Screening for inhibitors of the ubiquitin-proteasome pathway, considered to regulate important cellular events and linked to serious diseases as well, led to isolation of a new compound, panepophenanthrin, from the fermented broth of a mushroom strain, Panus rudis Fr. IFO 8994. This is the first inhibitor of the ubiquitin-activating enzyme, which is indispensable for the ubiquitin-proteasome pathway. The structure of panepophenanthrin was determined by NMR and X-ray crystallographic analyses as 1,3a,10-trihydroxy-10c-(3-hydroxy-3-methylbut-1-enyl)-5,5-dimethyl-1,2,3,3a,5,5a,8,9,10,10a,10b,10c-dodecahydro-4-oxa-2,3,8,9-diepoxyacephenanthrylen-7-one.

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Single-drop analysis of two different real sample solutions (2 microL) while simultaneously monitoring the activity of two sets of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip (CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane (PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5-8.2 (% RSD, n = 5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested the usefulness of the CAs-CHIP in the field of drug discovery.

Tyropeptins A and B, new proteasome inhibitors, were isolated from the culture broth of Kitasatospora sp. MK993-dF2.They were purified using ethyl acetate extraction, silica gel column chromatography, Sephadex LH-20 column chromatography and HPLC.Tyropeptin A inhibited the chymotrypsin-like (ChT-L) and trypsin-like (T-L) activities of 20S proteasome with IC50 values of 0.1^g/ml and 1.5/zg/ml respectively, but did not inhibit the peptidylglutamyl-peptide hydrolyzing (PGPH) activity of 20S proteasome at a concentration of 100 //g/ml. The inhibitory activities of tyropeptin A were about two times as strong as that of tyropeptin B. Taxonomyof the producing strain is also described.