Phosphorylation of the anti-HIV compound (S,S)-isodideoxyadenosine by human recombinant deoxycytidine kinase

doi:10.1016/S0006-2952(00)00462-7

Biochemical Pharmacology

Volume 60, Issue 10, 15 November 2000, Pages 1505-1508

https://doi.org/10.1016/S0006-2952(00)00462-7 Get rights and content

Abstract

(S,S)-Isodideoxyadenosine [(S,S)-isoddA] is an anti-HIV active compound discovered in our laboratory. However, its cellular mechanism of action, particularly the critical first stage of phosphorylation, is not understood. IsoddA is not phosphorylated by adenosine kinase. Also, because it is not a substrate for adenosine deaminase, it would not be activated by the pathway taken by ddA, i.e. via 5′-nucleotidase phosphorylation of ddI and conversion of ddIMP to ddAMP. However, we have discovered that human recombinant 2′-deoxycytidine kinase (dCK) phosphorylates (S,S)-isoddA. The enzyme kinetic data revealed that the extent of monophosphorylation of this l-related nucleoside was comparable to that found with ddA. (S,S)-IsoddATP is among the most potent inhibitors of HIV reverse transcriptase known, which suggests that the observed low efficiency of phosphorylation of this compound by dCK is a key factor that limits the capacity of human lymphocytes to make (S,S)-isoddA an exceptionally active anti-HIV agent.

Section snippets

Materials

Ni²⁺–nitrilotriacetic acid–agarose was purchased from Qiagen. Calf liver acetone powder, purine nucleoside phosphorylase, 5′-AMP–agarose, SDS–PAGE molecular weight marker kit, dC, dCMP, AMP, ADP, and ATP were procured from the Sigma Chemical Co. Bio-Rad protein assay reagent was purchased from Bio-Rad Laboratories. Purine nucleoside phosphorylase from calf spleen was purchased from Sigma. Blue-Sepharose was purchased from Pharmacia Biotech AB. The synthesis of (S,S)-isoddA has been described [1]

Results and discussion

dCK was purified to single band homogeneity using Ni²⁺–nitrilotriacetic acid–agarose affinity chromatography [12]. This enzyme was found to phosphorylate isoddA, an anti-HIV active compound, to isoddAMP with a catalytic efficiency of 0.33 nmol/min/mg/μM (Table 1). The catalytic efficiency was markedly less than for dA as substrate, but in the general range for that observed by us for ddA as substrate. The K_m values obtained suggested that ddA has a higher affinity for the enzyme than does its

Acknowledgements

We are grateful to Dr. Staffan Eriksson for the human recombinant dCK expressed in Escherichia coli. This research was supported by the National Institutes of Health (AI 32851).

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Citation Excerpt :
Moreover, based on its surprisingly broad substrate specificity regarding chemical modifications occurring either in the base or sugar moieties, dCK efficiently phosphorylates and thereby activates a wide range of nucleoside analogues, fundamental components of antileukemic, antitumour and antiviral therapeutic regimens [3,4]. The most widely used antimetabolites are: 2-chloro-2′-deoxyadenosine (Cladribine) [3], 2′,2′-difluorodeoxycytidine (Gemcitabine) and 1-β-d-arabinosylcytosine as antiproliferative agents [3] or 3′-azido-2′,3′-dideoxythymidine and (S,S)-isodideoxyadenosine as powerful compounds against HIV infection [4,5]. dCK is located in the proximity of the cytoplasmic membrane as well as in the perinuclear area [6] and shows a constitutive expression throughout the cell cycle [6–8], although 2- to 3-fold differences in its activity have been reported in different phases of the cell cycle [8].
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Phosphorylation of the anti-HIV compound (S,S)-isodideoxyadenosine by human recombinant deoxycytidine kinase

Abstract

Section snippets

Materials

Results and discussion

Acknowledgements

Biochem Biophys Res Commun

Biochem Pharmacol

Pharmacol Ther

Biochem Biophys Res Commun

Biochem Pharmacol

J Biol Chem

J Biol Chem

Biochim Biophys Acta

J Biochem Biophys Methods

Anal Biochem

Biochim Biophys Acta