Jason Nowak

NFkappaB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses

Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFkappaB-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure.

Structural plasticity in the oestrogen receptor ligand-binding domain

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand‐binding pocket. Here, we report the X‐ray crystal structure of the oestrogen receptor α (ERα) bound to an oestradiol derivative with a prosthetic group, ortho‐ trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand‐binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand‐binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand‐binding pocket.

Coupling of receptor conformation and ligand orientation determine graded activity

SUMMARY Small molecules stabilize specific protein conformations from a larger ensemble, enabling molecular switches that control diverse cellular functions. We show here that the converse also holds true, where the conformational state of the estrogen receptor can direct distinct orientations of the bound ligand. “Gain of allostery” mutations that mimic the effects of ligand in driving protein conformation allowed crystallization of the partial agonist ligand WAY-169916 with both the canonical active and inactive conformations of the estrogen receptor. The intermediate transcriptional activity induced by WAY169916 is associated with the ligand binding differently to the active and inactive conformations of the receptor. Analyses of a series of chemical derivatives demonstrated that altering the ensemble of ligand binding orientations changes signaling output. The coupling of different ligand binding orientations to distinct active and inactive protein conformations defines a novel mechanism for titrating allosteric signaling activity.

Resveratrol modulates the inflammatory response via an estrogen receptor-signal integration network

Resveratrol has beneficial effects on aging, inflammation and metabolism, which are thought to result from activation of the lysine deacetylase, sirtuin 1 (SIRT1), the cAMP pathway, or AMP-activated protein kinase. In this study, we report that resveratrol acts as a pathway-selective estrogen receptor-α (ERα) ligand to modulate the inflammatory response but not cell proliferation. A crystal structure of the ERα ligand-binding domain (LBD) as a complex with resveratrol revealed a unique perturbation of the coactivator-binding surface, consistent with an altered coregulator recruitment profile. Gene expression analyses revealed significant overlap of TNFα genes modulated by resveratrol and estradiol. Furthermore, the ability of resveratrol to suppress interleukin-6 transcription was shown to require ERα and several ERα coregulators, suggesting that ERα functions as a primary conduit for resveratrol activity. DOI: http://dx.doi.org/10.7554/eLife.02057.001

Ligand-binding dynamics rewire cellular signaling via estrogen receptor-α

Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands displayed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways, and reveals a novel role of the DBD in allosteric control of ERα-mediated signaling.

Structural mechanism for signal transduction in RXR nuclear receptor heterodimers

A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a significant clinical problem for estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.

Predictive features of ligand-specific signaling through the estrogen receptor.

Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.

The SERM/SERD Bazedoxifene Disrupts ESR1 Helix 12 to Overcome Acquired Hormone Resistance in Breast Cancer Cells

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER+ breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We find BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show that BZA’s selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations. Significance Bazedoxifene (BZA) is a potent orally available antiestrogen that is clinically approved for use in hormone replacement therapy (DUAVEE). We explore the efficacy of BZA to inhibit activating somatic mutants of ERα that can arise in metastatic breast cancers after prolonged exposure to aromatase inhibitors or tamoxifen therapy. Breast cancer cell line, biophysical, and structural data show that BZA disrupts helix 12 of the ERα ligand binding domain to achieve improved potency against Y537S and D538G somatic mutants compared to 4-hydroxytamoxifen.

Abstract 4854: Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Despite continued administration of antiestrogen therapies, approximately 50% of all estrogen receptor alpha (ERalpha) positive breast cancers will present new metastatic lesions. The acquisition of secondary hormone-resistant metastatic breast cancers represents a significant clinical barrier towards life-long disease free survival for the patient. Somatic mutations to the ERalpha gene (ESR1) Y537S and D538G represent a novel mechanism of acquired antiestrogen resistance because they confer hormone-free transcriptional activity and reduced selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD) potency. Fulvestrant, a SERD, was the only molecule that could completely ablate mutant ERalpha activity. Unfortunately, fulvestrant possesses poor pharmacologic profiles that limit its therapeutic utility. Bazedoxifene (BZA) is a potent mixed SERM/SERD and has improved pharmacokinetics and oral bioavailability compared to fulvestrant. We show that BZA inhibits Y537S and D538G ESR1 somatic mutation transcriptional activity with a greater potency than the SERMs 4-hydroxytamoxifen (TOT) and raloxifene (RAL). Further investigations into the biophysical and structural basis for BZA action suggest that BZA increases the conformational dynamics of helix 12, a key molecular switch that governs ERalpha action resulting in SERD-like properties and improved potency against the somatic mutations compared to TOT and RAL. Citation Format: Sean W. Fanning, Venkat Dharmarajan, Christopher G. Mayne, Weiyi Toy, Kathryn E. Carlson, Teresa A. Martin, Jason Nowak, Jerome Nwachukwu, David J. Hosfield, Emad Tajkhorshid, Sarat Chandarlapaty, Patrick Griffin, Yang Shen, John A. Katzenellenbogen, Geoffrey L. Greene. Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4854.