論文

The DEAD-box family of ATPases plays a critical role in nearly all stages of RNA metabolism, from transcription to degradation, and serves as a major regulator of biomolecular condensates. Dysregulation of DEAD-box proteins is well-established in a variety of diseases, including cancer and neurodegenerative disorders, making them attractive therapeutic targets. However, their classification as "undruggable" has historically hindered small-molecule-based modulation. In this study, we focus on DDX21, a member of the DEAD-box family involved in ribosome biogenesis and transcription regulation. As a proof of concept for targeting such RNA-binding proteins, we developed a lysate-based small-molecule microarray platform to identify compounds that directly bind DDX21. This screen led to the discovery of KI-DX-014, a small-molecule compound capable of inhibiting the interaction of DDX21 with RNA. KI-DX-014 modulated the RNA-dependent functions of DDX21, including its ATPase activity and biomolecular condensate formation. Furthermore, KI-DX-014 attenuated the DDX21-dependent release of P-TEFb from the 7SK snRNP complex in vitro, suppressed P-TEFb-dependent phosphorylation of the RNA polymerase II CTD, and induced developmental defects in zebrafish embryos. These findings reveal a previously unexploited therapeutic avenue and establish KI-DX-014 as a chemical probe for dissecting the biological functions of DDX21 in both normal physiology and disease states.

d-glycero-d-manno-Heptose 1,7-bisphosphate (HBP) is the precursor for heptose residues found in Gram-negative bacterial membrane surface glycoproteins and glycolipids. HBP beta-anomer was recently reported to be a pathogen-associated molecular pattern (PAMP) that regulates TIFA-dependent immunity. Herein, we report the chemical synthesis of HBP alpha- and beta-anomers, which highlights a C-7 carbon homologation via the Corey-Chaykovsky reaction, and the introduction of a phosphate group at the anomeric position using the Mitsunobu reaction. Furthermore, NF-kappaB reporter assaying revealed that HBP beta-anomer activates the NF-kappaB signaling pathway.

Efficient convergent chemical syntheses of digalactosyl diacylglycerols (DGDGs), which have both a galactose-galactose alpha(1-->6)-linkage and a galactose-glycerol beta-linkage along with a diacylglycerol containing various kinds of fatty acids, have been accomplished. In order to achieve a concise synthesis, we chose to use allylic protective groups as permanent protective groups. We have also achieved alpha- and beta-selective glycosylations for the respective linkages with high yields as the key steps.

The chemical synthesis of glycosyl inositol phospholipids from Entamoeba histolytica is reported. The key feature of this synthesis is a regioselective phosphorylation reaction that occurs through desymmetrization of a myo-inositol derivative with phosphoroselenoyl chloride. A new protecting-group strategy was developed that utilizes allyl and alloc groups to synthesize complex glycolipids bearing unsaturated lipids. These developments provided an efficient synthetic route for various complex inositol phospholipids and their analogues. Furthermore, the binding affinity of the synthetic inositol phospholipids with mouse CD1d molecules has been evaluated, as well as the immunostimulatory activity.

The CD1d protein is a nonpolymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self- and foreign-lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A' pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipid-protein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A' pocket could be used for the modulation of the lipid binding. A series of the ligands, alpha-galactosyl ceramide (alpha-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure-activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.

The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as alpha-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-gamma, IL-4, IL-17A, etc.). Herein, we report structure-activity relationship studies of alpha-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.

A regioselective phosphorylation method for myo-inositol was developed by utilizing readily preparable BINOL-derived phosphoramidites. The method also facilitated the complete separation of the diastereomeric products by simple chromatography. Based on this phosphorylation and Ni-catalyzed alkyl-alkyl cross-coupling reaction for long fatty acids, we achieved the first synthesis of a lysophosphatidylinositol, EhPIa having long fatty acid C30:1, as a partial structure of glycosylphosphatidylinositol (GPI) anchor from the cell membrane of a protozoa, Entamoeba histolytica.

The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA-binding proteins (RBPs) is present on the surface of living cells. These cell-surface RBPs (csRBPs) precisely organize into well-defined nanoclusters enriched for multiple RBPs and glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell-surface interaction for the cell-penetrating peptide trans-activator of transcription (TAT). Removal of RNA from the cell surface, or loss of RNA-binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.

Intracellular pathogens belonging to the genus Leishmania have developed effective strategies that enable them to survive within host immune cells. Immunostimulatory compounds that counteract such immunological escape mechanisms represent promising treatment options for diseases. Here, we demonstrate that a lipopeptidephosphoglycan (LPPG) isolated from the membrane of a protozoan parasite, Entamoeba histolytica (Eh), shows considerable immunostimulatory effects targeted against Leishmania (L.) major, a representative species responsible for cutaneous leishmaniasis (CL). Treatment led to a marked reduction in the number of intracellular Leishmania parasites in vitro, and ameliorated CL in a mouse model. We next designed and synthesized analogs of the phosphatidylinositol anchors harbored by EhLPPG; two of these analogs reproduced the anti-leishmanial activity of the native compound by inducing production of pro-inflammatory cytokines. The use of such compounds, either alone or as a supportive option, might improve the currently unsatisfactory treatment of CL and other diseases caused by pathogen-manipulated immune responses.

In recent years, as the immunomodulatory mechanisms have been greatly elucidated, examples of selective immunomodulatory actions have been found for a wide variety of small-molecule compounds, including natural products. In particular, with regard to immune activation through inhibition of immunosuppressive molecules, which has been the focus of recent attention, research on inhibitors of enzymatic immunosuppressive molecules has focused on small molecules. On the other hand, it is important to understand the molecules derived from microorganisms that activate or suppress the immune system, for understanding the mechanisms of infection or the protection against the microbes. This group of molecules is also a treasure trove of diverse immunomodulatory structures. In this article, we introduce the development of synthetic methods for immunomodulatory molecules based on natural compounds and their related compounds. Firstly, the development of IDO1 inhibitors, which are attracting attention as targets for anti-tumor agents by inhibiting immunosuppressive molecules, based on longamide B, a natural product derived from marine sponges, was introduced. In the latter half, the synthesis and development of the selective immune modulatory compounds, based on microbial innate immune receptor ligands. are introduced. The compounds include 1,7-bisphosphate heptose (HBP) as a novel factor involved in innate immune activation via TIFA, and also inositol phospholipids (EhPIb from Entamoeba histolytica and Ac1PIM1 from Mycobacterium tuberculosis) involved in regulating immune mechanisms were described with the characteristic methods of phosphate introduction.