Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents

Abstract

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

Keywords: DDX3; broad spectrum antivirals; coinfections; host factors; resistance.

Fig. 1.

2D structures of the known human DDX3 inhibitors targeting the RNA binding pocket.

Fig. 2.

Graphical representation of the DDX3 RNA binding site. The RNA strand is represented as yellow carbon sticks. The binding mode of compound 2 (green carbon sticks) was predicted by docking studies. Hydrogen bond interactions are visualized as black dashed lines. Compound 2 occupies a little part of the large pocket, and the two regions circled in magenta and cyan are unexplored by this ligand.

Fig. S1.

Synthesis of derivatives 9–12. Reagents and conditions: a, CH₂Cl₂, 5 h, room temperature (r.t.); b, HCl/AcOH, 6 h, 100 °C.

Fig. S2.

Synthesis of derivatives 16a–16g: a, CH₂Cl₂, 5 h reflux; b, H₂, Pd/C, MeOH, 1 h; c, t-BuONO, CH₃CN, 20 min, 0 °C, then TMSiN₃, CH₃CN, 2 h room temperature (r.t.); d, opportune alkyne, CuSO₄○5 H₂O, sodium ascorbate, H₂O t-BuOH (1:1), microwave (MW) 120 °C, 10 min; e, opportune alkynoic acid, CuCl, l-proline, K₂CO₃, DMSO, MW 65 °C, 20 min.

Fig. 3.

Binding mode of compound 16d (purple sticks). For comparison purpose, compound 2 (green sticks) was also visualized.

Fig. 4.

Dose–response curves for DDX3 helicase activity inhibition by 16d at increasing dsRNA substrate concentrations.

Fig. 5.

Representative images of the histological examination of H&E-stained sections of (A1–E1) livers, (A2–E2) kidneys, and (A3–E3) brains from three groups of rats. (A1–A3 and B1–B3) VG. (C1–C3 and D1–D3) TG. (E1–E3) WT group. Treated rats do not exhibit abnormal histopathological changes compared with the control group. The microphotographs were taken using a digital camera (Nikon SLR-D3000) at original magnifications of 100× and 200×. gt, Glomerular tuft; U, urinary space.

Fig. 6.

Pharmacokinetic parameters and tissue distribution in rats; 16d was administered as a single i.v. bolus injection of 10 mg/kg per group (n = 3). Data points represent the means ± SDs. (A) Plasma level curves of 16d. The semilogarithmic plot is shown in Inset. The elimination curve showed a first-order kinetic that showed an exponential decrease in the semilogarithmic plot. (B) Concentration levels of 16d in rat tissues at 24 h after single-dose administration.