ERα
June 2023
Estrogen receptor alpha (ERα)
Estrogen receptor alpha (ERα) is a transcription factor that is activated upon the binding of estrogens. ERα stimulates cell proliferation, therefore its dysregulation contributes to the development of various diseases, including osteoporosis, cardiovascular disorders, and breast cancer. If tumor cells express ERα in their nuclei, it is classified as an ERα-positive (ERα+) breast cancer. This subtype accounts for approximately 70% of all breast cancers. Many therapeutics have been developed over the years to inhibit ERα, however, patients often experience relapse and drug resistance.
There are two types of small-molecule ERα-binding compounds that target the pocket naturally binding estrogens:
selective estrogen receptor modulators (SERMs), which are ERα antagonists that prevent the binding of estrogen and the activation of the receptor
selective estrogen receptor degraders (SERDs) that, upon binding with the receptor, recruit the cellular degradation machinery, promoting the ERα disruption.
Both types of drugs have a chemical core that mimics the chemical structure of hormones and have a long side arm attached to one of the core rings.
Very recently, the FDA approved a new molecule targeting the ERα receptor: elacestrant, by Stemline Therapeutics (Menarini). Interestingly, in contrast with previously developed compounds, its side arm is attached to an unusual ring position. The clinical success of this molecule highlights that unconventional ERα antagonists could lead to novel and improved therapeutic strategies.
The researchers started their study by examining the structural differences between ERα in complex with its biological ligands (estrogens) and known SERMs and SERDs.
Estrogens interact with ERα in a region of the protein called the ligand binding domain (LBD). Analyzing the crystal structure of the estrogen-receptor complex, it can be seen that the helix 12 (H12) of the ERα receptor is located over the LBD.
This position of the H12 causes the exposure of an adjacent region of the protein that interacts with other regulatory proteins - activating function-2 (AF-2) cleft (Image 1A).
SERMs and SERDs show a similar binding pose of their estrogen-mimicking core in the LBD. However, their side-arm protrudes towards the H12 helix, with a T-shaped orientation, impeding it from positioning over the LBD. This causes the movement of H12 into the AF-2 cleft, which sterically precludes the binding of co-regulator proteins (Image 2B).
Both SERMs and SERDs induce a similar conformation of H12 upon binding with ERα, but previous studies demonstrated that SERDs cause an increased H12 mobility compared to the SERM-like analogs. The higher flexibility of H12 prevents stable H12 binding in the AF-2 cleft and destabilizes the receptor, favoring its degradation.
Image 1. Comparison of ERα in complex with an estrogen (orange, PDB: 1GWR), a SERD (white, PDB: 7R62) and a SERM (blue, PDB: 5W9C). A: Superposition of the complexes with an indication of LBD, H12, and AF-2 positions. The 3decision® highlight mode only shows the different portions of the superposed protein. In this way, it is easy to spot the big conformational change of the H12 helix induced by SERD and SERM binding, which moves from the top of LBD and occupies the AF-2 cleft. B: Comparison of the binding of the estrogen, SERD, and SERM molecules: the cores of the three ligands bind very similarly in the LBD, but the side arms on the SERD and SERM protrude towards the H12, inducing its conformational change. The pictures are produced with the 3decision® software.
To further investigate the effect of different ligands on the structural conformation of the LBD, the researchers determined the x-ray crystal structure of the receptor in complex with the newly discovered ligand elacestrant, which has an unconventional 2D chemical structure. Interestingly, they observed that, in contrast with SERMs and SERDs, the molecule adopts an “L-shaped” pose in the pocket and not the classic T-shaped one. The interaction network is similar in both structures, except for the D ring of the elacestrant, which is positioned closer to the helix H8, close to the LBD (Image 2).
Image 2. Overlay of ERα in complex with elacestrant (pink, PDB: 7TE7) and the SERM lasofoxifene (green, PDB: 6VJD). For clarity, the cartoon are hidden and only pocket residues are shown. Indications of H3, H8, H11 and H12 are reported, and the pocket surface is represented. Elacestrant adopts an “L-shaped” orientation and its D ring protrudes towards H8. Lasofoxifene binds with a classical T-shaped orientation. The ligand-protein interactions are automatically calculated by 3decision®. The picture is produced with the 3decision® software.
Inspired by structural insights, they developed a new chemical series of compounds having a tetrahydro-6-isoquinoline (T6I) core. At the position corresponding to the D ring, they tested several chemical groups and found that a benzamide gave the best potency results. Similarly to elacestrant, they observed that the benzamide (ring D) extended towards the helix H8, adopting a unique H3-8-11-12 ligand binding pose. They also tested several side arms of existing SERMs and SERDs. This portion of the molecule is particularly crucial because it highly influences the conformation and dynamics of the H12 helix. One of the developed compounds (T6I-29) carrying a pyrrolidine-fluoropropyl side arm showed hydrogen bonding that brought the side arm closer to the loop connecting H11 and H12 (Image 3).
Image 3. On the left: 2D chemical structure of the newly developed compound TI6-29. On the right: overlay of Erα in complex with elacestrant (pink, PDB: 7TE7), laso (green, PDB: 6VJD), and the newly developed TI6-29 (white, PDB: 8DVB). H3, H8, H11, and H12 are indicated. Only pocket residues and protein-ligand interactions from T6I-29 are shown for clarity. The ring D of the compound adopts a unique position towards H8, and the side arm protrudes towards the loop connecting H11-H12. Superposition of structures is done with the 3decision® software.
Interestingly, they observed that this compound T6I-29-1A showed lower inhibition of classic Erα target genes but had an inhibitory effect on other, unprecedently targeted pathways (CDK1 and SUMO1), which cause anti-breast cancer activity.
This study demonstrated that expanding the chemical diversity of the ligands with a structure-based approach led to different binding modes and, therefore, to different cellular responses, paving the way to novel therapeutic opportunities and drug development strategies.
Reference:
Hancock GR, Young KS, Hosfield DJ, Joiner C, Sullivan EA, Yildiz Y, Lainé M, Greene GL, Fanning SW. Unconventional isoquinoline-based SERMs elicit fulvestrant-like transcriptional programs in ER+ breast cancer cells. NPJ Breast Cancer. 2022 Dec 14;8(1):130. doi: 10.1038/s41523-022-00497-9. PMID: 36517522; PMCID: PMC9748900.