Structural Biology-Based Exploration of Subtype-Selective Agonists for Peroxisome Proliferator-Activated Receptors

Progress in understanding peroxisome proliferator-activated receptor (PPAR) subtypes as nuclear receptors that have pleiotropic effects on biological responses has enabled the exploration of new subtype-selective PPAR ligands. Such ligands are useful chemical biology/pharmacological tools to investigate the functions of PPARs and are also candidate drugs for the treatment of PPAR-mediated diseases, such as metabolic syndrome, inflammation and cancer. This review summarizes our medicinal chemistry research of more than 20 years on the design, synthesis, and pharmacological evaluation of subtype-selective PPAR agonists, which has been based on two working hypotheses, the ligand superfamily concept and the helix 12 (H12) holding induction concept. X-ray crystallographic analyses of our agonists complexed with each PPAR subtype validate our working hypotheses.

The physiological role of estrogen receptor functional domains

Estrogen receptor (ER) is a member of the nuclear receptor superfamily whose members share conserved domain structures, including a DNA-binding domain (DBD) and ligand-binding domain (LBD). Estrogenic chemicals work as ligands for activation or repression of ER-mediated transcriptional activity derived from two transactivation domains: AF-1 and AF-2. AF-2 is localized in the LBD, and helix 12 of the LBD is essential for controlling AF-2 functionality. The positioning of helix 12 defines the ER alpha (ERα) ligand properties as agonists or antagonists. In contrast, it is still less well defined as to the ligand-dependent regulation of N-terminal AF-1 activity. It has been thought that the action of selective estrogen receptor modulators (SERMs) is mediated by the regulation of a tissue specific AF-1 activity rather than AF-2 activity. However, it is still unclear how SERMs regulate AF-1 activity in a tissue-selective manner. This review presents some recent observations toward information of ERα mediated SERM actions related to the ERα domain functionality, focusing on the following topics. (1) The F-domain, which is connected to helix 12, controls 4-hydroxytamoxifen (4OHT) mediated AF-1 activation associated with the receptor dimerization activity. (2) The zinc-finger property of the DBD for genomic sequence recognition. (3) The novel estrogen responsive genomic DNA element, which contains multiple long-spaced direct-repeats without a palindromic ERE sequence, is differentially recognized by 4OHT and E2 ligand bound ERα transactivation complexes.

Altered Intracellular Trafficking of Mutant Thyroid Hormone Receptors in Resistance to Thyroid Hormone Syndrome

Resistance to Thyroid Hormone α (RTHα), a reduced sensitivity to thyroid hormone (T3) in peripheral tissues, is caused by mutations in thyroid hormone receptor α (TRα), a nuclear receptor that mediates T3-responsive gene expression. All mutations characterized in RTHα, to date, are in the ligand binding domain (LBD), resulting in reduced affinity for T3. In addition, some mutations result in truncated proteins lacking all or part of helix 12. Previously, we have used fluorescence recovery after photobleaching (FRAP) to examine the effects of select RTHα mutations on the intracellular trafficking of TRα. After transfecting HeLa cells with expression plasmids for green fluorescent protein (GFP)-tagged wild-type TRα and each of the mutants, we first assessed their intracellular distribution and initial intranuclear mobility. Although wild-type TRα is known to shuttle between the nucleus and cytoplasm, it is primarily localized to the nucleus at a steady state. We showed that TR-E403X, Ala382ProfsX7, and F397fs406X are also primarily localized to the nucleus, and FRAP revealed that wild-type TRα and the RTHα mutants are highly dynamic within the nucleus, indicating that the receptors rapidly dissociate and reassociate with DNA binding sites and/or other nuclear binding sites, independent of T3 (1). Some studies have shown that Nuclear Receptor Corepressor 1 (NCoR1), which interacts with the hinge region of TRα (the region between the DNA-binding domain and LBD) has a higher affinity for RTHα mutants compared to wild-type TRα, supporting the hypothesis that helix 12 of the LBD also functions to disassociate NCoR1 from TRα when it is bound to T3 (2). We proposed that this increased affinity for NCoR1 alters the mobility of TRα in the nucleus, impacting its function. Here, we show that NCoR1 has slower FRAP recovery kinetics compared with TRα, and we evaluate the intranuclear dynamics of RTH TRα1 mutants ΤR-E403X, Ala382ProfsX7, and F397fs406X in response to increased levels of NCoR1. Investigation of the effects of overexpression of NCoR1 will provide further insight into the impact of altered binding affinity for NCoR1 on the intranuclear dynamics of RTHα mutants. References: (1) Femia et al., Journal of Cellular Biochemistry, 2020 2020 Apr; 121(4), 2909-2926.(2) Bochukova et al., New England Journal of Medicine, 2012 Jan. 19; 366(3), 243–249.

Paradoxical androgen receptor regulation by small molecule enantiomers

Small molecules that target the androgen receptor (AR) are the mainstay of therapy for lethal castration-resistant prostate cancer (CRPC), yet existing drugs lose their efficacy during continued treatment. This evolution of resistance is due to heterogenous mechanisms which include AR mutations causing the identical drug to activate instead of inhibit the receptor. Understanding in molecular detail the paradoxical phenomenon wherein an AR antagonist is transformed into an agonist by structural mutations in the target receptor is thus of paramount importance. Herein, we describe a reciprocal paradox: opposing antagonist and agonist AR regulation determined uniquely by enantiomeric forms of the same drug structure. The antiandrogen BMS-641988, which has (R)-chirality at C-5 encompasses a previously uncharacterized (S)-stereoisomer that is, surprisingly, a potent agonist of AR, as demonstrated by transcriptional assays supported by cell imaging studies. This duality was reproduced in a series of novel compounds derived from the BMS-641988 scaffold. Coupled with in silico modeling studies, the results inform an AR model that explains the switch from potent antagonist to high-affinity agonist in terms of C-5 substituent steric interactions with helix 12 of the ligand binding site. They imply strategies to overcome AR drug resistance and demonstrate that insufficient enantiopurity in this class of AR antagonist can confound efforts to correlate structure with function.

One- and Two-Electron Reduction of Triarylborane-Based Helical Donor-Acceptor Compounds

One-electron chemical reduction of 10-(dimesitylboryl)-N,N-di-p-tolylbenzo[c]phenanthrene-4-amine (3-B(Mes)2-[4]helix-9-N(p-Tol)2) 1 and 13-(dimesitylboryl)-N,N-di-p-tolyldibenzo[c,g]phenanthrene-8-amine (3-B(Mes)2-[5]helix-12-N(p-Tol)2) 2 gives rise to monoanions with extensive delocalization over the annulated helicene rings and the boron pz orbital. Two-electron chemical...

Structural Mechanism Underlying Ligand Binding and Activation of PPARγ

ABSTRACTLigands bind to an occluded orthosteric pocket within the nuclear receptor (NR) ligand-binding domain (LBD). Molecular simulations have revealed several theoretical ligand entry/exit pathways to the orthosteric pocket, but experimentally it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Using NMR spectroscopy lineshape analysis, we show that ligand binding to the peroxisome proliferator-activated receptor gamma (PPARγ) LBD involves a two-step induced fit mechanism including an initial fast step followed by slow conformational change. Surface plasmon resonance and isothermal titration calorimetry heat capacity analysis support the fast kinetic binding step and the conformational change after binding step, respectively. The putative initial ligand binding pose is suggested in several crystal structures of PPARγ LBD where a ligand is bound to a surface pore formed by helix 3, the β-sheet, and the Ω-loop—one of several ligand entry sites suggested in previous targeted and unbiased molecular simulations. These findings, when considered with a recent NMR study showing the activation function-2 (AF-2) helix 12 exchanges in and out of the orthosteric pocket in apo/ligand-free PPARγ, suggest an activation mechanism whereby agonist binding occurs through an initial encounter complex with the LBD followed by transition of the ligand into the orthosteric pocket concomitant with a conformational change resulting in a solvent-exposed active helix 12 conformation.

Dynamic allosteric communication pathway directing differential activation of the glucocorticoid receptor

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

X-ray crystal structure analysis of elacestrant (RAD1901), a novel selective estrogen receptor degrader (SERD), bound to estrogen receptor alpha ligand binding domain.

e15647 Background: Antiestrogens are a mainstay of treatment for estrogen receptor positive (ER+) breast cancer in both the adjuvant and the advanced/metastatic settings. Elacestrant is a mixed activity selective estrogen receptor (SER) alpha (ERα) antagonist, acting as a SER modulator (SERM) at low doses and a SER degrader (SERD) at high doses. It has shown activity in hormone sensitive wild type (WT) ERα and insensitive estrogen receptor gene 1 (ESR1) mutation-harboring (Y537S and D538G) ERα breast cancer, both in preclinical models and in clinical studies. It also possesses a unique pharmacology compared to other competitive ER antagonists in its ability to cross the blood brain barrier. Competitive ERα antagonists are typically comprised of a core that sits in the ligand binding pocket and an arm that manipulates the structure to achieve SERM or SERD activities. In these molecules, the arm is attached in the same position as the triphenylethylene core of tamoxifen. However, elacestrant possesses a novel site of attachment. As such, we hypothesized that elacestrant adopts an alternative binding orientation in the ERα ligand binding pocket to achieve its unique pharmaceutical profiles. Methods: X-ray crystallography was used to solve a co-crystal structure of elacestrant in complex with WT ERα ligand binding domain to 2Å. Results: Overall, elacestrant promotes the formation of a canonical ERα ligand binding domain antagonist conformation, whereby helix 12 (H12) is docked into the activating function-2 cleft. However, elacestrant adopts a novel vector in the ERα ligand binding pocket that places it in close proximity to helix 12. As a result, it forms a bifurcated hydrogen bond that is not observed in other competitive antiestrogens and samples a chemical space known to increase H12 mobility and induce SERD activity. This novel vector also places it near positions 537 and 538, the two most common sites of somatic mutation. Conclusions: The high-resolution x-ray crystal structure of elacestrant suggests that the unique binding mode it adopts enables novel pharmacology and positions it to achieve potency in the WT and activating somatic ERα mutated breast cancer setting.

Delineation of the molecular determinants of the unique allosteric binding site of the orphan nuclear receptor RORγt

Nuclear receptors (NRs) are high-interest targets in drug discovery because of their involvement in numerous biological processes and diseases. Classically, NRs are targeted via their hydrophobic, orthosteric pocket. Although successful, this approach comes with challenges, including off-target effects due to lack of selectivity. Allosteric modulation of NR activity constitutes a promising pharmacological strategy. The retinoic acid receptor-related orphan receptor-γt (RORγt) is a constitutively active NR that positively regulates the expression of interleukin-17 in T helper 17 cells. Inhibiting this process is an emerging strategy for managing autoimmune diseases. Recently, an allosteric binding pocket in the C-terminal region of the ligand-binding domain (LBD) of RORγt was discovered that is amenable to small-molecule drug discovery. Compounds binding this pocket induce a reorientation of helix 12, thereby preventing coactivator recruitment. Therefore, inverse agonists binding this site with high affinity are actively being pursued. To elucidate the pocket formation mechanism, verify the uniqueness of this pocket, and substantiate the relevance of targeting this site, here we identified the key characteristics of the RORγt allosteric region. We evaluated the effects of substitutions in the LBD on coactivator, orthosteric, and allosteric ligand binding. We found that two molecular elements unique to RORγt, the length of helix 11′ and a Gln-487 residue, are crucial for the formation of the allosteric pocket. The unique combination of elements present in RORγt suggests a high potential for subtype-selective targeting of this NR to more effectively treat patients with autoimmune diseases.

OR12-07 Full Antagonism of Breast Cancer Cell Proliferation Can Result from Many Ligand-Induced Conformational Distortions of the Estrogen Receptor Ligand Binding Domain

Although most estrogen receptor alpha (ERα)-positive breast cancers initially respond well to endocrine therapies using aromatase inhibitors (AIs) or antiestrogens, after varying time periods the cancer frequently recurs as metastatic disease. A significant fraction of these recurrences are driven by ERs that have acquired activating mutations in their ligand binding domains (LBDs), giving them constitutive activity and thus resistance to AIs. Because these mutations also reduce the affinity and potency of SERMs and SERDs, expanded efforts have been made to vary the structure of antiestrogens to make them more potent. Typical antiestrogens are comprised of a core element that binds securely in the ligand binding pocket and from which extends a single ring (ring E) having a side chain that sterically interferes with the position of helix-12 by direct antagonism, reorienting it so that it occludes the activation function 2 (AF2) hydrophobic groove for coactivator binding. Through structural studies, we found that bridged oxabicycloheptene-sulfonamide (OBHS-N) core ligands have two rings (E and F) that can be poised to engage in both “direct antagonism” and “indirect antagonism”, the latter of which disrupts the orientation of helix-12 by impinging on helix-11 and the helix-11–12 loop. In this study, we have placed typical antiestrogen side chains on either the E or the F ring of OBHS-N core ligands and characterized their activities in ERα-positive breast cancer cells. All compounds have full antiproliferative activity and reverse estrogen-regulated gene expression, with the antiproliferative potency of each type of side chain having a distinct preference for E- vs F-ring attachment. Conformational analysis using a multiplexed coregulator peptide interaction assay shows that compounds with an E-ring substitution have interaction profiles similar to 4-hydroxytamoxifen and fulvestrant, whereas the F-ring substitution gives a very different pattern, suggesting that the antagonist activity of the two classes rely on different sets of coregulator proteins. A large number of high resolution (better than 2 Å) X-ray crystal structures reveal that this set of novel ER antagonists disrupt the conformation of the ER LBD in a variety of ways, several of which are distinct from those seen with previous antiestrogens such as Tamoxifen and Fulvestrant. Our findings expand design concepts by which ERα ligands can block the activity of this receptor and illustrate how direct and indirect modes of ER antagonism can be combined to facilitate the development of more efficacious antiestrogens for breast cancer treatment and possibly for regulating ER-mediated activities in other estrogen target tissues.