Note: Descriptions are shown in the official language in which they were submitted.
CA 02582179 2007-04-02
METHODS FOR IDENTIFYING COMBINATIONS OF
ENTITIES AS THERAPEUTICS
Background of the Invention
Many disease states are associated with a multitude of phenotypic changes.
This
has long been apparent in the clinic, but recent advances in genomics have
confirmed this
observation at the molecular level. Expression profiling of cancer cells, for
example, has
revealed hundreds of changes in gene expression caused by multiple somatic
mutations.
Furthermore, human cells and tissues have evolved homeostatic mechanisms such
that
they often contain redundant and self-buffering signaling systems. Natural
signals
causing a change in cellular state are often sent not to a single target, but
to the correct
combination of targets. Thus, modest changes in multiple variables can have a
highly
specific effect.
In contrast, for historical and technological reasons, the pharmaceutical,
chemical,
and biological communities have focused on single, individual molecules that
cause
biologic effects. This historical paradigm has resulted in the identification
of small
organic molecules that affect specific proteins, providing valuable reagents
for both
biology and medicine. These molecules are also useful as probes of the
biological
function of proteins that may have therapeutic relevance and have been
effective at
elucidating signal transduction pathways by acting as chemical protein
knockouts,
thereby causing a loss of protein function. Additionally, due to the
interaction of these
small molecules with particular biological targets and their ability to affect
specific
biological functions, they may also serve as candidates for the development of
therapeutics.
Because it is impossible to predict which small molecules will interact with a
biological target, intense efforts have been directed toward the generation of
large
numbers of small organic molecules. These are then gathered into what are
called
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CA 02582179 2007-04-02
"libraries" of such compounds, with the goal of building a "diverse library"
with the
appropriate desired characteristics. Such a diverse library may be built from
a pre-
existing collection of small molecules or may be generated using
"combinatorial
chemistry." These libraries can be linked to sensitive screens to identify
active molecules
(Stockwell et al. Chem. Biol. 1999, 6, 71-83).
In many cases, researchers have developed biased libraries, in which all
members
share a particular characteristic, such as an ability to interact with a
target protein, or a
characteristic structural feature designed to mimic a particular aspect of a
class of natural
compounds. For example, a number of libraries have been designed to mimic one
or
more features of natural peptides. Such "peptidomimetic" libraries include
phthalimido
libraries (WO 97/22594), thiophene libraries (WO 97/40034), and benzodiazepine
libraries (US Patent No. 5,288,514). One library that has structural features
reminiscent
of natural products and that is compatible with miniaturized cell-based assays
has been
synthesized (Tan et al. J. Am. Chem. Soc. 1998, 120, 8565).
The modem drug discovery process is largely built upon an ability to assay
rapidly
compounds for their effects on biological processes. In the pharmaceutical
industry,
attention has been focused on identifying compounds that block, reduce, or
even enhance
the interactions between biological molecules. Specifically, in biological
systems, the
interaction between a receptor and its protein ligand often may result, either
directly or
through some downstream event, in either a deleterious or beneficial effect on
that
system, and consequently, on a patient for whom treatment is sought.
Accordingly,
researchers have long sought compounds that reduce, block, or even enhance
such
receptor/ligand interactions.
High throughput screening systems were designed to overcome the practical
limitations on throughput for existing biochemical and cell-based assays.
Traditional
syntheses of organic compounds and traditional biological assays require
significant time,
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CA 02582179 2007-04-02
labor, and skill. Screening the maximum number of compounds necessitates
reducing the
time and labor requirements associated with each screen.
High throughput screening of diverse collections of molecules has thus played
a
central role in the search for lead compounds for the development of new
pharmacological agents. The inputs to these high throughput screens are
libraries of
compounds that have been assembled from preexisting chemically synthesized
molecules
(such as from a pharmaceutical company's proprietary library), natural
products (such as
microbial fermentation broths), and from novel libraries generated by
combinatorial
chemistry techniques (Tan et al. J. Am. Chem. Soc. 1998, 120, 8565). The
libraries
consist of up to a million compounds, which increases the likelihood of
finding one
compound with desirable properties to serve as a lead drug candidate (Tan et
al J. Am.
Chem. Soc. 1999, 121, 9073-9087).
Enhancing the traditional paradigm of drug discovery, combinatorial chemistry
has
resulted in a dramatic increase in the number of compounds that are available
for
screening, and human genome research has uncovered large numbers of new
molecular
targets for screening. Screens may use these new targets in a variety of ways,
searching
for enzyme inhibitors, receptor agonists or antagonists. The traditional goal
is to find
compounds that reduce, block, or enhance a single crucial interaction in a
biological
system (Weber et al., Angew. Chem. Int. Ed. Engl., 1995, 34, 2280-2282).
Additionally,
a number of researchers are adapting phenotype-based assay systems, where the
screening
is performed on whole, living cells, and the readout of the screen is some
detectable
property of the cell (Stockwell et al. Chem. Biol. 1999, 6, 71-83; Mayer et
al. Science,
1999, 286, 971-974).
The high throughput screening methods developed to date have been designed
based on the "one-drug-one-target" paradigm that dominates the pharmaceutical
industry.
For historical reasons, and because of regulatory considerations and perceived
risk
factors, the modern drug discovery process primarily looks to find one active
molecule at
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CA 02582179 2007-04-02
a time to serve as a drug candidate. Clinicians have long recognized that the
"one-drug-one-target" approach is not sufficient for the treatment of many
diseases. They
have tested some obvious combinations of agents that treat the same condition,
or that
have a clear logical connection. In HIV therapy and chemotherapy, for example,
combinations of multiple active agents have become de rigeur. One of the most
promising drugs of all times is a combination--Premarin7which is used to treat
the
complex changes in females after menopause, is composed of over 22 separate
and
important components.
Summary of the Invention
We have invented a powerful new method for perturbing biological systems. This
entails systematically performing high throughput screens of combinations of
compounds,
i.e. mixtures or non-chemically bonded combinations, to discover combinations
that
interact synergistically in biological systems. The methods of the invention
can identify
effective therapeutic combinations of compounds that exhibit previously
unknown
therapeutic effects even where each compound in the combination may have
previously
have had no recognized biological effect, and even where the compounds and the
combination would not, a priori, have been obvious candidates to use in
combination.
Accordingly, in one aspect, the invention provides a method of screening two-
entity or higher order combinations for biological activity using at least
seven entities in
at least a seven-by-seven combinational array that includes at least 49 unique
combinations of entities. The method includes the steps of (a) providing the
entities, (b)
creating the array of combinations of entities, (c) providing a test element
composed of
one or more distinct biological moieties, (d) contacting the array of
combinations of
entities with the test element under conditions that ensure that each
entity/test element
contacting is segregated from the others, (e) detecting or measuring a
property of the test
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element, and (f) identifying combinations of entities that cause an effect on
the test
element that is different from the effect of an entity of the combination by
itself.
In a second, related aspect, the invention features a method for screening two-
entity or higher order combinations for biological activity. The method
includes the steps
of= (a) creating an array of at least 200 unique combinations of two or more
entities, (b)
providing a test element that includes one or more distinct biological
moieties, (c)
contacting the array of combinations of entities with test element under
conditions that
ensure that each entity/test element contacting is segregated from the others,
(d) detecting
or measuring a property of the test element, and (e) identifying combinations
of entities
that cause an effect on the test element that is different from the effect of
an entity of the
combination by itself. The components of this assay can be varied as described
above in
connection with the first assay. In preferred embodiments, this method further
includes
the step of (f) repeating steps (a) through (e) at least twice, wherein, in
step (a), the array
of at least 200 combinations is different in each repetition. In other
preferred
embodiments, the array includes at least 400 or 1540 unique combinations.
Where two
repetitions of step (f) are carried out, preferably those occur within ten
days of each other.
In a third, related aspect, the invention features a method for screening two-
entity
or higher order combinations for biological activity. The method includes the
steps of
(a) creating an array of at least 49 unique combinations of two or more
entities, (b)
providing a test element that includes one or more distinct biological
moieties, (c)
contacting the array of combinations of entities with the test element under
conditions
that ensure that each entity/test element contacting is segregated from the
others, (d)
detecting or measuring a property of the test element, (e) identifying
combinations of
entities that cause an effect on the test element that is different from the
effect of an entity
of the combination by itself, and (f) repeating steps (a) through (e) at least
25 times over a
one week period, using a different array in each repetition. The components of
this assay
can be varied as described above for the previously described assays.
CA 02582179 2007-04-02
In a fourth, related aspect, the invention features a method for screening two-
entity
or higher order combinations for biological activity. The method includes the
steps of
(a) creating an array of at least 49 unique combinations of two or more
entities, (b)
providing a test element that includes one or more distinct biological
moieties, (c)
contacting the array of combinations of entities with the test element under
conditions
that ensure that each entity/test element contacting is segregated from the
others, (d)
detecting or measuring a property of the test element, (e) identifying
combinations of
entities that cause an effect on the test element that is different from the
effect of an entity
of the combination by itself, and (f) repeating steps (a) through (e) at least
100 times over
a one month period, using a different array in each repetition. The components
of this
assay can be varied as in the previously described assays.
In a fifth aspect, the invention features a method for screening two-entity or
higher
order combinations for biological activity. This method includes the steps of
(a) creating
an array of at least 10,000 unique two-entity or higher order combinations
from a set of
entities, (b) providing a test element comprising one or more distinct
biological moieties,
(c) contacting the array of combinations of entities with the test element
under conditions
that ensure that each entity/test element contacting is segregated from the
others, (d)
detecting or measuring a property of the test element, (e) identifying
combinations of
entities that cause an effect on the property of the test element that is
different from the
effect of an entity of the combination by itself, and (f) repeating steps (a)
through (e) at
least twice over a period of ten days or less, wherein, in step (a), the array
of at least
10,000 two-entity combinations is different in two or more repetitions.
In a sixth aspect, the invention features a method for screening combinations
of
entities for biological activity. This method includes the steps of (a)
providing a test
element comprising one or more distinct biological moieties, (b) contacting
the test
element with at least 100 entities under conditions that ensure that each
entity/test
element contacting is segregated from the others, (c) detecting or measuring a
property of
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CA 02582179 2007-04-02
the test element, (d) selecting entities that cause a change in the property
relative to said
property of the test element not contacted with the entities, (e) creating an
array of at least
49 unique two-entity or higher order combinations from the identified
entities, (f)
contacting the array of combinations of entities with a test element under
conditions that
ensure that each entity combination/test element contacting is segregated from
the others,
(g) detecting or measuring a property of the test element of step (f), and (h)
identifying
combinations of entities that cause an effect on the property of step (g) that
is different
from the effect of an entity of the combination by itself. Preferably, the
test element of
step (a) is the same as the test element of step (f), but it may be desirable
to use a different
test element in each step (e.g., cultured cells in step (a) and a whole animal
in step (f).
Similarly, the property of step (c) is the preferably the same as the test
element of step
(g), but it may be desirable to use a different property in each step.
For all of the assays, a number of activity read-out techniques can be used,
including a cytoblot assay, a reporter gene assay, fluorescence resonance
energy transfer
(FRET) assays, a fluorescent calcium binding indicator dye, fluorescence
microscopy, or
expression profiling. The assays preferably are automated, using robotics
systems and
384 and 1536 well plates. The assays may also be constructed so that parts or
all of the
process may be performed using microfluidics. Alternatively, the assays may be
executed
by using trained scientific labor and an efficient line process.
Preferred entities to be tested in combination according to the invention are
compounds (e.g., non-polymeric organic compounds, in particular, small
molecules;
lipids; carbohydrates; peptides; inorganic compounds; and oligonucleotides,
including
DNA and RNA molecules), ions, (e.g., metal ions), and radiation (e.g., visible
light, light
outside the visible range, microwave radiation, infrared radiation, or
ionizing radiation
such as x-rays and gamma rays). Where the entities being tested in combination
are
compounds, they are preferably used in purified form, but can also be provided
as
components of mixtures, e.g., as natural product extracts. For example, a
subset (one or
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CA 02582179 2007-04-02
more compounds) can be in purified form, each of the compounds can be employed
in
purified form. Preferably, each entity/test element contacting is in a volume
of less than
200 L, more preferably less than 100 L, and most preferably less than 50 L
or even
25 L. In certain circumstances, volumes as little as 10 L or even 500 nL or
less may be
used. Additionally, the compounds are preferably in volumes less than 100 L,
more
preferably less than 1 L, and most preferably less than 100 nL or even 50 nL.
One in the
art will recognize that smaller volumes (e.g., 10 nL or even 1 nL) may be
used.
Preferred test elements are whole cells (e.g., transformed cells or non-
transformed
cells) such as neurons, fibroblasts, smooth muscle cells, cardiac cells,
skeletal muscle
cells, glial cells, embryonic stem cells, hematopoeitic stem cells, mast
cells, adipocytes,
protozoans, bacterial cells, yeast cells, neural stem cells, and immune cells
including T
cells and B cells), clusters of cells, tissues, animals, and reconstituted
cell-free media; in
some instances, the test element can instead consist of a single biologically
relevant
molecule such as a protein or an oligonucleotide.
The effects displayed by desired combinations on the test element are
preferably
synergistic effects on a property of the test element, such as an increase or
decrease in
DNA synthesis. Alternatively, the combination may be additive in nature but
have fewer
side effects, or one entity may exert a negative, and useful effect, on
another, e.g., one
compound counteracting toxicity of another compound.
The entities can be contacted with the test element in any sequence, i.e., one
entity
can be added to the test element, followed by the addition of a second entity,
or
alternatively, the two entities can be combined prior to their being contacted
with the test
element.
The high throughput screening methods of the invention represent rapid and
powerful alternatives to traditional drug discovery methods employed by the
pharmaceutical industry. The invention can identify previously unknown, and
therapeutically potent combinations of, e.g., small molecules, some of which
may be
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newly synthesized and some of which may be known FDA-approved drugs. Where the
effective combinations are entirely made up of two, three, four, or more
drugs, all of
which are already FDA approved, the new combination has the further advantage
of being
easily moved through the FDA approval process.
Other features and advantages of the invention will be apparent from the
following
detailed description, and from the claims.
Definitions
"Combinatorial chemistry library": As used herein, a "combinatorial chemistry
library" is a plurality of complex molecules, preferably reminiscent of
natural products
synthesized from diversifiable scaffold structures by employing different
reactants at each
stage of diversification of the scaffold structure.
"Diverse library": As used herein, a "diverse library" is a plurality of
complex
molecules that have been assembled from any of multiple potential sources,
including
preexisting chemically synthesized molecules (such as from a pharmaceutical
company's
proprietary library), natural products (such as microbial fermentation
broths), and novel
libraries generated by combinatorial chemistry techniques.
"Diversifiable scaffold structure": As used herein, a' diversifiable scaffold
structure" is a compound synthesized from a template structure, which contains
latent or
active functionalities capable of being further reacted with synthetic
reagents to generate
at least one new functionality. As used herein a "latent functionality" is one
that is
present, but is temporarily inactive. Upon release with an activator reagent,
the latent
functionality becomes active, and is thus available for further
diversification.
"Test element": As used herein, a "test element" is the system to which the
combination of entities is contacted, and which is then observed for the
effects of the
entities. A test element preferably includes two or more biological moieties
inside of a
cell.
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"Compound printing": As used herein, "compound printing" refers to the
application of compounds to a surface (e.g., glass) using a high-precision
robot such as
that used in cDNA microarraying (J. Am. Chem. Soc. 1999, 121, 7967-7968). The
compound spots can be 250 microns in diameter or smaller, and the compounds
may be
either covalently linked or adhering to the surface through electrostatic or
hydrophobic
interactions.
"Microfluidics": As used herein, "microfluidics" devices are channeled
structures
made by any of the methods of photolithography, including conventional
photolithography (e.g. Caliper Technologies, Mountain View, CA)
or unconventional methods (such as soft lithography,
described, e.g., in Angew. Chem. Int. Ed. Engl. 1998, 37, 5 50-575).
"Ink jet": As used herein, "ink-jet" teclmology refers to both thennal ink-jet
as
well as piezoelectric spray technologies for delivery small volumes of
liquids.
"Small molecule": As used herein, "small molecule" refers to an organic
compound either synthesized in the laboratory or found in nature. Typically, a
small
molecule is characterized in that it contains several carbon-carbon bonds, and
has a
molecular weight of less than 1500 g/mole, although this characterization is
not meant to
be limiting for the purposes of the present invention. Examples of "small
molecules" that
occur in nature include, but are not limited to, taxol, dynemicin, and
rapamycin.
Brief Description of the Drawings
Fig. 1 is a conceptual diagram demonstrating how two different reagents could
act
synergistically inside of a cell, where the reagents bind to different targets
within the
same cell.
Fig. 2 is a conceptual diagram demonstrating how two different reagents could
act
synergistically inside of an organism, where the reagents bind to targets in
different cells
or tissues.
CA 02582179 2007-04-02
Fig. 3 is an example of the experimental data one would obtain in a
combinatorial
screen of the sort described herein. The results from five different 384 well
plates are
shown. The results are shown in plate format, where there are 16 rows labeled
A through
P, and 24 columns, labeled I through 24. The level of activity is shown in
each well as a
number, where 1 means basal activity (no effect) and 5 means an active
combination is
found.
Fig. 4 is a diagram of a method for performing combinatorial screening using
currently commercially available technology.
Detailed Description
The present invention provides methods of combining libraries of individual
molecules in 2-, 3-, 4-, and higher order combinations in a high throughput
screening
system using relevant biological assays to identify effects of the molecules
that are only
present in their unique combinations. Such combinations may then be used to
probe and
study additional biological systems, may have a direct biological use, and may
serve as
the active molecules for novel human therapeutics or other uses in humans.
These
combinations may also be useful for promoting the growth, fertility,
maturation, or other
characteristic in specific agricultural products, including animals or plants.
They may be
useful in creating cosmetic products, fragrances, food preservatives, or
nutritional
supplements. Thus, the invention provides powerful methods for systematically
performing high throughput screens of combinations of compounds to discover
combinations having improved properties in biological systems.
We posit that pharmaceuticals interacting with complex systems such as human
cells and tissues are likely to be most effective when they contain multiple
active agents
that interact with multiple molecular targets. This understanding of how
pharmaceuticals
operate forms one of the bases for our new strategy for how they should be
designed and
developed. This new approach promises to yield dramatic improvements in many
lt
CA 02582179 2007-04-02
existing treatments, and provide effective therapies for currently intractable
diseases,
particularly diseases requiring modest changes in multiple variables.
The current dominant paradigm of the pharmaceutical industry is one in which
one
drug is designed against one target. Our approach uses our understanding of
multivariable design requirements as the centerpiece of a framework for the
systematic
development of a new generation of pharmaceuticals.
Entities to be Tested in Combination
As is mentioned above, a wide variety of entities can be tested in combination
according to the invention. These are now discussed in greater detail.
Compounds and lons
The prevalent class of compounds that are screened according to the invention
are
small organic molecules. The compounds can be synthetic or naturally occurring
(e.g.,
prostaglandins, lectins, naturally occurring secondary metabolites, hormones,
etc.). Large
libraries of such molecules, in purified form, are available in pharmaceutical
companies,
chemicals companies, and academic laboratories; such libraries also can be
synthesized
using known combinatorial chemistry techniques. The compounds may or may not
have
known biological activity. The compounds and combinatorial libraries of the
invention
can be synthesized from diversifiable solid support bound scaffolds, e.g.,
compounds and
libraries that are generated from a diversifiable scaffold synthesized from a
shikimic
acid-based epoxyol template or from the pyridine-based template
isonicotinamide. In
addition to small organic molecules, other molecules that can be screened in
combination
according to the invention include biopolymers, including oligonucleotides
(DNA and
RNA); polypeptides (antibodies, enzymes, receptors, ligands, structural
proteins, mutant
analogs of human proteins, and peptide hormones); lipids; carbohydrates; and
polysaccharides. Inorganic molecules can be screened as well, as can
potentially
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biologically active ions such as metal ions, e.g., copper, iron, silver, zinc,
magnesium,
manganese, calcium, and gold ions.
Other Entities
Other, non-chemical, entities that potentially affect biological systems can
be
screened according to the invention as well, either in combination with other
non-
chemical entities, or with chemical entities, as described above. Non-chemical
entities
that can be screened include light (visible and outside the visible range,
e.g., infrared and
ultraviolet light); ionizing radiation such as x-rays and gamma rays;
hyperbaric pressure;
increased or decreased temperature or pH; gaseous substances such as oxygen,
nitrogen,
carbon dioxide, etc., and acoustic vibrations (sounds) of any frequency.
Test Elements
The biological assays used to detect the effects of the combinations will in
most
cases be composed of multiple components. In some assays, the test element is
a whole
cell, particularly where the assay is a phenotype-based assay. Such a whole
cell assay
provides the complete set of complex molecular interactions that are likely to
form the
basis of a multivariable therapeutic compound's efficacy. Other assays employ
a
reconstituted, cell free medium that contains many of the desired complex
systems, and
that may include some reporter effect that is based on the likely
combinatorial effect
being assayed for. Other assays employ higher order biological systems such as
clusters
of cells, tissues, and animal models for multivariable combinatorial
screening.
Any biological assay that is useful for assay of individual compounds is
readily
adapted to the combinatorial screening of the present invention. Assay
measurements can
include, for example, transport of a compound across the cell membrane,
electrical
potential, action potential generation, cell proliferation, cell death, cell
specification, cell
differentiation, cell migration, gene expression or protein levels (measured,
e.g., by
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CA 02582179 2007-04-02
detecting mRNA, protein, or a reporter gene), enzymatic activity,
phosphorylation,
methylation, acetylation, translocation of a protein to the cell nucleus (or
other change in
protein localization), ability to resist a pathogen (e.g., a virus or a
bacterium), and ability
to produce an immune response. In whole organism assays, animal behavior can
serve as
a reporter.
In one example, the assay is a non-destructive assay (e.g., a cell-based assay
in
which a measurement of the effect of a compound can be obtained without
harming the
cells). Such an assay allows assays to be performed on multiple concentrations
of
multiple combinations per well. For example, compound A is added at increasing
concentrations to a well and a measurement is taken after each addition of
compound.
When a desired concentration of compound A is reached (determined based on a
desired
assay response or on known properties (e.g., toxicity, solubility) of the
compound),
compound B is added in increasing concentrations, with an assay measurement
taken
after each addition. This process can be iterated many times in a single well,
allowing
hundreds, thousands, or even millions of assays to be performed in a single
plate.
It may be desirable to perform a comparison assay to identify possible side
effects
of combinations. For example, combinations of compounds that are screened for
their
ability to kill or decrease proliferation of tumor cells can be simultaneously
screened for
their effect on normal, non-tumor cells. Combinations of compounds that
display the
desired activity on tumor cells and that have little or no effect on normal
cells are
preferred combinations. Combinations that appear to have an undesired effect
on normal
cells are less preferred. These latter combinations (or combinations of
compounds having
similar structures) may, however, be effective at different concentrations and
should not
necessarily be excluded from additional study.
Cell-free media containing complex biomolecules such as proteins,
carbohydrates,
and lipids are made by known methods, e.g., by lysing mammalian, frog, yeast,
or
bacterial cells to provide a whole cell lysate, or by purifying a specific
fraction from such
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a cell lysate; by using a commercially available rabbit reticulocyte lysate
(conunonly used
for performing in vitro transcription and/or translation reactions), or by
harvesting the
culture supernatant from mammalian, yeast, or bacterial cells without lysing
the
underlying cells.
Cytoblot Assay
One method of detecting activity is the cytoblot assay. In this method, cells
are
seeded into wells of an assay plate. The cells are preferably adherent cells
so they attach
and grow on the bottom of the well. The compounds are added using the methods
described above. For example, the cytoblot can be performed to detect
proliferation by
measuring the incorporation of BrdU. In this example, the cells are incubated
for a set
period of time, (e.g., 4 to 72 hours). The medium is then aspirated using, for
example, a
robotic liquid transfer device or a sixteen or eight channel wand. The cells
are fixed by
the addition of 70% ethanol and phosphate-buffered saline (PBS) at 4 C for 1
hour. The
fixitive is removed and the cells are washed once with PBS. After the PBS
wash, 2N HCl
with 0.5% Tween 24 is added to each well for 20 minutes. The HCI is
neutralized with a
solution of Hank's balanced salt solution (HBSS) containing 10% by volume of
2N
NaOH. This solution is removed, the cells are washed twice with HBSS and then
once
with PBS containing 0.5% bovine serum albumin (BSA) and 0.1% Tween 20. The
wash
solution is removed and anti-BrdU antibody is applied as 0.5 g/mL mouse anti-
BrdU
antibody in PBS containing 0.5% bovine serum albumin (BSA) and 0.1% Tween 20.
This antibody solution also contains a secondary antibody (at a dilution of
1:2000) that
recognizes mouse. Ig antibody (e.g., the mouse anti-BrdU antibody); this
secondary
antibody is conjugated to the enzyme horseradish peroxidase (HRP). After one
hour of
incubation, the antibody solution is removed and the cells are washed twice
with PBS.
After the second PBS wash, the HRP substrate (which contains luminol, hydrogen
peroxide, and an enhancer such as para-iodophenol is added to each well. The
amount of
CA 02582179 2007-04-02
light in each well is then detected using either exposure to film (by placing
a piece of film
on top of the plate) or by reading the amount of luminescence from each well
using a
luminometer or luminescence plate reader using standard conditions (e.g., 0.3
seconds of
exposure per well). The amount of light output from each well indicates the
amount of
DNA synthesis that occurred in that well. A desirable combination of agents is
one in
which there is either increased or decreased light output compared to a
control. For
example, a combination that decreases light output would be decreasing the
rate of DNA
synthesis and, thus, may be effective in prohibiting or preventing the
proliferation of
tumor cells. Alternatively, an increase in light output represents an increase
in DNA
synthesis. For example, one could use primary cells in, for example, a 200 X 1
combinatorial array (in which the one fixed component blocked DNA synthesis
and, thus,
had a toxic effect on the cells). Using this array, one could screen for a
second reagent
that prevented this toxic effect of the first compound. In a variety of immune
deficiency
diseases, the immune cells do not proliferate in response to either an allo-
or an auto-
antigen. Using cultured immune cells and the foregoing method, one can screen
for
combinations of reagents that promote the proliferation of immune cells.
Alternatively,
one can screen for autoimmune suppressive agents. In this example, a
population of one
type of immune cell (a B-cell or a T-cell that has been isolated from
peripheral blood
cells) is stimulated by either allo- or auto-antigen. Combinations of
compounds that
specifically inhibit proliferation in response to auto-antigens but not allo-
antigens are
useful autoimmune therapeutic candidates.
Other Methods of Detecting Activity
The foregoing cytoblot assay is readily adapted to the detection of antigens
other
than BrdU. Moreover, one can detect a variety of post-translational
modifications within
cells. For example, an antibody against the phosphorylated version of
nucleolin or
histone H3 is useful for detecting cells that are in M (mitosis) phase of the
cell cycle.
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CA 02582179 2007-04-02
Combinations of compounds that cause an increase in phosphorylated nucleolin
or
histone H3 in the cytoblot assay would therefore be combinations that arrest
cells in M
phase and are possible anticancer agents. One could also use a cytoblot assay
to detect
increases in the acetylation of, for example, histone H4 using an antibody
that specifically
recognizes acetylated histone H4. Compounds or combinations of compounds that
cause
an increase in hyperacetylation of histone H4 could also be anticancer agents.
In the foregoing examples, the procedure may be altered in that the medium is
removed and a fixative (70% ethanol or 4% formaldehyde in PBS or Tris-buffered
saline)
is added. The membrane of the cells is then permeabilized by removing the
fixative and
adding a permeabilization agent (e.g., Tween 20, triton'X-100, or methanol).
The
membrane permeabilization agent is removed, the cells are then washed with PBS
or Tris-
buffered saline, and then the primary antibody is added. There is usually no
acid
denaturation step using these other cytoblot embodiments.
Reporter Gene Assays
Measurement of a property of the test element may be performed with the use of
a
reporter gene. This method provides the advantage that, once the reagents
(e.g., a stably
transfected cell line) are prepared, the assay is easy to perform and requires
less time
than, for example, the cytoblot assay. Reporter genes include a reporter
element,
encoding a polypeptide that is readily deiected due to a colorimetric,
fluorescent,
luminescent, or enzymatic property, and an enhancer/promoter element, which
confers
specificity to the expression of the reporter gene. Reporter elements include,
without
limitation, luciferase, beta lactamase, green fluorescent protein, blue
fluorescent protein,
chloramphenicol acetyltransferase (CAT), beta galactosidase, and alkaline
phosphatase.
Enhancer/promoter elements include, for example, those responsive to Nuclear
Factor of
Activated T Cells (NFAT), p53, TGF-beta, or any other signaling pathway or
stimulus for
which a responsive promoter/enhancer is known.
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CA 02582179 2007-04-02
NFkB
One commercially available reporter gene is pNFkB, a luciferase reporter gene
that
produces light when NFkB is activated (Stratagene, La Jolla, CA). This
reporter gene can
be transfected, either stably or transiently, into a cell line of interest
(i.e., A549 human
lung carcinoma cells). To generate a stably transfected cell line, the pNFkB
plasmid
DNA is mixed with a G418 resistance plasmid and a transfection reagent (e.g.,
lipofectamine, Life Technologies, Inc., Rockville, MD), and added to a 10 cm
petri dish
containing A549 or other cells attached to the dish at approximately 40%
confluency.
The plate with DNA/lipofectamine is incubated for 4-16 hours at 37 C with 5%
CO2. The
medium is removed, the cells are washed twice with serum-free medium, and then
medium with 10% fetal bovine serum (FBS) is added. G418 is added at a dose
(approximately 700 g/mL) that is just sufficient to kill all cells in a mock
transfection
(i.e., a transfection without a G418-resistance plasmid). The cells are
incubated at 37 C
with 5% CO2 for 2 weeks, with medium changes every three days. Stably-
transfected
clones will have grown as small colonies by the end of two weeks; these clones
are
separated by ring-cloning and propagated separately. G418-resistant clones are
screened
for the reporter gene of interest by measuring light output from cell lysates
with or
without transiently transfected NFkB present. Once a clone is identified with
stably-
transfected pNFkB, it is used for screening by plating the cells in the assay
plate.
For a transient transfection assay, the pNFkB plasmid DNA is mixed with a
transfection reagent, added to a 10 cm petri dish containing, for example,
A549 cells
attached to the dish at approximately 70% confluency. The plate with
DNA/lipofectamine is incubated for 4-16 hours at 37 C with 5% CO2: The medium
is
removed, the cells are washed twice with serum-free medium, and then medium
with
10% FBS is added. The cells are incubated at 37 C with 5% CO2 for 24 hours,
then
plated into assay plates for screening.
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CA 02582179 2007-04-02
The reporter gene can be introduced into the cells using other techniques,
including, without limitation, viral or retroviral infection, bolistic
injection, and cellular
uptake of naked DNA. One in the art will recognize that any method of
introduction of
the reporter gene into the cells to be assayed will be compatible with the
screening
methods described herein.
Once the cells with the reporter gene are available, they are seeded in assay
plates
(96 well, 384 well, etc) with a pipette, multichannel pipette, 384 well
Multidrop platefiller
(Labsystems, Franklin, MA), or other liquid handling device. Compounds are
added to
form combinations by one of several methods. After 4-72 hours, the medium is
removed,
the cells are washed twice with HBSS, a lysis buffer is added (see Stockwell
et al., J.
Amer. Chem. Soc. 1999, 121:10662-10663), ATP/luciferin added and luminescence
is
measured on a platereader or luminometer (e.g. LJL BioSystems Inc., Analyst
AD,
Sunnyvale, CA).
Fluorescence Resonance Energy Transfer Assays
In another example, fluorescence resonance energy transfer (FRET) is used to
detect and measure the interaction of two proteins of interest. In this
example, the first
and second proteins are fused with green fluorescent protein (GFP) and blue
fluorescent
protein (BFP), respectively, using standard molecular biological methods. The
DNA
constructs encoding the two fusion proteins are co-expressed in mammalian
cells, yeast,
worms, or other cell or organism using transfection techniques described above
or other
comparable methods. Combinations of compounds are added. The plate is placed
on a
platereader and fluorescence is measured as follows. The donor fluorophore
(i.e., BFP) is
excited and the emission of the acceptor fluorophore (i.e., GFP) is measured.
Increased
proximity of the two proteins will result in an increase in emission of the
acceptor
fluorophore. Thus, a combination of compounds that causes the two proteins of
interest
19
CA 02582179 2007-04-02
to be near each other is identified by an increase in fluorescence of the
acceptor
fluorophore.
For example, expression vectors containing Smad2 and Smad4 are obtained. The
cDNA for GFP is fused to the 5' end of Smad2 and the cDNA for BFP is fused to
the 5'
end of Smad4. These expression vectors are transfected into mammalian cells
stably or
transiently, cells are treated with combinations of compounds, and the plate
is irradiated
with light that excites BFP but not GFP. Fluorescence of GFP (e.g., 512 nm
light) is
measured and combinations of compounds that cause an increase in light
emission at this
wavelength are identified. Such combinations are causing Smad2 and Smad4 to
localize
near each other, and may be activating TGF-beta signaling, arid therefore may
be useful
for treating cancer chemotherapy mucositis, and autoimmune diseases.
Fluorescent Calcium Indicator Dyes
Another readout of a change in a property of a test element utilizes
fluorescent
calcium indicator dyes, such as fluo-3 (Molecular Probes, Eugene WA)
, fura-2, and indo- 1. Fluo-3 is essentially
nonfluorescent unless bound to Ca2+ and exhibits a quantum yield at saturating
Ca2+ of
-0.14. The intact acetoxymethyl ester derivative of fluo-3 AM (fluo-3 AM) is
therefore
also nonfluorescent. The green-fluorescent emission (525 nm) of Ca2+-bound
fluo-3 is
conventionally detected using optical filter sets designed for fluorescein
(FITC).
According to the manufacturer, fluo-3 exhibits an at least 100-fold Ca2+-
dependent
fluorescence enhancement.
Cells are seeded in wells, fluo-3 is added to the cells following the
manufacturer's
instructions, compounds are added, and fluorescence is measured using a plate
reader.
Combinations of compounds that result in an increase in fluorescence (but are
not
themselves fluorescent) are thus causing an increase in the concentration of
intracellular
calcium.
CA 02582179 2007-04-02
Fluorescence Microscopy
Another assay uses conventional fluorescence microscopy to detect a change in
the
level or localization of fluorescence in cells contacted with combinations of
compounds.
In one example, a stably transfected cell line expressing a GFP tagged Smad2
is used.
Cells are seeded, compounds are added and incubated for 1 hour, and a
fluorescence
microscope with an automated stage is used to image the cells in each well.
Combinations of compounds that cause a change in the localization of the
tagged protein
are identified. For example, combinations that cause GFP-Smad2 to translocate
from
outside of the nucleus to the interior of the nucleus can be identified in
this manner. These
combinations may be activating TGF-beta signaling and, thus, may be useful for
treating
cancer, autoimmune diseases, and mucositis.
Expression Profiling with cDNA Arrays
Another assay for the detection of compounds that, together, produce an
alteration
in a test element is expression profiling. In this example, cells are seeded,
combinations
of compounds are added, and the cells are incubated for 2-24 hours. PolyA RNA
is
harvested from each well using standard methods. The RNA is reverse
transcribed into
cDNA using standard methods, with the exception that a fluorescent dye (e.g.,
Cy3-dUTP) is incorporated during the reverse transcription. The Cy3-labeled
cDNA is
mixed with Cy5-labeled cDNA from untreated cells and hybridized to a DNA
microarray,
(e.g., a DNA microarray commercially available from Affymetrix, Santa Clara,
CA, or
Incyte, Palo Alto, CA reviewed in Nature Genet. Suppl. 21, Jan 1999
). The relative level of Cy3 and Cy5 fluorescence at each spot
in the microarray indicates which there has been a change in the expression of
each gene.
The method is used to identify combinations that cause a desired change in
gene
expression, such as reversion of a disease gene expression profile back to a
healthy
profile. Alternatively, the expression profile caused by a given signaling
molecule, such
21
CA 02582179 2007-04-02
as insulin, is determined, and combinations of compounds are found that cause
the insulin
profile to appear, indicating these combinations mimic the effects of insulin.
Whole Organisms
Another bioassay that is compatible with the screening assays described herein
utilizes a whole animal. In one example, the nematode C. elegans is placed
into
individual wells (preferably with more than one nematode per well), and the
activity of
compounds is detected by detecting a change in a property of the organism. For
example,
the nematode can be engineered to express green fluorescent protein at a
specific stage of
the life cycle, or only during the dauer state. An automated microscopy system
is used to
image the nematodes in each well and measure green fluorescent protein, or
detect
morphological changes in the worms caused by particular combinations of
compounds.
Another whole animal assay uses large animals, such as nude mice, that have
tumors on or near the skin surface. The combinations of compounds can be
mixtures in
DMSO that are rubbed into the skin, penetrate the skin, and reach the tumor.
Alternatively, the compounds can be administered intravenously,
intramuscularly, or
orally. Other whole-organism methods of detecting activity could include using
small
tadpoles derived from fertilized Xenopus oocytes that develop in defined
medium,
organotypic cultures (explants from mice or other animals) in which the organ
can be
cultured for a period of time in a defined medium, and eggs (fertilized or
unfertilized)
from a variety of animals. Another assay measures the tension of cardiac
tissue or muscle
tissue stretched between two springs; compound combinations that modulate
contraction
would result in increased or decreased in the tension on those springs.
Labeling of Compounds
Although some assays can be conducted without any of the combined entities
being labeled, in some embodiments one or more of the combined entities is
labeled so
22
CA 02582179 2007-04-02
that the effect of the combination on the test element can be detected or
measured. Any
of a wide variety of known labels can be used, e.g., techniques that have been
used widely
. in biochemistry for protein affinity chromatography using biotin-
streptavidin interactions
or hexahistidine tagged proteins (Janknect et al., Proc. Natl. Acad. Sci. USA
1991,
88:8972; Wilcheck et al Methods in Enzymology, Wilcheck, M; Bayer, E.A. Eds.
Academic Press Inc. San Diego, 1990; pp. 123-129).
Combining of the Molecules
The methods of the invention can use existing robotics systems, 96-well, 384-
well,
1536-well or other high density stock plates and 96-, 384-, or 1536-well or
other high
density assay plates, with which it is possible to screen up to 150,000
compounds or more
per week. The automation of this technology can be adapted according to the
invention
to screen combinations of molecules. The methods of the invention may also use
microfluidics systems made either by conventional photolithography or by
unconventional methods (such as soft lithography or near-field optical
lithography) to
miniaturize the process. The methods of the invention may also use ink-jet
printing or
compound printing technologies. Additionally, the methods of the invention may
use
trained technician labor to achieve the same results and throughput as the
robotics
systems. The combinations of compounds may be made prior to contact with the
test
element, or they may be in situ in the presence of the test element. These
plating methods
are described in more detail below.
Manual Plating
Compounds may be plated (i.e., added to the cells to be tested) manually. In
one
example, purified chemical compounds are manually combined and tested in a 7X7
combinatorial array. The compounds, which in this case include seven
compounds; are in
a stock plate. The compounds are combined in an assay plate. The operator
plates a first
23
CA 02582179 2007-04-02
compound (or plurality of compounds) from a well of the stock plate in one row
of the
assay plate and then plates one column in the assay plate. This is repeated
with a second
well from the stock plate, only the plating in the assay plate is one row over
and one
column over from those into which the first compound was plated. This process
is
repeated until the full set of combinations has been plated.
Robotic Plating
There are numerous methods for adding compounds to wells to form combinatorial
arrays, and one skilled in the art will recognize that the following examples
are for
illustrative purposes and are in no way limiting.
In one example of robotic plating, a robotic liquid transfer systems is used.
Transfer systems are commercially available from, for example, Beclanan
Coulter
(Fullerton, CA), Tecan (Research Triangle Park, NC) or Zymark (Hopkinton, MA).
The
robotic system plates specific volumes of the first set of compounds into each
well in a
given row, such that row 1 will have the same compound, row 2 will have the
same
compound, etc. Then, the liquid transfer device plates the same set of
compounds along
the columns such that each column will receive the same compound (although
different
columns will have different compounds). Transfer systems can be adapted for
transferring small volumes (e.g., 1 nL).
Another method for effective transfer utilizes ink-jet printing technology
(Gordon
et al., J. Med. Chem. 1994, 13:1385-1401; Lemmo et al., Curr. Opin.
Biotechnol. 1998,
9:615-617). Ink-jet printers draw from a plurality of vessels containing test
compounds;
each compound from a source well is printed out or injected onto the surface
in each
individual row and column for each compound. As describe above, the next
compound is
printed out onto the next row and the next column, and this is iterated until
the entire grid
is plated with combinations of compounds.
24
CA 02582179 2007-04-02
Yet another method for adding compounds to an assay plate utilizes a
microarray
spotter as developed by Patrick Brown at Stanford University for spoiting DNA.
This
device uses an eight-quill pen printing head and eight linear quillheads that
are dipped in
a stock plate and printed along every row. Subsequently, either the plate is
rotated ninety
degrees or the printing head is rotated ninety degrees; the printhead then
prints along the
columns. One could vary the size of the print head using, for example, two,
four or
sixteen printhead for standard 384 well plates, and higher numbers for higher
density
plates.
Yet another method for adding compounds to an assay plate uses a commercially
available instrument called the Hydra (Robbins Scientific, Sunnyvale, CA),
which can be
equipped with 384 separate syringes that are capable of dropping a known
volume from a
standard stock plate of compounds.
An alternate method for the plating of test compounds uses microfluidics
systems
such as those commercially available from Caliper Technologies (Mountain View,
CA)
and directly applying that system to creating an array that would create this
combination.
In this case, arrays of combinations at the microscale are created using
capillary flow to
distribute the compound solutions to the intersection points on a matrix.
Alternative Plating Methods
One in the art will recognize that any plate configuration can be adapted to
the
screening methods of the invention. For example, a 16 X 16 square plate would
have 256
wells instead of the 384 wells in a 16 X 24 plate. This would allow one to
adapt any
liquid addition system in which liquid is only added along the rows or only
along the
columns because one could simply rotate the square plate ninety degrees and
allow
addition in the other direction. One would also incorporate into this design,
a square plate
holder that would have the dimensions or footprint of a standard 96 well or
384 well
CA 02582179 2007-04-02
microtiter plate so that the adapted microsquare plate would fit within any
existing plate
liquid handling system.
Another method of providing the compounds or elements to be tested in
combination is to provide them in solid form rather than liquid form, e.g., as
small
amounts of dry powder. Thus, two dry components (or one wet and one dry
component)
can be mixed and then the combination added to the cells in the medium (in
which the
combined entities are soluble). Solid compounds include beads from a
combinatorial
synthesis on which a different compound is added. For example, beads could be
added
with a bead picker. In one example, the beads are magnetic and added using a
magnet.
In the high throughput assays of the robotics application of the invention,
each
well must be spatially addressable independently, and it is preferably
possible to
withdraw both large (up to 100 gL) and small (down to 1 nL) of each compound
from a
stock plate. An exemplary robotic platform for performing combinatorial
screening
assays of the invention is described below.
A two station robotic platform is created. The first station harbors a simple
XYZ
robotic arm with an attached pin transfer device such as is available through
VWR
(cat#62409-608). A stock plate and an assay plate enter the station and the
robotic arm
drops the pins into the stock plate and transfers these pins into the assay
plate, thereby
delivering 1-1000 nL, depending on pin size (most typically, 50 nL are
delivered).
Different pin devices allow transfer of different combinations of compounds,
as described
above in the example. The second station of the robot is a piezo electric
dispensor,
capable of withdrawing large volumes (up to 10 microliters) from a stock well
of a single
compound and then dispensing small volumes into each well of an assay plate.
For
example, the Ivek Digispense 2000 system has a
resolution of 10 nL and should be sufficient for this purpose.
26
CA 02582179 2007-04-02
Non-Compound Combination Reagents
If a combination reagent which is present across the entire plate is not a
compound
(i.e., if it is radiation of some form), then the plate that contains the
cells and the added
compound(s) can be passed by that source of radiation, thereby completing the
forming of
the combination. Other forms of non-compound combination reagents include, for
example, hyperbaric pressure and heat.
Some non-compound reagents can be varied in a manner similar to the addition
of
a compound. For example, to alter pH, different amounts of base or acid are
added to the
wells. Similarly, ions can be added using any method that is applicable for
the addition of
compounds.
Library Size
For small chemical libraries (<1000 compounds), all binary (up to one half
million
combinations) and tertiary combinations (up to several hundred million
combinations)
can be tested using the systems described herein.
The power of combinations quickly generates effective libraries to identify
multivariable therapeutics. The size of multivariable therapeutic libraries
quickly rivals
and then expands beyond any conventionally available diverse or non-diverse
libraries. A
library of 200 compounds in pair-wise combinations generates a multivariable
therapeutic
library of 19,900 combinations, larger in size than the natural product
library recently
used to identify a novel compound affecting cell division (Mayer et al.
Science, 1999,
286, 971-974). A library of 300 compounds in 3 way combinations generates
approximately 4.5 million distinct combinations of entities for a
multivariable therapeutic
library, larger than the largest currently generated combinatorial chemistry
libraries and
with potentially greater diversity.
27
CA 02582179 2007-04-02
Activity Rank-Order Screen
A diverse library may initially be tested individually using standard
methodologies. The compounds are screened in a biological assay and ranked by
activity.
The most active compounds (e.g., the twenty most active or the thousand most
active,
depending on the amount of combinatorial space to be assayed) in the library
are then
tested pair-wise and in three-way combinations. If desired, these combinations
can be
again ranked by activity and the process iterated for four-way combinations,
five-way
combinations, etc., until a desired number of effective combinations are
identified.
Genetic Algorithms
The use of genetic algorithms provides another method for identifying
combinations of agents with a desired biological activity that is distinct
from the single
agents themselves. In this method each single agent is assigned a unique
barcode, which
can be any series of numbers, letters, or other symbols. In a preferred mode
of
embodiment, the barcode is a binary code (zeros and ones). A specified number
(N, the
"population size") of pair-wise combinations are selected (randomly or based
on some
other characteristic) from the total pool of possible pair-wise combinations
and the
activity of each of these combinations is determined. The X most active
members of the
population are selected (where X is less than N). N new combinations are
formed from
the X active combinations using a series of operators which include (i)
replication (the
original combination is simply selected again) (ii) mutation (one bit of the
barcode is
mutated to a different value), and (iii) recombination (two barcodes are each
split at some
middle point and the opposing ends are joined to create two new hybrid
barcodes. This
process of selection, amplification, mutation, and recombination is repeated
over
numerous cycles, and the activity of each combination of agents determined at
the end of
each cycle. This provides a method of testing a limited number of
combinations, while
still allowing for the discovery of highly effective combinations of agents.
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CA 02582179 2007-04-02
A method for optimizing the parameters of the genetic algorithm (or other
algorithm) is as follows: C combinations of N agents are selected for study.
The activity
of each member of the full set of (N!)/((N-C)!(C!)) combinations is
determined.
Furthermore, all lower order combinations may be determined as well. With this
full data
set of activities, it is possible to evaluate the success rate and efficiency
of various
algorithms, or various permutations of a single algorithm without doing
additional "wet"
benchwork. That is, all strategies for finding active combinations can be
compared "in
silico" and their relative performance compared.
Stock Solutions
Two types of stock solutions are maintained. Each compound is deposited in
both
stock formats. The first stock solution format consists of compounds dissolved
in 100
microliters of dimethylsulfoxide (DMSO) at a final concentration of 4 mM and
stored in
384 well polypropylene plates (Matrix Technologies). The second type of stock
solution
consists of compounds dissolved in 1500 microliters of DMSO at a final
concentration of
4 mM and stored in deep-well 96 well polypropylene plates. Multiple copies of
each type
of plate are made. One copy of the stock plates is stored at 20 C for routine
use, and
backup copies are stored at -80 C for long-term storage.
Assay Plates
Three types of assay plates are used. The size of the pins in the pin transfer
devices described above is adapted to accommodate each size assay plate.
1) Commercial 384.we1l plates (e.g. NaigeNunc white opaque polystyrene cell
culture
treated sterile plates with lid, cat# 164610)
2) Commercial 1536 well plates (e.g. Greiner or Corning)
3) Custom prepared 1536 or 6144 well plates (made from polydimethysiloxane,
Dow
Coming) and deiran molds, and Omni irays.
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CA 02582179 2007-04-02
Software to Manage Compound Additions
Microsoft Visual Basic or programming language is used, using conventional
programming techniques, to write software to operate the instruments described
above.
The software will permit the instrument to read the barcode of a stock plate
or assay plate,
track the location of plates on the assay deck, and transfer the appropriate
volume of the
correct compound into the correct assay well. Thus all combinations to be
screened are
determined or selected beforehand, and the instrument carries out the
combination
screening in an automated format, requiring only simple operator steps, such
as placing
specified plates onto the assay deck and removing specified plates from the
assay deck to
an incubator.
Barcode Reader and Printer
A barcode printer is used, using standard techniques, to generate a unique
identification number for each plate, print the barcode on a label, and stamp
the label on
the assay or stock plate. Software records the identity of each compound in
each well of
each assay plate and stock plate. A barcode reader linked to the assay deck
scans each
plate as it enters and leaves the assay deck.
The following examples are provided for illustrative purposes, and are not
meant
to be limiting in any way.
Example 1
Fig. 1 is a conceptual diagram demonstrating how two different reagents could
act
synergistically inside of a cell, where the reagents bind to different targets
within the
same cell. In this figure, compound A 10 and compound B 12 cross the plasma
membrane 14 and diffuse freely into the cytosolic region of a mammalian cell.
Compound A binds to protein X 16, which is a kinase, inhibiting the activity
of this
kinase. Kinase X normally inactivates transcription factor Y 18 by adding a
phosphate
CA 02582179 2007-04-02
group to Y. Once compound A has inhibited kinase X, transcription factor Y is
activated,
and Y translocates into the nucleus, binding tightly to DNA in the enhancer
region of a
therapeutic gene, such as insulin. However transcription factor Y is unable to
activate
expression of insulin without the presence of a second transcription factor Z
20.
However, in the figure, compound B binds to transcription factor Z, removing
an
autoinhibitory loop on this transcription factor, thereby causing this
transcription factor Z
to translocate into the nucleus, and bind to transcription factor Y. Y and Z
together, but
neither alone, allow activation of expression of the therapeutic gene,
insulin.
Fig. 2 is a conceptual diagram demonstrating how two different reagents could
act
synergistically inside of an organism, where the reagents bind to targets in
different cells
or tissues. In this figure compound A 50 diffuses into beta islet cells 52 of
the pancreas
54. Compound A causes a therapeutic gene encoding insulin 56 to be expressed
in these
cells. However, insulin is ineffective without the presence of the insulin
receptor on
target adipocytes in fat tissue. Meanwhile, compound B diffuses into
adipocytes 58 in fat
tissue 60 and turas on expression of the insulin receptor 62 in these cells. A
and B
together, but neither one alone, allow insulin activity to be restored in this
individual.
Fig. 3 is an example of the experimental data one would obtain in a
combinatorial
screen of the sort we describe in this patent application. The results from
five different
384 well plates are shown. The results are shown in plate format, where there
are 16
rows labeled A through P, and 24 columns, labeled 1 through 24. The level of
activity is
shown in each well as a number, where I indicates basal activity (no effect)
and 5
indicates an active combination. Plate one shows the activity of compounds 1-
384, when
tested at 4 g/mL in a bromodeoxyuridine cytoblot assay for cell-cycle
arresting activity
in A549 human carcinoma cells (described below). Plate two shows the activity
of
compounds 1-384 when tested at 2 gg/mL in same assay. Plate three shows the
activity
of compounds 385-768, when tested at 4 gg/mL, in the same assay. Plate four
shows the
activity of 385-768, when tested at 2 gg/mL, in the same assay. Note that in
plates 1-4,
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CA 02582179 2007-04-02
none of the compounds shows any activity. Plate five shows the activity of 384
pair-wise
combinations of compounds 1-768, when tested at 2 g/mL (i.e., compounds 1-384
and
3 85-768 were both added simultaneously to the-assay plate at 2 g/mL each
compound,
creating 384 different random pair-wise combinations). Note that well Al shows
activity.
This means that compound 1 (in well A 1 of the plate with compounds 1-384) by
itself had
no activity and compound 385 (in well Al of the plate with compounds 385-768)
by itself
had no activity but together compound 1 and 385 synergize to create an active
combination.
Example 2
General Methods
Fig. 4 is an illustration of a method for performing combinatorial screening
using
currently commercially available technology. Human A549 lung carcinoma cells
are
obtained from the American Type Culture Collection (ATCC, catalog number
CCL185)
and cultured at 37 C with 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM)
with
10% FBS, 100 units/mL penicillin G sodium, 100 g/mL streptomycin sulfate, and
2 mM
glutamate (GibcoBRL, Rockville, MD) (referred to as 10% medium). Four thousand
cells are seeded in each well of a white, opaque 384-well plate 100 (Nalge
Nunc
International, Rochester, NY) using a Multidrop 3841iquid dispenser 110
(Labsystems
Microplate Instrumentation Division, Franklin, MA). The cells are seeded in 40
L of
10% FBS-containing medium.
After 16 hours at 37 C with 5% C02, 10 L of a 50 M stock of a compound of
interest in 10% medium is added to each well, bringing the total well volume
to 40 L
and the final concentration of this compound to 10 M. Either prior to,
immediately
afterwards, or several hours or days later, a second set of compounds is added
by dipping
small pins on a pin array 130 into each well of a stock plate 140 or 150 and
then into each
well of an assay plate 100. Matrix Technologies Pin Transfer device 130 (384
or 96 pins,
32
CA 02582179 2007-04-02
will suffice (catalog numbers 350500130 and 350510203). This two-step process
allows
for the testing of one specific compound against a large number of other
compounds in
many pair-wise combinations. It is necessary to also have a plate where the
set of pin-
transferred compounds is tested in the absence of the original compound (the
one present
in all wells) to determine whether a novel property has been achieved with the
combination.
A different method can also be used to provide combinations of compounds for
contacting with the test element. For example, instead of keeping one compound
fixed
throughout the pair-wise combinations, as described above, it is possible to
pin transfer a
set of compounds in such a way that all pairwise combinations of that set are
achieved. A
set of 16 compounds is pin transferred from stock plate 140 to the 16 rows of
the 384 well
assay plate 100. The same set of 16 compounds is then transferred to 16
columns of the
same assay plate, providing a 256 well matrix with different pairwise
combinations.
In each of the foregoing examples (or analogous versions), once the desired
compounds have all been added, the assay plate containing A549 cells and
compounds
are incubated for 48 hours at 37 C with 5% carbon dioxide in incubator 120. At
the end
of this time, 10 L of BrdU is added, using a Multidrop 384110, from a 50 M
stock in
medium (prepared from a 10 mM stock in PBS, pH 7.4) to a final concentration
of 10 M
BrdU. The cells are incubated for an additional 12-16 hours in incubator (120)
at 37 C
with 5% CO2. The supematant is removed from each well with a 24-channel wand
(V&P
Scientific), used throughout the protocol for aspiration, attached to a house
vacuum
source. The cells are fixed with 50 L of cold (4 C) 70% ethanol/30% PBS,
incubated for
one hour on ice, washed with 90 L of cold (4 C) PBS, and then 25 L of 2M HCI
/ 0.5%
Tween20 / ddH2O is added. The cells are incubated at room temperature for 20
minutes,
then washed with 90 gL of 10% 2M NaOH / 90% Hank's Balanced Salt Solution
(HBSS,
GibcoBRL), twice with 90 L of HBSS, and once with 75 L of PBSTB (PBS, 0.1%
Tween 20 (Sigma), 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO).
Then
33
CA 02582179 2007-04-02
20 L of antibody solution is added containing 0.5 g/mL mouse anti-BrdU
antibody
(1:1000 dilution of stock, (BD Biosciences-PharMingen San Diego, CA) and
1:2000
dilution of anti-mouse Ig antibody conjugated to HRP (Amersham Pharmacia
Biotech
Inc., Piscataway , NJ) in PBSTB. The cells are incubated for one hour at room
temperature, washed twice with 90 L PBS and then 20 L HRP substrate solution
is
added to each well. The HRP substrate solution is obtained by mixing equal
volumes of
solutions 1 and 2 from the Amersham ECL7detection kit. The plate is placed
into an LJL
Biosystems Inc. (Sunnyvale, CA) Analyst AD platereader 160 and luminescence is
detected in each well for 0.3 seconds. The activity of combinations are then
compared to
the activity of the single agents alone at both the original concentration and
at N times the
original concentration, where N is equal to the number of active compounds
found in the
screen. If a combination shows an activity that is greater than the activity
of the each of
the single agents alone at both the original concentration and N times the
original
concentration, then a synergistic effect has been observed. Even if synergy is
not
observed, a combination may have advantageous properties (e.g., reduced side
effects)
relative to the individual components that make up the combination.
The foregoing method is readily modified such that two different compounds are
added in the first step, allowing the testing of three-way combinations.
Similarly, three
compounds can be added to allow testing of four-way combinations, etc. Using
such an
approach, higher order combinations of compounds are tested.
The identification of a combination of compounds that inhibit proliferation is
described below. This description is for illustrative purposes, and should is
not limiting in
any way.
Seven compounds were tested alone and in all 21 possible pair-wise
combinations
in the BrdU cytoblot assay (see below) for their effect on cell cycle
progression. The
seven compounds (podophyllotoxin, paclitaxel, quinacrine, bepridil,
dipyridamole,
promethazine, and agroclavine; each purchased from Sigma Aldrich Corp., St.
Louis,
34
CA 02582179 2007-04-02
MO) were weighed into one dram glass vials and dissolved in dimethylsulfoxide
to create
= 4 mg/mL stock solutions. Six thousand A549 lung carcinoma cells were seeded
in each
well of a 384 white opaque NalgeNunc cell culture-treated plate (cat# 164610)
in 30 L
of 10% medium (Dulbecco's Modified Eagle Medium containing 10% fetal bovine
serum, 100 units/mL penicillin G sodium, 100 g/mL streptomycin sulfate, and 2
mM
glutamine, all obtained from Life Technologies). Each compound was diluted to
four
times the final assay concentration (final assay concentrations were 0.25%
DMSO, 240
nM podophyllotoxin, 60 nM paclitaxel, 420 nM quinacrine, 25 M bepridil, 400
nM
dipyridamole, 25 M promethazine, 840 nM agroclavine) in 10% medium. Fifteen
microliters of each 4X stock of compound in medium was added to one row and
one
column of an 8 column and 8 row square (the eighth lane containing only the
vehicle
DMSO), such that all possible binary combinations of the 7 compounds were
tested, as
well as the single agents themselves. The cells were incubated at 37 C with 5%
carbon
dioxide for 46 hours. BrdU was added to a final concentration of 10 M by
adding 15 L
of a 50 M solution of BrdU in 10% medium. The cells were incubated overnight
at
37 C with 5% carbon dioxide.
After 16 hours the medium was aspirated from each well with a 24 channel wand
(V&P Scientific), used throughout the protocol, attached to a house vacuum
source. Fifty
microliters of 70% ethanol/ 30% phosphate buffered saline (4 C) was added to
each well
with a Multidrop 384 plate filler (Labsystems), used for all subsequent liquid
addition
steps. The plate was incubated for one hour at room temperature, then the
wells were
aspirated, and 25 L of 2 M HCI with 0.5% Tween 20 was added to each well. The
plate
was incubated for 20 minutes at room temperature. Twenty five microliters of 2
M
NaOH was then added to each well. The liquid in each well was aspirated and
the wells
were washed twice with 75 L of Hank's Balanced Salt Solution. The wells were
washed
again with 75 L of PBSTB (phosphate buffered saline with 0.5% bovine serum
albumin
and 0.1 % Tween 20). Twenty microliters of antibody solution was added to each
well
CA 02582179 2007-04-02
(containing 0.5 g/mL anti-BrdU antibody (PharMingen) and 1:2000 dilution of
anti-
mouse Ig-HRP (Amersham). The plate was incubated with the antibody solution
for one
hour at room temperature, then the antibody solution was aspirated off and
each well was
washed once with phosphate buffered saline. Finally, 20 L of ECL detection
reagent
was added to each well (an equal mixture of solutions one and two frorn
Amersham's
ECL detection reagents). The luminescence in each well was read on an LJL
Analyst
platereader with 0.2 seconds integration time per well. The experiment was
repeated in
two plates and each pair-wise combination was tested in a total of sixteen
replicate
experiments. The data shown in Table 1 depict the mean antiproliferative
activity of
each combination of compounds. Five statisticaliy significant (p < 0.001)
combinations
are highlighted. All activity is normalized to a set of wells containing cells
that did not
receive any treatment. Thus, a value of one represents an inactive substance
and a value
greater than one indicates some level of antiproliferative activity.
Table 1- Antiproliferative activity of combinations of various FDA approved
drugs
~
o ~, a? m c m
p E . -J c
m >
~ ~, ~ ~ z m e m
~ a U C m ~ ~ U
oa a ~ m E 2
a a2.
: DMSO 1.0
Podophyiiotoxin 6.5 6.5
Paclitaxel 6.3 6.3 7.7
Quinacrine 1.7 6.6 6.9 2.3
Bepridil 1.8 15.2 3.1 3.0 14.4
Dipyridamoie 1.5 8.9 8.8 2.3 2.4 2.0
Promethazine 1.3 3.9 6.4 2.4 15.2 1.8 4.8
Agroclavine 1.0 5.7 5.8 1.6 1.9 1.5 1.2 1.0
The chart shows five combinations of existing FDA-approved drugs with
antiproliferative
activity that is distinct from that of the individual components.
Podophyllotoxin and
paclitaxel are both microtubule stabilizers that arrest cells in mitosis,
dipyridamole is an
anti-platelet agent, bepridil is a calcium channel blocker, and promethazine
is an Hl
36
CA 02582179 2007-04-02
histamine receptor antagonist and is also used as a CNS depressant and
anticholinergic
agent. Dipyridamole is generally considered to have a relatively high safety
profile as a
human therapeutic, particularly compared to the toxic side effects of
paclitaxel and
podophyllotoxin. Thus, in this assay, dipyridamole enhances the
antiproliferative effect
of both paclitaxel and podophyllotoxin on human lung cancer cells.
Furthermore, bepridil
enhances the effects of podophyllotoxin but inhibits the effect of paclitaxel.
This result
would not have been predicted a priori and highlights the importance of
empirical high
throughput testing of combinations to observe unexpected interactions among
drugs. For
example, bepridil and promethazine, neither of which is used as an
antiproliferative agent
in current therapeutic indications, combine to strongly inhibit the
proliferation of lung
cancer cells.
Other Embodiments
Various modifications and variations of the described method
and system of the invention will be apparent to those skilled in the art
without departing
from the scope and spirit of the invention. Although the invention has been
described in
connection with specific preferred embodiments, it should be understood that
the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed,
various modifications of the described modes for carrying out the invention
which are
obvious to those skilled in the field of drug discovery or related fields are
intended to be.
within the scope of the invention.
37
