Perturbation-Based Proteomic Correlation Profiling as a Target Deconvolution Methodology.

Molecular target identification of small molecules, so-called target deconvolution, is a major obstacle to phenotype-based drug discovery. Here, we developed an approach called perturbation-based proteomic correlation profiling (PPCP) utilizing the correlation between protein quantity and binding activity of compounds under cellular perturbation by gene silencing and successfully identified lanosterol synthase as a molecular target of TGF-ß pathway inhibitor. This PPCP concept was extended to the use of a cell line panel and provides a new option for target deconvolution.

Identification of metals from osteoblastic ST-2 cell supernatants as novel OGR1 agonists.

Ovarian cancer G-protein-coupled receptor 1 (OGR1) is a G-protein-coupled receptor (GPCR), which has previously been identified as a receptor for protons. It has been reported in this and previous studies that OGR1 expression was markedly up-regulated during osteoclast differentiation. We predicted the possibility of other molecules activating OGR1 in neutral pH, and that osteoblasts might release OGR1 agonistic molecules and activate OGR1 expressed in osteoclasts such as RANKL. We screened for cell supernatants and organ extracts and discovered OGR1 agonistic activity in ST-2 osteoblastic cell supernatants and pancreatic tissues. Finally, we partially purified and identified essential metals, Fe, Zn, Co, Ni and Mn, as novel OGR1 agonists. These OGR1 agonistic metals induce intracellular Gq-coupled inositol phosphate signals in OGR1-expressing cells and primary osteoclasts through OGR1. We also confirmed that these OGR1 agonistic metals activated OGR1 through the same residues which act with protons. Here, we demonstrate that metals, Fe, Zn, Co, Ni and Mn are the novel OGR1 agonists, which can singly activate OGR1 in neutral pH.

IDH1 and IDH2 have critical roles in 2-hydroxyglutarate production in D-2-hydroxyglutarate dehydrogenase depleted cells.

D-2-hydroxyglutaric aciduria (D-2HGA) is a hereditary metabolic disorder characterized by the elevated levels of D-2-hydroxyglutaric acid (D-2HG) in urine, plasma and cerebrospinal fluid. About half of the patients have autosomal recessive mutations in D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene. To analyze the origin of D-2HG in D2HGDH-depleted cells, we used small interfering RNA (siRNA) techniques. We found that knockdown of D2HGDH in MCF7 cells increased the levels of 2HG, mimicking D2HGDH mutant cells. Additional knockdown of isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) decreased the level of 2HG in D2HGDH knockdown MCF7 cells. Conversely, ectopic expression of IDH1 or IDH2 increased 2HG in MCF7 cells. These results suggest that IDH1 and IDH2 have roles in production of D-2HG in cells.

A-90289 A and B, new inhibitors of bacterial translocase I, produced by Streptomyces sp. SANK 60405.

During the course of screening for translocase I inhibitors, the new liposidomycin-related compounds, A-90289 A and B, were isolated from a culture broth of Streptomyces sp. SANK 60405. The structural elucidations were carried out by NMR and high-resolution mass spectral analyses, and they were classified as members of the liponucleoside antibiotics group with a sulfate group at the C-2' position. A-90289 A and B inhibited bacterial translocase I with IC(50) values of 36.5 ng ml(-1) and 33.8 ng ml(-1), respectively.

Synthesis and properties of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides.

Novel bicyclo nucleosides, 2'-O,4'-C-ethylene nucleosides and 2'-O,4'-C-propylene nucleosides, were synthesized as building blocks for antisense oligonucleotides to further optimize the 2'-O,4'-C-methylene-linkage of bridged nucleic acids (2',4'-BNA) or locked nucleic acids (LNA). Both the 2'-O,4'-C-ethylene- and propylene-linkage within these nucleosides restrict the sugar puckering to the N-conformation of RNA as do 2',4'-BNA/LNA. Furthermore, ethylene-bridged nucleic acids (ENA) having 2'-O,4'-C-ethylene nucleosides had considerably increased the affinity to complementary RNA, and were as high as that of 2',4'-BNA/LNA (DeltaT(m)=+3 approximately 5 degrees C per modification). On the other hand, addition of 2'-O,4'-C-propylene modifications in oligonucleotides led to a decrease in the affinity to complementary RNA. As for the stability against nucleases, incorporation of one 2'-O,4'-C-ethylene or one 2'-O,4'-C-propylene nucleoside into oligonucleotides considerably increased their resistance against exonucleases to an extent greater than 2',4'-BNA/LNA. These results indicate that ENA is more suitable as an antisense oligonucleotide and is expected to have better antisense activity than 2',4'-BNA/LNA.

Arborcandins A, B, C, D, E and F, novel 1,3-beta-glucan synthase inhibitors: physico-chemical properties and structure elucidation.

Arborcandins A, B, C, D, E and F, which possess potent 1,3-beta-glucan synthase inhibitory activity, were isolated from the cultured broth of a filamentous fungus, strain SANK 17397. The structures of arborcandins A, B, C, D, E and F were elucidated by a combination of NMR and mass spectrometry, and established to be novel cyclic peptides containing uncommon amino acid residues.

2'-O,4'-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug.

To develop antisense oligonucleotides, novel nucleosides, 2'-O,4'-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2'-O,4'-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.