pubmed.ncbi.nlm.nih.gov

Two-Metal Ion-Dependent Enzymes as Potential Antiviral Targets in Human Herpesviruses - PubMed

  • ️Sat Jan 01 2022

Two-Metal Ion-Dependent Enzymes as Potential Antiviral Targets in Human Herpesviruses

Katherine A DiScipio et al. mBio. 2022.

Abstract

The majority of drug discovery efforts against herpesviruses have focused on nucleoside analogs that target viral DNA polymerases, agents that are associated with dose-limiting toxicity and/or a narrow spectrum of activity. We are pursuing a strategy based on targeting two-metal ion-dependent (TMID) viral enzymes. This family of enzymes consists of structurally related proteins that share common active sites containing conserved carboxylates predicted to coordinate divalent cations essential for catalysis. Compounds that target TMID enzymes, such as HIV integrase and influenza endoribonuclease, have been successfully developed for clinical use. HIV integrase inhibitors have been reported to inhibit replication of herpes simplex virus (HSV) and other herpesviruses; however, the molecular targets of their antiviral activities have not been identified. We employed a candidate-based approach utilizing several two-metal-directed chemotypes and the potential viral TMID enzymatic targets in an effort to correlate target-based activity with antiviral potency. The panel of compounds tested included integrase inhibitors, the anti-influenza agent baloxavir, three natural products previously shown to exhibit anti-HSV activity, and two 8-hydroxyquinolines (8-HQs), AK-157 and AK-166, from our in-house program. The integrase inhibitors exhibited weak overall anti-HSV-1 activity, while the 8-HQs were shown to inhibit both HSV-1 and cytomegalovirus (CMV). Target-based analysis demonstrated that none of the antiviral compounds acted by inhibiting ICP8, contradicting previous reports. On the other hand, baloxavir inhibited the proofreading exonuclease of HSV polymerase, while AK-157 and AK-166 inhibited the alkaline exonuclease UL12. In addition, AK-157 also inhibited the catalytic activity of the HSV polymerase, which provides an opportunity to potentially develop dual-targeting agents against herpesviruses. IMPORTANCE Human herpesviruses (HHVs) establish lifelong latent infections, which undergo periodic reactivation and remain a major cause of morbidity and mortality, especially in immunocompromised individuals. Currently, HHV infections are treated primarily with agents that target viral DNA polymerase, including nucleoside analogs; however, long-term treatment can be complicated by the development of drug resistance. New therapies with novel modes of action would be important not only for the treatment of resistant viruses but also for use in combination therapy to reduce dose-limiting toxicities and potentially eliminate infection. Since many essential HHV proteins are well conserved, inhibitors of novel targets would ideally exhibit broad-spectrum activity against multiple HHVs.

Keywords: HCMV; HSV; ICP8; cytomegalovirus; herpes alkaline nuclease; herpes simplex virus; human cytomegalovirus; integrase inhibitors; proofreading exonuclease; two-metal ion-dependent enzymes; viral nucleases; viral polymerases.

PubMed Disclaimer

Conflict of interest statement

The authors declare a conflict of interest. L.R.W., D.L.W., and S.K.W. are cofounders of a small start-up antiviral company, Quercus Molecular Design. No funds were obtained from the company for this work.

Figures

FIG 1
FIG 1

Small molecule agents targeting two-metal ion-dependent enzymes (A) Blue highlighted regions indicate planar arrays of heteroatoms that can form two or more adjacent chelates. (B) Structure of raltegravir bound to the prototype foamy virus (PFV) intasome showing the coordination of drug to the two catalytic magnesium ions that are bound to the enzyme through a DDE motif (PDB ID no.

3OYA

).

FIG 2
FIG 2

Antiviral activity and cytotoxicity of compounds used in this study. (A) HFF cells were infected with KOS at an MOI of 0.1 PFU/cell in the presence of compound at concentrations of 2, 10, and 50 μM. At 48 hpi, cultures were collected, and viral titer was determined by plaque assay. The percentage of inhibition was calculated compared to the DMSO control. Values are the averages of data from three independent experiments. (B) Cytotoxicity of compounds on HFF cells after a 48-h incubation period was evaluated using the Promega Cell Titer-Glo assay, following the manufacturer’s instructions. (C) Dose-dependent cytotoxicity of BXA, AK-157, and AK-166 on HFF cells at 65 h posttreatment was measured using Promega Cell Titer-Glo. (D) HFF cells were infected with HSV (KOS strain) at an MOI of 0.1 PFU/cell, and viral titers were determined by plaque assay at 48 hpi. (E) HFF cells were infected with HCMV (Towne strain expressing pp28-luciferase) at an MOI of 2 PFU/cell, and viral infection was monitored at 65 hpi using Promega SteadyGlo. The data shown in panels D and E represent three duplicates within one experiment. (F) Calculated EC50 and 50% cytotoxic concentration (CC50) values for BXA, AK-157, and AK166 represent averages from three independent experiments. The selectivity index (SI) is given as CC50/EC50. *, P < 0.05 compared with the DMSO control by two-way ANOVA and Holm-Sidak multiple-comparison test. CC50 and EC50 values were compared with a nonlinear regression model, and groups were significantly different at P < 0.05.

FIG 3
FIG 3

Inhibition of ICP8-mediated activities. ICP8 (100 nM) was preincubated with 20 μM (A) or 100 μM (B) compound in 1.6% DMSO for 10 min at 37°C. Annealing reactions were initiated by addition of 50 ng linearized, heat-denatured plasmid DNA, and the mixtures were incubated at 37°C for 45 min. The presence of dsDNA was measured by PicoGreen fluorescence, and the data represent the average from three independent experiments. (C) ssDNA binding by ICP8 (200 nM) was assessed by EMSA in the presence of select compounds at the concentrations shown using 100 nM Cy5-labled 25-nt dT oligomer. Binding reactions (30 min at 37°C) were analyzed by 5% nondenaturing polyacrylamide gel electrophoresis. For panels A and B, the data represent the average from three independent experiments. *, P < 0.05 compared with DMSO control by one-way ANOVA and Holm-Sidak multiple comparisons test. For panel C, representative data from three independent experiments are shown. *, P < 0.05 compared with the DMSO control by one-way ANOVA and Holm-Sidak multiple-comparison test. # indicates that at these concentrations, compound precipitation was observed.

FIG 4
FIG 4

Inhibition of UL15C nuclease activity. A dual-probe assay was used to access inhibition of UL15C nuclease activity at 12.5 μM. The percentage of inhibition due to compounds was calculated compared to that of the uninhibited DMSO control. The data shown represent an average from three independent experiments in which each experiment is an average of three technical replicates. *, P < 0.05 compared with the DMSO control by one-way ANOVA and Holm-Sidak multiple-comparison test.

FIG 5
FIG 5

Inhibition of UL12 nuclease activity. UL12 nuclease activity was measured using the PicoGreen assay at a 20 μM inhibitor concentration. Any background fluorescence signals derived from inhibitors were subtracted from corresponding total RFU values, and the percentage of inhibition of UL12 nuclease activity was calculated compared to that of the uninhibited DMSO control. Data presented represent an average from three independent experiments in which each experiment is an average of three technical replicates. *, P < 0.05 compared with the DMSO control by one-way ANOVA and Holm-Sidak multiple-comparison test.

FIG 6
FIG 6

Inhibition of UL30 functions. (A) PolExo activity was measured using a gel-based assay to detect degradation of 5′-Cy5-tagged 25-mer oligo(dT) substrate using full-length His-tagged UL30 at a 20 μM drug concentration. (B) Polymerase activity was assayed using an N-terminally-deleted version of UL30 (UL30ΔN42) at a 20 μM drug concentration. A gel-based assay was used to monitor extension of a primer/template consisting of a 5′-FAM-labeled 15-nt primer annealed to a 35-nt oligonucleotide. The data shown are representative of three independent experiments, and representative panels are shown.

FIG 7
FIG 7

Viral gene expression. HFF cells were infected with KOS at an MOI of 5 PFU/cell in the presence of 4 μM compound ACV (A), AK-157 (B), or AK-166 (C) or DMSO alone for 3, 6, 9, and 12 h. Cell lysates were resolved by10% SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to proteins representing all three kinetic classes of viral gene expression: (i) ICP4 (immediate early), (ii) ICP8 and UL12 (early), and (iii) gC and UL32 (late).

FIG 8
FIG 8

Viral DNA synthesis as assessed by qPCR. HFF cells were infected with KOS at an MOI of 5 PFU/cell and incubated in the presence of either compound or DMSO only for 12 h. qPCR was used to determine the amount of viral DNA by measuring the copy number of the UL9 gene. The accumulation of the 200-nt fragment from the UL9 gene was monitored using SsoAdvanced Universal SYBR green supermix (Bio-Rad). Data represent an average from three independent experiments.

FIG 9
FIG 9

Replication compartment formation in the presence of compounds. (A) HFF cells were infected with KOS at an MOI of 5 PFU/cell in the presence of 0.5% DMSO or 4 μM AK-157 or ACV. At 6 hpi, cells were fixed for analysis by IF to detect ICP8 (green) or Hoechst staining to label nuclei (gray). (B) An average of 120 or more cells was counted from each treatment condition and classified according to the size of replication compartments. The prevalence of each category is presented as a percentage based on total ICP8-positive cells. Values represent an average from two independent experiments. *, P < 0.05 compared with the DMSO control by one-way ANOVA and Holm-Sidak multiple-comparison test.

Similar articles

Cited by

References

    1. Looker KJ, Magaret AS, May MT, Turner KME, Vickerman P, Gottlieb SL, Newman LM. 2015. Global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012. PLoS One 10:e0140765. doi:10.1371/journal.pone.0140765. - DOI - PMC - PubMed
    1. Gupta R, Warren T, Wald A. 2007. Genital herpes. Lancet 370:2127–2137. doi:10.1016/S0140-6736(07)61908-4. - DOI - PubMed
    1. Elion GB. 1982. Mechanism of action and selectivity of acyclovir. Am J Med 73:7–13. doi:10.1016/0002-9343(82)90055-9. - DOI - PubMed
    1. Epstein JB, Scully C. 1991. Herpes simplex virus in immunocompromised patients: growing evidence of drug resistance. Oral Surg Oral Med Oral Pathol 72:47–50. doi:10.1016/0030-4220(91)90188-I. - DOI - PubMed
    1. Bacon TH, Levin MJ, Leary JJ, Sarisky RT, Sutton D. 2003. Herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy. Clin Microbiol Rev 16:114–128. doi:10.1128/CMR.16.1.114-128.2003. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources