Summary /

The initial study tested the ability of ML324 to inhibit HSV viral IE infection in HFF cells (Hamada 2009). ML324, potently reduced IE gene expression (IC50 = 10 μM) compared to 0.75 mM for DMOG, without an impact on the expression of the cellular controls Sp1, S15 and TBP. Next, HFF cells were treated with various concentrations of ML324 and infected with HSV-1 (0.1 pfu/cell) for 24 hr. ML324 reduced viral yields in a dose-dependent manner (~4-5 logs at 25 μM, Figure 1B) whereas 1.5 mM of DMOG was required to achieve the same reduction (data not shown). HSV-1 infected MRC-5 cells were treated with DMSO or ML324 at various time points (2 hr pre-infection to 4 hr post infection) and mRNA levels of IE viral genes (ICP4, ICP22, ICP27) were measured relative to DMSO at 4 hr post infection. As shown in Figure 12A, ML324, markedly reduced the gene transcription of all 4 viral IE genes, even when added 2 hr post-infection (>50% reduction). ML324 was tested for its ability to reduce viral yields in MRC-5 cells infected at high multiplicity of infection (MOI) (Figure 2D), and was found to display comparable viral yield reduction at half the concentration needed for ACV (acyclovir), 50 μM vs. 100 μM respectively. Furthermore, ML324 is shown as an efficacious antiviral agent, by observing significant reduction in HSV plaque formation in Vero cells and MRC-5 with 50 μM, as compared to 100 μM ACV (Figure 2B-C).

Figure 1. (A) Viral IE (ICP4, ICP27, ICP22) and control (S15, Sp1, TBP) mRNA levels in HFF cells treated with DMSO or the indicated concentrations of ML324 for 3 hrs and infected with HSV-1 (0.1 pfu/cell) for 3 hr. (B) Viral yields from HFF cells treated with DMSO or ML324 for 3 hr and infected with HSV-1 (0.1 pfu/cell) for 24 hr in the presence of the drugs.

Figure 2. (A) MRC-5 cells were treated with DMSO or ML324 at the indicated time relative to infection with HSV-1 (1.0 MOI). Viral and cellular mRNA levels were determined at 4 hr post infection. (B) Plaque assay of Vero cells infected with HSV-1 at the indicated MOI for 12 hr, followed by the addition of DMSO, ACV (100 μM), or ML324 (50 μM) for 48 hr. (C) Representative fields of MRC-5 and Vero cells infected with HSV-1 for 12 hr followed by addition of DMSO, ACV (100 μM) or ML324 (50 μM) for 48 hr (D) MRC-5 cells treated with DMSO or 50 μM ML324 were infected with HSV-1 at the indicated MOI. Viral IE and cellular mRNA levels were quantitated at 2 hr post infection; viral yields were determined at 24 hr post infection.

Figure 3. MRC-5 cells were treated with DMSO or 50 mM ML324 for 3 hr, followed by infection with hCMV (0.1 pfu/cell) for 5 hr. mRNA levels of viral and cellular controls are expressed relative to cells treated with DMSO. Data shown are means +/- SEM.

Summary /

To investigate the effect of ML324 on the spread of infection to adjacent cells, MRC-5 cells were infected with HSV-1 and visualized by immunofluorescent staining for UL29 (HSV DNA replication protein) (Figure 4). ML324 blocked viral gene expression at an early stage prior to expression of the viral replication protein UL29, thereby reducing the number of cells exhibiting viral antigens.

Figure 4. Immunofluorescent staining for the HSV DNA replication protein UL29. MRC-5 cells were infected with HSV-1 (0.1 MOI) for 8 hr, followed by the addition of DMSO, ACV (100 μM), or ML324 (50 μM) for 12 hr. Cells were stained with anti-UL29 (green), Dapi (blue), and Phalloidin-647 (F-actin, red).

Summary /

ML324 exhibited significant antiviral activity in the mouse sensory ganglia explant model of HSV reactivation. First, ML324 reduced the viral yield per ganglia by 4.5 logs at 50 μM compared to DMOG which required concentrations up to 2 mM to achieve 1.5 log reduction (Figure 5A). Immunofluorescent staining of explanted ganglia sections (Figure 5B & D) revealed that ML324 was capable of suppressing the primary viral reactivation events. The spread of the reactivated viral infection of the ganglia was demonstrated in the DMSO control where the viral lytic gene (UL29) expression was observed in both isolated and clustered neurons (Figure 5D). However, upon treatment with ML324, significant reduction in both isolated and clustered UL29(+) neurons was observed (note: ML324 was more efficacious than ACV in single neurons). Importantly, robust viral replication was observed following ML324 withdrawal which indicates the reduction in reactivation by ML324 was not simply due to the inability of the ganglia to support viral replication (Figure 5C). In addition to the encouraging results obtained for the herpes simplex virus (HSV) reported herein, ML324 also demonstrated comparable activity towards β-herpes human Cytomegalovirus (hCMV) which causes mortality in immuno compromised patients and is the most significant cause for viral birth defects (Arvin 2007, Fields 2007).  These data support the notion that the JMJD2 demethylases are required for both HSV and hCMV IE gene expression and demonstrate the effectiveness of ML324 as an antiviral agent.

Figure 5. (A) Viral yields from HSV-1 latently infected trigeminal ganglia explanted in the presence of DMSO, or 2 mM DMOG or 50 μM ML324 for 48 hr. [DMSO vs DMOG; Wilcoxon paired 2-tailed t test, p = .0015, n = 12; DMSO vs ML324, p < .0001, n = 20]. (B & D) Latently infected trigeminal ganglia were explanted in the presence of DMSO, 100 μM ACV, or 50 μM ML324 for 48 hr. Sections were costained with anti-UL29 (red), neurofilament 200 (green), and DAPI (blue) and scored for UL29(+) cell clusters (Clusters) and individual neurons (Single). [ACV vs ML324; 2-tailed t test, n = 48 sections representing 8 ganglia). The total number of UL29(+) per ganglia are graphed (D) and representative sections illustrated (B). Data shown are means +/- SEM. (C) Latently infected ganglia were explanted in the presence of DMSO, ACV (100 μM), or ML324 (50 μM) for 48 hr. Viral yields were determined from one half of each ganglia at 48 hr (DMSO, ACV, ML324) and from the other half of each ganglia after drug reversal for an additional 72 hr (ACV-R, ML324-R).