Compounds That Act To Modulate Insect Growth And Methods And Systems For Identifying Such Compounds

Abstract

Disclosed are methods and systems for screening for compounds that act to modulate insect growth. Bioassays including cell culture and/or transgenic insects engineered with various components of the ecdysoid receptor (EcR) and/or the farsenoid-X receptor (RXR) systems to identify compounds that act as insecticides and/or hormone receptor activators are described. Also described are compounds, and compositions, identified as being putative insecticides based upon their ability to activate EcR and/or FXR mediated transcription.

1 . A method for testing the ability of a compound to act as a modulator of insect growth comprising determining whether the compound increases farnesoid X-activated receptor (FXR)-mediated transcription and/or ecdysone receptor (EcR)-mediated transcription. 
     
     
         2 . The method of  claim 1 , wherein the increase of EcR-mediated transcription comprises potentiation of hormone-activated EcR-mediated transcription. 
     
     
         3 . The method of  claim 2 , wherein the hormone comprises an ecdysteroid. 
     
     
         4 . The method of  claim 3 , wherein the hormone comprises muristerone A (murA) or 20-hydroxyecdysone (20E). 
     
     
         5 . The method of  claim 1 , comprising the steps of:
 (a) transfecting isolated cells with DNA comprising sequences that encode a functional isoform of the ecdysone receptor (EcR) or the farnesoid receptor (FXR);   (b) cotransfecting the cell with: (i) DNA comprising sequences that encode a functional heterologous binding partner for (a), wherein said heterologous binding partner complexes with either EcR or FXR, and wherein said complex binds to a hormone responsive element (HRE) on DNA to activate gene transcription; and (ii) DNA comprising a reporter construct comprising a hormone response element (HRE) linked to a gene;   (c) adding the compound to be tested; and   (d) measuring an increase in the synthesis of the reporter gene protein.   
     
     
         6 . The method of  claim 5 , wherein the increase in reporter gene protein is used to quantify the ability of the test compound to increase EcR-mediated transcription or FXR-mediated transcription. 
     
     
         7 . The method of  claim 6 , wherein the ability of the test compound to activate EcR-mediated transcription or FXR-mediated transcription is correlated to the potential insecticidal activity of the test compound. 
     
     
         8 . The method of  claim 5 , wherein the heterologous binding partner comprises Ultraspiracle (USP). 
     
     
         9 . The method of  claim 5 , wherein the heterologous binding partner comprises a retinoid X receptor (RXR). 
     
     
         10 . The method of  claim 5 , further comprising adding a modulator of EcR activity to the cell. 
     
     
         11 . A method for in situ testing for the presence of compound having the ability to act as a modulator of insect growth comprising:
 (a) transfecting a cell in an organism with DNA that encodes a functional isoform of the farnesoid X-activated receptor (FXR) and/or the ecdysone receptor (EcR);   (b) cotransfecting the cell with: (i) DNA that encodes a polypeptide comprising a functional retinoid X receptor (RXR) or Ultraspiracle (USP) isoform; and (ii) DNA comprising a hormone response element (HRE) linked to a reporter gene;   (c) optionally adding an ecdysteroid;   (d) exposing the organism to a compound to be tested; and   (e) measuring an increase in the protein encoded by the reporter gene.   
     
     
         12 . The method of  claim 11 , wherein the organism comprises a natural or modified eukaryotic cell. 
     
     
         13 . The method of  claim 11 , wherein the reporter construct comprises a promoter comprising multiple hormone response elements (HREs) linked to a gene encoding a detectable gene product. 
     
     
         14 . The method of  claim 11 , wherein the reporter gene comprises luciferase (LUC). 
     
     
         15 . The method of  claim 11 , wherein the hormone response element comprises a EcR response element. 
     
     
         16 . The method of  claim 11 , wherein the hormone response element comprises a FXR response element. 
     
     
         17 . A composition for use as an insecticide comprising a compound that increases FXR-mediated transcription or EcR-mediated transcription mixed with a suitable carrier for application to plants. 
     
     
         18 . The composition of  claim 17 , wherein the increase in EcR-mediated transcription comprises potentiation of hormone-activated EcR transcription. 
     
     
         19 . The composition of  claim 17 , wherein the hormone comprises an ecdysteroid.