Activation and deactivation of a broad-spectrum antiviral drug by a single enzyme: adenosine deaminase catalyzes two consecutive deamination reactions

Abstract

Ribavirin is an approved broad-spectrum antiviral drug. A liver-targeting prodrug of ribavirin, viramidine, is in clinical trial in an attempt to provide a better therapeutic index. The conversion of viramidine to ribavirin, and of ribavirin to an inactive metabolite through adenosine deaminase, is reported. Kinetic analysis indicates that adenosine deaminase is likely involved in activation of viramidine in vivo, and the process is highly pH sensitive. The differential activities of two consecutive deamination reactions are kinetically studied and interpreted based on adenosine deaminase structural information. A comprehensive understanding of the viramidine and ribavirin deamination mechanism should help in designing better nucleoside therapeutics in the future.

FIG. 1.

Adenosine deaminase catalyzes hydrolytic deamination of viramidine to ribavirin, and ribavirin to ICN3297, determined by UV spectral analysis. The difference of absorption at 240 nm from UV spectra of viramidine and ribavirin (0.2 mM) was utilized to monitor the first conversion. The insert shows the time course of conversion of viramidine (0.2 mM) to ribavirin by adenosine deaminase (0.2 μM) and the negative control (no enzyme).

FIG. 2.

Proposed deamination reaction mechanism of viramidine and ribavirin by adenosine deaminase. The interaction map between the ADA active site and viramidine (A) or ribavirin (B) is based on the cocrystal structure of adenosine deaminase with purine ribonucleoside (26).

FIG. 3.

The pH-rate profiles of adenosine deaminase with adenosine (□), viramidine (▵), or ribavirin (○) as a substrate. The experiments were conducted in a phosphate buffer. The initial velocities at various pH values were fitted to the equation given in the text. One representative set of experimental data is shown here.