Identification of inhibitors that block vaccinia virus infection by targeting the DNA synthesis processivity factor D4

Abstract

Smallpox was globally eradicated 30 years ago by vaccination. The recent threat of bioterrorism demands the development of improved vaccines and novel therapeutics to effectively preclude a reemergence of smallpox. One new therapeutic target is the vaccinia poxvirus processivity complex, comprising D4 and A20 proteins that enable the viral E9 DNA polymerase to synthesize extended strands. Five compounds identified from an AlphaScreen assay designed to disrupt A20:D4 binding were shown to be effective in: (i) blocking vaccinia processive DNA synthesis in vitro, (ii) preventing cellular infection with minimal cytotoxicity, and (iii) binding to D4, as evidenced by ThermoFluor. The EC(50) values for inhibition of viral infectivity ranged from 9.6 to 23 μM with corresponding selectivity indices (cytotoxicity CC(50)/viral infectivity EC(50)) of 3.9 to 17.8. The five compounds are thus potential therapeutics capable of halting smallpox DNA synthesis and infectivity through disruptive action against a component of the vaccinia processivity complex.

Figure 1

Hit compounds from HTS. Structures of the indentified hits represent unique chemical scaffolds.

Figure 2

Functional validation of hits. (A) Inhibition VACV processive DNA synthesis. Processive DNA synthesis was conducted using the rapid plate assay in which extended strand synthesis is quantified by the incorporation of DIG-dUTP. The percent inhibition was calculated relative to the uninhibited negative control (DMSO) and the completely inhibited positive control (EDTA). (B) Inhibition of VACV infection. BSC-1 cells were cultured to confluence and infected with vaccinia virus to achieve ~ 80 PFU/well on a 48-well well plate. DMSO was maintained at 1%, and the representative dose-response curves are shown for both assays. Experiments were performed in triplicates and repeated at least twice. For comparison, Fentichlor, a known processive DNA synthesis inhibitor, was employed.

Figure 2

Functional validation of hits. (A) Inhibition VACV processive DNA synthesis. Processive DNA synthesis was conducted using the rapid plate assay in which extended strand synthesis is quantified by the incorporation of DIG-dUTP. The percent inhibition was calculated relative to the uninhibited negative control (DMSO) and the completely inhibited positive control (EDTA). (B) Inhibition of VACV infection. BSC-1 cells were cultured to confluence and infected with vaccinia virus to achieve ~ 80 PFU/well on a 48-well well plate. DMSO was maintained at 1%, and the representative dose-response curves are shown for both assays. Experiments were performed in triplicates and repeated at least twice. For comparison, Fentichlor, a known processive DNA synthesis inhibitor, was employed.

Figure 3

Assessment of compound binding by ThermoFluor. (A) As heat is applied, protein denaturation is measured spectrofluorimetrically with the hydrophobic dye, Sypro Orange. Any perturbation produced when a compound is added is reflected in the thermal shift (ΔT_m), which is the difference in the unliganded (T_m1) and liganded complex (T_m2). (B) The thermograms of various compounds are shown, with the 1% DMSO mock indicated by the solid trace. (C) Structural representation of uracil (green) and compound 10 (yellow) docked to the VACV D4. The catalytic acid/base (H181/D68) of the UDG pocket and the amino acids shown to be important in processivity are presented. Both uracil and compound 10 conformations represent high-ranked poses as scored by ICM-Pro.

Figure 3

Assessment of compound binding by ThermoFluor. (A) As heat is applied, protein denaturation is measured spectrofluorimetrically with the hydrophobic dye, Sypro Orange. Any perturbation produced when a compound is added is reflected in the thermal shift (ΔT_m), which is the difference in the unliganded (T_m1) and liganded complex (T_m2). (B) The thermograms of various compounds are shown, with the 1% DMSO mock indicated by the solid trace. (C) Structural representation of uracil (green) and compound 10 (yellow) docked to the VACV D4. The catalytic acid/base (H181/D68) of the UDG pocket and the amino acids shown to be important in processivity are presented. Both uracil and compound 10 conformations represent high-ranked poses as scored by ICM-Pro.

Figure 3

Assessment of compound binding by ThermoFluor. (A) As heat is applied, protein denaturation is measured spectrofluorimetrically with the hydrophobic dye, Sypro Orange. Any perturbation produced when a compound is added is reflected in the thermal shift (ΔT_m), which is the difference in the unliganded (T_m1) and liganded complex (T_m2). (B) The thermograms of various compounds are shown, with the 1% DMSO mock indicated by the solid trace. (C) Structural representation of uracil (green) and compound 10 (yellow) docked to the VACV D4. The catalytic acid/base (H181/D68) of the UDG pocket and the amino acids shown to be important in processivity are presented. Both uracil and compound 10 conformations represent high-ranked poses as scored by ICM-Pro.