Structural insights into the nuclear import of the histone acetyltransferase males-absent-on-the-first by importin α1.

The histone acetyltransferase males-absent-on-the-first (MOF) acetylates the histone H4, a modification important for many biological processes, including chromatin organization, transcriptional regulation, DNA replication, recombination and repair, as well as autophagy. Depletion of MOF induces serious consequences because of the reduction of histone acetylation, such as nuclear morphological defects and cancer. Despite the critical roles of MOF in the nucleus, the structural or functional mechanisms of the nucleocytoplasmic transport of MOF remain elusive. Here, we identified novel importin α1-specific nuclear localization signals (NLSs) in the N-terminal of human MOF. The crystal structure of MOF NLSs in complex with importin α1 further revealed a unique binding mode of MOF, with two independent NLSs binding to importin α1 major and minor sites, respectively. The second NLS of MOF displays an unexpected α-helical conformation in the C-terminus, with more extensive contacts with importin α1 not limited in the minor site. Mutations of the key residues on MOF and importin α1 lead to the reduction of their interaction as well as the nuclear import of MOF, revealing an essential role of NLS2 of MOF in interacting with importin α1 minor site. Taken together, we provide structural mechanisms underlying the nucleocytoplasmic transport of MOF, which will be of great importance in understanding the functional regulation of MOF in various biological processes.

Structural basis for the hepatoprotective effects of antihypertensive 1,4-dihydropyridine drugs.

BACKGROUND: The 1,4-dihydropyridines (DHPs) are one of the most frequently prescribed classes of antihypertensive monotherapeutic agents worldwide. In addition to treating hypertension, DHPs also exert other beneficial effects, including hepatoprotective effects. However, the mechanism underlying the hepatoprotection remains unclear. METHODS: Biochemical AlphaScreen and cell-based reporter assays were employed to detect the activities of DHPs towards FXR. A crystallographic analysis was adopted to study the binding modes of four DHPs in complex with FXR. Acetaminophen (APAP)-treated wild-type and FXR knockout mice were used to investigate the functional dependence of the effects of the selected DHPs on FXR. RESULTS: A series of DHPs were uncovered as FXR ligands with different activities for FXR, suggesting FXR might serve as an alternative drug target for DHPs. The structural analysis illustrated the specific three-blade propeller binding modes of four DHPs to FXR and explained the detailed mechanisms by which DHPs bind to and are recognized by FXR. The results in mice demonstrated that cilnidipine protected the liver from APAP-induced injury in an FXR-dependent manner. CONCLUSIONS: This study reports the crystal structures of FXR in complex with four DHPs, and confirms that DHPs exert hepatoprotection by targeting FXR. GENERAL SIGNIFICANCE: Our research not only reveals valuable insight for the design and development of next-generation Ca2+ blocker drugs to provide safer and more effective treatments for cardiovascular disorders but also provides a novel and safe structural template for the development of drugs targeting FXR. Moreover, DHPs might be potentially repurposed to treat FXR-mediated diseases other than hypertension.

A Novel Class of Natural FXR Modulators with a Unique Mode of Selective Co-regulator Assembly.

The farnesoid X receptor (FXR) is an important target for drug discovery. Small molecules induce a conformational change in FXR that modulates its binding to co-regulators, thus resulting in distinct FXR functional profiles. However, the mechanisms for selectively recruiting co-regulators by FXR remain elusive, partly because of the lack of FXR-selective modulators. We report the identification of two natural terpenoids, tschimgine and feroline, as novel FXR modulators. Remarkably, their crystal structures uncovered a secondary binding pocket important for ligand binding. Further, tschimgine or feroline induced dynamic conformational changes in the activation function 2 (AF-2) surface, thus leading to differential co-regulator recruiting profiles, modulated by both hydrophobic and selective hydrogen-bond interactions unique to specific co-regulators. Our findings thus provide a novel structure template for optimization for FXR-selective modulators of clinical value.

Thyroid hormone resistance: Mechanisms and therapeutic development.

As an essential primary hormone, thyroid hormone (TH) is indispensable for human growth, development and metabolism. Impairment of TH function in several aspects, including TH synthesis, activation, transportation and receptor-dependent transactivation, can eventually lead to thyroid hormone resistance syndrome (RTH). RTH is a rare syndrome that manifests as a reduced target cell response to TH signaling. The majority of RTH cases are related to thyroid hormone receptor ß (TRß) mutations, and only a few RTH cases are associated with thyroid hormone receptor α (TRα) mutations or other causes. Patients with RTH suffer from goiter, mental retardation, short stature and bradycardia or tachycardia. To date, approximately 170 mutated TRß variants and more than 20 mutated TRα variants at the amino acid level have been reported in RTH patients. In addition to these mutated proteins, some TR isoforms can also reduce TH function by competing with primary TRs for TRE and RXR binding. Fortunately, different treatments for RTH have been explored with structure-activity relationship (SAR) studies and drug design, and among these treatments. With thyromimetic potency but biochemical properties that differ from those of primary TH (T3 and T4), these TH analogs can bypass specific defective transporters or reactive mutant TRs. However, these compounds must be carefully applied to avoid over activating TRα, which is associated with more severe heart impairment. The structural mechanisms of mutation-induced RTH in the TR ligand-binding domain are summarized in this review. Furthermore, strategies to overcome this resistance for therapeutic development are also discussed.

Discovery of a Highly Selective and H435R-Sensitive Thyroid Hormone Receptor ß Agonist.

The design and development of agonists selectively targeting thyroid hormone receptor ß (TRß) and TRß mutants remain challenging tasks. In this study, we first adopted the strategy of breaking the "His-Phe switch" to solve two problems, simultaneously. A structure-based design approach was successfully utilized to obtain compound 16g, which is a potent TRß agonist (EC50: 21.0 nM, 85.0% of the maximum efficacy of 1) with outstanding selectivity for TRß over TRα and also effectively activates the TRßH435R mutant. Then, we developed a highly efficient synthetic method for 16g. Our serials of cocrystal structures revealed detailed structural mechanisms in overcoming subtype selectivity and rescuing the H435R mutation. 16g also showed excellent lipid metabolism, safety, metabolic stability, and pharmacokinetic properties. Collectively, 16g is a well-characterized selective and mutation-sensitive TRß agonist for further investigating its function in treating dyslipidemia, nonalcoholic steatohepatitis (NASH), and resistance to thyroid hormone (RTH).

Structural Insights into the Specificity of Ligand Binding and Coactivator Assembly by Estrogen-Related Receptor ß.

Estrogen-related receptor ß (ERRß) is a nuclear receptor critical for many biological processes. Despite the biological and pharmaceutical importance of ERRß, deciphering the structure of ERRß has been hampered by the difficulties in obtaining a pure and stable protein for structural studies. In fact, the ERRß ligand-binding domain remains the last unsolved ERR structure and also one of only a few unknown nuclear receptor structures. Here, we report the identification of a critical single-residue mutation resulted in robust solubility and stability of an active ERRß ligand-binding domain, thereby providing a protein tool enabling the first probe into the biochemical and structural studies of this important receptor. The crystal structure reveals key structural features that have enabled the integration of the molecular determinants of signals transduced across the ligand binding and coregulator recruitment by all three ERR subtypes, which also provides a framework for the rational design of selective and potent ligands for the treatment of various ERR-mediated diseases.

Repositioning an Immunomodulatory Drug Vidofludimus as a Farnesoid X Receptor Modulator With Therapeutic Effects on NAFLD.

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disorder, and yet with no pharmacological treatment approved worldwide. The repositioning of old drugs provides a safe approach for drug development. Vidofludimus, an inhibitor for dihydroorotate dehydrogenase (DHODH) for the treatment of autoimmune disorders, is herein uncovered as a novel modulator for farnesoid X receptor (FXR) by biochemical and crystallographic analysis. We further revealed that vidofludimus exerts in vivo therapeutic effects on dextran sodium sulfate (DSS)-induced colitis in an FXR-dependent manner. Notably, vidofludimus also possesses remarkable beneficial effects in reducing NAFLD by targeting FXR, which may represent a unique approach in developing the treatment for NAFLD. Our findings not only reveal a promising template for the design of novel FXR ligands in treating autoimmune disorders, but also uncover a novel therapeutic effect for vidofludimus on NAFLD based on the newly established relationships among drugs, targets, and diseases.

Revealing a Mutant-Induced Receptor Allosteric Mechanism for the Thyroid Hormone Resistance.

Resistance to thyroid hormone (RTH) is a clinical disorder without specific and effective therapeutic strategy, partly due to the lack of structural mechanisms for the defective ligand binding by mutated thyroid hormone receptors (THRs). We herein uncovered the prescription drug roxadustat as a novel THRß-selective ligand with therapeutic potentials in treating RTH, thereby providing a small molecule tool enabling the first probe into the structural mechanisms of RTH. Despite a wide distribution of the receptor mutation sites, different THRß mutants induce allosteric conformational modulation on the same His435 residue, which disrupts a critical hydrogen bond required for the binding of thyroid hormones. Interestingly, roxadustat retains hydrophobic interactions with THRß via its unique phenyl extension, enabling the rescue of the activity of the THRß mutants. Our study thus reveals a critical receptor allosterism mechanism for RTH by mutant THRß, providing a new and viable therapeutic strategy for the treatment of RTH.