U.S. patent application number 12/123426 was filed with the patent office on 2008-10-09 for methods of identifying therapeutic compounds in a genetically defined setting.
Invention is credited to Bernhard O. Palsson.
Application Number | 20080248483 12/123426 |
Document ID | / |
Family ID | 26973872 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080248483 |
Kind Code |
A1 |
Palsson; Bernhard O. |
October 9, 2008 |
METHODS OF IDENTIFYING THERAPEUTIC COMPOUNDS IN A GENETICALLY
DEFINED SETTING
Abstract
The invention provides a method of identifying therapeutic
compounds in a genetically defined setting. The method consists of
contacting a cell indicative of a pathological condition from a
diseased individual and a cell from a genetically related normal
individual with a plurality of candidate therapeutic compounds
under suitable assay conditions, and identifying a compound that
preferentially alters a predetermined property of the cell from the
diseased individual.
Inventors: |
Palsson; Bernhard O.; (La
Joll, CA) |
Correspondence
Address: |
MCDERMOTT, WILL & EMERY
4370 LA JOLLA VILLAGE DRIVE, SUITE 700
SAN DIEGO
CA
92122
US
|
Family ID: |
26973872 |
Appl. No.: |
12/123426 |
Filed: |
May 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10357556 |
Feb 3, 2003 |
7374880 |
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12123426 |
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09568595 |
May 10, 2000 |
6524797 |
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10357556 |
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60304193 |
May 10, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/29 |
Current CPC
Class: |
G01N 2500/00 20130101;
G01N 33/5011 20130101; G01N 33/5008 20130101; G01N 33/502
20130101 |
Class at
Publication: |
435/6 ;
435/29 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. A method of identifying a therapeutic compound potentially
effective against a predetermined pathological condition,
comprising: (a) contacting ex vivo a cell indicative of said
pathological condition from a diseased individual with a plurality
of candidate therapeutic compounds under suitable assay conditions;
(b) contacting ex vivo a control cell from a normal individual
genetically related to said diseased individual with said plurality
under said assay conditions, said genetically related normal
individual having a common ancestor within 7 to 10 generations with
said diseased individual, and (c) identifying a compound from said
plurality that preferentially alters one or more predetermined
properties of said cell from said diseased individual, said
compound being characterized as a therapeutic compound potentially
effective against said pathological condition.
2. The method of claim 1, wherein step (a) comprises contacting a
cell from each of two or more genetically related diseased
individuals.
3. The method of claim 2, wherein said two or more genetically
related diseased individuals exhibit a range of disease severity or
risk.
4. The method of claim 1, wherein step (b) comprises contacting a
control cell from each of two or more genetically related normal
individuals having a common ancestor within 7 to 10 generations
with said diseased individual.
5. The method of claim 1, wherein step (a) comprises contacting a
cell from each of two or more genetically related diseased
individuals and step (b) comprises contacting a control cell from
each of two or more genetically related normal individuals having a
common ancestor within 7 to 10 generations with said diseased
individual.
6. The method of claim 1, wherein said normal individual having a
common ancestor within 7 to 10 generations with said diseased
individual and said diseased individual are members of a
genetically homogeneous population.
7. The method of claim 6, wherein said genetically homogeneous
population is an Icelandic population.
8. The method of claim 1, wherein said plurality of candidate
therapeutic compounds comprises greater than 10.sup.5
compounds.
9. The method of claim 1, wherein said plurality of candidate
therapeutic compounds are sequentially contacted with said cell
from said diseased individual or said control cell from said normal
individual having a common ancestor within 7 to 10 generations with
said diseased individual.
10. The method of claim 1, wherein said plurality of candidate
therapeutic compounds are sequentially contacted with said cell
from said diseased individual and said control cell from said
normal individual having a common ancestor within 7 to 10
generations with said diseased individual.
11. The method of claim 1, wherein said plurality of candidate
therapeutic compounds are simultaneously contacted with said cell
from said diseased individual or said control cell from said normal
individual having a common ancestor within 7 to 10 generations with
said diseased individual.
12. The method of claim 1, wherein said plurality of candidate
therapeutic compounds are simultaneously contacted with said cell
from said diseased individual and said control cell from said
normal individual having a common ancestor within 7 to 10
generations with said diseased individual.
13. The method of claim 1, wherein said pathological condition is
selected from the group consisting of diseases of the
cardiovascular system, nervous system, immune system, respiratory
system, gastrointestinal system, endocrine system, and cancer.
14. The method of claim 1, wherein said method is automated.
15. The method of claim 1, wherein step (a) comprises contacting a
cell from each of 10 or more genetically related diseased
individuals.
16. The method of claim 1, wherein step (a) comprises contacting a
cell from each of 100 or more genetically related diseased
individuals.
17. The method of claim 1, wherein step (b) comprises contacting a
control cell from each of 10 or more genetically related normal
individuals.
18. The method of claim 1, wherein step (b) comprises contacting a
control cell from each of 100 or more genetically related normal
individuals.
19. The method of claim 1, wherein said plurality of candidate
therapeutic compounds comprises greater than 50 compounds.
20. The method of claim 1, wherein said pathological condition is
cancer.
21. The method of claim 1, wherein said predetermined property is
selected from the group consisting of proliferation, adhesion,
differentiation, motility and apoptosis.
22. The method of claim 1, wherein said cell is obtained from a
tissue selected from the group consisting of breast, prostate,
colon, lung, brain and ovary.
23. The method of claim 1, wherein said cell from said diseased
individual is an unaffected cell.
24. The method of claim 1, wherein said cell from said diseased
individual and said cell from said normal individual are propagated
in culture.
25. The method of claim 1, wherein said cell from said diseased
individual and said cell from said normal individual are transduced
or transfected with a nucleic acid molecule.
26. The method of claim 1, further comprising: (d) repeating steps
(a) and (b) with said therapeutic compound obtained from step (c),
and determining the ability of said therapeutic compound to
preferentially alter a second or more predetermined property of
said cell from said diseased individual.
Description
[0001] This application is a continuation of U.S. Ser. No.
10/357,556, filed Feb. 3, 2003, which is a continuation of U.S.
Ser. No. 09/568,595, filed May 10, 2000, which claims the benefit
of priority of U.S. Provisional Application No. 60/304,193, filed
May 10, 1999, which was converted from U.S. Ser. No. 09/309,468,
each of which the entire contents is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to the fields of
medicine and pharmacology and, more specifically, to methods of
identifying therapeutic compounds in a genetically defined
setting.
[0003] In the past, the process of discovering novel therapeutic
compounds was slow and laborious, and usually involved
administering individually synthesized compounds to experimental
animals in the hope of observing a therapeutic effect. Recently,
significant advances have been made in medicinal chemistry,
resulting in the development of combinatorial chemistry methods
that allow the rapid production of enormous libraries of
structurally distinct compounds. Additionally, due to recent
progress in understanding the underlying molecular mechanisms of
many diseases, it has become possible to develop in vitro assays to
rapidly screen candidate therapeutic compounds. Automation of these
assays using computer-controlled robotic systems in high throughput
screening methods has made it possible for biotechnology companies
to screen millions of compounds per year.
[0004] The identification of therapeutic compounds using automated
screening methods requires the development of in vitro assays that
accurately predict the therapeutic potential of a compound
identified by the assay for treatment of the particular
pathological condition. So far, current drug screening methods have
fallen short of this goal. For example, a variety of cell-free
assays have been developed that focus on interactions of candidate
compounds with isolated target molecules. Such assays have been
shown to be of limited value, since neither the binding properties
nor the expected biological properties of the compounds have
usually proven to be relevant in vivo.
[0005] In an attempt to overcome the limitations of cell-free
assays, a variety of cell-based assays have recently been
developed. Such assays detect particular cellular functions
believed to be relevant to the underlying disease mechanism. To
date, however, most cell-based assays for screening candidate
therapeutic compounds have used established cell lines. Established
cell lines, as evidenced by their ability to be propagated
indefinitely in culture, are highly abnormal and are often
neoplastically transformed. Therefore, screening assays using such
abnormal cell lines are poorly predictive of the therapeutic
efficacy of compounds for affecting cell function in an
individual.
[0006] Additionally, current cell-based assays to identify
therapeutic compounds generally use cell lines established from a
single individual, or cell lines established from unrelated normal
and diseased individuals. Screening assays using cells from
unrelated individuals are likely to identify compounds that alter a
cellular function related to the genetic differences between the
individuals, rather than compounds that alter a cellular function
relevant to the underlying disease mechanism.
[0007] Therefore, there exists a need for improved methods of
screening candidate therapeutic compounds. Ideally, such methods
would use relevant cells and assay conditions so as to be highly
predictive of the therapeutic efficacy of the compounds. The
present invention satisfies this need and provides related
advantages as well.
SUMMARY OF INVENTION
[0008] The invention provides a method of identifying a therapeutic
compound potentially effective against a predetermined pathological
condition. The method consists of contacting a cell indicative of
the pathological condition from a diseased individual with a
plurality of candidate therapeutic compounds under suitable assay
conditions, and also contacting a cell indicative of the
pathological condition from a normal individual, who is genetically
related to the diseased individual, with the plurality of compounds
under the same assay conditions. A compound from the plurality of
compounds that preferentially alters a predetermined property of
the cell from the diseased individual is identified, and
characterized as a therapeutic compound potentially effective
against the pathological condition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows a signal transduction pathway in a normal
individual (Panel A) and two diseased individuals (Panels B and
C).
[0010] FIG. 2 shows two signal transduction pathways that together
produce a signal in an assay of a predetermined property.
[0011] FIG. 3 shows a method of mapping disease-associated gene
products to loci of a signal transduction pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The invention is directed to a method of identifying
therapeutic compounds effective against a variety of pathological
conditions. The method is advantageous in using primary cells
obtained from genetically related normal and diseased individuals
to screen candidate compounds. Therefore, variables due to genetic
heterogeneity and abnormalities of established cell lines are
minimized, and compounds identified by the method will have a high
likelihood of being therapeutically effective in patients.
[0013] Specifically, the invention provides a method of identifying
a therapeutic compound potentially effective against a
predetermined pathological condition. The method consists of
contacting a cell indicative of the pathological condition from a
diseased individual with a plurality of candidate therapeutic
compounds under suitable assay conditions, and also contacting a
cell indicative of the pathological condition from a normal
individual, who is genetically related to the diseased individual,
with the plurality of compounds under the same assay conditions. A
compound from the plurality of compounds that preferentially alters
a predetermined property of the cell from the diseased individual
is identified, and characterized as a therapeutic compound
potentially effective against the pathological condition.
[0014] As used herein, the term "plurality of candidate therapeutic
compounds" is intended to mean 2 or more different candidate
therapeutic compounds. Such compounds can be used in a screening
assay to identify one or more compounds that affect a predetermined
property of a cell from a diseased individual to a greater extent
than a cell from a normal individual. The number of different
compounds in the plurality of compounds can be determined by those
skilled in the art depending on the application of the method. For
example, a smaller number of candidate compounds can be
advantageous if the type of compound that is likely to affect a
predetermined property of the cell is known or can be predicted.
Additionally, if the method is used to compare the efficacy of
compounds against cells from multiple normal or diseased
individuals, it can be desirable to practice the method using a
smaller number of compounds. Therefore, a plurality of compounds
can include 2, 3, 4, 5, 10 or more, 20 or more, or 50 or more
candidate compounds. Those skilled in the art also understand that
the method can be practiced with a single compound, if desired.
[0015] However, when the type of compound that is likely to affect
a predetermined property of the cell is unknown, it is generally
understood that the larger the number of candidate therapeutic
compounds, the greater the likelihood of identifying a therapeutic
compound. Additionally, when the method is practiced using cells
from only one or several diseased individuals, and one or several
normal individuals, it may be desirable to screen a large number of
different compounds. Therefore, a plurality of candidate
therapeutic compounds can contain, for example, greater than about
10.sup.3 different compounds, preferably greater than about
10.sup.5 different compounds, more preferably, greater than about
10.sup.7 different compounds.
[0016] A candidate therapeutic compound can be a naturally
occurring macromolecule, such as a polypeptide, nucleic acid,
carbohydrate, lipid, or any combination thereof. A candidate
therapeutic compound also can be a partially or completely
synthetic derivative, analog or mimetic of such a macromolecule or,
a small, synthetic molecule, such as an organic molecule prepared
by combinatorial chemistry methods. A candidate compound can be
detectably labeled or attached to a solid support, if desired, in a
particular assay.
[0017] Methods for producing pluralities of compounds, including
chemical or biological molecules such as simple or complex organic
molecules, metal-containing compounds, carbohydrates, peptides,
proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic
acids, antibodies, and the like, are well known in the art and are
described, for example, in Huse et al., U.S. Pat. No. 5,264,563;
Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et
al., Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94
(1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the
like. Libraries containing pluralities of candidate therapeutic
compounds also can be obtained from commercial sources.
[0018] A therapeutic compound identified by a method of the
invention is potentially effective in preventing or treating a
predetermined pathological condition. As used herein, the term
"pathological condition" is intended to mean a disease state
characterized by aberrant physiological function or organization of
cells, tissues or organs. A pathological condition can result, for
example, from genetic or developmental abnormalities, nutritional
or environmental factors, infection, neoplasia, aging, altered
immune or endocrine function, tissue damage, or any combination of
these factors. The invention is particularly amenable to
identifying therapeutic compounds potentially effective against
pathological conditions with a known hereditary component, and
which affect a significant proportion of the population, such as,
for example, asthma, cardiovascular disease, many types of cancer,
schizophrenia, dementia, obesity, and diabetes. The invention can
also be practiced with respect to rarer or monogenetic diseases
such as, for example, diseases described in the Online Mendelian
Inheritance in Man database (Center for Medical Genetics, Johns
Hopkins University (Baltimore, Md.) and National Center for
Biotechnology Information, National Library of Medicine (Bethesda,
Md.) (1998)).
[0019] The methods of the invention are applicable to pathological
conditions that affect all systems of the body, including, for
example, the cardiovascular system, immune and hematopoietic
system, respiratory system, hepatobiliary system, gastrointestinal
system, endocrine system, urinary system, genital system, nervous
system and musculoskeletal system. Certain pathologies are
considered multisystem diseases and include, for example, systemic
lupus erythematosus, systemic sclerosis, diabetes mellitus, and
other inflammatory and metabolic disorders. Other diseases
primarily or initially affect a single tissue or organ, such as a
benign or malignant tumor of the breast, prostate, colon, lung,
brain or ovary.
[0020] As used herein, the term "diseased individual" is intended
to mean an individual exhibiting, or is considered to be at
elevated risk, compared to the general population, of exhibiting
signs or symptoms of a pathological condition. In contrast, as used
herein, the term "normal individual" is intended to mean an
individual who does not exhibit, or is considered to be at low risk
of exhibiting, signs or symptoms of a pathological condition. The
signs, symptoms and genetic and environmental risk factors
associated with different pathological conditions are known in the
art and are described, for example, in Stevens et al., ed.,
Pathology, Mosby: London (1995).
[0021] As used herein, the term "genetically related individual" is
intended to mean an individual with a common ancestor within
several generations. A more distantly genetically related
individual can share an ancestor within 10 or fewer generations,
such as 8 or fewer generations, preferably 6 or fewer generations.
A more closely genetically related individual can share an ancestor
within 4 or fewer generations, more preferably 3 or fewer
generations, most preferably 2 or fewer generations. Genetically
related individuals can be of the same generation, such as
siblings, first cousins or distant cousins, or of different
generations, such as a grandparents and grandchildren, parents and
children, and aunt or uncle and niece or nephew.
[0022] The method is advantageous in that the effect of a compound
on a cell from a diseased individual and a cell from a normal
individual, genetically related to the diseased individual, is
compared. Therefore, genetic variables unrelated to the
pathological condition are minimized, which could otherwise
complicate the interpretation of the screening assay. The more
closely related a diseased and a normal individual are, the more
likely it is that any difference observed is indicative of an
effect on the pathological condition, rather than an effect of the
compound on an irrelevant parameter. Therefore, when a method of
the invention is practiced using a cell from a single diseased and
a single normal individual, it is preferable to obtain samples from
closely related individuals, such as siblings, more preferably
fraternal twins, most preferably identical twins.
[0023] The invention can also advantageously be practiced using
cells from multiple individuals selected from a large family or
genetically homogeneous population that includes both normal and
diseased individuals of varying degrees of relatedness. Using
methods known in the art, the degree of relatedness of the
individuals within the pedigree, and the relative risk for each
individual within the pedigree of exhibiting the disease, can be
established. The number of normal and diseased individuals and
their degree of genetic relatedness can be determined by those
skilled in the art for a particular application of the method. For
example, the method can be practiced using cells from 2, 3, 4, 5,
10 or more, 20 or more, 50 or more, or 100 or more normal
individuals, depending on the number of compounds being screened,
the assay format, the ease of obtaining the samples, and the
statistical significance required. Similarly, the method can be
practiced using cells from 2, 3, 4, 5, 10 or more, 20 or more, 50
or more, or 100 or more diseased individuals.
[0024] In one embodiment, the invention can be practiced by
obtaining cells from diseased individuals who each exhibit the same
degree of severity of the pathological condition. In another
embodiment, the diseased individuals can exhibit a range of
severities of the pathological condition ranging, for example, from
mildly affected to moderately affected to severely affected. For
example, with regard to a disease such as cancer, cells could be
obtained from diseased individuals who exhibit differing disease
severities ranging from, for example, benign hyperplasia or
dysplasia, to neoplasia, to a highly metastatic tumor.
[0025] In a further embodiment, the diseased individuals can
exhibit a range of risk of developing the pathological condition
ranging, for example, from a moderate risk of developing the
pathological condition, to a high risk of developing the
pathological condition, to actually exhibiting symptoms of the
pathological condition. For example, in diseases with a hereditary
component, the degree of risk can be related to the degree of
relatedness to an affected individual. Furthermore, in diseases in
which a susceptibility locus or gene has been identified, the
degree of risk can be associated with the presence of none, one or
two alleles of the susceptibility locus or gene. Similarly, in
diseases with an environmental component, such as exposure to
cigarette smoke, toxins, radioactivity, nutritional factors and the
like, the degree of risk can be associated with the amount or
extent of exposure to the environmental component.
[0026] Those skilled in the art can readily assess the degree of
severity of the pathological condition for each individual and the
degree of risk of developing the pathological condition for each
individual, using knowledge of the risk factors, pathological
mechanisms, and clinical signs and symptoms of a given disease.
Those skilled in the art can also determine for a given application
of the method the appropriate range of disease severity or risk,
and the appropriate number of individuals with each degree of
disease severity or risk. Factors involved in determining the
number of individuals include, for example, the statistical
significance of the data required, the qualitative or quantitative
nature of the data obtained, and the availability of cells from a
range of individuals for a given disease.
[0027] A method of the invention can advantageously be practiced
using cells from individuals from genetically homogeneous
populations, such as geographically isolated populations with
relatively few founder individuals, or populations that are
isolated for cultural or religious reasons. Isolated populations
have had relatively little inward migration or intermarriage, and a
result, most of the population is descended from the original
founder individuals. Therefore, there is an increased likelihood
that differences in the ability of therapeutic compounds to alter a
property of cells of diseased and normal individuals will be
related to the disease mechanism, rather than to cell-to-cell
variations resulting from different genetic backgrounds.
Additionally, environmental variation is likely to be minimal
within isolated populations. Genetically homogeneous populations
also advantageously include individuals with varying degrees of
genetic relatedness, from distantly related to closely related.
Therefore, the relative effect of a therapeutic compound on a
property of a cell can be statistically analyzed and correlated
with the degree of genetic relatedness.
[0028] Examples of genetically homogeneous populations of
individuals are known in the art and include, for example,
geographically isolated populations, such as island populations.
Preferably, genetically homogeneous populations of individuals have
extensive and accurate medical records and detailed genealogical
records. Genetically homogeneous populations with extensive medical
and genealogical records are well known in the art and include, for
example, the population of Iceland, populations of the Scandinavian
countries, the Mormon population of Utah, and the Amish and
Hutterite populations of North America.
[0029] For certain diseases, epidemiological studies have been
conducted among genetically homogeneous populations with an
increased incidence of the disease. Therefore, a method of the
invention can be practiced using cells obtained from diseased and
normal individuals within such populations to identify
therapeutically effective compounds against such diseases. As
several non-limiting examples, it is known in the art that the
population of Tristan de Cunha has an increased prevalence of
asthma, as described in Zamel et al., Am. J. Respir. Crit. Care
Med. 153:1902-1906 (1996); that the Pima Indians have an increased
frequency of non-insulin dependent diabetes mellitus, as described
in Bogardus et al., J. Cell Biochem. 48:337-343 (1992); that the
population of Finnish North Karelia has an increased incidence of
hypercholesteremia and coronary heart disease, as described in
Vuorio et al., Arterioscler. Thromb. Vasc. Biol. 17:3127-3138
(1997); and that the population of the Central Valley of Costa Rica
has increased prevalence of bipolar disorder, as described in
Sheffield et al., Trends in Genetics 14:391-396 (1998). Other
genetically homogeneous populations susceptible to particular
pathological conditions of interest are known or can be determined
by those skilled in the art.
[0030] The method is practiced by contacting a cell indicative of
the predetermined pathological condition with the plurality of
candidate therapeutic compounds under suitable assay conditions. As
used herein, the term "cell indicative of a pathological condition"
is intended to mean a cell that has, or can be made to have, one or
more properties in a diseased individual that is detectably altered
relative to a cell of the same histological origin from a normal
individual. The term "a cell" is intended to include single cells,
as well as pluralities of cells of the same or different
histological type present in a cell suspension, cell culture, or
tissue sample. The type and number of cells to use to identify a
therapeutic compound will depend on the particular pathological
condition and the assay used, and can be determined by those
skilled in the art for a given application of the method.
[0031] A cell indicative of a pathological condition can be
selected from a tissue or organ affected, or most affected, in the
particular disease. Alternatively, a cell indicative of a
pathological condition can be selected from an apparently
unaffected tissue of a diseased individual. Many diseases, such as
a genetic or multisystemic disorders, will be manifested in a
variety of different tissues and cell types. Accordingly, it is not
always necessary to know or to determine an affected, or the most
affected, cell or tissue. Therefore, a cell indicative of many
pathological conditions can be obtained from any convenient source.
A factor to be determined in obtaining cells, particularly from a
large number of normal and diseased individuals, is the ability to
obtain the cells using minimally invasive methods. Therefore, cells
from both normal and diseased individuals can readily be obtained,
for example, from fluids such as the blood, lymph, urine or breast
milk, or from accessible tissues such as the skin, hair follicles,
cervix or cheek. Additionally, cells can readily be obtained from
both normal and diseased individuals using slightly more invasive
procedures, such as punch biopsies of the breast or muscle, or from
the bone marrow or cerebrospinal fluid. Depending on the need and
the availability of an appropriate surgical procedure, cells from
essentially any organ or tissue of the body can be obtained from
genetically related individuals and used in the methods of the
invention.
[0032] Those skilled in the art can readily determine which cells
are indicative of a pathological condition, which cells are
appropriate control cells from normal individuals, and what is the
most desirable source of such cells. Additionally, methods of
obtaining, storing, culturing, and manipulating cells to ensure the
introduction of the minimal amount of irrelevant variations between
samples are well known in the art.
[0033] A cell indicative of a pathological condition can be a
primary cell or tissue sample obtained directly from an individual.
If desired, depending on the assay conditions employed, a cell
indicative of a pathological condition can also be modified or
altered from how it was initially obtained from the individual. For
example, a cell indicative of a pathological condition can be a
primary cell disaggregated from connective tissue and irrelevant
cells using known methods, such as, for example, enzymatic
digestion and biochemical separation. Likewise, a cell indicative
of a pathological condition can be a cell separated from other
cells using affinity separation methods known in the art. As an
example, flow cytommetry or antibody panning methods can be used to
select a population of cells expressing a detectable surface marker
such as, for example, CD4, CD8, CD34 or CD38.
[0034] Additionally, a cell indicative of a pathological condition
can be a cell propagated in culture, using methods known in the
art, for several generations. Depending on the assay conditions
employed, and as described below, such a cell can also be a cell
that has been transduced or transfected with a nucleic acid
encoding an expressible reporter construct, or contacted with a
detectable molecule such as a radiolabeled compound or
fluorochrome.
[0035] A cell from one or more diseased individuals and a cell from
one or more normal individuals, selected as described above, are
each contacted, either sequentially or simultaneously, with a
candidate therapeutic compound under suitable assay conditions. As
used herein, the term "suitable assay conditions" is intended to
mean conditions under which a particular assay, such as an assay
described below, will identify a compound that alters a
predetermined property of a cell. Suitable assay conditions take
into account factors such as the concentration of the compound, the
duration of contact with the compound, the temperature and buffer
conditions, the method of contact, whether or not cell viability is
required, and the detection format. Suitable assay conditions
depend on the cell type, the predetermined property to be detected,
the pathological condition, and the number of compounds being
screened. Assay conditions to identify compounds that alter
predetermined properties of cells are known in the art or can be
readily determined for a particular application of the method.
[0036] The method involves contacting a cell from a diseased
individual and a cell from a genetically related normal individual
under suitable assay conditions with a candidate compound, such
that the effect of the compound on the predetermined property can
be determined and a compound that preferentially alters the
predetermined property of the cell from the diseased individual can
be identified. The term "preferentially alter," as used herein, is
intended to mean qualitatively or quantitatively changing the
predetermined property of a cell from a diseased individual,
relative to the same property of a cell from a normal
individual.
[0037] As used herein, the term "predetermined property" is
intended to mean a property that is known to be, or is considered
by those skilled in the art to be, a credible indication of the
particular pathological condition. The predetermined property to be
detected can be chosen by those skilled in the art using knowledge
of the underlying pathological mechanisms that are associated with
the pathological condition. A predetermined property consistent
with a method of the invention can be a biological process, a
functional activity, or a structural property of the cell, so long
as it is considered to be associated with the pathological
condition and can be qualitatively or quantitatively detected in a
cell-based in vitro assay.
[0038] For example, a predetermined property associated with a
pathological condition can be a biological process such as cell
proliferation, adhesion, differentiation, motility or apoptosis.
Therefore, an appropriate assay would detect the ability of a
compound to preferentially increase or inhibit such a process in
cells from a diseased individual. Such biological assays generally
involve initially viable cells, and assay conditions consistent
with cell viability would be chosen.
[0039] An as example, a therapeutic agent potentially effective
against cancer could be identified by screening candidate compounds
against cells from an individual having, or a risk of developing, a
neoplastic tumor, and against normal cells from related normal
individuals. Compounds that preferentially exhibit cytotoxic or
cytostatic activity against cells from the diseased individual, as
determined by a reduction in cell number or viability, would be
characterized as therapeutic compounds that are potentially
effective against cancer.
[0040] A predetermined property associated with a pathological
condition can also be a functional activity, such as, for example,
altered production or turnover of a second messenger, GTP
hydrolysis, influx or efflux of ions or amino acids, altered
membrane voltage, increased or decreased protein phosphorylation,
altered activity of an enzyme, altered protein-protein
interactions, relocalization of a protein within the cell, or
induction of gene expression, in response to contacting the cell
with a therapeutically effective compound. Assays to detect
alterations in these functional activities are well known in the
art or can be readily adapted to a novel predetermined property
relevant to a disease of interest. Such assays are described, for
example, in Gonzalez et al., Curr. Opin. in Biotech. 9:624-631
(1998) and in Jayawickreme et al., Curr. Opin. Biotech. 8:629-634
(1997), and in references reviewed therein.
[0041] Often assays to detect a relevant functional activity
involve first contacting the cell with a detectable biosensor, such
as a fluorescent calcium indicator, green fluorescent protein, a
fluorophore, a radiolabeled compound, or a chemiluminescent
indicator, either alone or linked to another molecule such as an
amino acid, peptide, oligosaccharide, nucleotide or nucleic acid.
Additionally, such assays can involve first transducing the cells
with a promoter-reporter nucleic acid construct such that, for
example, .beta.-lactamase, luciferase, green fluorescent protein or
.beta.-galactosidase will be expressed in response to contacting
the cell with a therapeutically effective compound. Appropriate
assays to detect functional activities preferentially altered in a
diseased cell by a therapeutic compound can be determined by those
skilled in the art based on knowledge of the underlying
pathological mechanisms, such as knowledge of the signal
transduction pathway or molecular interactions that underlie the
pathology.
[0042] A predetermined property associated with a pathological
condition can also be a structural property, such as the altered
ability of a compound to bind a cell from a diseased individual.
Therefore, a method of the invention can detect a compound that
interacts with receptors present in increased or decreased
abundance on the surface of cells from diseased individuals, or a
compound that interacts with increased or decreased affinity with
receptors present in equal abundance on the surface of cells from
normal and diseased individuals. Such compounds can be either
agonists or antagonists of the receptor.
[0043] Assays suitable for detecting various binding interactions
are known in the art and include, for example, fluorescence
correlation spectroscopy (FCS) and scintillation proximity assays
(SPA), which are reviewed, for example, in Major, J. Receptor and
Signal Transduction Res. 15:595-607 (1995); and in Sterrer et al.,
J. Receptor and Signal Transduction Res. 17:511-520 (1997). Other
assays for detecting binding interactions include, for example,
ELISA assays, FACS analysis, and affinity separation methods which
are described, for example, in Harlow and Lane, Eds., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory (1988). Such
assays can often be performed with either viable or non-viable
cells.
[0044] If desired, a method of the invention can be practiced using
an assay wherein one or several steps, such as cell manipulation,
culture plate manipulation, contacting the cells with the
compounds, detection of the predetermined property, or statistical
analysis of the data, are automated. Such automation advantageously
provides for high throughput screening of candidate therapeutic
compounds, often using smaller numbers of cells and smaller amounts
of compounds and reagents than manual assays. Those skilled in the
art can determine for a particular application of the method
whether it would be advantageous to automate one or more steps of
the screening assays. Methods of automating the assays described
herein are well known in the art.
[0045] When the invention is practiced with a large number of
compounds or with cells from a large number of individuals, or
both, such as in a high-throughput screening format, the efficacy
of compounds in altering a predetermined property in cells from
normal and diseased individuals can be rank ordered and analyzed
using known statistical methods. For example, when the invention is
practiced with regard to a panel of cells from individuals with a
range of disease severities, or with a range of disease risk, the
effect of a compound in altering a predetermined property can be
correlated with the disease severity or risk of the cells it
affects, using, for example, statistical methods known within the
art. Therefore, the method provides a means of rapidly identifying
compounds that are potentially effective in preventing or treating
a particular pathological condition in all individuals, most
individuals, or only those individuals with a given degree of
disease severity or risk. Additionally, the method can provide a
means of comparing and ranking the potential of multiple compounds
to be effective against a particular pathological condition in
multiple individuals with varying degrees of disease severity or
risk.
[0046] Statistical methods for analyzing the data obtained using
the methods of the invention can be similar to methods used to map
disease associated genes. Such statistical methods are described,
for example, in Weeks et al., Trends in Genetics 11:513-519 (1995),
in Taylor et al., Methods Mol. Biol. 68:11-25 (1997), and in Lynch
and Walsh, Genetics and Analysis of Quantitative Traits, Sinauer
Associates, Inc., Sunderland, Mass. (1998).
[0047] The method of identifying a therapeutic compound that
preferentially alters the predetermined property of a cell from a
diseased individual, from the plurality of candidate compounds,
will depend on the number of compounds being tested and the
particular assay employed. For example, the method of the invention
can be repeated by subdividing pools of compounds into smaller
pools, until a single compound that reproducibly preferentially
alters a predetermined property of a cell from a diseased
individual is identified. Alternatively, a compound that
preferentially alters a predetermined property of the cell can be
isolated away from the cell it affects and its identity determined.
Additionally, a compound that preferentially alters a predetermined
property of the cell can be identified by virtue of an inherent
characteristic structural or functional property, or by virtue of a
distinguishing label. These and other methods of identifying a
potentially effective therapeutic compound resulting from practice
of the method of the invention are known in the art.
[0048] A therapeutic compound identified by a method of the
invention is potentially effective against the predetermined
pathological condition. As used herein, the term "potentially
effective" is intended to mean that a compound identified by a
method of the invention has an increased likelihood, relative to a
randomly chosen compound, to be effective in preventing or treating
the pathological condition in vivo. Determining the actual efficacy
of a potentially effective therapeutic compound is beyond the scope
of the invention, as it is appreciated by the inventors that the
safety and therapeutic efficacy of a compound must ultimately be
determined by clinical trials in humans.
[0049] However, those skilled in the art can practice a method of
the invention so as to increase the likelihood that a therapeutic
compound will actually be effective in preventing or treating the
disease in a clinical setting. For example, the efficacy of a
therapeutic compound can be generalized by repeating the method
using cells from additional genetically related normal and diseased
individuals. Such individuals, if desired, can be from the same
family or genetically homogeneous population initially used, or
from different families or genetically related populations. Such
individuals can exhibit, if desired, the same or varying degrees of
disease severity or risk as the individuals whose cells were
initially used in the method.
[0050] Additionally, the efficacy of the compound can be further
validated by repeating the method using assays that detect
alterations in one or more different predetermined properties
associated with the pathological condition that the predetermined
property initially assayed. Moreover, the method can be repeated
using varying concentrations of a compound to determine the
minimally effective and least toxic concentration. Therefore, the
method can be used to identify those compounds that are most likely
to be safe, effective and practical as therapeutics to prevent or
treat a pathological condition.
[0051] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
EXAMPLE I
Identification of Therapeutic Compounds in a Genetically Defined
Setting
[0052] Many human diseases have their origin in malfunctioning
signal transduction pathways. A simple signal transduction pathway
in a normal individual is shown in FIG. 1A, with an interaction
between a ligand and a receptor initiating a cascade of
intracellular signals that results in a normal response in an assay
of a predetermined property. A defect in any one of these
molecules, such as a ligand, a receptor, or an intracellular
signaling molecule, can cause a pathological condition, which can
be detected by an alteration in the predetermined property
indicative of the pathological condition. The genetic relatedness
between a normal individual and a diseased individual greatly
enhances the probability that all the molecular components in the
signaling cascade are similar or identical, except the component
that causes the pathological condition.
[0053] As shown in FIG. 1B, a pathological condition can result
from a defective receptor, indicated by the altered shape of the
receptor as compared to the normal receptor in FIG. 1A. A compound
that binds the abnormal receptor and allows it to interact with
ligand, shown as a triangle, can restore normal signaling, as
determined by a more normal response in an assay of a predetermined
property. Other compounds in a plurality of candidate therapeutic
compounds that do not restore interaction between the ligand and
receptor will not affect the readout of the predetermined property
in the diseased cell.
EXAMPLE II
Identification of Therapeutic Compounds Specific for Different
Subtypes of a Pathological Condition
[0054] A phenotypically similar pathological condition in
individuals of genetically heterogeneous backgrounds can be caused
by different underlying molecular mechanisms. For example, as shown
in FIG. 1C, a phenotypically similar pathological condition to the
pathological condition depicted in FIG. 1B can result from a defect
in a different component of the signal transduction pathway, as
indicated by the altered (non-square) shape of the ligand. A
compound effective in a patient with the type I subtype of the
pathology, indicated by a triangle in FIG. 1B, would not be able to
restore normal signaling to a cell from a patient with the type II
subtype of the pathology, and would not be an effective therapeutic
compound for treating the type II subtype of the pathology.
However, a compound that allows the defective ligand to interact
with the receptor in a cell from a patient with the type II subtype
of the pathology would restore normal signaling, and is a
potentially effective therapeutic compound.
[0055] The methods of the invention can readily be applied to
identifying potential therapeutic compounds that are effective
against each of the different subtypes of the pathology, without
prior knowledge of the molecular mechanisms that underly the
different subtypes of the pathology. Thus, in the above example,
cells from a genetically related population consisting of normal
individuals and diseased individuals exhibiting the type I form of
the pathology would be used in a screen to identify a therapeutic
compound potentially effective against the type I form of the
pathological condition. Likewise, cells from a genetically related
population consisting of normal individuals and diseased
individuals exhibiting the type II form of the pathology would be
used in a screen to identify a therapeutic compound potentially
effective against the type II form of the pathological
condition.
[0056] The compounds so identified can be used as diagnostic
reagents to determine the subtype of a pathology in a patient
exhibiting clinical indications of the pathology. For example, the
ability of a cell from a patient to give a normal response in an
assay of a predetermined property in the presence of a compound
that has been determined, as described above, to restore normal
signaling in the type I subtype of the pathology, indicates that
the patient has that subtype of the pathological condition. In
contrast, the inability of a cell from a patient to give a normal
response in an assay of a predetermined property in the presence of
the same compound indicates that that patient has a different
subtype of the pathology. As described above, compounds that
restore normal signaling in each of the different subtypes of the
pathology can be determined, and these compounds can be used in
diagnostic assays to identify the particular subtype of the
pathology in each patient.
EXAMPLE III
Development of Combination Therapies
[0057] Most cellular functions involve multiple signal transduction
pathways. Thus, the response in an assay of a predetermined
property often requires the function of several convergent
signaling pathways. Such a situation is shown in FIG. 2, where two
ligands each bind their own receptor and initiate signaling
cascades that converge to produce a signal in an assay of a
predetermined property. In this example, the product of gene X,
which carries out a process involved in one signaling transduction
pathway, is a known disease associated gene. The products of genes
Y and Z carry out processes involved in a second signal
transduction pathway, and can be considered modifying factors of
the effect of gene product X in an assay of a predetermined
property.
[0058] In a genetically homogeneous population in which the
diseased individuals have disease allele X, and normal individuals
have wild-type allele X, the activities of gene products Y and Z
are likely to be constant within the population, and the
measurement of the predetermined property thus reflects the
activity of X in each individual in that population. In contrast,
in an outbred population, the measurement of the predetermined
property reflects a varying contribution of the activities of X, Y
and Z in each individual. Therefore, in a genetically homogeneous
population, a therapeutic compound that targets gene product X can
be identified. Additional screens in the presence of this compound
can then be performed to identify compounds that affect the
activities of modifying factors Y or Z, as measured by a further
alteration in the readout in an assay of the predetermined
property. Therefore, the invention provides a method of developing
combination therapies that target both disease associated genes and
their modifying factors.
[0059] In a different genetically homogeneous population, having
different alleles or activities of modifying factors Y and Z, a
similar screen performed in the presence of a compound that targets
gene product X would likely identify different compounds that
affect the activities of the modifying factors Y or Z in that
population. Therefore, the invention provides a method of
developing combination therapies that are specific for a patient's
particular X, Y, and Z allele or activity combination.
EXAMPLE IV
Pathway Mapping of Disease-Associated Genes
[0060] In searching for a genetic basis for disease, methods have
been developed to scan the genes of multiple individuals in a
pedigree and to associate a marker on a locus with the disease
history of the members of the pedigree. Such association are then
used to map the disease-associated genes to a particular locus. The
methods described herein can be used in a similar manner to map
disease-associated genes to a "locus" of a signal transduction
pathway.
[0061] As described herein, assays are known in the art that can be
used to measure a predetermined property indicative of an
alteration in any step of a signaling pathway, such as
receptor/ligand interaction, second messenger signaling,
phosphorylation events, gene expression and the like. For example,
as shown in FIG. 3, three different predetermined properties
representing three different loci within of a signaling pathway can
be assayed. In a genetically homogeneous population, normal
individuals and diseased individuals will have few differences in
the molecules that carry out signaling steps, apart from the
molecule responsible for the disease pathogenesis. Thus, by
comparing several predetermined properties in normal and diseased
cells from individuals in a genetically homogeneous population, the
step in the signaling pathway that is altered in diseased
individuals can be determined, and the disease-associated gene
mapped to that "locus" of the signaling pathway. The
disease-associated gene or gene product is likely to be an
appropriate target in a high-throughput drug screening assay to
identify compounds that can be used to treat the disease.
[0062] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference in this application
in order to more fully describe the state of the art to which this
invention pertains.
[0063] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific experiments detailed are only
illustrative of the invention. It should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *