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Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti‐ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU‐approved anthelmintic drug for domesticated animals, binds to the T4‐binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone‐related proteins, including serum albumin and thyroid hormone receptor β. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone‐related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid‐disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors. [ABSTRACT FROM AUTHOR]

Death‐associated protein kinase 1 (DAPK1), a Ca2+/calmodulin‐regulated serine/threonine kinase, regulates cell apoptosis and autophagy and has been implicated in the pathogenesis of Alzheimer's disease (AD). Targeting DAPK1 may be a promising approach for treating AD. In our previous study, we found that a natural polyphenol, resveratrol (1), is a moderate DAPK1 inhibitor. In the present study, we investigated the interactions between natural and synthetic derivatives of 1 and DAPK1. Binding assays including intrinsic fluorescence quenching, protein thermal shift and isothermal titration calorimetry indicated that oxyresveratrol (3), a hydroxylated derivative, and pinostilbene (5), a methoxylated derivative, bind to DAPK1 with comparable affinity to 1. The enzymatic assay showed that 3 more effectively inhibits the intrinsic ATPase activity of DAPK1 compared with 1. Crystallographic analysis revealed that the binding modes of the methoxylated derivatives were different from those of 1 and 3, resulting in a unique interaction. Our results suggest that 3 may be helpful in treating AD and provide a clue for the development of promising DAPK1 inhibitors. [ABSTRACT FROM AUTHOR]

Landomycin A (LaA) is the largest member of the landomycin group of angucyclic polyketides. Its unusual structure and strong anticancer properties have attracted great interest from chemists and biologists alike. This, in particular, has led to a detailed picture of LaA biosynthesis in Streptomyces cyanogenus S136, the only known LaA producer. LanK is a TetR family repressor protein that limits the export of landomycins from S136 cells until significant amounts of the final product, LaA, have accumulated. Landomycins carrying three or more carbohydrate units in their glycosidic chain are effector molecules for LanK. Yet, the exact mechanism that LanK uses to distinguish the final product, LaA, from intermediate landomycins and sense accumulation of LaA was not known. Here, we report crystal structures of LanK, alone and in complex with LaA, and bioassays of LanK's interaction with synthetic carbohydrate chains of LaA (hexasaccharide) and LaE (trisaccharide). Our data collectively suggest that the carbohydrate moieties are the sole determinants of the interaction of the landomycins with LanK, triggering the latter's dissociation from the lanK–lanJ intergenic region via structure conversion of the helices in the C‐terminal ligand‐binding domain. Analysis of the available literature suggests that LanK represents an unprecedented type of TetR family repressor that recognises the carbohydrate portion of a natural product, and not an aglycon, as it is the case, for example, with the SimR repressor involved in simocyclinone biosynthesis. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus expresses several hemolytic pore‐forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane β‐barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one‐component α‐hemolysin (α‐HL) and two‐component γ‐hemolysin (γ‐HL). The α‐HL forms a homo‐heptamer, whereas γ‐HL is an octamer composed of F‐component (LukF) and S‐component (Hlg2). Because PFTs are used as materials for nanopore‐based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a β‐barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the β‐barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α‐HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one‐component PFT, whereas another participates as both a heptameric one‐ and heptameric/octameric two‐component PFT. All chimeric mutants intrinsically assemble into SDS‐resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT β‐barrels for use in further promising applications. [ABSTRACT FROM AUTHOR]

Bromodomain‐containing protein 4 (BRD4) recognizes the acetylated lysine of histone H4 via its bromodomains, leading to the recruitment of positive transcription elongation factor b. Small molecules that inhibit BRD4 have potential as anticancer agents by leading to the downregulation of specific oncogenes. Using X‐ray crystallographic screening, we identified the BRD4 inhibitory activity of isoliquiritigenin (ISL), a natural chalcone found in licorice. Structural analysis revealed that ISL bound to BRD4 with a novel binding mode and squeezed out one of the six conserved water molecules that form a strong hydrogen bond network. The thermodynamic analysis revealed that the binding of ISL is enthalpy driven, suggesting that strong hydrogen bonds would compensate for the desolvation penalty. Neutron protein crystallography further suggested that the favorable binding enthalpy originates from the stabilization and optimization of the hydrogen bond network of the conserved water molecules. Here, we describe the novelty and potential of ISL as a template for new BRD4 inhibitors. [ABSTRACT FROM AUTHOR]