Search

Amyloid fibrils of transthyretin (TTR) consist of full‐length TTR and C‐terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C‐terminal fragment remains unclear. Here, we investigated trypsin‐induced aggregation and urea‐induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less‐bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin‐induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin‐induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion. [ABSTRACT FROM AUTHOR]

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]

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]