# Patent

## Title
Drug discovery methods

## Abstract

Methods for identifying disease-related pathways that can be used to identify drug discovery targets, to identify new uses for known drugs, to identify markers for drug response, and related purposes.

## Background
The application is a continuation-in-part of and claims the benefit of priority under 35 USC §120 of International Application No. PCT/US03/03006 filed Feb. 3, 2003, which further claims the benefit of priority under 35 USC 119(e) to Provisional Applications Ser. Nos. 60/421,772 filed Oct. 29, 2002 and 60/353,176 filed Feb. 4, 2002.


FIELD OF INVENTION
Background of the Invention
The last 5 years or so has seen an explosion in the availability of data relating to genomics, i.e., information related to genes, their nucleic acid sequences, the proteins these genes encode for, the biological effect of the proteins, and other related information. The availability of this data has opened up unprecedented opportunities for understanding disease pathways and for identifying new therapies and prophylaxes based on these understandings.
There are multiple routes to modern drug discovery. In general, these require identification of a gene or gene product (i.e., an RNA, polypeptide or protein) that is associated with a given disease. After this association has been made, researchers can design drugs that antagonize or inhibit, or agonize or enhance, the expression of or activity (i.e., function) of the gene or gene product in order to treat or prevent the disease.
Preferably, researchers will have not only knowledge of the association of a given gene or gene product with a disease but a fuller understanding of the entire disease pathway, i.e., the series of biochemical processes within the body that result in disease. Researchers also desire to have a fuller understanding of other pathways that may comprise the given gene or gene product, as well as other pathways, i.e., pathways that do not comprise the gene or gene product, that lead to the same disease. Even more preferably, researchers would wish to have a fuller understanding of additional indicators of safety and efficacy, such as genotypic or phenotypic “markers” or biochemical or environmental factors that are associated with responses to specific drugs, which responses vary among subsets of a patient population.
So, for example, the knowledge that a hypothetical protein, referred to now for illustrative purposes as Protein A, is associated with inflammation suggests to researchers that Protein A is a likely target for drug intervention because a drug that inhibits Protein A is likely to have a positive effect on Protein A-related inflammation.
Researchers would prefer to have a fuller understanding of the association of Protein A to inflammation. For illustrative purposes, researchers would want to know, hypothetically:

    
    
        Up regulation of Gene A results in expression of Protein A
        Protein A phosphorylates Protein B in certain cell types
        Protein B, upon phosphorylation, up-regulates Gene C
        Up-regulation of Gene C results in expression of Protein C
        Protein C activates T cells
        Activation of T cells causes inflammation.
    
    


More preferably, the researchers would also have a fuller understanding of additional pathways that may comprise Protein A, as such information would help researchers predict side effects. Also, researchers would wish to have a fuller understanding of alternative pathways that result in the same disease because such information would help them better predict the efficacy of inhibiting Protein A. As noted above, researchers would also want to understand more fully additional factors that would help them predict safety or efficacy in given patients. Genotypic markers typically comprise specific polymorphisms, such as repeats, SNPs, insertions or deletions; phenotypic markers can include a number of factors such as race, gender, ethnicity, age, weight, etc.; environmental factors can include, e.g., behaviors such as smoking or drinking alcohol, exposure to toxins, etc.; biochemical markers can include, e.g., cholesterol levels, etc.
A great deal of such information is available from public sources, e.g., scientific publications. However, the sheer volume of such data is overwhelming such that the data cannot be accessed and correlated in an efficient and effective manner. Compounding the problem is that the data are in disparate sources making it extremely hard to piece together in order to derive a fuller picture.
There have been several attempts to address this problem by creating search tools, such as, MEDLINE® Chemical Abstracts®, BIOSIS Previews®, etc., that permit computer searching of large numbers of scientific journals or abstracts, such as Science, Nature, Proceedings of the National Academy of Sciences, etc. Searching these journals is still a problem because there are hundreds of such journals and many can only be searched by key words (and searching is sometimes restricted to key word fields or abstracts) or by reading full abstracts, which in either case is very time-consuming and inefficient such that important articles are easily missed.
Another partial solution is databases of genomics data. One example is GenBank®, which is maintained by the National Center for Biotechnology Information (NCBI). Gene sequences entered in such databases are usually annotated with information that may include, e.g., the type of cell in which a given gene sequence is expressed, the probable function of the sequence, etc.
While these databases are enormously helpful, they miss some data that appear in scientific publications and, more problematically, they cannot readily be used to determine disease pathways because the data are not structured in a way that allows computer analysis of complex relations between different genes and gene products.
SUMMARY OF THE INVENTION
The present invention relates to methods for identifying pathways for particular phenotypic traits. In a particular representative embodiment, the invention relates to methods of identifying drug discovery targets by defining disease pathways by computer analysis of direct as well as complex relations among different genes, gene products, or processes. In other embodiments, the invention provides methods for identifying new uses for known drugs, methods for predicting likely side effects of treatment with a given drug, and methods of predicting efficacy of a given drug in a given individual.
The invention makes use of a structured database representation of information concerning genes, gene products, processes, and phenotypic traits of interest, and optionally other information (including for example information concerning SNPs, non-genomic DNA sequences, allelic variations, etc,.) such that relationships that are several steps removed and that may be multi-directional, can be identified. The information that is stored typically comprises data from public sources such as databases and scientific publications. It can also be proprietary data or a mix of proprietary and public data. The phenotypic trait of interest is typically a disease, a susceptibility to a disease, or a drug response, e.g., a side effect or a degree of efficacy.
A structured database representation of information will be able to define biological relationships that are at least one step removed. For example, information that may be acquired from one data source, e.g., a scientific journal article, might conclude that Protein A phosphorylates Protein B. Information from a second data source, e.g., a second scientific journal article, might conclude that Protein B, upon phosphorylation, up regulates Gene C. The relationship between Protein A and Gene C is one step removed. Each such “step” can actually involve a number of biological interactions between or otherwise affecting the relationship between or among two or more components of the body. Preferably, the system will be able to define biological relationships that are 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more steps removed. The biological relationships that can be defined will often times be complex, or multi-directional, relationships in the sense that one or more genes or gene products in a given pathway may also appear to be parts of multiple other pathways so that many of the genes or gene products in the database will be related to others in a complex, “spiderweb-like” relationship. A biological relationship exists when a component (i.e., concept) of a pathway has a biological effect upon, or is biologically affected by, another component of the pathway. So, with reference to the simple illustration provided above, a biological relationship exists between any two of, and among all of, Protein A, the gene that expresses protein A, Protein B, the gene that expresses Protein B, Gene C, and the gene product of Gene C. Thus, a preferred database for use in the invention may be referred to as a “biological relationships database,” i.e., one that identifies related biological concepts and that specifies what the functional biological relationship (or functional biological relationships) between or among the concepts is (or are).
There are several uses for a structured database representation of genomics information. In one such use, a method for identifying a drug discovery target includes the steps of querying the database to identify a disease-related pathway whereby each of the “actor concepts” in the pathway (as described hereinbelow) is an actual or putative candidate drug discovery target. The genomics information may comprise information relating to the biological interactions of each of the “concepts” in the pathway, both within the pathway as well as external to the pathway. Such external information can be used to select, de-select, or prioritize certain “steps” as drug discovery targets.
The candidate drug discovery targets in the disease related pathway may be prioritized based on factors that include function and complexity, a presence of markers for side effects and patient responsiveness, and “drugability” (this term is used in the field of drug discovery to indicate the likelihood that the activity of a particular biological entity can be affected by use of a pharmaceutical agent, e.g., by looking at the protein family class (e.g. GPCR family members generally considered more easily target-able because they sit on the cell surface), through structural analysis, or other experiences. Results of querying the database may be combined with the results of additional data obtained from one or more additional methods for identifying candidate drug discovery targets (e.g., differential gene expression studies).
The database may include the use of an “ontology” as this particular form of structured information may be used to infer classifications based upon the biological interactions of interest. This classifying one or more findings using an ontology may further include determining a likelihood that the one or more findings residing in a particular biological classification in the ontology is statistically significant (e.g., by testing a null hypothesis).
In another aspect, there is a method for identifying a new use for a known therapy including the steps of providing a means for querying the database to identify a disease-related pathway comprising a known therapy target; selecting at least one of such disease-related pathways wherein the known therapy target is also comprised within a second disease-related pathway; and identifying treatment of the second disease as a new use for the known therapy.
In another aspect, a method for prioritizing candidate development compounds for further development is provided. In this embodiment, the method includes the steps of querying the database to identify all pathways associated with the target of each candidate development compound and giving higher priority to development compounds on the basis of whether or not they are likely to result in an undesirable effect based on their involvement in other biological pathways.
In another aspect, a method for identifying disease-related pathways wherein the disease is a side effect of drug therapy is provided. In this embodiment, the method includes the steps of identifying the disease-related pathway affected by a drug or drug discovery target and providing a means for querying the database to identify alternative pathways that are also affected by the drug or the drug discovery target and that result in the undesirable phenotype.
In another aspect of the invention, a method for identifying or validating a genotypic marker for a disease state includes providing a means for querying the database to identify a genotypic marker that is associated with a disease state.
In another aspect of the invention, a method for evaluating user-supplied genomics data is provided. In this embodiment, the steps include (a) defining a profile model based on one or more profile definition criterion; (b) building a collection of profiles according to the profile model; (c) identifying one or more profiles that overlap at least a portion of the user-supplied genomics data and determining, for each such overlapped profile, whether the overlap is statistically significant; and (d) analyzing one or more statistically significant profiles together with the user-supplied genomics data including inspecting database-asserted biological interactions embodied in the one or more statistically significant profiles. The building step may further include building profile libraries containing a plurality of profiles, each one of which being based upon a unique profile model. The profiles may correspond to static profile models, i.e., pre-generated, or dynamic, i.e., created on an as-needed basis by direct queries of the database. In the former case, a separately stored, structured representation of profiles is the primary focus for subsequent analysis, rather than the database or a copy of the database.
Profiles may be generated using one of a data-driven and model-driven approach and each of the profiles may be generated by building a profile about a central genomic data type, e.g., gene, gene product, process. Statistical significance may be measured in other ways, such as the statistical significance of one or more biological associations that appear to correlate with the overlapped profiles.
A fuller description of these embodiments of the invention, as well as other embodiments of the invention, which will become apparent from the following detailed description, follows. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation without limiting the scope of the invention as claimed.



BRIEF DESCRIPTION OF THE FIGURES
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate preferred embodiments of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 illustrates the positioning within an ontology of the finding, “Human Bax protein accelerated the death by apoptosis of rat DRG neurons after infection with Sindbis Virus.”
FIG. 2 illustrates a graphical example of the complex relationships among concepts involved in disease-related pathways.
FIG. 3 is a schematic illustrating a method for analyzing gene microarray expression data according to a method of the invention.
FIG. 4 is Venn Diagram illustrating a conceptual framework for determining whether a set or subset of user supplied gene expression data that is also present in a profile found in a Knowledge Base is statistical significant and therefore potentially related to an underlying biological process of interest.
FIG. 5 is a graphical illustration of the statistical significance (as measured by the probability under a null hypothesis) that one, two, three, four or five user genes overlapping a profile from the Knowledge Base is a random occurrence.



DETAILED DESCRIPTION
Definitions
As used in the description that follows:
“Disease” means any phenotype or phenotypic trait of concern, including by way of example a disease or disease state, a predisposition or susceptibility to a disease, or an abnormal drug response. Illustrative and non-limiting examples of disease states include high cholesterol levels, congestive heart failure, hypertension, diabetes, glucose intolerance, depression, anxiety, infectious disease, toxic states, drug therapy side effects, inefficacy of drug therapy, alcoholism, addiction, etc.
A “disease-related pathway” is a series of biochemical reactions in the body that result in disease, i.e., it is a series, linear or branched, of biological interactions in the body that collectively have an effect on a disease state, e.g., initiation, progression, remission, or exacerbation. Such biological interactions, i.e., biological effects or functional relationships, are the biological processes that occur within the body, e.g., binding, agonizing, antagonizing, inhibiting, activating, modulating, modifying, etc.
“Therapy” and “therapeutic” include prophylaxis and prophylactic and encompass prevention as well as amelioration of symptoms associated with a disease state, inhibition or delay of progression of a disease state and treatment of a disease state.
“Protein” or “gene product” means a peptide, oligopeptide, polypeptide or protein, as translated or as may be modified subsequent to translation. A gene product can also be an RNA molecule.
“Findings” are the data that is used to build an information database. This data may come from public sources, such as databases and scientific publications, but it may also include proprietary data or a mix of proprietary and public data. In preferred embodiments, findings are derived from natural language (e.g., English language) formalized textual content according to methods outlined in greater detail below.
“Biological effect” includes the molecular effects of a given biological concept as well as the effects of such concept at the level of a cell, tissue or organism.
Unless otherwise specified, “include” and “includes” mean including but not limited to and “a” means one or more.
The Database
In a preferred embodiment, information is stored in, and accessed using two databases. The first database is a knowledge base (“KB”) of the scientific findings structured according to predetermined, causal relationships that generally take the form of effector gene (and/or product) ->object gene (and/or product) type relationships (hereinafter the “Findings KB”). The preferred database structure for this Findings KB is a frame-based knowledge representation data model, although other database structures may alternatively be used for structuring the scientific findings. The second database type is an ontology. An ontology is a multiple-hierarchical representation of the taxonomy and formal concepts and relationships relevant to the domain of interest, preferably organized in a frame-based format. The Findings KB and ontology are herein collectively referred to as a knowledge representation system (“KRS”). Other database structures, comprising one or more knowledge bases comprising a KRS, may be employed for representing a body of knowledge when practicing the invention. However, when an ontology is used together with other KBs to form a KRS, or solely as a KRS, the methods of the invention can leverage the taxonomy and formal concepts and relationships defined in an ontology for purposes of inferring conclusions about scientific findings which may not otherwise be readily apparent, especially where findings form part of a complex, or multi-directional series of causal events. Accordingly, provided below is a further description of a preferred ontology that may be used to practice the invention.
With respect to the preferred embodiments, the principal domain of interest is genomic information, which comprises at a minimum information relating to genes, their DNA sequences, mRNA, the proteins that result when the genes are expressed, and one or more biological effects of the expressed proteins but which can include other, related information. It will be clear to the reader that the genomics information can also be information relating to other genomics, proteinomics, metabolic and behavioral information, as well to other biological processes and to biological components other than proteins and genes, such as cells, including, e.g., the biological effects of cells. A preferred ontology structure stores its contents in a frame-based format, which allows searching of the ontology to find relationships between or to make inferences about items stored in the ontology. In this illustrative ontology, the primary organizational grouping is called a class. A class represents a group of things that share similar properties. For example, in the ontology described herein, one class is human cells, which class includes lung cells, skin cells, brains cell and so on. Each of the members of a class is an “instance” of that class, which instances represent single items or elements belonging within the specified class. Thus, an individual blood cell is an instance of the class of human cells.
The relationships between different instances in the ontology are defined by “slots.” Slots can be thought of as the verbs that relate two classes. For example, pancreatic Beta cells have a slot, “produce,” linking them to insulin. A “facet” represents more detailed information about a “slot” and can in some cases restrict the values that a slot can have when related to specific instances of a class. The slots and facets define and structure the taxonomic relationships and partonomic relationships between classes.
When scientific findings are entered into the ontology, each finding is separated into its discrete components, or “concepts.” So, for example, in the finding: “Human Bax protein accelerated the death by apoptosis of rat dorsal root ganglion (“DRG”) neurons after infection with Sindbis Virus,” each of the following bracketed phrases is a concept: [Human Bax protein] [accelerated] the [death] by [apoptosis] of [rat] [DRG neurons] after [infection] with [Sindbis Virus]. The actor concepts are the physical biological components of the pathway that cause or lead to another reaction in the pathway. In the instant example, the actor concepts are Human Bax protein and Sindbis Virus. Actor concepts, each of which is a putative drug discovery target, are likely to be genes or gene products (including, e.g., receptors and enzymes) but can also be, e.g., other DNA sequences (including, e.g., DNA that is not transcribed or that is not transcribed and translated,) RNA (including, e.g., mRNA transcripts,) cells, and bacteria, viruses or other pathogens.
FIG. 1 illustrates how these concepts are structured in a preferred ontology. As illustrated, Human Bax protein is a subclass of protein; apoptosis is a subclass of death, and DRG is a subclass of neuron. This figure also illustrates how the concepts in this simple, illustrative finding are related to each other, making it easier to visualize how each of these concepts can be further linked to other concepts in other findings, at the same level and at higher and lower levels. In a preferred embodiment of the invention, findings are structured to represent causality, thus permitting the discovery of unidirectional sets of findings that are likely to lead, collectively, to a given biological effect.
Clearly, for the ontology to be effective, it is preferable to develop a common set of terms for like things. It is a well-recognized problem in fast moving scientific fields, like genomics, for different terms to be applied by different laboratories to the same genes, proteins or other biological materials, and for terminologies to change over time as conventions develop. Thus, the storing and accessing of genomics information will preferably be organized to ensure semantic consistency. For example, data entry could be limited to a pre-set, or glossary of terms, inclusion of a scientific thesaurus that automatically converts inputted terms into accepted terms, and human review to update the thesaurus or glossary.
Regardless of the subject matter captured and described by the ontology, whether genomics or toxicology, it is necessary to examine closely the body of knowledge that comprises the subject matter so that the knowledge can be organized into the proper classes and linked by the appropriate slots and facets and finally stored in a form that will allow the contents and the relationships contained in the ontology to be properly represented, searched, accessed and maintained.
The selection of sources for the information or “facts” that will be included in the ontology and the methods used to digest those sources so that the facts can be supplied to the ontology in proper form are described in commonly-assigned patent applications: (1) Ser. No. 09/733,495, filed on 8 Dec. 2000 and entitled, “Techniques for Facilitating Information Acquisition and Storage;” and (2) Ser. No. 10/038,197, filed on 9 Nov. 2001, entitled “Method and System for Performing Information Extraction and Quality Control for a Knowledge base, the contents of all of which are incorporated by reference herein for all purposes.
As described more fully in those references and below, scientists who read the articles that comprise a data source for the ontology may abstract the facts contained in those articles by filling in fact templates. An abstracted fact refers to a fact retrieved from an information source that is rewritten (e.g., by using a template) in the computational information language of the ontology. A completed fact template is called an instantiated template. The contents of the instantiated templates are placed in the ontology. The type and format of these fact templates are dictated by the content and structure of the ontology. The information contained in these facts are also stored in the Findings KB, which, as mentioned above, is used to store scientific findings. Although all information in the Findings KB is also contained in the ontology, it is preferred to use the Findings KB when specific findings are later retrieved as this can facilitate computational efficiency for searches of multiple findings where information about the classification of, e.g., the effector and/or object in the finding within the ontology is not needed.
Each type of permitted fact of the ontology can also be associated with a fact template that is created to facilitate the proper entry of the information or data comprising that particular type of fact into the ontology. These fact templates are presented to scientists as they abstract information from the sources. Pull-down menus within the template present the scientist with the appropriate classes, slots and facets for the particular fact type.
The process of abstracting information is called structuring knowledge, as it places knowledge into the structure and architecture of the ontology. The method for structuring the knowledge is based on formalized models of experimental design and biological concepts. These models provide the framework for capturing a considerable portion of the loosely articulated findings typically found in academic literature. The specific level of experimental results that is of greatest value to industrial and academic scientists can be particularly targeted for capture. So, for example, in the field of genomics, knowledge that focuses on the effects that both perturbation to genes, gene products (RNA and proteins) and small molecules and various physical stimuli have upon biological systems is singled out. These perturbations and stimuli form the backbone of the ontology and provide the necessary framework for developing a more sophisticated representation of complex biological information.
Examples of the types of facts and biological relationships that can be translated into the ontology are: a) an increase in the amount of Fadd protein increases apoptosis; b) a decrease in Raf levels increases activation of Rip2; and c) the allele delta32 of CCR5, compared to the wild-type allele, decreases HIV transmission. In a preferred embodiment, biological systems are defined in terms of processes and objects. Discrete objects are physical things such as specific genes, proteins, cells and organisms. Processes are actions that act on those objects. Examples of processes include phosphorylation, which acts on discrete objects such as proteins, and apoptosis, which acts on cells. Perturbation of an object can have an effect on a process or on an object. Using these concepts of objects and processes, the information in the ontology may be represented by a variety of fact types.
As mentioned above, templates are associated with each fact type. In a preferred embodiment, there are five template types used for fact entry into the ontology. The corresponding fact types may be described as observational facts, comparison facts, case control facts, case control modifier facts, or case-control comparison facts. Of course, the structure and variety of fact types depend on the field of knowledge of the ontology, all of which will be known to those skilled in the art.
Examples of each of the aforementioned fact types of a preferred embodiment follow. Observational facts (OFs) are observations about something. An example of an OF is “Tyrosine phosporylation of INRS-1 was observed.” Comparison facts (CFs) compare a property of one thing to a property of another thing. An example of a CF is “The size of a lymphocyte in one organism is greater than the size of a lymphocyte in another organism.” Case control facts (CCFs) describe an alteration in something which causes changes to a property aspect of something. An example of a CCF is “Mouse-derived Brca-1 increased the rate of apoptosis of 293 cells.” Case control comparison facts (CCCFs) compare the effect that something has in a first fact to the effect that something has in a second fact. An example of a CCCF is “Fas increases total apoptosis of 293 cells with Brd4 (introduced by vector transformation) more than it increases total apoptosis of 293 cells without Brd4.” Case control modifier facts (CCPMFs) express an alteration in something that causes changes to a property of a modifier of a process. An example of a CCPMF is “Mouse-derived BRCA-1 increased the rate of the induction of 293 cell apoptosis.”
Despite the restraints imposed by a template pull-down menu system and the template's isomorphic relationship with the ontology structure for each of the above fact types, there may still exist an enormous number of permutations of values for each type. The consequences of an incorrectly instantiated template are potentially serious, as erroneous entries in the ontology would necessitate a quality control process to address the incorrectly entered fact. This process can be expensive and time-consuming. Moreover, for those who are relatively inexperienced in the field of knowledge engineering, it is not always an easy task to recognize subtle differences between a correct and incorrect fact abstraction when facts are represented in the structured language of the ontology. This is especially true when an instantiated template represents a complex fact. To meet this need, natural language fact verification by a scientist may be included as part of knowledge acquisition. In a preferred embodiment, a fact verification scheme includes a natural language display of the fact derived from the template so that a scientist can verify, by reviewing the natural language representation of the structured fact entered into the template, whether the fact entered into the template was the fact as intended.
Alternatively, or additionally, information is extracted automatically by use of a computer to “read” and analyze papers and to extract data therefrom for inclusion in the ontology. In these embodiments, a natural language (e.g., English) source text is first interpreted using computational linguistics to determine, to the extent possible, the precise meaning of the “fact” contained in the natural language source. After this “fact” has been determined, it may be reviewed and then abstracted according to an automated procedure, manual procedure (i.e., human involvement) or a combination of both. Preferably, a combination manual and automated procedure is used to verify that the fact extracted from the source text is both a fact of interest, that it accurately reflects the intended meaning of the source text, and that it is appropriately structured for storage in the ontology. The data sources are not restricted to journal articles. Other data sources include, e.g., public databases, private databases, and proprietary data such as confidential data developed within and confined to a particular laboratory.
With data from multiple sources acquired and stored in the database, such as is described above, it is possible to determine relationships among genes and gene products that previously would have been exceedingly difficult or even impossible to identify because, e.g., of the number of sources from which data are required and the use of inconsistent language (e.g., different names for the same protein are used simultaneously or over time.) So, while it may be possible for one or a small number of individuals to stay abreast of all or most publications relating to a very narrowly defined field, it is impractical to think of scouring public data sources to identify disease pathways that comprise drug discovery targets without the aid of a structured database, such as is described above. Even with respect to particular diseases, genes or gene products, this task can be enormously difficult and time-consuming without the aid of a structured database.
Findings information may come from informal sources, as well as the more formalized documents and publication sources discussed above. For example, findings may be extracted using a network search tool that searches a network and then attempts to extract information contained in pages that seem to be about a biological concept of interest (e.g., a web-crawler that searches over the internet). Alternately, or additionally, a search engine may be used to scan corporate email, discussion groups, PowerPoint presentations, etc., to try to identify and then extract information relating to biological functions. Of course, one should expect a lower quality of results from these sources, both because the data parsing would be automatic, there would likely be higher error rates than manually entered content, and the content sources will more likely be informal or invalidated discussions, rather than peer-reviewed journals and the like.
Findings need not be limited to literature-based private or public information. For example, findings could include findings derived from, e.g., a company's microarray chip experiments. In this case, the array data could be reviewed to try to identify which genes are being co-expressed and/or co-regulated, from which a “A<-->B” relationship could be deduced. These findings could then go into the KB directly or into a graph structure directly. The data may also include findings that scientists enter directly, or could be data straight from experiments (i.e. w/out interpretation by scientists). The findings acquisition process discussed above may also be useful as a tool for publication, in addition to a data extraction or entry process. Much in the way that authors need to include abstracts and indexing keywords when proposing a publication for submission, they might also be required to write down their key conclusions in “findings format”. In this contemplated use, the author or a 3rd party may perform the findings extraction (e.g., as in the way the National Library of Medicine is currently responsible for approving, if not creating, the keywords associated with paper abstracts). KRS technology is not required for creating a structured database. While KRS technology may be preferred as it can simplify certain tasks in the data acquisition and data structuring process, it is also possible to create a KB using existing relational, object or XML database technology.
With an ontology such as described above, it is practical to query the knowledge representation system for actor concepts, e.g., genes and gene products, related to a disease and thereby to construct a disease-related pathway that extends back several steps, and that branches out to identify overlapping disease-related pathways, as described above. Each gene or gene product in the pathway is a candidate drug discovery target because it is at least theoretically possible to treat the disease state by interrupting the disease-related pathway at any point. It will be clear to persons of skill in the art that further validation of such targets may be appropriate prior to incorporating such targets into a drug discovery program. Such further validation, if any, can be done in an number of ways including by correlating the targets with other relevant data, such as differential gene expression data as described below, or by use of animal models, including but not limited to transgenic knockouts. So, with respect to the findings illustrated in FIG. 1, human Bax protein is a candidate drug discovery target because inhibiting the expression of or activity of the protein will potentially avoid acceleration of apoptosis of DRG neurons after infection with Sindbis Virus. FIG. 2 illustrates slightly more complex relationships in disease-related pathways.
In general, the database is queried to identify pathways to a phenotypic trait, e.g., a disease state or a predisposition to a disease state or other phenotypic trait of interest, by constructing a query designed to produce a response, following computational analysis of the database (or ontology), that reveals all concepts that are biologically related to the phenotypic trait state or to a biological component of the body that is already known to be biologically related to the phenotypic trait. The query can also fix the number of steps removed from the phenotypic trait or other biological component. So, with reference to the simple illustration provided in FIG. 1, a query might be, e.,g., “Identify all concepts that are related to apoptosis of DRG neurons.”
The means for storing and accessing genomics information and the means for computational analysis of complex relationships among the stored concepts will typically comprise a computer system, i.e., any type of system that comprises stored, e.g., digitized, data and a means to query the stored data. Such computer system can be a stand alone computer, a multicomponent computer, e.g., one in which the stored data are physically remote from the user interface, networked computers, etc. Any known means for querying the database will also be useful, e.g., software and hardware for electronically searching fields, categories or whole databases.
Thus, in one aspect, the invention comprises a method for identifying a candidate drug discovery target by (a) providing a means for storing and accessing genomics information wherein said means permits computational analysis of complex relationships among the stored concepts; (b) querying the database to identify a disease-related pathway; and (c) identifying the biochemical reactions in the disease-related pathway whereby each of the actor concepts involved in each such reaction is a candidate drug discovery target.
In a preferred embodiment, the candidate drug discovery targets are prioritized based on their function and complexity. For example, gene products that phosphorylate or activate a second gene product may be of special interest, as may gene products that are “simple” in the sense that they are involved in few other pathways and therefore are less likely to produce undesirable physiological effects. On the other hand, “two-hybrid data” might be considered to have a lower likelihood of representing an actual functional or physiological effect because two-hybrid experiments measure only simple protein-protein interactions and therefore provide a relatively impoverished representation of biological function and state. In addition, two-hybrid experiments have a relatively high false positive rate, resulting in noisy data that might further reduce the likelihood of representing true biological function. Similarly, genes that have highly redundant links, i.e., are involved in multiple other pathways, may be deprioritized because as targets their disruption may be expected to disrupt a number of pathways. Similarly, pathways to disease for which there are multiple pathways are also, in general, less preferred.
More preferably, candidate drug targets are also prioritized based on involvement in other pathways that are unlikely to result in unwanted side effects. For example, inhibiting a gene product such that a desirable biochemical pathway is unintentionally inhibited is likely to result in unwanted side effects and should, in certain cases, be avoided. Thus, in an aspect of the invention, the means for storing and accessing the genomics information may be used to predict side effects or non-responsiveness by queries that identify all known pathways linked to a candidate drug discovery target, which would include pathways of genes and gene products that would be undesirably affected by affecting the candidate drug discovery target.
In cases in which there are multiple pathways to a disease, the invention may also be used to identify multiple drug discovery targets leading to development of an adjunctive therapy that may include administering more than one drug, whereby multiple pathways to the same disease are interrupted. In some cases, there may be an existing known drug for one or more of the alternative pathways.
In some cases, it is not clear how a drug undergoing pre-clinical or clinical development is effective in treating a disease because the association between the drug target and the disease is not well-understood. In one aspect of the invention, there is provided a method for determining or validating the mechanism of action of a drug which comprises using the means for storing and accessing genomics information to define the pathway or pathways between the drug target and the disease.
The invention can be used to aid target validation by elucidating other pathways that may be affected by agonism or antagonism of a candidate drug discovery target and by showing alternative pathways that might complement or replace the pathway affected by drug intervention at the point of a given candidate drug discovery target.
In another aspect, this invention comprises a method for identifying diagnostic markers for a given disease. In this aspect, the invention comprises: (a) providing a means for storing and accessing genomics information wherein said means permits computational analysis of complex relationships among the stored concepts and (b) querying the database to identify markers that are associated with the disease. The markers that are associated with the disease are typically genetic markers, i.e., polymorphisms such as repeats, inserts, deletions, SNP's, etc. They can also be protein markers, i.e., proteins that are expressed or not expressed, relatively under expressed or over expressed, post-translationally processed differently or mutated. Other markers are also useful, e.g., antibodies, mRNA, biochemical markers such as enzyme or metabolite levels, etc.
The present invention is also useful in the growing field of pharmacogenomics. For example, in another aspect, the invention provides a method for identifying diagnostic markers specifically for drug response, i.e., unwanted side effects or non-responsiveness. By identifying markers for side-effects or non-responsiveness, a population of patients having a given disease can be stratified into sub-populations based on likelihood of having a serious adverse event or for not responding to a given therapy, for purposes of enrollment in clinical trials or for treatment.
The invention in yet another aspect comprises a method for identifying new uses for known drugs. In this aspect, the invention comprises using the means for storing and accessing genomics information to identify all pathways in which the target of the known drug is involved, additional to the pathway for the disease for which the drug is indicated, and then determining which if any of the additional pathways result in a different disease. In this way, it is possible to identify different diseases, i.e., new uses, for the known drug.
The method of the invention for predicting disease pathways and targets for drug discovery may be enhanced by leveraging the information obtained by querying a database with data obtained from other methods for identifying disease pathways or targets for drug discovery. For example, the method of the invention may include, additionally, the use of differential expression data in conjunction with relationships asserted in the database.
The invention also contemplates use of drug discovery targets for drug discovery. How to use drug discovery targets identified through the use of the invention (optionally following further validation) in drug discovery will be apparent to persons of ordinary skill in the art. A typical means includes screening a diverse library of compounds against the target and using knowledge gained thereby to iteratively design and screen new compounds having greater potency.
Analysis of Microarray Expression Data
The following provides examples of how a KRS may be used in conjunction with user-provided differential gene expression data to analyze, understand or validate candidate drug discovery pathways according to the principles of invention. This detailed description of preferred, exemplary embodiments of the invention, like the preceding description, is intended for illustrative purposes only and is not limiting on the invention. Rather, the limitations of the invention are set forth in the appended claims.
An example of a process flow for analysis of microarray data in accordance with the invention is illustrated in FIG. 3. A knowledge base (KB) (3) including structured scientific findings taken from the research literature (1) and from other sources (22), as discussed earlier, is stored in the Findings KB and are structured according to the ontology (embedded in 3). The ontology and Finding KB, which form the KB, are stored in a KRS, and can be retrieved and manipulated using an KRS Application Program Interface (API) and/or querying language, as discussed above.
FIG. 3 shows the conceptual components of the analysis. The data structures, algorithms, and software components used to perform the analysis may form a stand alone software tool or they may be integrated with an existing platform and/or suite of applications that are used to access information stored in the KRS. The analysis may include two steps. A first step involves a series of computations over a copy of the KB to identify profiles, and a second step that involves scoring these profiles against user provided data. In the following description and in reference to FIG. 3, an example of the analysis uses user-supplied expression array data. A library (7) of profiles is preferably generated according to a user data set, e.g., user-supplied differential gene expression data, but in other embodiments profiles may be pre-generated independently of the user data. The nature of the generated profiles may vary considerably based on the goals of the analysis, as is explained in greater detail below. In an alternative embodiment, a pre-generated “library” of profiles, mapping an entire KB, may be preferred for the sake of performance—pre-generate all of these maps so that retrieving them later will be faster. The user-supplied data may include array data provided from a third party product, e.g., an Affymetrix GeneChip®, online service or proprietary database.
Profile Definition
A “profile” may include information about, and be defined according to concepts such as a particular combination of genes or gene products that appear to act in a biologically coordinated manner, e.g., form all or part of a disease related pathway, cells and/or cellular components, anatomical parts, molecular, cellular or disease processes, and the relationships between them. An overview of a preferred profile generation and profile-to-data scoring algorithm is presented below. However, before turning to this example, it is important to emphasize that a “profile” as used in this discussion refers to a subset of the data contained in the database that is defined according to criterion(s) suited to the researcher's goals. As such, criteria (or a criterion) means any attribute of a profile that is determined, at least in part, by the researcher's needs. This may include criterion defined in terms of one or more biological concepts, the size of the profile (e.g., graph size), or the findings connectivity in the profile. It should therefore be remembered that the examples of profile criteria enumerated below are intended only as exemplary embodiments of profile defining criteria. In general, it is understood and indeed expected that profile defining criteria will vary from one application of the invention to another since a profile structure according to the invention is driven by research goals.
Thus, the effectiveness of one or more profiles in communicating information depends upon the criterion (or criteria) used to define the profile(s), which naturally depends upon the particular scientific goal for which information is being sought. For example, if it is believed that information relating to a particular cellular process would be highly informative of a targeted pathway, then findings relating to this cellular process would be a factor to consider when selecting a profile criterion. In another situation, the source of the findings (e.g., tissue type) or the size of the profile (e.g., the size of a graph structure illustrating the profile) may be effective profile selection criterion.
Profile Generation
As mentioned above, a first step of the analysis generates computational models for biological pathways. These models, referred to as “profiles”, become tools for interrogating and interpreting genomic data sets like microarray expression data. They are constructed from findings in the KB, and consist of sets of gene (product) abstractions, together with their known macromolecular interactions, and various biological processes the KB asserts the genes to play roles in.
The gene abstractions consist of official LocusLink gene symbols to which are mapped known instances of gene and gene products in the KB, potentially from both human and non-human species. The intermolecular interactions consist of specific instances of effector gene (product)→object gene (product) relations; the mapping of gene (product) instances to the more abstract gene symbols thus allows inferred generalized effector gene symbol→object gene symbol relationships (as discussed earlier). Borrowing concepts from graph theory, the available genes and gene interactions can be represented computationally as collections of “nodes” (for genes) connected by directed “edges” (for interactions), with various properties being associated with each node (e.g. gene properties), and various properties associated with each edge (e.g. molecular process types, direction of process changes, number of findings/publications asserting the interaction, etc). In addition, various properties can be associated with the entire profile, including for example, biological processes, the number of genes in the profile, the method of construction, etc.
The ability to associate a rich set of node, edge, and graph properties with profiles provides opportunities to apply a variety of selection criterions on the profiles:

    
    
        Criterions applied during selection of nodes and/or edges can provide diversity in the composition and structure of the profiles produced.
        Criterions applied after profile construction but prior to scoring against expression data can reduce unproductive false ‘hits’ or provide a more focused analysis.
        Criterions applied after profile construction and after expression scoring can provide additional ranking of profiles (by criteria other than expression scoring) for review by researchers.
    
    


Referring again to FIG. 3, step one, profile generation, begins with a dynamic pre-calculation of a master graph (or network) that fits a certain set of criteria. The criteria may be pre-set by the system or defined by the user and may pertain to any category in the database, e.g., genes or gene products, chemicals, protein complexes, protein families, processes, sources of findings, experimental techniques, organism context, or other criteria, e.g., genes that are absent according to the user's data. Then profiles are created from this graph based on further criteria pre-set by the system or defined by the user, eg. genes of particular interest to the user, maximum number of nodes per profile, etc.
Conceptually, each profile is a response to a query against the KB to find networks of findings that meet the criteria. These profiles may be pre-built off of a copy of the KB to optimize performance (producing a library of pre-built profiles), or the profiles may be built directly against the KRS, so as to allow profiles to incorporate recently discovered findings as they are stored in the KB. Profiles could also be built using something of a “bootstrap approach”: an initial set of profiles could be built, then tested for sensitivity in detecting expression changes, and the best profiles could be enlarged (by adding more gene members, by merging profiles, or by otherwise changing the criteria that define the profile model), and the sensitivity test repeated. Eventually profiles that are optimal in detecting gene expression changes (the per gene-member sensitivity measure would be optimal) but not too large could emerge from this process.

    
    
        The profiles are generated by first extracting a subset of the KB findings (4) and then converting findings (4) into a large graph data structure (5). This is essentially a simplified version of the KB that is amenable to high-performance graph data structure operations. Part of this simplification may include converting findings from a literature-based representation, where each finding represents a result from a performed experiment, to a biology-based representation, where each finding represents a conclusion about biology.
        The profile generation algorithm then processes this graph (6) to produce a collection of subnetworks (profiles) (7) that may be analysis-specific, e.g., user-provided array expression data (10) input as parameters (6) to a profile generation algorithm, and that match input criteria. Examples of input criteria are the size of the profile (number of nodes in each profile), whether they are differentially regulated in the user's experiment or otherwise flagged as of interest to the user, the processes involved (e.g., “activation+cleavage” or “phosphorylation”), and/or the source of a finding (e.g., only observed in human liver cells).
        Many such collections can be pre-generated given a profile generation algorithm and a set of parameters. If the profile collections are built upon a copy of the KB, they must be re-built when the KB changes (e.g. when new findings are added) if the profiles are to be up-to-date. The collections may also be dynamically built, i.e., as the KB changes or as new user-provided genomics data becomes available. Either configuration is contemplated and considered within the scope of invention.
    
    


Many profile generation algorithms could be used, such as a gene-centric algorithm. In this embodiment, the algorithm creates one profile for each gene in the KB. Each gene's profile consists of the gene that “anchors” the profile and a set of “nearby” genes that match a certain criteria. By “nearby” it is meant those genes that are most directly related to the anchor (or “seed”) gene through some process, in terms of the number findings linking the gene to the anchor gene. This approach is termed “model-driven” because the profiles are based on a pre-defined algorithmic model. Alternatively, a “data-driven” model may be used, where the profile is not pre-generated but instead is assumed to be the set of genes of interest to the user (eg. genes identified as differentially-regulated based on the user's data or the major genes in the user's field of research) together with their known interactions as revealed by the KB. This essentially takes all the user genes and connects them using findings from the KB.
In a preferred embodiment, a hybrid model and data-driven approach is used. This approach will be described by using the example of a user-supplied set of differentially expressed genes. In this example, profiles are built about the differentially expressed user-genes such that a maximum number of clustered genes supportable by the graph is established among each of the user-genes and, in certain cases, their neighbors in the master graph. By maximizing the connectivity (number of interactions) among the clustered genes, the specificity and biological cohesiveness of the subnetwork is generally increased. A preferred algorithm for accomplishing this task is summarized as follows:

    
    
        1. Based on the user's genes of interest (eg. by applying a expression fold-change magnitude threshold to the user's data), partition the user's genes into two sets: differentially expressed genes, and non-differentially expressed genes. Note that users may use other criteria to select their genes of interest. For example, a user may select genes of interest based upon expression levels derived from a microarray experiment (e.g. by picking a fold-change cutoff, or a p-value significance cutoff); results generated by another gene analysis algorithm (e.g. initial clustering or data cleaning algorithms); or other criteria (e.g. user's favorite genes).
        2. Using the differentially expressed set of genes, (hereinafter “user-genes”), and the master graph, compute a total “triangles” score for each user-gene. A “triangle” consists of the edges (eg. direct physical interactions) that connect the user-gene to two of its neighboring genes as well as an edge connecting those two neighboring genes to each other. Together the 3 genes define a path that has the same topology as a triangle or loop. As such, it identifies a unit of local connectivity that is the maximum possible for the 3 neighboring genes. The total count of loops or triangles a user-gene is one potential metric for its sensitivity to perturbations among its neighboring genes. Other alternative ways of ordering genes of interest, such as by magnitude of fold-change or p-value significance, could be used. Thus, genes could instead be prioritized according to the magnitude of fold change, regardless of the connectivity among other genes.
        3. Sort the user-genes by decreasing total triangle count.
        4. Using the user-gene with the highest triangle count, start a “seed set”.
        5. Iteratively examine each triangle-sorted user-gene in the list by computing a “specificity” score for its relationship to the seed set genes. The specificity score is used to determine which of the user-genes exhibit the highest degree of activity (i.e., connectivity) within the seed set relative to its activity outside of the seed set. A specificity score may be defined as the ratio of the number of interactions between a user-gene and its immediate neighbors in the seed set over the total number of unique seed set genes, neighbors of seed set genes, and the user-gene plus its neighbors: i.e. (A∩B)/(A∪B) where A is the user-gene and its immediate ‘direct’ neighbors, and B is the seed set genes and their ‘direct’ neighbors. The candidate user-gene corresponding to the highest specificity score (i.e., most activity within the seed set relative to the master graph) is added to the seed set. This added user-gene is then removed from the list of candidate user-genes for subnetwork formation. Note that in this example of a specificity score a gene that has more connections within the seed set but a low specificity score is given lower priority to a user-gene having a higher specificity score because genes that are predominately active within the seed set are believed more likely to be of interest to the user.
        6. If a user-gene is added to the seed set, then repeat step 5 until there are no remaining user-genes that have at least one direct connection to the seed set or a maximum profile size for the seed set is reached. Hence, for a subsequent iteration, re-calculate a specificity score for each remaining user-gene not included in the seed set. Then, based upon the revised specificity scores, select the user-gene having the highest specificity score for inclusion in the seed set and remove this gene from the candidates of user-genes for subnetwork formation. If the maximum profile has not been reached, and there are other user-genes that have at least one direct connection to the seed set, then repeat this process.
        7. Repeat, starting from step 4, until all user-genes in the triangle-sorted user-gene list have been either allocated to seed sets, or left over as unincorporated genes, i.e., user-genes that have no direct connections with a seed set. The latter are used to create single-gene seed sets and added to the collection of other seed sets.
        8. Separate the seed sets into two groups: those that are already at target profile size, and those that are smaller than the target size.
        b 9. Using a queue of the smaller seed sets sorted in increasing size, perform pair-wise comparisons among the created seeds sets to identify those pairs that are optimal for merging via a “linker” gene. The linker gene is any gene that according to the master graph has one or more direct connections to both seed sets under consideration. To compute the optimal pairs of seed sets and the optimal linker gene to use, compute an “edge sum” score for each combination of seed sets and linker. The edge sum is the total number of interactions (edges) that exist between the linker gene and genes contained in the two seed sets. The higher the edge sum, the more connectivity will be increased if the two seed sets are merged. Combine the best mergeable combination of seed sets and linker gene, and create a new merged seed set. If the newly merged seed set is large enough, set aside with the other full-size seed sets. If it is still too small, add it to the queue for potential merging with additional seed sets.
        10. Repeat step 9 until no more seed sets can be merged, or all merged sets are large enough to meet the maximum profile size.
        11. Convert each assembled seed set (large and small) into a subnetwork by recruiting additional connected genes and gene interactions from the master network in such a way as to maximize the biological focus. To do this, detailed criteria are applied in the selection of genes from among the surrounding candidates in the network neighborhood, including: a) sufficient connections to the existing members of the seed set (e.g. requiring 2 or more connections to the seed genes in cases where there are multiple seed genes); b) a high specificity for interactions with the seed genes, which is expressed as the ratio of the number of seed gene interactions over the total number of gene interactions it has with all KB genes; and c) a high level of differential regulation (the sum of the magnitudes of dysregulation values of the gene and its neighbors divided by the number of corresponding nodes with such values). In the preferred embodiment, criterion a) is used as a minimum acceptance criterion, and criterion b) and c) are given equal weight in picking the best new network gene to add to the profile subnetwork. After all member genes have been selected for the subnetwork, all known direct interactions among all the member genes, together with supporting findings, citations, molecular processes, etc. are assembled from the master graph and a subnetwork (profile) is created.
        12. Store each subnetwork in a library for processing and scoring against user data.
    
    


The above algorithm is one possible algorithmic approach for generating a library of profiles from biological information known to the KB. In this approach, the profiles contain biological information which is believed to be most closely related to the user-supplied biological data. It is understood that various modifications to the above algorithm, or additional algorithms, may be employed to arrive at a library of analysis-specific profiles without departing from the scope of invention.
As noted above, the profiles may alternatively may be pre-generated independently of the user-data. In this approach, called a “model-driven” approach, profiles are built based upon a pre-defined algorithmic model independent of user-supplied biological data. In this embodiment, the algorithm creates one profile for each gene in the KB. Each gene's profile consists of the gene that “anchors” the profile and a set of “nearby” genes that match a certain criteria. By “nearby” it is meant those genes that are most directly related to the anchor (or “seed”) gene through some process, in terms of the number findings linking the gene to the anchor gene. A more detailed discussion of this alternative approach for profile generation is discussed below.
Profile Scoring
Step two of the analysis, Profile Scoring (12), is the process of computing a P-value that ranks a profile (9) against the user-supplied data, e.g., gene expression data (10). In a particular application, there may be many profile libraries generated, each of which contains profiles matching the user or system specified criteria. Profile Scoring described herein will work for any of these libraries. In one embodiment, the algorithm makes two simplifying assumptions.

    
    
        1. The expression array data is converted into a list of dysregulated genes (11) (i.e., abnormally up or down regulated) by selecting only those genes that show an N-fold or greater difference in regulation (in one embodiment, N=2 or greater). This is a common initial simplification for expression analysis. However, in other embodiments a more sophisticated continuous distribution approach that uses the full distribution of expression values over all the genes in the experiment rather than a cutoff threshold may be used.
        2. For the purposes of scoring, profiles are considered to simply be a particular set of genes from the KB, e.g., the aforementioned Findings KB. In particular, the relationships between these genes are not used for scoring purposes, only for the purpose of generating the profile and subsequently for display and annotating it during results creation (see below). Scoring algorithms may also take gene connections into account as well, leveraging directionality in the gene connections and/or the molecular process nature of the connections to score the “fit” between the profile and expression data set.
    
    


Several other embodiments of the invention are contemplated. In one embodiment, a continuous measure of dysregulation is used, rather than an expression level cutoff when comparing microarray data to profiles.
In another embodiment, one may develop an aggregate scoring metric that includes graph-theoretic metrics, either as a compound score or a coarser ranking for profiles that match based on the existing score. For example, for N profiles that score equally well using a first metric, rank them further based on, e.g., graph connectivity metrics under the assumption that the more connected the genes, more likely they are working together.
In another embodiment, the system could allow user annotation to indicate (hypothesized) dependencies within the expression dataset. Specifically, if users have a priori knowledge about dependencies between the genes in their experiment, allow this to be included (e.g. as edge annotations, additions of new edges, or removal of edges whose evidence is hypothesized to be weak) in the set of genes to be analyzed. This feature, which is preferred, would require that the analysis gene sets have edge drawings (if it is desirable to display this information in graph form) which use the same semantics of directness as those underlying the profile edges, i.e., a data-driven profile can be constructed from user-supplied information. Alternatively, forms may be provided to input edges and tables provided for visual output for the edges. Thus, in addition to findings from the literature, users can add their own findings, or modify existing ones by, e.g., specifying a confidence measure. These user findings could be modifications to the KB itself (add custom findings to the KB, which are then converted to the graph (5) format) or to the graph itself (convert KB→graph as usual, but then modify graph (5)). Updates to the KB may use templates to enter these new findings, as discussed above. If these findings are added to the graph, then templates customized for graph edits may be used. This resulting data or model driven profile (or profiles, if there is more than one hypothesized dependency for a gene set) may then be used to further rank existing profiles by, e.g., doing an isomorphism comparison with model-based profiles. Thus, in this embodiment, data- or model-driven profiles are ranked against both the prior knowledge asserted in the KRS and the user's personal knowledge assumptions about the data.

    
    
        Both of the above approaches (or any other graph theoretic) could be refined by increasing the semantics of nodes, edges, etc and refining the corresponding isomorphism algorithm to reflect the particular semantics of nodes. For example, an edge “type”comparison in the isomorphism calculation.
        Expand the ranking notion to explain all dysregulated genes in the expression dataset rather than only those genes that are mapable. For example, if one only can map 10% of all dysregulated genes, score all profiles lower under the assumption that none of them will do a particularly good job covering the biology of the full set of dysregulated genes. A similar profile weight could be calculated by comparing the ratio of mappable genes in the entire expression dataset against the set of genes covered by the KRS in order to estimate the relative coverage of the KRS against a given expression dataset.
        Given models of chains of reactions that may underlie the observed gene expression, one can determine which models best fit the data. One method to compute this is to permute the user-supplied dysregulated gene expression values thousands of times and estimate the P-value based on the proportion of randomized data trials that score as well or better than the observed data (ie. Monte Carlo simulations). These mechanistic models (pathways) can either be pre-specified by users or generated automatically by searching over the knowledge in the KB to find biologically plausible paths between causative events (eg. binding of a ligand to its receptor) and biologically relevant effects (eg. transcriptional activation of a gene). The highest scoring models are the ones most likely to explain the data given the computationally-available information and provide users with actionable hypotheses.
        Take into account the context of a user's experiment to adjust relevant content in the computation (eg. what type of cell line did they use, whether they know that certain genes are knocked out or transfected in, etc.). This would allow one to score profiles based on how well they matched up against this background knowledge about the experiment.
        Take into account medium-throughput data to refine expectations of what is ‘normal’ for different cells, what proteins potentially can interact, etc. This would provide a normalized baseline across various biological contexts and refine the sensitivity with which one can distinguish statistically significant results.
    
    


Results from the analysis may be presented to the user in various forms. In one embodiment, three types are presented:

    
    
        1. The first is a list of profiles ranked according to a profile score (14), generated by calculating the P-value for each profile (13) in the library and sorting the resulting list. Each profile lists the gene central to the profile, and any genes from the expression dataset that also appear in the profile. Users can view this list and pick profiles that appear to be interesting to look at them in greater detail. This output may be viewed using a spreadsheet program.
        2. The second is one or more profile diagrams (17) for each of the profiles. These diagrams show all the genes from the profile and the key relationships between them in the form of a “circles and arrows” diagram. Different symbols, colors, labels, and positions are used to encode additional information about the profile which is extracted (16) from the KB. Different diagrammatic representations may be used to display the same underlying profile but highlighting different characteristics. For example, one diagram may use a layout algorithm that highlights the subcellular localization of the gene product by grouping symbols together if they share the same localization. Another diagram may use a layout algorithm that highlights the interrelationships between gene products by grouping symbols together if they share many interactions. An example of such information is the subcellular localization of the gene product (information that can be stored in the KB but is not used for profile generation or profile scoring in a preferred embodiment). The diagram itself may be generated (15) using an open source/freely-available 3rd party diagramming tool from AT&T Research called GraphViz. The output may be a printout of a diagram or a web-accessible graphic (image file or Scalable Vector Graphics—SVG-file).
        3. The third is algorithmic association of biological processes with pathway profiles (18). This step involves generating a description or summary of the biology manifested by a given profile by performing algorithmic analyses of the findings relating the genes in the profile. Conceptually this is analogous to automatically generating a set of labels or captions (18) that describe the molecular, cellular, organismal and/or disease processes that best represent the function(s) of the genes in this profile. For example, while many cellular processes may be involved in the various genes in the profile, “apoptosis” may stand out as statistically significant among them. Inferred processes can be derived from the findings to collect findings that support the involvement of genes in more general processes using the ontology hierarchy. For instance, some genes may ‘increase apoptosis’ and others may affect ‘apoptosis of T cells’, yet all of those genes can be inferred to be involved in ‘apoptosis.’ This aspect of results creation is particularly powerful since it leverages the unique structure of an ontology. These process annotations—e.g., the most representative or highest scoring ones—may appear on the diagram itself, or may be supported by a more complete list on a separate page, or via a web display that supports iterative “drill down” to reveal additional details. The output may be a text printout, but may also be presented to the user in a GUI interactive form. Specifically, the findings in the KB structure information about processes such that, for example, the process, the location(s) in which the process occurs, when the process occurs, the molecule(s) that initiated or affected the process, and the objects acted on by the process are distinguished from each other. The association between molecules and the processes they are involved in can be constructed by first building a graph (tree) of processes, starting with nodes that represent the detailed processes (e.g., ‘increases arrest in G2 phase of fibroblasts’), and then deriving (more general) parent processes by successively removing (e.g.., ‘increases’) or generalizing (e.g., ‘fibroblasts’ are ‘cells’ based on relationships in the ontology) details of those process. These generalized processes are not necessarily stated explicitly in any findings, but instead are inferred from the specific processes that are stated explicitly in the findings and inference rules based on relationships in the ontology. Thus, the presence of an ontology allows a single stated set of relationships (the finding) to imply a much larger set of relationships that can still be used for computation and display to users. After the process tree is constructed, genes and the findings that support the detailed processes are also relevant to their parent processes so they are inferred up the process tree. Therefore, very general processes at the top of the tree (e.g., ‘apoptosis’) may be associated with all the genes and findings for all their more-specific child processes (e.g., apoptosis of specific cells, directions of effect on apoptosis, etc.). Thus, the process tree aggregates information at different levels of detail, from specific to general, and the molecules associated with each process annotation are compared to the molecules in each profile to score them.
    
    


The results output may be delivered to the user online as part of an integrated site that makes available all related KB applications. This is advantageous because every piece of information generated in all of the outputs is based on concepts and findings stored in the KB, which can also be made available to clients located on a network (e.g., the internet) for purposes of interrogating the KB for more detailed information related to the profile summaries. Thus, embodiments of the invention can be tightly integrated with supporting content, for example by allowing “click-thru” and “drill-down” functionality to take users from the high-level profile summaries to the detailed supporting evidence. One example of such a network adapted for this use is Ingenuity's LifeSciences web site where users may click on a node representing a gene to take the user to a “GeneView” page for that gene.
Other types of results may be provided to the user:

    
    
        Annotation of profiles with drug target information by visually highlighting those genes that are known drug targets (i.e. for which a targeting molecule has been found or created) or for which there is evidence that suggests that they may be good drug targets based on e.g. gene family membership. Drug target information may be integrated into the results by simply highlighting the genes on a profile diagram, or drug target information could be taken into account when scoring the profiles.
        Similar annotations and scoring modifications could be based on unwanted side effects for the drug, tissue specificity (e.g. increasing the score of profiles where most of the genes are known to be overexpressed in the tissue in which the experiment was performed), or IP (e.g. scoring profiles based on the number of patented genes in the profile).
    
    


Alternative Pathway Profile Generation Algorithms
In the previously discussed preferred embodiment, a hybrid model and data driven approach is used which determines the nature of the constructed profiles based, at least in part, on a user-prescribed set of data, e.g., gene expression data. Profiles may alternatively be constructed using a purely model-driven approach. This approach may be regarded as “gene centric” in nature: A pathway profile is constructed around each of the gene symbols in the KB, using each as a “seed” gene, and including other genes with which it is known in the KB to interact. In this way, the profiles come to represent the “interaction neighborhood” or “sphere of influence” of the seed gene. Profiles may alternatively be constructed using non-gene concepts as the “seeds”. For example, a cellular process like Apoptosis could be selected as a seed, and then all or some subset of the genes the KB implicates in Apoptosis could be added to the profile, together with their known inter-molecular interactions (as edges). But regardless of the nature of the “seed” in the profile, the rationale behind profile construction about a “seed” is that if a particular profile can be significantly correlated with a genomic data set (e.g. expression data 10), then the “seed” becomes the focus of interpretation.
Beyond the “seed” node and edges connecting the seed to other nodes, profiles may be constructed in a myriad of ways. All of these approaches attempt to deal with the following concerns: The complete set of macromolecular interactions represented by a KRS will usually be too large and too diverse to be compared in its entirety with a genomics data set. Hence, an algorithm is needed to “carve up” this large “macromolecular interaction space” into numerous practical-sized interaction neighborhoods to support a finer-grained probing of genomic data sets. This carving up should be done with considerable gene overlap among the different profiles to minimize the chance that a rare combination of genes might be missed. On the one hand, profiles should be modest in size so that the set of biological functions that might be ascribed to the profile are not too diverse or heterogeneous. Smaller size profiles also aid in human review and interpretation. On the other hand, profiles should be sufficiently large (i.e., they should include, e.g., a sufficient number of genes) so that there will be enough statistical power when computing correlations with genomic data sets and/or with biological associations, such as molecular, cellular, organismal, and/or disease processes defined in the KB (as discussed below). Another concern is that a profile should be relatively symmetrical in the collection of genes connected to the central “seed” gene. In other words, a highly interconnected “1st tier” gene (i.e., a gene connected directly to the seed) should not swamp the profile with 2nd-tier genes (i.e., genes one step removed from the seed) because this can change the seed-gene-centricity of the profile.
One example of an alternative algorithm developed to address the above goals is referred to as a “spiral” algorithm. In this algorithm, profiles are generated from a fully-extended master graph (5) of all known interactions. Graph (5) is constructed from a complete set of the pair-wise macromolecular interactions held in the KB, and will naturally differ in density (i.e., connectedness among nodes) in different parts of it. For each gene or gene product concept represented by a node in the master graph:

    1) Designate the gene or its product as the “seed” node.
    2) Add all immediate neighbor nodes (genes known to participate in interactions with the seed gene) as long as the number of findings supporting the claim that the seed and the neighbor interact is greater than 1, or stop adding if the maximum number of nodes has been reached. The elimination of interactions based on only a single finding is thought to weed out unconfirmed or weakly-substantiated findings. These are the 1st tier nodes and the connections from the seed to the nodes are 1st tier edges.
    3) For each 1st tier node, compile a list of nodes and edges (besides the seed) that are neighbors of the 1st tier node, as long as the number of findings supporting the interactions is 4 or more. This increases the stringency for scientific confidence in the interactions, which as explained above is consistent with assumptions about a decrease in the degree of influence of one gene over another when there are intervening genes between them. These additional nodes and edges are considered “2nd tier” candidates.
    4) Sort the 2nd tier candidate edges by decreasing findings counts.
    5) After all 2nd tier edge candidates have been enumerated and sorted by the findings count, begin adding 2nd tier candidates to the profile in a round-robin fashion, picking one 2nd tier edge candidate for each of the 1st tier nodes by selecting the 2nd tier edge with the highest number of findings.
    6) Repeat the round-robin edge addition in step 5) until either the number of 2nd tier edge candidates is exhausted, or the maximum number of nodes for the profile has been reached. This results in a profile based on edges with the largest number of scientific findings substantiating the interactions.


The above “spiral” approach (essentially a breadth-first search of available nodes) aims to enlarge the profile in a symmetrical fashion. Second tier edges are added from 1st tier nodes with equal opportunity (but preferentially those with more findings counts), reducing the chance that a highly-connected 1st tier node (with lots of 2nd tier edges) will swamp the profile with its connections. Thus, the sphere of influence surrounding the seed gene is optimally represented. Additional profile assembly algorithms may also be used.
The above algorithm, when applied to each gene or product in the KB, results in a profile library where a model of each gene's sphere of influence is collected. Profile Libraries may be constructed which use specific edge types/molecular process criterions [these criteria can be more general: can be based on cellular process types, disease states, etc] (e.g. binding only, functional interactions only, or all types) when selecting from available edges. Then, when analyzing a genomic data set (e.g. expression data set), each and every model in the profile library (or libraries) may be used to interrogate the data set, and the corresponding fit between the model and the data set is computed. This approach is referred to as “model-driven”. As mentioned above, a fundamentally different, “data-driven” approach to profile construction may also be performed. In this case, the nodes from which the profiles are built consist of only those genes (or products) that are observed to be altered (e.g. dysregulated) in a genomic data set. When performed with data obtained from a time-series, interesting “spreading activation”patterns of profile enlargement can be seen.
Uses of the assembled profiles have focused on interrogating and interpreting large scale genomic data sets where the profiles are treated as static models. Additional uses of the profiles are also possible. For example, the pathway profiles could be fed to simulation software that could allow the dynamic behavior of the interacting genes to be explored. The process nature and directionalities (increases/decreases) of the inter-molecular interactions can be used to track “what if” scenarios regarding the changes (abundance) in one or more genes in the profile and the consequences of that change on the other members of the profile. Boolean networks and Petri nets offer some technologies that might be used in such simulations. Another example of how the pathways could be used is in the generation of testable hypotheses. Computational systems could be devised to generate experimentally verifiable predictions about the molecular interactions, and perhaps even report on reagents available (e.g. mouse knockouts in some of the profile's genes) and additional information for performing the experiments. There could also be computational support for the revision/fine-tuning of the profile models to reflect new knowledge obtained from those experimental verifications.
Pathway Profile Graphics & Biological Annotations
To facilitate understanding the gene composition, connectivity, and dynamics of pathway profiles, and how they overlapped with expression data patterns, a system according to the invention may be constructed to automatically annotate profiles with biological associations and render the profiles as interactive graphics.
Biological annotations consist of biological processes thought to be emergent properties of the set of interacting genes in the profile. These biological processes correspond to concepts defined in the Knowledge Base (KB), and can span different levels of biological abstraction/granularity:

    
    
        Molecular processes, involving a macromolecule acting on another macromolecule
        Cellular processes, involving a change in the state of cells
        Organismal processes, involving a change in the state of an organism or organismal component
        Disease processes, involving an abnormal change in the state of an organism or organismal component
    
    


The linked biological processes are those determined to be shared among a statistically significant fraction of the genes in a pathway profile. A “P-value” significance measure may be computed for each profile—biological process association to provide a means to rank different associations, and to flag particular associations as outstanding. The ranked list of biological associations can be presented to a user, together with lists of specific genes linked to those biological processes. In this way, a user is provided with biological “readouts” of a profile, which can aid in assessing the fit of the profile to the known biology of a tissue sample, or alternatively, reveal new insights about the biology underlying an uncharacterized tissue sample. In one embodiment, annotations are limited to biological process concepts; however, other embodiments of the system could leverage additional types of concepts in a KB (e.g. cell types, specific organs, increases/decreases in processes, and other combinations of biological concepts) to compute statistically significant associations for pathway profiles. Moreover, the system may be extended, or easily modified to include additional kinds of statistical analyses. A preferred algorithm for enumerating and statistically ranking the potential biological processes linked to a genomic data set is described below. Biological annotations of pathway profiles can occur either before or after the scoring of profiles against expression data. In the former case, the biological annotations can be used in pre-filtering the set of profiles based on biological criteria. In the latter case, the biological annotations can be used in ranking the various scored profiles according to biological weightings.
Graphical rendering of profiles aims to convert the extensively integrated information of a pathway profile into something that is quickly interpretable by a user. For example, genes (or gene products) in the profile may be rendered as nodes, and inter-molecular interactions are rendered as lines connecting the nodes. In both cases, labels accompany the renderings (nodes are labeled internally with gene symbols, and edges are labeled with molecular process abbreviations). The central “seed” gene may be graphically distinguished from other nodes (e.g., by using an octagon shape), and the protein structural class of each gene product may be conveyed by a unique node shape. The overlap detected between the expression data set and the genes in the profile may also be conveyed in the graphic as follows: dysregulated genes are labeled with their fold change (a + or − floating point value), and colored such that down-regulated genes are red, up-regulated genes are green, and the intensity of color parallels the magnitude of the dysregulation. Interactions between dysregulated genes may be highlighted visually by color and/or line thickness and/or line density and/or labeling of the line. All intermolecular interactions are preferably labeled with a series of single-letter abbreviations indicating interaction types, such as activation, deactivation, binding, transcriptional effects, modifications, cleavage, etc. This use of single-level abbreviations allows multiple processes to be summarized without creating over-crowded labeling. Lines connecting gene (or product) nodes may take the form of arrows, so that an “effector” gene is connected at the ‘tail’ end of the arrow, and “object” genes are connected at the ‘head’ end of the arrows. When reciprocal interactions exist between two genes (gene products), two arrows of reciprocal direction may be drawn between the gene nodes. Subcellular localization of the gene products may be conveyed by placing the gene nodes into labeled boxes corresponding to each of 5 main locations (nucleus, cytosol, cell surface, cell periphery, and unknown). The arrangement of the subcellular location boxes may or may not follow the convention of nucleus at the bottom, cell periphery and cell surface at the top, and cytosol and unknown in the middle of the graphic. Information about known or suspected drug targets are conveyed graphically using gene nodes that are highlighted in color and/or shape and/or labeling.
Using a ranked list of biological processes compiled as in the examples provided above, the top 3 or so most statistically significant biological processes may also be rendered as features on the profile graphic displays. For example, a biological process graphical feature may include a box containing the name of the biological process, the number of genes from the profile that are implicated in the process, and a computed P-value reflecting the statistical significance of the association with the member genes. In addition, the biological process box may be connected by dotted lines (to distinguish from intermolecular interaction lines which are solid) to those nodes depicting genes in the profile which the KB asserts are implicated in the biological process. Using an interactive GUI display of the profiles, the user may have an option to dynamically control the types and amounts of information conveyed. In addition, elements in the graphic profile display can be hyperlinked to detailed views into the KB for the concepts to which those elements correspond (e.g. a GUI summarizing all available knowledge about a particular gene).
The combination of extensive knowledge integration (connectivity, directionality, interaction types) within profiles, computed biological annotations, computer-generated graphical displays of the profiles, and superimposition of known pharmacological targets result in a system that can support rational strategies for drug target selection. The knowledge of connectivity and directionality of interactions among member genes in a profile can reveal the potential for information flow through the set of genes. The integrated knowledge regarding protein structural classes (drug target opportunities) as well as prior known drug targets (e.g., IP obstacles) can help in the selection of appropriate drug target candidates. The biological process annotations and connections to genes can help in predicting the biological consequences of modulating specific genes in the profiles. Taken together, topological knowledge, target candidacy, and biological consequences can support the selection and evaluation of novel pharmacological intervention strategies.
Algorithm for Computing Statistically Significant Biological Process Associations for Pathway Profiles
The goal is to reveal biological phenomena from the KB that is associated with the collection of genes in profiles in a statistically significant fashion. Although the 20 or 40 genes in a profile are each likely to be associated with many biological processes, the ones of most interest are those that are shared by many of the genes in the profile. To be statistically significant, the shared biological associations should occur at a frequency that is higher than that expected by chance alone. Not only do we want to find these significant associations, we would like a measure of the significance of the association. This statistical measure of significance is called a “P-value”. It is a probability measure (with values in the range of 0 to 1) that indicates the likelihood that the observed biological associations are simply due to chance. The lower the P-value, especially when below 0.05 (i.e. more than 95% confidence), the less likely the associations can be explained as mere chance events.
Let's assume that Profile X has 20 genes, and of those 20 genes 12 are known (from the KB) to be associated with the cellular process “migration”. The question to be answered is: could the 12 out of 20 genes linked to “migration” be explained as simply reflecting the frequency of “migration” cellular processes among the set of genes in the entire KB, or is this concentration of “migration” genes unusual. To answer this question, you need to know the probability (p) that any randomly-selected gene in the KB will be associated with “migration”. This probability can be determined by computing the distribution of KB genes across the various cell processes represented in the KB. This distribution may then be made available for quick access by the analysis software by storing the information in a database. In the case of the information available in the KB of a preferred embodiment, it was found that 386 genes are linked to the cellular process of “migration” out of a total of 10,500 genes in this KB. This means the probability that any randomly selected gene will be a “migration” gene is 386 ÷10,500 or 0.0368. The probability of 12 out of 20 randomly selected genes being linked to “migration” may be computed using the Binomial Distribution:




  
    
      
        
          
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From the example above,p would be 0.0368. From (1), and p=0.0368, we can calculate the probability that 12 out of a random selection of 20 genes would be linked to “migration” as:




  
    
      
        
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It is important to note that this computes the probability of exactly 12 genes out of 20 being linked to “migration”. In judging the significance of this, we are interested in the cumulative probability of 12 “or more” genes out of 20. This is computed from (1) by summing the binomial probabilities:




  
    
      
        
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For the “migration” cellular process, this gives the cumulative probability that any observation of 12 or more genes out of a profile of 20 occuring by chance of: 1.9e-12. This is the P-value, and in this case gives 1 in 1.0e12 chance that the results are due to chance.
This test is commonly referred to as the “Fischer Sign Test”, and in the preferred embodiment is automatically performed on a profile for any of the cellular, organismal, and disease associations linked to the genes in the KB.
For display purposes, the preferred embodiment only displays the more specific annotation(s) when both a more general (parent) and more specific (child in process annotation tree hierarchy) have the same overlapping profile genes. The more general annotations in these cases always have a p-value at best as significant a child annotation p-value since the genes associated with a more general annotation are always a superset of the genes associated with its more specific child annotations in the process tree. This display pruning eliminates less informative processes that share genes and findings with the remaining more specific annotations.
Scoring Statistics for Profiles
An example of an expression scoring statistical analysis based on profiles generated from the KB is presented next. The following, generalized assumptions were made concerning this statistical analysis:

    
    
        1. The knowledge base contains one or more findings about each of zero or more (KB) distinct genes.
        2. Each of the generated profiles is a set of (BCP=Biologically Coordinated Pathway) genes from the KB
        3. The user assays a set of genes (USR distinct genes).
        4. The genes that the user assayed that map to the genes (MAP) is in the range [0,KB].
        5. Genes that the user assays may be dysregulated (DYS), in the range [0,USR].
        6. The significant genes are the ones that are dysregulated and mapped to the genes (SIG) which is in the range [0,MAP]
        7. Some of the SIG genes may also be genes in a particular profile. For a particular BCP, this overlap (OVP) is in the range [0,min(BCP,SIG)]
    
    


FIG. 4 illustrates the relationships among the above sets in the form of a Venn diagram. The statistical approach described herein is concerned with determining whether an overlap (OVP) of some subset of a BCP with the SIG is statistically significant based upon the probability that OVP is a random event. Two possible approaches for determining this probability of randomness are presented.
Approach 1: Exact Probability of Overlap
The initial approach calculates the exact probability of observing an overlap of size OVP given a fixed KB, MAP, BCP, and SIG. It computes what would be expected if the algorithm that generated the profiles randomly picked sets of BCP genes from the set of KB total genes (i.e., ignored all information we have about how the genes are related to each other, and blindly picked every combination of BCP total genes) and if the dysregulated genes in the assay are also random (i.e., every assayed gene has an equal probability of being dysregulated). The purpose of this statistic is to indicate how likely it is to observe the overlap if everything, both the matched profile and assay results, were completely random. So the closer the computed value is to one (100%), the more likely the overlap occurred by chance, and the closer the value is to zero, the better since ‘random chance’ as an explanation of how the overlap occurred (the null hypothesis) becomes less likely.
Note that whether there are 0 or 10,000s of USR genes that are not mapped (represented by the light green area in the USR gene box of FIG. 4) does not matter, since we have no knowledge about them; they are not in the universe of KB genes from which the profiles are picked. Likewise, only the genes that the user considers significantly dysregulated (DYS) that are mapped to KB genes matter, since if they're not mapped, we have nothing to say about them. However, the proportion of mapped dysregulated genes (SIG) does matter since we're also computing the likelihood that the particular dysregulated genes that overlap a particular profile happened to be blips—ie. not biologically coordinated. For the null hypothesis to be true, every combination of SIG genes that could be picked from the total MAP genes is equally likely. Treating the assay results as random makes the probability more robust since it does not assume the user's data is noiseless (averaging many repetitions of the experiment reduces noise, but often only a single microarray experiment is done for each condition/timepoint, resulting in significant undetected noise in the results) or that the genes that the user considers ‘dysregulated’ are actually biologically coordinated.
The formula for computing this exact overlap probability under the null hypothesis that both the profiles and dysregulated genes are random is:




  
    
      
        
          
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(or alternatively, “Choose (N,K)”) refers to the mathematical operation (1/K!)*(N!/(N−K)!) for N, K integers. The first quotient enclosed within square brackets in Eq. 5 computes the fraction of the different profiles containing BCP total genes randomly picked from the KB total possible genes that also include the overlap OVP genes. The Choose (N,K) function computes how many distinct ways K items can be chosen from N total items (note that it evaluates to 1 if K=0 or K=N) without replacement (i.e., each item can only be chosen once—since the profiles are sets, the same gene appears at most once in each profile).

To visualize this, look at the conceptual framework diagram of FIG. 4. Imagine moving the BCP box (vertical lines representing one profile) around in the KB box (clear box, actually it is the constant proportion of the BCP box in the KB box that is relevant, not that it is a box). Each different location of the BCP box would be a different profile that could be randomly picked. Choose(KB,BCP) computes how many possible distinct combinations of BCP genes are possible. However, the OVP genes are fixed, so only some of all the possible random profiles would also contain the OVP genes. That's what the numerator calculates—how many different profiles consisting of BCP total genes that include the specific OVP genes could be picked randomly from all KB total genes.
The second quotient enclosed within square brackets in Eq. 5 computes the probability that the overlapping gene(s) are dysregulated but occurred by chance in the user's data. Suppose that only one mapped dysregulated gene (SIG) existed in a particular experiment, out of 1000 mapped user genes (MAP). The probability of P(OVP=1) would be 1/1000, since for an overlap of one, the overlap gene would have to be the single mapped dysregulated gene (ie. Choose (SIG=1,OVP=1)=1). However, there are 1000 ways a different single mapped gene could be chosen (ie. one way for each of the 1000 MAP genes). So there is a 1/1000 chance in this case that a single randomly chosen gene is dysregulated (SIG) and in the overlap (OVP).
Note that (Choose (SIG,OVP)/Choose (MAP,OVP))=((Choose (MAP−OVP,SIG−OVP)*Choose (OVP,OVP))/Choose (MAP,SIG)) in Eq. 5; the former was used above for simplicity, and the quotient appearing in the first square brackets can be rewritten equivalently. Both parts of the formula for P(OVP) assume a fixed set of overlap genes.
Multiplying the first and second bracketed quotients in Eq. 5 computes P(OVP), the probability that a given set of overlap genes would occur in randomly-chosen profiles (each containing BCP genes) and that the overlap genes happened to be randomly ‘dysregulated’ genes—the null hypothesis. For reference, the Eq. 5 simplifies to:

P(OVP)=(SIG!*BCP!*(KB−OVP)!*(MAP−OVP)!)/((SIG−OVP)!*(BCP−OVP)!*KB!*MAP!)

Some implications to keep in mind:

    
    
        1. For a fixed number of KB genes and a fixed number of SIG genes:
        
            a. The larger the profile (>BCP), the MORE likely the match occurred by chance
            b. The larger the overlap (>OVP), the LESS likely the match occurred by chance
        
        
        2. For a fixed number of OVP genes and a fixed number of BCP genes:
        
            a. The more dysregulated mapped genes (>SIG), the MORE likely the match occurred by chance
            b. The more genes we know about (>KB), the LESS likely the match occurred by chance
        
        
        3. If BCP=KB, then if OVP is non-zero, P(OVP)=1 (ie. 100%)
        4. If SIG=KB, then if OVP is non-zero, P(OVP)=1, since this means that every gene in the KB is a dysregulated user gene, so OVP=BCP for every possible profile
        5. If MAP<KB, then P(OVP) in general is greater (ie. more likely to be random) than if MAP=KB
    
    


In order of effect, the following parameters minimize P(OVP) the most (ie. reduce the chances the observed outcomes are random):

    
    
        1. KB>>BCP (ie. Profiles only contain a small subset of all genes in the KB)
        2. OVP>>1 (ie. The more dysregulated user genes that overlap the profile, the less likely it is to occur by chance)
        3. MAP=KB (ie. All user genes are mapped to genes in the KB)
        4. BCP=OVP (ie. Every gene in a profile is a dysregulated gene)
        5. SIG=OVP (ie. All the mapped dysregulated genes overlap the profile)
    
    


FIG. 5 is a graph which shows the dominant effect (#2 above—the greater the number of dysregulated user genes that overlap a profile, the less probable the overlap is to occur by random chance. Note that the y-axis is on a log scale, so each additional overlapping gene decreases the probability by several orders of magnitude. Note also that this effect is still dramatic even for larger profiles (ie. where the percentage of genes in the overlap as a fraction of total genes in the BCP is smaller). In this example, the values 7000 KB genes, 1500 MAP genes, and 70 SIG genes were used.
Keep in mind that although a profile with a large overlap may have a really low probability of occurring by chance, the value of the profile to a user depends not only on the low likelihood of being an artifact, but also on the explanation of how the genes in the profile are related to each other. The more believable the explanation of how the algorithm determined that the set of genes in the profile act in a biologically coordinated manner, and the more plausible that explanation is given the user's particular assay conditions, the more valuable the match, since it increases the probability that the decisions the user makes based on the insight provided by the profile explanation will be biologically sound.
Also note that this approach is computing the exact probability, which permits all profiles to be compared in relative terms against each other for a given KB and assay. However, this exact probability is not as good as measure for comparing best-scoring profiles across multiple assays, since the number of dysregulated mapped genes and maximum overlap, which have a significant effect on the exact probability value, can vary dramatically across experiments.
Approach 2: Cumulative Probability of Overlap (P-value)
A preferred statistic for comparing overlaps across experiments, as well as getting a better intuitive feel for the significance of an overlap uses a cumulative probability distribution, instead of a single value from a probability distribution function (i.e., which is Approach 1). This can be computed by summing all of the individual probability values that are less than or equal to the exact probability value, and determining what fraction of the total sum of all the possible probability values it represents; this measure is usually called the ‘P-value’.
Computing P-values over multi-variable distributions is usually complex. The typical approach is to fix as many variables as possible, determine whether the calculation can be reduced to an integral, and then solve the integral for the free variables. Note that: 0<=OVP<=BCP<=KB; and OVP<=SIG<=MAP<=KB. So to make this calculation tractable, let us make the following assumptions:

    
    
        1. When comparing P-values across experiments, the number of KB genes is constant. Since KB is already a large number, even if this assumption is not strictly adhered to, in general the difference will be minimal.
        2. When comparing P-values across experiments, the number of mapped genes (MAP) is constant. This is less stringent than requiring that the user assay all of the same genes for each experiment, although presumably that would be the norm. Users only need to assay the same mapped genes with each assay; but if they compare general assay results to a targeted or different assay with only a fraction of the mapped genes in common, the P-value results would not be directly comparable.
        3. The number of mapped, dysregulated genes (SIG) may vary across experiments (ie. 0<=SIG<=MAP). However, for any given experiment it is assumed that the total number of SIG genes is non-random, although the particular SIG genes are assumed to be random.
    
    


Note that, unlike the familiar ‘normal’, one-dimensional bell-shaped distributions, this distribution is five-dimensional. Also, the probability density function (PDF) of this distribution decreases rapidly as OVP increases and is discrete (i.e., each dimension has integer ranges from 0 to a fixed number, not a real-valued range of −infinity to +infinity like the normal distribution). These features make it challenging to develop a formula that directly computes P-values; reducing it to three dimensions by requiring that KB and MAP are constant helps, but the integration (computation of the probability for each of KB*SIG*MAP possible outcomes) still requires a lot of CPU cycles (unless a closed-form integral exists that can directly compute the values). For this reason, it may be preferable to, e.g., pre-compute a table of probability values once, and then compare the probability value from the first approach to the tabular values for each BCP profile when determining a P-value. Nevertheless, if KB=10,000 and SIG is treated as a random variable, this lookup table could require about 100 GB of memory—a supercomputer. But if the total number of SIG genes is non-random, this calculation may be easily done once per experiment (i.e., so the same lookup could be used for scoring all profiles against it), which would require a lookup table with KB*SIG entries (KB since BCP size range is 1 . . . KB; SIG since OVP size range is 1 . . . SIG). This approach is preferred as it limits the demands on computational resources. For example, using this approach in the case where SIG=KB and KB=10,000, only a 100 MB lookup table would be needed.
The P-value is computed by summing all probability values that are less than or equal to the probability value computed by the first approach and dividing by the sum of all possible probability values. Note that since the outcomes most likely to occur by chance involve an overlap of one gene, for cases where the observed OVP is greater than one, the P-value will tend to be quite small. So unlike the normal distribution, where a P-value<0.05 is generally ‘significant’, a lower threshold is preferably imposed for this distribution. A metric that computes the percentage of all possible outcomes that are less than or equal to the observed probability value may be better for assessing profile scores for single experiments than by using a straight probability value, but would not be suitable for comparing across experiments since the weight (probability value) for each outcome may vary significantly.
Approach 3: Approximate Cumulative Probability of Overlap (P-value)
A third approach is to approximate the p-value with a simpler model since it is quicker to compute dynamically. Since a random set (SIG) drawn from a random set (MAP) is still random, the MAP term does directly play a role in the probabilities (it just makes them smaller by taking possible noise in the user's data set into account). A simpler model still uses the hypergeometric distribution, but without MAP:




This is still expensive to calculate unless we assume that bcp is small compared to both kb and sig. Then we can approximate the hypergeometric distribution (without replacement) with the binomial distribution (with replacement):



  
    
      let
      ⁢
      
        
      
      ⁢
      p
    
    =
    
      sig
      kb
    
  




  
    
      prob
      ⁡
      
        (
        ovp
        )
      
    
    =
    
      
        (
        
          
            
              bcp
            
          
          
            
              ovp
            
          
        
        )
      
      ⁢
      
        
          
            p
            ovp
          
          ⁡
          
            (
            
              1
              -
              p
            
            )
          
        
        
          bcp
          -
          ovp
        
      
    
  



Where the p-value is the sum of these binomial coefficients for overlaps up to and including ovp. These p-values can be displayed as the—log 10 (p-value) to covert them to an integer score (corresponding to p-value exponent) which is easier for users to quickly read and compare.
Pathway Quality Attributes
The believability of a pathway expressed in a profile and its relevance to user-provided genomics data depends on (1) the ability of the KB to accurately represent characteristics of biological pathways, and (2) the extent to which any given pathway in the KB represents the true biological pathway underlying the user supplied data. These metrics are referred to as Pathway Quality Attributes (PQAs). The examples of profile scoring discussed earlier would fall under a Category 2-type PQA and the KB's degree of knowledge about a particular pathway contained in a profile model would correspond to a Category 1-type PQA.
Table 1 provides several examples of PQAs. Each row refers to a favorable attribute of a pathway in a profile. A pathway in a profile that has one or more of these favorable attributes may tend to reflect either the KB's ability to accurately represent a true biological pathway (Category 1) and/or the pathway's ability to explain the true biological pathway in the user data (Category 2). Referring to Table 1, PQA nos. 1-10, 13-15 and 18 refer to Category 1-type PQAs and PQA nos. 11, 12, 16 and 17 refer to Category 2-type PQAs. Under column heading “Attribute”, the attribute type, or quality, is summarized and under the column “Description”, there is an example (or examples) of how this attribute may appear in a profile, or be implemented as a profile model criterion.







TABLE 1







Pathway Quality Attributes (PQAs)










Attribute
Description












1
Contains tight sub-
Assume three genes minimum to form a notion of a pathway. Want the



network of
profile to include a network of at least three highly-interconnected genes



recognizable pathway
for the recognizable pathway (the more, the better).



genes



2
Dysregulated genes
Prefer sub-networks that have a large number of dysregulated genes, and



mutually highly-
prefer even more those networks where such genes are further highly-



connected in the
interconnected and dysregulated as a whole (profiles can contain both



network
dysregulated genes - those that were active in the experiment - and non-




dysregulated genes. This is a major benefit since these additions can




provide additional insight not obvious from just the dysregulated genes). In




general, prefer to see more dysregulated genes. Given 2 profiles that each




have 20 genes, one may have 3 out of those 20 dysregulated, and the other




will have 10 out of those 20 dysregulated, the latter is preferable.


3
Findings connectivity
Prefer high level of connectedness of sub-network of genes, where




connectedness is measured by # of findings supporting a given relationship




or edge (e.g., pick profile size of 4 nodes that have at least 5 findings




connecting nodes or prefer profiles where all pairs of nodes (genes) are




related by 5 or more findings on average).


4
Edge connectivity
Prefer high level of connectedness of sub-network of genes, where




connectedness is measured by # of edges (e.g., if there are 4 nodes in a




network, then require minimum of 3 edges connecting each node to other




nodes).


5
Journal source
Prefer high level of connectedness of sub-network of genes, where



connectivity
connectedness is measured by # journal sources.


6
Finding quality
Prefer high level of connectedness of sub-network of genes, where



believability
connectedness is defined by canonical or high-confidence profile edges




given preference. This refers to a preference for findings that come from




content sources that are more trusted. For example, 1 finding from a




review article may be considered as trustworthy as 5 similar findings from




original research papers because the latter are more likely to be shown to




be wrong over time.


7
Consistent cellular
Prefer genes known to be involved in same cellular function (A →



function
apoptosis and B → apoptosis then A   B). For example, a preference for




genes known to be involved in same cellular function with same direction




of influence (A → increase apoptosis and B → increase apoptosis then A




  B, but A → increase apoptosis and C → decrease apoptosis does not




relate A to C).


8
Preponderance of 
Generalization of attribute 7 across all genes in a profile, i.e., if you have



evidence towards
an entire pathway where ALL genes are known to be involved in, e.g.,



specific pathway
apoptosis, then this is a highly favorable attribute. This features may be



function
thought of as an extension of the “seed” concept to include a “seed




process” or “seed function” as the central element of a profile.


9
Tissue consistency
Prefer genes consistent with studied tissue: either shown to be dysregulated




in experiment, or known to be expressed in tissue being studied (for non-




dysregulated genes).


10
Direct (physical
Prefer connections/edges that are supported by evidence of direct physical



interactions are more
molecular relationship, as opposed to only high-level/cellular/disease



reliable/robust)
type associations. Direct physical interactions are considered better




because they describe an actual mechanistic molecular interaction rather




than the higher level result of that interaction (e.g. symptoms of a disease)


11
Consistent with
Prefer profiles where profile description of regulation relationships and



experimental
dependencies is consistent with KB finding expression directional effects.



expression change
This attribute may be evaluated for expression changes provided as a time



pattern
series or without (since array results show evidence of past cellular events




as well). So if A - inhibits → B, and A is down-regulated, then you might




expect to see B up-regulated. If your expression data shows this (A down,




B up) then this finding is a potential explanation.


12
Consistency with
Prefer high aggregate magnitude of expression change (four 3-fold



experimental
dysregulated genes are more interesting than four 2-fold dysregulated



expression levels
genes). Aggregation may be measured by, e.g., average, sum, absolute




values, etc.


13
Intermediate genes
Avoid genes in a profile that are not able to be linked to a process that is



that don't appear tied
central to the other profile genes (i.e., if statistics suggest that 3 out of 4



into pathway function
nodes relate to process A, but the remaining node does not, then avoid




including this node in profile).


14
No findings or
Avoid connectivity metrics that may be biased by coverage in the



literature bias
literature, or the coverage of a particular group of biological concepts (e.g.,




a particular group of genes) in the literature during KB data acquisition.




This bias may be accounted for by, e.g. a normalization based on an




understanding of the scope of content coverage in the KB, or the scope of




content (e.g., genes types) studied in the literature.


15
One or more seed
A “seed” approach to associating a pathway to a profile (e.g., a seed gene)



concepts for profiles
is one of various ways in which a story or significance can be drawn out of




profiles for users. This allows profiles to have e.g. one or more seed genes




that are considered to be the most central to the profile function. Allow




users to specify these seeds OR allow the system to pick them e.g. by




iterating over all combinations of genes in the KB. While a seed approach




to profile creation may be computationally useful, other attributes of a




profile, which may not focus on the seed, may be equally insightful.


16
Connectable to
Good if a pathway function is related to something to do with the



experimental context
experimental context because it can validate that you are “in the right




space”.


17
Completeness of
Two proteins may be discovered to interact with one another, but the



knowledge of function
significance of that interaction or what happens during that interaction may



of interactions
be unknown. So interactions that define what happens (e.g. one protein




chemically modifies the other when they interact) provide a more complete




description of knowledge about the function of the interaction. So




generally biological roles (activation/inhibition effect of one protein on




another) is more informative than functional interactions (enzymatic




effects carried out by one protein on another), which are in turn more




informative than physical interactions (two proteins co-localize or can bind




to each other). So profiles whose interactions convey a high degree of




knowledge are preferred.


18
Pathway function is
Good if a pathway function is related to something with interesting



relatable to research
implications on system/disease target being studied in the experiment. A



goals
pathway may be biologically accurate and be associated with several




biological functions. The function that is closest e.g. to the disease being




studied may be of more interest to a research than those that are true but




incidental to the central research question at hand.


19
Gene sensitivity to
Discriminate between findings/connectivity that suggest a high or low



neighbors
likelihood that a gene and its neighbors will influence each other's




activities. Can normalize against excessively high or low influence genes.









As will be appreciated by those of ordinary skill in the art, the examples of PQAs above, in addition to those discussed earlier, are informative of the possible scope of profile definition criteria that will allow the creation of profiles best suited for research goals. As mentioned earlier, the lists enumerated above are provided only as examples of possible profile criteria and should not be understood as limitations of the invention.
System Configuration
A system for practicing the methods of the invention need not be limited to a single entity, e.g., a private company, which, for example, builds and interrogates a KB for biological pathway information and provides the user interface for inspecting results. Rather, a system may be created as a result of combined efforts from one or more entities, which when combined (e.g., by a customer or through a systems integrator) provides a system capable of being used to practice methods of the invention. In the following, an example of how each of the tasks associated with developing components of this system is provided. Reference is made to a “Company A”, “Company B”, etc. These entities may correspond to public entities, private entities, public-private entities or a combination thereof.

    
    
        Company A builds and sells a KB (possibly by acquiring content from 3rd parties or creating the content themselves. This endeavor could itself be split, i.e., one company could build the KRS software system, another could build the ontology, a third could build tools to enter data, and a fourth could use the preceding to actually enter the data (findings).
        Company B, a data conversion/translation company, transforms the KB into a graph network.
        Company C, an analyst or systems integrator company, figures out what profile characteristics are important for a set of users/customers.
        Company D, software developers, build an algorithm that constructs profiles based on criteria provided by Company C.
        Company E builds and/or sells visualization and browsing tools to view Company D profiles.
        Company F, software developers, build an algorithm to rank the profiles against various experimental datasets. Company F1 could do it for expression data, company F2 for protein-protein interaction data, etc.
        Company G, systems integrations, integrates all of the above into a system that takes expression data and predicts functional pathways based on scoring profiles built from the KB through the graph.
        Company H, an analyst or systems integrator company, possibly in conjunction with company C, figures out what additional pathway information would be useful to users for interpreting the pathway. This could include characteristics identified by company C but that were not used by company D to create the profile. For example, a particular profile generation algorithm may not try to build profiles around a central biological process automatically, but users will still want to know what process(es) are more or less central to the profile.
        Company I, software developers, build a second set of algorithms to calculate and/or display additional attributes of these profiles (for example, our process annotations).
        Company J, a content company, manually enters existing pathways, replacing companies A-D, so that company G can now build/integrate a system that uses the same profile scoring algorithm, visualization, GUI, and attribute calculations, but uses them against manually created profiles rather than computer-generated profiles.
        Company K, software developers, might help company J by creating a “pathway editor” software package that lets users create their own profiles by drawing pathway-like diagrams. This is “reverse visualization”: draw the picture, and infer the biological relationships by seeing which circles are connected to which arrows, etc.
    
    


Although the present invention has been described in detail with reference to its preferred embodiments, it is understood that various modifications can be made without departing from the spirit of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. All publications, patent applications and patents mentioned in this specification are hereby incorporated as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference.

## Claims

1. A computer system for identifying one or more drug discovery targets, the system comprising:
a knowledge representation system comprising an ontology, and a knowledge base of scientific findings; wherein:
(a) the knowledge base comprises genomics information, and findings derived from natural language, wherein the genomics information comprises information relating to genes, their DNA sequences, their mRNA, proteins expressed from said genes and the biological effects of the expressed proteins;
(b) the ontology is organized so that:
(i) each gene, mRNA, protein expressed from said gene, and biological effect is given an identifier which is related to synonyms for the identifier;
(ii) each gene, mRNA, protein expressed from said gene, and biological effect is categorized by class; and
(iii) the relationship of each gene, mRNA, protein expressed from said gene and disease state is defined by slots and facets; and

(c) the computer system is configured to:
(i) receive a profile definition criterion from a user identifying a subset of biological objects and processes that act on biological systems in a disease-related pathway;
(ii) generate a profile using the knowledge base, wherein the profile comprises a subset of genomics information contained in the knowledge base comprising information relating to genes, their DNA sequences, their mRNA, proteins expressed from said genes and the biological effects of the expressed proteins;
(iii) perform computational analysis of biological relationships among the subset of genomics information in the profile based upon the profile definition criterion; and
(iv) identify additional biological objects and processes that act on those objects associated with the disease-related pathway using the analysis of biological relationship; whereby each identified object or process is a drug discovery target,


wherein the computer system is a stand alone computer, a multicomponent computer, or a networked computer, and wherein the computer system farther comprises hardware for electronically searching the knowledge base.

2. The computer system of claim 1 wherein said genomics information comprises data extracted from multiple public sources.

3. The computer system of claim 1 wherein said genomics information comprises proprietary data.

4. The computer system of claim 1 wherein said genomics information comprises data extracted from a combination of proprietary and public data sources.

5. The computer system of claim 1 wherein drug discovery targets in the disease-related pathway are prioritized based on factors that include function and complexity.

6. The computer system of claim 1 wherein drug discovery targets are prioritized based on markers for side effects and patient responsiveness.

7. The computer system of claim 1 wherein the genomics information comprises information relating to genotype and the disease-related pathway comprises a gene, mRNA or protein expressed from said gene associated with a particular genotype.

8. The computer system of claim 1, wherein the genomics information comprises a name of each gene, mRNA or protein expressed from said gene, and their biological effects, and the computer identifies relationships between genes and/or proteins expressed from said genes that are at least two steps removed from each other in a disease-related pathway.

9. The computer system of claim 1, further comprising a second knowledge base of scientific findings.

10. The computer system of claim 9, wherein the knowledge base is a frame-based knowledge base.

11. The computer system of claim 1, wherein the system is further configured to compare disease-related pathways with data obtained from gene expression studies or a manually entered gene list.

12. The computer system of claim 11, wherein the gene expression studies comprise differential gene expression studies or microarray studies.

13. The computer system of claim 11, wherein the comparison of the disease-related pathways with user-defined data is made using a statistical model.

14. The computer system of claim 13, wherein the statistical model calculates the probability that overlaps between the disease-related pathways and user-defined data is a random event.

15. The computer system of claim 1, wherein the identification step further comprises storing the identified biological objects and processes according to the ontology.

16. The computer system of claim 1, wherein performance of computational analysis of biological relationships comprises generating one or more subsets of genomics information.

17. The computer system of claim 16, wherein the one or more subsets of genomics information are pre-generated from the knowledge base.

18. The computer system of claim 16, wherein the one or more subsets of genomics information are generated by one of a data-driven and model-driven approach.

19. The computer system of claim 16, wherein the one or more subsets of genomics information are generated based upon information contained in the knowledge base and user supplied genomics information.

20. The computer system of claim 16, further comprising the step of providing a user-supplied set of gene expression data for identifying a particular subset of genomics information, wherein the gene expression data are selected based on one or more of expression levels derived from inicroarray experiments, a prior analysis algorithm, and a user's preferred gene set.

21. The computer system of claim 16, wherein the one or more subsets of genomics information are gene-centric being derived about a central gene for all genes in the knowledge base.

22. The computer system of claim 16, wherein the generation of one or more subsets of genomics information further comprises deriving one or more subsets of genomics information of related user-selected genes.

23. The computer system of claim 22, wherein the one or more subsets of genomics information are generated so as to be non-overlapping by ensuring that user-selected genes do not appear in more than a predetermined maximum threshold number of subset of genomics information.

24. The computer system of claim 22, wherein the one or more subsets of genomics information are generated so as to be based on connections between a first known drug target gene and a second drug target gene of interest.

25. A networked computer system for identifying one or more drug discovery targets comprising:
a communication network; and
a computer system coupled to the communication network comprising a database for storing and accessing genomics information, wherein the genomics information comprises information relating to genes, their DNA sequences, their mRNA, proteins expressed from said genes and the biological effects of the expressed proteins;
wherein the computer system is configured to:
(i) receive a profile definition criterion from a user identifying a subset of biological objects and processes that act on biological sytems in a disease-related pathway;
(ii) generate a profile using the knowledge base, wherein the profile comprises a subset of the information contained in the database comprising information relating to genes, their DNA sequences, their mRNA, proteins expressed from said genes and the biological effects of the expressed proteins;
(iii) perform computational analysis of biological relationships among the subset of genomics information in the profile based upon the profile definition criterion; and
(iv) identify additional biological objects and processes that act on those objects associated with the disease-related pathways using the analysis of biological relationships; whereby each identified object or process is a drug discovery target,

wherein the computer system comprises hardware for electronically searching the database.