Addressing the selectivity and toxicity of antiviral nucleosides

Feng, Joy Y

As most nucleoside/tides analogs need to be transformed into 5'-TP to exert on-target and off-target effects, their TP forms are necessary for elucidating these mechanisms ( Table 1 ). The typical initial enzymatic assay for polymerases is a radioactivity-based polymerization assay, in which the inhibition by nucleotides is measured as an IC 50 value. The inhibition IC 50 assays can be easily adapted to a 96-well filter binding assay, and multiple compounds can be accessed simultaneously. However, based on inhibition studies alone, one cannot establish whether the nucleotide was actually incorporated. Therefore, a single nucleotide incorporation (SNI) assay can be particularly useful to determine if a NTP analog actually served as a substrate for the polymerases. Under a high concentration of NTP analogs (e.g. 500 mM ribose nucleotide TP (rNTP) or 50-100 mM 2 0 -deoxyribose nucleotide TP (dNTP)), the relative rate of incorporation of an analog can be compared to that of its natural NTP counterpart. For further mechanistic insight, a pre-steady state kinetic analysis of the NTP incorporation offers detailed kinetic parameters such as pre-steady state polymerization rate constant k pol and NTP dissociation constant K d . In these studies, incorporation rates are studied using multiple time points and nucleotide concentrations, often using quenchflow techniques, making these studies reagent-and time-consuming. Finally, the mechanism of a nucleotide analog-derived enzyme inhibition can be revealed by an elongation assay, in which a stable elongation complex is formed, and the incorporation of the next few natural NTPs is measured. Normally, an NTP analog can be characterized into one of the three groups: chain-terminators, delayed chain-terminators, or stably incorporated analogs. Stably incorporated NTP analogs are difficult to study for toxicity as they could lead to little or no quantitative changes in DNA, RNA, or protein expression. 36 The quality of the above-mentioned biochemical assays relies heavily on the availability of high quality TP active forms, which can be time-and resourceconsuming to synthesize, and the TP salts are subject to degradation overtime. At times, certain inorganic contaminants could lead to apparent inhibition of polymerases.