It is well-established that phytochemicals serve as recruitment signals resulting in the symbiotic activation of plant growth signals by soil bacteria, and that the presence of endocrine disrupting compounds (EDCs) contained in pesticides and other industrial by-products can disrupt this process [38
]. The direct production of xenoestrogenic compounds by soil bacteria is not as well established and therefore represents an opportunity for discovery of new chemical scaffolds with possible utility as, or the potential for optimization of, ER modulators. Secondary metabolites have been identified through drug discovery methods and include valuable compounds such as antitumor and antibacterial agents. The University of Wisconsin's WDL contains crude bacterial extracts consisting of various natural products and was therefore screened using three highly optimized BRET assays to identify novel inducers of ERα/α and ERβ/β homodimerization as well as ERα/β heterodimerization. The application of these BRET assays to screen identify new ER modulators from crude extracts obtained from actinomycetes originating from unique ecological niches resulted in the identification of a novel, previously uncharacterized, dimer selective ER agonist named actinopolymorphol A.
High throughput, mechanism-based assays are on call to advance the discovery of xenoestrogens and drug leads at a rapid pace. Two types of high throughput assays are popularly used for large-scale screening of ER structural scaffolds and agonists or antagonist ligands. A fluorescence polarization (FP) method that measures the capacity of a competitor chemical to displace a high affinity fluorescent E2 from purified, recombinant ERα or ERβ have been adapted for testing environmental chemicals for ER binding interactions [36
]. However, this method requires pure preparation of receptor, and the fluorescence from the test compounds could interfere with fluorescence readout [36
]. Thus far this method has been restricted to use with pure compounds and has not been applicable to whole cell extracts or bioassay-guided fractionation efforts.
Transcriptional reporter assays can be applied to library extracts or compounds but require 18-24 h incubations. Thus, these extracts or compounds must be sterile and of high-quality tissue culture grade to avoid contamination and concomitant ablation of the transcriptional output signal. The BRET assays described herein circumvent this issue because the library extract or compound in question needs only to be incubated with cells expressing ER fusion proteins for 1 hour to induce dimerization. Furthermore, the three possible ER dimer pairs (ERα/α homodimers, ERβ/β homodimers, and ERα/β heterodimers) may be directly examined in parallel yet in isolation from each other, thus providing an added layer of sensitivity and complexity to the library extract or compound's ability to act as an agonist or antagonist ligand. Thus, the utility of this cell-based assay for high throughput crude extract screening lies in its rapid assay time frame. In addition, this method does not require sterile tissue-culture grade extracts. Using this BRET screening method, the crude extract from A. rutilus was found to selectivity induce formation and transcriptional activity of ERβ/β homodimers and ERα/β heterodimers. This screening method allowed assay-guided fractionation of the extract, and the pure compound responsible for induction of dimerization and subsequent transcriptional activity was identified as actinopolymophol A, a previously unknown natural product. However, estrogenic compounds identified by BRET assays may activate both genomic and non-genomic signaling pathways. Different from the classical genomic signaling through EREs, these non-genomic signaling pathways initiated at the cell membrane may also require receptor dimerization and couples with a variety of other signaling partners that can eventually culminate in the phosphorylation of transcription factors and their partners, ultimately influencing transcriptional outcome, and thus physiological effects such as cell division and apoptosis. Thus, the physiological effects of BRET identified compound await further characterization.
It is worth noting that BRET assays measure the ligand's ability to induce receptor dimerization and the FP method measures ligand replacement of E2 in ER pocket; neither of these assays can distinguish agonists from antagonists. Transcriptional reporter assays measure the ability of the lead compound to induce or inhibit (in the presence of E2) transcription of ER subtypes, allowing determination of agonist or antagonist activity of a ligand. For example, while a low level of ERα/α homodimerization was induced by this compound and substantiated via the BRET assay, these proliferative dimers were not transcriptionally active in ERE-luciferase reporter assays. Because of the limitation of each assay, all three were employed in the present study leading to the discovery of the estrogenic natural product.
The structure of this novel ER dimer-selective natural product from A. rutilus was determined by NMR and mass spectroscopic analysis and its absolute stereochemistry was established by total synthesis using an optically pure starting material ((S)-2-hydroxy-3-(4-hydroxyphenyl)-propionic acid). (Huang et al., submitted for publication). Combined, the results of structural characterization and coordinated BRET assays reveal that actinopolymorphol A represents a novel scaffold for estrogenic small molecule design. Molecular modeling suggests that, although the phenolic hydroxyl group of actinopolymorphol A mimics the C-3 hydroxyl group of 17β-estradiol and makes the same hydrogen bond interactions with residues Glu and Arg in both ERs' binding pockets (), other structural elements of the natural product do not strictly adhere to predictions likely to be made on the basis of other ER ligands such as tamoxifen or raloxifene. The modeled structure explains how actinopolymorphol A may compete with E2 in binding to the same LBD in FP assay while also displaying a lower binding affinity due to the absence of functionalities needed to H-bond with histidine distal to the Glu and Arg end of the ER LBD. This modeling indicates that the agonist or partial agonist conformation is adopted by ERα and ERβ and that their ligand binding cavities are shaped into a low-energy conformation by actinopolymorphol A. Perhaps most significantly, actinopolymorphol A is, to the best of our knowledge, the first ER dimerization modulator identified from actinomycetes. As such, discovery of this natural product and subsequent association with ER modulatory function unveils a new molecular scaffold with a novel and potentially useful bioactivity. This structure may serve as a molecular scaffold upon which chemical modifications may be made in order to increase the selectivity and efficacy of this novel compound.
The application of ER BRET assays for high throughput screening of crude natural product extracts and subsequent bioassay-guided fractionation leading to the identification of actinopolymorphol A showcases a new molecular scaffold but also highlights the utility of BRET assays in discovering new, otherwise difficult to detect, natural products with ER modulatory activity. These ER modulatory compounds may function through genomic or non-genomic signaling pathways. Follow-up assays showed that actinopolymorphol A is able to act as an agonist on ERs and can decrease the growth of ERα and ERβ positive cell lines while not adversely affecting their viability. Despite its low activity on ERs, this novel structure is able to compete with endogenous E2 for LBD binding in both ERα and ERβ. Competitive LBD binding by actinopolymorphol A is rationalized on the basis of molecular modeling which suggests the natural product can induce an agonist conformation upon binding to both ERs. Combined, these data reveal the unique application of BRET assays to find new ER modulators and reveal actinomycetes as a potentially rich source of such bioactive natural products, the apparent first example of which, highlights a unique molecular scaffold which may serve as a lead for drug discovery and therapeutic intervention in ER dependent diseases such as breast and prostate cancers.