62(P-35) 放線菌Streptomyces sp. K99-5041株由来新規ラノステロール合成酵素阻害活性物質(ポスター発表の部)

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Hypercholesterolemia is recognized as the risk factor of atherosclerotic diseases. For pharmaceutical therapy for hypercholesterolemia, inhibitors of HMG-CoA reductase are currently used in clinics. Since HMG-CoA reductase is located in the upstream of the cholesterol biosynthetic pathway, inhibition of this enzyme may cause the simultaneous reduction of the physiologicaly essential non-steroidal isoprenoid metabolites such as dolichol, ubiquinone and prenylated proteins, and the long-term administration of the inhibitors of this enzyme may cause unexpected side effects. We have chosen lanosterol synthase as a more selective target for suppression of cholesterol biosynthesis, since this enzyme locates at more downstream than HMG-CoA reductase in the pathway. We previously succeeded in cDNA cloning of human lanosterol synthase and functional expression of the enzyme in yeast. Using recombinant human enzyme, screening of inhibitors from natural origin has been carried out. From an Actinomycete strain, Streptomyces sp. K99-5041, two inhibitors, K99-5041-C1x (1) and K99-5041-C2x (2), have been isolated as new natural products and their structures elucidated by spectroscopic method. The crude extract (5.5g) from the whole broth of this strain was applied to a SiO_2 column and fractionated. Inhibitory activity was concentrated to one fraction which showed a single spot on TLC. This fraction (named as Fr. N) was analyzed with LC-APCIMS (TSKgel ODS-80T_M (TOSOH)). In the UV and total ion monitoring of this fraction, ten peaks (A1, A2, B1, B2, C1, C2, D1, D2, E1 and E2) were observed. Further investigation revealed that each of the peaks was not composed of a single component but of two compounds, which differ in molecular mass by 26 mass units, and thus the Fr. N eventually contained 20 kinds of homologous compounds. Fr. N was separated by the two-step preparative RP-LC (Lobar column (Merck)→TSKgel ODS-80T_M (TOSOH)), first to obtain fractions C1 and C2, and further purification with the third preparative HPLC (TSKgel ODS-120T (TOSOH)) afforded compounds C1x (1) (3.5mg) and C1y (3) (0.6mg) from fraction C1 and compounds C2x (2) (2.3mg) and C2y (4) (0.5mg) from fraction C2. The IC_<50> values of these compounds were 15 (1), 18 (2), 33 (3), 41 (4)μM, respectively, and their potency was comparable to the known oxidosqualene cyclase inhibitor, lauryldimethylamine N-oxide (LDAO (5); IC_<50>=0.84μM). Detailed spectroscopic analyses of isolated compounds led to the identification of their structures. Compounds 1 and 2 were (3E)-isohexadecylmethylidene-2-methyl-1-pyrroline and (3E)-hexadecylmethylidene-2-methyl-1-pyrroline, respectively, and the compounds 3 and 4 were the homologs whose molecular mass was 26-mass unit larger with the insertion of an cis-double bond in the side chain of 1 and 2, respectively. All other homologs have 2-methyl-1-pyrroline moiety as a common building block and differ in the length of side chains. The investigation of the effect of these compounds on cellular lipid metabolism is underway.
天然有機化合物討論会の論文
2002-09-01

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