Mechanism of Action of the Novel Anticancer Agent 6-Fluoro-2-(2'-fluoro-l,l'- biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic Acid Sodium Salt (NSC 368390): Inhibition of de Novo Pyrimidine Nucleotide Biosynthesis

Exposure of cultured clone A human colon tumor cells to 25 to 75 MM of NSC 368390 |6-fluoro-2-(2'-fluoro-l,l'-biphenyl-4-yl)-3-inethyl-4quinolinecarboxylic acid sodium salt, DuP 785) for 48 to 72 h resulted in a 99.9% cell kill as determined by clonogenic assay. Cells exposed to NSC 368390 became depleted in intracellular pools of uridine S'-triphosphate and cytidine S'-triphosphate. Both uridine S'-triphosphate and cytidine S'-triphosphate were decreased to 50% of levels in control cells at 3 h and were undetectable at 15 h after addition of 25 MMof NSC 368390 to the cultures. Similar effects were observed in LI 210 leukemia cells. Addition of 0.1 HIMof uridine or cytidine restored intracellular pools of uridine S'-triphosphate and cytidine S'-triphosphate to control levels and rescued clone A cells from NSC 368390 cytotoxicity. Addition of uridine circumvented NSC 368390 cytotoxicity in LI 210 cells, but addition of cytidine did not. This result is consistent with the fact that LI 210 cells hick cytidine deaminase and thus cannot form uridine or its anabolites from cytidine. These results indicated that NSC 368390 inhibits a step in the de novo biosynthetic pathway leading to uridine 5'monophosphate. Therefore, the effects of NSC 368390 on the six enzymes that comprise the de novo pathway leading to the formation of uridine S'-monophosphate were examined. The results showed that NSC 368390 was a potent inhibitor of dihydroorotate dehydrogenase, the fourth en zyme in the pathway; thus, this study demonstrates that NSC 368390 exerts its tumoricidal effect by inhibiting a step in de novo p>rimirimi' biosynthesis resulting in the depletion of critical precursors for RNA and DNA synthesis. INTRODUCTION The novel anticancer agent NSC 3683902 (Fig. 1) inhibits the growth of a broad spectrum of human solid tumors im planted in nude mice; the water soluble compound is equally efficacious whether administered p.o. or paren terally (1). Be cause of its activity against experimental tumors, NSC 368390 has recently been entered into phase 1 clinical trials. The structural novelty of the compound as an antitumor agent has precluded any a priori prediction of its mode of action; there fore, studies were conducted to elucidate the mechanism of action of NSC 368390. Our results demonstrate that NSC 368390 exerts its tumoricidal effect by inhibiting dihydrooro tate dehydrogenase, an enzyme in the de novo pyrimidine nucleotide biosynthetic pathway. Portions of this work have been presented in a preliminary form (2). MATERIALS AND METHODS Materials and Chemicals. NSC 368390 was synthesized by the Me dicinal Chemistry Section, Pharmaceuticals Division, Du Pont BiomedReceived 2/11/86; revised 5/6/86; accepted 6/27/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Du Pont Company, Glenolden Laboratory, 500 S. Ridgeway Avenue, Glenolden, PA 19036. 2The abbreviations used are: NSC 368390, 6-nuoro-2-(2'-f1uoro-l,l'-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid sodium salt, DuP 785; 1)11, I>l ilith¡ot lire iml; PALA, A^phosphonacetyO-L-aspartate; HPLC, high-perform ance liquid chromatography; RPMI-C, RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, buffers, and antibiotics; RPMI-L, RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, gentamicin (25 ng/ml), and 55 MM2-mercaptoethanol. ical Products Department. The reagents for tissue culture were pur chased from GIBCO, Grand Island, NY. These included RPMI 1640 medium, fetal bovine serum, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, tricine buffer, sodium bicarbonate, fungizone, antipleuropneumonia-like organism agent, penicillin-streptomycin, trypsin, and trypan blue. Gentamicin was purchased from Valley Biologicals, State College, PA. Glycerol, dimethyl sulfoxide, NaF, and HPLC grade potassium phosphate monobasic were obtained from Fisher Scientific Co., King of Prussia, PA. DEAE-Sephadex A25 was purchased from Pharmacia, Piscataway, NJ. Acetaldehyde was obtained from Aldrich Chemical Co., Milwaukee, WI. Whatman DE81 and 3 MM chroma tography papers were purchased from VWR Scientific, Philadelphia, PA. Kodak X-Omat AR film was obtained from PX Imaging Inc., Plymouth Meeting, PA. BALB/c x DBA/2 F, (hereafter called CD2F,) mice were purchased from Charles River, Wilmington, MA. Dowex 50 W (8X), pyruvate kinase (rabbit muscle), ornithine transcarbamylase (Streptococcus faecalis), dihydroorotate dehydrogenase (Zymobacteriurn oroticum), alcohol dehydrogenase (equine liver), and all other chemicals were purchased from Sigma Chemical Company, St. Louis, MO. En3 Hance spray, Biofluor, L-[t/-I4C]aspartic acid (specific activity, 231.0 mCi/mmol), [carboxyl-l*C]oTotk acid (specific activity, 60 mCi/ mmol), and [l4C]sodium bicarbonate (specific activity, 51.5 mCi/mmol) were purchased from New England Nuclear Research Products, Boston, MA. Synthesis and Purification of L-[care<«y/-14C]Dihydroorotate.L-[carboxyl-'*C]Dihydroorotate was synthesized enzymatically from [car¿0x)>/-l4C]orotate,and purified from other radiolabeled by-products using a DEAE-Sephadex A25 column (16 x 330 mm) eluted with 0.2 M ammonium formate (pH 7.0) (3). The fractions containing i.-[carftary/-l4C]dihydroorotate were pooled and concentrated to dryness by lyophilization. The residue was reconstituted in water and applied to a Dowex 50 W (8X) column (25 x 140 mm), which was eluted with glass distilled water. The radioactive fractions were pooled and concentrated again. The purified L-[carAoArv/-l4C]dihydroorotate was reconstituted with glass distilled water and stored at -20°C. Assuming its specific activity to be the same as that of the starting [car6o;ry/-'4C]orotate, the concentration of L-[car6ary/-14C]dihydroorotate was determined from its radioactivity. L-[caroojcy/-14CJDihydroorotate was identified by its mobility on DES l Chromatographie paper as compared to a nonradioactive standard as well as by its ability to be converted enzymatically to orotate. Cell Cultures. Clone A human colon cancer cells were isolated from the heterogeneous DLD-1 colon tumor line established from a surgical specimen of primary colon adenocarcinoma (4). Clone A cells are hyperploid, highly clonogenic in soft agar, and produce poorly differ entiated carcinomas when injected s.c. into nude mice (5). The colon cancer cells were grown in 60mm tissue culture dishes (Falcon Plastics, Oxnard, CA) in RPMI-C as reported previously (5); cells were passaged by trypsin treatment. Murine leukemia I.I210 cells were cultured in RPMI-L. Both cell lines were incubated at 37°Cin a humidified atmosphere of 5% CO2-95% air. For routine maintenance, the cells were subcultured every 4 days. Cell numbers and viability were deter mined by trypan blue dye exclusion using a hemocytometer. Clonogenic Assay. Exponentially growing cultured clone A cells were incubated with graded concentrations of NSC 368390 for different time intervals. The cells were then harvested with trypsin (0.033%) and centrifuged. The cell pellets were resuspended in RPMI-C and the cell concentrations were adjusted for appropriate seeding density (depend ing on NSC 368390 concentration and exposure time). The cells were