Fluorescent GFP chromophores as potential ligands for various nuclear receptors

Nuclear receptors are ligand activated transcription factors, where upon binding with small molecule ligands, these proteins are involved in the regulation of gene expression. To date there are approximately 48 human nuclear receptors known, involved in multiple biological and cellular processes, ranging from differentiation to maintenance of homeostasis. Due to their critical role in transcriptional regulation, these receptors are implicated in several diseases. Currently, 13% of prescribed drugs in the market are NR ligands for diseases such as cancer, diabetes and osteoporosis. In addition to drug discovery, the mechanism of function, mobility and trafficking of these receptors is poorly understood. Gaining insight into the relationship between the function and /or dysfunction of these receptors and their mobility will aid in a better understanding of the role of these receptors. The green fluorescent protein (GFP) has revolutionized molecular biology by providing the ability to monitor protein function and structure via fluorescence. The fluorescence contribution from this biological marker is the chromophore, formed from the polypeptide backbone of three amino acid residues, buried inside 11-stranded â-barrel protein. Synthesis of GFP derivatives of is based on the structure of the arylmethyleneimidazolidinone (AMI), creating a molecule that is only weakly fluorescent. Characterizing these AMI derivatives for other proteins can provide a powerful visualization tool for analysis of protein function and structure. This development could provide a very powerful method for protein analysis in vitro and in vivo. Development of such fluorescent ligands will prove beneficial for the nuclear receptors. In this work, libraries of AMIs derviatives 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 á (ERá). These ligands are showed to activate the receptor with an EC50 of value 3 ìM and the 10-fold activation with AMI 1 and AMI 2 in comparison to the 21-fold activation observed with natural ERá ligand, 17â-estradiol. These novel ligands were not able to display the fluorescence upon binding the receptor. However, fluorescence localized in nucleus was observed in case of another AMI derivative, AMI 10, which does not activate the receptor. Such ligands open new avenues for developing fluorescent probes for ERá that do not involve fluorescent conjugates attached to a known ERá ligand core. AMIs were also characterized for non-steroidal receptors,specifically the pregnane x receptor (PXR) and retinoic acid receptor á (RARá). 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 EC50 of value 6.3 ìM and the 154-fold activation in comparison to the 90-fold activation and an EC50 value of 1.3 ìM seen with rifamipicin. This ligand is not only able to activate PXR but also displays fluorescence upon binding to the receptor. The fluroscence 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 with AMI 109 in comparison to an EC50 value of 0.8 nM with the natural ligand for RARá. 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.Ph