell types. Cells which are
not naturally adherent can often be made adherent by chemically
modifying the substrate (e.g., treating the substrate with silane
to provide OH groups, or with amine reagents to provide amine
groups) or by expressing cell surface receptor molecules on the
cell (e.g., recombinantly) and providing an appropriate ligand
fixed on the substrate. In one embodiment, the cell is a hybridoma
or a pre-B cell. In another embodiment, the cell is a mammalian or
mouse cell, e.g., a 293 cell, a HeLa cell, a HepG2 cell, a K562
cell, or a 3T3 cell.
Inhibitors and High Throughput Techniques
[0087] A. Inhibitors
[0088] The compounds tested as inhibitors and modulators of
premature translation termination and NMD can be any small chemical
compound, or a biological entity, such as a protein, sugar, nucleic
acid or lipid. Typically, test compounds will be small chemical
molecules and peptides. Essentially any chemical compound can be
used as a potential modulator in the assays of the invention. The
compounds can be dissolved in aqueous or organic solutions (e.g.,
methanol, DMSO, or a mixture of organic solvents). The assays are
designed to screen large chemical libraries by automating the assay
steps and providing compounds from any convenient source to assays,
which are typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland) and the like.
[0089] In one embodiment, high throughput screening methods involve
providing a combinatorial chemical or peptide library containing a
large number of potential therapeutic compounds (potential
modulator or ligand compounds). Such "combinatorial chemical
libraries" or "ligand libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0090] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0091] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091),
benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., J.
Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses
of small compound libraries (Chen et al., J. Amer. Chem. Soc.
116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303
(1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger
and Sambrook, all supra), peptide nucleic acid libraries (see, e.g.
U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et
al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. Nos. 5,506,337; benzodiazepines,
5,288,514, and the like).
[0092] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa.,
Martek Biosciences, Columbia, Md., etc.).
[0093] B. Solid State and Soluble High Throughput Assays
[0094] In one embodiment the invention provides in vitro soluble
assays in a high throughput format. In another embodiment, the
invention provides soluble or solid phase based in vivo assays in a
high throughput format, where the cell or tissue is attached to a
solid phase substrate. Optionally, the in vitro assay is a solid
phase assay.
[0095] In the high throughput assays of the invention, it is
possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter
plate can be used to run a separate assay against a selected
potential modulator, or, if concentration or incubation time
effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 100- about 1500 different
compounds. It is possible to assay several different plates per
day; assay screens for up to about 6,000-20,000 different compounds
is possible using the integrated systems of the invention. More
recently, microfluidic approaches to reagent manipulation have been
developed.
[0096] The molecule or cell of interest can be bound to the solid
state component, directly or indirectly, via covalent or non
covalent linkage of a tag and or a tag binder. A number of tags and
tag binders can be used, based upon known molecular interactions
well described in the literature. For example, where a tag has a
natural binder, for example, biotin, protein A, or protein G, it
can be used in conjunction with appropriate tag binders (avidin,
streptavidin, neutravidin, the Fc region of an immunoglobulin,
etc.) Antibodies to molecules with natural binders such as biotin
are also widely available and appropriate tag binders; see, SIGMA
Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
[0097] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody which recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs. For example, agonists and antagonists of cell
membrane receptors (e.g., cell receptor-ligand interactions such as
transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherein family, the integrin
family, the selectin family, and the like; see, e.g., Pigott &
Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins
and venoms, viral epitopes, hormones (e.g., opiates, steroids,
etc.), intracellular receptors (e.g. which mediate the effects of
various small ligands, including steroids, thyroid hormone,
retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic
acids (both linear and cyclic polymer configurations),
oligosaccharides, proteins, phospholipids and antibodies can all
interact with various cell receptors.
[0098] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0099] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly gly sequences of between about 5 and 200 amino acids. Such
flexible linkers are known to persons of skill in the art. For
example, poly(ethelyne glycol) linkers are available from
Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally
have amide linkages, sulfhydryl linkages, or heterofunctional
linkages.
[0100] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent which fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example, groups which are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature. See, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank
& Doring, Tetrahedron 44:60316040 (1988) (describing synthesis
of various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry
39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
Labels and Means of Detection
[0101] Detectable labels and moieties can be primary labels (where
the label comprises an element which is detected directly or which
produces a directly detectable element) or secondary labels (where
the detected label binds to a primary label, e.g., as is common in
immunological labeling). An introduction to labels, labeling
procedures and detection of labels is found in Polak & Van
Noorden (1997) Introduction to Immunocytochemistry (2.sup.nd ed.
1977) and Handbook of Fluorescent Probes and Research Chemicals, a
combined handbook and catalogue Published by Molecular Probes,
Inc., Eugene, Oreg. Primary and secondary labels can include
undetected elements as well as detected elements.
[0102] The particular label or detectable group used in the assay
is not a critical aspect of the invention, as long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means.
[0103] Useful primary and secondary labels in the present invention
can include spectral labels such as fluorescent dyes (e.g.,
fluorescein and derivatives such as fluorescein isothiocyanate
(FITC) and Oregon Green.TM., rhodamine and derivatives (e.g., Texas
red, tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin,
biotin, phycoerythrin, AMCA, CyDyes.TM., and the like), radiolabels
(e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, .sup.33P,
etc.), enzymes (e.g., horseradish peroxidase, alkaline phosphatase
etc.), spectral calorimetric labels such as colloidal gold or
colored glass or plastic (e.g. polystyrene, polypropylene, latex,
etc.) beads.
[0104] The label may be coupled directly or indirectly to a
component of the detection assay according to methods well known in
the art. Non-radioactive labels are often attached by indirect
means. Generally, a ligand molecule (e.g., biotin) is covalently
bound to the molecule. The ligand then binds to another molecules
(e.g., streptavidin) molecule, which is either inherently
detectable or covalently bound to a signal system, such as a
detectable enzyme, a fluorescent compound, or a chemiluminescent
compound. As indicated above, a wide variety of labels may be used,
with the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0105] In general, a detector which monitors a particular probe or
probe combination is used to detect the recognition reagent label.
Typical detectors include spectrophotometers, phototubes and
photodiodes, microscopes, scintillation counters, cameras, film and
the like, as well as combinations thereof Examples of suitable
detectors are widely available from a variety of commercial sources
known to persons of skill. Commonly, an optical image of a
substrate comprising bound labeling nucleic acids is digitized for
subsequent computer analysis.
[0106] Preferred labels include those which utilize enzymes such as
hydrolases, particularly phosphatases, kinases, esterases and
glycosidases, or oxidotases, particularly peroxidases;
chemiluminescence (e.g., enzymes such as horseradish peroxidase or
alkaline phosphatase with substrates that produce photons as
breakdown products; kits available, e.g., from Molecular Probes,
Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL);
color production (using, e.g., horseradish peroxidase,
.beta.-galactosidase, or alkaline phosphatase with substrates that
produce a colored precipitate; kits available from Life
Technologies/Gibco BRL, and Boehringer-Mannheim); hemifluorescence
(using, e.g., alkaline phosphatase and the substrate AttoPhos
(Amersham) or other substrates that produce fluorescent products);
fluorescence (e.g., using Cy-5 (Amersham), fluorescein, and other
fluorescent tags, and fluorescent proteins such as Green and Red
Fluorescent Protein); antibodies bound to a detectable moiety, and
radioactivity. Other methods for labeling and detection will be
readily apparent to one skilled in the art. For example, phenotypic
changes such as drug resistance can be used as a "label" in the
present invention.
[0107] One preferred example of detectable secondary labeling
strategies utilizes an labeled antibody which recognizes a cell
surface molecule such as an immunoglobulin or a channel molecule
such as CFTR. The antibody is detected using FACS. In another
embodiment, an antibody is used in an ELISA assay to detect a
reporter molecule of the invention.
[0108] Preferred enzymes that can be used as reporters or
detectable moieties include, e.g., .beta.-galactosidase,
luciferase, green or red fluorescent protein, kinase, peroxidase,
e.g., horse radish peroxidase, phosphatase, e.g., alkaline
phosphatase, and chloramphenicol transferase. The chemiluminescent
substrate for luciferase is luciferin. One embodiment of a
chemiluminescent substrate for .beta.-galactosidase is
4-methylumbelliferyl-.beta.-D-galactoside. Embodiments of alkaline
phosphatase substrates include p-nitrophenyl phosphate (pNPP),
which is detected with a spectrophotometer;
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
(BCIP/NBT) and fast red/napthol AS-TR phosphate, which are detected
visually; and 4-methoxy-4-(3-phosphonophenyl)
spiro[1,2-dioxetane-3,2'-adamantane], which is detected with a
luminometer. Embodiments of horse radish peroxidase substrates
include 2,2'azino-bis(3-ethylbenzthiazoline-6 sulfonic acid)
(ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, and
o-phenylenedianiine (OPD), which are detected with a
spectrophotometer; and 3,3,5,5'-tetramethylbenzidine (TMB),
3,3'diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and
4-chloro-1-naphthol (4C1N), which are detected visually. Other
suitable substrates are known to those skilled in the art. The
enzyme-substrate reaction and product detection are performed
according to standard procedures known to those skilled in the art
and kits for performing enzyme immunoassays are available as
described above.
[0109] In one embodiment, inhibition of premature translation
termination or NMD is measured by quantitating the amount of label
in a cell fixed to a solid support. Typically, presence of a test
compound during cell incubation will increase or decrease the
amount of label relative to a control incubation which does not
comprise the test compound, or as compared to a baseline
established for a cell type and culture condition (e.g., with a
reporter molecule). Means of detecting and quantitating labels are
well known to those of skill in the art. Thus, for example, where
the label is a radioactive label, means for detection include a
scintillation counter or photographic film as in autoradiography.
Where the label is optically detectable, typical detectors include
microscopes, cameras, phototubes and photodiodes and many other
detection systems which are widely available.
[0110] RNA expression can also analyzed by techniques known in the
art, e.g., reverse transcription and amplification of mRNA, e.g.,
RTQ-PCR, isolation of total RNA or poly A.sup.+ RNA, northern
blotting, dot blotting, in situ hybridization, RNase protection,
probing DNA microchip arrays, and the like. In one embodiment, high
density oligonucleotide analysis technology (e.g., GeneChip.TM.) is
used to identify reporter RNA molecules of the invention, see,
e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869-876
(1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al.,
Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat.
Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.
8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866
(1998).
Kits
[0111] The present invention also provides for kits for screening
for inhibitors of premature translation termination and NMD. Such
kits can be prepared from readily available materials and reagents.
For example, such kits can comprise any one or more of the
following materials: in vitro translation cellular extract; a
nucleic acid encoding a polypeptide, wherein the coding sequence
for the polypeptide comprises a premature stop codon, reaction
tubes, and instructions for testing inhibitor activity. A wide
variety of kits and components can be prepared according to the
present invention, depending upon the intended user of the kit and
the particular needs of the user.
[0112] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0113] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0114] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of noncritical parameters that could be
changed or modified to yield essentially similar results.
Example I
Cell-based Assay
[0115] On day 1, clone 55-31, a 293 cell line stably transfected
with pMLT 55 (see FIG. 1) was plated in 96 well plates in the
morning in DMEM media with 10% fetal bovine serum. Approximately 80
plates are plated at 40,000 cells per well in 100 .mu.L. The cells
were incubated overnight at 37.degree. C.
[0116] On day two, test compounds (1 .mu.L of 1 mM stock solution
in 100 .mu.L DMSO; 10 .mu.M final concentration) were added. G418
(1 mg/ml) was added to column 12 of each plate as a control. The
plates were incubated at 37.degree. C. for 12-16 hours.
[0117] On day three, luciferase substrate was added to the cells
and the plates were read on a Torcon automated luciferase assay
reader for 96 well plates (see, e.g., FIGS. 3-4). Luciferase
substrate is commercially available, e.g., from Promega.
[0118] Optionally, days one and two can be combined, with plating
early in the day and compounds added late in the day, with
overnight incubation and then assaying for luciferase activity on
the following day.
Example II
Biochemical Assay
[0119] In vitro synthesized reporter RNA (see constructs in FIG. 5)
was added to 15 .mu.L of fully supplemented rabbit reticulocyte
lysate according to standard methodology (prepared by the method of
Jackson & Hunt, Methods in Enzymology 96:50-75 (1983). 0.5 to 1
.mu.g of in vitro transcribed RNA was used per reaction. The
reaction was mixed with 15 .mu.L of a 2% DMSO solution containing
20 .mu.M of test compound. The reaction was incubated for 90
minutes at 30.degree. C. in a humidified incubator. Luciferase
substrate was added and luciferase activity was read on a Torcon
automated reader (see FIGS. 6-7).
* * * * *