Synthesis and antitumor activity of an inhibitor of fatty acid synthase

Abstract

Compared to normal human tissues, many common human cancers, including carcinoma of the colon, prostate, ovary, breast, and endometrium, express high levels of fatty acid synthase (FAS, EC ), the primary enzyme responsible for the synthesis of fatty acids. This differential expression of FAS between normal tissues and cancer has led to the notion that FAS is a target for anticancer drug development. Recent studies with C75, an inhibitor of fatty acid synthesis, have shown significant antitumor activity with concomitant inhibition of fatty acid synthesis in tumor tissue and normal liver. Importantly, histopathological analysis of normal tissues after C75 treatment showed no adverse effects on proliferating cellular compartments, such as bone marrow, gastrointestinal tract, skin, or lymphoid tissues. In this study, we describe the de novo synthesis of C75 based on the known mechanism of action of cerulenin and the theoretical reaction intermediates of the beta-ketoacyl synthase moiety of FAS. In addition, we demonstrate that C75 is a synthetic, chemically stable inhibitor of FAS. C75 inhibits purified mammalian FAS with characteristics of a slow-binding inhibitor and also inhibits fatty acid synthesis in human cancer cells. Treatment of human breast cancer cells with [5-(3)H]C75 demonstrates that C75 reacts preferentially with FAS in whole cells. Therefore, we have shown that the primary mechanism of the antitumor activity of C75 is likely mediated through its interaction with, and inhibition of, FAS. This development will enable the in vivo study of FAS inhibition in human cancer and other metabolic diseases.

Figure 1

Proposed mechanism of chain elongation in fatty acid biosynthesis and the synthesis of 3-carboxy-4-alkyl-2-methylenebutyrolactones. (A) Malonate, bound as its thioester to ACP, enters the active site where the elongating fatty acid chain is covalently linked to a reactive cysteine residue. (B) Decarboxylation of the malonyl-ACP results in a reactive enolate anion. (C) Enolate anion attacks the acyl-ACP in a thioester Claisen condensation to form the transiently generated tetrahedral oxyanion. (D) This rapidly decomposes to generate the resting state of KAS and the homologated β-ketoacyl intermediate bound to ACP. 1, Deprotonation of p-methoxybenzyl itaconate at low temperature produces the dianion 2, which underwent aldol reaction with a series of aldehydes, RCHO, to give on strongly acidic workup a mixture of γ-lactones 3 and 4. Separation on silica gel and crystallization afforded the pure diastereomers. Note the structural differences and similarities between cerulenin [(2S, 3R) 2,3-epoxy-4-oxo-7E, 10E-dodecadienamide] and C75. Although C75 lacks the highly reactive epoxide moiety, it retains the 8-carbon lipid tail without the two double bonds.

Figure 2

C75 inhibits FAS and fatty acid synthesis. (A) C75 has characteristics of a slow-binding inhibitor of type I FAS. With increasing preincubation time of FAS and C75, there is greater inhibition of FAS activity (solid line) compared with control (dotted line). (B) C75 inhibits fatty acid synthesis in HL60 cells. With increasing doses of C75, [U-¹⁴C]acetate incorporation into acylglycerides is inhibited by 80%. Incorporation into the free fatty acid pool is reduced by about 50%.

Figure 3

C75 labels FAS. SDS/PAGE and fluorography of SKBr3 cell lysate after 4 h exposure of cells to ³H-C75 [5 μg/ml]. Lane 1, whole cell lysate. Lane 2, FAS immunoprecipitation. Note the labeling of the approximately 250-kDa band, which represents human FAS.