Investigating N-arylpyrimidinamine (NAPA) compounds as early-stage inhibitors against human cytomegalovirus

Abstract

Human cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

Keywords: Congenital CMV and transplant recipients; Early-stage infection inhibitors; High-throughput screening; Human cytomegalovirus; NAPA compounds.

Figure 1:. Screening strategy to identify early stage CMV inhibitors.

(A) The schematic and criteria of the high-content screen to identify inhibitors of CMV infection using AD169^IE2-YFP/fibroblast infection. (B) Structure of MBXC-4302.

Figure 2.. MBXC-4302 inhibits CMV infection.

CMV strains AD169^R (A and C) and TB40/E (B and D) infection (MOI=0.2) of fibroblasts (A-B) and ARPE-19 cells (C-D) pretreated with 0, 1, 5, and 20 μM MBXC-4302 were assessed 24hpi based on GFP fluorescence (A,C) or IE1 (B,D) expression by an anti-IE1 immunostain. Relative % infection was determined using DMSO-treated cells as 100% infection. The data points are the averages of three replicates and the error bars are the standard deviation. Statistical tests were performed using one-way ANOVA with multiple comparisons to DMSO-treated cells as a control and a Dunnett’s post-test; ****, p<0.0001.

Figure 3.. NAPA compound MBX-4992 significantly limits CMV infection in vitro.

CMV strains AD169^R (A,C) and TB40/E (B,D) infection (MOI=0.2 or 0.4, respectively) of fibroblasts (A-B) and ARPE-19 cells (C-D) pre-treated with 0, 1, 5, and 20 μM MBX-4992 were assessed 24hpi based on GFP fluorescence (A,C) or anti-IE1 immunostain (B,D). Relative % infection was determined using DMSO-treated cells as 100% infection. The data points are the averages of three replicates with error bars indicating standard deviation. Statistical tests were performed using one-way ANOVA with multiple comparisons to DMSO-treated cells as a control and a Dunnett’s post-test; ***, p<0.001; ****, p<0.0001. (E) Total cell lysates from AD169^R-infected (MOI=0.2) NHDF cells pretreated with DMSO, MBX-4992 (1-10 μM), ganciclovir (5 μM) or convallotoxin (0.01 μM) collected at 24hpi were subjected to Western blot analysis using a rabbit anti-IE1 antibody (Lanes 1-6) and an anti-GAPDH antibody (Lanes 7-12) as a loading control. The molecular weight markers and respective proteins are indicated.

Figure 4.. NAPA compounds target an early, post-attachment step.

(A) MBX-4992 (10 μM) was added along a time course following infection with AD169 virus (MOI=0.2) from −60 mins prior to infection to 90 mpi on NHDF cells. Infection at 24 hours was quantified with anti-IE1 immunostain and untreated virus-infected cells was normalized to 100%. (B) NHDFs were preincubated with DMSO, MBX-4992, or heparin for 30 mins at 37°C followed by the addition of AD169^R (MOI=0.2) for 30 minutes at 4°C. Cell media was either unchanged (No Wash) or removed (Wash) and replaced with media containing DMSO, MBX-4992, or heparin. (C) AD169^R (MOI=0.2) pre-incubated with DMSO, MBX-4992 (1 μM), or heparin (50μg/ml) for 30 mins at 37°C was added to fibroblasts for 30 min at 4°C. Cells were not washed (No Wash) or media was removed (Wash) and replaced with media containing either DMSO, MBX-4992, or heparin shifting cells to 37°C. Virus infection was quantified at 24hpi by GFP fluorescence with DMSO-treated virus normalized to 100%. The data points are the averages of three replicates with error bars. Statistical tests were performed using ordinary one-way ANOVA with multiple comparisons to DMSO-treated cells as a control and a Dunnett’s post-test: *, p<0.05; ****, p<0.0001.