AU2011256908A1 - Methods for identifying new drug leads and new therapeutic uses for known drugs - Google Patents

Abstract

The instant invention provides a method for establishing safety profiles for chemical compounds, as well as pharmacological profiling said method comprising (A) testing the 5 effects of said chemical compounds on the amount and/or post-translational modifications of two or more macromolecules in intact cells; (B) constructing a pharmacological profile based on the results of said tests; and (C) comparing said profile to the profile(s) of drugs with established safety characteristics. Additionally, the invention is also directed to a composition comprising an assay panel, said panel comprising at least one high-content 10 assay for the amount and/or post--translational modification of a protein and at least one high-content assay for the amount and/or subcellular location of a protein-protein interaction. 00t m C (Um o Eu > 0

Description

AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention title: Methods for identifying new drug leads and new therapeutic uses for known drugs The following statement is a full description of this invention, including the best method of performing it known to us: METHODS FOR IDENTIFYING NEW DRUG LEADS AND NEW THERAPEUTIC USES FOR KNOWN DRUGS This application claims the priority benefit under 35 U.S.C. section 119 of U.S. 5 Provisional Patent Application No. 60/611,715 entitled "Methods For Identifying New Drug Leads And New Therapeutic Uses For Known Drugs", filed September 22, 2004, which is in its entirety herein incorporated by reference. BACKGROUND OF THE INVENTION 10 Known drugs that are marketed for various therapeutic indications often have 'hidden phenotypes' resulting from unexpected or unintended activities on biochemical pathways in human cells. These observations of new and useful properties of known drugs are relatively infrequent and are usually discovered by serendipity. A classical example is that of rapamycin (sirolimus), marketed as Rapamune@, which 15 was approved for the treatment of immunosuppression in 1999. In the 1970's, rapamycin was found by the NCI to have potential anticancer properties. Now, over 30 years later, an analog of rapamycin known as CCI-779 is finally in clinical trials for the treatment of breast and renal carcinoma. As recently as 2000, rapamycin was also found to prevent restenosis when used in coated (rapamycin-eluting) stents; the stents are sold by Cordis Corp. for that purpose. These 20 properties result from the underlying activity of rapamycin on biochemical pathways: namely, its ability to activate a pathway leading to apoptosis. Another example is that of thalidomide, the drug originally developed as an anti-emetic. Thalidomide, which is in clinical development for the treatment of multiple myeloma, is likely to have a broad spectrum of anti-cancer activity as a result of its ability to block a pathway that is a hallmark of the cancer cell. 25 If there were a reliable and systematic method to identify such useful biological properties, such observations could be made on a large scale. This would have important benefits not only for patients in need of new remedies, but also for the pharmaceutical industry. Perturbations in signal transduction pathways are known to underlie the mechanisms of action of most if not all drugs. In principle, unexpected biologic activities of drugs could be 30 found by testing their activities directly against the pathways that control the behavior of the living cell. If an unexpected activity is found against a pathway that is known to be linked to a 2 disease, the drug can be tested in phenotypic assays, model organisms and other model systems to determine if it has an effect on that disease. Since known drugs have established safety profiles and pharmacodynamic properties, if the drug shows promise of being effective in the new disease indication it can be rapidly advanced into clinical trials for the treatment of patients 5 with that disease. Therefore we sought to develop a rapid, pathway-based system in living cells for rapidly identifying unexpected activities of drugs on a large scale. The advantages of using a cell-based system are that drugs can be studied in the context of the complex biology of the whole cell. To date, cell-based screening approaches have relied either on phenotypic screens, 10 reporter gene assays, or mRNA profiling. For a summary of such approaches, see the Disease Proteomics reference. For example, cells can be treated with individual drugs or elements of chemical libraries and a phenotype can be measured, such as growth, apoptosis, migration, cell cycle arrest, etc. Phenotypic screens have been widely used in recent years but do not provide an indication of the underlying mechanism by which drugs cause the phenotypic change. 15 Reporter gene assays have also been used to identify the activities of compounds and drugs against biochemical pathways in living cells. Reporter gene assays couple the biological activity of a target to the expression of a readily detected enzyme or protein reporter, allowing monitoring of the cellular events associated with signal transduction and gene expression. Based upon the fusion of transcriptional control elements to a variety of reporter genes, these 20 systems "report" the effects of a cascade of signaling events on gene expression inside cells. Synthetic repeats of a particular response element can be inserted upstream of the reporter gene to regulate its expression in response to signaling molecules generated by activation of a specific pathway in a live cell. Such assays have proven useful in primary and secondary screening of chemical libraries and drug leads. However, such assays only measure the consequence of 25 pathway activation or inhibition and not the site of action of the compound. Microarrays allow measurements of gene expression patterns on a large scale. Following a drug treatment, messenger RNA is isolated from a cell or tissue; and the expression patterns of the mRNA in the absence and presence of the drug are compared. Identifying groups of genes that are stimulated or repressed in response to specific conditions or treatments is a useful way to 30 begin to unravel the cellular mechanisms of drug response. However, changes in the level of particular mRNA molecules do not always correlate directly with the level or activity of any 3 corresponding protein at a single point in time. Furthermore, many proteins undergo post translational modifications and protein-protein interactions, which may affect the functions and activities of proteins within a tissue or cell. Consequently, gene chip experiments are not always predictive of biological activity. 5 In sum, such approaches do not enable an understanding of the mechanisms of action of drugs at their site of activity within biochemical pathways. It would be preferable to directly probe the networks of living human cells. Direct measures of specific events within signaling pathways would eliminate the problems associated with interpretation of transcriptional profiles. Unlike transcriptional reporter assays, the information obtained by monitoring a protein 10 modification or its interactions reflects the effect of a drug on a particular branch or node of a cell signaling pathway, not its endpoint. In making the present invention, our central premise was that (a) the biological and biochemical effects of drugs can be studied with living cells; (b) one can determine the unexpected effects of drugs on cellular pathways by probing those pathways directly, at the level 15 of specific proteins, following drug treatment; (c) the effects of drugs on their targets will propagate through or between functional modules, inducing spatial and temporal changes in proteins downstream of the target of a drug (Figure 1); (d) such changes can be quantified by measuring changes in protein-protein complexes or interactions 'downstream' of the site(s) of action of the drug; and (e) such changes will be dynamic, that is, they will occur transiently within 20 minutes - or at most, within hours - after drug treatment of the cells . Changes in protein-protein complexes (interactions) could be effected by a variety of biochemical events within the module (e.g. post-translational modification, allosteric transition, protein degradation or de novo protein synthesis, protein stabilization or destabilization, or protein translocation), wherein the change propagates through or between modules from the drug 25 target, resulting in a perturbation of a protein-protein complex. In the context of the invention, we use the terms "protein-protein interaction" and "protein-protein complex" interchangeably. An interaction between proteins is reflected in the presence of a complex between the proteins, and the amount and/or location of the complex is altered by biochemical events that stimulate or inhibit the pathways that influence the proteins in question. We hypothesized that temporal, 30 drug-induced changes in the amount, subcellular location, or post-translational modification status of proteins within a dynamic complex within a pathway could be detected by directly 4 measuring particular complexes, as protein-protein pairs, in human cells following drug treatment. We systematically applied this strategy to the known pharmacopeia in order to identify drugs that are capable of modulating the activity of the oncogenic pathways underlying the 5 cancer phenotype. By 'known drug' and 'known pharmacopeia' we mean drugs currently or previously administered to patients. We screened a portion of the known pharmacopeia and identified dozens of drugs, previously or currently marked for a variety of indications, with surprising and previously-unsuspected activity against 'hallmark' cancer pathways. We then showed that over 20 of these drugs indeed have anti-proliferative activity in tumor cells, 10 underscoring the utility and predictability of the screening approach. The drugs we identified represent potential new treatments for cancer in man. Importantly, the strategy and methods presented herein represent an entirely novel system for therapeutic discovery on a large scale. SUMMARY OF THE INVENTION 15 We have invented a powerful new method for identifying compounds with new and useful biological activities. The methods of the invention can be used to identify previously unknown drug activities, even for drugs that have been well characterized with standard biochemical assays. The screening system utilizes dynamic measurements of pathway activity to detect the 20 activities of drugs within cellular pathways. The invention has commercial importance for the pharmaceutical industry. If drugs that are known to be safe can be found to have new indications, they can be rapidly advanced to clinical trials to demonstrate efficacy for the new indications. Also, if drugs that are known to be safe have failed to demonstrate efficacy for their originally intended indication, they may still be rescued for use in a new therapeutic indication. 25 Finally, drugs that have adverse effects when used at specific doses or in chronic administration may still be tolerable if used in a new indication or a new dosing regimen. The methodology will extend the utility of the current pharmacopeia and provide the basis for de novo discovery of drugs with a broad range of therapeutic indications. In the present invention we identified antiproliferative activities for over 20 known drugs including drugs 30 previously used for the treatment of congestive heart failure, hypertension, hypercholesterolemia, 5 asthma, infection (antibiotic, antiprotozoan, antihelminthic, antifungal), emesis, migraine, psychosis, dementia, and other common conditions. The drugs we identified as having activity on cancer pathways include sertraline (Zoloft), terfenadine (Seldane), atorvastatin (Lipitor), fenofibrate (Tricor) and other well-known drugs currently or previously marketed for a wide 5 range of non-cancer indications. Some of these activities have been previously suspected whereas others are completely unsuspected. These activities are linked to the ability of these drugs to inhibit one or more of the key pathways contributing to the cancer phenotype. Our success in identifying drugs with potential anti-cancer activities is likely due to the 10 uniquely informative whole-cell assay approaches we employed here; in particular, the ability to assess multiple pathway activities of drugs in human cells that have the requisite intracellular machinery.. The approach can be applied to a wide range of chronic and acute diseases in man by simply probing other pathways linked to those diseases. 15 OBJECTS AND ADVANTAGES OF THE INVENTION It is an object of the present invention to provide methods for the identification of new therapeutic uses for existing drugs. A further object of this invention is to provide methods, assays and compositions useful for drug discovery on a large scale. 20 The present invention has the advantage of being broadly applicable to any disease or medical condition, drug target class or agent. The present invention has the advantage of being independent of the primary or intended or original target of a drug or drug candidate. The present invention has the advantage of being applicable to any therapeutic indication. 25 The present invention has the advantage of being applicable to any cell type or disease model system or organism on a genome-wide scale. The present invention has the advantage of being performed in high throughput and can be completely automated. 30 6 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic of the relationship between a drug target and a cellular assay in the present invention. Effects of drugs on cellular pathways can be determined by measuring protein interactions and/or modifications 'downstream' of a drug target. Shown in red are 5 network 'nodes'; wherein each node is a protein-protein complex. Figure 2 Pathways probed in the present Invention. Protein-protein complexes, comprised of the signaling proteins that are outlined in red, formed the basis for the construction of assays in living cells. Physical interactions between proteins are indicated by arrows. Figure 3A Examples of drug effects on BCL-xL:BIK and PINI:JUN complexes in 10 human cells. Photomicrographs show the effects of fenofibrate on BclxL:BIK complexes and the effect of niclosamide on PIN1:fJN complexes, as assessed with protein-fragment complementation assays. The drugs cause a decrease in the level of the protein-protein complexes as assessed by a decrease in the intensity of the fluorescence in the assays. Figure 3B Examples of drug effects on p27:Ubiquitin and CyclinD1:CDK4 15 complexes in human cells. Photomicrographs show the effects of fenofibrate on p27:Ubiquitin complexes and on CyclinDl:CDK4 complexes, as assessed with protein-fragment complementation assays. Fenofibrate caused a decrease in the level of the protein-protein complexes Fenofibrate caused a decrease in the level of these protein-protein complexes as assessed by a decrease in the intensity of the fluorescence in the assays. 20 Figure 3C Examples of drug effects on AKT1:p27 and Cofilin:LIMK2 in human cells. Photomicrographs show the effects of fenofibrate on the protein-protein complexes, as assessed with protein-fragment complementation assays. Fenofibrate caused a decrease in the level of these protein-protein complexes as assessed by a decrease in the intensity of the fluorescence in the assays. 25 Figure 3D. Examples of drug effects on HSP90:CDC37 and HSP90:Eef2k in human cells. Photomicrographs show the effects of niclosamide on the protein-protein complexes as assessed with protein-fragment complementation assays. Niclosamide caused a decrease in the level of these protein-protein complexes as assessed by a decrease in the intensity of the fluorescence in the assays. 30 Figure 3E Examples of drug effects on Ras:Raf in human cells. Photomicrographs show the effects of promazine, sanguinarine, desispramine, metergoline, and tamoxifen citrate 7 on Ras:Raf complexes, as assessed with protein-fragment complementation assays. The drugs caused a decrease in the level of the Ras:Raf complexes, as assessed by a decrease in the intensity of the fluorescence compared with the vehicle alone; the drugs also caused a change in the subcellular location of the complexes, as assessed by an obvious change in the subcellular 5 patten of the fluorescence. In the control cells (vehicle only) the Ras:Raf complexes were localized at the cell membrane; the drugs caused a redistribution of the complexes to an intracellular structure. Figure 3F Examples of drug effects on CDC42:PAK4 in human cells. Photomicrographs show the effects of terfenadine, bepridil, and metergoline, on CDC42:PAK4 10 complexes, as assessed with protein-fragment complementation assays. The drugs caused a decrease in the level of these protein-protein complexes as compared with the vehicle alone, as assessed by a decrease in the intensity of the fluorescence in the assays. Figure 3G. Examples of drug effects on the post-translational modification status of ERK (MAPK) in the presence of VEGF. Photomicrographs show the effects of fenofibrate 15 and niclosamide on phospho-ERK, as assessed with immunofluorescence assays using phospho ERK-specific antibodies. The drugs caused a decrease in the level of phospho-ERK as compared with the vehicle alone, as assessed by a decrease in the intensity of the fluorescence in the assay. Figure 4 Antiproliferative activity of fenofibrate vs. an analog. The ability of fenofibrate to reduce the proliferation of PC-3 cells is shown. An analog, WY-14643, had no 20 effect on proliferation as assessed by a MTT assay, demonstrating a structure-activity relationship in these cellular assays. Dose dependence for fenofibrate (triplicate assays) is shown in the proliferation assay as compared to the DMSO control. Also shown are images of the MTT assay wells, and phase contrast images of the treated cells vs. the untreated (DMSO) control showing that fenofibrate reduced the cell count as compared with the control. 25 Figure 5 Antiproliferative activity of terfenadine (Seldane). Dose dependence for terfenadine (triplicate assays) is shown in the MTT proliferation assay as compared to the untreated (DMSO) control. Also shown are images of the MTT assay wells and phase contrast images of the treated cells vs. the untreated (DMSO) control, showing that terfenadine reduced the cell count as compared with the control. 30 Figure 6 Antiproliferative activity of sertraline (Zoloft). Dose dependence for sertraline (triplicate assays) is shown in the MTT assay as compared to the DMSO control. Also 8 shown are images of the MTT assay wells and phase contrast images of the treated cells vs. the untreated (DMSO) control, showing that sertraline reduced the cell count as compared with the control. Figure 7 Antiproliferative activity of cinnarazine. Dose dependence for cinnarizine 5 (triplicate assays) is shown in the MTT assay as compared to the DMSO control. Also shown are images of the MTT assay wells and phase contrast images of the treated cells vs. the untreated (DMSO) control, showing that cinnarazine decreased the cell count as compared with the control. Figure 8 Antiproliferative activity of isoreserpine. Dose dependence for isoreserpine 10 (triplicate assays) is shown in the MTT assay as compared to the DMSO control. Also shown are images of the MTT assay wells and phase contrast images of the treated cells vs. the DMSO control, showing that isoreserpine decreased the cell count as compared with the control.. Figure 9 Antiproliferative activity of clotrimazole. Dose dependence for clotrimazole (triplicate assays) is shown in the MTT assay as compared to the DMSO control. Also shown 15 are images of the MTT assay wells and phase contrast images of the treated cells vs. the DMSO control, showing that clotrimazole decreased the cell count as compared with the control. Figure 10 Antiproliferative activity of atorvastatin (Lipitor). Dose dependence for atorvastatin (triplicate assays) is shown in the MTT assay as compared to the DMSO control. Also shown are images of the MTT assay wells and phase contrast images of the treated cells vs. 20 the DMSO control, showing that atorvastatin decreased the cell count as compared with the control. Figure 11 Positive predictive value of the process. 960 individual drugs were tested in 4 different assays in human cells according to the present invention. Drugs that showed activity in any one of the four assays were then tested at a single concentration to determine if they were 25 capable of reducing the proliferation of PC-3 cells, as assessed by a reduction in cell number to less than 80% of or less than 50% of the control (untreated) cells. The number of assay hits that proved to have antiproliferative activity is shown. 9 DETAILED DESCRIPTION OF THE INVENTION Perturbations in signal transduction pathways are known to underlie the mechanisms of action of most if not all drugs. Drugs that are capable of re-routing the abnormal circuits of the disease cell should in principle be capable of restoring the phenotype of the normal or healthy 5 cell. The phenotype of a cell is, in turn, controlled at a high level by biochemical pathways that regulate the expression and activity of proteins. Hence, the key to identifying drugs that are capable of re-routing cellular circuits is the ability to detect specific activities of drugs within, and on, the pathways of living cells. Our objective was to establish a systematic, high-throughput process for the identification of agents capable of rerouting the circuits of living cells. 10 The invention is based on the concept of a cellular network as a series of interconnected pathways involving physical connections between proteins, as depicted in Figure 1. The rationale underlying the invention is as follows. A pathway is a series of steps, with each step occurring at a particular point in time (with the first step preceding the second step which precedes the third step, etc.) and in space (for example, starting with a receptor at the cellular 15 membrane and proceeding to a transcription factor in the cell nucleus). In our model, each step involves the association or dissociation of proteins. If the pathway is a signal transduction pathway, activation of the pathway - for example, by binding of an agonist to a receptor initiates a cascade of events by which an external signal is transduced to the nucleus. These events involve the interactions of proteins which modify their activities. These changes are 20 dynamic, beginning within minutes of drug treatment. The ultimate consequence of these biochemical events is a change in cell behavior: growth, division, apoptosis, migration, differentiation, metastasis, or another behavior that is characteristic of the cell under study. Drugs that block a particular step would lead to inhibition of the steps 'downstream' of the original site of action of the drug. Conversely, drugs that activate a particular step would lead to 25 activation of the steps 'downstream' of the original site of action of the drug. It follows that dynamic measurements of the proteins within selected functional pathways should enable the identification of drugs that affect, in a desired way, the activities of those pathways. Typically, assessing the activity of individual proteins involves in vitro measurements of enzyme activity (kinases, phosphatases, proteases, hydrolases, etc.) and/or ligand binding, in the 30 case of receptors. We sought instead to measure the physical/chemical changes in proteins that precede or coincide with such activity changes. Such physical/chemical changes involve 10 interactions between proteins that lead to the formation and movement of protein-protein complexes; and the post-translational modifications that result from those interactions and movements. Moreover, we sought to make such measurements in intact cells before and after treating the cells with a drug. 5 Cancer cells have defects in regulatory circuits that govern the behavior of healthy cells. For cancer, these defects occur in the regulatory circuits that govem normal cell proliferation and homeostasis. Hanahan and Weinstein have outlined the essential alterations in cell physiology that collectively dictate malignant growth. These include: self-sufficiency in growth signals; insensitivity to growth-inhibitory (antigrowth) signals; evasion of programmed cell death 10 (apoptosis); limitless replicative potential; sustained angiogenesis; and tissue invasion and metastasis. Each of these physiologic changes is a result of alterations in the behavior of the proteins that control the underlying biochemical pathways. Such alterations are due to mutational changes as well as changes in the expression level of various proteins. As a result, the pathways controlling mitogenesis, apoptosis, the cell cycle, invasion and metastasis are 15 abnormally regulated. It follows that a successful therapeutic strategy involves re-routing the abnormal circuits of the cancer cell such that normal cellular behavior is restored. Therefore we sought to construct live cell-based assays for key cancer pathways (Figure 2) and to use these assays to screen for drugs capable of regulating these pathways. We started with the known pharmacopeia in order to identify potential hidden phenotypes of known drugs. The strategy and 20 the methods provided herein will also be useful for de novo drug discovery. Experimental Design The strategy involved constructing dynamic, pathway-specific screening assays in live cells; treating the cells with a drug; and assaying for previously-unsuspected activities of drugs 25 on the pathway(s) of interest. We constructed assays in human cells to 'read out' the activity of pathways known to be involved in the pathways of interest. The pathways were selected to represent the key hallmarks of the cancer cell outlined above. These include such well characterized pathways as the MAP kinase pathway (phosphoprotein ERK); the ras/raf oncogenic pathway (ras oncogene and raf kinase); cell cycle 30 pathways (Cyclin Dl, cyclin-dependent kinase CDK4, cell cycle progression kinase CDC2, transcription factor c-MYC and cell cycle regulatory protein p27); apoptotic pathways (BID, Il BAD and BCL-xL); the proteasome and chaperone systems (Ubiquitination; heat shock protein HSP90); and the actin cytoskeleton (cofilin, the CDC42 effector kinase PAK4, and the LIM kinase LIMK2). The proteins selected for assay construction are shown in Table I with a description of 5 their function. Although the biochemical functions of these proteins have been well characterized, the prior art is silent on the use of live cell assays for these proteins in the context of drug discovery. To achieve our objectives, it was necessary to satisfy the following criteria : (1) Assays must be constructed that are sufficiently sensitive to detect the dynamic or transient changes in 10 individual proteins, or protein-protein complexes, that occur upon pathway activation or inhibition; (2) The chosen assays must be capable of reading out the activity of a pathway in an intact cell, but the assay should not disrupt the biology of the cell or the pathway of interest; (3) Activation or inhibition of a particular pathway must be readily detectable and quantifiable; (4) Ideally, the methods will be suitable for scale-up and automation. 15 The effects of drugs on cellular pathways of interest were determined by measuring changes in the level and/or the subcellular location and/or post-translational modification status of protein complexes in cells following drug treatment. The assays we used can be performed with automated instrumentation, using automated microscopy, automated image analysis, or alternative fluorescence instrumentation which is described in detail below. 20 We used these methods to mine the known pharmacopeia for drugs with previously unsuspected activity against cancer-related pathways. Specifically, we screened drugs that have been used to treat patients for diseases other than cancer and screened for novel activities on cancer pathways. Hits from our cell-based screens were tested for antiproliferative activity in up to 5 different human tumor cell lines, as follows. First, drugs with significant activity in the 25 cellular screen(s) were tested for their ability to block proliferation of a human tumor cell line (PC-3) at an initial concentration of 10 micromolar. For drugs with antiproliferative activity in PC-3 cells, dose-response curves to determine the IC50 for antiproliferative activity and additional tumor cell lines were tested to determine the breadth of activity. Methods for assessing the antiproliferative activity of the cell-based drug 'hits' are described below. 30 Based on our initial screens of 960 known drugs and natural products, we identified over 20 drugs with unexpected activity on cancer-related pathways. For the majority of these, the 12 observed activity on cancer pathways represents a completely novel finding. These drugs are being further developed for potential clinical use in the treatment of cancer and other neoplasms. Methods for the construction of cell-based assays 5 To construct dynamic assays for protein-protein complexes, we used a protein-fragment complementation assay strategy (PCAs) in intact human cells. (A variety of other suitable assays, and reporter options, are described in detail below.) The principle of PCA is that complementary polypeptide fragments (F[l] and F[2]) of a reporter protein or enzyme will fold into an active form only if fused to two proteins which interact and bring the complementary 10 fragments of the reporter protein into proximity. Reconstitution of the fragments of the reporter protein generates a fluorescent signal that can be quantified in intact cells, and the amount of the signal is proportional to the amount of the protein-protein complex used in constructing the assay. Given a suitable reporter type, the subcellular location of the complex can also be measured. 15 We studied protein-protein interactions within key pathways regulating cell homeostasis. Specifically, we studied interacting proteins within pathways representing the processes of cell cycle control, DNA damage response, apoptosis, molecular chaperones, cytoskeletal regulation, proteasomal degradation, mitogenesis, inflammation, and nuclear hormone receptor activation. Proteins used to construct these particular assays are summarized in Tables 1 and 2. For 20 reporting out protein-protein interactions, we constructed PCAs based on fragments of fluorescent proteins; however, many other reporter proteins are suitable for use with PCA (see US 6,270,964 and the References herein) including enzymatic reporters such as dihydrofolate reductase (DHFR), beta-lactamase, luciferase, beta-galactosidase and others which are discussed in more detail below. 25 We also studied post-translational modifications of particular proteins within the same pathways in response to drugs. An ever-increasing assortment of phospho-specific antibodies enables probing of the phosphorylation status of individual proteins in tissues or cells, either in whole tissues or cells or in vitro in cell or tissue extracts. Phospho-specific antibodies are distributed by life sciences product companies including BD Biosciences 30 (www.bdbiosciences.com), Cell Signaling Technology (www.cellsignal.com) and a variety of other companies. These antibodies are directed specifically against phosphorylated antigens and 13 do not recognize the unphosphorylated form of the protein. Methods suitable for such assays are well known to those skilled in the art of cell biology. Immunofluorescence methods - combined with robotic systems and automated fluorescence microscopy - offer the additional potential for the development of high-throughput screens that combine the biological advantages of intact cell 5 detection with scalable, automated, high-throughput methods. Although the individual techniques we employed have been widely used in mechanistic biochemical research, the prior art is silent on the use of such methods to identify hidden phenotypes of drugs. We sought to apply such methods to identify new indications for known drugs and to carry out de novo drug discovery on a broad scale. 10 Protein-fragment complementation assays (PCA) Reporter fragments for PCA were generated by oligonucleotide synthesis (Blue Heron Biotechnology, Bothell, WA), starting with the sequence of yellow fluorescent protein (YFP). First, oligonucleotides coding for polypeptide fragments YFP[1]and YFP[21 (corresponding to 15 amino acids 1-158 and 159-239 of YFP) were synthesized. Next, PCR mutagenesis was used to generate the mutant fragments IFP[1] and IFP[2]. The IFP[1] fragment corresponds to YFP[1] (F46L, F64L, M153T) and the IFP[2] fragment corresponds to YFP[2]-(V163A, S175G). These mutations have been shown to increase the fluorescence intensity of the intact YFP protein (Nagai et al., 2002). The YFP[1], YFP[2], IFP[1] and IFP[2] fragments were amplified by PCR 20 to incorporate restriction sites and a linker sequence, described below, in configurations that would allow fusion of a gene of interest to either the 5'- or 3'-end of each reporter fragment. The reporter-linker fragment cassettes were subcloned into a mammalian expression vector (pcDNA3.]Z, Invitrogen) that had been modified to incorporate the replication origin (oriP) of the Epstein Barr virus (EBV). The oriP allows episomal replication of these modified vectors in 25 cell lines expressing the EBNA1 gene, such as HEK293E cells (293-EBNA, Invitrogen). Additionally, these vectors still retain the SV40 origin, allowing for episomal expression in cell lines expressing the SV40 large T antigen (e.g. HEK293T, Jurkat or COS). The integrity of the mutated reporter fragments and the new replication origin were confirmed by sequencing. PCA fusion constructs were prepared for a proteins known to participate in cellular 30 pathways that have been described in the scientific literature as being linked to cancer. The selection of protein-protein complexes used, and the rationale for their use, is provided in Table 14 1 and the gene identifiers for the cDNAs used in assay construction are provided in Table 2. The full coding sequence for each gene of interest was amplified by PCR from a sequence-verified full-length cDNA. Resulting PCR products were column purified (Centricon), digested with appropriate restriction enzymes to allow directional cloning, and fused in-frame to either the 5' or 5 3'-end of YFP[1], YFP[2], IFP[1] or IFP[2] through a linker encoding a flexible 10 amino acid peptide (Gly.Gly.Gly.Gly.Ser)2. The flexible linker ensures that the orientation or arrangement of the fusions is optimal to bring the reporter fragments into close proximity (Pelletier et al., 1998). Recombinants in the host strains DH5-alpha (Invitrogen, Carlsbad, CA) or XLI Blue MR (Stratagene, La Jolla, CA) were screened by colony PCR, and clones containing inserts of the 10 correct size were subjected to end sequencing to confirm the presence of the gene of interest and in-frame fusion to the appropriate reporter fragment. A subset of fusion constructs were selected for full-insert sequencing by primer walking. DNAs were isolated using Qiagen MaxiPrep kits (Qiagen, Chatsworth, CA). PCR was used to assess the integrity of each fusion construct, by combining the appropriate gene-specific primer with a reporter-specific primer to confirm that 15 the correct gene-fusion was present and of the correct size with no internal deletions. Transfections and cell preparation HEK293 cells were maintained in MEM alpha medium (Invitrogen) supplemented with 10% FBS (Gemini Bio-Products), 1% penicillin, and 1% streptomycin, and grown in a 37*C 20 incubator equilibrated to 5% C02. Approximately 24 hours prior to transfections cells were seeded into 96 well ploy-D-Lysine coated plates (Greiner) using a Multidrop 384 peristaltic pump system (Thermo Electron Corp., Waltham, Mass) at a density of 7,500 cells per well. Up to 100ng of the complementary YFP or IFP-fragment fusion vectors were co-transfected using Fugene 6 (Roche) according to the manufacturer's protocol. A list of the selected protein-protein 25 complexes (PCA pairs) screened in this study, is in Table 2. Following 24 or 48 hours of expression, cells were screened against the selected drugs as described below. For several PCAs, stable cell lines were generated. HEK293 cells were transfected with a first fusion vector and stable cell lines were selected using 100 pg/ml Hygromycin B (Invitrogen). Selected cell lines were subsequently transfected with the second, complementary 30 fusion vector, and stable cell lines co-expressing the complementary fusions were isolated following double antibiotic selection with 50 pg/ml Hygromycin B and 500 pg/ml Zeocin. For 15 all cell lines, the fluorescence signals were stable over at least 25 passages (data not shown). Approximately 24 hours prior to drug treatments, cells were seeded into 96 well ploy-D-Lysine coated plates (Greiner) using a Multidrop 384 peristaltic pump system (Thermo Electron Corp., Waltham, Mass). 5 Assessing drug activity on protein-protein complexes Drugs were screened in duplicate wells at a concentration of 10 micromolar. All liquid handling steps were performed using the Biomek FX platform (Beckman Instruments, Fullerton, CA). Cells expressing the PCA pairs were incubated in cell culture medium containing drugs for 10 90 min.and 8 hours, or in the case of pre-stimulation with camptothecin (CPT) for 16-18 hours. For some assays cells were treated with known pathway agonists immediately prior to the termination of the assay. Following drug treatments cells were stained with 33 micrograms/ml Hoechst 33342 (Molecular Probes) and fixed with 2% formaldehyde (Ted Pella) for 10 minutes. In some cases cells were simultaneously stained with Hoechst and with 15 micrograms/ml 15 TexasRed-conjugated Wheat Germ Agglutinin (WGA; Molecular Probes), and then fixed. Cells were subsequently rinsed with HBSS (Invitrogen) and maintained in the same buffer during image acquisition. YFP, Hoechst , and Texas Red fluorescence signals were acquired using the Discovery-I automated fluorescence imager (Molecular Devices, Inc.) equipped with a robotic arm (CRS 20 Catalyst Express; Thermo Electron Corp., Waltham, Mass). The following filter sets were used to obtain images of 4 non-overlapping populations of cells per well: excitation filter 480/40nm, emission filter 535/50nm (YFP); excitation filter 360/40nm, emission filter 465/30nm (Hoechst); excitation filter 560/50nm, emission filter 650/40nm (Texas Red). All treatment conditions were run in duplicate yielding a total of 8 images for each wavelength and treatment condition. 25 16 Table 1. Assays used to demonstrate the invention and their rationale Assay Brief Assay Description Key node for apoptotlc signaling. Bad complexes with BclxL and BcI-2 block the anti-apoptotic activity of the latter two proteins Indicates apoptotic activity V Key node for apoptotic signaling. Bid complexes with BclxL and BcI-2 block the anti-apoptotic activity of the latter two proteins HSP90 is key chaperone regulating protein stability/activity/half-life. CDC37 Is co-chaperone; determines vHW a 0WeD ,3 activity and client protein selectivity small GTPase/kinase signaling node. PAK4 is CDC42 effector; transmits the signal from the molecular CDC PAK4-. switch to downstream substrates such as LIMK. BAD - $' key cell cycle control node Chk kinases regulate CDC25 phosphatases; activation Indicates cell cycle checkpoint activation: CPT Chk1.M P +eb (camptothecin) topolsomerase inhibitor causes DNA damage and activates checkpoints 1CD W~ Chk kinases regulate CDC25 phosphatases: activation Indicates cell cycle checkpoint activation hki'.CbC25C ~ Chk kinases regulate CDC25 phosphatases: activation Indicates cell cycle checkpoint activation .Coflliin~lMK ~ LIM kinases phospohorylate cofiln and regulate cytoskeletal dynamics Hsp9O Eef2k ;.. translation factor-controlling kinase Eef2k is HSP client protein ER receptor tyrosine kinase:adaptor protein complex: indicates activated receptor ERK mitogen-activated protein kinase interacts with and phosphorytates the Elk-1 (Ets family) transcription factor small GTPase/kinase signaling node. Ras is commonly mutated human oncogene; activates ERK/MAP kinase path among others; downstream from receptor tyrosine kinases and some G-proteins .MY c-Myc is a transcription factor and human proto-oncogene. Activity correlates with cell cycle progression - C complex of upstream activator PAK4 with downstream effector collin; regulates actin cytoskeleton *%dA z TGF beta responsive transcription factor Smad3 in nuclear with histone deacetylase C 2 vkinase Weel Is negative regulator of Cdc2 (cell cycle progression kinase) Intersection of key anti-apoptotic (Akt) and cell cycle regulatory (p27) signaling nodes. Both targets Invovled in human tumors. p27 Is key cell cycle regulator loss is associated with human tumor progression. p27 levels are controlled by ubiquitination. C C . Phosphatase/kinase complex; activity leads to cell cycle progression E 5 indicates p53 primed for proteasomal degradation increased Interaction and dimerization of p53 Indicates heightened activity of this node ERK mitogen-activated protein kInase is activated by signaling through the vascular endothelial growth ERkPVEGF factor pathway 17 Table 2. Description, Genbank identifiers and reporter fusion orientations for protein fragment complementation assays PCA Descripti Stimulation Genbank Reporter rusion Genbank Renorter Gene 01 orientation Gene 02 fion orientation BAD:BID NM_004322 N NM_001196 C Bcl-xL: Bad NM 138578 C NM_004322 N Bel-xL: BIK NM 138578 N NM 001197 N Cdc2:Cdc25A +CPT 500nM CPT; 16 hrs NM_001786 N NM_001789 C Cdc2:CDC25C NM_001786 N NM_001790 C Cdc2:CDC25C +CPT 500nM CPT; 16 hrs NM_001786 N NM_001790 C Cdc2:WeeI NM_001786 N NM_009516 N CDC42:PAK4 NM_001791 N NM_005884 C ChkI:CDC25A +CPT 500nM CPT; 16 hrs NM_001274 N NM_001789 C Chklc:CDC25C NM_001274 N NM_001790 C ChkI:CDC25C+CPT 500nM CPT;16 hrs NM 001274 N NM 001790 C Cofilin:LMK2 NM_005507 C NM_005569 N CyclinD:Cdk4 NM053056 N NM001791 C CDC25C:CDC2 NM_001274 N NM_001786 N E6:p53 AJ388069 N NM000546 N H-Ras:Raf NM_005343 N NM_002880 C Hsp9O:CDC37 NM_007355 C NM_007065 N Hsp9O:Eef2k NM007355 C NM_007908 N MAX: MYC NM_002382 C NM 002467 C CDC2: p21 NM_001786 N NM_000389 N P27:UbiquitinC NM004064 N NM_021009 N (CDS 69..296) p53:Chkl NM_000546 C NM_001274 N p53:Chkl +CPT 500nM CPT; 16 hrs NM_000546 C NM_001274 N p53:p5 3 NM000546 C NM_000546 C p53:p53 +CPT 5O0nM CPT; 16 hrs NM_000546 C NM000546 C PAK4:Coflin NM 005884 C NM_005507 C Smad3:HDAC NM 005902 N NM_004964 C 18 Immunofluorescence methods Immunofluorescence was performed on drug-treated cells to assess the post-translational modification status of proteins involved in the pathways of interest. We constructed assays designed to measure the phosphorylation status of key signaling proteins in the absence and 5 presence of a growth factor stimulus. A drug capable of blocking or inhibiting the pathway leading to the signaling protein would in principle cause a decrease in the phosphorylation of that signaling protein in response to the selected growth factor. Such a change in phosphorylation status could be measured by a decrease in fluorescence in the presence of the drug. To exemplify the approach, we studied changes in the phosphorylation status of the protein kinase, 10 ERK (mitogen activated protein kinase) in the MAP kinase pathway that is linked to the angiogenic growth factor, VEGF (vascular endothelial growth factor). HEK293T cells were seeded at a density of 7,500/well in poly-D-lys coated, blackwalled 96 well plate (Greiner). After 24 hours, cells were transfected with 1Ong/well mVEGFR2 in the pCDNA3.1 expression vector. Forty-eight hours following transfection, the cells were incubated 15 in the absence or presence of indicated drugs for 90 min. The cells were stimulated with 50ng/ml mVEGF (R & D Systems) during the last 5 min of drug treatment and fixed with 4% formaldehyde in PBS for 15 min. For antibody staining, cells were permeabilized with 0.25% Triton X-100 for 6 min and non-specific staining was blocked by incubating the cells with 3% BSA in PBS for 15 min. Phosphorylated ERK was detected by incubating the fixed cells with 20 rabbit phospho-ERK (T202/Y204) - specific antibodies (Cell Signaling Technologies) followed by Alexa488 conjugated goat anti-rabbit antibody (Molecular Probes). Cell nuclei were stained with Hoechst 33342 (Molecular Probes). A solution of 5% glycerol (in PBS) solution was used to overlay the cells. Fluorescence images were acquired on a Discovery-1 imaging station (Molecular Devices) as described above. 25 Background fluorescence due to nonspecific binding by the secondary antibody was established with the use of cells that were incubated with BSA/PBS and without primary antibodies. Fluorescence image analysis 30 To allow quantitation of the drug effects observed by fluorescence microscopy in the PCA and immunofluorescence assays, we applied image analysis algorithms to the images 19 acquired by automated microscopy. A variety of commercial software packages for the analysis of cell-based 'high content' assays are suitable for this purpose and are commercially available (Cellomics; GE Medical/Amersham; BectonDickinson/Atto Bioscience; Beckman Coulter/Q3DM; and others). We used publicly-available software (ImageJ API/library 5 (http://rsb.info.nih.gov/ij/, NIH, MD) to analyze the raw images in 16-bit grayscale TIFF format. First, images from the fluorescence channels were normalized using the ImageJ built-in rolling ball algorithm [S.R. Stenberg, Biomedical image processing. Computer, 16(1), January 1983). Next a threshold was established to separate the foreground from background. An iterative algorithm based on Particle Analyzer from ImageJ was applied to the thresholded Hoechst 10 channel image (HI) to obtain the total cell count. The nuclear region of a cell (nuclear mask) was also derived from the thresholded HI. The positive particle mask was generated from the thresholded YFP image (YI). To calculate the global background (gBG), a histogram was obtained from the un-thresholded YI and the pixel intensity of the lowest intensity peak was identified as gBG. Masks from different fluorescence channels were overlapped to define the 15 correlated sub-regions of the cell. The mean pixel intensity for all positive particles within each defined sub- region was calculated, resulting in multiple parameters: MT, the mean intensity of the total fluorescence); Ml, the mean intensity of the Hoechst defined region); M2, the mean intensity of the WGA-defined region, where used; and M3, the mean intensity of the pixels excluded from the other regions). All means were corrected for the corresponding gBG. 20 For each set of experiments (assay + drug treatment + treatment time), fluorescent particles from eight images were pooled. For each parameter,- an outlier filter was applied to filter out those particles falling outside the range (mean * 3SD) of the group. The sample mean or control mean for each parameter was obtained from each filtered group. 25 MTT Proliferation Assays Human non-small cell lung carcinoma (A549, ATCC # CCL-185), colon adenocarcinoma (LoVo, ATCC # CCL-229), pancreatic carcinoma (MIA PaCa-2, ATCC # CRL-1420 ), prostate adenocarcinoma (PC-3, ATCC # CRL-1435) , and glioblastoma (U-87 MG, ATCC # HTB-14) cells were acquired from American Type Culture Collection (ATCC, Manassas, VA). Cells were 30 maintained in various media as follows: A549, LoVo and PC-3 (Ham's F12K medium with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate ), MIA PaCa-2 (Dulbecco's modified Eagle's 20 medium with 4 mM L-glutamine and 4.5 g/L glucose), U87-MG (MEM + Earle's BSS). Medium for each cell line was supplemented with 10% FBS and 100mg/ml Penecillin/Streptomycin. All cells were grown in incubators set at 37*C, 5% CO 2 . Thiazolyl Blue Tetrazolium Bromide (MTT) based proliferation assays were performed to assess the anti-proliferative activities of the 5 compounds on these cells. Cells were seeded in 96 well plates at a density of 750 cells/well 24 hours prior to compound treatment. The cells were incubated with varying concentrations of compounds for 120 hours. Compound concentrations range from 0.03 to 100 microM (half log increments) except for alpha-Tomatine (0.001 - 100 microM, half log increments), Neriifolin (0.0002-100 microM) and Peruvoside (0.01-100 microM). Drug treatment was performed in 5 10 replicate wells. Background absorbance was established by wells containing medium but no cells. Vehicle (DMSO) only was used as control. MTT (Sigma-Aldrich, St. Louis, MO) was added to each well at a final concentration of 0.5mg/ml. Following a 2 hour incubation at 37 0 C, medium in the wells was replaced with 0.15 ml DMSO. The plates were agitated for 15 min using a microtiter plate shaker. Absorbance at 560nM was measured using SpectraMax Plus 15 (Molecular Devices). Mean absorbance values were calculated from 5 replicate wells of each drug treatment following subtraction of background absorbance from blank samples and plotted as a percentage of control. 21 Table 3. Results of screening known drugs to identify new activities A4.& T-. C.0 pooihod RIAW"o Pak.m Ooroks Pp Y M~oo~d VS IC40 m1ghuI D," t. 0-!b.. Os.~u BOduaftI (Y"~ A~f Amn odloo f -10 kn WWof NSCILC 4.74 C42-YN.. C*, 3.60 nodonlos PW* W; 1 58 008080' Co.10 &.~onl0', P ~o 809 IMI VAIMS WOW I FD Apmdlqe NO llwmetsf 11C420PAK4 111lasiololo 51 75 m S5O.C 5.1 Won ?. 14 PM~km1 Po 1 0.1 AS-. CW'o.t*. Mao.bw Jo s OULS PM Wp b CCC42,PMC4 GIOn 13.s5 P"UPI* ?800.C 0.004 D"* CO.S= NOMA 0-0 75o. rcodo. 7 calm08 0.58 111 875.008 04588P5088 0186 7.ob" S08lo11 Wk9W8 ______ 03- -m* M A______ _____ ____0 U.S. N- N. pad Cm d Rm AMM 0.34*8Z ~ Q INkCLC 0.58 Cob' 0.00 lftS0 poa"Ift 0.15 llmoW&. O r-kn I Plomm 1. 3 f..l T4,00,.8f Mew"b ""Cho0 W____ mk No ____o ICDC4ZOMA4 GSMbb,,, IS28 py -Mm MI 500SVSWo". NGO.C ;I.8 l~50sGo" ISO 5080055PaumSad 1.93 P5*658,5CA*" WLogffo.0I. A"S*6 FD~W~ Ek Ya es ~ d RnRO G~OMOMa 5.0 W Cmmtia am.M58K 0211128. NSCILC ;5,5 KT111o2. CWb 12 llYp.,dkb0 CkIXM. PS88ot 14 p,,I8; NSCr 50. 3 PIO.k 1.0 a 85n,50 NSCLC I.0 Cob, 3. Pan80o.1c 5.53 MSQG. 5.23 Colon 1.13 paa8c 15 PAX44V*1 PMUM8 15 dtn'O,01 00000 0,.,- Q0.1" US Ma #0 ,o8 O4A4 C0AO., 02 01ooo.bo 22 Table 3 (continued) Tw.01 CaO p10004.60 Mmll'ng Pst418 Gwk. path"'." 1.00.8 . IC40 04031 C.PU4Mls Sr-!m. Drll beu 000106 64610103 b~.l (Y A..ydo01ol T~4M~d00 ________ OSOC I., Nee wkm h,.uS4. I MR*.~ P.1141 6.10 n'Aa, 0,04 _______ olole1 U.S. No8 ffwutS1 COC421AC4 00680635,4. 460 pce b NSPOW.139 NSQ..C 2 .6 COMh~UU63. cow8 .4. PINIJUN. P.-.148 0.50 P000516103. P0016 0.3 GelOC 0 i I A~L pmsko aal 0,1,1104l 04H18,W0400AlW01 NOI In U.S. Nm 11W 1.04s6060 1C42F4414 -b.a6 1480 NS2C 1.23 Ck~ Ctess'e C.IO tio8g.. 340. Pac flh3 Cf.mApoe - I PAK4MFL Ps001*. P.043 of p.14.bk0. j-.0, .06.w4 P Us N-w 318 -Unsl CDC42/P*J04 81464664 sees_ NS=L 0.092 0010' 007 P6..f i 131 046100mb 81.~IP 009 P016460k 0.16 A106146Z06 Atoms owo48u 0m. FA Dn E.9h No8 .0.14 C42/PA4 37nU01.W __ Ar48091810 Pg.0.06 2.13 IPloaw 027 8008041 V6,. ______104"6 FDAp.. PE~k Ye O~a PAK4CFL I0180461066 44.4.909 340.0 0.35 N=~u6C 9,3 95020 10.6 Cob. Pabciadc 06.21 NOela ________ Oftge61 F90060 In ml0t .0e44 41PAK4 0101mm wtalelli________ N2CLC .40 AN-MOM41 .1146 _______ FDA.P0 AOO-w.4 Protected 96. ff014014616 I7FLIIAK4 PG18018000.. 4.0 I'm 080. 1 .4 EGOO,09 P090646 2.77 SIP814: he$ M-606.es.01 PUM06. P.61* 200 k-0 6. 011g4Japadlop Hz___ 04 U.& 3048 "a 1001.040 CG0I8 00408601.0 2.86 cancet1 aoy CD0C25C. Me=. ;.22 ECFRIC92 cow 2.21 E602lftPOD. Paweaf 2.4? .030O3. P0418 .8 23 RESULTS Photomicrographs showing specific assay results are shown in Figure 3A-3G. Antiproliferative activities of selected drugs are shown in Figs. 4-10. A summary of drugs that 'hit' specific pathways is shown in Table 3, together with the assay activities and the activity of 5 each drug on the proliferation of human tumor cell lines as assessed in the MTT assay. The IC50 for proliferation (concentration of each drug that inhibits proliferation by 50%) is shown in Table 3 for each tumor cell line that was tested. A variety of drug mechanisms of action may underlie the activities seen here. For example, the amount of a protein complex may vary as a result of increased formation, decreased 10 formation, or a decrease in stability of one or more of the components. Biochemical mechanisms underlying the changes include changes in post-translational modification status of one or more proteins in the complex; inhibition of chaperone function; proteasome inhibition; pathway inhibition in the presence of a stimulus; direct inhibition of a protein-protein interaction; and other potential mechanisms. Any of these mechanisms may result in a change in the amount, 15 subcellular location, or post-translational modification status of the cognate complexes, as measured here. There are several features of our results that are particularly interesting. First, the entire screening process is extremely fast: the identification.of drugs with promising antiproliferative activity could be completed in two weeks following assay construction. Second, the pathway 20 based screens we constructed were remarkable indicators of antiproliferative activity with individual assays giving positive predictive values that in most cases exceeded 50% (Figure 11). Combinations of assays gave positive predictive values of 70-80%. Third, many of the drugs hit two or more cancer pathways. This supports our notions of the interconnectivity of cellular networks and also shows that even well-characterized drugs have significant and surprising 'off 25 pathway' effects. Fourth, the number of drugs found to have anti-proliferative activity in this proof-of-principle study was 23/960 or over 2%, which is a substantial rate in terms of reindicating drugs for new therapeutic uses. This also suggests that the overall strategy presented here, if applied to a broad range of disease pathways, will provide a powerful strategy for identifying medicines that can be fast-tracked for the treatment of human disease. Since 30 these drugs have well-characterized safety profiles they can quickly be advanced into clinical trials for the new therapeutic indications. 24 Remarkably, in most cases, the doses of drugs that inhibited proliferation of tumor cells were in the low-micromolar range and in fact well within the range of plasma levels for that drug (where documented in the literature). It will be appreciated by one skilled in the art that the exact sentinels (proteins, and 5 protein-protein interactions) to be used for this strategy will depend upon the disease of interest and that the invention is not limited to the particular pathways, proteins, or sentinels provided herein or to a particular mechanism by which a drug affects that pathway. For example, to identify anti-proliferative agents, we used pathways that contribute to the cancer phenotype. However, the methodology applied in the current invention is not limited to the identification of 10 anti-cancer activities of drugs. For other diseases we are probing pathways characteristic of those disorders: e.g. for diabetes, we are studying pathways involved in glucose transport, glycogenesis, insulin receptor regulation and insulin signaling pathways. For bone disease, we are using pathways that are involved in bone remodeling and the differential activity of osteoclasts and osteoblasts. For neurological disorders we are using pathways that are 15 downstream of the dopamine receptor and the serotonin receptor. The invention can also be applied to cell types other than mammalian cells. For example, the invention can be applied to the discovery of antibiotic agents, antifungal agents, antiviral agents, and other infectious diseases. In these cases the cell of interest (bacterial, fungal, etc.) can be used to construct the assays, in conjunction with the pathways/proteins of interest for the 20 disease in question. For example, an agent that disrupts a pathway that is key for the survival of a bacterial cell may be a useful antibiotic agent. In the case of antiviral agents, mammalian cells can be used for the readout and viral/host interacting proteins can be used to construct the assays. For example, an agent that disrupts a host protein receptor or a viral/host protein-protein interaction - either directly or indirectly - may have utility in the prevention or treatment of viral 25 infection. Many ideas for new assays can be gleaned from the biochemical literature and applied, in conjunction with the methods provided herein, to identifying new indications for known drugs. The genes to be used in the assays may code either for known or for novel interacting proteins. The interacting proteins may be selected by one or methods that include bait-versus-library 30 screening; pairwise (gene by gene) interaction mapping; and/or prior knowledge or a hypothesis regarding a pathway or an interacting protein pair. In addition, novel pathways that are useful 25 for drug discovery can be identified empirically by constructing assays for novel protein protein interactions; determining if these are responsive to agents known to affect the pathway(s) of interest; and using the resulting novel assays to screen for known drugs as well as for new chemical entities with desired activities. 5 METHODS SUITABLE FOR USE WITH THE SCREENING SYSTEM The screening system presented here can be used in several different modes including high-throughput screens (HTS) and high-content screens (HCS). In the case of purely quantitative assays (HTS), the signal generated in the assay is quantified with a microtiter 10 fluorescence plate reader, flow cytometer, fluorimeter, microfluidic device, or similar devices. The intensity is a measure of the quantity of the protein-protein complexes formed and allows for the detection of changes in protein-protein complex formation in live cells in response to agonists, antagonists and inhibitors. In the case of high-content assays (HCS), cells are imaged by automated microscopy, confocal, laser-based, or other suitable high-resolution imaging 15 systems. The total fluorescence per cell as well as the sub-cellular location of the signal (membrane, cytosol, nucleus, endosomes, etc.) can be detected. Cell fixation offers advantages over live cell assays for purposes of laboratory automation, since entire assay plates can be fixed at a specific time-point after cell treatment, loaded into a plate stacker or carousel, and read at a later time. 20 The choice of HTS or HCS formats is determined by the biology and biochemistry of the signaling event and the functions of the proteins being screened. It will be understood by a person skilled in the art that the HTS and HCS assays that are the subject of the present invention can be performed in conjunction with any instrument that is suitable for detection of the signal that is generated by the chosen reporter. 25 In addition to the use of live (fixed or unfixed) cells, cell lysates can be prepared following drug treatment and can be used in the present invention. Finally, for specific protein protein interactions of interest, in vitro assays can be constructed using the methods described herein and used to further study the mechanism of action of any assay hits; to facilitate studies of structure-activity relationships; and to enable de novo discovery of new chemical entities with 30 desired activities. 26 Methods for the detection or measurement of protein-protein interactions PCA represents a preferred embodiment of the invention. PCA enables the detection and quantitation of the amount and/or subcellular location of protein-protein complexes in living cells. With PCA, proteins are expressed as fusions to engineered polypeptide fragments, where 5 the polypeptide fragments themselves (a) are not fluorescent or luminescent moieties; (b) are not naturally-occurring; and (c) are generated by fragmentation of a reporter. Michnick et al. (US 6,270,964) taught that any reporter protein of interest can be used for PCA, including any of the reporters described in Table 4. Thus, reporters suitable for PCA include, but are not limited to, any of a number of monomeric or multimeric enzymes; and fluorescent, luminescent, or 10 phosphorescent proteins. Small monomeric proteins are preferred for PCA, including monomeric enzymes and monomeric fluorescent proteins, resulting in small (-150 amino acid) fragments. Since any reporter protein can be fragmented using the principles established by Michnick et al., assays can be tailored to the particular demands of the cell type, target, signaling process, and instrumentation of choice. Finally, the ability to choose among a wide range of 15 reporter fragments enables the construction of fluorescent, luminescent, phosphorescent, or otherwise detectable signals; and the choice of high-content or high-throughput assay formats. It will be apparent to one skilled in the art that the choice of expression vector depends on the cell type for assay construction, whether bacterial, yeast, mammalian, or other cell type; the desired expression level; the choice of transient versus stable transfection; and other typical 20 molecular and cell biology considerations. A wide variety of other useful elements can be incorporated into appropriate expression vectors, including but not limited to epitope tags, antibiotic resistance elements, and peptide or polypeptide tags allowing subcellular targeting of the assays to different subcellular compartments (e.g. A Chiesa et al., Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signaling). The incorporation of a 25 different antibiotic resistance marker into each of the two complementary constructs would allow for the generation of stable cell lines through double antibiotic selection pressure, whereas subcellular targeting elements would allow for the creation of assays for pathway events that occur within a particular subcellular compartment, such as the mitochondria, Golgi, nucleus, or other compartments. 30 A variety of standard or novel expression vectors can be chosen based on the cell type and desired expression level; such vectors and their characteristics will be well known to one 27 skilled in the art and include plasmid, retroviral, and adenoviral expression systems. In addition, there is a wide range of suitable promoters including constitutive and inducible reporters that can be used in vector construction. If an inducible promoter is used, the signal generated in the assay will be dependent upon activation of an event that turns on the transcription of the genes encoded 5 by the PCA constructs. The general characteristics of reporters suitable for PCA have previously been described (References incorporated herein). A preferred embodiment of the present invention involves cell-based assays generating a fluorescent or luminescent signal are particularly useful. Examples of reporters that can be used in the present invention have been provided in the 10 References. It will be appreciate by one skilled in the art that the choice of reporter is not limited. Rather, it will be based on the desired assay characteristics, format, cell type, spectral properties, expression, time-course and other assay specifications. For any reporter of interest various useful pairs of fragments can be created, for example using the methods taught in US 6,270,964 and the References incorporated herein, and then engineered in order to generate 15 fragments that produce a brighter signal or a specific color readout upon fragment reassembly. It will be obvious to one skilled in the art that various techniques of genetic engineering can be used to create useful fragments and fragment variants of any of the reporters that are the subject of this invention. It will be appreciated by a person skilled in the art that the ability to select from among a 20 wide variety of reporters makes the invention particularly useful for drug discovery on a large scale. In particular, reporters can be selected that emit light of a specific wavelength and intensity that may be suitable for a range of protein expression levels, cell types, and detection modes. The flexibility is an important feature of the invention because of the wide range of signaling events, or biochemical processes, that may be linked to drug activity. For some 25 biochemical events, activation of a pathway - for example, by the binding of an agonist - will lead to an increase in the association of a receptor and a cognate binding protein, or of two elements 'downstream' in the pathway, such as a kinase and its substrate. An increase in the association of the two proteins that form the PCA pair leads to an increase in the signal generated by the reassembled reporter fragments. In that case, a high-throughput assay format can be used 30 to measure the fluorescent signal that is proportional to the amount of the complex of interest. For quantitative assays, where the readout is an increase or decrease in signal intensity, any of 28 the reporters discussed in the present invention can be used and each reporter has various pros and cons that are well understood by those skilled in the art of cell biology. Enzymes - for which the catalytic reaction generates a fluorescent, phosphorescent, luminescent or other optically detectable signal - may be best suited for purely quantitative assays. Upon fragment 5 complementation, the reconstituted enzyme acts upon a substrate to generate a fluorescent or luminescent product, which accumulates while the reporter is active. Since product accumulates, a high signal-to-noise can be generated upon fragment complementation. Such assays are particularly amenable to scale-up to 384-well or 1536-well formats and beyond, and are compatible with standard and ultra high-throughput laboratory automation. 10 Preferred reporters for the present invention include but are not limited to a beta lactamase PCA or a luciferase PCA such as with a firefly luciferase or Renilla luciferase. Each of these enzymes has been successfully used as a cell-based reporter in mammalian systems (S Baumik & SS Gambhir, 2002, Optical imaging of renilla luciferase reporter gene expression in living mice, Proc. Natl. Acad. Sci., USA 2002, 99(1): 377-382; Lorenz et al., 1991, Isolation and 15 expression of a cDNA encoding renilla reniformis luciferase, Proc. Nati. Acad. Sci. USA 88: 4438-4442; G. Zlokarnik et al., 1998, Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter, Science 279: 84-88). As an example of the construction of a PCA, beta-lactamase PCAs have been constructed with cell-permeable substrates that generate a high signal to background upon cleavage (A Galameau et al., 2000, 20 Nature Biotechnol. 20: 619-622). The beta-lactamase PCA is a sensitive and quantitative assay suitable for HTS. This PCA has been used with CCF2/AM, a green fluorescent molecule which becomes blue upon cleavage of the beta-lactarn ring by beta-lactamase; the blue-green ratio is therefore a measure of the activity of beta-lactamase which is reconstituted upon protein fragment complementation. Luciferase PCAs can also be used with cell-permeable substrates to 25 generate HTS assays suitable for the present invention (e.g. R Paulmurugan et al., 2002, Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies, Proc. Natl. Acad. Sci. USA 99: 15608-15613). With suitable modifications, any of these PCAs can also be used in vivo or in vitro for the present invention. It will be apparent to one skilled in the art that PCAs based on inherently 30 fluorescent, phosphorescent or bioluminescent proteins can be read either in high-content formats or in high-throughput formats. These PCAs have the advantage of not requiring the 29 addition of substrate; however, the signal generated is usually lower than that generated by an enzymatic reporter. Calcium-sensitive photoproteins would be useful as PCAs for such assays. These could be based on fragments of aequorin, obelin; or any other calcium-sensitive protein (e.g. MD 5 Ungrin et al., 1999, An automated aequorin luminescence-based functional calcium assay for G protein-coupled receptors, Anal Biochem. 272: 34-42; Rizzuto et al., 1992, Rapid changes of mitochondrial calcium revealed by specifically targeted recombinant aequorin, Nature 358 (6384): 325-327; Campbell et al., 1988, Formation of the calcium activated photoprotein obelin from apo-obelin and mRNA in human neutrophils, Biochem J. 252 (1):143-149). Aequorin, a 10 calcium-sensitive photoprotein derived from the jellyfish Aequorea victoria, is composed of an apoprotein (molecular mass -21 kDa) and a hydrophobic prosthetic group, coelenterazine. Calcium binding to the protein causes the rupture of the covalent link between the apoprotein and the coelenterazine, releasing a single photon. The rate of this reaction depends on the calcium concentration to which the photoprotein is exposed. Intact aequorin with coelenterazine 15 has been used to monitor calcium flux in cell-based assays. Obelin is a 22-kDa monomeric protein that also requires coelenterazine for signal generation. Construction of an aequorin PCA or an obelin PCA would enable assays in which photon release only occurs if the reporter fragments are associated as a result of a ligand-protein interaction or a protein-protein interaction. Such an assay would combine measures of pathway activation with calcium flux, 20 making the assays extraordinarily sensitive for pathway-based studies. Although small monomeric reporters are preferred for this invention due to the small size of the reporter fragments, it will be apparent from the prior art that multimeric enzymes such as beta-galactosidase, beta-glucuronidase, tyrosinase, and other reporters can also be used in the present invention. A number of multimeric enzymes suitable for PCA have previously been 25 described (US 6,270,964). Fragments of multimeric proteins can be engineered using the principles of PCA described in the prior art; alternatively, naturally-occurring fragments or low affinity subunits of multimeric enzymes can be used including the widely-used beta galactosidase ot and o complementation systems. Beta -galactosidase (beta-gal) is a multimeric enzyme which forms tetramers and octomeric complexes of up to I million Daltons. Beta-gal 30 subunits undergo self-oligomerization which leads to activity. This naturally-occurring phenomenon has been used to develop a variety of in vitro, homogeneous assays that are the 30 subject of over 30 patents. Alpha- or omega-complementation of beta-gal, which was first reported in 1965, has been utilized to develop assays for the detection of antibody-antigen, drug protein, protein-protein, and other bio-molecular interactions. The background activity due to self-oligomerization has been overcome in part by the development of low-affinity, mutant 5 subunits with a diminished or negligible ability to complement naturally, enabling various assays including for example the detection of ligand-dependent activation of the EGF receptor in live cells (Rossi and Blau). These low-affinity subunits can be used to construct assays in conjunction with the present invention. For some pathways, activation of the pathway leads to the translocation of a pre-existing 10 protein-protein complex from one sub-cellular compartment to another, without an increase in the total number of protein-protein complexes. In that case, the fluorescent signal generated by the reassembled reporter at the site of complex formation within the cell can be imaged, allowing the trafficking of the complex to be monitored. Such "high-content" PCAs can be engineered for any suitable reporter for which the signal remains at the site of the protein-protein complex. 15 Examples include the DHFR PCA, which has been used for high-content assays of signal transduction pathways (I Remy & S Michnick, 2001, Visualization of Biochemical Networks in Living Cells, Proc Natl Acad Sci USA, 98: 7678-7683) and also for high-throughput assays (I Remy et al., 1999, Erythropoietin receptor activation by a ligand-induced conformation change, Science 283: 990-993). Reconstituted DHFR binds methotrexate (MTX); if the MTX is 20 conjugated to a fluorophore such as fluorescein, Texas Red, or BODIPY, the PCA signal can be localized within cells. Additional reporters particularly useful for high-content assays are described in US 6,270,964 and include the green fluorescent protein (GFP) from Aequorea victoria. PCAs based on GFP, YFP, and other inherently fluorescent, luminescent or phosphorescent protein reporters are preferred embodiments of the present invention. Any 25 number of fluorescent proteins have been described in the scientific literature (e.g. RY Tsien, 1998, The Green Fluorescent Protein, in: Annual Reviews of Biochemistry 67: 509-544; J Zhang et al., 2000, Creating new fluorescent probes for cell biology, Nature Reviews 3: 906 918). Any mutant fluorescent protein can be engineered into fragments for use in the present invention. Suitable reporters include YFP, CFP, dsRed, mRFP, 'citrine', BFP, PA-GFP, 'Venus', 30 SEYFP and other AFPs; and the red and orange-red fluorescent proteins from Anemonia and Anthozoa. 31 Reporters generating a high signal-to-background are preferred for the present invention. For example, PCAs based on YFP, SEYFP, or 'Venus' (T Nagai et al., 2002, A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications, Nature Biotech. 20: 87-90) are particularly suitable for the present invention. PCAs based on proteins 5 for which the signal can be triggered, such as a kindling fluorescent protein (KFPI) (DM Chudakov et al., 2003, Kindling fluorescent proteins for precise in vivo photolabeling, Nat. Biotechnol. 21, 191-194), a photo-converting fluorescent protein such as Kaede (R Ando et al., 2002, An optical marker based on the uv-induced green-red photoconversion of a fluorescent protein, Proc. Natl. Acad. Sci. USA, 2002, 99 (20): 12651-12656), or a photoactivatable protein 10 such as PA-GFP (GH Patterson et al., 2002, A photoactivatable GFP for selective labeling of proteins and cells, Science 297: 1873-1877) may have advantages, particularly in cases where it is necessary to capture very rapid signaling events. KFPI is derived from a unique GFP-like chromoprotein asCP from the sea anemone Anemonia sulcata. asCP is initially nonfluorescent, but in response to intense green light irradiation it becomes brightly fluorescent (kindles) with 15 emission at 595 nm. Kindled asCP relaxes back to the initial nonfluorescent state with a half-life of <10 seconds. Alternatively, fluorescence can be "quenched" instantly and completely by a brief irradiation with blue light. The mutant (asCP A148G, or KFPI) is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form that has 30 times greater fluorescent intensity than the unkindled protein, making it particularly 20 suitable for live cell PCAs. Alternative techniques for measuring protein-protein interactions are equally suitable for this invention. The most widespread fluorescent, cell-based protein-protein interaction assays to date are based on the phenomenon of fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). In a FRET assay the genes for two different 25 fluorescent reporters, capable of undergoing FRET are separately fused to genes encoding of interest, and the fusion proteins are co-expressed in live cells. When a protein complex forms between the proteins of interest, the fluorophores are brought into proximity if the two proteins possess overlapping emission and excitation, emission of photons by a first, "donor" fluorophore, results in the efficient absorption of the emitted photons by the second, "acceptor" 30 fluorophore. The FRET pair fluoresces with a unique combination of excitation and emission wavelengths that can be distinguished from those of either fluorophore alone in living cells. As 32 specific examples, a variety of GFP mutants have been used in FRET assays, including cyan, citrine, enhanced green and enhanced blue fluorescent proteins. With BRET, a luminescent protein- for example the enzyme Renilla luciferase (RLuc) -is used as a donor and a green fluorescent protein (GFP) is used as an acceptor molecule. Upon addition of a compound that 5 serves as the substrate for Rluc, the FRET signal is measured by comparing the amount of blue light emitted by Rluc to the amount of green light emitted by GFP. The ratio of green to blue increases as the two proteins are brought into proximity. Newer methods are in development to enable deconvolution of FRET from bleedthrough and from autofluorescence. In addition, fluorescence lifetime imaging microscopy (FLIM) eliminates many of the artifacts associates 10 with quantifying simple FRET intensity. Alternative methods for the measurement of protein protein interactions can also be used for this invention. These include assays based on split ubiquitin, as well as two-hybrid and three-hybrid assays and similar approaches for the detection and measurement of protein-protein complexes. Many of these systems have been adapted for the generation of fluorescence signals in intact cells. 15 Methods for the detection of post-translational modifications of proteins The present invention teaches that cell-based fluorescence or luminescence assays for post-translationally modified proteins can be used to identify new activities of drugs and new therapeutic uses for known drugs. By applying antibodies to fixed cells, one can measure the 20 absolute level and the subcellular location of a particular protein or class of proteins, as well as specific post-translational modifications (e.g. phosphorylation, acetylation, ubiquitination, sumoylation, methylation, nitrosylation, glycosylation, myristoylation, palmitoylation, farnesylation, etc.) that occur in response to drug treatment. In making the present invention, cell-based assays using modification state-specific antibodies were used to monitor the dynamic 25 changes that occur in cells in the presence of a drug of interest. In addition to phospho-specific antibodies, other modification-state-specific antibodies can, in principle, be generated for any macromolecule that undergoes a post-translational modification in the cell. Such novel reagents can be used in conjunction with this invention. Such post-translational modifications include methylation, acetylation, farnesylation, 30 glycosylation, myristylation, ubiquitination, sumoylation, and other post-translational modifications that may occur in response to drug effects. 33 Such post-translational modifications may be detected using antibodies in conjunction with immunofluorescence, as described herein; however, the method is not limited to the use of antibodies. It is important to note that the invention is not limited to specific reagents or classes or reagents, or protocols for their use. Alternative (non-antibody) probes of target or pathway 5 activity can be used, so long as they (a) bind differentially upon a change in a macromolecule in a cell, such that they reflect a change in pathway activity, cell signaling, or cell state related to the effect of a drug; (b) can be washed out of the cell in the unbound state, so that bound probe can be detected over the unbound probe background; and (c) can be detected either directly or indirectly, e.g. with a fluorescent or luminescent method. A variety of organic molecules, 10 peptides, ligands, natural products, nucleosides and other probes can be detected directly, for example by labeling with a fluorescent or luminescent dye or a quantum dot; or can be detected indirectly, for example, by immunofluorescence with the aid of an antibody that recognizes the probe when it is bound to its target. Such probes could include ligands, native or non-native substrates, competitive binding molecules, peptides, nucleosides, and a variety of other probes 15 that bind differentially to their targets based on post-translational modification states of the targets. It will be appreciated by one skilled in the art that some methods and reporters will be better suited to different situations. Particular reagents, fixing and staining methods may be more or less optimal for different cell types and for different pathways or targets. In addition to proteins, a variety of macromolecules are modified post-translationally, 20 including DNA and lipids. Methylation of DNA is important in the sequence-specific and gene specific regulation of transcription. Phosphorylation of lipids is important in the control of cell signaling; for example, the balance between inositol polyphosphates is crucial in regulating the level of the second messenger, inositol trisphosphate (IP3); and the fatty acid composition of phospholipids such as phosphatidylcholine, phosphatidylinositol and phosphatidylserine 25 regulates membrane fluidity and permeability. As the toolbox of modification-state-specific reagent expands, such assays will be added into the panels we are constructing for pharmacological profiling. 30 34 TABLE 4. Examples of modification-state-specific reagents that may be used In conjunction with the present Invention Akt (pS472/pS473), Phospho-Specific (PKBa) Antibodies Caveolin (pY14), Phospho-Specific Antibodies 5 Cdkl/Cdc2 (pY15), Phospho-Spedfic Antibodies eNOS (pS1177), Phospho-Specific Antibodies eNOS (pT495), Phospho-Specific Antibodies ERK1/2 (pT202/pY204), Phospho-Specific Antibodies (p44/42 MAPK) FAK (pY397), Phospho-Specific Antibodies 10 IkBa (pS32/pS36), Phospho-Specific Antibodies integrin b3 (pY759), Phospho-Specific Antibodies JNK (pT1 83/pYl 85), Phospho-Specific Antibodies Lck (pY505), Phospho-Specific Antibodies p38 MAPK (pT18O/pY182), Phospho-Specific Antibodies 15 p120 Catenin (pY228), Phospho-Specific Antibodies p120 Catenin (pY280). Phospho-Specific Antibodies p120 Catenin (pY96), Phospho-Specific Antibodies Paxillin (pY1 18), Phospho-Specific Antibodies Phospholipase Cg (pY783), Phospho-Specific Antibodies 20 PKARilb (pS114), Phospho-Specific Antibodies 14-3-3 Binding Motif Phospho-specific Antibodies 4E-BP1 Phospho-specific Antibodies AcCoA Carboxylase (Acetyl CoA) Phospho-specfc Antibodies Adducin Phospho-specific Antibodies 25 AFX Phospho-specific Antibodies AIK (Aurora 2) Phospho-specific Antibodies Akt (PKB) Phospho-specific Antibodies Akt (PKB) Substrate Phospho-specific Antibodies ALK Phospho-speclfic Antibodies 30 AMPK alpha Phospho-specific Antibodies AMPK betal Phospho-specific Antibodies APP Phospho-specific Antibodles Arg-X-Tyr / Phe-X-pSer Motif Phospho-specific Antibodies Arrestin 1, beta Phospho-specific Antibodies 35 ASKI Phospho-specific Antibodies ATF-2 Phospho-specific Antibodles ATM / ATR Substrate Phospho-specific Antibodies Aurora 2 (AIK) Phospho-specific Antibodies Bad Phospho-specific Antibodies 40 Bel-2 Phospho-specific Antibodies Bcr Phospho-speelfic Antibodies Bim EL Phospho-specific Antibodies BLNK Phospho-specific Antibodies BMK1 (ERK5) Phospho-specific Antibodies 45 BRCA1 Phospho-specific Antibodies Btk Phospho-specific Antibodies C/EBP alpha Phospho-specific Antibodies C/EBP beta Phospho-specific Antibodies c-Abi Phospho-specific Antibodies 50 CAKb Phospho-specific Antibodies Caldesmon Phospho-specific Antibodies CaM Kinase li Phospho-specific Antibodies Cas, p130 Phospho-specific Antibodies Catenin, beta Phospho-specific Antibodies 55 Catenin. p120 Phospho-speclfic Antibodies Caveolin 1 Phospho-specific Antibodies Caveolin 2 Phospho-specific Antibodies Caveolin Phospho-specific Antibodies c-Cbt Phospho-specific Antibodies 60 CO117 (c-Kit) Phospho-specific Antibodies C01 9 Phospho-specific Antibodies cdc2 p34 Phospho-specific Antibodies cdc2 Phospho-specific Antibodies cdc25 C Phospho-specific Antibodles 65 cdk1 Phospho-specific Antibodies cdk2 Phospho-specific Antibodies CDKs Substrate Phospho-specific Antibodies CENP-A Phospho-specific Antibodies c-erbB-2 Phospho-speclfic Antibodies 35 Chki Phospho-specific Antibodies Chk2 Phospho-specific Antibodies c-Jun Phospho-specific Antibodies c-Kit (CD 117) Phospho-specific Antibodies 5 c-Met Phospho-specific Antibodies c-Myc Phospho-specific Antibodies Cofilin 2 Phospho-specific Antibodies Cofilin Phospho-specific Antibodies Connexin 43 Phospho-specific Antibodies 10 Cortactin Phospho-speclic Antibodies CPI-17 Phospho-specific Antibodies cPLA2 Phospho-specific Antibodies c-Raf (Rafi) Phospho-specific Antibodies CREB Phospho-specific Antibodles 15 c-Ret Phospho-specific Antibodies Cdli Phospho-specific Antibodies CrkL Phospho-specfic Antibodies Cydin B1 Phospho-specific Antibodles DARPP-32 Phospho-specific Antibodies 20 DNA-topolsomerase il alpha Phospho-specific Antibodies Dok-2, p56 Phospho-specific Antibodles eEF2 Phospho-specific Antibodies eEF2k Phospho-specific Antibodies EGF Receptor (EGFR) Phospho-specific Antibodies 25 elF2 alpha Phospho-specific Antibodies elF2B epsilon Phospho-specific Antibodies elF4 epsilon Phospho-specific Antibodies elF4 gamma Phospho-specific Antibodies Elk-i Phospho-specific Antibodies 30 eNOS Phospho-specific Antibodies EphA3 Phospho-specific Antibodies Ephrin B Phospho-specific Antibodies erbB-2 Phospho-specific Antibodies ERKI / ERK2 Phospho-specific Antibodies 35 ERK5 (BMK1) Phospho-specfic Antibodies Estrogen Receptor alpha (ER-a) Phospho-specific Antibodies Etk Phospho-specific Antibodies Ezrin Phospho-specific Antibodies FADD Phospho-specific Antibodies 40 FAK Phospho-specific Antibodies FAK2 Phospho-specific Antibodies Fc gamma Rilb Phospho-specific Antibodies FGF Receptor (FGFR) Phospho-specific Antibodies FKHR Phospho-specific Antibodies 45 FKHRL1 Phospho-specific Antibodies FLT3 Phospho-specific Antibodies FRS2-alpha Phospho-speciflc Antibodles Gab1 Phospho-specific Antibodies Gab2 Phospho-specific Antibodies 50 GABA B Receptor Phospho-specific Antibodies GAP-43 Phospho-specific Antibodles GATA4 Phospho-specfic Antibodies GFAP Phospho-specfic Antibodies Glucocorticold Receptor Phospho-specific Antibodies 55 GluRI (Glutamate Receptor 1) Phospho-specific Antibodies GluR2 (Glutamate Receptor 2) Phospho-specific Antibodies Glycogen Synthase Phospho-spedific Antibodles GRB10 Phospho-specific Antibodies GRK2 Phospho-specific Antibodies 60 GSK-3 alpha / beta Phospho-specific Antibodies GSK-3 alpha Phospho-specific Antibodies GSK-3 beta (Glycogen Synthase Kinase) Phospho-specific Antibodies GSK-3 beta Phospho-specific Antibodies GSK-3 Phospho-specific Antibodies 65 H2A.X Phospho-specific Antibodies Hck Phospho-specific Antibodies HER-2 (ErbB2) Phospho-specific Antibodies Histone HI Phospho-specific Antibodies Histone H2A.X Phospho-specific Antibodies 70 Histone H2B Phospho-specific Antibodies 36 Histone H3 Phospho-specific Antibodies HMGN1 (HMG-14) Phospho-specific Antibodies Hsp27 (Heat Shock Protein 27) Phospho-specific Antibodies IkBa (i kappa B-alpha) Phospho-specific Antibodies 5 Integrin alpha-4 Phospho-specific Antibodies Integrin beta-1 Phospho-specific Antibodies Integrin beta-3 Phospho-specific Antibodies IR (Insulin Receptor) Phospho-speciflc Antibodies IR / IGF1R (Insulin/Insulin-Like Growth Factor-I Receptor) Phospho-specific Antibodies 10 IRS-1 Phospho-specific Antibodies IRS-2 Phospho-specific Antibodies Jak1 Phospho-specific Antibodies Jak2 Phospho-specific Antibodies JNK (SAPK) Phospho-specific Antibodies 15 Jun Phospho-specific Antibodies KDR Phospho-spedfic Antibodies Keratin 18 Phospho-specific Antibodies Keratln 8 Phospho-specific Antibodies Kinase Substrate Phospho-specific Antibodies 20 Kip1, p27 Phospho-specific Antibodies LAT Phospho-specific Antibodies Lck Phospho-specific Antibodies Leptin Receptor Phospho-specific Antibodies LKB1 Phospho-specific Antibodies 25 Lyn Phospho-specific Antibodies MAP Kinase / CDK Substrate Phospho-spedfic Antibodies MAP Kinase, p38 Phospho-speclfic Antibodies MAP Kinase, p44 / 42 Phospho-specific Antibodies MAPKAP Kinase Ia (Rski) Phospho-specific Antibodies 30 MAPKAP Knase 2 Phospho-spedfic Antibodies MARCKS Phospho-speclfic Antibodies Maturation Promoting Factor (MPF) Phospho-specific Antibodies M-CSF Receptor Phospho-specific Antibodies MDM2 Phospho-specific Antibodies 35 MEK1 I MEK2 Phospho-specific Antibodles MEK1 Phospho-specific Antibodies MEK2 Phospho-specific Antibodles MEK4 Phospho-specific Antibodies MEK7 Phospho-specific Antibodies 40 Met Phospho-specific Antibodies MKK3 / MKK6 Phospho-specific Antibodies MKK4 (SEKI) Phospho-specific Antibodies MKK7 Phospho-spedfic Antibodies MLC Phospho-specific Antibodies 45 MLIK3 Phospho-specific Antibodies Mnki Phospho-specific Antibodies MPM2 Phospho-specific Antibodies MSK1 Phospho-specific Antibodies mTOR Phospho-specific Antibodies 50 Myelin Basic Protein (MBP) Phospho-specific Antibodies Myosin Light Chain 2 Phospho-specific Antibodies MYPT1 Phospho-specific Antibodies neu (Her2) Phospho-specific Antibodies Neurofilament Phospho-specific Antibodies 55 NFAT1 Phospho-specific Antibodies NF-kappa B p65 Phospho-specific Antibodies Nibrin (p95 / NBSI) Phospho-spedfic Antibodies Nitric Oxide Synthase, Endothelial (eNOS) Phospho-specific Antibodies Nitric Oxide Synthase, Neuronal (nNOS) Phospho-specific Antibodies 60 NMDA Receptor 1 (NMDAR1) Phospho-specific Antibodies NMDA Receptor 28 (NMDA NR2B) Phospho-specfic Antibodies nNOS Phospho-specific Antibodies NPM Phospho-specific Antibodies Opioid Receptor, delta Phospho-specific Antibodies 65 Opioid Receptor, mu Phospho-specific Antibodies p53 Phospho-specific Antibodies PAKI / 2 / 3 Phospho-specific Antibodies PAK2 Phospho-specific Antibodies Paxilin Phospho-specific Antibodies 70 Paxillin Phospho-specific Antibodies 37 PDGF Receptor alpha / beta Phospho-specific Antibodies PDGF Receptor alpha Phospho-specific Antibodies PDGF Receptor beta Phospho-specific Antibodies PDGFRb (Platelet Derived Growth Factor Receptor beta) Phospho-specific Antibodies 5 PDK1 Docking Motif Phospho-specific Antibodies PDK1 Phospho-specific Antibodies PDKI Substrate Phospho-specific Antibodies PERK Phospho-specific Antibodies PFK-2 Phospho-specific Antibodies 10 Phe Phospho-specific Antibodies Phospholamban Phospho-spedfic Antibodies Phospholipase C gamma-i Phospho-spedfic Antibodies Phosphotyrosine IgG Phospho-specific Antibodies phox. p40 Phospho-specific Antibodies 15 P13K Binding Motif, p85 Phospho-specific Antibodies Pini Phospho-specific Antibodles PKA Substrate Phospho-specific Antibodies PKB (Akt) Phospho-specific Antibodies PKB (Akt) Substrate Phospho-specific Antibodies 20 PKC alpha / beta 1i Phospho-spedfic Antibodies PKC alpha Phospho-specfic Antibodies PKC deta / theta Phospho-specific Antibodies PKC delta Phospho-specific Antibodies PKC epsilon Phospho-spedfic Antibodies 25 PKC eta Phospho-specfic Antibodies PKC gamma Phospho-specific Antibodies PKC Phospho-specific Antibodies PKC Substrate Phospho-specific Antibodies PKC theta Phospho-specific Antibodies 30 PKC zeta / lambda Phospho-spedflic Antibodies PKD (PKC mu) Phospho-specfic Antibodies PKD2 Phospho-speciflc Antibodies PKR Phospho-specific Antibodies PLC beta 3 Phospho-speciflc Antibodies 35 PLC gamma 1 Phospho-specific Antibodies PLC gamma 2 Phospho-specific Antibodies PLD1 Phospho-specific Antibodies PP1 alpha Phospho-specific Antibodies PP2A Phospho-specific Antibodies 40 PPAR Alpha Phospho-specific Antibodies PRAS40 Phospho-specific Antibodies Presenllin-2 Phospho-specific Antibodies PRK2 (pan-PDKI phosphorylation site) Phospho-specific Antibodies Progesterone Receptor Phospho-specific Antibodies 45 Protein Kinase A, Ril (PKARII) Phospho-specific Antibodies Protein Kinase B Phospho-specific Antibodies Protein Kinase B Substrate Phospho-specific Antibodies Protein Kinase C, alpha (PKCa) Phospho-specific Antibodies Protein Kinase C, epsilon (PKCe) Phospho-specific Antibodies 50 PTEN Phospho-specific Antibodies Pyk2 Phospho-specific Antibodies. Rac1 / cdc42 Phospho-specific Antibodies Rac-Pk Phospho-specific Antibodies Rac-Pk Substrate Phospho-specific Antibodies 55 Rad 17 Phospho-specific Antibodies Rad17 Phospho-specific Antibodies Raf-I Phospho-specific Antibodies Ras-GRF1 Phospho-specific Antibodies Rb (Retinoblastoma Protein) Phospho-specific Antibodies 60 Ret Phospho-specific Antibodies Ribosomal Protein S6 Phospho-specific Antibodies RNA polymerase 1i Phospho-specific Antibodies Rsk, p90 Phospho-specific Antibodies Rski (MAPKAP Kia) Phospho-specific Antibodies 65 Rsk3 Phospho-specific Antibodies S6 Kinase Phospho-specific Antibodies S6 Kinase, p70 Phospho-specific AntibodIes S6 peptide Substrate Phospho-specific Antibodies SAPK (JNK) Phospho-spedfic Antibodies 70 SAPK2 (Stress-activated Protein Kinase, SKK3, MKK3) Phospho-specific Antibodies 38 SEK1 (MKK4) Phospho-specfic Antibodies Serotonin N-AT Phospho-specific Antibodies Serotonin-N-AT Phospho-specific Antibodies SGK Phospho-specific Antibodies 5 Shc Phospho-specific Antibodies SHIP1 Phospho-specific Antibodies SHP-2 Phospho-specific Antibodies SLP-76 Phospho-specfic Antibodies Smad1 Phospho-specific Antibodies 10 Smad2 Phospho-specific Antibodies SMCI Phospho-specific Antibodies SMC3 Phospho-specific Antibodies SOX-9 Phospho-specific Antibodies Src Family Negative Regulatory Site Phospho-specific Antibodies 15 Src Family Phospho-specific Antibodies Src Phospho-specific Antibodies Stati Phospho-specific Antibodies Stat2 Phospho-specific Antibodies Stat3 Phospho-specific Antibodies 20 Stat4 Phospho-specific Antibodies Stat5 Phospho-specific Antibodies Stat5A / Stat59 Phospho-specific Antibodies Stat5ab Phospho-specific Antibodies Stat6 Phospho-specific Antibodies 25 Syk Phospho-specific Antibodies Synapsin Phospho-specific Antibodies Synapsin site 1 Phospho-specific Antibodies Tau Phospho-spedfic Antibodies Tie 2 Phospho-specific Antibodles 30 Trk A Phospho-specifilc Antibodies Troponin 1. Cardiac Phospho-spedfic Antibodies Tuberin Phospho-specific Antibodies Tyk 2 Phospho-speclfic Antibodies Tyrosine Hydroxylase Phospho-specific Antibodies 35 Tyrosine Phospho-specific Antibodles VASP Phospho-spedfic Antibodies Vav1 Phospho-specific Antibodies Vav3 Phospho-specific Antibodies VEGF Receptor 2 Phospho-specific Antibodies 40 Zap-70 Phospho-specific Antibodies Instrumentation The assays described above generate optically detectable signals that can be read on 45 commercially available instrumentation, including fluorescence plate readers, luminometers, and flow cytometers. Such instrumentation is widely available from commercial manufacturers, including Molecular Devices, Packard, Perkin Elmer, Becton Dickinson, Beckman Coulter, and others. All such assays can be constructed in multiwell (96-well and 384-well) formats. The high-content assays described above, including the protein-fragment complementation assays 50 and immunofluorescence assays, generate optically detectable signals that can be spatially resolved within subcellular compartments. The resulting images can be captured with automated microscopes, confocal imaging systems, and similar devices. Suitable imaging instrumentation is widely available from a variety of commercial manufacturers including Molecular Devices (Universal Imaging), Amersham Bioscience, Cellomics, Evotec, Zeiss, Q3DM, Atto, and others. 39 Image analysis software such as MetaMorph, the publicly available IMAGE software from the National Institutes of Health (http://rsb.info.nih.gov/nih-image/) and various proprietary software packages are used to distinguish the signal emanating from different subcellular compartments (membrane, cytosol, nucleus) and to quantitate the total fluorescence per cell. In 5 addition, multi-well PCA formats for the present invention can be constructed for array-based or slide-based assay formats (Sabatini et al.) allowing the rapid, simultaneous processing of a large number of different PCAs on a single array. Suitable pairs of interacting molecules for assay construction can be identified by any one of the methods outlined in Figure 1. PCA enables a systematic characterization of the 10 interactions made among theproteins in living cells by first examining whether different pairs of proteins generate a PCA signal in a cell type of interest and then determining whether the signal amount or subcellular location is affected by drugs that modify cell signaling. Systematic screening can also be performed to identify pathway elements; for example, a protein tagged with Fl of a suitable reporter can be tested individually against other proteins tagged with 15 complementary fragment F2 (gene-by-gene analysis). The presence of a PCA signal indicates an interaction between the two proteins tagged with the complementary fragments. The advantage of the present invention is that, once an interaction has been identified, an assay is in hand that can be used to screen for drugs that modulate the pathway of interest by using a high-content or high-throughput PCA as a screen. 20 The components of many important cellular pathways and disease-related pathways have been partially elucidated, and the known or hypothesized interactions can readily be used to design assays according to the present invention. The present invention encompasses assays for a variety of steps in cancer-related pathways. A few of these steps are listed in Table 1. Any of the protein-protein interactions reported to date can be used as the basis for the construction of 25 protein-fragment complementation assays enzyme-fragment complementation assays, FRET or BRET assays. All of the assays that are the subject of the present invention are of general use as validation assays or in basic experimental biology research as well as in drug discovery. The following patents, published patent applications as well as all their foreign counterparts and all cited references therein are incorporated in their entirety by reference herein 30 as if those references were denoted in the text: 40 US 20040161787 Protein fragment complementation assays for high-throughput and high-content screening US 20040137528 Fragments of fluorescent proteins for protein fragment complementation assays 5 US 20040038298 Protein fragment complementation assays for the detection of biological or drug interactions US 20030108869 Protein fragment complementation assay (PCA) for the detection of protein-protein, protein-small molecule and protein nucleic acid interactions based on the E. coli TEM- I beta-lactamase 10 US 20030049688 Protein fragment complementation assays for the detection of biological or drug interactions US 20020064769 Dynamic visualization of expressed gene networks in living cells US 20010047526 Mapping molecular interactions in plants with protein fragments complementation assays 15 US 6,428,951 Protein fragment complementation assays for the detection of biological or drug interactions * US 6,294,330 Protein fragment complementation assays for the detection of biological or drug interactions US 6,270,964 Protein fragment complementation assays for the detection of biological or 20 drug interactions 41

Claims (24)

1. A method for identifying-a new therapeutic use for a test entity, wherein said test entity is a drug or a drug candidate, said method comprising (A) selecting a test entity; (B) 5 testing the activity of said test entity against a protein complex in a cell; (C) using the results obtained from (B) to identify a new activity of said test entity.

2. A method for screening a test entity, wherein said test entity is a drug or a drug candidate, said method comprising: (a) constructing an assay for a protein complex in a cell; (b) 10 testing the effects of said test entity on one or more properties of said assay(s); (c) using the results of (b) to identify an activity of said test entity.

3. A method for identifying an entity that modulates the activity of a cellular pathway, said method comprising: (a) providing (i) a test entity and (ii) an assay for a protein complex in 15 a cell; (b) contacting said assay with said test entity; and (c) detecting or measuring one or more properties of said assay; wherein a change in one or more properties of said assay in the presence of said test entity, relative to the absence of said test entity, indicates that the test entity modulates the activity of said cellular pathway. 20

4. A method according to any of claims 1-3 wherein at least one assay property that is measured is (a) the amount of said protein complex or (b) the subcellular location of said protein complex.

5. A method according to any of claims 1-3 wherein said test entity produces either (a) 25 an overall increase in said protein complex, (b) an overall decrease in said protein complex, (c) a change in the subcellular location of said protein complex or (d) a change in the subcellular amount of said protein complex.

6. A method according to any of claims 1-3 wherein a property of said protein complex is 30 altered by said test entity via one or more of the following mechanisms: (a) increasing or decreasing the formation of said complex; (b) stabilizing or destabilizing said complex; (c) 42 increasing or decreasing the dissociation of said complex; (d) increasing or decreasing the rate of degradation or proteolysis of one or more proteins in said complex; (e) increasing or decreasing or modifying the post-translational modification status of one or more proteins in said complex; (f) increasing or decreasing the amount of one or more proteins in said complex. 5

7. A method for identifying a new therapeutic use for a test entity, wherein said test entity is a drug or drug candidate, said method comprising (A) selecting a test entity; (B) testing the ability of said test entity to alter the amount and/or post-translational modification status of one or more proteins in a cell or a collection of cells; (C) using the results obtained from (B) to 10 identify a new activity of said test entity.

8. A method for screening a test entity, wherein said test entity is a drug, said method comprising: (a) constructing an assay for the amount and/or post-translational modification status of one or more proteins in a cell or a collection of cells; (b) testing the effects of said test entity 15 on one or more properties of said assay(s); (c) using the results of (b) to identify an activity of said test entity.

9. A method for identifying an entity that modulates the activity of a cellular pathway, said method comprising: (a) providing (i) a test entity and (ii) an assay for the amount and/or 20 post-translational modification status of one or more proteins in a cell or a collection of cells; (b) contacting said assay with said test entity; and (c) detecting or measuring one or more properties of said assay; wherein a change in one or more properties of said assay in the presence of said test entity, relative to the absence of said test entity, indicates that the test entity modulates the activity of said cellular pathway. 25

10. A method according to any of claims 1-3 or claims 7-9 wherein said test entity is a drug that is either (a) approved by a governmental regulatory agency for administration to a patient or (b) not approved by a governmental regulatory agency. 30

11. A method according to any of claims 1-3 or claims 7-9 wherein an immunofluorescence assay is used 43

12. A method according to any of claimsl-3 or claims 7-9 wherein at least one assay property that is detected or measured is selected from the group comprising (a) the phosphorylation status of one or more proteins; (b) the ubiquitination status of one or more proteins; (c) the sumoylation status of one or more proteins; (d) the methylation status of one or 5 more proteins; (e) the acetylation status of one or more proteins; (f) the nitrosylation status of one or more proteins; (g) the myristoylation status of one or more proteins; (h) the palmitoylation status of one or more proteins; (i) the farmesylation status of one or more proteins; (j) the geranylation status of one or more proteins; or (k) the glycosylation status of one or more proteins. 10

13. A method according to any of claims 1-3 or claims 7-9 wherein said test entity results in an increase or decrease in the level or type of one or more post-translational modifications of one or more proteins, relative to the level or type of said post-translational modifications of said protein(s) in the absence of said test entity. 15

14. A method for either (a) reducing the proliferation of a mammalian cell or (b) increasing the proliferation of a mammalian cell, said method comprising administering to a patient an entity that modulates either the amount, the subcellular location, or the post translational modification status of a protein complex in said cell, such that the entity modulates 20 intracellular signaling in said cell.

15. A method according to any of claims 13-14 wherein said entity is a drug that is either approved by a governmental regulatory agency for administration to a patient or that is not approved by a governmental regulatory agency. 25

16. A method according to any of claims 13-14, said method comprising modulating a protein complex in a mammalian cell, said protein complex selected from the group consisting of (a) a RAS:RAF complex; (b) a PAK4:Cofilin complex; (c) a CDC42:PAK4 complex; (d) a CDC37:HSP90 complex; (e) a Cofilinl:LIMK2 complex; (f) a Cofilinl:PAK4 complex; (g) an 30 EGFR:Grb2 complex; (h) a p53:E6 complex; (i) a p53:p53 complex; () a CDC25C:Chkl 44 complex; (k) a BAD:BID complex; (k) a CDC2:WEEI complex; (1) a BCL-xL:BAD complex; (m) a BCL- xL:BIK complex; (n) a Cdc2:CDC25C complex; (o) a Chkl:CDC25A complex; (p) a CyclinD:CDK4 complex; (q) a CyclinE:CDK2 complex; (r) an HSP90:Eef2k complex; (s) a MAX:MYC complex; (t) a Cdc2:p21 complex; (u) a p27:ubiquitin complex; (v) a 5 protein:ubiquitin complex; (w) a p53:Chkl complex; or (x) a Smad3:HDAC complex.

17. An assay for a protein complex or protein complexes, said assay comprising either (a) whole proteins or (b) domains of proteins, wherein at least one of said proteins is selected from the group consisting of CDC2, CDK4, PAK4, COFILIN, WEEl, BAD, BID, BIK, BCL 10 xL, Chkl, CDC25C, CDC25A, E6, EGFR, GRB2, RAS, RAF, CDC37, LIMK2, HSP90, AKTI, p27, UBIQUITIN, p53, Smad3, HDAC, MAX, MYC, ERK, CyclinD or Cyclin E.

18. A collection of cells or a cell line, wherein said cells or cell line contains (a) whole proteins or (b) domains of proteins, wherein said proteins or said domains of proteins are 15 separately linked to complementary fragments of a reporter, and wherein at least one of said proteins is selected from the group consisting of : CDC2, CDK4, PAK4, COFILIN, WEEl, BAD, BID, BCL-xL, CHK1, CDC25C, E6, EGFR, GRB2, RAS, RAF, CDC37, LIMK2, HSP90, AKTI, p27, UBIQUITIN, p53, Smad3, HDAC, MAX, MYC, ERK, CyclinD or Cyclin E. 20

19. An assay according to any of Claims 2, 3, or 18 wherein said assay is selected from the group comprising (a) a protein-fragment complementation assay, (b) an enzyme-fragment complementation assay, (c) a subunit complementation assay, (d) a fluorescence resonance energy transfer assay (FRET), (e) a bioluminescence resonance energy transfer assay (BRET), () a split ubiquitin assay, (g) a split intein assay, (h) a two-hybrid assay (i) a three-hybrid assay or 25 (j) an immunofluorescence assay.

20. A method according to any of claims 1- 3 or 18 wherein said proteins are detected or measured with one or more of the following methods: (a) a protein-fragment complementation assay, (b) an enzyme-fragment complementation assay, (c) a subunit complementation assay, (d) 30 a fluorescence resonance energy transfer assay (FRET), (e) a bioluminescence resonance energy 45 transfer assay, (f) a split ubiquitin assay, (g) a split intein assay, (h) a two-hybrid assay; (i) a three-hybrid assay; or (j) an immunofluorescence assay.

21. An assay according to any of claims 2-3 or 18 wherein said assays comprise one or 5 more reporters selected from the group consisting of a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), a red fluorescent protein (RFP), a luciferase, a beta-galactosidase, a dihydrofolate reductase, an RNAse, an intein, a ubiquitin, a beta-lactamase, or a mutant of any of the foregoing reporters. 10

22. An assay according to any of Claims 1-3, 7-9 or 18 said assay comprising the use of one or more of the following methods: fluorescence microscopy, flow cytometry, fluorescence spectroscopy, luminometry, and/or automated image analysis.

23. A method for identifying drug leads and useful pharmacophores from a known test 15 entity, wherein said entity is a drug or a drug candidate, said method comprising (A) selecting an entity; (B) testing the activity of said entity against a protein complex in a cell; (C) using the results obtained from (B) to identify a new drug lead based on said entity.

24. A method for identifying a drug lead compound that modulates the activity of a 20 cellular pathway, said method comprising: (a) assembling a library of candidate drugs that modulate a cellular pathway; (b) screening the library of candidate drugs that modulate a cellular pathway by providing (i) a library of candidate drugs and (ii) an assay for a protein complex or a post-translationally-modified protein in a cell; (c) contacting said assay with said candidate drugs; and (d) detecting or measuring one or more properties of said assay; wherein a change in 25 one or more properties of said assay in the presence of said drug candidates, relative to the absence of said drug candidates, identifies a drug lead that modulates a cellular pathway. 46