Inactivation of human S-adenosylhomocysteine hydrolase by covalent labeling of cysteine 195 with thionucleoside derivatives

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Abstract

A new series of 5′-thioadenosine derivatives 14 were synthesized for selectively targeting 195Cys of human AdoHcy hydrolase. Their incubation with the enzyme resulted in time- and concentration-dependent inactivation, without major modifications of the NAD+/NADH ratio. The electrospray mass analysis of the inactivated enzyme with 1, 2, 3, and 4b showed that inhibition was accompanied by the formation of a specific and covalent labeling of each AdoHcy hydrolase subunit. Proteolytic cleavage (endo-Lys-C) and subsequent peptide characterization of the labeled enzyme revealed that 195Cys was the residue modified during the inactivation process.

Graphical abstract

Thionucleosides 14 were synthesized for selectively targeting 195Cys of human placental AdoHcy hydrolase.

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Introduction

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyses the interconversion of AdoHcy into adenosine (Ado) and l-homocysteine (Hcy).1 Inhibition of this enzyme results in intracellular accumulation of AdoHcy which in turn provokes feed back inhibition of S-adenosylmethionine-dependent methylation reactions (i.e., viral mRNA methylation) which are essential for viral replication.2 AdoHcy hydrolase also controls levels of Hcy, which appear to be a risk factor for cardiovascular diseases.3 Therefore, AdoHcy hydrolase has emerged as a target for molecular design of antiviral agents as well as therapeutic inhibitors that may restore normal plasma levels of Hcy.

The recent success in the determination of the X-ray structures of rat liver4 and human5 AdoHcy hydrolase has led to the identification of the essential amino acids involved in the different steps of the catalytic cycle. Among them, the 194Cys residue in rat liver enzyme (195Cys in human enzyme) was proposed to modulate the oxidation state of the bound cofactor NAD+.4a This proposal is consistent with the results obtained by Yuan et al. in their pioneer study on chemical modification and site-directed mutagenesis of cysteine residues in AdoHcy hydrolase.6 These two details led us to consider that specific covalent modification of 195Cys should lead to the inactivation of human AdoHcy hydrolase. This possibility was examined with a series of thionucleosides 14.

These sulfur-containing nucleosides fulfill the basic requirements of being active-site-directed inhibitors for selectively targeting 195Cys. Disufide bond formation resulting from their interaction with 195Cys at the active site might provoke covalent inactivation of AdoHcy hydrolase (Fig. 1).

We describe here the synthesis of thionucleosides 14 and their interactions with human placental AdoHcy hydrolase.

Section snippets

Chemistry

5′-Deoxy-5′-thioadenosine 1 was obtained from Ado by a described procedure.7 The corresponding disulfide 2 was prepared by iodine oxidation of 1.8 The disulfide 3, which was synthesized according to the general procedure described by Décout and co-workers,9 was a gift from these authors. The epithionucleosides 4a and 4b were obtained from an unique precursor: the N6-benzoyl-2′,3′-O-isopropylidene-9-(β-d-allofuranosyl)adenine 8, which was prepared from diacetone-d-glucose 5 by the procedure

Results and discussion

Recombinant human placental AdoHcy hydrolase purified to homogeneity was used in this study.15 AdoHcy hydrolase (10 nM) was assayed in the synthetic direction in the presence of [8-14C]-Ado or [2,8-3H]-Ado (15 μM, 300 Bq) and Hcy (5 mM) in 20 mM potassium phosphate buffer pH 7.5, 1 mM EDTA. Incubation of the enzyme with 1, 2, 3, 4a, and 4b resulted in time- and concentration-dependent inactivation. Compounds 14 are stable under assay conditions whereas thiol 1 became contaminated with 15% of

Conclusion

We have identified a new series of inhibitors of AdoHcy hydrolase, which covalently modify the enzyme by interactions with 195Cys. These results confirm the crucial role of 195Cys at the catalytic center of AdoHcy hydrolase. Docking experiments with the epithionucleosides 4a and 4b into the active site of AdoHcy hydrolase are under progress to understand their different behaviors.

Acknowledgements

The authors thank Pr. Jean Luc Décout, Université Joseph Fourier—Grenoble I, for providing us a sample of 5′-deoxyadenosin-5′-yl methyldisulfide and le Conseil Général de la Marne for a doctoral grant for C.G.

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