Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acid esters
Reexamination Certificate
2002-02-21
2004-10-12
Killos, Paul J. (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acid esters
C560S022000
Reexamination Certificate
active
06803479
ABSTRACT:
STATEMENT OF INVENTION
This invention is a photosensitive and photolabile precursor of a non-steroidal ecdysone mimetic (NSE), or a “caged” NSE. A caged NSE is biologically inactive until it is photochemically transformed by light to yield free NSE, which is biologically active. A caged NSE can be introduced into living cells and tissues having the appropriate protein machinery and genes under ecdysone promoter control. When illuminated, biologically active NSE will be generated photochemically in situ, and activate expression of the genes placed under ecdysone promoter regulation. Caged NSE thus allows the researcher to turn on recombinant gene expression with unprecedented spatial and temporal control.
BACKGROUND INFORMATION ON THE CHEMICAL STRUCTURES ON WHICH THIS INVENTION IS BASED
NSEs are molecules that act biologically as does the insect hormone ecdysone, which controls the molting process during insect growth and development. NSEs have been put to two uses: 1) as an insecticide against agriculturally damaging insect pests (it acts by disrupting the normal endocrine regulation of insect growth); and 2) as an activator of transgene expression in transgenic cells in which an exogenous gene is placed under transcriptional regulation of the ecdysone promoter. Insecticidal use of the bisacylhydrazine class of NSEs was developed primarily by researchers at Rohm and Haas. The use of one of the bisacylhydrazine NSEs for ecdysone promoter-regulated transgene activation was introduced by Invitrogen (Invitrogen markets a bisacylhydrazine compound under the name “GS-E,” under license from Rohn and Haas). The same advantage of NSE underlies both its insecticidal and transgenic gene expression applications. The bisacylhydrazines are non-toxic and environmentally benign, and because they are mechanism-based drugs, work only against certain classes of agricultural pests without affecting other lifeforms. Similarly, because the ecdysone molting hormone system is unique to insects, when the ecdysone promoter system is used to control transgene expression in non-insect organisms, it can be activated only by applying ecdysone steroid or its bioactive mimetics. The NSEs thus afford a non-toxic method for activating transgene expression in organisms other than insects.
SUGGESTED SCOPE OF INVENTION
The primary intended use of caged NSEs is for activating transgene expression with high temporal and spatial resolution in transgenic cells, tissues, or developing animals.
RESULTS DEMONSTRATING THE CONCEPT IS VALID
Caged Molecules Our laboratory has been designing, synthesizing caged molecules, as well as developing their applications in biological systems for over ten years. The concept of using light to rapidly transform a biologically inactive molecule into a biologically active one in situ has been repeatedly proven in a variety of reagents.
Synthesis and Characterization of a NSE, upon which the Caged NSE is Based
We have synthesized and characterized a NSE of the bisacylhydrazine family, namely, 1-(3,5-dimethylbenzoyl)-1-tert-butyl-2-(2-ethyl-3-methoxybenzoyl)hydrazine (hereafter referred to as NSE-1). The chemical synthesis is shown in Schemes 1 and 2. Physical properties are shown in Table 1.
TABLE 1
Physical properties of 1-(3,5-dimethylbenzoyl)-1-tert-
butyl-2-(2-ethyl-3-methoxybenzoyl) hydrazine
Chemical Formula
C
23
H
30
N
2
O
3
Molecular Weight
382.51
Aqueous Solubility*
47 &mgr;M
Extinction Coefficient at 280 nm (&egr;
280
)*
2,550 ± 80 L · mol
−1
· cm
−1
*Measured in 100 mM KCl, 10 mM K · HEPES, pH 7.4
Biological Testing of NSE-1
Toxicity of NSE-1 in Cell Culture
Cellular toxicity of NSE-1 was tested in cell culture. NIH3T3-ER fibroblasts (Stratagene, La Jolla, Calif.) were seeded into 96-well plates, allowed to attach for 24 hours in DMEM supplemented with 10% v/v fetal bovine serium (FBS). The medium was then replaced with DMEM-10 FBS containing ecdysoid drugs or solvent vehicle, and the cells were allowed to grow for 48 hours, at the end of which the cells were lysed and the amount of DNA in each well quantified through the CyQuant assay (Molecular Probes, Inc. Eugene, Oreg.). Neither Ponasterone A at 10 &mgr;M, nor NSE-1 at 10, 30 or 100 &mgr;M significantly affected the growth capacity of the cells. The data are summarized in FIG.
1
. These data suggest that NSE-1 is not cytotoxic.
Toxicity of NSE-1 in Animals
NSE-1 was suspended (32.5 mg/ml) in carrier vehicle consisting of 15% w/v Pluronic F127 surfactant (BASF Corp., Wyandotte, Mich.) and 3% v/v dimethylsulfoxide in phosphate buffered saline (PBS, pH 7). Intraperitoneal injections of this formulation (six 200-&mgr;l aliquots administered at 12-hour intervals) into 8 mice did not produce any signs of intoxication. These observations give at least preliminary data showing that NSE-1 is not acutely toxic to mammals.
Induction of Transgene Expression in Transiently-Transfected Cells
NIH3T3-ER cells were transiently transfected with a plasmid pEGSH-GFP carrying the reporter eGFP gene under ecdysone promoter control. The transfected cells were treated with 10 &mgr;M ponasterone A, or 30 &mgr;M NSE-1, or plain medium for 48 hours and then screened for induction of eGFP expression by single-cell microfluorimetry. The results are shown in FIG.
2
. These results demonstrate the efficacy of NSE-1 in inducing transgenes in cultured cell lines.
Chemical Synthesis of New Cage
Synthesis of the photosensitive caging group to be used for the caged NSE invention is nearing completion. The structure of the caging reagent is shown to the right.
Caging Reaction of NSE
We have completed a caging reaction of NSE-1 with a known cage which shares structural features with the proposed new cage shown above. This model reaction is shown in the scheme below. That this model reaction was successful suggests that when the new cage is synthesized, its caging reaction with NSE-1 will mostly likely be successful.
VARIATIONS AND ALTERED FORMS OF THE INVENTION
There are two other transgene induction methods in current use. One relies on compounds in the tetracycline class of antibiotics, the other depends on the antiprogestin mifepristone (commonly known as RU486). Potentially the caging approach could be applied to yield caged tetracyclines and caged antiprogestins for use in those inducible gene expression systems, and thus extend their power and utility.
NOVEL FEATURES
No comparable method for rapidly and spatio-selectively activating gene expression.
REFERENCES:
patent: 4985461 (1991-01-01), Hsu et al.
patent: 632016 (1995-01-01), None
Monroe et al,J. of Biol. Chem., 274(30):20895-20900.
Cambridge et al,Science, 277:825-828 (1997).
Freilich David Ari
Kao Joseph Pao Yung
Killos Paul J.
University of Maryland Biotechnology Institute
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