Kendall W. Nettles

Prenylation of phenolic and aromatic natural products across plants, bacteria, and higher organisms contributes to their lipophilicity and biological function. The process also enhances host defense and serves as a building block for chemical diversity, with medicinal chemistry mimicking these effects to create more effective compounds. While phytoestrogens, particularly those from licorice, are established as key natural regulators of estrogen receptor (ER) biology, the specific mechanisms by which prenylated polyphenolics in licorice regulate ER ligand binding and activity remain unknown. Herein, we show that the widespread prenylation of stilbenes from two commonly used licorice species reveals a broad spectrum of ER interaction profiles in the context of different scaffolds. To identify structural rules for how prenylation modulates ligand binding, we synthesized 16 mono- or diprenylated stilbennoid analogs based on common resveratrol or dihydroresveratrol cores. These variously prenylated stilbenoid compounds exhibited ER agonistic effects on cell growth with a remarkable 5-log range of cellular potencies. X-ray crystal structures of these compounds with ERα showed that this wide range of activities were found in essentially identical ligand–receptor structural states, except for differing side chain rotamers in one region of the binding pocket that accommodates the prenyl substituents. Thus, chemical diversity from a simple class of stilbenoids generated biological diversity through the modular combination of prenyl substitutions that revealed localized structural flexibility in an otherwise highly stable ER agonist conformation. The emergence of this prenylation code provides guidance for understanding and predicting the effect of prenyl-like substitution on phytoestrogens and other ER ligands.

Glucocorticoids (GCs) coordinate immunity, inflammation, and metabolism through allosteric regulation of the glucocorticoid receptor (GR) transcription factor. GCs are indispensable anti-inflammatory drugs yet linking specific ligand–receptor structural states to specific biological outcomes has remained a major barrier to designing safer, more selective therapies. Using structure-based design, we developed selective glucocorticoid receptor modulators (SGRMs) of immune function by extending a steroidal scaffold from the ligand-binding pocket into an adjacent solvent channel. These SGRMs suppressed T cell pro-inflammatory cytokines and promoted differentiation of memory precursor T cells while showing minimal induction of M2 macrophage polarization or T cell checkpoint proteins PD-1 and CTLA-4, all key targets of immunotherapy. Molecular dynamics simulations revealed that solvent-channel substituents function as a lever arm to drive dynamic oscillations in the steroid core, thereby allosterically tuning GR activity states. Systematic perturbation of immune cells with a graded series of ligands enabled a l igand p erturbation with m achine l earning (LPML) framework to map coregulated responses across cell types and identified effector T cell gene networks tightly coupled with immune checkpoint induction. This approach outlines a general strategy for decoding the logic of allosteric drug action, enabling the rational design of SGRMs with tailored immunomodulatory profiles.

Oligomerization of proteoforms produces functional protein complexes. Characterization of these assemblies within cells is critical to understanding the molecular mechanisms involved in disease and to designing effective drugs. Here we present a native top-down proteomics (nTDP) strategy to identify protein assemblies (≤70 kDa) in breast cancer cells and in cells that overexpress epidermal growth factor receptor (EGFR), which serves as a resistance model of estrogen receptor-alpha (ER)-targeted therapies. This nTDP approach identified ~104 complexoforms from 17 protein complexes, which revealed several molecular features of the breast cancer proteome, including EGFR-induced dissociation of nuclear transport factor 2 (NUTF2) assemblies that modulate ER activity. We found that the K4 and K55 post-translational modification sites discovered with nTDP differentially impact the effects of NUTF2 on the inhibition of the ER signaling pathway. The characterization of endogenous proteoform–proteoform/ligand interactions revealed the molecular diversity of complexoforms and their role in breast cancer growth.

Bronchopulmonary dysplasia, the most prevalent chronic lung disease of prematurity, is often treated with glucocorticoids (GCs) such as dexamethasone (DEX), but their use is encumbered with several adverse somatic, metabolic, and neurologic effects. We previously reported that systemic delivery of the GC prodrug ciclesonide (CIC) in neonatal rats activated glucocorticoid receptor (GR) transcriptional responses in lung but did not trigger multiple adverse effects caused by DEX. To determine whether limited systemic metabolism of CIC was solely responsible for its enhanced safety profile, we treated neonatal rats with its active metabolite desisobutyryl-ciclesonide (Des-CIC). DEX but not Des-CIC caused a reduction in body weight as well as reduced insulin-like growth factor-1 serum levels and chronic hyperglycemia in neonatal rats. However, Des-CIC was as effective as DEX in reducing the expression of various bleomycin-induced proinflammatory cytokine mRNAs. In vitro studies with various cell types demonstrate the potent GR transactivation and transrepression activity of Des-CIC, although genome-wide transcriptomic analyses reveal differences in DEX vs. Des-CIC responses in neonatal rat lung and liver tissue. Des-CIC is a GR super-agonist as revealed by an in vitro coregulator peptide binding assay. In addition, molecular dynamics simulations revealed unique Des-CIC-dependent allosteric signaling pathways between specific residues in the GR ligand-binding domain and receptor surfaces interacting with coregulator peptides. Thus, Des-CIC is a potential novel selective GR modulator that could impart a favorable therapeutic index for CIC use for even modest durations of GC exposure which could have long-lasting adverse somatic, metabolic, or neurologic effects.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations—receptor conformational heterodimers—mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

The estrogen receptor-α (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations —receptor conformational heterodimers—mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. Significance The estrogen receptor-α (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

: Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ’s mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP₂) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP₂ clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP₂ clusters, and HCQ moves ACE2 away from PIP₂ clusters—erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

The Creb‐Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1‐mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle‐specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge‐eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2‐driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1‐mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

Significance: To address the unmet clinical need for effectively suppressing estrogen receptor (ER) activity with both de novo resistance and in advanced ER-positive breast cancers that are resistant to standard-of-care antiestrogens, we have developed dual-mechanism ER inhibitors (DMERIs) that employ two distinct ER-targeting moieties. These DMERI elicited noncanonical structural perturbations of the receptor ligand-binding domain and stabilized multiple antagonist substates within the dimer to generate highly efficacious antagonism of proliferation in ER-positive breast cancer cells and in allele-specific resistance models. This work reveals conformational modes by which the activity of ER can be effectively suppressed to block breast cancer proliferation.