N-arylpyrimidinamine (NAPA) compounds are broadly acting inhibitors of human cytomegalovirus infection and spread

Abstract

Human cytomegalovirus (HCMV) is a β-herpesvirus that contributes to the disease burden of immunocompromised and immunomodulated individuals, including transplant recipients and newborns. The FDA-approved HCMV drugs can exhibit drug resistance and severe side effects including bone marrow toxicity, gastrointestinal disruption, and nephrotoxicity. In a previous study, we identified the N-arylpyrimidinamine (NAPA) compound series as a new class of HCMV inhibitors that target early stages of infection. Here we describe the inhibitory activity of two potent NAPA analogs, MBXC-4336 and MBX-4992, that broadly block infection and spread. MBXC-4336 and MBX-4992 effectively inhibited infection by diverse HCMV strains and significantly prevented virus spread in fibroblast and epithelial cells as evaluated by quantifying infected cells and viral genome levels. Further, the NAPA compounds limited replication of clinical HCMV isolates, including a ganciclovir-resistant strain. Importantly, combination studies of NAPA compounds with ganciclovir demonstrated additive or synergistic inhibition of HCMV spread. Collectively, NAPA compounds have therapeutic potential for development as a novel class of anti-HCMV drugs.

Keywords: Broad spectrum antivirals; Combination therapy; Human cytomegalovirus; NAPA compounds; Synergy.

Fig. 1.. NAPA analogs effectively prevent HCMV infection.

HCMV strain AD169R treated with NAPA compounds MBX-4325, MBX-4330, MBXC-4336, and MBXC-4992 followed by infection (MOI = 0.2) of NHDF cells were analyzed 24hpi with the virus infectivity assay. The relative infection (%) was determined using DMSO-treated cells as 100% infection. Error bars represent standard deviation from the mean. Statistical tests were performed using ordinary two-way ANOVA with multiple comparisons to DMSO treated cells as a control and a Dunnett’s post-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; lack of * indicates not significant. The chemical structure for each compound is shown below the graph.

Fig. 2.. NAPA compounds MBXC-4336 and MBX-4992 broadly limit HCMV infection.

HCMV strains TB40/E, TR, and Towne pretreated with 0, 1, 5, and 20 μM MBXC-4336 (A) or MBX-4992 (B) followed by infection (MOI = 0.2) of NHDF or ARPE-19 cells were analyzed 24hpi with the virus infectivity assay. The relative infection (%) was determined using DMSO-treated virus as 100% infection. Error bars represent standard deviation from the mean. Statistical tests were performed using ordinary two-way ANOVA with multiple comparisons to DMSO treated cells as a control and a Dunnett’s post-test: *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; lack of * indicates not significant.

Fig. 3.. MBXC-4336 inhibits HCMV infection post-attachment to cells.

MBXC-4336 (5 μM), MBX-4992 (5 μM), and anti-gH antibody 1C10 (10 μg/ml) were either preincubated with TB40/E (MOI = 0.2) or added at relative time post-infection (A) on ARPE-19 or NHDF cells. Relative infection (%) was quantified at 24hpi via an anti-IE1 immunostain and untreated infected cells was normalized to 100% (B). TB40/E (MOI = 0.2) pretreated with MBXC-4336 or MBX-4992 (5 μM) and heparin (100 μg/mL) was added to cells or diluted 50-fold before infection of NHDF cells. Virus was also pretreated with 0.1 μM of MBXC-4336 and MBX-4992 and 2 μg/mL of heparin prior to infection (C). At 24hpi, infection was assessed using the virus infectivity assay, with relative infection (%) determined using DMSO-treated virus as 100% infection for each respective condition (D). Error bars represent standard deviation from the mean.

Fig. 4.. NAPA compounds reduce virus foci upon addition post-infection.

MBXC-4336 (A) and MBX-4992 (B) (0–50 μM) were added to TB40/E (MOI = 0.1) infected NHDF cells 48 hpi. Controls include TB40/E preincubated (PI) with DMSO (−), MBXC-4336 and MBX-4992 (5 μM), and ganciclovir (GCV, 2.5 μM). At 9dpi, virus foci (~5,000 μm²) were evaluated based on IE1 staining. The relative virus foci (%) were designated to 100% based on virus foci from DMSO treated cells. Statistical tests were performed using ordinary two-way ANOVA with multiple comparisons to DMSO treated cells as a control and a Dunnett’s post-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; lack of * indicates not significant.

Fig. 5.. NAPA compounds limit HCMV spread.

TB40/E pretreated with 0, 1.25, 2.5, 5, 10, and 20 μM MBXC-4336 (A) and MBX-4992 (B), or 5, 10 μM ganciclovir (GCV) (A and B) followed by infection (MOI = 0.1) of ARPE-19 or NHDF cells prior to addition of 1% SeaPlaque agarose in which compound treatments were replaced at 3 and 6dpi. At 9dpi, infected cells were determined and relative infection (%) was calculated based on DMSO-treated virus as 100%. Error bars represent standard deviation from the mean. Statistical tests were performed using ordinary two-way ANOVA with multiple comparisons to DMSO treated cells as a control and a Dunnett’s post-test: ***, p < 0.001; ****, p < 0.0001.

Fig. 6.. NAPA compounds limit HCMV replication and spread.

TB40/E pretreated with 0, 1.25, 2.5, 5, 10, and 20 μM MBXC-4336 (A and B) and MBX-4992 (C and D) or 5, 10 μM ganciclovir (GCV) followed by infection (MOI = 0.1) of ARPE-19 (A and C) or NHDF (B and D) cells were assessed for HCMV genome levels at 9dpi after drug replenishment at days 3 and 6pi. HCMV clinical isolates sensitive (PT37, E) or resistant (CH7, F) to GCV proliferating in ARPE-19 cells were treated with 5 and 20 μM of MBXC-4336, MBX-4992, or GCV for up to 9 days while replacing the drugs at days 3 and 6. Letermovir (LTV, 0.02 μM) was used to treat the GCV-resistant clinical isolate as a positive control. The HCMV relative genome levels (2^ΔΔCt) were quantified for the UL83 gene relative to wild-type, DMSO-treated cells after normalizing to housekeeping gene RPS11 expression. The bar represents the relative mean. Statistical tests were performed using ordinary two-way ANOVA with multiple comparisons to DMSO treated cells as a control and a Dunnett’s post-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; lack of * indicates not significant.

Fig. 7.. NAPA/ganciclovir combinations effectively inhibit HCMV.

Combinations of MBXC-4336 (A,C) or MBX-4992 (B,D) with ganciclovir were evaluated for limiting spread of TB40/E (MOI = 0.05) in ARPE-19 epithelial (A, B) and NHDF (C, D) cells at 9 dpi. All treatments were replaced at 3 and 6dpi. Relative infection (%) was calculated based on DMSO-treatment as 100% and these values were used to determine the Loewe synergy scores (depicted in accompanying heat maps). Error bars represent standard deviation from the mean. Statistical tests were performed using ordinary two-way ANOVA with multiple comparisons to DMSO treated cells as a control and a Dunnett’s post-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. p values were based on DMSO-treatment in NAPA series.