Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1998-12-21
2003-04-15
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S008000, C800S009000
Reexamination Certificate
active
06548733
ABSTRACT:
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. Drug discovery processes are extremely lengthy. A conventional process involves the screening of thousands of individual compounds for a desired biological/therapeutic activity. Historically, less than 1 in 10,000 of the synthetic compounds have actually been approved by the Food and Drug Administration (FDA), at a cost of greater than $200 million per drug (see e.g., Ganellin, et al., 1992. In: Medicinal Chemistry for the 21
st
Century. pp. 3-12 (Blackwell Publications, London, England)).
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.
An alternative methodology for the development of novel pharmacological compounds has been to screen individual compounds which have been previously synthesized and saved in “libraries” within drug/chemical companies or research institutions. The compounds in these libraries were often initially chosen for synthesis or screening due to the fact that they possessed a particular functionality thought to be relevant to a specific biological activity.
More recently, peptide or oligonucleotide libraries have been developed which may be screened for a specific biological function (see e.g., Musser, 1992. In: Medicinal Chemistry for the 21
st
Century. pp. 3-12 (Blackwell Publications, London, England); U.S. Pat. Nos. 5,593,853 and 5,639,603). For example, recombinant peptide libraries have been generated by the insertion of degenerate oligonucleotides into the genes encoding the capsid proteins of filamentous bacteriophage and the DNA-binding protein Lac I (see e.g., Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:6378-6382; Cull, et al., 1992. Proc. Natl. Acad. Sci. U.S.A. 89:1865-1869 and PCT Publications Nos. WO 91/19818 and WO 93/08278). These random libraries may contain more than 109 different peptides, each fused to a larger protein sequence which is physically-linked to the genetic sequences encoding it. The libraries are subsequently screened by allowing the peptide to interact with its specific ligand (e.g., receptor, nucleic acid, etc.) via several rounds of affinity purification and the selected exposition or display vectors are then amplified in, for example,
E. coli
, and the nucleic acid contained therein is sequenced to reveal the identity of the peptide responsible for the ligand interaction.
(i) Screening Methodologies
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 the precise knowledge of both the molecular defect underlying a given disease and the knowledge 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 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.
Currently, numerous drug screening protocols rely upon high-throughput screening (HTS) of compound libraries using cell-free or in vitro cell-based assay systems. Several drugs (e.g., cyclosporine A and mevastatin) have emerged directly from utilization of this methodology. HTS is a process by which large numbers of compounds with putative biological activity may be tested, preferably 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). It should be noted, however, that HTS does not actually identify a drug, but rather, the primary goal of HTS is to identify high-quality “hits” or “leads” (i.e., compounds which affect the target molecule in the desired manner) which are active at a relatively low concentration and that possess a novel structure or sequence and to supply directions for their potential optimization. Although HTS is a powerful screening tool, it possesses 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. These limitations exist because many of the properties critical to the development of a drug typically can not be directly assessed by HTS; therefore, the final compound which eventually becomes a drug is unlikely to have been the molecule present in the initial small molecular weight compound library. Generally, the greater the number and diversity of the compounds which are analyzed, the more successful the screening is likely to be, a fact which has markedly accelerated the development of HTS.
A well-designed HTS screening assay may also provide information regarding the potency of a compound of interest. Generally, the lower the concentration at which the compound of interest exhibits activity, the more likely it will exhibit specificity and, as a corollary, the less likely that it will have undesirable or deleterious side-effects. Information on specificity may also be obtained by concomitantly performing a counter-screen with a related target molecule (e.g., an HIV protease verses a cellular aspartyl protease or the serotonin 2A receptor verses the serotonin 2C receptor). Compounds which exhibit activity only against the primary target are deemed most likely to possess the necessary selectivity. If different chemotypes may be identified using the same screen, then medicinal chemists will have a broader range of options for modification of the novel, lead compound. In addition, the spectrum of compounds which score positive (and to some extent those compounds which score negative) may help to pinpoint those structural characteristics and motifs of the molecules which are responsible for their efficacy and specificity.
The HTS methodology requires four distinct elements: (i) suitably arrayed compound libraries; (ii) as assay methodology amenable to automation; (iii) a robotics workstation and (iv) a computerized system for input and analysis of incoming data from the screening assay. Currently, the 96-well microtiter plate is the standard format for automated HTS assay, although arrays of co
Crouch Deborah
Elrifi Ivor R.
Konzak Kristin E.
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
The Genetics Company, Inc.
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