Resistance to endocrine therapies in breast cancer highlights the need to explore new modes for antagonizing the activity of estrogen receptor alpha (ERα) that might lead to clinically more effective antiestrogens. Most clinically approved antiestrogens, however, have conceptually similar overall structures. Typically, this includes a lipophilic ligand core with a single side chain attached to an E-ring that uses direct steric contact to displace helix-12 of the ER ligand binding domain (LBD) from the agonist conformation. Unlike conventional antiestrogens, certain structurally bridged oxabicycloheptene-sulfonamide (OBHS-N) ER ligands have two rings (E and F) onto which side chains can be attached, thereby probing two distinct regions and modes of antagonism within the ER LBD. We attached characteristic ER antagonist side chains onto each of these two rings seeking to displace helix-12 sterically (“direct antagonism”) and/or to distort the ligand binding pocket and thereby alter the conformation of the helix-11/12 loop (a distinct mode, termed “indirect antagonism”). Several of these OBHS-N compounds have full antiproliferative activity on ERα-positive breast cancer cells and reverse estrogen-regulated gene expression, and the inhibitory potency of compounds with each type of side chain shows a distinct preference for E- or F-ring of attachment. Conformational analysis of these ER-ligand complexes using a multiplexed coregulator peptide interaction assay shows that sidechain substitution on the E-ring gives interaction profiles similar to that for hydroxytamoxifen and fulvestrant, whereas F-ring substitution gives a very different pattern. X-ray crystallographic structure analysis confirms that F-ring substituted OBHS-N compounds use a combination of direct and indirect modes of antagonism that both displaces and disorders helix-12 of the ER LBD. Our findings expand design concepts by which ERα ligands can block the activity of this receptor in breast cancers and structurally how direct and indirect modes of inhibition can be combined. This could facilitate the development of more efficacious antiestrogens for breast cancer treatment. Supported by the Breast Cancer Research Foundation (BCRF 18-084 to JAK and BSK) and the National Institutes of Health (R01 CA220284 to JAK, BSK, TI, and KWN; T32 GM070421 Fellowship to VSG).

Citation Format: John A Katzenellenbogen, Jian Min, Sathish Srinivasan, Erumbi S. Rangarajan, Jerome C. Nwachukwu, Valeria Sanabria Guillen, Yvonne Ziegler, Shunchao Yan, Kathryn E. Carlson, Yingwei Hou, Sung Hoon Kim, Tina Izard, Rene Houtman, Kendall W. Nettles, Benita S. Katzenellenbogen. Combining direct and indirect modes of antiestrogen ligand antagonism of estrogen receptor activity in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-10.