Structural elucidation of A-74528, an inhibitor for 2',5'-phosphodiesterase isolated from Streptomyces sp.

A-74528 (1) is a metabolite of Streptomyces sp. discovered in the screening for 2',5'-oligoadenylate phosphodiesterase inhibitors. The planar structure of 1 was mainly elucidated by NMR techniques including natural abundance INADEQUATE, and the relative configuration and the conformation were elucidated by the analyses of NOEs and assessment of dihedral angles predicted by QUANTA/CHARMm computations and coupling constants. It was proved that 1 is a highly fused polyketide with a side-chain branching site that never appeared before from the nature.

Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2',5'-oligoadenylate synthetases (2',5'-OASs), inactivated by 5'-phosphatase and completely degraded by 2'-phosphodiesterase (2'-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2',5'-OASs and RNase L have been molecularly cloned and studied well, the identification of 2'-PDE has remained elusive. Here, we describe the first identification of 2'-PDE, the third key enzyme of the 2-5A system. We found a putative 2'-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2'-PDE by mass spectrometry. Based on the full-length sequence of the human 2'-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2'-PDE expressed in mammalian cells certainly cleaved the 2',5'-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2'-PDE were found, the human 2'-PDE was considered to be a unique enzyme without isoform. Suppression of 2'-PDE by a small interfering RNA and a 2'-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2'-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2'-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

Bovine 5-oxo-L-prolinase: simple assay method, purification, cDNA cloning, and detection of mRNA in the coronary artery.

RS-7897 is a novel antianginal nitrate containing an L-2-oxothiazolidine-4-carboxylic acid (OTCA) and unlike other organic nitrates does not induce nitrate tolerance. OTCA is known to be converted to L-cysteine (L-Cys) by rat 5-oxo-prolinase (5-OPase) and was detected in the plasma of RS-7897-treated dogs. Nitrate tolerance is considered to develop mainly through sulfhydryl depletion. We hypothesized that RS-7897-derived OTCA was converted into L-Cys by 5-OPase and supplied sulfhydryl groups in vascular smooth muscle cells, the targets of the nitrate. As the initial step for clarifying the presumed role of 5-OPase in RS-7897 metabolism, we established a non-radiochemical assay method highly specific for 5-OPase. Using this assay method, we purified 5-OPase from bovine kidney cytosol until homogeneous results were obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The catalytic activity of bovine 5-OPase to convert OTCA to L-Cys was confirmed, as was the case for the rat enzyme. Then the cDNA encoding bovine 5-OPase was cloned. The deduced amino acid sequence revealed that bovine 5-OPase was highly homologous to rat 5-OPase. Based on the bovine cDNA sequence, oligonucleotide primers were synthesized and used for RT-PCR. 5-OPase mRNA was significantly detected in the RT-PCR product of the bovine coronary artery, a major target organ of RS-7897. These results suggest that OTCA may be converted to L-Cys by 5-OPase in the mammalian coronary artery when treated with RS-7897, and thus generated L-Cys may reduce sulfhydryl depletion and contribute to the prevention of the development of nitrate tolerance.