Transformation of thiols to disulfides by epolactaene and its derivatives.

In this paper we report a disulfide formation of thiols induced by epolactaene and its derivatives. We previously reported the disulfide formation of N-acetylcysteine methyl ester by epolactaene in a 1:1 MeOH/0.5M NaHCO(3) aq solution. The present studies reveal that the disulfide formation proceeds under mild conditions such as in PBS at pH 7.3, suggesting that epolactaene may induce disulfide formation of cellular thiols. This compound induces the disulfide formation of several thiols in a 1:1 MeOH/0.5M NaHCO(3) aq solution at room temperature. Moreover, our results show that the acyl side-chain of epolactaene greatly influences the products of the reaction. We analyzed the reaction mechanism by using thiolysis products of epolactaene derivatives and propose a new reaction mechanism.

Syntheses and applications of fluorescent and biotinylated epolactaene derivatives: Epolactaene and its derivative induce disulfide formation.

Epolactaene, isolated from cultured Penicillium sp. BM 1689-P mycelium, induces neurite outgrowth and arrests the cell cycle of the human neuroblastoma cell line, SH-SY5Y, at the G1 phase. We have found that epolactaene and its derivatives induce apoptosis in the human leukemia B-cell line, BALL-1. In this study, we prepared fluorescent and biotinylated epolactaene derivatives. We characterized the cellular location and the identification of BALL-1 proteins that reacted with these compounds. The results obtained from the reaction of epolactaene or its derivative with N-acetylcysteine methyl ester indicate that these compounds induce the disulfide formation and the alpha-position of the epoxylactam core is the reactive site.

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

BACKGROUND: Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Screening of paclitaxel-binding molecules from a library of random peptides displayed on T7 phage particles using paclitaxel-photoimmobilized resin.

Paclitaxel (Taxol), an effective anticancer agent, is known to bind to tubulin and induce tubulin polymerization. Several other binding proteins of paclitaxel, such as Bcl-2, heat shock proteins, and NSC-1, have also been reported. Here, we describe a T7 phage-based display to screen for paclitaxel-binding molecules from a random peptide library using paclitaxel-photoimmobilized TentaGel resin. Specific phage particles that bind the paclitaxel-immobilized resin were obtained. Among them, two phage clones included the same consensus amino acid sequence (KACGRTRVTS). Analysis of the protein database using BLAST revealed that a portion of this sequence is conserved in the zinc finger domain of human NFX1. Binding affinity of paclitaxel against the partial recombinant protein of NFX1 (424aa-876aa) was confirmed by pull-down assays and surface plasmon resonance analyses.

Apoptosis-inducing effect of epolactaene derivatives on BALL-1 cells.

Epolactaene, a neuritogenic compound in human neuroblastoma SH-SY5Y, induces apoptosis in a human leukemia B-cell line, BALL-1. The apoptosis-inducing activities of 34 epolactaene derivatives, including those of the newly synthesized alpha-alkyl-alpha,beta-epoxy-gamma-lactam derivative and cyclopropane derivatives, were also tested. The structure-activity relationships of the epolactaene derivatives as an inducer of apoptosis are described. The alpha-acyl-alpha,beta-epoxy-gamma-lactam moiety as well as the hydrophobicity derived from the long alkyl side chain are both important for activity. Compound 1e displayed the strongest activity among all the synthesized compounds with an IC50 value of 0.70 microM.

Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis.

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.