Novel selective human mitochondrial kinase inhibitors: Design, synthesis and enzymatic activity

doi:10.1016/j.bmc.2007.01.049

Bioorganic & Medicinal Chemistry

Volume 15, Issue 8, 15 April 2007, Pages 3065-3081

https://doi.org/10.1016/j.bmc.2007.01.049 Get rights and content

Abstract

Selective and effective TK2 inhibitors can be obtained by introduction of bulky lipophilic chains (acyl or alkyl entities) at the 2′ position of araT and BVaraU, nucleoside analogues naturally endowed with a low TK2 affinity. These derivatives showed a competitive inhibitory activity against TK2 in micromolar range. BVaraU nucleoside analogues, modified on the 2′-O-acyl chain with a terminal N-Boc amino-group, conserved or increased the inhibitory activity against TK2 (7l and 7m IC₅₀: 6.4 and 3.8 μM, respectively). The substitution of an ester for a carboxamide moiety at the 2′ position of araT afforded a consistent reduction of the inhibitory activity (25, IC₅₀: 480 μM). On the contrary, modifications at 2′-OH position of araC and araG, have provided inactive derivatives against TK2 and dGK, respectively. The biological activity of a representative compound, 2′-O-decanoyl-BVaraU, was also investigated in normal human fibroblasts and was found to impair mitochondrial function due to TK2 inhibition.

Graphical abstract

Introduction

Deoxyribonucleoside kinases (dNKs) are key enzymes in the salvage pathway of mammalian cells.¹ dNKs catalyze the conversion of the nucleosides derived from DNA degradation, or from the extra-cellular environment, into the corresponding deoxyribonucleoside monophosphates. Human dNKs, thymidine kinase 1 (TK1), thymidine kinase 2 (TK2), deoxycytidine kinase (dCK), and deoxyguanosine kinase (dGK), have different sub-cellular localization and variable substrate specificities.2, 3, 4 TK2 and dGK are key enzymes in the mitochondrial nucleoside salvage pathway and are crucial in the maintenance of balanced mitochondrial deoxyribonucleotide pool especially in non-replicating tissues.⁵ Mutated TK2 and dGK are associated with decreased copy number of mitochondrial DNA (mtDNA) and mitochondrial respiratory chain (MRC) dysfunction.6, 7

TK2 is homologous with dCK, dGK, the recently discovered multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK).⁸ TK2 is able to activate several antiviral nucleoside analogues such as (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU), 1-β-d-arabinofuranosyl-5-(2-bromovinyl)uracil (BVaraU), 3′-azido-2′-,3′-dideoxythymidine (AZT), and 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-5-iodouracil (FIAU).9, 10, 11, 12, 13 Thus, treatment with these analogues may be accompanied with mitochondrial toxicity of terminally differentiated cells (AZT and FIAU).14, 15 Because of this it has been suggested to exploit TK2 in gene therapy against malignancies.16, 17

The development of potent and selective inhibitors of TK2 may be helpful to unravel a variety of metabolic processes, including the role of TK2 in the metabolic activation of some antiviral and anticancer nucleoside analogues, the role of TK2 in the maintenance of the mitochondrial dNTP pool, mtDNA synthesis and repair, and in the homeostasis of mitochondria. Also, specific TK2 inhibitors may elucidate the differential activities of cytosolic versus mitochondrial thymidine kinase in different cell lines or in the different phases of the cellular replication cycle.

So far only few literature reports have described selective and effective TK2 inhibitors. Among these, the 5′-O-substituted derivatives of thymidine, BVDU, and their acyclic analogues are able to selectively inhibit the mitochondrial enzyme, therefore their structural features can contribute to desiging new TK2 inhibitors.18, 19, 20 Also some ribofuranosylnucleoside analogues highly functionalized at the 3′-position are unexpectedly able to inhibit the mitochondrial enzyme. This is in complete disagreement with the available data that the enzyme recognize, as an efficient substrate, only natural deoxyribonucleosides and unnatural deoxyribonucleoside analogues.²¹

Recently, we have described the inhibitory activity against TK2 of a series of 2′-O-alkylester and ether derivatives of arabinofuranosyl nucleosides. The bulky lipophylic entities introduced at the 2′-OH of 1-β-d-arabinofuranosylthymine (araT) and BVaraU, both not preferential substrates for the TK2 enzyme, converted the araT and BVaraU nucleoside analogues to selective and purely competitive TK2 inhibitors in the micromolar concentration range.22, 23

Intrigued by these interesting preliminary results, further investigations were performed to complete the series of 2′-O-acyl/alkyl substituted arabinofuranosyl nucleosides and to synthesize a new class of compounds. In this study we report in detail: (a) the synthetic approaches applied to achieve the desired chemical structures; (b) the inhibitory effect of synthesized derivatives on phosphorylation by deoxyribonucleoside kinases; (c) the biological effect of one of the most effective TK2 inhibitors (7e) in tissue culture.

Preliminary data aimed to identify potential dGK inhibitors, able to provide information about substrate-specificity and the physiological role of the mitochondrial enzyme will be also reported.

Section snippets

Chemistry

2′-O-Acyl derivatives of BVaraU (compounds 5a, b, d, and e) and araT (compounds 6d–g) were prepared introducing the suitable acyl chain, in refluxing pyridine on the 3′,5′-O-TIPDS protected arabinonucleosides 3²⁴ and 4, as previously reported by us (compound 5c.²⁴) Compound 4 was synthesized as described for the derivative 3.

2′-O-(N-Boc-aminoalkanoyl) derivatives of BVaraU (compounds 5h–m) were obtained in quantitative yield by reaction of the N-Boc amino acid derivatives with 3′,5′-O-TIPDS

Biological results and discussion

We started the present study with several aims: (a) to design selective TK2 inhibitors; (b) to gain insights into structure–activity relationships (SAR) of the inhibitors for TK2 versus other nucleoside kinases; (c) to reveal whether the newly designed TK2 inhibitors can be uptaken by intact mitochondria; (d) to investigate the impact of TK2 inhibitors on mitochondrial (dys) function.

New synthetic work has been directed to modify the more interesting BVaraU and araT acyl derivatives by

Conclusion

We have demonstrated that nucleoside analogues such as araT and BVaraU bearing bulky lipophylic substituents at the 2′ position of the sugar can be efficiently recognised by TK2 in a highly selective manner. This finding suggests that TK2, in contrast with the HSV-1 TK, VZV-TK, and Dm-dNK, must contain a lipophilic pocket or cleft in which the 2′-substituents may fit. Since there is no crystal structure available for TK2, it is currently unclear how this lipophylic pocket is created in the

Chemistry

Reaction courses were routinely monitored by thin-layer chromatography (TLC) on silica gel precoated Macherey-Nagel durasil-25, with detection under a 254-nm UV lamp and/or by spraying the plates with 10% H₂SO₄/CH₃OH and heating. Column chromatography was performed with Macherey-Nagel 0.063–0.2 mm/70–230 mesh silica gel. MALDI-TOFMS (Matrix-assisted laser desorption ionization time-of-flight) spectra were obtained on a Hewlett–Packard HPG2025A mass spectrometer operative on a positive linear

Enzyme assays

The radiolabeled substrate [methyl-³H]dThd (70 Ci/mmol) was obtained from Amersham Pharmacia Biotech. The cDNAs of TK1, TK2, Dm-dNK, and HSV-1 TK were inserted in the pGEX-5X-1 vector, expressed as fusion proteins to glutathione S-transferase, and purified. The activity of the purified recombinant nucleoside kinases was assayed in a 50 μl reaction mixture containing 50 mM Tris–HCl, pH 8.0, 2.5 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), 3

Acknowledgments

A part of this work was supported by research grants from the Israeli Academy of Sciences No. 406/02 and Israeli Ministry of Health No. 5307 (A.S.) and by the University of Ferrara (FAR 2005-2006, project CAN2005-2006) Also, the European Commission (QLRT-2001-01004) (J.B. and A.K.) and the Flemish ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO G-0267.04) (J.B.) provided financial support to J.B. and A.K.

References and notes (39)

E.S.J. Arner et al.
Pharmacol. Ther.
(1995)
C.Y. Zhu et al.
J. Biol. Chem.
(1998)
P. Hatzis et al.
J. Biol. Chem.
(1998)
M. Johansson et al.
J. Biol. Chem.
(1999)
J. Balzarini et al.
Biochem. Pharmacol.
(1988)
J. Balzarini et al.
J. Biol. Chem.
(1989)
J. Wildiers et al.
Eur. J. Cancer Clin. Oncol.
(1984)
J.M. Colacino
Antiviral. Res.
(1996)
R.M. Blaese et al.
Eur. J. Cancer
(1994)
A.-I. Hernandez et al.
Bioorg. Med. Chem. Lett.
(2003)

S. Manfredini et al.

Bioorg. Med. Chem. Lett.

(2001)

C.B. Reese et al.

Tetrahedron Lett.

(1980)

F. Hansske et al.

Tetrahedron

(1984)

B. Munch-Petersen et al.

J. Biol. Chem.

(1991)

B.H. Robinson

Methods Enzymol.

(1996)

A. Saada et al.

Mol. Genet. Metab.

(2003)

D.C. Wharton et al.

Methods Enzymol.

(1967)

J.C. Fischer et al.

Clin. Chim. Acta

(1986)

W.K. Pogozelski et al.

Mitochondrion

(2003)

Cited by (7)

Photoreactivities of 5-Bromouracil-containing RNAs
2013, Bioorganic and Medicinal Chemistry
Citation Excerpt :
They then were analyzed by HPLC comparing the retention time with authentic material of A, C, G, U, ketoA, and ketoG. In order to confirm the generation of keto adenosine and keto guanosine after photoirradiation, they were synthesized according to the previous method with a slight modification.27–30 Pfitzner–Moffatt oxidation procedure was used instead of using Dess–Martin periodinane reagent.
5-Bromouracil (^BrU) was incorporated into three types of synthetic RNA and the products of the photoirradiated ^BrU-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA^BrUGC)₂ and r(CGAA^BrUUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine (^ketoA) product at the 5′-neighboring nucleotide, such as r(GC^ketoAUGC) and r(CGA^ketoAUUGC), respectively. The photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in Z-form RNA produced the 2′-keto guanosine (^ketoG) product r(CGC^ketoGUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.
Enzymatic regioselective and complete deacetylation of two arabinonucleosides
2010, Journal of Molecular Catalysis B: Enzymatic
Candida antarctica lipase B (CAL-B)-catalysed regioselective deacetylation of 2′,3′,5′-tri-O-acetyl-1-β-d-arabinofuranosyluracil (1) and 2′,3′,5′-tri-O-acetyl-9-β-d-arabinofuranosyladenine (2) was studied. The choice of the reaction medium allowed the regioselective formation of products bearing different degree of acetylation: in isopropanol, CAL-B catalysed the formation of the corresponding 2′-O-acetylated arabinonucleosides, while hydrolyses afforded the 2′,3′-di-O-acetylated products. In particular, the procedure herein described allows a simple and efficient preparation of the reported vidarabine prodrug 2′,3′-di-O-acetyl-9-β-d-arabinofuranosyladenine, avoiding the utilisation of protective groups. Moreover, to achieve full deacetylation of the assayed substrates, a set of commercial hydrolases and fungal keratinases from Doratomyces microsporus (DMK) and Paecilomyces marquandii (PMK) were tested. While only PMK and DMK catalysed the quantitative complete deacetylation of 1, DMK accomplished full deacetylation of 2 in shorter time than the other assayed enzymes.
Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion
2014, Biochemistry
Phosphorylation of nucleoside-metallacarborane and carborane conjugates by nucleoside kinases
2013, Nucleosides, Nucleotides and Nucleic Acids
Recent advances in thymidine kinase 2 (TK2) inhibitors and new perspectives for potential applications
2012, Current Pharmaceutical Design
Human mitochondrial thymidine kinase is selectively inhibited by 3′-thiourea derivatives of β-thymidine: Identification of residues crucial for both inhibition and catalytic activity
2009, Molecular Pharmacology

View all citing articles on Scopus

View full text

Novel selective human mitochondrial kinase inhibitors: Design, synthesis and enzymatic activity

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Biological results and discussion

Conclusion

Chemistry

Enzyme assays

Acknowledgments

Pharmacol. Ther.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Eur. J. Cancer Clin. Oncol.

Antiviral. Res.

Eur. J. Cancer

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Tetrahedron Lett.

Tetrahedron

J. Biol. Chem.

Methods Enzymol.

Mol. Genet. Metab.

Methods Enzymol.

Clin. Chim. Acta

Mitochondrion