Screening of microbial inhibitors of mammalian ornithine decarboxylase.

Sir: In the course of searching for inhibitors of mammalian ornithine decarboxylase (ODC), an active compound was isolated from the culture broth of an unidentified fungus and identified as citrinin, a known antibiotic1,2). Citrinin showed cytotoxicity in vitro to mouse leukemia cells. Structurally related compounds were examined for possible inhibition of ODC and cytotoxicity, and we found that N°'-hydroxy-L-ornithine, a degradation product of vanoxonin3), is an inhibitor of ODC. ODC was prepared as follows at below 4~C unless otherwise mentioned. To a 8-week old female rat (SPF-Donryu) weighing 180 to 200 g, thioacetamide was administered at 150 mg/kg intravenously 24 hours before sacrifice. The liver was removed and homogenized in 0.25 M sucrose solution. The homogenate was centrifuged at 10,000 x g for 20 minutes, and the supernatant was centrifuged again at 100,000 x g for 60 minutes. The supernatant was used as crude enzyme for the screening. For kinetic analysis, the crude enzyme was purified 10-fold by successive application of DEAE-cellulose column chromatography, ammonium sulfate fractionation and dialysis. Protein was determined by the method of LOWRY et al. 4). The reaction mixture contained the followings in I ml: 50 mm sodium phosphate (pH 7.2), 0.2 mm pyridoxal-5-phosphate, 5 mm dithiothreitol, 0.2 MM L-ornithine, D,L-[5-14C]ornithine (0.05 t4Ci, Amersham International, plc), either inhibitor solution or water (100 1d) and enzyme solution (200 «l, added last). The reaction progressed for 90 minutes at 37°C, and was terminated by heating in a boiling water bath. After cooling, the reaction mixture was diluted with 4 ml of water and centrifuged at 2,000 rpm for 15 minutes. The supernatant was applied to a 0.5-m1 column of Amberlite CG-50 (Na+ type). Unreacted D,L-[5-14C]ornithine was eluted first with 5 ml of 0.4 N NH,OH, and thereafter [114C]putrescine, the reaction product, was eluted with 2 ml of 5 N NH4OH. The eluate of [1-14C]putrescine was collected in a via], mixed with 6 ml of scintillation solution (Atomlight, New England Nuclear) and submitted to radioactivity measurement with a Beckman scintillation counter (Model LS9800). By this assay method, Km for ornithine was 0.4 mm. The fungus was grown in flasks with shaking at 180 rpm for 4 days at 27°C. The culture medium was composed of tomato paste 2.4%, dextrin 2.4% yeast extract 1.2%, and CoCI2 0.0006% in distilled water (pH 7.0). The ODC inhibitory activity in the broth filtrate was precipitated at pH 2 and crystallized from methanol to obtain citrinin as yellow needles (250 mg per liter of the broth)2). Both citrinin and N°'-hydroxy-L-ornithine inhibited rat liver ODC competitively with respect to ornithine, with Ki values of 0.15 mm and 0.04 mm, respectively (Table 1). Citrinin inhibited the growth of mouse leukemia cells L1210, L5178Y and P388 with IC50 values of 8.4, 4.3 and 3.3 Itg/ml, respectively (Table 2). N°'-HydroxyL-ornithine also inhibited the growth of L1210 cells with a IC;, value of 44.0 pg/ml (Table 2).