J. Nat. Prod. 2002, 65, 1491-1493
1491
Panepophenanthrin, from a Mushroom Strain, a Novel Inhibitor of the Ubiquitin-Activating Enzyme Ryuichi Sekizawa,*,† Souichi Ikeno,‡ Hikaru Nakamura,† Hiroshi Naganawa,† Susumu Matsui,§ Hironobu Iinuma,† and Tomio Takeuchi† Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan, Showa Pharmaceutical University, 3-3165 Higashitamagawa-gakuen, Machida-shi, Tokyo 194-8543, Japan, and TaKaRa Agri Co., Ltd., 2257 Noji-cho, Kusatsu-shi, Shiga 525-0055, Japan Received March 8, 2002
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,10trihydroxy-10c-(3-hydroxy-3-methylbut-1-enyl)-5,5-dimethyl-1,2,3,3a,5,5a,8,9,10,10a,10b,10cdodecahydro-4-oxa-2,3,8,9-diepoxyacephenanthrylen-7-one. The ubiquitin-proteasome pathway (UPP) has been determined to regulate important cellular functions via the degradation or processing of targeted proteins. Ubiquitin is first activated by a ubiquitin-activating enzyme (E1) prior to conjugating to a substrate through the ubiquitinconjugating enzymes (E2s) and ubiquitin ligases (E3s) following degradation by proteasome.1-3 E1 activates the C-terminal glycine of ubiquitin to an adenylated intermediate and transfers it to a thiol site in the enzyme.4,5 This high-energy intermediate of ubiquitin is further transferred to the thiol site of an E2 and finally to the targeted protein by combination with an E3.1 Recent observations suggested that ubiquitination of a protein is not only the tag for proteasomal degradation but also a modification state of the signal mediators. Some signal transducers are regulated by ubiquitination in response to activation signals for cellular functions.6-10 Hence, E1 activity is indispensable for UPP and some signal transductions, and an inhibitor of E1 could be expected to block the UPP and ubiquitin functions that are linked to serious disease.11 To identify new microbial secondary metabolites that are biologically, as well as structurally, interesting, relevant to certain diseases or functions, and potential drug candidates, we screened the cultured broth of mushrooms, which include a variety interesting metabolic pathways,12 for inhibition of E1, which is indispensable for the ubiquitinyl cascade. As a result of this study, we have identified a novel compound named panepophenanthrin (1) from the mushroom strain Panus rudis Fr. IFO8994. This first reported inhibitor of E1 from a natural source has a unique structure that was determined from NMR experiments and X-ray crystallographic analysis. We describe herein the isolation and structural determination of 1, as well as its biological activity. The UPP is initiated by the ubiquitin-activating enzyme called E1. To discover the inhibitors of the UPP process, we constructed E1-bearing Escherichia coli in which the human E1 gene was expressed via the pTV-118N vector and utilized the active enzyme to screen for inhibitors in * To whom correspondence should be addressed. Tel: +81-3-3441-4173. Fax: +81-3-3441-7589. E-mail:
[email protected]. † Institute of Microbial Chemistry. ‡ Showa Pharmaceutical University. § TaKaRa Agri Co., Ltd.
10.1021/np020098q CCC: $22.00
microbial broths. The mushroom strain IFO8994 produced an inhibitory substance for the E1-ubiquitin complex formation, and a novel inhibitor, namely, panepophenanthrin (1), was isolated and identified as the first inhibitor of the ubiquitination process. The molecular formula of 1 was determined as C22H28O8 from its HRFABMS. The 1H and 13C NMR data of 1 are shown in Table 1. The 13C NMR and DEPT spectra indicated the presence of 22 carbons including four methyl groups, four olefinic carbons, and a carbonyl group. No methylene group was observed. On the basis of the chemical shifts in the 13C NMR spectra, there were six methines and two quaternary carbons bearing oxygen atoms. The analyses of the 1H-1H COSY spectrum suggested a partial structure composed of eight methine signals from 3-H to 8-H as shown in Figure 1. In the HMBC spectrum, the signal of 8-H was coupled to C-6a, and an olefinic proton of 6-H correlated to C-7 and C-10a. The signal of 3-H showed cross-peaks with signals for C-3a and C-10c, and 5a-H correlated with C-10b and C-10c. Other HMBC correlations were observed from 11-H to C-3a and from 10b-H to C-11. The presence of two oxirane rings was indicated by the large 1JC,H values of 185 Hz for C-2 (57.1 ppm) and C-3 (57.2 ppm) and 188 Hz for C-8 (55.1 ppm) observed in the HMBC spectrum. A hydroxyisopentenyl moiety was deduced from the long-range couplings from methyl groups 14 and 15. Connection of the hydroxyisopentenyl moiety at C-10c was revealed by the HMBC correlations of H-11 to C-10b, C-10c, and C-3a. A methyl signal of an oxyisopropyl (16-Me, δH 1.45 ppm) showed long-
© 2002 American Chemical Society and American Society of Pharmacognosy Published on Web 08/01/2002
1492 Journal of Natural Products, 2002, Vol. 65, No. 10 Table 1.
13C
and 1H NMR Data of 1 in CD3ODa
position
δC mult.
δH mult.
1 2 3 3a 5 5a 6 6a 7 8 9 10 10a 10b 10c 11 12 13 14 15 16 17
69.0 d 57.1 d 57.2 d 102.6 s 79.2 s 57.3 d 139.9 d 138.8 s 196.2 s 55.1 d 60.7 d 66.2 d 51.2 d 50.0 d 55.6 s 129.2 d 143.0 d 71.8 s 29.4 q 30.3 q 26.1 q 32.3 q
4.35 br t 3.50 br t 3.32 d
2.0, 2.0 4.0, 4.0 4.0
3.35 dd 6.81 dd
2.0, 5.0 3.0, 5.0
3.42 d 3.84 br t 4.55 br t 2.33 m 2.03 br d
4.0 4.0, 4.0 2.0, 2.0 10.0, 2.0, 2.0, 2.0, 3.0 10.0, 2.0,
