| In this work, libraries of arylmethyleneimidazolidinone (AMI) derivatives were synthesized by manipulating various R groups around the core structure, and tested for their ability to serve as nuclear receptor ligands with the ability to fluoresce upon binding. The fluorogens are developed for steroidal and non-steroidal receptors, two general classes of nuclear receptors. Specific AMIs were designed and developed for steroid receptor estrogen receptor alpha (ERalpha). These ligands are showed to activate the receptor with an EC 50 value of 3 muM and the 10-fold activation with AMI 1 and AMI 2 in comparison to the 21-fold activation observed with natural ERalpha ligand, 17beta-estradiol. These novel ligands were not able to display the fluorescence upon binding the receptor. However, fluorescence localized in nucleus was observed in the case of another AMI derivative, AMI 10, which does not activate the receptor. Such ligands open new avenues for developing fluorescent probes for ERalpha that do not involve fluorescent conjugates attached to a known ERalpha ligand core.;AMIs were also characterized for non-steroidal receptors, specifically the pregnane x receptor (PXR) and retinoic acid receptor alpha (RARalpha). To date, fluorogens which turn fluorescence upon binding and activate the receptor have not been developed for these receptors. With respect to PXR, several AMI derivatives were discovered to bind and activate this receptor with a fold-activation better than the known agonist, rifampicin. The best characterized AMI derivative, AMI 4, activates the receptor with an EC 50 of value 6.3 muM and the 154-fold activation in comparison to the 90-fold activation and an EC50 value of 1.3 muM seen with rifamipicin. This ligand is not only able to activate PXR but also displays fluorescence upon binding to the receptor. The fluorescence pattern was observed around the nucleus. Besides AMI 4, 16 other AMI derivatives are identified that activate PXR with different activation profiles. Thus, a novel class of PXR ligands with fluorescence ability has been developed. The AMI derivatives able to bind and activate RAR, also displayed activation profiles that were comparable to the wild-type ligand, all trans retinoic acid. These ligands activated the receptor with an EC50 value of 220 nM for AMI 109 in comparison to an EC50 value of 0.8 nM with the natural ligand for RARalpha. When these ligands were tested for fluorescence in yeast, the yeast were able to fluoresce only in the presence of the receptor and the AMI derivative, indicating that these agonists also have the ability to fluoresce.;To provide the opportunity to gain further insight into interaction between the ligand and the receptor, these nuclear receptors were also subjected to rounds of mutagenesis, engineering receptors with the ability to bind AMI derivatives. The most promising of these rounds of mutagenesis was the discovery of an estrogen receptor alpha variant, E380D that showed an increase in the fold activation from two fold to five fold with AMI 2 in comparison to the wild-type receptor. This variant also showed an enhanced fluorescence with AMI 1 and 2 in comparison to the wild-type receptor. Interestingly, the variants have mutations outside of the ligand binding pocket, showing the importance of residues that are not in direct contact with the ligand and how the secondary shell residues and even those on the surface of the protein could have an allosteric effect on ligand binding.;The discovery of this novel class of ligands, AMIs, for various nuclear receptors indicates that they can serve as strong candidates for these receptors, in terms of drug development and resources for visualizing nuclear receptor mobility and trafficking. These fluorogens hold great promise as perhaps the next generation of novel ligands for various nuclear receptors and will perhaps allow a better understanding of the functionality of these proteins and their mechanism to certain diseases. (Abstract shortened by UMI.). |
