Development of Benziodarone Analogues with Enhanced Potency for Selective Binding to Transthyretin in Human Plasma.

Transthyretin amyloidosis is a fatal disorder caused by transthyretin amyloid aggregation. Stabilizing the native structure of transthyretin is an effective approach to inhibit amyloid aggregation. To develop kinetic stabilizers of transthyretin, it is crucial to explore compounds that selectively bind to transthyretin in plasma. Our recent findings demonstrated that the uricosuric agent benziodarone selectively binds to transthyretin in plasma. Here, we report the development of benziodarone analogues with enhanced potency for selective binding to transthyretin in plasma compared to benziodarone. These analogues featured substituents of chlorine, bromine, iodine, a methyl group, or a trifluoromethyl group, at the 4-position of the benzofuran ring. X-ray crystal structure analysis revealed that CH···O hydrogen bonds and a halogen bond are important for the binding of the compounds to the thyroxine-binding sites. The bioavailability of benziodarone analogues with 4-Br, 4-Cl, or 4-CH3 was comparable to that of tafamidis, a current therapeutic agent for transthyretin amyloidosis.

Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol.

Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.

Trichomes on female reproductive tract: rapid diversification and underlying gene regulatory network in Drosophila suzukii and its related species.

BACKGROUND: The ovipositors of some insects are external female genitalia, which have their primary function to deliver eggs. Drosophila suzukii and its sibling species D. subpulchrella are known to have acquired highly sclerotized and enlarged ovipositors upon their shifts in oviposition sites from rotting to ripening fruits. Inside the ovipositor plates, there are scale-like polarized protrusions termed "oviprovector scales" that are likely to aid the mechanical movement of the eggs. The size and spatial distribution of the scales need to be rearranged following the divergence of the ovipositors. In this study, we examined the features of the oviprovector scales in D. suzukii and its closely related species. We also investigated whether the scales are single-cell protrusions comprised of F-actin under the same conserved gene regulatory network as the well-characterized trichomes on the larval cuticular surface. RESULTS: The oviprovector scales of D. suzukii and D. subpulchrella were distinct in size and spatial arrangement compared to those of D. biarmipes and other closely related species. The scale numbers also varied greatly among these species. The comparisons of the size of the scales suggested a possibility that the apical cell area of the oviprovector has expanded upon the elongation of the ovipositor plates in these species. Our transcriptome analysis revealed that 43 out of the 46 genes known to be involved in the trichome gene regulatory network are expressed in the developing female genitalia of D. suzukii and D. subpulchrella. The presence of Shavenbaby (Svb) or svb was detected in the inner cavity of the developing ovipositors of D. melanogaster, D. suzukii, and D. subpulchrella. Also, shavenoid (sha) was expressed in the corresponding patterns in the developing ovipositors and showed differential expression levels between D. suzukii and D. subpulchrella at 48 h APF. CONCLUSIONS: The oviprovector scales have divergent size and spatial arrangements among species. Therefore, these scales may represent a rapidly diversifying morphological trait of the female reproductive tract reflecting ecological contexts. Furthermore, our results showed that the gene regulatory network underlying trichome formation is also utilized to develop the rapidly evolving trichomes on the oviprovectors of these flies.

A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-ß-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.