Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay

Abstract

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

Keywords: E3 ubiquitin ligase; HSV; ICP0; high-throughput screening assay; small-molecule inhibitors.

FIG 1

ICP0 HTS assay. (A) GST-ICP0 fusion proteins used for the HTS assay. ICP0 exon II spans amino acids 20 to 241 of ICP0 from HSV-1(F), while the portion of exon III of ICP0 that was used as a control spans amino acids 543 to 768 (ICP0 exon III-C). (B and C) HTS assay measuring the proximity of the ubiquitin (Ub) moiety to ICP0.

FIG 2

ICP0 ubiquitination assay optimization. The HTRF based ubiquitination assay was performed as discussed in Materials and Methods. (A) Specificity and time dependence of ubiquitination. (Upper) Ubiquitination was found to be specific for the WT GST-ICP0 exon II protein. The RF mutant protein, GST-ICP0 exon II (RF), showed more than 50% reduction in ubiquitination, whereas little or no ubiquitination was observed with GST-ICP0 exon III-C or with the GST protein alone. GST-Mdm2, a control protein, was ubiquitinated to much lower extent than the exon II protein, indicating that the assay parameters were highly specific for ubiquitination of the exon II protein. (Lower) Only a slight increase (1.5-fold) in ubiquitination activity of exon II (RF) was observed after 3 h of incubation, whereas ubiquitination of other proteins remained unchanged over time. ΔRatio was obtained after reads from ICP0 control reactions and UbcH5a control reactions were subtracted from the reads of reactions containing both ICP0 and UbcH5a. (B) Signal uniformity. The uniformity of the HTRF signal was tested in three independent experiments, using low-volume 384-well plates. A window of 5.5 ± 0.2 between complete ubiquitination reactions and controls (lacking exon II) was observed across the three experiments, with an average coefficient of variation of 3.9% and an average Z′ score of 0.8 ± 0.02. (C) Distribution of Z′ scores across all of the plates screened using the optimized assay (∼5,000 compounds). The assay gave an average Z′ score of 0.84 ± 0.04, indicating good separation between the positive and negative controls on each plate.

FIG 3

Selectivity of the hits for GST-ICP0 exon II ubiquitination. The hits identified from the primary screen were picked from HTS library stocks, and their activities were confirmed in HTRF assays containing either GST-ICP0 exon II or GST-Mdm2 protein. Except for compound B, all of the hits showed greater activity against ICP0 exon II than against Mdm-2.

FIG 4

Effects of compounds on accumulation of ICP0 substrates and on viral gene expression. HEp-2 cells were infected with HSV-1(F) (5 PFU/cell). The compounds were added to the cultures at 10 or 100 μM at the time of infection, and the cells were harvested at 8 h postinfection. Equal amounts of proteins from total cell lysates were analyzed by immunoblot analysis using antibodies against ICP0, ICP4, UL38, β-actin, and USP7 (left). Uninfected cells (lanes 1, 9, 15, and 21) and infected but untreated cells (lanes 2, 10, 16, and 22) served as controls. Quantification of the protein bands was performed using Image J software (right).

FIG 5

Effects of selected hits on HSV-1 growth. HEL cells were infected with HSV-1(F) (0.01 PFU/cell) or ΔICP0 virus (0.01 PFU/cell) and either not treated or treated with compound A, C, G, or H at 10 or 100 μM, added to the cultures at the time of infection. PAA was added at 500 μg/ml at the time of infection. Infection with the ICP0 RF mutant was performed at 0.01 PFU/cell. Cells were harvested at 3, 24, and 48 h postinfection, and titration of progeny viruses was performed in Vero cells. **, P ≤ 0.01.

FIG 6

Effects of selected hits on HSV-1 and ΔICP0 virus gene expression. (A) U2OS cells were infected with HSV-1(F) or ΔICP0 virus (0.1 PFU/cell) and either not treated or treated with compound A, C, G, or H at 10 or 100 μM, added to the cultures at the time of infection. The cells were harvested at 2, 4, and 8 h postinfection, and viral gene expression was assessed by qPCR analysis. The 18S rRNA primers were used for normalization. (B) HEp-2 cells were treated as in Fig. 4 and pictures were acquired at 24 h postinfection, using an inverted Nikon Eclipse TE2000-S microscope equipped with a Nikon camera.

FIG 7

Effects of the compound A analogues on HSV-1 viral gene expression and the stability of ICP0. (A) HEL cells were infected with HSV-1(F) or ΔICP0 virus (0.1 PFU/cell) and either not treated or treated with compound A, A-1, A-2, or A-3 at 20 μM, added to the cultures at the time of infection. The cells were harvested at 8 h postinfection, and viral gene expression was quantified by qPCR analysis. The 18S rRNA primers were used for normalization. Error bars represent standard deviations. Statistical analysis was performed using unpaired t tests. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) HEp-2 cells were infected with HSV-1(F) or an ICP0 RF mutant virus (5 PFU/cell). CHX (100 μg/ml) was added to the cultures at 7 h postinfection. The cells were harvested at the time of CHX addition or at 1, 2, 3, 4, and 5 h posttreatment, and ICP0 protein was assessed in equal amounts of cell lysates by immunoblot analysis. β-Actin served as a loading control. (C) HEp-2 cells were infected with HSV-1(F) (2 PFU/cell), compound A-1, A-2, or A-3 (50 μM) was added to the infected cultures at 4.5 h postinfection, and CHX (100 μg/ml) was added to the cultures 0.5 h later. The cells were harvested at 0, 1, 2, 3, and 4 h after the addition of CHX, whereas an infected untreated sample harvested at 9 h after infection served as a control. The amounts of ICP0 protein were assessed in equal amounts of cell lysates by immunoblot analysis. β-Actin served as a loading control. (D) HEp-2 cells were infected with HSV-1(F) (2 PFU/cell) for 4 h and incubated with the indicated compounds (50 μM) for an extra 1 h. Cells were then collected and CETSA was performed. ICP0 temperature stability was assessed in the soluble fraction by immunoblot analysis. (E) The experiment was as in panel D except that HEL cells were used and compound A was added to the cultures at 20 μM for 1 h at 16 h postinfection.