FUNCTION-BASED SMALL MOLECULAR WEIGHT COMPOUND SCREENING SYSTEM IN DROSOPHILA MELANOGASTER

Abstract: A methodology for screening libraries of compounds for desirable biological/therapeutic activities in a system that may be automated for the microinjection of compound(s) of interest into the open circulatory system (i.e., hemolymph) of Drosophila larvae genetically modified to sensitize a particular biochemical pathway, such as those related to a human disease, either by expression of a human disease gene or by the activation of a Drosophila gene that in the adult fly results in the development of an easily detectable phenotypes such that compounds that selectively interfere with this specific biochemical pathway will modify or suppress the phenotype and can be identified rapidly and efficiently.

Description: FUNCTION-BASED SMALL MOLECULAR WEIGHT COMPOUND SCREENING SYSTEM IN DROSOPHILA MELANOGASTER FIELD OF THE INVENTION The present invention relates to the use of genetically-modified strains of Drosophila melanogaster in high-throughput screening (HTS) of small molecular weight compounds. The present invention also relates to the systematic identification of small molecules which interfere with specific disease pathways using Drosophila as the genetic model system. Additionally, the present invention relates to the use of automated screening systems employing microinjection of small molecular weight compounds, possessing putative biological activity, into the open circulatory system (i.e., the hemolymph) of genetically-modified Drosophila larva. BACKGROUND OF THE INVENTION The identification and subsequent characterization of new therapeutics are the primary rate limiting step in pharmacological research. Pharmacological compounds have been sought from natural products for many years. In general, complex mixtures derived from cells, or their secondary metabolites, are screened for biological activity. Subsequently, when the desired biological activity is identified in such a complex mixture, the specific molecule which possesses the activity has been purified, using the biological activity as the means for identifying the component of the mixture which possesses the desired biological activity. Many of the existing therapeutics on the market to date have been identified in an accidental manner, and frequently their mechanism of action is poorly understood. A more direct approach towards the identification of new small molecular weight compounds effective against various disease conditions requires some knowledge of both the molecular defect underlying a given disease and of the cellular pathways and processes in which the defective component is acting. In fact, such knowledge of the pathways involved is essential since the defective gene product may not be the best target for a small molecular weight compound. In addition, despite the great value that large libraries of molecules can have for identifying useful compounds or improving the properties of a lead compound, the difficulties of screening such libraries, particularly extremely large libraries, has limited the impact that access to such libraries should have made in reducing the costs of drug discovery and development. This is, in a large part, due to the weaknesses inherent in the current screening methodologies of compound libraries which employ both cell-free and in vitro cell-based assay systems. Accordingly, there remains an as yet unfulfilled need within the relevant fields for a rapid, quantitative HTS screening methodology that may be fully automated and that utilizes a genetic model possessing, but not be limited to, the following characteristics: (i) a high degree of conservation of the various signaling pathways involved in the etiology of disease, especially human disease; (ii) the ability to be grown rapidly in large numbers with little effort; (iii) a stable genetic mutation(s) and (iv) an easily discernible genetic outcome for use in the screening procedure. SUMMARY OF THE INVENTION The present invention targets the ubiquitous nature of the biochemical pathways present in most every cell and couples them to an assay system of choice within the appropriately modified Drosophila strain. More specifically, the present invention utilizes genetically sensitized Drosophila strains that possess mutations within a selected biochemical pathway in the construction of a high-throughput screen (HTS) of small molecular weight compounds to facilitate the identification and characterization of novel, lead drug candidates that dominantly modify the phenotypic effects of the chosen sensitized Drosophila biochemical pathways. The present invention discloses a methodology for the screening of compounds for desirable biological/therapeutic activities that involves the screening of individual chemical compounds which have been synthesized and cataloged in libraries of drug or chemical companies or research institutes. The active "lead" compounds and novel chemical entities identified and characterized by the present invention may be utilized for the development of bioactive "leads" in small molecule libraries for pharmaceuticals, agrochemicals and the like. The high degree of conservation of morphogenetic processes between Drosophila and humans has made Drosophila a prime model system for the identification of new putative drug targets using function-based genetic approaches. Genetically sensitized Drosophila systems, wherein the gene modification results in a dose-sensitive phenotype, permit detection of a mere two-fold effect of small compounds on specific signaling pathways related to human diseases. Such Drosophila strains may be genotypic (+/null), and hence are hemizygous for a dose sensitive gene within a given biochemical pathway. The present invention is a novel combination of an automation-capable, in vitro HTS assay with an in vivo "readout" system comprised of Drosophila melanogaster that are genetically-sensitized for a specific chosen biochemical pathway, preferably a human disease pathway. Hence, the present invention discloses a methodology that serves to "bridge the gap" of the presently-utilized screening systems by use of a well-established model for human disease {Drosophila) for the screening of large numbers of compounds in a rapid, quantitative and highly efficacious manner. The expression of human disease genes or their homologs within any chosen stage of the developing Drosophila larva should model the effects of these genes in human cells and subsequently produce phenotypes that are modified by either the mutations within these interacting genes, or by compounds that block the function of the corresponding gene product. These gene products are prime candidates as targets for small compounds which interfere with their function. The preferred embodiment of the present invention employs an automation-capable system for the microinjection of compound(s) of interest into the open circulatory system (i.e., hemolymph) of Drosophila larvae, in particular the Drosophila first, second, or third instar larvae, that have been previously genetically-modified in a gene that is specific to a signaling pathway involved in human disease. The modified gene may be involved in any biochemical pathway, such as the Ras signaling pathway, and have a functional phenotype that is readily scorable, such as by observation of the developing Drosophila eye. The invention may use any biochemical pathway to monitor phenotypic changes involved in the development of any imaginal disk. Therefore, any stage of Drosophila larval development may be used in the analysis. Following maturation of the microinjected Drosophila larvae, the biological effect of the injected compound(s) are assessed, preferably in adult flies. In addition, the screening methodology disclosed by the present invention may allow the simultaneous observation of: (i) the general biological toxicity of the microinjected compound(s) through 50% lethal dose (LD50) computations: (ii) the specificity of the modification of the specific Drosophila phenotype (i.e., suppression of the rough eye phenotype) and (iii) the non-specific interference with other well-defined developmental and physiological pathways. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a series of scanning electron micrographs of the Drosophila compound eye (Panels A-D) and light micrographs of tangential cross-sections of the Drosophila compound eye (Panels E-H) following genetic modification of the Raf' orY9 phenotype by Su{Raf) and two E{Raf) mutations. Panels A and E illustrate the wild-type genotype; Panels B and F illustrate the Raf' orY9 /+ genotype; Panels C and G illustrate the Raf' orY /+:Su{Raf)3 A 9 " genotype and Panels D and H illustrate the Raf'° rn /+ , +/E(Rα }2A ,6T ' genotype. It should be noted that the Su{Raf) and two E{Raf) mutations dominantly-modify the overall degree of Drosophila compound eye texture roughening in Raf' orY9 flies. In contrast, in the case of Su{Raf)3A, all ectopic R7 cells were eliminated and the eye regained the smooth texture observed in wild-type Drosophila. FIG. 2 is a schematic illustration of the known or estimated cytological chromosomal locations of all six Su{Raf) and two E{Raf) complementation groups (loci). All loci were mapped to either chromosome 2 or 3. In addition, the approximate chromosomal breakpoints of selected deficiencies are also shown. Deficiencies that both modified the Raf' orY9 phenotype and failed to complement EMS-induced Su{Raf) and two {Raf) mutations are indicated by the solid black box (■). The two deficiencies that failed to complement Su{Raf)3A but did not appreciably suppress Raf' orY9 are indicated by the lightly-shaded box (D). The deficiencies completely spanning the interval to which Su{Raf)3A was mapped, but that failed to modify either the Raf' orY9 phenotype or display an obvious phenotype in trans to Su{Rqf)3A liA2 , are indicated by the non-shaded box (D). FIG. 3 is a graphic illustration of the survival rates of third instar RIO larvae injected with 10 μM rapamycin (dark bars) or a control solution (light bars). DETAILED DESCRIPTION OF THE INVENTION The present invention provides an efficient methodology for the quantitative, systematic identification of small molecular weight molecules which interfere with specific disease conditions in an animal model system. Genetically-modified stains of the fruitfly, Drosophila melanogaster, have played a pivotal role in the identification of new genes whose products act in signaling pathways that are altered in human diseases (see e.g., Dickson, et al., 1996. Genetics 142:163-171; Dominguez, et al., 1996. Dev. Biol. 7:219-226). Such genetically sensitive backgrounds have been used previously to identify mutations in genes coding for rate limiting components in a particular signaling pathway. The present invention makes use of such genetically-modified Drosophila strains to screen small molecular weight compounds of interest for biological activity which functions to interfere with specific disease-related pathways. Currently, numerous drug screening protocols rely upon high-throughput screening (HTS) of compound libraries using cell-free or in vitro cell-based assay systems. HTS is a process by which large numbers of compounds with putative biological activity may be tested, for example in an automated manner, for activity as inhibitors (antagonists) or activators (agonists) of a specific biological target (e.g., cell-surface receptor or a metabolic enzyme). However, current HTS methods possess a number of limitations such as: (i) bioavailability; (ii) pharmacokinetics; (iii) toxicity and (iv) absolute specificity. Hence, subsequent medicinal chemical and pharmacological studies are required to convert a compound which emerges from an initial HTS screening into a therapeutically useful drug. The potent and specific biological activities of many low molecular weight peptides make these molecules attractive starting points for therapeutic drug discovery (see e.g., Hirschmann, et al., 1991. Angew. Chem. Int. Ed. Engl. 30:1278-1301). The present invention discloses a methodology for the screening of compounds for desirable biological/therapeutic activities that involves the screening of individual chemical compounds that have been synthesized and cataloged in libraries of drug or chemical companies or research institutes. The active "lead" compounds and novel chemical entities identified and characterized by the present invention may be any chemical compound of interest, and may be utilized for the development of bioactive "leads" in small molecule libraries for pharmaceuticals, agrochemicals and the like. With respect to the generation of small molecular weight compound libraries of the present invention, the combination of biochemical diversity is often synergistic with the metabolic diversity obtained from the in vivo production of "natural products". Collections of naturally or synthetically produced chemical or oligomeric compounds, for example peptides, can be administered to cultures of microorganisms. In accord, each microbial strain may potentially create numerous modified chemical derivatives, thus generating a "metabolite library". Because each of these aforementioned cultures would (potentially) contain a very complex mixture of metabolites, a highly efficacious method of screening would be required (i.e., HTS). An aliquot of the library is incubated with each of the many strains typical of a microorganism fermentation screening program, and the media screened utilizing an HTS- based assay. In another aspect of the invention, natural product diversity is screened by creating a mixture of combinatorially-tagged liposomes; wherein each liposome encapsulates, for example, only one member or a simple mixture of a natural product compound library. The libraries which are generated by the methodologies disclosed herein may be screened for any biological activity known within the art. These include, but are not limited to: anti-microbial activity, anti-tumor activity, enzyme inhibiting activity, receptor binding, growth promotion activity, and in vitro and in vivo tests for biological responses. With the advent of genomics, combinatorial paradigms and high-throughput screening (HTS) assay-based pharmacological testing, the number of compounds possessing biological/therapeutic activity is likely to markedly increase. HTS assays are, preferably, based upon automation, validation and integration of in vitro absorption-metabolism models and database management (see e.g., Rodrigues, et al., 1997. Pharm. Res. 14:1504-1510). Complementary to this tenet is the need to generate a taxonomy of known compounds, identifying those with similar mechanisms, preferably in a way that provides clues as to the nature of those mechanism. The present invention discloses a screening methodology that is based upon the utilization of genetically-sensitized Drosophila as a model system with a new method for drug administration that permits both high-throughput and automation. Based upon previous experience with genetic screens for dominant modifier mutations, genetically-sensitized systems comprising specific pathways that are sensitive to such modifier mutations will permit detection of mere two-fold effects of small molecular weight compounds on specific signaling pathways related to human diseases. This assay sensitivity results from the ability to differentiate dose-dependent phenotypes of the modified Drosophila related to the presence of none, one, or two modified alleles within its genome. The methodology disclosed by the present invention is both more powerful and more sensitive than traditional, cell-free or in vitro cell-based assay systems. In addition, the Drosophila-based screening assay disclosed herein is based upon a biological "readout" related to human disease, and its intrinsic ability to test for both the overall specificity and toxicity of a compound(s) of interest. Utilization of Drosophila melanosaster as a Model System for the Genetic Dissection of Cellular Processes The fruitfly, Drosophila melanogaster, has been used as an ubiquitous model for the characterization of cellular processes (e.g., signaling pathways) involved in a variety of human diseases. A high degree of conservation of morphogenetic processes between Drosophila and humans has made Drosophila a prime model system for the identification of putative drug targets using function based genetic approaches. Drosophila eye development is a prime example of a model system for the study of well defined and functionally integrated genetic controls, and it has been used extensively for the systematic genetic dissection of conserved signaling pathways. The Drosophila compound eye is composed of a hexagonal array of approximately 800 identical units, called ommatidia. Each of these units consists of eight photoreceptor cells (R1-R8), four lens-secreting cone cells, and pigment cells that optically insulate each ommatidium. This highly organized structure develops from a single layer epithelial sheet, the eye imaginal disc, during larval and pupal stages by the stepwise recruitment of cells into the ommatidial clusters. During this process, differentiating cells specify the fate of neighboring, but still undetermined, cells by inductive signals. Drosophila eye development has provided most of the important genetic information regarding evolutionarily conserved mechanisms. While the concept of wide-ranging evolutionary gene conservation is not new, it has been critical in elucidating many complexities in, for example, the Ras and tyrosine kinase signaling pathways via the analysis of Drosophila eye mutants. These genes encode components of a highly conserved signaling cascade that has in part been described in vertebrate cells. Activation of the sev {sevenless) receptor by boss {bride of sevenless) protein presumably results in receptor dimerization and subsequent autophosphorylation on tyrosine residues. This autophosphorylation creates binding sites for the Drk SH2/ SH3 adaptor protein (see e.g., Raabe, et al., 1995. EMBO J 14:2509-2518). Drk binds to sev via its SH2 domain and to the C-terminus of Sos {son of sevenless) protein via its SH3 domains, and thereby brings the Sos protein to the membrane. Sos is a guanine nucleotide releasing factor that activates Rasl protein by facilitating the conversion from GDP-Rasl to GTP-Rasl. Rasl in turn activates a cascade of three cytoplasmic kinases, namely, the homologs of Raf, MEK (MAPK kinase) and MAP kinase encoded by the genes ra Dsorl, and rolled, respectively (Dickson, et al., 1996 Genetics 142:163-171). All of these cytoplasmic components are also required for signaling by other RTKs in Drosophila, including both torso and DER (see e.g., Chang, et al., 1994. Cold Spring Harbor Symp. Quant. Biol. 59:219-226; Dominguez & Hafen, 1996. Drosophila Sem. Cell Dev. Biol. 7:219-226). The elucidation of these signaling pathways are of great importance as they control various aspects of human developmental regulation, hormone action, and neoplasia. Genetically manipulated Drosophila have served as a powerful tool for dissecting and characterizing gene pathways. Recombinant methodologies used to modify specific Drosophila genes are well known in the art, as are methodologies used for the maintenance of said Drosophila strains, said strains being homozygous, hemizygous, or heterozygous for defined allelic combinations of the gene or genes of interest. One such phenotype is an irregular, rough eye surface induced upon activation of a genetically modified raf gene in the Ras signaling pathway. In such a genetically sensitized background, mutations in genes coding for rate limiting components in a particular biochemical and/or signaling pathway can be identified as modifiers of the phenotype (see e.g., Dickson, et al., 1996. Genetics 142:163-171). These mutations thus identify gene products whose activity or function is critical for the development the disease-related phenotype. A variety of cell-fate decisions are controlled by the activation of receptor tyrosine kinases (RTKs). One such interaction involves the Drosophila rough eye phenotype which is an irregular, rough eye surface induced upon activation of a genetically-modified raf gene (a downstream effector of the Rasl oncogene) in the Ras signaling pathway (see e.g., Dickson, et al., 1996. Genetics 142:163-171). In such a genetically-sensitized background, mutations in genes encoding rate limiting components in a particular signaling pathway may be identified as modifiers of the phenotype. These mutations thus identify gene products whose activity or function is critical for the development the disease-related phenotype. The formation of R7 photoreceptor cells is a model system where scientists have a complete knowledge of all genes acting in the signal transmission pathway that extends from the cell membrane to the nucleus (see e.g., Dominguez, et al., 1997. Dev. Biol. 7:219-226). Although extensive genetic screens for recessive, viable mutations affecting the development of the R7 cells have been carried out, mutations in only four genes apart from sev and boss have been identified. Flies homozygous for mutations in the sina gene (which encodes a nuclear protein) lack R7 cells and are non- viable. Mutations in Gapl (encoding a homolog of a GTPase activating protein), yan (encoding a transcription factor of the ETS-family) and tramtrack {ttk) have been shown to cause the formation of multiple R7 cells in each ommatidium (see e.g., Dominguez, et al., 1997. Dev. Biol. 7:219-226). These results tends to suggest that Gapl, yan and ttk act as inhibitors of the R7 specification; whereas sina acts as an activator. Mutations in genes whose products are also involved in other developmental decisions prior to the formation of the eye may cause less informative phenotypes such as lethality. Using current methodologies, their role in R7 development cannot be tested directly and alternative genetic strategies are thus required to identify them. The most successful approach to date utilizes a hypomorphic sev mutation that encoded a partially functional, temperature-sensitive sev receptor that, at an intermediate temperature, provides barely sufficient activity to specify R7 cells. See, Simon et al., 1991, Cell 67:701-716. In this sensitized genetic background, normally recessive mutations acted dominantly in the R7 decision. A similar system utilizing the multiple R7 phenotypes in sev gain of function (GOF) mutations {sev 00 ) determines the overall number of cells of the R7 equivalence group which assume R7 cell fate, and so provides a sensitive measure of the sev kinase activity and signal transduction efficiency. The recruitment of extra R7 cells disrupts the hexagonal array of the ommatidial units and causes a roughening of the external surface of the eye. Mutations that reduce the efficiency of sev signaling are thus detected as suppressors of the rough eye phenotype of sev 00 21 . The value of Drosophila as a screening system for evaluating the biological activities of chemicals has been well-documented (see e.g., Schulz, et al., 1955. Cancer Res. 3 (suppl.): 86- 100; Schuler, et al., 1982. Terat. Carcin. Mutag. 2:293-301). Small numbers of chemical substances are administered to larvae or flies by feeding, and flies are then analyzed for survival and for phenotypic alterations. These conventional tests do not permit high- throughput screens, nor permit a directed search for small molecular weight compounds that interfere with a specific morphogenetic pathway related to a human disease condition. Application of compounds by feeding requires relatively large amounts of the substance, and its uptake by the larvae and thus its final concentration is, at best, difficult to control. Furthermore, application by feeding does not permit automation of the procedure necessary for high-throughput analysis. Use of Drosophila in the present invention It is contemplated that any biochemical pathway may be utilized in the present invention, including but not limited to metabolic and signaling pathways. Many, if not all, metabolic and signaling pathways are present in every living cell. However, only a subset of pathways are active at any given time during cellular development and differentiation. The present invention would deliver small molecule compounds to be tested at the development stage when the activity of the desired pathway is required. Any stage in Drosophila development is contemplated, including but not limited to all larval stages such as the first, second and third instar larval stage of development. In Drosophila, the most sensitive phenotypic changes occur in the developing imaginal disks in the third instar larva, as most differentiation occurs at this stage. Metabolic and signaling pathways, which play important roles in human diseases, may be the "targets" of this screening assay. Cell specificity is conferred upon activation of a specific signaling pathway by effector genes located downstream of a given biochemical pathway. It is these downstream effector genes that determines the cell- or tissue-specific phenotype, even though the signaling pathway may actually be indigenous to all cells. These signaling pathways may utilized intracellular "signals" that are normally turned on within the cells of interest, or they may utilize ectopic pathways. Ectopic expression of a component of a given pathway may be induced during development in either a temporally or spatially restricted manner. If the intact signaling pathway to be targeted is present within a cell which ectopically expresses the induced component, the component will activate the targeted signal transduction pathway. The prime target of a signaling pathway is ideally the component which is the most sensitive to alterations within the signaling pathway. The assay methodology of the present invention relies upon the screening of small molecular weight compounds to identify lead compounds that modify essential components in a given signaling pathway, resulting in an observably altered phenotype in comparison to untreated, wild-type Drosophila. One experimental approach for the identification of new components of any given biological process is to search for mutations which dominantly modify the effects of another mutation disrupting the same biological process. This technique allows these aforementioned mutations to be recovered in a simple one-generation (F,) screening assay. More importantly, by sensitizing only a single biological pathway, one may create conditions in which even a slight reduction of gene activity (e.g., due to the loss of only one functional copy of the gene) can result in a detectable phenotype. This is of particular utility when the gene is involved in many other cellular processes, and a more severe loss of function may therefore produce an experimentally less-informative phenotype (e.g., such as lethality). This approach has been successfully used to identify components of the sevenless {sev) RTK signal transduction pathway involved in the induction of the R7 fate during eye development (see e.g., Simon, et al., 1991. Cell 67:701-716). The present invention comprises the use of Drosophila strains that are genetically- modified within the Ras proto-oncogene signaling pathway. A component of this pathway, the Raf serine/ threonine kinase, has been demonstrated to play a critical role in the signal transduction pathways activated by receptor tyrosine kinases (RTKs) across a broad phylogenetic spectrum. Within these signaling pathways, Raf acts to couple Ras activation to the mitogen-activated protein kinase (MAPK) cascade, which consists of the protein kinases MEK (MAPK kinase) and MAPK (see e.g., Marshall, 1995. Cell 80:179-185). The roles of these proteins in Raf signaling have been well-established by both biochemical and genetic studies. Less well-understood, however, are the roles of other R ^binding proteins such as 14-3-3 proteins (see e.g., Fanti, et al., 1992. Nature 371 :612-614; Freed, et al., 1994. Science 265:1713-1716; Fu, et al., 1994. Science 266:126-129), hsp90 (see e.g., Stancato, et al,. 1993. J. Biol. Chem. 268:21711-21716) and immunophilins (see e.g., Stancato, et al., 1994. J. Biol. Chem. 269:22157-22161), as well as the overall extent to which signal transduction via Raf is further regulated by as yet uncharacterized proteins acting within this, or parallel, signaling pathways. In genetically modified Drosophila strains, specific signaling pathways involved in human diseases can be activated at a threshold to produce an easily detectable altered phenotype. It is preferred that such strains are genotypic (+/null), and hence are hemizygous for a dose sensitive gene within a given signaling pathway. These gene products are prime candidates as targets for small compounds that interfere with their function. Activation of the Ras/MAP kinase cascade by Torso RTK results in the specification of head and tail regions in embryonic cells while activation of the same cascade by the sev RTK in the developing eye results in the specification of photoreceptor cell fate. It is possible that each of these receptors activates specific pathways in addition to the common Ras/MAP kinase pathway. The cell-type specific response may depend on which of these parallel pathway is activated by a given receptor. In vertebrate cell culture systems, it has for example been shown that the platelet derived growth factor (PDGF) receptor activates multiple signaling pathways. Identification of the general signaling components downstream of sev, namely Torso and DER {Drosophila homolog of the EGF (epidermal growth factor) receptor), is based on the study of loss-of-function (LOF) mutations, and indicate that the corresponding gene products thus identified are necessary for signaling. Indeed the complete removal of Drk, Sos, or Rasl function in the Torso pathway produces a less severe phenotype than removal of either Torso or Raf function. This suggests that Torso can activate Raf independently of Drk, Sos and Rasl. Additionally, Drosophila possessing the rolled gain of function mutation Sevenmaker {rl SEM ) display a number of additional phenotypes that resemble those of gain of function mutations in torso and DER. Embryos derived from rl SEM females resemble those produced by females carrying a weak torso GOF mutation since they lack to a variable degree the central segmented region. Similarly, the formation of extra veins on the wing of rl SEM flies is reminiscent of the Elp phenotype caused by a gain of function mutation in DER. Therefore, hyperactivation of MAP kinase is not only sufficient to activate the sev pathway but also the developmental pathways controlled by other RTKs. The activation of RTK-specific signaling pathways that act in parallel to the general Ras/MAP kinase pathway is a possible way of maintaining the specificity of the inducing signal from the receptor to the nucleus. The decision of how a cell responds to the generic signal is taken in the nucleus and the nature of the response is determined to a large extent by the combination of nuclear factors present in the different cells at the time of induction. Hence, it may be an evolutionary advantage to use the same universal signaling cascade which can be activated by a number of different cell surface receptors to elicit a limited set of responses at any given stage in development and thereby successively restrict the developmental potential of cells. It will be readily apparent to those individuals skilled in the art that other genetically- modified Drosophila strains may be used in the practice of the present invention. An important attribute in choosing other appropriate Drosophila strains is that the strains have an easily monitored phenotype which is detectably-altered in response to the modification of genes related to disease pathways. Appropriate disease pathways include, but are not limited to, signaling pathways controlled by the Ras proto-oncogene; the WNT tumor suppressor gene; Rb (retinoblastoma tumor suppressor gene); HH (hedgehog development regulator) or the HH vertebrate homolog SHH (sonic hedgehog developmental regulator); activated protein kinase B (PKB/AKT); insulin receptor; insulin receptor substrates (IRS); c-src proto-oncogene; c-Jun proto-oncogene; c-myc proto-oncogene; p53; Janus kinases (JAK STAT pathway); nitric oxide (NO); calmodulin; cAMP dependent protein kinase (PKA); Ca 2+ dependent protein kinase (PKC); growth factors such as GΗ, TGF, PDGF and the like; receptor tyrosine kinases (RTKs); interferons (IFN); lipid metabolites; steroid hormones; phosphatidylinositol; G-protein coupled receptors; c-abl proto-oncogene; TGF-β and Smad gene family members; interleukins; GTPases; and ionophores. This list has been provided by way of example, for purposes of illustration only, and is not intended to be limiting with respect to scope, either real or implied. The present invention discloses a methodology for the screening of genetically- modified Drosophila systems which are sensitive to gene dosage. The Drosophila phenotype which is to be assayed may involve the development of the eye, wing, or any other structure that develops from the imaginal discs in the fly. It should be noted that all previous assays to test the role of small molecular weight compounds have been performed with non-genetically- modified, wild-type Drosophila. Small molecular weight compounds will be administered by microinjection into the open circulatory system (i.e., hemolymph) of genetically-modified Drosophila first, second, or third instar larvae for subsequent use in high-throughput screening (ΗTS) assays of small molecular weight compound libraries and/or in the identification of the dose response curve (or other pharmacological parameters) of lead compounds of interest. Any stage in Drosophila development may be used in the present invention. The Drosophila third instar larval stage may be chosen for both practical as well as specific considerations, which include, but are not limited to: (i) the third instar larvae are easily manipulated at this stage of development; (ii) the third instar larvae are large enough to facilitate automated microinjection in a high-throughput screening (ΗTS) assay; (iii) third instar larvae have an open circulatory system (i.e., hemolymph) through which there is rapid diffusion of the administered compound of interest; and (iv) in the third instar larvae myriad cellular signaling pathways are active in the growth and patterning of imaginal discs, which give rise to the adult structure(s). At this stage any one of these pathways can be genetically sensitized in a way that small perturbation in its activity lead to readily detectable phenotypes in the adult. Perturbation may occur by mutations in genes coding for essential components or as in the case of this invention, by selecting small compounds on the basis of their ability to specifically interfere the adult phenotype. Of equal importance is the fact that the myriad cellular signaling pathways are extremely active during this stage of Drosophila development due to imaginal disc development. As previously discussed, the present invention discloses the utilization of a genetically- modified Drosophila strain which is specifically modified such that it possesses a gene (within a specific signaling pathway known to be involved in human diseases) which can be activated at a defined threshold level to produce an easily-detectable phenotype (e.g., the generation of an irregular, rough eye surface phenotype). This genetic modification may be produced by a naturally-occurring, non- wild-type allele of a specific gene which is isolated from a genetic mutagenesis screening assay well-known to those individuals skilled within the art. Alternatively, the genetic modification may be produced by genetic manipulation using genetic recombination/molecular biological techniques known to those skilled within the art. One contemplated genetic modification is a non- wild-type allele which, when present in the heterozygous state, results in an altered phenotype that is dose dependent. As utilized herein, the term "dose dependent" is designated as meaning that the genetically-sensitized Drosophila strain exhibits an observably different phenotype for each specific genetic state when it possesses either none, one or two copies of the modified allele of the gene of interest. The present invention utilizes the methodology of microinjection into the instar larval developmental stage of genetically-modified Drosophila, for example the third instar, thus permitting high-throughput screening (HTS) with subsequent automation of the assay procedure. This microinjection administration methodology offers several advantages over the conventional application by feeding. These advantages include, but not limited to: (i) both the time and developmental stage of the microinjection may be controlled in a precise manner; (ii) the amount, and thus the final concentration of the compound in vivo can be determined; (iii) the compound is rapidly dispersed through the open circulatory system and quickly reaches the target tissue, the imaginal discs; and (iv) the compounds are only administered at the time when the activity of the pathway is required to develop the easily scorable phenotype. The HTS-based small molecular weight compound screening assay of the present invention is designed to ascertain (i.e., scores for) both the ability of the compound-treated larvae to undergo subsequent development (i.e., pupation) and for their eventual phenotype upon eclosion, in comparison to the mock-treated control Drosophila strain. The use of such in vivo assays allows for the highly relevant analysis of the bioavailability, biological/therapeutic function and toxicity of the compound(s) being tested. The present invention differs from conventional, HTS assays (based upon both cell-free or in vitro cell-based assay systems) in a number of ways including, but not limited to: (i) its overall degree of versatility; (ii) its indigenous screening capacity for compound specificity and non-toxicity and (iii) its lack of bias for specific classes of drug targets. Without automation, the HTS-based small molecular weight compound screening assay of the present invention is capable of screening up to 100,000 compounds in a 1-2 month period. In contrast, with the use of an automated assay procedure, approximately 100,000 compounds may be screened in as little as 1-2 weeks. It should also be possible to scale up throughput with automated screening involving computer-assisted pattern recognition. A wide variety of small molecular weight compounds may be used in the screen. Such compounds include, but are not limited to, any compositions which are being tested for lead drug discovery or development. Compounds may be aqueous- or lipid-soluble. Compounds may be delivered individually to separate Drosophila larvae or may be delivered to separate larvae as one of a plurality of different chemical compounds contained within a reagent solution, such as is performed within a multiplexing schema. Compounds may be dissolved or suspended within solution, or affixed to a solid-support. Solid supports may include, but are not limited to, insoluble polymer beads or a polymeric matrix coated with one or a plurality of individual compounds, or with combinatorial chemistries. Dosages and volumes which are microinjected into the Drosophila larvae may be varied so as to optimize dosages for further studies or to rank compounds as to their toxicity and/or potency. Information resulting from said variations in conditions may be used to prioritize chemicals for further study, to delineate the relative toxicities of structurally related chemicals, and/or to identify the proper dose range for subsequent toxicity studies (see e.g., Harris, et al., Fundam. Appl. Toxicol. 19:186-196). In one embodiment of the present invention, recombinant DNA methodologies will be utilized to express exogenous genes which are functionally-linked to cell-specific transcriptional regulatory sequences. In an additional embodiment, exogenous genes which 10 encode human homologs of the genes involved in the signaling pathway of interest will be utilized, so as to enable "humanization" of the aforementioned disease pathways. An embodiment of the present invention involves the targeting of cell-specific expression of the incorporated exogenous genes to the cells of the Drosophila imaginal discs. Such genetic alterations possess the ability to greatly vary the genetic capabilities of the cells. Compounds that are screened by use of the methodology disclosed in the present invention may be useful as analgesics and/or for the treatment of inflammatory disease, especially in the case of the azotricyclic compounds acting as antagonists of the neurokin 1/brandykin receptor. Members of the benzodiazopine library may be useful as a muscle relaxant and/or tranquilizer and/or as a sedative. Members of the 23 million Mixed Amide Library may be of use in the treatment of hypertension on endothelin antagonists or Raynaud's syndrome. The carbon-carbon backbone of the compounds of the present invention may be saturated or unsaturated, cyclic or linear. These aforementioned compounds include, but are not limited to, carbohydrates, polyalcohols (e.g., ethylene glycol and glycerol) and polyphenols (e.g., hydroquinones and tetracylines). Carbohydrate- and polysaccharide-transformed compounds are defined herein so as to include all chemical moieties possessing a saccharide unit or which are transformed from a saccharide. These compounds may also include glycopeptides, glycolipids and other biopolymers (or biomacromolecules) containing saccharides, either in their entirety or as part of the molecular framework. The term carbohydrates merely represent a portion of a much larger family of polyhydroxylated organic compounds which are within the scope of the present invention. In addition, the carbohydrated/polyhydroxylated organic compounds of the present invention include, but are not limited to: monomeric acyclic compounds (e.g., ethylene glycol, glycerol and 1,2,3- trihydroxy pentane); polymeric acyclic compounds (e.g., di- or tri-ethylene diglycol; monomeric cyclic compounds (e.g., inositol and 1,2,3-trihydroxycyclopentane); polymeric cyclic compounds (e.g., di-inositol); polymeric and monomeric unsaturated compounds (e.g., tetrahydroxy-l,4-quinone) and polyphenols (e.g., tetracyclines) and derivatives, analogs and fragments thereof. With respect to the generation of the small molecular weight compound libraries of the present invention, the combination of biochemical diversity is often synergistic with the metabolic diversity obtained from the in vivo production of "natural products". Collections of starting compounds, for example peptides, can be administered to cultures of microorganisms. In accord, each microbial strain may potentially create numerous modified peptides or peptide byproducts, thus generating a "metabolite library". Because each of these aforementioned cultures would (potentially) contain a very complex mixture of metabolites, a highly efficacious method of screening would be required (i.e., HTS). An aliquot of the library is incubated with each of the many strains typical of a microorganism fermentation screening program, and the media screened utilizing an HTS-based assay. In another aspect of the invention, natural product diversity is screened by creating a mixture of combinatorially-tagged liposomes; wherein each liposome, for example, encapsulates only one member or a simple mixture of a natural product compound library. The libraries which are generated by the methodologies disclosed herein may be screened for any biological activity known within the art. These include, but are not limited to: anti-microbial activity, anti -tumor activity, enzyme inhibiting activity, receptor binding, growth promotion activity, and in vitro and in vivo tests for biological responses. Compounds may be based on naturally occurring extracellular or intracellular signaling molecules or their derivatives or the like (see, e.g., Alberts, et al., 1989. "Chapter 12: Cell Signaling." 2 nd Edition. Garland Publishing, Inc., New York, NY, pp. 681- 726). Unfortunately, many peptidic-based compounds possess unfavorable pharmacodynamic properties such as poor oral bioavailability and rapid clearance in vivo, which have tended to limit the more widespread development of these compounds as potential therapeutic agents. This realization, however, has recently inspired workers to extend the concepts of combinatorial organic synthesis beyond peptide chemistry to create libraries of known pharmacophores (e.g., benzodiazepines; see e.g., Bunin, et al., 1992. J. Amer. Chem. Soc. 114:10997-10998), as well as polymeric molecules such as oligomeric N-substituted glycines (i.e., peptoids) and oligocarbamates (see e.g., Dower, et al., U.S. Patent No. 5,639,603). The invention does not preclude the use of any type of compound library. Each library has its own specific advantages and disadvantages. EXAMPLES Example 1: Microinjection of Third Instar Drosophila melano aster Larvae Drosophila melanogaster which had been previously genetically-modified to activate a disease-related morphogenetic pathway in the developing compound eye are mated. Eggs are collected on a nylon mesh by use of standard techniques known within the art and placed onto standard fly food. Approximately three to five day old larvae (third larval instar) are then collected and placed in the reservoir for the automated injection robot. Larvae are transported on a conveyer belt and immobilized through cooling to 10°C. Larvae are injected through the cuticle into the hemolymph with defined amounts of each compound using a hypodermic needle of 20 μm internal diameter. Following injection, the larvae are placed into glass vials for completion of their development. After eclosion, the adult flies are anesthetized with CO 2 and visually inspected utilizing a dissecting microscope. The parameters which are scored include: (i) the overall toxicity of the compound (determined by the number of survivors compared to mock injected larvae); and (ii) the modification of the genetically-modified Drosophila phenotype. For example, in the case of the rø/phenotype, the genetic modification which is scored is the suppression or enhancement of the irregular rough eye morphology. Example 2: Generation of Drosophila rα ' Mutants Su(Raf) and E(Raf) mutations were initially isolated by virtue of their ability to dominantly modify the rough eye phenotype in transgenic Drosophila carrying the activated Rα/construct Rα/ torY9 (see e.g., Dickson, et al, 1996. Genetics 142:163-171). Figure 2 represents a schematic illustration of the known or estimated cyto logical chromosomal locations of all six Su{Raf) and two E(Raf) complementation groups (loci) (see e.g., Dickson, et al., 1996. Genetics 142:163-171). The Raf ,orY9 fusion produced was identical to that previously described (see e.g., Dickson, et al. 1992. Nature 360:600-603) with the exception that it contained the weakly-activating Y9 mutation in the Torso extracellular domain (see e.g., Sprenger & Nusslein-Volhard, 1992. Cell 71:987-1001). Two different Raf'° m transgenic lines were used in this screening assay. The first was comprised of a second chromosomal insertion of a construct in which Raf toτY9 is expressed under the control of a single sev enhancer and the heat-inducible hsp70 promoter. This insertion is lethal in homozygotes. Drosophila carrying this chromosome balanced over the CyO balancer were used for the first rounds of mutagenesis and the Cy + progeny were scored. Subsequent rounds were performed using Drosophila homozygous for a chromosome 2 carrying a Raf torY9 fusion construct expressed under a duplicated sev enhancer and the sev promoter (see e.g., Dickson, et al., 1996. Genetics 142:163- 171). The two Raf torY9 constructs were found to produced phenotypes of identical strength in heterozygotes. The first number of each allele indicated the round of mutagenesis in which the allele was isolated (i.e., numbers from 1-7 were isolated using the first construct and numbers 8-22 with the second construct). The phyl alleles 3G6 1 7L1 and 20Q1 have also been referred to as phyl 1 , phyl 2 andphyP, respectively (see e.g., Dickson, et al., 1995. Cell 80:453-462). The first rounds (rounds 1-7) of mutagenesis were performed by mutagenizing w 118 males isogenized for chromosome 2; whereas subsequent rounds (rounds 8-22) were performed with males reisogenized for the major autosomes. Males were treated with EMS (see e.g., Lewis & Bacher, 1968. Drosophila Info. Service 43:193-194) and crossed to Raf torY9 transformants. The efficiency of mutagenesis, as estimated by the induction of sex-linked lethal mutations, was consistently demonstrated to be on the order of ~0.60 lethal hits per major chromosomal arm. Example 3: Isolation and Analysis of Modifiers of the Raf XmY9 Phenotype by Genetic Means The following example shows the method by which genes that modify a given pathway of choice can be identified using genetic means. In the manner of the invention described supra, corresponding small molecular weight compounds that modify a given pathway of choice will be used with the resulting genetically modified Drosophila strains to identify biologically active "lead" compounds. The Raf kinase may be activated either by truncation of its amino-terminal domain (see e.g., Stanton, al., 1989. Mol. Cell Biol. 9:639-647) or by relocation of the entire protein to the cell membrane following post-translational modification at an artificial carboxyl-terminal CAAX site (see e.g., Leevers, et al., 1994. Nature 369:411-414; Stokoe, et al., 1994. Science 264:1463-1467). Similarly, the Drosophila Raf kinase is activated following amino-terminal truncation and relocation to the cell membrane via fusion to the extracellular and transmembrane domains of the Torso protein, a receptor tyrosine kinase (RTK) (see e.g., Dickson, et al. 1992. Nature 360:600-603). It should be noted that the Raf kinase activity of this aforementioned fusion protein may be further increased by introducing point mutations in the Torso extracellular domain which are analogous to dominantly activating mutations in the Torso RTK, tor γQ and tor 4021 (see e.g., Sprenger & Nusslein-Volhard, 1992. Cell 71:987-1001). Transgenic Drosophila possessing the activated Raf construct Raf torY9 expressed under the transcriptional control of an enhancer element of the sevenless {sev) gene were generated (see e.g., Basler, et al., 1991. Cell 64:1069-1081). In these transgenic Drosophila, the Raf kinase was found to be constitutively activated in the five cells of the developing eye which choose between the alternative fates of development as an R7 photoreceptor or a non-neuronal cone cell. Typically, Raf is activated in only one of these five cells (i.e., the R7 precursor) via the local activation of the sev RTK. Hence, activation of Raf is both necessary and sufficient for this cell type to select the R7 fate (see e.g., Dickson, et al., 1992. Genetics 142:163-171). In Rα/ ,orY9 Drosophila mutants, ectopic Raf activity within the cone cell precursors results in some of these cells also developing as R7 cells. As a result, most of the 800 ommatidia found within the Drosophila compound eye were found to contain several, instead of just one, R7 photoreceptors. This result is illustrated in Figure 1 (panel F) and these abnormal ommatidia disrupt the regular hexagonal ommatidial lattice. The Drosophila eye thus acquires a "roughened" external appearance, readily observed in live, anaesthetized flies (see Figure 1, panel B). The degree of roughening generally reflects the average number of additional R7 cells per ommatidium, and thus provides an indirect measure of the efficiency of Raf signaling within the cone cell precursors. This observation would thus allow for the identification of "lead" compounds which dominantly-modify the degree of roughening and thus potentially affect some step within the cellular signaling pathway between R«/activation and the selection of the R7 fate. Example 4: Interactions with a Hvpomorphic raf Allele The following example shows the method by which genes that modify a given pathway of choice can be identified using genetic means. In the manner of the invention described supra, corresponding small molecular weight compounds that modify a given pathway of choice will be used with the resulting genetically modified Drosophila strains to identify biologically active "lead" compounds. A complementary screen was performed in which mutations that dominantly-modify the phenotype of a gain-of-function (GOF) raf allele affecting only R7 cell development were isolated. The primary advantage of this approach is that, by specifically-addressing R7 development, it was possible to avoid complications with the formation of lethal genetic Drosophila mutations. Additionally, this type of approach also permitted simultaneously screening for both suppressor Su{Raf) and enhancer E{Raf) mutations. Thus, both positive and negative regulators may potentially be identified with a far greater degree of efficiency by the recovery of LOF alleles. The gain-of-function (GOF) construct Rα/ ,orY9 , provided a convenient means for isolating interacting mutations. There is some evidence, however, that these aforementioned mutations may interact specifically with this construct (e.g., with either the sev enhancer or Torso domain) and not with the Raf ' kinase itself. It was therefore necessary to test each Su{Raf) and E{Raf) locus for genetic interactions with the hypomorphic raf ' allele, raf™ 1 ' '. The raf™ 11 allele produces reduced levels of the wild-type protein, sufficient for Drosophila survival at 18°C but not at 25°C (see e.g., Melnick, et al., 1993. Development 118:127-138). Raised at the permissive temperature of 18°C, only 50% of ommatidia in the eyes of hemizygous raf™ 11 Drosophila males were found to contain an R7 cell (see e.g., Dickson, et al., 1992. Nature 360:600-603). Various result scenarios are possible. For example, if it was demonstrated that the Su{Raf) mutations impaired signaling via Raf it would be expected that they would enhance the raf™ 11 phenotype. If it was found that the Su{Raf) mutations are generally required for Raf function, this may result in a synthetic lethality (i.e., non-allelic, non-complementation) at 18°C. On the other hand, if the Su{Raf) mutations were demonstrated to be specifically required for Rfl function within the Drosophila eye, it would be expected that they would enhance the raf™ 11 eye phenotype without affecting the viability of these flies. Conversely, E{Raf) mutations, if they were demonstrated to relieve negative influences on Raf signaling, it would be expected that they would suppress the raf™ 11 phenotype. Table 1, entitled "Genetic interactions with raf** 1 ", illustrates the genetic interactions between raf HM" and Su{Raf) and two E{Raf) loci. For viable combinations following mating, the eyes of both classes were compared for any modification of the mild degree of roughening caused by the raf HM~ mutation, alone. Four of the Su{Raf) loci were found to enhance the raf ™ 11 phenotype in accordance with these aforementioned predictions. Of these loci, three {rl, Su{Raf)3A and Su{Raf)3B) are synthetically lethal in combination with raf™ 11 , thus suggesting that they encode proteins generally required for signal transduction via Raf. In agreement with this prediction, rl has also been shown to encode a MAPK homologue required in several signaling events involving Raf {see e.g., Biggs, et al., 1994. EMBO J. 13:1628-1635; Brunner, et al., 1994. Nature 370:386-389). The fourth of these \oci,phyl, enhances only the eye phenotype of raf™ 11 , which was demonstrated to significantly increases the number of ommatidia lacking R7 cells (see e.g., Dickson, et al., 1995. Genes Dev. 6:2327-2339). This finding is consistent with the interpretation disclosed herein of phyl as a target gene transcriptionally-regulated only within R7 cells and two other photoreceptors, in response to activation of the Raf ' pathway. It should be noted that, at present, there is no evidence for phyl acting as a target gene for Rα/signaling in any other tissue. The other two Su{Raf) loci - Su{Raf)2A and Su{Raf)3C - show no dominant genetic interaction with raf™ 11 . Somewhat surprisingly, neither of the E{Raf) loci were found to suppresses raf™ 11 , but rather enhance the hypomorphic rø/phenotype, resulting in synthetic lethality. The E{Raf)2A allele 1401 appears to be weaker than the other two alleles at this locus, and a small number of flies of the genotype raf™ 11 ^; 1401/+ eclose. Additionally, the raf™ 11 eye phenotype is also slightly enhanced in these flies. Since the E{Raf) mutations enhance the phenotypes of both LOF and GOF ra/alleles, it is considered to be unlikely that these genetic interactions reflect direct biochemical interactions between the proteins these genes encode and Raf. Genetic interactions with raf •HM7 Allele No. of raf HM7 ; Su/+ No. of raf HM7 ; Relative Modification of raf HM7 Locus tested or τaf HM? ; E/+ males Pw + /+ males viability (%) eye phenotype Su(Raf)2Λ 3E8 73 136 54 None 4P5 54 45 120 None rolled 2L1 0 61 0 6L1 0 63 0 phyllopod 3G6 50 53 94 Enhanced 17L1 156 118 132 Enhanced Su(Raf)3A Ψ 0 83 0 19F2 0 60 0 Su(Raf)3B 18Λ2 0 174 0 Su(Iϊaf)3C 17 Ml 127 121 105 None E(Raf)2A 1401 13 72 18 Enhanced 16H1 0 135 0 16T1 0 157 0 Star 0 50 0 Genetic interactions between raf HM? and Su(Raf) and E(Raf) loci. raf HM? , w"/FM 7 virgins were mated to Su(Raf)/Pιυ + or E(Raf)/ Pιυ + males, and the total number of raf lm7 progeny surviving to adulthood were scored for each class. For viable combinations, the eyes of both classes were compared for any modification of the mild roughening caused by the raf' mι mutation. Example 5; Homozygous Su(Raf) and E(Raf) Phenotypes The following example shows the method by which genes that modify a given pathway of choice can be identified using genetic means. In the manner of the invention described supra, corresponding small molecular weight compounds that modify a given pathway of choice will be used with the resulting genetically modified Drosophila strains to identify biologically active "lead" compounds. The recessive phenotypes of rl, phyl and Star mutations have been reported elsewhere (see e.g., Heberlein, et al., 1993. Dev. Biol. 144:353-361; Biggs, et al., 1994. EMBO J. 13:1628-1635; Chang, et al., 1995. Cell 80:463-472; Dickson, et al., 1995. Genes Dev. 6:2327- 2339). The rl phenotype appears to be required for the development of all eight photoreceptors; whereas phyl is required specifically for Rl, R6 and R7. However, there is only circumstantial evidence for a role of Star in R7 development (see e.g., Kolodkin, et al., 1994. Development 120:1731-1745), but in any case the recovery of a Star allele in our screen is more likely due to its dominant rough eye phenotype than a specific interaction with Raf torY9 . Surprisingly, three of the Su{Raf) genes do not appear to be absolutely required for development either of R7 or any other photoreceptor. The single Su{Raf)3B and Su{Raf)3C alleles are both viable in the homozygous condition and show no obvious defects in eye development. Su{Raf)2A mutations are homozygous lethal, but patches of homozygous mutant tissue could readily be recovered in heterozygous animals using the FLP/FRT technique to induce site-specific mitotic recombination (see Xu & Rubin, 1993. Development 117:1223- 1237). Examination of such homozygous Su{Raf)2A clones shows that, although required for viability, this gene is dispensable for normal Drosophila eye development. Su{Raf)3A mutations were also demonstrated to be lethal in the homozygous state, and it proved to be impossible to generate Su{Raf)3A mutant clones by mitotic recombination by use of the methodologies of the present invention. This suggests that Su{Raf)3A, like raf ' and rl, may also be required for cell proliferation (see e.g., Nishida, et al., 1988. EMBO J. 7:775-781; Biggs, et al, 1994. EMBO J. 13:1628- 1635). The requirement of Su{Raf)3A in the early proliferative phase of eye development precludes a direct examination of its role in the later stages of eye development during which cell fates are determined. In conclusion, E{Raf)2A has been demonstrated to be required for both viability and eye development Homozygous mutant ommatidia were found to be of variable composition, often lacking either R7 or one or more other photoreceptors, but also occasionally containing extra photoreceptors of either class. Example 6: Su(Raf)2A is Required for Ectopic R7 Development The following example shows the method by which genes that modify a given pathway of choice can be identified using genetic means. In the manner of the invention described supra, corresponding small molecular weight compounds that modify a given pathway of choice will be used with the resulting genetically modified Drosophila strains to identify biologically active "lead" compounds. Interestingly, it was found that the elimination of one copy ofSu{Raf)2A severely impairs signaling via Raf ,orY9 ; whereas elimination of both copies of the gene did not appear to affect signaling via the endogenous Raf kinase. One possible explanation of this result would be that Su{Raf)2A encodes a protein that interacts specifically with the activated Raf ,orY9 fusion protein but not at all with the endogenous Raf. To test this hypothesis, Su{Raf)2A clones were generated in genetic backgrounds in which the Raf ' pathway is constitutively activated at points further upstream, i.e., at Sev in Sev su transformants; (see e.g., Basler, et al., 1991. Cell 64:1069- 1082) and at Rasl in Rαϊl V12 transformants (see e.g., Fortini, et al., 1992. Nature 355:559- 561). This aforementioned assay resulted in the formation of ectopic, but not endogenous R7 cells is completely blocked within the Su{Raf)2A clone. This observation argues strongly against a specific interaction between Su(Raf)2A and Raf torY9 and suggests that the protein encoded by Su{Raf)2A is indeed important for signaling via the wild-type Raf kinase, at least for the generation of ectopic R7 cells due to constitutive activation of the pathway at or above Raf Ectopic R7 cells are also formed in yan mutants in an independent manner from the Raf pathway (see e.g., Lai, et al., 1992. Cell 70:609-620). It is believed that the Raf/MAPK pathway acts in part to overcome the inhibitory influence on R7 development normally exerted via the Yan protein (see e.g., Brunner, et al., 1994. Nature 370:386-389; O'Neill, et al., 1994. Cell 78:137-147; Rebay, et al., Cell 81:857-866). The formation of extra R7 cells inyan mutants is also independent of Su(Raf)2A, thus suggesting that in Su{Raf)2A mutant tissue the pathway leading to ectopic R7 development is blocked at some point between Raf and Yan. Example 7: Injection of Rapamycin as a Demonstrative Example To demonstrate the effectiveness of the injection system we developed a protocol for small scale experiments. As a model we used Drosophila larvae genetically modified to express constitutively active PI3 kinase (PI3K) in the compound eye, and Rapamycin, a well known inhibitor in vitro and in vivo of the TOR kinase. Both PI3K and TOR are downstream components of the insulin receptor regulated growth pathway. As a result of the over-activation of this pathway, the eyes of these flies, called RIO, are bigger and more irregularly shaped than wild type eyes. Early phase third instar larvae were anaesthetized for 2 minutes with ether and injected manually with increasing amounts of either a 50 μM solution of Rapamycin in DMSO (dark bars) or DMSO solvent only (light bars) using an Eppendorf microinjector. The injected larvae were transferred to culture tubes and allow to develop at 25°C to adult flies. Flies were then scored for phenotypic changes under the microscope. As shown in FIG. 3, 70 % of the larvae survived the injection procedure, with an injection mortality of about 20%. However, Rapamycin caused a high mortality even at quite low dosages. Larval survival was dose-dependent and reproducible. In addition, 100-500 pi resulted in a significant rapamycin effect (survival and size) without significant unspecific toxicity. R10 flies surviving the injection normally had eyes that were significantly smaller in 80% of rapamycin-injected flies. At higher injection doses only, flies treated with 100-500 pi were also up to 30% smaller than control injected flies. Therefore, the specific reversion of the big eye phenotype at lower dosages demonstrates that genetically modified Drosophila larvae can be used to screen for specific inhibitors of a particular biochemical pathway. EQUIVALENTS Specific embodiments have been exemplified herein for purposes of illustration only, and are not intended to be limiting with respect to the scope of the appended claims. In particular, various substitutions, alterations, and/or modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

Claims: WHAT IS CLAIMED IS: 1. A method of using genetically-modified Drosophila strains in a high-throughput screening assay to screen for compounds putatively affecting a biochemical pathway involving said genetic modification whose activity or function is critical for the development of a phenotype and which can be activated so as to generate an easily detectable phenotype within said genetically-modified Drosophila strains.. 2. The method of claim 1, wherein said genetic modification of Drosophila is comprised of modification of a chromosomal sequence which is related to, and functions in, a disease system or pathway. 3. The method of claim 1, wherein said disease system or pathway is a human disease system or pathway. 4. A method of using a larva from a chosen genetically-modified Drosophila strain for high-throughput screening of one or more compounds which putatively affect the biochemical pathway involving said genetic modification, whereby said genetic modification involves the development of an imaginal disk into its mature structure during pupation; comprised of the following steps: (a) microinjecting at least one compound, alone or in combination, into the open circulatory system (hemolymph) of a genetically-modified Drosophila larva; (b) allowing said microinjected genetically-modified Drosophila larva to undergo pupation; and (c) analyzing the assay results by interpretation of the physiological or morphological effect of said microinjected compound on the insect's adult structure formed from the imaginal disk as seen in the Drosophila imago following eclosion of the mature imago from the pupa. 5. The method of claim 4, where in the genetically-modified Drosophila larva is chosen from a first, second, or third instar larva. The method of claim 4, where in the Drosophila larva is a third instar larva. 7. The method of claim 4, wherein the insect's adult structure formed from the imaginal disk is the compound eye. 8. The method of claim 4, wherein said physiological or morphological effect is the effect of said microinjection compound on the irregular rough eye phenotype of the Drosophila compound eye. 9. The method of claim 4, wherein said compounds are comprised of small molecular weight compounds. 10. The method of claim 4, wherein said compounds, which may be natural products, non- natural products or combinations thereof, are selected from the group consisting of: amino acids, polypeptides, proteins, nucleotides, carbohydrates, lipids, steroids, coenzymes, enzyme inhibitors, ligands and pharmaceuticals such as anti-hypertensive agents, anti-ulcer drugs/H2- receptor antagonists, anti-fungal agents, cholesterol-demethylase inhibitors, anxiolytic agents, analgesics, antibiotics, anti-inflammatory agents, contraceptives, abortifacients, anti- histamines, anti-tussive agents and sedatives, as well as modifications, derivatives and analogs thereof. 11. The method of claim 4, wherein said compounds are attached to an insoluble, solid support matrix by linkages which are cleavable by alteration of the physical environment of said matrix. 12. The method of claim 4, wherein said genetic modification of Drosophila is comprised of modification of a chromosomal sequence which is related to, and functions in, a disease system or pathway. 13. The method of claim 12, wherein said disease system or pathway is a human disease system or pathway. 14. The method of claim 4, wherein said genetic modification of Drosophila is comprised of modification of one or more gene(s) selected from the group comprising: a Raf gene; a Ras proto-oncogene system or pathway; a WNT tumor suppressor system or pathway; a hedgehog development regulator (HH) system or pathway; a sonic hedgehog development regulator (SHH) system or pathway; a retinoblastoma tumor suppressor (Rb) system or pathway; an activated Drosophila protein kinase B (PKB/AKT) gene; an activated human PKB/AKT gene; an activated insulin receptor gene; an insulin receptor substrates (IRS) gene or pathway; c-src proto-oncogene or pathway; c-Jun proto-oncogene or pathway; c-myc proto-oncogene or pathway; p53; Janus kinases (JAK/STAT pathway); nitric oxide (NO); calmodulin; cAMP dependent protein kinase (PKA) or pathway; Ca 2+ dependent protein kinase (PKC) or pathway; growth factors such as GΗ, TGF, PDGF and the like; receptor tyrosine kinases (RTKs) and pathways; interferons (IFN) or pathways; lipid metabolites or pathway; steroid hormones or pathway; phosphatidylinositol or pathway; G-protein coupled receptors or pathways; c-abl proto-oncogene or pathway; TGF-β and Smad gene family members; interleukins or pathway; GTPases or pathway; and ionophores or pathway. 15. A genetically modified Drosophila strain expressing one or more native or modified genes selected from the group comprising: a Raf gene; a Ras proto-oncogene system or pathway; a WNT tumor suppressor system or pathway; a hedgehog development regulator (HH) system or pathway; a sonic hedgehog development regulator (SHH) system or pathway; a retinoblastoma tumor suppressor (Rb) system or pathway; an activated Drosophila protein kinase B (PKB/AKT) gene; an activated human PKB/AKT gene; an activated insulin receptor gene; an insulin receptor substrates (IRS) gene or pathway; c-src proto-oncogene or pathway; c-Jun proto-oncogene or pathway; c-myc proto-oncogene or pathway; p53; Janus kinases (JAK STAT pathway); nitric oxide (NO); calmodulin; cAMP dependent protein kinase (PKA) or pathway; Ca 2+ dependent protein kinase (PKC) or pathway; growth factors such as GΗ, TGF, PDGF and the like; receptor tyrosine kinases (RTKs) and pathways; interferons (IFN) or pathways; lipid metabolites or pathway; steroid hormones or pathway; phosphatidylinositol or pathway; G-protein coupled receptors or pathways; c-abl proto-oncogene or pathway; TGF-β and Smad gene family members; interleukins or pathway; GTPases or pathway; and ionophores or pathway. 16. The method of claim 4, wherein said microinjection procedure is automated. 17. The method of claim 4, wherein said high-throughput screening procedure is automated.

Inventors: HAFEN ERNST, HAFEN, ERNST
Applicants: GENETICS COMPANY INC, HAFEN ERNST, HAFEN, ERNST, THE GENETICS COMPANY, INC.
Application year: 1999
Main classification: G01N33
IPC codes: A01K67, A61K49, G01N33