In silico design of a novel nucleotide antiviral agent by free energy perturbation

Abstract

Nucleoside analogs are the backbone of antiviral therapies. Drugs from this class undergo processing by host or viral kinases to form the active nucleoside triphosphate species that selectively inhibits the viral polymerase. It is the central hypothesis that the nucleoside triphosphate analog must be a favorable substrate for the viral polymerase and the nucleoside precursor must be a satisfactory substrate for the host kinases to inhibit viral replication. Herein, free energy perturbation (FEP) was used to predict substrate affinity for both host and viral enzymes. Several uridine 5'-monophosphate prodrug analogs known to inhibit hepatitis C virus (HCV) were utilized in this study to validate the use of FEP. Binding free energies to the host monophosphate kinase and viral RNA-dependent RNA polymerase (RdRp) were calculated for methyl-substituted uridine analogs. The 2'-C-methyl-uridine and 4'-C-methyl-uridine scaffolds delivered favorable substrate binding to the host kinase and HCV RdRp that were consistent with results from cellular antiviral activity in support of our new approach. In a prospective evaluation, FEP results suggest that 2'-C-dimethyl-uridine scaffold delivered favorable monophosphate and triphosphate substrates for both host kinase and HCV RdRp, respectively. Novel 2'-C-dimethyl-uridine monophosphate prodrug was synthesized and exhibited sub-micromolar inhibition of HCV replication. Using this novel approach, we demonstrated for the first time that nucleoside analogs can be rationally designed that meet the multi-target requirements for antiviral activity.

Keywords: RNA-dependent RNA polymerase; alchemical free energy perturbation; flavivirus; nucleoside antiviral agents; structure-based drug design; viral polymerase.

Fig. 1

Activation of uridine analogs to inhibit HCV replication

Fig. 2

Analysis of compound 3 species interacting with UMPK and HCV RdRp from molecular dynamics simulations. A) 3-MP (elemental) bound to UMPK model active site (electrostatic surface) from the final frame of the 100 ns simulation. Hydrogen bond interactions are shown as yellow dashes, and the catalytic Mg²⁺ is shown as a pink sphere. B) Simulation interaction diagram for 3-MP bound to UMPK. Cationic residues are shown as purple circles, anionic residues are red, hydrophobic are green, and polar are cyan. Arrows indicate hydrogen bonds present in the provided percentage of analyzed frames. C) 3-TP (elemental coloring) in the active site of HCV RdRp from the final frame of the 50 ns simulation. Yellow dash lines show canonical base pairing to the template ADE. D) Simulation interaction diagram for 3-TP bound to HCV RdRp using the same scheme as (B). E) Conformation of the 3 scaffold bound to UMPK and HCV RdRp. Dihedrals were measured and averaged during the 600 equilibrated frames of each molecular dynamics simulation: β (P-O5’-C5’-C4’), γ (O5’-C5’-C4’-C3’), δ (C5’-C4’-C3’-O3’), and χ (O4’-C1’-N1-C2).

Scheme 1.

Synthesis of compounds 3 and 3-MP (prodrug). Reagents and conditions: (a) Ethyl-2-bromoisobutyrate, Zn, THF, 80 °C, 2 h, 42%; (b) 80% AcOH in H₂O, ACN, 70 °C, 3 h, 72%; (c) BzCl, Et₃N, 1,4-dioxane 0 °C to rt, 30 min, 56%; (d) LiAl(OtBu)₃H, THF, 0 °C to rt, 2 h, 86%; (e) MsCl, Et₃N, DCM, 0 °C to rt, 2 h, 82%; (f) Uracil, BSA, TMSOTf, DCE, rt, 16 h, 78%; (g) NH₃, MeOH, rt, 12 h, 70%; (h) NMI, THF, 4 °C, 12 h, 28%.

Scheme 2.

Synthesis of compound 6 and 6-MP (prodrug). Reagents and conditions: (a) 2,2-dimethoxypropane, p-TSA, DMF, acetone, 65 °C, 20 h, 86%; (b) (i) t-BuMgCl, THF, 0 °C to rt, 20 h (ii) 70% HCOOH, 50 °C, 1 h, 42% over two steps.