Bioavailable diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex
View Patent ↗The present invention relates to non-steroidal ligands for use in nuclear receptor-based inducible gene expression system, and a method to modulate exogenous gene expression in which an ecdysone receptor complex comprising: a DNA binding domain; a ligand binding domain; a transactivation domain; and a ligand is contacted with a DNA construct comprising: the exogenous gene and a response element; wherein the exogenous gene is under the control of the response element and binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene.
1. A method of modulating the expression of a target gene in a host cell, wherein the host cell comprises recombinant DNA encoding:
(i) a transactivation domain;
(ii) a DNA-binding domain; and
(iii) a Choristoneura fumiferana ecdysone receptor ligand binding domain;
and a gene expression cassette comprising:
(i) a response element capable of binding to said DNA binding domain;
(ii) a promoter that is activated by the transactivation domain; and
(iii) said target gene;
the method comprising contacting said host cell with a compound
selected from the group consisting of:
3,5-Dimethyl-benzoic acid N′-(2-ethyl-3-methoxy-benzoyl)-N-(2-hydroxyimino-1,1-dimethyl-ethyl)-hydrazide;
Acetic acid 2-[N-(3,5-dimethyl-benzoyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazino]-2-methyl-propyl ester;
2-Ethyl-3-methoxy-benzoic acid N′-tert-butyl-N′-(1-methyl-1H-indole-2-carbonyl)-hydrazide;
3-(H 2 NC(O)NHN═CH-5-methyl-benzoic acid N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
3-(H 2 NC(O)C(O)NHN═CH-5-methyl-benzoic acid N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
2-Methoxy-6-trifluoromethyl-nicotinic acid N-tert-butyl-N′-(3-methoxy-2-methyl-benzoyl)-hydrazide;
3-Methoxy-2-methy-benzoic acid N′-tert-butyl-N′-(1-methyl-2-oxo-6-trifluoromethyl-1,2-dihydro-pyridine-3-carbonyl)-hydrazide;
1H-Benzoimidazole-5-carboxylic acid, N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
1-Trityl-1H-benzoimidazole-5-carboxylic acid N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
3-Trityl-1H-benzoimidazole-5-carboxylic acid N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
1H-Indazole-3-carboxylic acid N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
1-Trityl-1H-indazole-3-carboxylic acid N-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
3,5-Dimethyl-benzoic acid N-[2-(tert-butyl-dimethyl-silanyloxy)-1,1-dimethyl-ethyl]-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide;
3,5-Dimethyl-benzoic acid N′-(2-ethyl-3-methoxy-benzoyl)-N-[2-(2-hydroxy-ethylimino)-1,1-dimethyl-ethyl]-hydrazide;
3,5-Dimethyl-benzoic acid N-[(H 2 NC(O)NHN═CH)(CH3) 2 C-]N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide; and
3,5-Dimethyl-benzoic acid N-[(H 2 NC(O)C(O)NHN═CH)(CH3) 2 C-]-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide.
2. The method of claim 1 , wherein said recombinant DNA encodes GAL4 DBD(1-147)-CfEcR(DEF)/VP16AD-βRXREF-LmUSPEF, GAL4 DBD-CiEcR(DEF)/VP16AD-MmRXRE, GAL4 DBD(1-147)-CfEcR(DEF)/HsRXRβ-EF-LmUSPEF, or CfEcR D, E and F domains corresponding to SEQ ID NO:1 wherein valine (V) at position 107 is mutated to isoleueine (I) and tyrosine (Y) at position 127 is mutated to glutamic acid (E).