Enzyme-catalyzed therapeutic agent (ECTA) design: activation of the antitumor ECTA compound NB1011 by thymidylate synthase1
Introduction
1A major problem in the chemotherapeutic treatment of cancer is the development of resistance. Resistance develops when drug exposure favors the growth and reproduction of those tumor cells overexpressing enzyme(s) targeted for inhibition by the drug. For example, drug-associated enzyme overexpression in tumor cells can result from transcriptional derepression subsequent to loss of functional tumor suppressor elements such as p53, RB, and p16 [1], [2], [3], [4]. Elevated expression also can be mediated by gene amplification in vivo following chemotherapy with a regimen containing 5-FU [5]. It would be particularly advantageous to capitalize on the elevated enzyme levels by administering an ECTA drug, a relatively non-toxic compound specifically designed to generate a toxic species as a result of enzymatic processing. The differential in enzyme levels between tumor (high/sensitive) and normal (low/resistant) cells should provide ECTA drugs with a beneficial therapeutic index.
TS is an enzyme critical for DNA synthesis in all organisms and is the target for both fluoropyrimidine and antifolate-based cancer chemotherapies. TS inhibitors such as 5-FU can result in more than 4-fold [6] elevation of TS, and antifolates can result in still higher levels of TS expression in tumor cells [7]. Overexpression of TS can have other consequences within cells, including suppression of p53 levels [8]. Because of the well-documented overexpression response to inhibitor drugs and the extensive background of structural and mechanistic characterization [9], [10], we selected TS as the focus for the development of an ECTA approach to dealing with the problem of enzyme-mediated drug resistance.
(E)-5-(2-Bromovinyl)-2′-deoxy-5′-uridyl phenyl l-methoxyalaninylphosphoramidate (NB1011, 3, Fig. 1) was designed as a pronucleotide to demonstrate the ECTA concept of drug design because neutral 5′-phosphoramidates, especially phenyl l-alaninylphosphoramidate esters, are effective agents for intracellular delivery of 2′,3′-dideoxyribose-based 5′-mononucleotide antiviral agents [11]. Furthermore, (E)-5-(2-bromovinyl)-2′-deoxyuridine 5′-monophosphate (BVdUMP, 2, Fig. 1) has been shown to be an alternative, competitive substrate for Lactobacillus casei TS in vitro, having a similar Km but a lower kcat than dUMP. By forming a covalent intermediate with 2, TS converts the inert vinylic bromide into a nucleophilic displacement-reactive allylic bromide; in the presence of 2-mercaptoethanol, this intermediate gives rise to 5-[2-(2-hydroxyethyl)thioethyl]-based dUMP derivatives in a reaction catalyzed by TS in vitro[12].
Based upon recent information about the active site structure of human TS [13], we predicted that the 5′-monophosphate of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU, 1, Fig. 1) was likely to be converted by intracellular TS to cytotoxic reaction products without inactivating the enzyme. In addition, because TS productively binds a variety of 5′-monophosphates of uracil 2′-deoxyribonucleosides as substrates, including those with moderately sized substituents at the pyrimidine 5-position, this system offers the opportunity to explore the ECTA concept by designing and testing a variety of 5-substituted deoxyuridine derivatives.
Section snippets
General methods
BVdU (1), prepared by the method of Dyer et al.[14], was dried in vacuo at 75° adjacent to P2O5 immediately prior to use. Radial chromatography was performed on a Chromatotron instrument (Harrison Research), using Merck silica gel-60 with a fluorescent indicator as adsorbent. BVdUMP (2) was prepared by standard chemical phosphorylation of BVdU.
NMR
1H NMR spectra were recorded on a Varian Associates Gemini spectrometer at 300 MHz, using hexadeuterio-dimethyl sulfoxide (C2H3)2SO solutions. Chemical
Chemical synthesis
The synthesis of NB1011 (pronucleotide 3) required the development of reaction conditions that yield primarily the 5′-phosphoramidate, while leaving the 3′-OH free. Attempts to prepare 3 along a regioselective route involving phosphoramidation of the O3′-TBDMS derivatives of 1 failed when the 5′-phosphoramidate group proved sensitive to the mild conditions (tetrabutyl ammonium fluoride on silica gel, 23°, tetrahydrofuran) used to effect removal of the O3′ protecting group. Loss of the
Discussion
The LC/MS analysis of cell extracts, combined with HPLC fluorescence detection and UV spectra, demonstrated that NB1011 treatment results in the appearance of BVdUMP in cell extracts. In addition, a number of fluorescent products were detected in extracts prepared from cells treated with NB1011. We suggest that the selective tumor cell cytotoxicity of NB1011 may be due, at least in part, to the eventual production of compounds similar to 4(Fig. 1), a 5,O4-ethenodeoxyuridine nucleotide. The
References (24)
- et al.
Inhibition of mouse thymidylate synthase promoter activity by the wild-type p53 tumor suppressor protein
Exp Cell Res
(1997) - et al.
Thymidylate synthasestructure, inhibition, and strained conformations during catalysis
Pharmacol Ther
(1997) - et al.
Thymidylate synthetase-catalyzed conversions of E-5-(2-bromovinyl)-2′-deoxyuridylate
J Biol Chem
(1983) - et al.
Aryl phosphate derivatives of AZT retain activity against HIV1 in cell lines which are resistant to the action of AZT
Antiviral Res
(1992) - et al.
Expression of human thymidylate synthase in Escherichia coli
J Biol Chem
(1989) - et al.
Isolation and characterization of a thymidylate synthase-deficient human colon tumor cell line
Biochem Pharmacol
(1999) - et al.
Genetic instability as a consequence of inappropriate entry into and progression through S-phase
Cancer Metastasis Rev
(1995) - et al.
S100A4 involvement in metastasisderegulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme
Cancer Res
(1999) - et al.
Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines
Proc Natl Acad Sci USA
(1995) - et al.
Higher frequency of gene amplification in breast cancer patients who received adjuvant chemotherapy
Cancer
(1996)
Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines
Br J Cancer
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Abbreviations: BVdU, (E)-5-(2-bromovinyl)-2′-deoxyuridine; BVdUMP, (E)-5-(2-bromovinyl)-2′-deoxyuridine 5′-monophosphate; NB1011, (E)-5-(2-bromovinyl)-2′-deoxy-5′-uridyl phenyl l-methoxyalaninylphosphoramidate; COSY, correlated spectroscopy; DCI, direct current ionization; DMF, N,N-dimethylformamide; dUMP, 2′-deoxyuridine 5′-monophosphate; ECTA, enzyme-catalyzed therapeutic agent; 5-FU, 5-fluorouracil; 5-FdUMP, 5-fluoro-2′-deoxyuridine 5′-monophosphate; RFU, relative fluorescence units; THF, N5,N10-methylene tetrahydrofolate; TBDMS, tert-butyldimethylsilyl; and TS, thymidylate synthase (EC 2.1.1.45).