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`US 20060160109A1
`
`119) United States
`112) Patent Application Publication
`MacDonald et al.
`
`110) Pub. No. : US 2006/0160109 A1
`Jul. 20, 2006
`143) Pub. Date:
`
`154) HARNESSING NETWORK BIOLOGY TO
`IMPROVE DRUG DISCOVERY
`
`175)
`
`Inventors: Marnie L. MacDonald, Pleasanton, CA
`1US); John K. Westwick, San Ramon,
`CA (US); Brigitte Keon, Castro Valley,
`CA (US); Jane Lamerdin, Livermore,
`CA (US); Stephen W. Michnick,
`Montreal 1CA)
`
`Correspondence Address:
`Isaac A. Angres
`Suite 301
`2001 Jefferson Davis Highway
`Arlington, VA 22202 (US)
`
`173) Assignee: Odyssey Thera, Inc. , San Ramon, CA
`1US)
`
`121) Appl. No. :
`
`11/282, 745
`
`122) Filed:
`
`Nov. 21, 2005
`
`Related U. S. Application Data
`
`160) Provisional application No. 60/629, 558, filed on Nov.
`22, 2004.
`
`Publication ClassiTication
`
`151) Int. Cl.
`C408 40/08
`12006. 01)
`C408 40/10
`12006. 01)
`152) U. S. Cl. . . . . . . . . . . . . . . . 435/6; 435/7. 1; 977/702; 977/926
`
`157)
`
`ABSTRACT
`
`This invention provides principles, methods and composi-
`the mechanism of action of pharma-
`tions for ascertaining
`in the context of network
`cologically
`important compounds
`biology, across the entire scope of the complex pathways of
`the principles, methods and com-
`living cells. Importantly,
`allow a rapid assessment of the on-
`positions provided
`effects of lead compounds
`and off-pathway
`and
`pathway
`in living cells, and comparisons of lead
`drug candidates
`compounds with well-characterized
`drugs and toxicants
`to
`identify patterns associated with efficacy and toxicity. The
`invention will be useful
`the drug discovery
`in improving
`process, in particular by identifying drug leads with desired
`safety and efficacy and in effecting early attrition of com-
`pounds with potential adverse effects in man.
`
`Beckman Exhibit 1056, Page 1
`
`

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`Patent Application Publication
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`

`

`US 2006/0160109 A1
`
`JU1. 20, 2006
`
`HARNESSING NETWORK BIOLOGY TO
`IMPROVE DRUG DISCOVERY
`
`[0001] This application claims the priority benefit under
`35 U. S. C. section 119 of U. S. Provisional Patent Application
`No. 60/629, 558 entitled "Harnessing network biology
`to
`improve drug discovery" filed Nov. 22, 2004, which is in its
`entirety herein incorporated by reference.
`
`BACKGROUND OF THE INVENTION
`
`[0002] The central challenge of the pharmaceutical
`indus-
`that are both safe and eflective
`try is to develop drugs
`in
`man. Even an exquisitely selective chemical compound
`that
`target may have completely unex-
`to a therapeutic
`binds
`pected or 'ofl'-pathway'
`efl'ects
`to
`in living cells, leading
`expensive pre-clinical and clinical failures. Regardless of
`whether a drug or drug candidate
`is an agonist, antagonist,
`inhibitor or activator of a target, drugs exert their actions by
`the function of that
`to a target protein and altering
`binding
`'ofl'
`protein. For the purposes of this invention, we define
`pathway' activity as any activity of a compound on a cellular
`target of the
`target or pathway
`other
`the
`intended
`than
`compound.
`[0003] As evidenced by the 75% failure rate of drugs
`in
`clinical trials, the development of new drugs is a costly and
`unpredictable process, despite the number of research tools
`industry. The US Food and
`to the pharmaceutical
`available
`that even a 10%
`has estimated
`Drug Administration
`adverse eflects of compounds,
`improvement
`in identifying
`prior to clinical trials, could save $100 Million in develop-
`ment costs per drug (reference white paper). Our central
`efl'ects of new drugs are
`the ofl'-pathway
`is that
`premise
`for many if not all of the failures
`responsible
`in new drug
`of the full spectrum of
`development. An understanding
`biological activity of any new chemical entity would help to
`identify potentially adverse eflects of drugs prior to clinical
`to
`trials. Therefore, we sought
`to establish a rapid method
`assess the activity of any new chemical entity in the context
`of the complex networks of living cells.
`
`[0004] Numerous
`are
`in vivo and
`in vitro approaches
`the selectivity of lead compounds. Typi-
`aimed at assessing
`cal methods are briefly described here.
`[0005] The selectivity of a compound can be assessed by
`constructing panels of in vitro assays to measure the activity
`of the compound against
`in the target
`individual proteins
`is the target class comprised of protein
`class. An example
`kinases. There are over 500
`(tyrosine and serine/threonine)
`in the mammalian genome, making
`distinct protein kinases
`the development of selective
`chal-
`inhibitors particularly
`lenging. A variety of companies
`(e. g. PanLabs, Kinexus)
`kinase
`have established
`inhibitor profiling products
`and
`the selectivity of lead com-
`to assess
`services designed
`in vitro against panels of
`pounds by testing each compound
`individual, purified kinases. The completion of the mapping
`of the
`and the availability of full-length
`'kinome'
`genes
`encoding human kinases has aided in the development of
`such assay panels.
`
`[0006] Although
`such assay panels exist for kinases, as
`well as for many other common drug target classes such as
`G-protein-coupled
`receptors (GPCRs), such panels are only
`capable of assessing drug activity against
`the proteins
`that
`are directly assayed. Even if it were possible to construct an
`
`assay for every kinase in the kinome, the approach would be
`limited in its ability to identify ofl'-pathway effects of kinase
`leads. The most significant
`is that even a highly
`limitation
`inhibitor of a kinase may be capable of binding,
`selective
`activating, or inhibiting a plethora of other proteins
`that are
`not even in the same target class. Such ofl'-target/ofl'-path-
`and cannot be assessed in
`way activities are unpredictable,
`a comprehensive way with in vitro assays. Since the number
`of proteins
`in the proteome exceeds 30, 000, a comprehen-
`testing every compound of
`sive analysis would
`require
`interest against over 30, 000 proteins. First,
`it would be
`necessary to purify each of the thousands of proteins
`in the
`human proteome; and then to construct a biochemical assay
`to measure the activity of that particular protein; and finally,
`to assay each chemical compound of interest
`in 30, 000
`discrete assays. This is not practical or even feasible in the
`near future.
`
`involve panning bio-
`[0007] Other profiling approaches
`that are capable of
`logical extracts or lysates for proteins
`to a compound of interest. Such approaches
`typi-
`binding
`cally involve contacting a cell lysate or tissue extract with a
`test compound
`that is bound to a bead or other solid surface
`to the bead. The
`the proteins bound
`and then analyzing
`proteins bound to the bead can be analyzed by mass spec-
`or flow cytometry. Unfortu-
`troscopy, immunoprecipitation,
`nately, such methods often require concentrations of com-
`levels of any
`pound that are far higher than the physiological
`drug. In addition, artifacts can occur as a result of removing
`the proteins from their cellular milieu and subcellular con-
`text. For example, when a cell is lysed, proteins are released
`from their normal subcellular compartments
`and a particular
`protein may be capable of binding
`to a compound on a bead;
`'see' that
`in the living cell, the drug would never
`whereas,
`protein.
`
`[0008] To date,
`to pharmacological
`in vivo approaches
`tissues or whole
`living cells,
`involve
`treating
`profiling
`organisms with a test compound;
`then measuring changes in
`one or more phenomenological,
`functional or gene expres-
`sion patterns of the cells, tissues or organisms
`in response to
`the test compound. Each of these is discussed below.
`
`[0009] Phenomenological
`and functional assays allow an
`assessment of the spectrum of functional consequences of
`in living cells and a comparison of those
`drug activity
`to that of agents with known characteristics. For
`responses
`example, Dunnington et al. described methods for studying
`the function patterns of pharmacologically
`important com-
`the efl'ects of compounds on a plethora
`pounds by measuring
`of physiological measurements
`in a variety of cell types (US
`20030100997). They specified assays for cellular membrane
`transport, cell pro-
`potential, gene expression, physiological
`secretion,
`liferation,
`toxicity,
`scattering,
`light
`apoptosis,
`chemotaxis, adhesion, and similar parameters
`morphology,
`which represent measurable parameters of cell behavior or
`the majority of these parameters are
`response. Importantly,
`per se. The phenomenological
`not molecular parameters
`approach has the advantage of determining a broad scope of
`functional properties of compounds.
`desired and undesired
`it has the disadvantage of being purely descrip-
`However,
`of the biochemical
`the determination
`tive, not allowing
`mechanism of action of any undesired properties or of
`func-
`that may not have immediate
`identifying properties
`tional consequences. Also, unlike molecular parameters
`in the tens of thousands,
`is a limited
`which number
`there
`
`Beckman Exhibit 1056, Page 26
`
`

`

`US 2006/0160109 A1
`
`JU1. 20, 2006
`
`that
`
`parameters
`
`number and variety of phenomenological
`in any particular cell.
`can be measured
`[0010] The use of gene microarrays
`for pharmacological
`profiling has become routine in the pharmaceutical
`industry.
`For this purpose, cells~r even whole animals
`are treated
`with the drug or compound of interest. Following a period
`of time (usually 24-48 hours) messenger RNA is isolated
`from the cell or tissue. The pattern of expression of thou-
`sands of individual mRNAs
`in the absence and presence of
`to
`have been used
`the drug are compared. Microarrays
`risk of side
`have
`predict which compounds
`the greatest
`specific kinds of therapeutic
`eflects, and also to identify
`et al. (Proc Natl Acad Sci USA 100:
`activity. Gunther
`9608-9613) first produced a series of gene expression pro-
`to act on the
`files by treating cells with chemicals known
`that a small number of genes were good
`CNS and found
`markers of antipsychotic,
`or opiate efl'ects.
`antidepressant,
`U. S. Pat. No. 6, 372, 431 describes the use of gene microar-
`in order to
`rays to test samples
`treated with drug candidates
`the gene expression pattern associated with drug
`elucidate
`treatment. This gene pattern can be compared with gene
`expression patterns associated with compounds which pro-
`duce known metabolic and toxicological responses. Simi-
`larly, U. S. Pat. No. 5, 569, 588 discloses methods
`for drug
`a plurality of separately
`isolated
`screening by providing
`cells, each having an expression
`system with a diflerent
`regulatory element. Contacting
`transcriptional
`this plurality
`of cells with a drug candidate and detecting reporter gene
`product signals from each cell provides a profile of response
`to this multiplicity of regulatory
`to the drug with regard
`elements.
`
`[0011] In sum, high-throughput
`gene expression measure-
`ments using DNA microarrays provide global snapshots of
`the dynamics of gene networks at the RNA level. However,
`the ultimate conse-
`transcription
`only reveal
`experiments
`quences of pathway perturbation,
`rather than the cause or
`mechanism. Gene network reconstruction
`from microarray
`the so-called
`problem'
`'dimensionality
`from
`data suflers
`the number of genes
`because
`is much greater
`the
`than
`number of microarray experiments. Thus, simply identifying
`all of the mRNA species present and the levels at which they
`are present at a particular
`time, may not yield a complete
`picture of a particular drug. Moreover, changes
`in the level
`of individual mRNA molecules do not always correlate
`level or activity of the corresponding
`the
`directly with
`protein at a single point in time. Many proteins and other
`macromolecules
`undergo post-translational modifications
`interactions, which may afl'ect
`and macromolecular
`the
`functions and activities of proteins within a tissue or cell
`independent of gene expression.
`[0012] An alternative approach to pharmacological profil-
`ing is to analyze proteins
`the signaling path-
`that regulate
`ways that, in turn, control gene expression and cell behavior.
`For example, cells or whole animals can be treated with the
`test compound, a cell lysate or tissue extract prepared, and
`status of proteins
`modification
`the post-translational
`in the lysate or extract. The proteins
`in the cell
`assessed
`lysates are typically either separated by 2-dimensional
`gel
`electrophoresis
`then probed using Western blotting
`and
`arrays of phos-
`techniques, or are analyzed by multiplexed
`pho-specific antibodies on beads or on antibody arrays (e. g.
`Nielsen et al. , 2003, PNAS 100: 9330-9335). Janes et al.
`(Molecular & Cellular Proteomics 2: 463-473, 2003) used
`
`to develop
`a microtiter-plate-based
`assay
`this approach
`for multiple kinase activities. Following
`treatment
`panel
`with agonists, cell lysates were assayed
`in microtiter wells
`precoated with anti-kinase
`and kinase activity
`antibodies
`measured with P2]P-ATP. These and similar biochemical
`information on what types of proteins are
`methods provide
`their level of expression, and
`in a given pathway,
`involved
`the way they interact with each other; but rarely can resolve
`where and when such proteins are activated within a cell.
`Traditional biochemical
`techniques are laborious, difficult to
`the use of radioactive reagents.
`automate, and may require
`such techniques are not amenable
`to multiplex-
`Importantly,
`ing with other types of assays, or to assaying
`thousands of
`drug candidates
`simultaneously.
`in which a multitude of
`[0013] Cells are complex systems
`biochemical
`reactions and molecular events
`take place at
`one time and need to be finely orchestrated
`to preserve
`the
`and direct the cell-specific
`cell homeostasis
`functions.
`In
`the flow of information
`from many diflerent
`particular,
`to a diverse set of physiological
`cellular
`functions
`inputs
`rely on a precise organization of the
`intracellular
`must
`and on their
`timely and coordinated
`signaling networks
`activation. In recent years, as a result of the development of
`imaging of fluorescent
`new technologies based on real-time
`the direct visualization of individual molecular
`indicators,
`events taking place in intact cells has become possible. The
`living cells opens a new path
`to work with
`to
`ability
`obtaining basic
`critical
`to understanding
`the
`information
`cell's molecular processes. These tools have been success-
`to
`and high-throughput
`targets
`individual
`fully applied
`reso-
`screening campaigns. The high spatial and temporal
`lution that such methodologies provide opens the possibility
`to accurately measure quantitative
`and dynamic parameters
`of signaling networks
`in their complex cellular environment.
`However, with the exception of our own work and a single
`study of protein localization with respect to toxicology (see
`the prior art is silent on the application of
`References)
`profiling of lead com-
`network biology to pharmacological
`pounds or drug candidates. U. S. Pat. No. 6, 673, 554 provided
`localization assays for toxicity based on a single
`protein
`translocation of proteins,
`intracellular
`phenomenon, namely,
`in particular protein kinase C isozymes. While this reference
`embodies the use of whole cell assays it is limited to a single
`that is restricted
`to a relatively
`phenomenon
`small propor-
`tion of all the macromolecules of the cell.
`[0014] The principles of network biology
`in living cells
`have never before been applied to drug discovery on a large
`scale. There are a number of factors that may have prevented
`its application until now. First, it is generally believed
`that
`it will take 20-30 years to solve the problem. In particular,
`'systems biology' is perceived as a computational
`challenge
`which can only be solved when masses of descriptive
`information are in hand some years in the future. Second,
`current dogma holds that cell signaling events occur within
`seconds or even milliseconds,
`that dynamic
`suggesting
`events are difficult to capture except in rare circumstances
`techniques. Third, biomo-
`and with the most sophisticated
`lecular interactions
`that control pathways
`such as protein-
`are generally perceived as events that
`protein interactions
`can only be identified with static methods
`such as yeast
`screens. Fourth, the vast majority of small mol-
`two-hybrid
`inter-
`ecule drugs do not themselves disrupt protein-protein
`to study drug action by
`actions; which means that attempts
`complexes are often perceived as
`studying biomolecular
`
`Beckman Exhibit 1056, Page 27
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`US 2006/0160109 A1
`
`JU1. 20, 2006
`
`3
`
`to perturb
`themselves.
`the interactions
`misguided
`attempts
`the majority of bio-
`Finally, because of budget
`limitations,
`studying drug action in cells do not
`chemical
`researchers
`to do so.
`utilize high throughput
`instrumentation
`[0015] We sought to apply network biology to drug dis-
`covery in living cells on a large scale. The present invention
`a comprehensive
`rationale, principles,
`provides
`strategy,
`the mecha-
`for investigating
`compositions and methodology
`nism of action of any molecule
`in any living cell, including
`eflects of
`the identification of unintended or 'ofl'-pathway'
`the molecule of interest. The invention enables the creation
`of quantitative
`profiles of
`and predictive pharmacological
`regardless of their
`drugs, and toxicants
`lead compounds,
`intended mechanisms of action; and an assessment of unin-
`and/or adverse efl'ects of molecules of
`tended, ofl'-pathway
`interest. The invention enables early attri-
`pharmacological
`tion of lead compounds with undesirable properties, poten-
`tially saving millions of dollars spent on compounds with
`or adverse eflects
`in the clinical
`subsequent
`unintended
`setting.
`
`OBJECTS AND ADVANTAGES OF THE
`INVENTION
`
`[0016] It is an object of the present
`to provide
`invention
`profiling of chemical com-
`for pharmacological
`principles
`pounds, drug candidates, established drugs and toxicants on
`a global scale.
`[0017] It is a further object of the invention
`to provide
`methods for assessing the activity, specificity, potency, time
`course, dose response and mechanism of action of chemical
`in living cells.
`compounds
`[001S] It is also an object of the
`to allow
`invention
`determination of the selectivity of a chemical compound
`the biological context of any cell.
`within
`[0019] It is an additional object of the present invention
`to
`allow detection of the potential ofl'-pathway
`efl'ects, adverse
`eflects, or toxic eflects of a chemical compound within
`the
`biological con