Elsevier

Biochemical Pharmacology

Volume 61, Issue 2, 15 January 2001, Pages 179-189
Biochemical Pharmacology

Enzyme-catalyzed therapeutic agent (ECTA) design: activation of the antitumor ECTA compound NB1011 by thymidylate synthase1

This work has been presented in preliminary form at the NCI-EORTC Symposium “Chemotherapeutic Strategies for Treatment of Colorectal Cancer: Present and Future Developments,” February 10–12, 1999, Amsterdam, The Netherlands.
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Abstract

The in vivo administration of enzyme-inhibiting drugs for cancer and infectious disease often results in overexpression of the targeted enzyme. We have developed an enzyme-catalyzed therapeutic agent (ECTA) approach in which an enzyme overexpressed within the resistant cells is recruited as an intracellular catalyst for converting a relatively non-toxic substrate to a toxic product. We have investigated the potential of the ECTA approach to circumvent the thymidylate synthase (TS) overexpression-based resistance of tumor cells to conventional fluoropyrimidine [i.e. 5-fluorouracil (5-FU)] cancer chemotherapy. (E)-5-(2-Bromovinyl)-2′-deoxy-5′-uridyl phenyl l-methoxyalaninylphosphoramidate (NB1011) is a pronucleotide analogue of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdU), an antiviral agent known to be a substrate for TS when in the 5′-monophosphorylated form. NB1011 was synthesized and found to be at least 10-fold more cytotoxic to 5-FU-resistant, TS-overexpressing colorectal tumor cells than to normal cells. This finding demonstrates that the ECTA approach to the design of novel chemotherapeutics results in compounds that are selectively cytotoxic to tumor cell lines that overexpress the target enzyme, TS, and therefore may be useful in the treatment of fluoropyrimidine-resistant cancer.

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

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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).

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