Nudix hydrolase 18 catalyzes the hydrolysis of active triphosphate metabolites of the antivirals remdesivir, ribavirin, and molnupiravir

Figure 1. Structure, intracellular metabolism, and mode of action of remdesivir. Figure was created with BioRender.

Figure 2. NUDT18 catalyzes the hydrolysis of remdesivir-TP.A, hydrolysis activity of MTH1, NUDT15, NUDT18, NUDT5, and NUDT9 (200 nM) with 100 μM remdesivir-TP was tested in assay buffer (20 mM Tris–acetate pH 8.0, 40 mM NaCl, 10 mM Mg(Ac)2, 1 mM DTT, and 0.05% Tween20) by incubation at 22 °C for 30 min. Formed Pi was detected by addition of malachite green reagent and measurement of absorbance at 630 nm. B, activity of 200 nM MTH1, NUDT15, NUDT18, and NUDT5 was also tested in the presence of PPase (0.2 U/ml) that converts formed PPi to Pi. C, activity of the human pyrophosphatases ITPase, dUTPase, and dCTPase with 100 μM remdesivir-TP in the presence of PPase was assayed and compared to activity of NUDT18 with and without PPase and to NUDT18 activity with 100 μM 8-oxo-dGDP. D, hydrolysis products of NUDT18-catalyzed hydrolysis of remdesivir-TP in the presence and absence of NUDT18. Bars and error bars represent average and standard deviation determined from the mean of produced phosphate in two independent experiments in which phosphate production was assayed in duplicate. MTH1, MutT Homolog 1; NUDT, Nudix hydrolase; PPase, pyrophosphatase.

Figure 3. NUDT18 catalyzes the hydrolysis of ribavirin-TP and molnupiravir-TP.A, hydrolysis activity of 100 nM NUDT18 with 100 μM ribavirin-TP or remdesivir-TP, in the presence and absence of 0.2 U/ml Escherichia coli PPase, was monitored in assay buffer (20 mM Tris–acetate pH 8.0, 40 mM NaCl, 10 mM Mg(Ac)2, 1 mM DTT, and 0.05% Tween20) by incubation at 22 °C for 30 min. Hydrolysis activity of 10 nM NUDT18 was assessed with 100 μM ribavirin-TP, remdesivir-TP, or 8-oxo-dGDP in assay buffer with E. coli PPase (0.2 U/ml) by incubating reactions at 22 °C for 30 min. B, hydrolysis activity of 20 nM NUDT18, MTH1, and NUDT15 was tested with 100 μM molnupiravir-TP, in the presence and absence of 0.2 U/ml E coli PPase by incubating reactions at 22 °C for 30 min. Formed Pi was detected by addition of malachite green reagent and measurement of absorbance at 630 nm. MTH1, MutT Homolog 1; NUDT, Nudix hydrolase; PPase, pyrophosphatase.

Figure 4. Kinetic analysis of NUDT18 with triphosphates of antiviral drugs. Enzyme saturation curves of NUDT18 with (A) remdesivir-TP, (B) ribavirin-TP, (C) molnupiravir-TP, and (D) 8-oxo-dGDP. E, specific activity of 20 nM NUDT18 with 100 μM of triphosphates of the antivirals ganciclovir, aciclovir, sofosbuvir, and the cytostatic drug gemcitabine was tested by incubation for 30 min at 22 °C in assay buffer (20 mM Tris–acetate pH 8.0, 40 mM NaCl, 10 mM MgAcetate, 1 mM DTT, and 0.01% Tween20), in the absence and presence of Escherichia coli PPase (0.2 U/ml). Formed Pi was detected using the addition of a malachite green reagent followed by measurement of absorbance at 630 nm. Figure shows formed [Pi] (μM) per [NUDT18] (nM). F, enzyme saturation curves of NUDT18 with sofosbuvir-TP, aciclovir-TP, and dGTP. Graphs with saturation curves show average and standard deviation of initial rates, determined from measurements of produced phosphate at 0, 10, 20, and 30 min performed in duplicate, from two independent experiments. MTH1, MutT Homolog 1; NUDT, Nudix hydrolase; PPase, pyrophosphatase.

Figure 5. Relative catalytic efficiency and turnover numbers of NUDT18 with tested substrates and their chemical structures. NUDT, Nudix hydrolase.

Figure 6. Inhibition of NUDIX enzymes by Remdesivir and its metabolites. MTH1 and NUDT15 was tested for inhibition by 100 μM remdesivir-TP, remdesivir, or remdesivir metabolite (GS-441524) in an activity assay using 100 μM dGTP as substrate and 10 nM NUDT15 or MTH1. The potency of remdesivir and remdesivir metabolite (GS-441524) to inhibit NUDT18 was tested in an assay using 100 μM 8-oxo-dGDP as substrate and using 100 nM NUDT18. NUDT15 is modestly inhibited by remdesivir, and activity of all three enzymes are reduced by approximately 20% in the presence of the remdesivir metabolite (GS-441524). MTH1, MutT Homolog 1; NUDT, Nudix hydrolase.