Green fluorescent protein inspired chromophore as estrogen receptor agonist-synthesis, biological evaluations and cellular application

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Walker, Christopher L.
Williams, Loren D.
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Nuclear receptors are ligand activated transcription factors that are widely distributed throughout the mammalians. There are 48 known human nuclear receptors within the body located in various systems. While some nuclear receptors can be located wholly within certain regions and tissues the clear majority are widely distributed, overlapping expression in the same locations. The role of nuclear receptors as transcription factors has caused them to be implicated in a vast number of diseases including metabolic, cardiovascular and neurological. The role of nuclear receptors in diseases and the potential to promote ligand activated transcription makes nuclear receptors of pharmaceutical significance. Currently it is estimated that 33% of nuclear receptors are targeted by the pharmaceutical industry resulting in ~20% of pharmaceutical development worldwide. The potential to control physiological responses via introduction of a ligand to nuclear receptors has continued the interest in development of new ligands to further the understanding of nuclear receptor behavior. The challenge nuclear receptors present is to develop ligands that are selective in targeting within families and among different classes of nuclear receptors. At the core, ligand activated nuclear receptor modulation is chiefly centered around the relationship between the ligand binding pocket of the receptor and the ligand. Composed primarily of non-polar amino acid residues the ligand binding pocket is the cavity by which small hydrophobic molecules bind. Demonstrating large variance across classes of nuclear receptor and little divergence within families the ligand binding pocket serves as the focal point for targeting selectivity. Successful binding and thus receptor response is contingent upon the ligand meeting criteria established by the ligand binding pocket such as satisfactory size/volume of the ligand and key ligand-receptor amino acid residue interaction. Research conducted by Katzenellenbogen was paramount in understanding the relationship between the estrogen receptors and its ligands. His established pharmacophore unraveled features for potential ligands that are exchangeable from those that are indispensable. The commercial success of estrogen receptor ligands has fueled the interest in not only understanding ligand-receptor binding interactions but its subcellular movement. The Green Fluorescent Protein completely revolutionized the way in which cellular probing is conducted. The chromophore internally synthesized by the protein through a series of folding of amino acid residues afforded the opportunity to monitor cellular movements with the aid of fluorescence. Commonly utilized in visualization as a fusion protein, the GFP chromophore provided the ideal tool for understanding protein cellular movement and interaction. Simply put due to the chromophore that resides at the center, GFP provides the perfect technique for cellular probing. Here in we report the use of GFP-chromophore inspired ligands for utility as estrogen receptor agonist. By utilizing the GFP chromophore skeleton as a template for ligand development the potential arises for the molecule to co-function as a receptor binder and probe. The use of the GFP-chromophore skeleton boasts several advantages in addition to synthetic amenable features the arymethyleneimidazolone core maintains the same frame work as Katzenellenbogen’s proposed pharmacophore. Through the lens of Katzenellenbogen’s consideration and the use of a simple but elegant synthesis a small library of GFP chromophore inspired arylmethyleneimidazolone ligands were synthesized, screened for selective estrogen receptor activation and tested both in vivo and in vitro for cellular probing applications. Through this work we identified a set of 10 ligands that serve as agonist for the estrogen receptor. Although of the 10 ligands several demonstrate activation for both ERα and ERβ, a high degree of preference for ERα is observed. Of the ligands screened all estrogen receptor active ligands were nuclear receptor selective failing to activate other receptors such as RAR and RXR. Biological screening also uncovered a super agonist in CW32 that demonstrated the highest level of activation. Though a structure activity relationship model was established for top activators and additional generations synthesized no compound was found to be more active than CW32. While the majority of ligands displayed a preferential affinity for ERα ligand CW72 demonstrated complete specificity for ERα. All ligands were confirmed through TRFRET as binding in the same ligand binding pocket as estradiol. Computational modeling supports the rationale that the following three criteria governed the ligands ability to successfully bind: 1) hydrogen bonding network, 2) ligand size/volume and 3) molecular topology. Embracing the ligands skeleton originating from the Green fluorescent chromophore ligands that demonstrated ER activation were visualized under confocal both with in vivo and in vitro systems. Several ligands successfully demonstrated the ability to turn on fluorescence in responds to binding in vitro. While other ligands failed to display fluorescence in conjunction with binding. Despite all binders displaying fluorescence this represent a class of ligand that can serve as a activator and probe.
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