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. 2014 Jul;58(7):3636-45.
doi: 10.1128/AAC.02666-14. Epub 2014 Apr 14.

Efficiency of incorporation and chain termination determines the inhibition potency of 2'-modified nucleotide analogs against hepatitis C virus polymerase

Amy Fung  1 , Zhinan Jin  2 , Natalia Dyatkina  1 , Guangyi Wang  1 , Leo Beigelman  1 , Jerome Deval  2
Affiliations

Efficiency of incorporation and chain termination determines the inhibition potency of 2'-modified nucleotide analogs against hepatitis C virus polymerase

Amy Fung et al. Antimicrob Agents Chemother. 2014 Jul.

Abstract

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2'F-2'C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2'F-2'C-Me-UTP was efficiently incorporated by HCV polymerase with apparent Kd (equilibrium constant) and kpol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s(-1), respectively, giving an overall substrate efficiency (kpol/Kd) of 0.0059 ± 0.0015 μM(-1) s(-1). We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2' position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH2 > F-C-Me > C-Me > N3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2'F-2'C-Me-, 2'C-Me-, and 2'ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2'-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2'-modified NTP analogs against HCV polymerase.

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Figures

FIG 1
FIG 1
Two-step nucleotide incorporation mechanism. (A) ERn is defined as the enzyme/primer/template complex, NTP is the nucleotide triphosphate substrate, and ERn + 1 is the product of the reaction that contains the primer extended by one nucleotide. For the simulation, k1 = 100 μM−1 s−1, k−1 = 10,000 s−1, k2 = 1 s−1, and k−2 = 0 were applied. The initial concentration for ERn was set at 0.1 μM, and the starting concentrations for NTP were set at various points (800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μM). The output for the simulation is the percentage of ERn + 1 in the initial concentration of ERn. The simulated progress curves of the percentage of ERn + 1 increase then were generated at up to 60 s and are shown in Fig. 5A. (B) Classic two-step reaction mechanism for a polymerase reaction.
FIG 2
FIG 2
Isolating the NS5B elongation complex (EC). (A) Principle of the initiation, labeling, and elongation reactions. Initiation of RNA synthesis by NS5B was performed in the presence of a GG-dinucleotide primer together with ATP and GTP for 2 h. Because of the template sequence, the NS5B EC was stalled at the 9-mer position. To track the RNA in the EC by phosphorimaging, 3′-end isotope labeling was performed by incorporation of [33P]CMP at the 10-mer position. The fast elongation of RNA synthesis could resume by adding UTP (1 min or less). The boldfaced “C” represents the radiolabeled nucleoside. (B) Processivity of NS5B complex during initiation and elongation. The enzyme processivity during 10-mer and 20-mer formation was monitored by adding heparin either at the beginning of the initiation reaction or together with UTP after the 10-mer EC was already formed. The error bars represent the standard deviations from two independent experiments. (C) High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the formation of the radiolabeled 10-mer RNA product of NS5B activity in the presence of the [33P]CTP tracer (lane 1). After centrifugation of the reaction product for 5 min at 13,000 × g, the supernatant did not contain any 10-mer product (lane 2). The EC containing the 10-mer product formed a precipitate that could be washed two times with low-salt buffer and then redissolved in high-salt buffer (lane 3).
FIG 3
FIG 3
Pre-steady-state kinetics of incorporation of 2′F-2′C-Me-UMP. (A) Principle of the reaction. Once the stalled 10-mer EC was isolated, increasing concentrations of 2′F-2′C-Me-UTP at 0.46 (●), 1.4 (■), 4.1 (▲), 12.4 (▼), 37 (◆), 111 (○), 333 (□), and 1,000 μM (◇) were added to start the 10- to 11-mer extension reactions. The reactions were quenched by adding formamide with 50 mM EDTA at various time points. The boldfaced and underlined “C” represents the radiolabeled nucleoside. The box around “A” represents the opposing nucleoside to the incoming nucleotide. (B) Time courses of 11-mer formation at various 2′F-2′C-Me-UTP concentrations. Each time course was fit to a single exponential equation to obtain the rate of analog incorporation at each concentration. (C) The rates of incorporation were plotted against analog concentrations, and the data were fit to a hyperbola equation to derive the maximum rate of 2′F-2′C-Me-UMP incorporation, with a kpol of 0.67 ± 0.05 s−1 and a dissociation equilibrium constant (Kd) of 113 ± 28 μM. The calculated overall catalytic efficiency (kpol/Kd) for 2′F-2′C-Me-UMP incorporation is 0.0059 ± 0.0015 μM−1 s−1. The error bars represent the standard deviations from two independent experiments.
FIG 4
FIG 4
Chain termination with 2′F-2′C-Me-UMP. (A) Principle of the reaction. Once the stalled 10-mer EC was isolated, adding either UTP (40 s) or 2′F-2′C-Me-UTP (5 min) enabled the formation of the 11-mer product. After this step, GTP and ATP were added for 20 s as the next correct nucleotides to monitor 20-mer formation. The boldfaced and underlined “C” represents the radiolabeled nucleoside. The box around “A” represents the opposing nucleoside to the incoming nucleotide. (B) High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with 2′F-2′C-Me-UTP, followed by the addition of the next correct nucleotides (+). The formation of RNA products longer than the 11-mer was expressed as the percentage of the initial amount of 11-mer formed for either UTP or 2′F-2′C-Me-UTP in the absence of GTP and ATP (−). “/” indicates that no product was detected.
FIG 5
FIG 5
Single-turnover kinetics of nucleotide incorporation. (A) Simulated progress curves of product formation at various nucleotide concentrations. Single-nucleotide incorporation was simulated using arbitrary values of Kd of 100 μM and kpol of 1 s−1, resulting in a theoretical specificity or substrate efficiency, kpol/Kd, of 0.01 s−1 μM−1. (B) Single incorporation of UMP opposite template A. High-resolution Tris-borate-EDTA–urea gel electrophoresis shows the extension of 10- to 11-mer RNA products. Increasing concentrations of UTP ranging from 0.0015 to 10 μM were added to start the extension reaction. Four different time points were measured to evaluate the effect of time on K1/2 and on kpol/Kd. The boldfaced “C” represents the radiolabeled nucleoside. The box around “A” represents the opposing nucleoside to the incoming nucleotide. (C) Quantitative analysis of product formation. For each time point, percent UMP incorporation was calculated and plotted as a function of UTP concentration. The data were fit to a sigmoidal dose-response equation (equation 1) to obtain K1/2 values, as reported in Table 1.
FIG 6
FIG 6
Differences between substrate efficiency and inhibition potency of nucleotide analogs. (A) Chemical structures of nucleotide analogs. 3′dUTP was used as a control for chain termination. All of the other molecules contain a 3′OH group, with a modification at the 2′ position: OH, F, NH2, C-Me, N3, or ara. (B) Substrate efficiency (kpol/Kd) for each NTP analog was measured from single-nucleotide incorporation experiments, and discrimination levels were calculated as (kpol/Kd,UTP)/(kpol/Kd,UTP analog). Inhibition potency, expressed as IC50, was measured with a long RNA template under steady-state conditions where each UTP analog competes against natural UTP. The discrimination values and IC50s for each nucleotide can be found in Table 3.
FIG 7
FIG 7
Chain termination in the presence of the next correct nucleotide. High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with UTP or UTP analogs, followed by the addition of the next correct nucleotides (+), following the same scheme as that explained in the legend to Fig. 4A. Formation of RNA products longer than the 11-mer was expressed as the percentage of the initial amount of 11-mer formed for each UTP analog in the absence of GTP and ATP (−). “/” indicates that no product was detected.
FIG 8
FIG 8
Kinetics of next-correct-nucleotide incorporation. (A) Principle of the reaction. Once the stalled 10-mer EC was isolated, adding either the natural UTP (40 s) or a UTP analog (5 min) enabled the formation of the 11-mer product. After this step, GTP at various concentrations was added and reacted for a fixed time. The boldfaced “C” represents the radiolabeled nucleoside. The box around “A” represents the opposing nucleoside to the incoming nucleotide. (B) Gel image showing the GTP concentration-dependent incorporation of one or two consecutive GMPs into the 11-mer RNA containing a UMP or 2′F-UMP at its 3′ end. The sum of the 12- and 13-mer RNA products was expressed as a percentage of the initial amount of 11-mer. The percentage of GMP incorporation product was plotted as a function of GTP concentration after UMP (left) and 2′F-UMP (right). The data were fit to a sigmoid dose-response equation (equation 1) to obtain K1/2 values. The kpol/Kd values for GMP incorporation after UMP and 2′F-UMP were calculated from K1/2 values using equation 2, and the means ± standard deviations from more than two repeats are reported in Table 4. (C) GTP incorporation after 2′F-2′C-Me-UMP and 2′C-Me-UMP terminated RNA. Once the stalled 10-mer EC was isolated, it was mixed with 100 μM UTP analogs for 5 min to form the 11-mer product. After this step, GTP at various concentrations was added and reacted for 20 min. The gel image shows that no GMP incorporation (12-mer) was detected up to 1 mM GTP reaction for 20 min, resulting for both compounds in a discrimination level of >2 × 106 compared to GTP incorporation after natural UMP (Table 4).
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