Novel opportunities for thymidylate metabolism as a therapeutic target

Abstract

For over 40 years, the fluoropyrimidine 5-fluorouracil (5-FU) has remained the central agent in therapeutic regimens employed in the treatment of colorectal cancer and is frequently combined with the DNA-damaging agents oxaliplatin and irinotecan, increasing response rates and improving overall survival. However, many patients will derive little or no benefit from treatment, highlighting the need to identify novel therapeutic targets to improve the efficacy of current 5-FU-based chemotherapeutic strategies. dUTP nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi, providing substrate for thymidylate synthase (TS) and DNA synthesis and repair. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA as uracil is lethal. Importantly, uracil misincorporation represents an important mechanism of cytotoxicity induced by the TS-targeted class of chemotherapeutic agents including 5-FU. A growing body of evidence suggests that dUTPase is an important mediator of response to TS-targeted agents. In this article, we present further evidence showing that elevated expression of dUTPase can protect breast cancer cells from the expansion of the intracellular uracil pool, translating to reduced growth inhibition following treatment with 5-FU. We therefore report the implementation of in silico drug development techniques to identify and develop small-molecule inhibitors of dUTPase. As 5-FU and the oral 5-FU prodrug capecitabine remain central agents in the treatment of a variety of malignancies, the clinical utility of a small-molecule inhibitor to dUTPase represents a viable strategy to improve the clinical efficacy of these mainstay chemotherapeutic agents.

Figures

Figure 1. Mechanism of 5-FU-induced DNA Damage

5-Fluorouracil is converted to active metabolites: fluorodeoxyuridine monophosphate (FdUMP) and fluorodeoxyuridine triphosphate (FdUTP). FdUMP binds to, and inhibits the enzyme thymidylate synthase (TS) by formation of a ternary complex with the methyl donor co-factor. Inhibition of TS induces a metabolic blockade, resulting in depletion of thymidylate and the accumulation of dUMP which can be phosphorylated to dUTP. When dUTP pools expand, dUTPase activity can become saturated resulting in uracil misincorporation into DNA. In tumors with elevated expression of dUTPase, saturation of enzyme activity is unlikely to occur and dUTP misincorporation is eliminated as a mechanism of cytotoxicity. Generation of FdUTP and subsequent incorporation into DNA has also been demonstrated to induce significant cytotoxicity and dUTPase has been demonstrated to possess affinity for FdUTP, catalyzing its hydrolysis and preventing the misincorporation of FdUTP into DNA.

Figure 2. Variation in dUTPase Expression in Cell Lines

Western blot analysis of dUTPase expression in a panel of colon (SW620, HCT-8, HCT116, HT29, LoVo) and gastric (AGS), mitochondrial and nuclear isoforms are indicated. β-actin was used to control for loading. dUTPase relative enzyme activity assay from corresponding Western blot cell lysates, Histogram bars represent the mean ±SEM of corresponding Western lysates analyzed in duplicate..

Figure 3. Elevated dUTPase Protects Breast Cancer Cells from 5-FU

A, Western blot and analysis of dUTPase expression and corresponding dUTPase enzyme activity assay following 72 h transfection with pTre-Tight:DUT-N in the presence and absence of 0.5 μg/ml dox, Histogram bars represent the mean ±SEM of corresponding Western lysates analyzed in duplicate. B, Analysis of dUTP accumulation in pTre-Tight:DUT-N-transfected MCF-7 psTet-off cells with basal (+ dox) and induced (− dox) dUTPase expression and treated with 10 μM 5-FU, 1 μM FUdR and taxol (paclitaxel) 5 nM for 24 h, % dUTP was calculated as described in ‘Materials and Methods’, Histogram bars represent the mean ±SEM of two independent treatments, † = dUTP not detected. C, Growth inhibition measured by MTS assay in pTre-Tight:DUT-N-transfected MCF-7 pTet-off cells following treatment with increasing concentrations (in μM) of 5-FU for 72 h. Histogram bars represent the mean ±SEM of three independent treatments, *=p

Figure 4. Immunohistochemical Analysis of dUTPase in Human Breast Adenocarcinoma

Formalin-fixed, paraffin-embedded breast cancer tumor specimens were routinely processed and stained using the DUT415 monoclonal antibody. A represents low dUTPase expression, B and C represent elevated nuclear and cytoplasmic expression. Photomicrographs A and B are 20X magnification, photomicrograph C is 40X magnification.

Figure 5. Shape-merged pharmacophore model derived from Dud778

A, Dud778 mapping onto the feature model. Green sphere represents the H-bond acceptor, and magenta represents H-bond donor. B, Shape-merged feature model of Dud778 mapping onto the shape query. The gray area represents the shape constraint generated from the crystallized conformation. C, Dud778 mapping onto the shape-merged feature model as shown in B.

Figure 6. Docking validation of Dud778-dUTPase co-crystal structure

A, Dud778 as observed in the x-ray structure (1Q5H) is shown in green, and the atom-type colored conformation of Dud778 is predicted by GOLD. B, superimposition of a representative compound, colored by atom type, with x-ray determined Dud778 conformation (green). The compound was selected from database screening that favorably interacts with the ligand binding domain by efficiently filling the deep cavity.